Studies in Environmental Science 27
FLUORIDE RESEARCH 1985 Selected Papers from the 14th Conference of the Internation...
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Studies in Environmental Science 27
FLUORIDE RESEARCH 1985 Selected Papers from the 14th Conference of the International Society for Fluoride Research, Morioka, Japan, 12-15 June 1985
Edited by
Humio Tsunoda Department of Hygiene & Public Health, School of Medicine, I wate Medical University, 19-1 lchimaru, Morioka, 020 Japan
Ming-Ho Yu Huxley College of Environmental Studies, Western Washington University, Bellingham, WA 98225, U.S.A.
ELSEVIE R
1986
Amsterdam - New York - Oxford - Tokyo
E L S E V I E R SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O.B o x 21 1, 1000 A E Amsterdam, T h e Netherlands
Distributors for the United States and Canada: E L S E V I E R SCIENCE P U B L I S H I N G C O M P A N Y INC. 52, Vanderbilt Avenue New York, N Y 10017, U.S.A.
ISBN 0-444-42678-7 ( V o l . 2 7 ) ISBN 0-444-41696-X (Series)
0 Elsevier Science Publishers B.V., 1986 All rights reserved. N o part o f this publication may be reproduced, stored in a retrieval system o r transmitted i n any f o r m or b y any means, electronic, mechanical, photocopying, recording o r otherwise, w i t h o u t the p r i o r w r i t t e n permission o f the publisher, Elsevier Science Publishers B.V./Science & Technology Division, P.O. B o x 330, 1000 A H Amsterdam, The Netherlands. Special regulations f o r readers i n the U S A - This publication has been registered w i t h the Copyright Clearance Center Inc. (CCC), Salem Massachusetts. I n f o r m a t i o n can be obtained f r o m the CCC about conditions under which photocopies o f parts o f this publication may be made in the USA. A l l other copyright questions, including photocopying outside o f the USA, should be referred t o the copyright owner, Elsevier Science Publishers B.V., unless otherwise specified. Printed in The Netherlands
In Memory of
NORYKO TSUNODA
XI11
PREFACE T h i s volume c o n t a i n s f o r t y - n i n e p a p e r s . They c o n s i s t o f a s e l e c t i o n o f t h e research papers,
and some r e v i e w p a p e r s ,
t h a t were presented a t t h e
1 4 t h C o n f e r e n c e o f t h e I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e R e s e a r c h (ISFR) h e l d f r o m 1 2 - 1 5 June 1985,
i n Morioka,
w i t h a n a l y t i c a l methods f o r f l u o r i d e : inorganic fluorides;
Japan.
The p a p e r s a r e c o n c e r n e d
environmental
p o l l u t i o n caused b y
and t h e e f f e c t s o f f l u o r i d e on p l a n t s ,
animals,
and
humans. F l u o r i d e i s a common e l e m e n t and i t i s known t o be w i d e l y d i s t r i b u t e d i n nature. With regard t o t h e p u b l i c h e a l t h i m p l i c a t i o n s o f f l u o r i d e , appears toxic
that
a t t e n t i o n has been f o c u s e d
effects
of
this
substance
l a r g e l y on t h e b e n e f i c i a l
i n d r i n k i n g water
and
its
effect
t e e t h . Because o f i t s w i d e s p r e a d and g r o w i n g use i n i n d u s t r i e s ,
it
and on
however,
f l u o r i d e i s now f o u n d i n i n c r e a s i n q amounts i n t h e s u r r o u n d i n g s o f many
I n s e v e r e l y p o l l u t e d a r e a s , f l u o r i d e has
i n d u s t r i a l areas i n t h e world.
been shown t o cause i n j u r i e s t o p l a n t s and a n i m a l s ;
i t c o u l d a l s o con-
c e i v a b l y cause a d v e r s e e f f e c t s on humans. I n t h e past,
as a consequence o f i n d u s t r i a l d i s c h a r g e , Japan has u n f o r -
t u n a t e l y e x p e r i e n c e d such p o l l u t i o n - r e l a t e d
e p i s o d e s as Minamata d i s e a s e
and I t a i - I t a i d i s e a s e . I t i s w i d e l y known t h a t f l u o r i d e ,
l i k e m e t h y l mer-
c u r y and cadmium w h i c h p r o d u c e d t h e s e e p i s o d e s , c a n a c c u m u l a t e i n l i v i n q organisms, mental
c a u s i n g i n j u r i e s . To u n d e r s t a n d f u l l y t h e e f f e c t s o f e n v i r o n -
p o l l u t a n t s on l i v i n g systems,
i n c l u d i n g humans,
an i n t e r d i s c i p -
l i n a r y approach t o r e s e a r c h i s needed. We
feel
strongly
that
the
basis
for
understanding f l u o r i d e - r e l a t e d
p r o b l e m s l i e s i n h a v i n g sound knowledge c o n c e r n i n g a l l phases o f f l u o r i d e occurrence,
i n c l u d i n g not o n l y t h e f l u o r i d e
b u t also
i n a i r and w a t e r ,
t h a t i n o r g a n s and t i s s u e s o f l i v i n g o r g a n i s m s . The s e c t i o n " A n a l y t i c a l Methods f o r F l u o r i d e " was i n c l u d e d as one o f t h e m a i n themes i n t h e conference
based o n t h i s
consideration.
developed a n a l y t i c a l
methods
widely
furthering
employed
medicine,
in
reported
l e a r n ways
w h i l e newly
i n t h e c o n f e r e n c e m i g h t be m r e
fluoride
d e n t i s t r y and b i o c h e m i s t r y ,
t h i s opportunity t o
I t was hoped t h a t , research
in
such
fields
as
a n a l y t i c a l c h e m i s t s m i g h t also use
i n which t h e y c o u l d c o n t r i b u t e
to the
endeavor. Thanks
to
the
interest
C o n f e r e n c e was w e l l
shown
attended:
by
many
scientists,
the
14th
ISFR
n e a r l y 200 p a r t i c i p a n t s came f r o m e l e v e n
d i f f e r e n t c o u n t r i e s . They r e p r e s e n t e d such d i v e r s e f i e l d s
as m e d i c i n e ,
dent i s t r y ,
biology,
veterinary
environmental science. b
science,
pharmacology,
Altogether,
chemistry,
and
n e a r l y 100 p a p e r s were p r e s e n t e d i n
XIV the oral tations
sessions
and i n t h e p o s t e r s e s s i o n s .
Both forms
g e n e r a t e d i n t e r e s t i n q and w i d e - r a n g i n g d i s c u s s i o n s .
t h e s e p a p e r s were c o n c e r n e d w i t h n e w l y - d e v e l o p e d analysis.
These
methods
can no doubt
research,
and t h e p a p e r s
addition,
we a r e p l e a s e d t o i n c l u d e s e v e r a l
case s t u d i e s
of
airborne
are thus
be
fluoride
i n this
that
"Fluoride",
are
included
in
pollution, this
publication.
In
skeletal
will
volume
fluorosis,
and
The m r e t h a n t h i r t y be
published
in
t h e o f f i c i a l j o u r n a l o f t h e ISFR.
The success of t o the s k i l l greatly
not
i n many areas o f
o t h e r papers d e a l i n q w i t h
r e p o r t s on t h e b i o c h e m i c a l e f f e c t s o f f l u o r i d e . papers
Several of
methods f o r f l u o r i n e
utilized
included
o f presen-
t h e m e e t i n g was due u l t i m a t e l y t o t h e p a r t i c i p a n t s and
and a s s i s t a n c e o f t h e c h a i r m e n o f t h e s e s s i o n s .
We a r e
i n d e b t e d t o each o f t h e c o n t r i b u t o r s o f t h e s e p r o c e e d i n g s who
c o o p e r a t e d i n many ways c o n c e r n i n g t h e p u b l i c a t i o n . I n conclusion,
we g r a t e f u l l y acknowledge t h e c o n t r i b u t i o n made b y t h e
l a t e D r N o r i k o Tsunoda,
D i r e c t o r o f N i s h i Matsuzono C l i n i c ,
M o r i o k a , who
n o t o n l y gave s u b s t a n t i a l f i n a n c i a l a s s i s t a n c e t o t h e c o n f e r e n c e , b u t was a l s o i t s h o s t e s s and o r g a n i z e d a l l o f t h e s o c i a l programs. away o n 18 J u l y 1985,
N o r i k o Tsunoda was t h e d e a r w i f e o f one o f us, H. Tsunoda, f r i e n d o f M.H.
Yu and h i s w i f e ,
Conference
and
Petersburg,
Florida,
Noriko
Tsunoda
the
and i n Logan,
f l u o r i d e research.
acquainted
Fluoride Utah,
with
Dr
and a s p e c i a l
S i n c e a t t e n d i n q t h e 1 2 t h ISFR
Ervena.
International
was
She passed
a p p r o x i m a t e l y one month a f t e r t h e m e e t i n g .
USA,
many
Symposium
held
respectively, investigators
in
St.
i n 1982, D r involved
in
I t was h e r s i n c e r e hope t h a t t h e ISFR C o n f e r e n c e h e l d
i n M o r i o k a , where she l i v e d , w o u l d be s u c c e s s f u l .
She made e v e r y e f f o r t
t o a s s i s t h e r husband and f r i e n d s p r i o r t o and t h r o u g h o u t t h e c o n f e r e n c e t o ensure i t s success. F o r t h e s e r e a s o n s we d e d i c a t e t h i s volume,
i n memoriam,
t o D r Noriko
Tsunoda.
Humi o Tsunoda Ming-Ho Yu
Tokyo January 1986
xv ACKNOWLEDGMENTS On b e h a l f o f t h e O r g a n i z i n g C o m m i t t e e o f t h e 1 4 t h C o n f e r e n c e o f t h e I S F R , we w i s h t o e x p r e s s o u r deep g r a t i t u d e t o t h e many i n s t i t u t i o n s , organizations.
and i n d i v i d u a l s f o r t h e i r s u p p o r t ,
f i n a n c i a l assistance.
and
cooperation
which c o n t r i b u t e d t o t h e success o f t h e Conference.
In
particular,
we w o u l d l i k e t o t h a n k D r . K i j u r o O b a r a ,
Medical University, University, Ohbori,
Dr.
Yoshito Tsuji,
and Dr.
Public Health,
S a n s h i Abe,
Chief Director, President,
President.
Iwate
Japanese S o c i e t y o f
Higashi
N i p p o n Gakuen
who s e r v e d a s C o n f e r e n c e H o n o r a r y A d v i s o r s ; and D r . T s u t o m u
I w a t e M e d i c a l U n i v e r s i t y , Dr. S e i k o I s h i k a w a .
S h u n i c h i Sato,
Iwate Medical University,
Iwate University,
and D r . Y a s u h i r o S u z u k i ,
Dr.
Yamagata
U n i v e r s i t y , who s e r v e d as C o n f e r e n c e A d v i s o r s . We g r e a t l y a c k n o w l e d g e t h e f o l l o w i n g i n s t i t u t i o n s and o r g a n i z a t i o n s w h i c h sponsored t h e Conference; I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e Research: I w a t e M e d i c a l U n i v e r s i t y : Japanese S o c i e t y f o r Hygiene: Japanese S o c i e t y o f P u b l i c H e a l t h : A s s o c i a t i o n o f I n d u s t r i a l Health:
Japan S o c i e t y o f A i r P o l l u t i o n ;
Japan
and Japan
S o c i e t y f o r A n a l y t i c a l Chemistry. Our s i n c e r e a p p r e c i a t i o n i s e x t e n d e d t o members o f t h e C o n f e r e n c e E x e c u t i v e C o m m i t t e e and S e c r e t a r i a l S t a f f f o r o r g a n i z i n g and h a n d l i n g t h e many d e t a i l s i n v o l v e d i n t h e C o n f e r e n c e . t h a n k Dr. Masanobu T a t s u m i ,
I n particular,
we w o u l d l i k e t o
D r . S h i g e n a o Nakaya, D r .
Dr. S h i r o Sakurai,
M i t s u o M i t a , D r . K a z u y o s h i I t a i , D r . K e i k o Nagayama, M r . T o r u Sato.
Mr.
T o s h i t a k a H o r i u c h i , D r . Y o i c h i I i j i m a , D r . K o s u k e Okada, and Mrs. M i c h i k o M a t s ud a. We
are deeply grateful
f o r the financial
Conference by t h e f o l l o w i n g i n s t i t u t i o n s , Iwate Prefecture, Association,
City o f
Morioka,
Iwate Dental Association,
assistance given t o t h i s
organizations. City o f
and i n d i v i d u a l s :
Sakata,
Department o f P u b l i c H e a l t h a t Fukushima Medical College, Co-op,
Iwate Medical
I w a t e Hea1,th S e r v i c e A s s o c i a t i o n , Morioka Medical
Keiryokai (Alumni Association o f Iwate Medical University):
and
Professor Emeritus Tadashi I g a r i ( I w a t e Medical U n i v e r s i t y ) , Prof. Teru Takanohashi ( I w a t e U n i v e r s i t y ) . (Oshuku S p r i n g H o s p i t a l ) , Osamu T a k a h a s h i
(Hanamaki
( K a w a s a k i S t e e l Co.).
M r . A k i o Yamaraki and Dr. Yasuo Kuboya
Dr. Sadayoshi Ichinohe ( I c h i n o h e C l i n i c ) , Mental Hospital),
and Dr.
Yoshinori
Dr.
Shoji
XVI we w o u l d l i k e a l s o t o t h a n k t h e f o l l o w i n g members o f t h e A l u m n i
Lastly,
A s s o c i a t i o n o f t h e D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h ,
Dr.
Susumi Atsumu,
Dr.
Fujino,
I w a t e Medical
f o r t h e i r c o o p e r a t i o n and d e v o t i o n :
University,
Dr.
M r s . T o m o k o Endo.
S h o i c h i r o Fusa,
Dr.
Dr.
Y a s u h i r o Kudo,
D r . Susumu Motoyama.
Sadakatsu Naito, Kyoko Odashima,
Sekiya,
Dr.
Dr.
Dr.
Kazuhiro
Dr. S h i g e r u Nagasawa.
M r . YUJI
Mrs. Yoko Nakaya, Dr. H i d e o N i h e i , Dr. K e n i c h i Ohsawa.
Fumihiko Saito,
Mr.
Ken S a i t o ,
D r . H i n a Sato, Dr. N o b u h i s a Sato, Mrs.
Mr.
M a t s u o Kumagai,
H i r o s h i Mural,
Dr.
Miss
Noru,
D r . S h u n p e i O i k a w a , Dr. T a d a s h i O g i w a r a , D r . K e i k o Onodera,
Dr. K e n i c h i Onodera, Saka,
Dr.
Dr. Yoshim1
D r . Y u t a k a I s h i z a w a , D r . T a k a s h i I t o , D r . S a i c h i Kon, D r .
Ichinowatari,
T a k a t s u g u Kudo,
Orikasa,
Yutaka
S e t s u H a s h i m o t o , Dr. C h i s a t o Hamajima.
T o k u z o I k e n o , D r . S a d a t o I c h i n o h e , Dr. K a z u k o I c h i n o w a t a r i ,
Matsuda,
Dr.
Shoko F u j i m o r i ,
D r . T a k e o Ohsawa, D r . Choshun Mrs. M l h o k o S a i t o ,
Dr. M a s a t a k a
Mr. Shigeki Sasaki,
Mrs. A t s u k o
D r . T s u n e a r i Sugawara,
Re1 k o S h i r u k u i s h i ,
Mr.
Mamoru S u z u k i .
D r . Y o s h i o S h i m a z a k i , D r . H i d e k o Sonoda, D r . I k u r o T a k a h a s h i , D r . Susumu Takahashi.
Dr.
K e n i c h i Takeuchi,
Mitsugu Tobari,
Dr. Kazuo T o d o r i ,
Dr.
Ryozo Tanaka,
Mr.
Dr. M i n o r u Uematsu.
S e n j i r o Tanaka,
Dr.
Dr. Masao Yamada, Dr.
K e n j i Y a z a k i , Dr. Y u t a k a Y a s u i , D r . Masao Y o s h i d a . D r . H i d e k o Y o s h i m u r a , Mrs. Y o s h i k o Y o s h i o k a .
and Dr. T a k e h i s a Yoshiyama.
H. Tsunoda
M.
H. Yu
O r g a n i z i n g C o m m i t t e e o f t h e 1 4 t h C o n f e r e n c e o f t h e ISFR. Japan, Dr.
Humio Tsunoda ( C o n f e r e n c e Chairman), Public Health,
h e l d i n Morioka,
1985.
J u n e 12-15,
Professor,
Dept. o f Hygiene
-
Iwate Medical U n i v e r s i t y
Dr. K e i i c h i r o Fuwa,
Professor,
Dr. T s u y o s h i Katayama,
Dept. o f C h e m i s t r y ,
Professor,
U n i v e r s i t y o f Tokyo
Dept. o f P r e v e n t i v e D e n t i s t r y ,
Iwate
Medical U n i v e r s i t y
D r . J i r o Matsushima, P r o f e s s o r , Dr.
Kan-ichi
Ohshima,
Faculty o f Agriculture, Mie University
Professor,
Dept.
o f Veterinary Pathology,
Iwate
University
D r . S h i g e r u Ono, P r o f e s s o r , D e p t . o f B i o c h e m i s t r y ,
Dr. Y o s h i n o r i Takaesu,
Professor,
Iwate Medical U n i v e r s i t y
Dept. o f P r e v e n t i v e D e n t i s t r y ,
Tokyo
Dental College
Dr.
F u m i y o s h i Yanagisawa, Un iv e r s it y
Professor Emeritus,
Tokyo Medical - Dental
XVII Dr. Yasuhisa Yoshida,
P r o f e s s o r , Dept. o f H y g i e n e
- Public
H e a l t h , Osaka
Medical College
D r . Ming-Ho Yu.
Professor,
Washington U n i v e r s i t y
Huxley College o f Environmental Studies,
Western
3
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 3-14 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands
ANALYTICAL C H E M I S T R Y AND BIOGEOCHEMISTRY OF FLUORINE: AN HISTORICAL V I E W
K E I I C H I R O FUWA Department o f Chemistry, F a c u l t y o f Science, Bunkyo-ku, Tokyo 11 3, J a p a n
The U n i v e r s i t y o f Tokyo,
ABSTRACT The a n a l y s i s o f f l u o r i n e i s one o f t h e m o s t d i f f i c u l t e l e m e n t a l a n a l y s e s i n t h e h i s t o r y o f a n a l y t i c a l chemistry.
Each o f t h e r e p r e s e n t a t i v e i i l e t h o d s
i n t h e f i e l d o f f l u o r i d e a n a l y s i s was d e s c r i b e d w i t h p a r t i c u l a r e m p h a s i s on c o l o r i m e t r y and s p e c t r o c h e m i s t r y .
Some b i o g e o c h e m i c a l a n d e n v i r o n m e n t a l
aspects o f f l u o r i n e were a l s o mentioned. INTRODUCTION F l u o r i n e . t h e f i r s t member o f t h e h a l o g e n s , i s an e l e m e n t w i t h u n i q u e p h y s i c a l and c h e m i c a l p r o p e r t i e s . t h e most n o n - m e t a l l i c
nature,
I t has t h e h i g h e s t e l e c t r o n a f f i n i t y o r
and t h e r e f o r e ,
i t s compounds o f t e n d i s p l a y
d i f f e r e n t p r o p e r t i e s f r o m t h o s e o f o t h e r halogens.
For instance,
fluorides
o f a l k a l i n e e a r t h s s u c h as CaF2 and S r F 2 h a v e l o w s o l u b i l i t y i n w a t e r ,
in
c o n t r a s t t o c h l o r i d e s o r b r o m i d e s s u c h a s CaC12 a n d S r C 1 2 o r C a B r 2 a n d
SrBr2.
Whereas s i l v e r f l u o r i d e (AgF) i s e a s i l y s o l u b l e i n w a t e r o r h y g r o -
scopic.
s i l v e r c h l o r i d e (AgC1) o r s i l v e r b r o m i d e ( A g B r ) a r e t h e l e a s t
s o l u b l e compounds. and i r o n ,
F l u o r i d e g i v e s complex anions w i t h s i l i c o n .
aluminum
A l F g 3 and FeFg3, w h i c h a r e s p e c i e s o f t e n f o u n d i n
forming SiFi2,
n a t u r a l w a t e r s b e s i d e s f l u o r i d e (F-)
itself.
The g e o c h e m i s t r y ,
biogeochem-
i s t r y and b i o c h e m i s t r y o f f l u o r i d e a r e s p e c i a l and a r e o f p a r t i c u l a r i n t e r e s t because o f t h e s e fundamental s p e c i a l L h a r a c t e r i s t i c s i n b o t h p h y s i c s and c h e m i s t r y .
The a n a l y t i c a l c h e m i s t r y o f f l u o r i n e i s a l s o u n i q u e
i n t h a t i t i s one o f t h e m o s t d i f f i c u l t e l e m e n t s t o h a n d l e i n t e r m s o f b o t h c h e m i c a l and i n s t r u m e n t a l t e c h n i q u e s u s e d i n i t s a n a l y s i s . A n a l y t i c a l Chemistry o f Fluorine A l m o s t a l l c l a s s i c a l and i n s t r u m e n t a l
a n a l y t i c a l methods have been
a p p l i e d t o t h e a n a l y s i s o f f l u o r i n e (Table I). summary
of
each method
s p e c t r o c h e m i c a l methods,
with
special
The f o l l o w i n g i s a b r i e f
e m p h a s i s o n c o l o r i m e t r y and
w h i c h h a v e been s t u d i e d i n t h i s l a b o r a t o r y and a r e
considered most u s e f u l f o r f l u o r i n e analysis. Gravimetry.
Calcium fluoride,
lanthanum f l u o r i d e ,
a
LaF3,
CaFZ,
lead chlorofluoride,
PbClF, and
a r e common i n s o l u b l e s p e c i e s f o r t h e g r a v i m e t r i c
4
TABLE I METHODS OF FLUORINE ANALYSIS Species etc.
Method
CaF2, PbClF, LaF3 i zari ne S; AgN03/PbCl F
Gra v i met ry
Ti tration
Th( N03)4/A1
Electrochemical Ion Chromatography
F-,
Radiochemical
"F(r1,2n)~*F,
Colorimetry Spectrochemical
Zr-Alizarin, Al-Hematoxylin La-ALC(TRLCC) Emission; CaF, SrF, BaF, F
Ion Selective Electrode, LaF, SiFg2, AlFi3 19F(n,p)190
Absorption; AIF, InF
analysis of fluoride (1).
A well-known classical Berzelius-Hillebrand
method of analyzing fluoride in rocks is based on dissolving fluoride (CaF2) after alkaline fusion o f the sample powder followed by the addition of calcium chloride in excess to the solution. Titration. Fluoride i n aqueous solution can be titrated by thorium nitrate, Th(N03)4, using alizarin S as an indicator, or by argentimetric titration of chloride after separating the fluoride as PbClF (1.2). Electrochemical method. Among several electrochemical techniques, the one using an ion selective electrode for fluoride with LaF3 membrane is the most useful (3). This method i s simple, and can distinguish fluoride ions from other complexed species, and the sensitivity is high enough, 20 ppb of F-,
so that it can be applied to most determinations. Ion chromatography. Ion chromatography has been developed recently and
has become one o f the most convenient methods for anion determinations in natural water (4). Fluoride can be determined by ion chromatography, with proper caution against interfering elements. Radiochemical method. Activation analysis of fluorine can be achieved by nGutron bombardment of samples in an atomic pile and counting the resulting r a d i o a c t i v e species. T h e reaction m a y be either 19F(n,2n)18F or ''F(n,~)~'0. The half-lives of each atom are 1.87h for 1 8 F and 29.5s for "0, respectively (5.6) _Colorimetry. ____
The color bleaching effect by fluoride on yellowish
titanium color, developed with hydrogen peroxide in an acidic solution was one of the early colorimetric methods used for fluoride determination (7). As a more sensitive fading c o l o r i m e t r y f o r fluoride, z i r c o n i u m -
5 a l i z a r i n a t e (8) o r a l u m i n u m h a s e m a t o x y l i n e ( 9 ) w e r e u t i l i z e d i n o r d e r t o determine f l u o r i d e c o n t e n t o f n a t u r a l water i n c l u d i n g sea water. a b s o r p t i o n s p e c t r a o f t h e s e compounds w e r e i n v e s t i g a t e d (Fig.
F i g u r e 1.
The
1) (10).
Colorimetry o f f l u o r i d e
Although use o f t h e c o l o r f a d i n g e f f e c t s o f f l u o r i d e f o r i t s d e t e r m i n a t i o n was s o m e w h a t s u c c e s s f u l , and West ( I I ) ,
i.e..
(ALC.1.2-dihydroxy
a c o l o r d e v e l o p i n g compound was f o u n d b y B e l c h e r
t h e r e d d i s h lanthanum complex w i t h a l i z a r i n complexon
anthraqhinonyl-3-methylamine-N, N - d i a c e t i c a c i d ) changes
t o blue by reacting w i t h fluoride,
r e s u l t i n g 7n a c o m p l e x w i t h an a b s o r p -
t i o n maximum a t 620 nm. T h i s La-ALC m e t h o d has been e m p l o y e d i n m o s t o f t h e c o l o r i m e t r y o f f l u o r i d e i n r e c e n t years. t i v i t y o f t h e method,
I n order t o increase t h e sensi-
a technique using t o t a l a r e f l e c t i o n long c a p i l l a r y
was a p p l i e d (12). T h i s t e c h n i q u e u s e d a " l i q u i d c o r e o p t i c a l f i b e r " as a n absorption cell,
t h e core l i q u i d being t h e s o l u t i o n i n which t h e m a t e r i a l
i s d i s s o l v e d . so t h a t t h e a b s o r p t i o n c e l l c o u l d be v e r y l o n g , and consequently,
o f high analytical sensitivity.
the solution,
and t h e r e f o r e ,
The s o u r c e l i g h t t r a v e l s t h r o u g h
the r e f r a c t i v e index o f the solution,
o u g h t t o be g r e a t e r t h a n t h a t o f t h e c e l l w a l l , n1
' n2
n1,
n2, o r
(1)
Then t h e c r i t i c a l a n g l e i i s e x p r e s s e d b y S i n i = n2/nl and t h e i n c i d e n t s o u r c e l i g h t , can t r a v e l intensity, i n Figure
through
w h i c h h a s a l a r g e r i n c i d e n t a n g l e t h a n i,
the c e l l by t o t a l
as S n e l l ' s l a w holds.
2,
(2) reflection without
losing i t s
A t y p i c a l i n s t r u m e n t a l d i a g r a m i s shown
i n w h i c h an o p t i c a l f i b e r t y p e c a p i l l a r y c e l l i s seen a t t h e
6 center.
S i n c e a g l a s s o r p y r e x c a p i l l a r y c e l l i s used,
w i t h a r e f r a c t i v e index higher than t h a t of pyrex, C a r b o n d i s u l f i d e , w h o s e i n d e x i s 1.66, and c a r b o n d i s u l f i d e ,
an o r g a n i c s o l v e n t
1.48,
has t o be used.
o r a m i x t u r e of a n o r g a n i c s o l v e n t
i s t h e b e s t f o r t h i s purpose.
A diethylaniline
d e r i v a t i v e o f ALC was f o u n d t o be more s u i t a b l e f o r t h e s o l u b i l i z a t i o n o f i t s l a n t h a n u m and f l u o r i d e d e r i v a t i v e s
i n t o carbon d i s u l f i d e .
t o t a l r e f l e c t i o n long c a p i l l a r y technique.
0.01 p p b F i n w a t e r c a n b e
A d e t a i l e d d e s c r i p t i o n o f t h e a n a l y t i c a l procedure, instrumenta-
measured. tion,
With t h i s
a s w e l l as some e x p e r i m e n t a l r e s u l t s a r e r e p o r t e d e l s e w h e r e i n t h i s
paper.
LENS F I LTER "OPT I CAL FIBER"
-
SAMPLE
RECORDER
F i g u r e 2.
D e t e c t i o n system w i t h " h o l l o w f i b e r "
Spectrochemical analysis.
long c a p i l l a r y c e l l
Emission spectrochemical analysis o f f l u o r i n e
b y D C a r c u s i n g a CaF b a n d a t 5 2 9 nm w a s f i r s t e m p l o y e d b y P a p i s h ( 1 4 ) . T h i s p a r t i c u l a r b a n d a s w e l l a s o t h e r CaF b a n d s a n d t h e m o n o f l u o r i d e s o f strontium author (15).
and b a r i u m
i n a DC a r c
were
further
investigated by the
T a b l e I 1 shows v a r i o u s m e t a l l i c m o n o f l u o r l d e s ,
which give
e i t h e r e m i s s i o n o r a b s o r p t i o n bands i n a h i g h t e m p e r a t u r e medium (16).
All
e m i s s i o n b a n d s o f a l k a l i n e e a r t h m o n o f l u o r i d e s i n v e s t i g a t e d i n a DC a r c b y e i t h e r naked e y e o b s e r v a t i o n o r s p e c t r o p h o t o g r a p h i c t e c h n i q u e s w e r e f o u n d t o be enriched i n t h e negative flame region. n e g a t i v e f l a m e e n r i c h m e n t o f CaF bands.
Figure 3 illustrates the
T h i s phenomenon c a n be c o n s i d e r e d
e q u i v a l e n t t o t h e cathode l a y e r enrichment f o r m e t a l l i c species s t u d i e d by G o l d s c h m i d t a n d S t r o c k (17).
By o b s e r v i n g w i t h t h e n a k e d e y e t h e g r e e n i s h
y e l l o w CaF b a n d a t 5 2 9 nm e n r i c h e d i n t h e n e g a t i v e f l a m e n e a r t h e l o w e r cathode,
i n w h i c h 20-30
mg o f CaC03 d i l u t e d s a m p l e i s l o a d e d ,
O.OOl%F was
TABLE I 1 BAND HEADS OF METAL-MONOFLUORIDES Wavelength (nm)
D.E. (ev)
10
M9F
539.42V
-
CaF
606.47V
8
10
SrF
4.5
529.10R
10
10
324.54v
10
-
663.27V
10
10 8
5.45
5.8
577.95R
7
352.98V
10
5.4
711.60R
10
495.08V
4
8 -
380.99V
10
10
BF
195.74
10
-
7.85
A1 F
227.47v
10
-
6.85
GaF
211.02R
10
-
6.1
InF
233.73v
10
-
5.35
T1F
219.80R
10
-
4.5
CuF
493.20V
10
10
3.5
MnF
351.78V
10
-
242.41V
10
-
BaF
F r o m : R. Spectra"
F.E.
Abs.
5.2
6. P e a r s e a n d A. G. G a y d o n : " T h e I d e n t i f i c a t i o n o f M o l e c u l a r
W.
d e t e c t e d (15).
S r F a t 577 nm a n d BaF a t 6 9 4 nm g i v e s i m i l a r o r s l i g h t l y
l o w e r s e n s i t i v i t i e s (18). available,
e.g.,
b u t t h e y a r e u s e f u l w h e n t h e CaF b a n d i s n o t
w h e n t h e r e g i o n a t 5 2 9 nm i s i n t e r f e r e d w i t h b y o t h e r
e l e m e n t s such as barium.
F i g u r e 4 shows a r e p r o d u c t i o n o f BaF bands.
As a t o m i c a b s o r p t i o n s p e c t r o s c o p y b e c a m e p o p u l a r , fluorine a t
95 nm
fluorine
atomic
I t was f o u n d t h a t t h e r e s o n a n c e l i n e o f
a b s o r p t i o n was i n v e s t i g a t e d .
was t o o s h o r t f o r r e g u l a r a p p l i c a t i o n s (19).
f l u o r i d e species were i n v e s t i g a t e d i n t h i s laboratory,
Diatomic
and aluminum
m o n o f l u o r i d e ( A l F ) w i t h a b a n d h e a d a t 2 2 7 nm w a s f o u n d t o b e t h e m o s t p r o m i s i n g (20).
A t t h i s wavelength,
a b s o r p t i o n peak ( F i g .
5). Thus,
A1F g i v e s a " l i n e - l i k e ' '
f l u o r i n e has become a u s e f u l method. apparatus
sharp
h i g h temperature absorption spectrometry o f
can be used e x c e p t
that
the
c o n t i n u o u s s p e c t r a f r o m a h y d r o g e n lamp.
Any r e g u l a r a t o m i c a b s o r p t i o n light
source
should
provide
The g r a p h i t e f u r n a c e was f o u n d
negative flame
I -
center of arc column
in
CaF
(bh529 1 A )
B
F i g u r e 3. F l u o r i n e d e t e r m i n a t i o n b y DC a r c (CaF s p e c t r a ) A p p r o x . 30 mg s a m p l e i s d i l u t e d w i t h CaC03. DC 1 0 0 V, 8 - 1 0 A i s u s e d . CaF ( b h 529.1 nm) i s e n r i c h e d m o s t i n t h e n e g a t i v e f l a m e n e a r t h e l o w e r e l e c t R e p r o d u c e d f r o m : Fuwa K ( 1 9 5 1 ) J J p n Chem SOC 71:341 r o d e i n B.
_ _ _ _ B a F bh 5000 6A ---- B a F bh 4992 1A
---- B a F bh 4950 8 A
'
F i g u r e 4. F l u o r i n e d e t e r m i n a t i o n b y DC a r c ( 5 a F ) . A i s t a k e n on t h e photo p l a t e and 5 i s t h e s p e c t r u m observed BaS04/5aF2. R e p r o d u c e d f r o m : Fuwa b y t h e n a k e d eye. A p p r o x i m a t e l y O.l%F i s d e t e c t e d . K ( 1 9 5 5 J J J p n Chem SOC 76:14
9
W a v e l e n g t h (nm) F i g u r e 5.
Absorption s p e c t r u m o f A1F
p a r t i c u l a r l y s e n s i t i v e and t h e d e t e c t i o n l i m i t has r e a c h e d a s s m a l l a q u a n t i t y a s 0.021 n g
F.
For most p r a c t i c a l purposes,
a platinum atomic
l i n e f r o m a P t h o l l o w c a t h o d e l a m p t o g e t h e r w i t h a D2 l a m p f o r b a c k g r o u n d c o r r e c t i o n seems t o b e t h e b e s t , a l t h o u g h t h e f o r m a t i o n o f t h e A1F m o l e c u l e i n s i d e t h e medium i s o f t e n d i s t u r b e d b y e x i s t i n g f o r e i g n m a t t e r (21.22).
A
d e t a i l e d d e s c r i p t i o n and i t s a p p l i c a t i o n t o some b i o l o g i c a l s a m p l e s h a v e g i v e n e l s e w h e r e i n t h i s paper. I n t h e l a s t t e n years,
e m i s s i o n s p e c t r o m e t r y has regained a t t e n t i o n i n
the f i e l d o f fluorine analysis since
s p e c i a l l y designed i n d u c t i v e l y
c o u p l e d a r g o n p l a s m a was f o u n d e x c e l l e n t i n t e r m s o f b o t h p r e c i s i o n and s e n s i t i v i t y (23).
I t g i v e s s i g n a l s as s e n s i t i v e as a t o m i c a b s o r p t i o n does
f o r most m e t a l l i c elements.
I n addition, a simultaneous determination
s y s t e m has been c o n v e n i e n t l y d e s i g n e d w i t h a p o l y c h r o m a t o r d e t e c t o r combined w i t h a computerized read-out
I t has s i n c e r e p l a c e d a
system.
large p a r t o f the f i e l d o f elementary analysis previously c a r r i e d o u t by atomic absorption.
However,
most o f t h e non-metallic
f l u o r i n e a r e excluded f r o m t h i s t e c h n i q u e again, h i g h e x c i t a t i o n energies. most energetic region, However, atom,
Only between t h e i n d u c t i o n c o i l s ,
when h e l i u m g a s i s e m p l o y e d a s t h e p l a s m a gas,
w h i c h has a s u f f i c i e n t l y h i g h energy l e v e l , investigated
determination.
and
apparently the
was some a t o m i c f l u o r i n e e m i s s i o n o b s e r v e d (24).
i s i l l u s t r a t e d i n F i g u r e 6. been
elements i n c l u d i n g
m a i n l y because o f t h e i r
t h e m e t a s t a b l e He
c o u l d e x c i t e f l u o r i n e as
Use o f m i c r o w a v e i n d u c e d p l a s m a ( M I P ) ,
established
as
a
novel
method
for
has
fluoride
By s i m p l y s h a k i n g an aqueous s a m p l e c o n t a i n i n g f l u o r i d e
w i t h tetramethyl chlorosilane, f l u o r o s i l a n e , TMFS.
TMCS,
f l u o r i d e i s converted t o tetramethyl
The l a t t e r i s e x t r a c t e d i n t o an o r g a n i c s o l v e n t and
10
F i g u r e 6.
E n e r g y d i a g r a m o f f l u o r i n e and z i n c
t h e e x t r a c t i s t h e n analyzed b y gas chromatography,
w i t h M I P as an e l e m e n t
s e l e c t i v e d e t e c t o r (25). H i s t o r y o f spectroscopy.
It i s said t h a t h i s t o r y repeats r t s e l f .
In
s c i e n t i f i c h i s t o r y man c a n a l s o f i n d r e p e a t e d d e v e l o p m e n t s i n some f i e l d s . S p e c t r o s c o p y i s p e r h a p s one o f t h e b e s t examples. described i n t h i s section,
As h a s a l r e a d y b e e n
t h e r e c u r r e n c e o f e m i s s i o n and a b s o r p t i o n I n T a b l e 111,
spectrometry i n h i s t o r i c a l terms i s indeed most s t r i k i n g .
t h e e s s e n c e o f t h i s r e p e a t e d h i s t o r y o f s p e c t r o s c o p y i s shown.
It i s
i n t e r e s t i n g t h a t b o t h a t o m i c a b s o r p t i o n and p l a s m a e m i s s i o n h a v e r e g a i n e d t h e i r r e p u t a t i o n and p r o v e d i n v a l u a b l e i n t h e a r e a o f f l u o r i d e a n a l y s i s . Fluorine i n Hokutolite H o k u t o l i t e i s a n i s o m o r p h o u s m i x t u r e o f BaS04 and PbS04, m i n e r a l s produced i n b o t h Hokuto h o t spring, h o t s p r i n g , A k i t a , J a p a n ( F i g . 7). studied i n detail.
Taipei,
one of t h e r a r e
T a i w a n and Tamagawa
H o k u t o l i t e f r o m T a m a g a w a h o t s p r i n g was
It i s t h e d e p o s i t from a unique h o t spring,
t e m p e r a t u r e w a s 97°C.
pH 1.2,
d u e m a i n l y t o HC1.
r a d i o a c t i v i t y d u e t o ThX, Ra ( F i g . 8). f l u o r i d e as a m i n o r c o n s t i t u e n t ,
Since the spring water contained
the deposition o f fluoride i n Hokutolite
w a s i n v e s t i g a t e d b y t h e e m i s s i o n s p e c t r o m e t r y o f BaF (18). F i g u r e 8, source,
0.01-0.05%
where t h e
and w h i c h c o n t a i n e d
As s h o w n i n
F was f o u n d i n H o k u t o l i t e d e p o s i t e d f a r f r o m t h e
near t h e j u n c t i o n o f Shibukuro River,
where b o t h t h e t e m p e r a t u r e
and t h e a c i d c o n c e n t r a t i o n o f t h e s p r i n g w a t e r w e r e s u b s t a n t i a l l y l o w e r e d . The H o k u t o l i t e f r o m Tamagawa h o t s p r i n g h a s been named one o f t h e n a t i o n a l
11 TABLE I 1 1 HISTORY OF SPECTROSCOPY Year
Name
Fact
Emission o r absorption -
1666
S i r I . Newton
D i s c o v e r y o f c o l o r and v i s i b l e spectrum
emission absorption
1817
J. F r a u n h o f e r
Fraunhofer Lines
1859
G.R.
K i r c h h o f f ' s Law
R. Bunsen
D i s c o v e r y o f Rb, Cs, T1
emission
1955
S i r A. Walsh
Atomic Absorption
absorption
1974
V.A.
P l a s m a emission
erni s s i o n
F i g u r e 7.
Kirchhoff
Fassel
H o k u t o l i t e producing areas
Hokuiollte 2
D i a g r a m s h o w i n g t h e g e o g r a p h y of Tamagawa H o t S p r i n g a r e a ( A k i t a F i g u r e 8. Pref., Japan)
12 s p e c i a l p r o d u c t s f o r c o n s e r v a t i o n i n Japan and m o s t o f i t i s b e i n g k e p t a t t h e Geological Department of A k i t a U n i v e r s i t y ,
Japan.
Biogeochemistry o f F l u o r i n e Some c r u c i a l
f a c t s about t h e biogeochemistry o f f l u o r i n e a r e reproduced
i n T a b l e I V (26). bioelement,
Fluorine i s not
c o n s i d e r e d an e s s e n t i a l e l e m e n t o r
w h i c h i s needed f o r t h e n o r m a l b i o l o g i c a l f u n c t i o n o f any
l i v i n g matter.
I t i s o f t e n considered toxic,
on many enzymes.
as i t has i n h i b i t o r y e f f e c t s
But, as i s shown i n T a b l e I V ,
g r o w t h o f mammalian teeth,
i t i s needed f o r h e a l t h y
b e s i d e s b e i n g a c o n s t i t u e n t o f bones and t e e t h .
O D t i m a l u p t a k e and o p t i m a l c o n c e n t r a t i o n s i n aqueous m e d i a , f o o d and b o d i e s a r e obviously t h e most important.
Many s u b j e c t s r e l a t e d t o t h e s e a r e
described elsewhere i n t h i s paper, t h e r e f o r e o n l y a few r e c e n t environm e n t a l problems o f f l u o r i n e a r e mentioned here: F l u o r i d a t i o n o f d r i n k i n q water. s t i l l a social,
0.5-1.0
A l t h o u g h t h i s p r o b l e m i s n o t new,
e n v i r o n m e n t a l a n d h e a l t h p r o b l e m f o r humans.
ppm i n d r i n k i n g w a t e r i s c o n s i d e r e d o p t i m a l .
it i s
Fluoride a t
Recommended concen-
t r a t i o n s may depend on f a c t o r s s u c h a s t h e c o n s t i t u e n t s o f w a t e r and t o t a l c o n t e n t o f f l u o r i n e i n foods. Ozone d e p l e t i o n i n t h e s t r a t o s p h e r e .
O r g a n o f l u o r i n e g a s e s s u c h a s Freon,
CC12F2, a r e f o u n d t o d e c o m p o s e o z o n e i n t h e o z o n e l a y e r o f t h e s t r a t o -
TABLE I V EIOGEOCHEMISTRY OF FLUORINE Abundance i n t h e U n i v e r s e : l g n e o u s Rocks: 625ppm; Sandstones: F r e s h Water:
Air:
270ppm; 0.09ppm;
3D0-900OF/1O6Si Shales:
740ppm
L i m e s t o n e s : 330ppm Seawater:
1.3ppm
0.01pgm-3
S o i l s : 200ppm: f i x e d i n many c l a y m i n e r a l s and i n a p a t i t e . The v e g e t a t i o n f r o m F - r i c h s o i l s i s t o x i c t o g r a z i n g mammals. F - d e f i c i e n c y i s a s s o c i a t e d w i t h d e n t a l decay. M a r i n e P l a n t s : 4.5ppm;
Land P l a n t s : 0.5-40ppm
M a r i n e A n i m a l s : 2ppm; Land A n i m a l s : s o f t t i s s u e s , 1500ppm i n bones.
150-500ppm i n mammalian
Function: n o t proved e s s e n t i a l , moderately t o x i c t o a l l organisms, b u t a i d s p r o d u c t i o n o f sound t e e t h i n mammals. From Bowen, H.J.M.:
1966
T r a c e E l e m e n t s i n B i o c h e m i s t r y , Academic P r e s s ,
London,
13 sphere.
As a r e s u l t ,
UV r a d i a t i o n f r o m t h e s u n r e a c h i n g t h e e a r t h w i l l be
more i n t e n s e and w i l l i n c r e a s e t h e i n c i d e n c e o f human s k i n c a n c e r . deemed a g l o b a l e n v i r o n m e n t a l p r o b l e m .
This i s
The m a n u f a c t u r e o f F r e o n h a s been
s t o p p e d i n t h e U.S.A. F l u o r i n a t i o n o f chemicals.
R e c e n t l y , c h l o r i n a t e d h y d r o c a r b o n s s u c h as
PCB, DDT, BHC. a n d D i o x i n , h a v e c a u s e d m u c h e n v i r o n m e n t a l c o n c e r n . result,
As a
f l u o r i n a t i o n o f i n d u s t r i a l o r g a n i c c h e m i c a l s has r e p l a c e d c h l o r i n a -
t i o n t o some e x t e n t .
Consequently,
there i s a trend t o increasing the
f l u o r i n e b u r d e n i n t h e e n v i r o n m e n t , w h i c h i s c o n s i d e r e d t o be one o f t h e f u t u r e environmental problems.
REFERENCES 1 . H i l l e b r a n d WF,
L u n d e l l GEF (1953)
Applied Inorganic Analysis.
Wiley,
New Y o r k
2. K o l t h o f f
IM, Sandell M a c M i l l a n , New Y o r k
EB ( 1 9 6 9 )
Q u a n t i t a t i v e Chemical Analysis.
3.
R o s s J r JW, R i s e m a n JH, K r u e g e r J A ( 1 9 7 3 ) Electrodes. Butterworths
4.
S m a l l H,
S t e v e n s TS,
Selective Ion-sensitive
Bauman WC ( 1 9 7 5 ) A n a l Chem 47:1801-1809
5. L e o n h a r d t W ( 1 9 6 3 ) K e r n e n e r g i e 6:45-46
6. B l a c k b u r n R (1964) A n a l Chem 36:669-671 7. S a n d e l l EB ( 1 9 5 9 ) C o l o r i m e t r i c D e t e r m i n a t i o n o f T r a c e s o f M e t a l s . I n t e r s c i e n c e , New Y o r k 8.
B o l t z DF ( 1 9 5 8 ) C o l o r i m e t r i c D e t e r m i n a t i o n o f N o n m e t a l s . New Y o r k
9.
Fuwa K (1954) J a p a n A n a l y s t 3:98-104
10. Fuwa K ( 1 9 5 4 ) PhD t h e s i s , 11. B e l c h e r R.
12. Fuwa K.
L e o n a r d MA,
We1 L.
14. A b r e n s LH, R e a d i ng
U n i v e r s i t y o f Tokyo
West TS ( 1 9 5 8 ) J Chem Soc:2390-2393
F u j i w a r a K (1984)
13. We1 L ( 1 9 8 2 ) MS t h e s i s ,
Interscience.
A n a l Chem 56:1640-1644
U n i v e r s i t y o f Tokyo
T a y l o r SR (1961)
Spectrochemical Analysis.
Addison-Wesley.
15. Fuwa K (1950) J J a p a n Chem SOC 71:341-343 16. P e a r s e RWB, Gaydon AG (1950) Chapman and H a l l , London
The I d e n t i f i c a t i o n o f M o l e c u l a r S p e c t r a .
17. S t r o c k LW ( 1 9 3 6 ) S p e c t r u m A n a l y s i s w i t h t h e C a r b o n A r c C a t h o d e Laser. Adam H i l g e r , London 18. Fuwa K (1955) J J a p a n Chem SOC 76:14-17 19. S l a v i n W ( 1 9 6 8 ) A t o m i c A b s o r p t i o n S p e c t r o s c o p y . I n t e r s c i e n c e . 20. Tsunoda K.
F u j i w a r a K.
21. Tsunoda K.
C h i b a K,
22. F u j i m o r i S,
a
I t a i K,
New Y o r k
Fuwa K (1977) A n a l Chem 49:2035-2039
H a r a g u c h i H,
Fuwa K (1979) A n a l Chem 51:2059-2061
Tsunoda H ( 1 9 8 4 )
F l u o r i d e 17:27-35
14
23. 24.
F a s s e l VA,
K n i s e l e y RN
(1974)
F r y RC, N o r t h w a y SJ. B r o w n RM,
46:lllOA-l120A SK (1980) A n a l Chem 52:1716-1722
A n a l Chem Hughes
25. C h i b a K, Y o s h i d a K, T a n a b e K, O z a k i M, H a r a g u c h l H, (1982) Anal Chem 54:761-765 26. B o w e n H J M (1966) T r a c e E l e m e n t s i n B i o c h e m i s t r y . London
W T n e f o r d n e r JD Academic Press,
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 15-23 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
SPECTROCHEMICAL ANALYSIS
OF
15
FLUORINE BY ALUMINUM MONOFLUORIDE ABSORPTION I N
H I G H TEMPERATURE
KIN-ICHI
TSUNODA, H I R O K I HARAGUCHI,
Department o f Chemistry, 113, J a p a n
AND K E I I C H I R O FUWA
U n i v e r s i t y o f Tokyo,
7-3-1
Hongo, Bunkyo-ku,
Tokyo
ABSTRACT Spectrochemical a n a l y s i s of f l u o r i n e by aluminum monofluoride absorption i s described i n detail. method a r e reviewed.
Firstly, the analytical characteristics o f this Then,
t h e e f f e c t s o f m a t r i x m o d i f i e r s such as
a l k a l i n e e a r t h m e t a l s o n A1F m o l e c u l a r a b s o r p t i o n a r e d i s c u s s e d o n t h e b a s i s o f t i m e - r e s o l v e d measurements o f t h e m o l e c u l a r a b s o r p t i o n s o f v a r i o u s monofluorides.
I n addition, t h e determination o f f l u o r i n e i n urine,
serum,
and m i l k s a m p l e s b y A1F m o l e c u l a r a b s o r p t i o n i s shown a s an a p p l i c a t i o n o f t h i s m e t h o d t o b i o l o g i c a l samples. INTRODUCTION S e v e r a l y e a r s ago,
t h e a u t h o r s d e v e l o p e d a new s p e c t r o c h e m i c a l m e t h o d f o r
f l u o r i n e d e t e r m i n a t i o n , t h a t i s , a1 u m i n u m m o n o f l u o r i d e (A1 F) m o l e c u l a r absorption spectrometry,
where m o l e c u l a r a b s o r p t i o n o f aluminum monofluo-
r i d e p r o d u c e d i n a h i g h t e m p e r a t u r e g r a p h i t e f u r n a c e was m e a s u r e d a t 227.45 nm u s i n g a d e u t e r i u m l a m p o r a p l a t i n u m h o l l o w c a t h o d e l a m p as t h e l i g h t s o u r c e (1,Z).
T h i s m e t h o d has some s u p e r i o r a n a l y t i c a l f e a t u r e s s u c h a s
h i g h s e n s i t i v i t y (0.021
ng o f 1% a b s o r p t i o n ) ,
a p p l i c a b i l i t y t o s m a l l volume
(5-20 VL) o f s a m p l e s , and s i m p l i c i t y o f s a m p l e p r e t r e a t m e n t . The m e t h o d has s i n c c been s t u d i e d f u r t h e r and u t i l i z e d f o r t h e d e t e r m i n a t i o n o f t r a c e amounts o f f l u o r i n e i n d i f f e r e n t samples n o t o n l y i n t h i s l a b o r a t o r y b u t a l s o i n s o m e o t h e r s (3-8).
Tsunoda
aJ.,
i n p a r t i c u l a r , have success-
f u l l y u t i l i z e d t h i s method f o r t h e d e t e r m i n a t i o n o f f l u o r i n e i n b l o o d serum s a m p l e s (6).
More r e c e n t l y ,
been r e p o r t e d b y I t a i
a s u b s t a n t i a l improvement of t h i s method has
gtd. ( 7 ) .
These s t u d i e s h a v e made i t p o s s i b l e t o
e v a l u a t e t h e p o t e n t i a l i t y of t h i s method as w e l l as i t s l i m i t a t i o n . I n t h i s paper,
r e c e n t s t u d i e s on b o t h a f r a m e w o r k and an a p p l i c a t i o n o f
A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y w i t h an e l e c t r o t h e r m a l g r a p h i t e f u r nace a r e i n t r o d u c e d a f t e r a b r i e f r e v i e w o f t h e a n a l y t i c a l c h a r a c t e r i s t i c s o f t h i s method.
16 Analytical Features o f Aluminum Monofluoride S p e c t r o m e t r y w i t h an E l e c t r o t h e r m a l G r a p h i t e F u r n a c e T h e m o l e c u l a r a b s o r p t i o n s p e c t r u m o f A1F.
Molecular
Absorption
which i s produced i n a h i g h
t e m p e r a t u r e m e d i u m s u c h a s f l a m e s o r an e l e c t r o t h e r m a l
g r a p h i t e furnace,
p r o v i d e s a s h a r p a b s o r p t i o n b a n d n e a r 227.45 nm a s shown i n F i g u r e 1. When an excess amount o f aluminum compared t o f l u o r i n e i s i n t r o d u c e d i n t o t h e t h e i n t e n s i t y o f m o l e c u l a r a b s o r p t i o n o f A1F t h u s f o r m e d i s p r o p o r -
medium.
t i o n a l t o t h e a m o u n t o f f l u o r i n e p r e s e n t i n t h e sample.
Thus,
b e d e t e r m i n e d b y m e a s u r i n g t h e A1F m o l e c u l a r a b s o r p t i o n .
f l u o r i n e can Because t h e
f o r m a t i o n o f a l u m i n u m m o n o f l u o r i d e m o l e c u l e s i n an e l e c t r o t h e r m a l g r a p h i t e f u r n a c e i s so e f f i c i e n t , m i n e d (1). 227.438
Moreover,
and 227.485
Consequently,
subnanogram q u a n t i t i e s o f f l u o r i n e c a n be d e t e r -
i t h a s been shown t h a t t h e p l a t i n u m a t o m i c l i n e s a t nm a r e g o o d a b s o r b e r s o f t h e A1F m o l e c u l a r band.
when a p l a t i n u m l a m p i s u s e d a s a l i g h t s o u r c e ,
background c o r r e c t i o n u s i n g a
d e u t e r i u m lamp,
simultaneous
w h i c h i s t h e m o s t commonly
u s e d c o n v e n t i o n a l a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r , c a n be a p p l i e d w i t h o u t a n y i n s t r u m e n t a l m o d i f i c a t i o n (2). I n t h i s study, a commercial a t o m i c a b s o r p t i o n spectrometer w i t h a s i m u l taneous background c o r r e c t i o n system (Model
0.3
0.2
170-50 f r o m H i t a c h i Co.,
Ltd..
: A1 I 2 2 6 3
.
P)
0
m C
f
'
u)
n 4:
0.1
.
ol
*
226
227
228
Wavelength. nm
F i g u r e 1. M o l e c u l a r a b s o r p t i o n s p e c t r u m o f A1F i n an e l e c t r o t h e r m a l g r a p h i t e furnace. -A-: B a c k g r o u n d s p e c t r u m o b t a i n e d w i t h 5 111 o f 0.01 M a l u m i n u m n i t r a t e s o l u t i o n ; -0-: s p e c t r u m o b t a i n e d w i t h 4.5 n g F a d d e d t o t h e d r i e d b a c k ground solution.
17 Japan) e q u i p p e d w i t h an e l e c t r o t h e r m a l g r a p h i t e f u r n a c e a t o m i z e r (FLA 1 0 0 f r o m N i p p o n J a r r e l l A s h Co.. molecular absorption.
L t d . ) w a s u s e d f o r t h e m e a s u r e m e n t o f A1F
The a n a l y t i c a l p r o c e d u r e and t h e i n s t r u m e n t a l c o n d i -
t i o n s a r e s u m m a r i z e d i n T a b l e I. The a n a l y t i c a l p r o c e d u r e i s b a s i c a l l y t h e same a s t h a t o f g r a p h i t e f u r n a c e a t o m i c a b s o r p t i o n s p e c t r o m e t r y , e x c e p t t h a t a d d i t i o n o f a l u m i n u m and m a t r i x m o d i f i e r s s u c h a s F e ( I I 1 ) and S r ( I 1 ) were r e q u i r e d (see below).
The a n a l y t i c a l w o r k i n g r a n g e f o r A1F m o l e c u l a r
a b s o r p t i o n s p e c t r o m e t r y i n t h i s s y s t e m w a s ca. 0.1-1.0 v o l u m e r e q u i r e d was 5-20 uL.
ng and t h e s a m p l e
This s e n s i t i v i t y is s l i g h t l y b e t t e r o r almost
t h e same a s a n y o t h e r m e t h o d f o r f l u o r i n e d e t e r m i n a t i o n s u c h as t h e f l u o r i d e i o n s e l e c t i v e e l e c t r o d e ( I S E method), f l u o r i d e b l u e method.
and t h e l a n t h a n u m / a l i z a r i n
I t s h o u l d be n o t e d , h o w e v e r , t h a t t h e s e n s i t i v i t y o f
t h i s method v a r i e s w i t h t h e t y p e o f f u r n a c e , a p p a r e n t l y more so t h a n t h e measurement o f a t o m i c a b s o r p t i o n .
M
0.05
B a c k g r o u n d a b s o r p t i o n due t o NaCl up t o
c o u l d be c o r r e c t e d b y t h e p r e s e n t s y s t e m .
c o r r e c t i o n s y s t e m was u t i l i z e d ,
Even when t h e b a c k g r o u n d
m e a s u r e m e n t o f A1F a b s o r p t i o n o f t e n s u f -
fered from a high blank signal.
Recently,
i t was f o u n d t h a t t h i s b l a n k
s i g n a l was m a i n l y due t o t h e m o l e c u l a r a b s o r p t i o n o f a l u m i n u m o x i d e enhanced b y t h e p r e s e n c e o f o x y g e n a s an i m p u r i t y i n a r g o n s h e a t h gas. b l a n k s i g n a l can,
therefore,
The
be decreased c o n s i d e r a b l y b y p u r i f i c a t i o n o f
t h e a r g o n g a s u s i n g an o x y g e n t r a p p i n g column.
TABLE I EXPERIMENTAL PROCEDURES AND CONDITIONS FOR FLUORINE DETERMINATION BY A j F MOLECULAR ABSORPTION SPECTROMETRY WITH AN ELECTROTHERMAL GRAPHITE FURNACE A p p l i c a t i o n o f aluminum s o l u t i o n ' (0.01
M, 2 0 u l ) D r y i n g I (150"C,
20 s )
A s h i n g I (600"C,
15 s )
Cooling o f furnace A p p l i c a t i o n o f sample s o l u t i o n ( 5 u l ) D r y i n g I 1 (150"C,
20 s )
A s h i n g 11 (600"C,
30 s )
A t o m i z a t i o n and measurement (2500°C.
7 s)
' E x p e r i m e n t a l c o n d i t i o n s a t e a c h s t a g e a r e shown i n p a r e n t h e s e s . 'Aluminum n i t r a t e s o l u t i o n c o n t a i n i n g f e r r i c n i t r a t e (0.01 n i t r a t e ( 0 . 0 1 M) 1
M) and s t r o n t i u m
18
This method can detect not only inorganic fluoride but also organic fluorine. that is, covalently bound fluorine (8). However, the application of this method is limited to nonvolatile fluorine compounds as pointed out by Venkateswarlu (8). Volatile fluorine compounds may escape from the furnace before the formation of AlF molecules and predecomposition is required for such samples. Effects of Cations on the Formation of A1F Molecules in an Graphite Furnace
Electrothermal
As described previously, the nature of A1F molecular absorption spectrometry is similar t o t h a t o f graphite furnace atomic absorption spectrometry. However, the former method also has other aspects because of the complexity of the molecular formation process of A1F in a graphite furnace. In particular, the effects of different cations on this method are noteworthy. It has been shown that some transition metals such as iron, nickel, and cobalt reduced the background absorption due to aluminum oxide (1). In this figure, background
This situation can be seen i n Figure 2.
absorption due to aluminum solution was considerably reduced by the addition of Co(I1). This is interpreted as being due to Co(I1) cutting the chemical bond of aluminum and oxygen (9). Moreover, alkaline earth metals, especially strontium, were found to act as a spectral buffer for enhancing the signals and eliminating the influence of concomitants in the A1F molecular absorption method (1). Thus, i n practice, Sr(I1) and Fe(II1) have been added to the aluminum solution as matrix modifiers for the determination of fluorine
in
actual samples (1-4).
Figure 2. Background absorption spectra i n an electrothermal graphite furnace. -0-: Aluminum nitrate solution (0.01 M); -A-: Mixed solution of aluminum nitrate (0.01 M) and strontium (0.01 M); -0-: Mixed solution of aluminum nitrate (0.01 M). and cobalt nitrate (0.01 M).
19 TABLE I 1 S E N S I T I V I T I E S O F FLUORINE DETERMINATION BY DIATOMIC MOLECULAR ABSORPTION SPECTROMETRY WITH AN ELECTROTHERMAL GRAPHITE FURNACE Species
Wavelength (nmf
Sensitivity
(1% Abs.,ng)
C o m p o s i t i o n of A d d i t i v e s
A1 F
227.45
0.021
Fe(II1).
Sr(I1)
GaF
211.23
0.16
Fe(III),
Sr(I1)
InF
233.75
0.11
Fe( 1 1 1 ) . K ( I )
M9F
358.8
1.5
0.085
CaF
606.44
0.50
SrF
662.94
0.38
BaF
500.5
1.5
none
To u n d e r s t a n d t h e e f f e c t s o f c o e x i s t i n g c a t i o n s .
these s t u d i e s were
extended t o t h e molecular absorption o f o t h e r monohalides o f a l k a l i n e e a r t h m e t a l s and Group I I I B e l e m e n t s . m o l e c u l e s w e r e o b t a i n e d (10.11).
F i r s t , the absorption spectra o f those Table I 1 summarizes t h e s e n s i t i v i t i e s o f
the molecular absorptions o f various monofluorides.
Although aluminum
monofluoride molecular absorption provides the highest sensitivity,
other
m o n o f l u o r i d e m o l e c u l e s a l s o h a v e r e l a t i v e l y s h a r p and s t r o n g band s p e c t r a . These s p e c t r a c a n a l s o b e u t i l i z e d f o r f l u o r i n e d e t e r m i n a t i o n i n t h e same way a s t h e a l u m i n u m m o n o f l u o r i d e spectrum.
Furthermore, time-resolved
signals o f the molecular absorption o f those monofluorides w i t h various c o e x i s t i n g c a t i o n s were o b t a i n e d u s i n g a r a p i d response measurement system i n o r d e r t o i n v e s t i g a t e t h e i n f l u e n c e of t h e s e c a t i o n s .
The t i m e - r e s o l v e d
s i g n a l p r o f i l e s o f GaF m o l e c u l a r a b s o r p t i o n a r e s h o w n i n F i g u r e 3 a s a n example.
P r o f i l e a) shows GaF m o l e c u l a r a b s o r p t i o n w i t h t h e s o l u t i o n o f
G a ( l 0 mM) and F ( 2 u g / m l ) , ug/ml), tion,
p r o f i l e b ) w i t h Ga ( 1 0 m M ) .
p r o f i l e c ) w i t h Ga ( 1 0 m M ) ,
Na ( 1 0 m M ) a n d F ( 2
S r (10 mM) and F ( 2 ug/ml).
I n addi-
1 0 m M Fe was added t o e a c h o f t h e s a m p l e s t o m i n i m i z e t h e m o l e c u l a r
absorption o f g a l l i u m oxides. s p l i t i n t o t w o peaks.
I n p r o f i l e c,
t h e a b s o r p t i o n s i g n a l was
The f i r s t peak c o r r e s p o n d s t o t h a t o f p r o f i l e a,
t h e s e c o n d peak a p p e a r e d i n t h e h i g h e r t e m p e r a t u r e r e g i o n .
but
The t e m p e r a t u r e
r e g i o n o f t h e second peak i n p r o f i l e c was c l o s e t o t h a t o f S r F m o l e c u l a r a b s o r p t i o n ( p r o f i l e d).
S i m i l a r r e s u l t s w e r e o b t a i n e d f o r a l l o t h e r mono-
f l u o r i d e s o f Group I I I B e l e m e n t s .
These o b s e r v a t i o n s s u g g e s t t h e c o m p l e x -
f o r m a t i o n o f f l u o r i d e w i t h c o e x i s t i n g c a t i o n s on t h e g r a p h i t e s u r f a c e and t
20
Time, s
F i g u r e 3. T i m e - r e s o l v e d s i g n a l p r o f i l e s o f GaF m o l e c u l a r a b s o r p t i o n . a, Ga and Fe ( e a c h 0.01 M) and 2 u g / m l o f F; b, Ga, Fe and Na ( e a c h 0.01 M ) and 2 u g / m l o f F; c , Ga, Fe and S r ( e a c h 0.01 M ) and 2 u g / m l o f F: d, S r (0.01 M ) and 2 u g / m l o f F ( S r F m o l e c u l a r a b s o r p t i o n p l o t t e d ) . C a t i o n s w e r e added as n i t r a t e . GaF a n d S r F m o l e c u l a r a b s o r p t i o n s w e r e o b t a i n e d a t 211.45 nm and 663.1 nm, r e s p e c t i v e l y .
t h e e f f e c t o f t h e c a t i o n s on c o n t r o l l i n g t h e v a p o r i z a t i o n o f f l u o r i d e . Moreover,
i t has been observed t h a t t h e a p p a r e n t t e m p e r a t u r e o f a S r F
m o l e c u l e i s a l m o s t t h e s a m e a s t h a t o f a n A1F m o l e c u l e i n t h e g r a p h i t e furnace.
Thus, t h e e n h a n c i n g e f f e c t o f S r ( I 1 ) i s i n t e r p r e t e d as a r e s u l t
of p r o t e c t i o n o f f l u o r i d e b y S r ( I 1 ) u n t i l t h e t e m p e r a t u r e o f A1F f o r m a t i o n on t h e g r a p h i t e s u r f a c e i s r e a c h e d . D e t e r m i n a t i o n o f F l u o r i n e i n B i o l o g i c a l S a m p l e s b y A1F M o l e c u l a r A b s o r p t i o n Spectrometry F l u o r i n e i n u r i n e , serum, t i o n o f th!s tion of
and m i l k s a m p l e s w e r e d e t e r m i n e d a s an a p p l i c a -
m e t h o d t o b i o l o g i c a l samples.
fluorine
i n a freeze-dried
M a t e r i a l 2671 f r o m NBS i n U.S.A.)
(3).
T a b l e I 1 1 shows t h e d e t e r m i n a -
u r i n e sample (Standard Reference The u r i n e s a m p l e s w e r e d i l u t e d w i t h
d i s t i l l e d w a t e r b y v a r i o u s d i l u t i o n f a c t o r s and a p p l i e d d i r e c t l y t o t h e A 1 F m o l e c u l a r a b s o r p t i o n measurements. a s d e s c r i b e d i n S e c t i o n 2.
The a n a l y t i c a l p r o c e d u r e was t h e same
As c a n be seen f r o m t h e t a b l e , f l u o r i d e concen-
t r a t i o n s o b t a i n e d b y t h i s m e t h o d w e r e i n good a g r e e m e n t w i t h b o t h c e r t i f i e d
21
TABLE 111 DETERMINATION OF FLUORINE I N FREEZE-DRIED U R I N E FROM NBS (SRM 2671) Samples
Dilution factor
Found, r.cg/mL
I SE*
AI ~1
Certified values w/ml
elevatedlevel sample
100 40 20
7.1 f 0.1 6.90 f 0.18 7.10 f 0.18
7.6 k 0.3 7.00 f 0.24 7.00 f 0.24
7.14 f 0.48
low-level sample
10
0.89 f 0.03 0.81 f 0.05
0.80 f 0.03 0.80 f 0.05
0.84 5 0.08
5
' A l F molecular absorption spectrometry 2 F l u o r i d e i o n s e l e c t i v e e l e c t r o d e method
NBS v a l u e s and t h o s e o b t a i n e d by t h e I S E method.
I n addition,
the f l u o r i n e
c o n t e n t i n b l o o d serum samples was a l s o d e t e r m i n e d by t h i s method,
and t h e
r e s u l t s were compared w i t h t h o s e o b t a i n e d by t h e I S E method ( T a b l e I V ) .
In
t h i s e x p e r i m e n t , t h e serum samples were d i l u t e d 10 t i m e s w i t h d i s t i l l e d water,
and A 1 F m o l e c u l a r a b s o r p t i o n s were measured i n t h e same manner as
w i t h u r i n e s a m p l e s . R e c o v e r y v a l u e s o f ca. 100% i n T a b l e I V i n d i c a t e t h a t A 1 F m o l e c u l a r a b s o r p t i o n method i s a r e l i a b l e method f o r t h e d e t e r m i n a t i o n o f f l u o r i n e i n b l o o d serum.
On t h e o t h e r hand,
t h e v a l u e s o b t a i n e d by t h i s
method were much h i g h e r t h a n t h o s e by t h e I S E method.
As aluminum mono-
f l u o r i d e m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y can d e t e c t n o t o n l y i o n i c f l u o r i d e , b u t a l s o c o v a l e n t l y bound f l u o r i n e ( s e e S e c t i o n 2). t h e d i f f e r e n c e
TABLE I V
D E T E R M I N A T I O N OF FLUORINE I N BLOOD SERUM
Sample
A1F method' Added Recovery3 (r.cg/mL) (Z)
Found (w/mL)
Found (ug/mL)
I
0.13 f 0.02
0.34 f 0.04
105
0.076 f 0.002
I1
0.12 f 0.02
0.31 f 0.04
95
0.025 f. 0.001
111
0.21 f 0.03
0.40
f 0.04
95
0.020 f 0.001
IV
0.16 ? 0.02
0.35 f 0.04
95
0.024 f 0.001
' A l F molecular absorption spectrometry 2 F l u o r i d e i o n s e l e c t i v e e l e c t r o d e method 3 F l u o r i d e was added a t 0.2 vg/ml I
I S E method2
22 between t h e t w o methods may be a t t r i b u t a b l e t o t h e e x i s t e n c e o f s o - c a l l e d "nonionic
fluorine"
"nonionic fluorine,"
i n serum samples (6,12). however,
The a c t u a l c h e m i c a l f o r m s o f
r e m a i n unclear.
A l t h o u g h t o t a l f l u o r i n e i n b o t h u r i n e and serum samples can be d e t e r m i n e d b y d i r e c t m e a s u r e m e n t u s i n g t h e A1F m o l e c u l a r a b s o r p t i o n ,
t h i s method
cannot be a p p l i e d d i r e c t l y t o t h e d e t e r m i n a t i o n o f f l u o r i n e i n bovine m i l k As shown i n T a b l e V,
samples.
l o w e r a n a l y t i c a l v a l u e s were o b t a i n e d w i t h
t h e d i r e c t a n a l y s i s o f t w i c e d i l u t e d m i l k samples by t h i s method compared t o t h o s e f o r t h e d i s t i l l e d samples.
F u r t h e r i n v e s t i g a t i o n suggested t h a t
t h i s d i f f e r e n c e was m a i n l y due t o t h e i n t e r f e r e n c e o f o r g a n i c m a t t e r s c o n t a i n e d i n m i l k samples.
A l t h o u g h i n t e r f e r e n c e by o r g a n i c m a t t e r s w i t h
t h i s m e t h o d was p a r t i a l l y r e m o v e d b y t h e a d d i t i o n o f a l a r g e amount o f m a t r i x m o d i f i e r such as S r ( I I ) ,
p r e t r e a t m e n t w i l l be necessary f o r samples
containing high concentrations o f organic matters.
TABLE V
D E T E R M I N A T I O N OF FLUORIDE I N B O V I N E MILK BY A1F MOLECULAR ABSORPTION SPECTROMETRY
Method Direct-' D r y ashing'
F l u o r i d e Content (ng/ml)
33.6 ?r 15.9
105.3 f 19.8
'The samples were d i l u t e d t w i c e w i t h d i s t i 1 l e d water. 'The samples were dry-ashed w i t h t h e a d d i t i o n o f sodium carbonate and t h e n d i s t i l l e d w i t h a steam d i s t i l l a t i o n s ys tern.
CONCLUSION AluminiAm m o n o f l u o r i d e m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y has e x c e l l e n t a n a l y t i c a l f e a t u r e s as w e l l as some l i m i t a t i o n s .
T h i s method i s a p p l i c a b l e
t o t h e d e t e r m i n a t i o n o f f l u o r i n e i n v a r i o u s b i o l o g i c a l and e n v i r o n m e n t a l samples,
a l t h o u g h i t may need o c c a s i o n a l sample p r e t r e a t m e n t such as d r y
a s h i n g f o l l o w i n g d i s t i l l a t i o n o r d i f f u s i o n techniques.
It i s hoped t h a t
t h e p o t e n t i a l i t y o f t h e method becomes w i d e l y known and t h a t i t can cont r i b u t e t o d i f f e r e n t areas o f f l u o r i d e research.
23
REFERENCES 1. Tsunoda K,
F u j i w a r a K,
2. Tsunoda K,
C h i b a K, H a r a g u c h i H,
3. C h i b a K, Tsunoda K.
Fuwa K (1977) A n a l Chem 49:2035-2039 Fuwa K (1979) A n a l Chem 51:2059-2061
H a r a g u c h i H,
Fuwa K (1980) A n a l Chem 52:1582-1585
4. T a k a t s u A. C h i b a K, O z a k i M, F u w a K, H a r a g u c h i H ( 1 9 8 4 ) S p e c t r o c h i m A c t a 39B:365-370
5. D i t t r i c h K (1979)
A n a l Chim A c t a 111:123-135
6. F u j i m o r i S, I t a i K, 7. I t a i K.
Tsunoda H (1984)
F l u o r i d e 17:27-35
Tsunoda H (1985) A n a l Chim A c t a 171:293-301
8. V e n k a t e s w a r l u P, 55: 2232-2236
W i n t e r LD,
9. Tsunoda K.
F u j i W a r a K,
10. Tsunoda K.
H a r a g u c h i H.
P r o k o p RA,
H a g e n D F ( 1 9 8 3 ) A n a l Chem
Fuwa K (1978) A n a l Chem 50:861-865 Fuwa K (1984) S p e c t r o c h i m A c t a 35B:715-729
11. Tsunoda K, C h i b a K. H a r a g u c h i H, C h a k r a b a r t i CL, Fuwa K (1982) Canadian J S p e c t r o s c 27:94-97
12. Taves DR (1968)
N a t u r e 220:582-583
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 25-29 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
25
DETERMINATION OF SUBMICROGRAM QUANTITIES OF FLUORIDE BY A R A P I D AND HIGHLY SENSITIVE METHOD
K A Z U Y O S H I I T A I AND HUM10 TSUNODA D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , U n i v e r s i t y , M o r i o k a , I w a t e 020, J a p a n
School o f Medicine,
Iwate Medical
ABSTRACT
A new m e t h o d h a s b e e n d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f s u b m i c r o g r a m q u a n t i t i e s o f f l u o r i d e . It c o n s i s t s o f s e p a r a t i n g f l u o r i d e f r o m t h e t e s t sample b y t h e p y r o h y d r o l y t i c s e p a r a t i o n method and d e t e r m i n a t i o n o f t h e r e s u l t i n g aqueous HF s o l u t i o n b y e l e c t r o t h e r m a l m o l e c u l a r a b s o r p t i o n spect r o m e t r y as a l u m i n u m m o n o f l u o r i d e ( A l F ) . sensitive,
T h i s method i s rapid,
highly
and c a n be used f o r t h e d e t e r m i n a t i o n o f t o t a l ( o r g a n i c and
i n o r g a n i c ) f l u o r i d e i n v a r i o u s o r g a n i c compounds and b i o l o g i c a l m a t e r i a l s .
INTRODUCTION
A number o f a n a l y t i c a l m e t h o d s h a v e been d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f f l u o r i n e i n o r g a n i c f l u o r i d e compounds
and b i o l o g i c a l m a t e r i a l s (1-3).
Many o f t h e s e m e t h o d s a r e r a t h e r c o m p l e x and t i m e - c o n s u m i n g .
I n addition,
when t h e f l u o r i n e c o n c e n t r a t i o n s o f t h e s a m p l e s a r e l o w , t h e y c a n n o t b e a c c u r a t e l y determined.
A r a p i d and h i g h l y s e n s i t i v e m e t h o d f o r f1uo:ide developed.
d e t e r m i n a t i o n h a s been
I t combines t h e p y r o h y d r o l y t i c s e p a r a t i o n method w i t h
electro-
t h e r m a l a l u m i n u m m o n o f l u o r i d e (A1F) m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y . The p y r o h y d r o l y t i c s e p a r a t i o n method i s v a l u a b l e f o r r a p i d i s o l a t i o n o f f l u o r i n e f r o m i n o r g a n i c r e f r a c t o r y compounds, many o r g a n i c m a t e r i a l s (4-7).
s u c h as c a l c i u m f l u o r i d e ,
o r from
I n a d d i t i o n , t h i s method i s f r e e f r o m conta-
m i n a t i o n by extraneous f l u o r i d e s .
By u s e o f an a u t o s a m p l e r ,
f l u o r i n e can
be a c c u r a t e l y and p r e c i s e l y d e t e r m i n e d a t t h e p p b l e v e l b y e l e c t r o t h e r m a l A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y . MATERIAL AND METHODS Apparatus F i g u r e 1 shows t h e a p p a r a t u s u s e d i n t h e p y r o h y d r o l y s i s . t i o n tube,
A quartz reac-
550 m m l o n g w i t h an i n t e r n a l d i a m e t e r o f 1 3 m m i s m a i n t a i n e d b y
26 a main-heater
a t a p p r o x i m a t e l y 1100°C.
The t u b e i s g r a d u a l l y
heated t o
500°C w i t h a s u b - h e a t e r . O x y g e n g a s i s s c r u b b e d w i t h 0.2N KOH s o l u t i o n , s i l i c a g e l , a n d a c t i v e charcoal,
and p a s s e d t h r o u g h t h e t u b e w i t h w a t e r v a p o r a t a r a t e o f a p p r o x -
i m a t e l y 1 l/min.
P l a t i n u m gauze,
p l a c e d a t t h e end o f t h e t u b e .
used a s a c a t a l y s t f o r t h e o x i d a t i o n .
is
A p i e c e o f q u a r t z wool i s placed behind t h e
p l a t i n u m gauze f o r t r a p p i n g v o l a t i l e m e t a l s and m e t a l o x i d e s (6).
A suit-
a b l e a m o u n t o f c o n d e n s a t e i s w e i g h e d w i t h an e l e c t r i c b a l a n c e i n a p o l y s t y r e n e b o t t l e w i t h cover. The a p p a r a t u s as f o l l o w s :
used f o r d e t e r m i n a t i o n o f f l u o r i n e i n t h e condensate a r e
a t o m i c a b s o r p t i o n s p e c t r o m e t e r m o d e l AA-855.
equipped w i t h a
d e u t e r i u m s i m u l t a n e o u s b a c k g r o u n d c o r r e c t i o n s y s t e m ; g r a p h i t e f u r n a c e atomi z e r , m o d e l FLA-100; sampler,
m i c r o p r o c e s s o r r e a d o u t s y s t e m , m o d e l MC-100;
model AS-301
( a 1 1 m a n u f a c t u r e d b y N i p p o n J a r r e l l - A s h Co.,
and a u t o Ltd.).
F i g u r e 1. A d i a g r a m s h o w i n g a p p a r a t u s u s e d f o r p y r o h y d r o l y s i s 1: 0.2" KOH s o l u t i o n : 2: S i l i c a g e l ; 3: A c t i v e c h a r c o a l : 4: F l o w m e t e r : 5: W a t e r f l a s k ( 9 0 ° C ) ; 6: R e a c t i o n t u b e ( Q u a r t z ) ; 7: S u b h e a t e r (0-500°C); 8: M a i n h e a t e r ( 1 1 0 0 ° C ) : 9: S a m p l e b o a t ; 1 0 : P l a t i n u m g a u z e ; 1 1 : Q u a r t z w o o l : 12: Condenser; 13: S t y r e n e b o t t l e : 14: E l e c t r i c b a l a n c e .
Reagents
A s t a n d a r d f l u o r i d e s o l u t i o n was p r e p a r e d b y d i s s o l v i n g a n a l y t i c a l - g r a d e s o d i u m f l u o r i d e (Wako P u r e C h e m i c a l s ) i n d i s t i l l e d w a t e r .
A 1% a l u m i n u m
s o l u t i o n w a s p r e p a r e d b y d i s s o l v i n g 5 g o f a l u m i n u m m e t a l p o w d e r (99.5%. Wako) i n 1 0 m l o f c o n c e n t r a t e d n i t r i c a c i d a n d d i l u t i n g t o 5 0 0 m l w i t h d i s t i l l e d water.
A 2% b a r i u m s o l u t i o n was p r e p a r e d b y d i s s o l v i n g 38.06 g
o f a n a l y t i c a l - g r a d e b a r i u m n i t r a t e (Wako)
i n d i s t i l l e d w a t e r and d i l u t i n g
t o 1000 m l . Procedure S a m p l e s r a n g i n g f r o m s e v e r a l mg t o g w e r e w e i g h e d i n t o t h e s a m p l e b o a t , and w e r e i n t r o d u c e d i n t o t h e s u b - h e a t e r a t u r e o f t h e sub-heater 4
p a r t o f t h e tube.
was g r a d u a l l y r a i s e d t o 500°C,
i n t r o d u c e d i n t o t h e c e n t e r o f t h e main-heater.
When t h e t e m p e r -
t h e s a m p l e b o a t was
F l u o r i n e i n t h e s a m p l e was
27 i s o l a t e d a s HF a n d w a s c o n d e n s e d w i t h w a t e r v a p o r b y t h e c o n d e n s e r .
AP
a p p r o p r i a t e a m o u n t o f t h e c o n d e n s a t e was w e i g h e d i n t o a p o l y s t y r e n e b o t t l e C o n d e n s a t e s r a n g i n g f r o m 5 t o 20 g ( m l ) w e r e u s u a l l y c o l -
w i t h cover. lected.
For very resistant materials,
tungsten t r i o x i d e ,
s u c h a s CaF2,
t h e a d d i t i o n o f some
w h i c h a c t e d a s an a c i d f l u x , was r e q u i r e d (7).
Measurement o f F l u o r i n e i n C o n d e n s a t e The p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f f l u o r i n e i n t h e c o n d e n s a t e b y e l e c t r o t h e r m a l A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y i s shown i n F i g u r e 2
(5).
To 800 p 1 o f t h e c o n d e n s a t e o r a s t a n d a r d s o l u t i o n i n a p o l y e t h y l e n e
c u p was added 200 p l o f t h e a l u m i n u m s o l u t i o n c o n t a i n i n g 0.1% a l u m i n u m and
1% b a r i u m a s n i t r a t e , r e s p e c t i v e l y . autosampler.
The p o l y e t h y l e n e c u p was s e t i n t h e
The m i x t u r e c o n t a i n i n g t h e c o n d e n s a t e and t h e a l u m i n u m s o l u -
t i o n was a u t o m a t i c a l l y i n J e c t e d i n t o t h e g r a p h i t e f u r n a c e . m e a s u r e m e n t a r e s h o w n i n F i g u r e 2.
Conditions f o r
T h e a b s o r b a n c e o f A1F g e n e r a t e d w a s
a u t o m a t i c a l l y m e a s u r e d and t h e f l u o r i n e c o n c e n t r a t i o n o f t h e c o n d e n s a t e was determined by a microprocessor readout system from a c a l i b r a t i o n curve obtained by using standard f l u o r i d e solutions.
Condensate
1 -
8 0 0 , ~l
; At I
U I
soIution'200p
e
P o l y e t h y l e n e cup
I
Autosampler (20,u Q )
I
D r y i n g (,l50V 30sec) I C h a r r i n g ( 8 3 O C 15sec)
I I
A t o m i z i n g (2800C 7 s e e ) Measurement F i g u r e 2. P r o c e d u r e f o r d e t e r m i n a t i o n o f f l u o r i d e b y A l F m o l e c u l a r a b s o r p t i o n spectrometry. A1 s o l u t i o n c o n s i s t s o f 0.1% A1 and 1% BA a s a n i t r a t e .
RESULTS AND D I S C U S S I O N Recoveries o f f l u o r i n e f r o m several standard m a t e r i a l s by use o f t h e p r e s e n t m e t h o d a r e shown i n T a b l e I. I n a l l t h e f l u o r i d e compounds t e s t e d , t h e f l u o r i n e contents found were i n agreement w i t h t h e c a l c u l a t e d values, a n d t h e r e c o v e r y w a s n e a r l y 100%.
As observed by Warf gt
r i d e s c a n be g r o u p e d i n t o t w o c a t e g o r i e s : and a s l o w l y p y r o h y d r o l y z a b l e group. alkaline-earth 1
aj.
(4).
fluo-
a r a p i d l y p y r o h y d r o l y z a b l e group,
The f l u o r i d e s o f t h e a l k a l i n e and
m e t a l s b e l o n g t o t h e l a t t e r g r o u p (6).
F l u o r i n e compounds
28 TABLE I
OF
RECOVERIES
FLUORINE FROM STANDARD MATERIALS
% F calculated
% F found
Recovery (%)
NaF
45.2
45.5
CaF2
48.7
48.2'
107 99.0
N ( CF2CF2CF2CF3)3
76.4
76.1
99.6
Cg IH 5 C6 H4 F
11.0
10.7
97.3
'W03 was added
o f the alkaline-earth
metals,
such as c a l c i u m f l u o r i d e .
are very heat
F l u o r i n e i n t h e s e m a t e r i a l s was s e p a r a t e d b y t h e u s e o f W03 as
resistant.
A c c o r d i n g t o Leuven Q t d l (7).
an a c i d f l u x .
i n t h e absence o f t h e f l u x
t h e r e c o v e r y o f f l u o r i n e i n t h e s e m a t e r i a l s was p o o r , b e l o w 50%, b u t i n t h e p r e s e n c e o f t h e f l u x t h e r e c o v e r y w a s n e a r l y 100%.
I n t h i s method,
the
r e c o v e r y o f f l u o r i n e i n CaF2 w i t h t h e u s e o f W03 was n e a r l y 100%. T a b l e I 1 shows t h e f l u o r i n e c o n t e n t s o f v a r i o u s o r g a n i c m a t e r i a l s d e t e r m i n e d b y t h i s method.
I n order f o r rapid pyrohydrolytic separation o f
f l u o r i n e i n t h e s e m a t e r i a l s a s HF,
W03 w a s a d d e d a s a n a c i d f l u x .
f l u o r i n e c o n t e n t o f orchard leaves,
NBS S t a n d a r d R e f e r e n c e M a t e r i a l 1571,
w a s r e p o r t e d t o b e 4 ppm.
The
W i t h t h i s m e t h o d , i t w a s f o u n d t o b e 4.6 ppm.
T h e a v e r a g e f l u o r i n e c o n t e n t i n t h e same s a m p l e d e t e r m i n e d b y A.O.A.C. m e t h o d ( 1 ) was 5.320.61
11.5%.
ppm (n=7),
and t h e r e l a t i v e s t a n d a r d d e v i a t i o n was
The a v e r a g e v a l u e o f u n p o l i s h e d r i c e o b t a i n e d b y t h i s m e t h o d was
0.57 ppm.
The a v e r a g e f l u o r i d e v a l u e i n t h e same s a m p l e a s d e t e r m i n e d b y
t h e A.O.A.C.
m e t h o d ( 1 ) was 0.7020.13
ppm ( n = 5 ) ,
and t h e r e l a t i v e s t a n d a r d
TABLE I 1 DETERMINATION
OF
Sample
FLUORIDE I N V A R I O U S MATERIALS n
x f S.D. (uLl/g)
0.21
c.v
(%)
O r c h a r d 1e a v e s
8
4.6
f
Unpolished r i c e
6
0.57
2 0.02
3.4
Human h a i r
5
2.4
f 0.24
10.0
Human serum
4
0.043 f 0.006
4.6
14.0
29 d e v i a t i o n was 18.6%.
Sakurai
& &.
(9) r e p o r t e d t h a t t h e average f l u o r i n e
c o n t e n t i n u n p o l i s h e d r i c e i n J a p a n was 0.89 ppm. The f l u o r i n e c o n t e n t o f human h a i r , available,
f o r which l i t t l e information i s
was d e t e r m i n e d b y t h i s method.
r e l a t i v e s t a n d a r d d e v i a t i o n w a s 10%. t h o s e f o r t h e p l a n t s a m p l e s shown,
The a v e r a g e was 2.4 ppm and t h e
The l a r g e v a r i a t i o n , c o m p a r e d t o
i s p r e s u m a b l y due t o t h e h a i r b e i n g
s c a t t e r e d b y a n e l e c t r o s t a t i c f o r c e w h i l e t h e s a m p l e was p l a c e d i n t h e r e a c t i o n tube.
The f l u o r i n e c o n c e n t r a t i o n o f human s e r u n i s s o l o w t h a t
a c c u r a t e d e t e r m i n a t i o n h a s been d i f f i c u l t .
A l a r g e volume o f serum sample
i s needed f o r t h e d e t e r m i n a t i o n .
W i t h t h i s method,
done w i t h o n l y 1 m l o f sample.
The r e l a t i v e l y h i g h r e l a t i v e s t a n d a r d
d e v i a t i o n seen i n T a b l e I 1 i s due t o
t h e d e t e r m i n a t i o n was
the extremely low concentration o f
f l u o r i n e i n t h e sample.
CONCLUSION T h e c o m b i n e d u s e o f p y r o h y d r o l y s i s a n d e l e c t r o t h e r m a l A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y h a s made i t p o s s i b l e t o d e t e r m i n e f l u o r i n e i n many m a t e r i a l s r a p i d l y and a c c u r a t e l y .
I n t h e presence o f a c a t a l y s t such as
W03, a r a p i d s e p a r a t i o n o f f l u o r i n e f r o m i n o r g a n i c r e f r a c t o r y c o m p o u n d s (e.g.
CaF2) c a n b e a c c o m p l i s h e d .
Aluminum m o n o f l u o r i d e (AlF) m o l e c u l a r
a b s o r p t i o n s p e c t r o m e t r y c a n be u s e d t o d e t e r m i n e t h e l e v e l o f f l u o r i n e i n a s a m p l e o f 20 g l w i t h h i g h a c c u r a c y . hydrolysis,
When s a m p l e s a r e o b t a i n e d f r o m
pyro-
c o n t a m i n a t i o n b y extraneous f l u o r i d e i s avoided.
ACKNOWLEDGEMENT T h i s research i s supported by Grant-in-Aid R e s e a r c h (No.
60480193) f o r 1985-1987
f o r Co-operative S c i e n t i f i c
from t h e M i n i s t r y o f Education,
S c i e n c e and C u l t u r e o f t h e G o v e r n m e n t o f Japan
REFERENCES 1.
O f f i c i a l Methods o f A n a l y s i s (1970) 6.023
1 1 t h Ed AOAC.
2.
B a i l e y JJ,
3.
V e n k a t e s w a r l u P ( 1 9 7 7 ) M e t h B i o c h e m A n a l 24:93
4.
W a r f JC, C l i n e WD,
5.
K a k a b a d s e GJ, M a n o h i n N a t u r e 229:626
G e h r i n g DG ( 1 9 6 1 )
6.
B e r n s EG,
7.
Van L e u v e n HCE, 296: 36
8.
I t a i K,
9.
I
A n a l Chem 33:1760
Tevebaugh RD (1954) A n a l Chem 26:342
B,
B a t h e r JM, W e l l e r EC.
Woodbridge
P
(1971)
Van D e r Zwaan PW (1972) A n a l C h i m A c t a 59:293
Tsunoda H.
S a k u r a i S.
W a s h i n g t o n , USA,
I t a i K,
R o t s c h e i d GJ, B u i s WJ ( 1 9 7 9 ) F r e s e n i u s Z A n a l Chem I k e d a M (1985) A n a l C h i m A c t a 171:293 Tsurfoda H (1983) F l u o r i d e 16:175
H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 31-42 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
31
PLASMA E M I S S I O N SPECTROMETRY OF FLUORINE AND I T S ENVIRONMENTAL APPLICATION
H I R O K I HARAGUCHI Department o f Chemistry, F a c u l t y o f Science, Bunkyo-ku, T o k y o 11 3, J a p a n
The U n i v e r s i t y o f Tokyo,
ABSTRACT Spectrochemical a n a l y s i s o f f l u o r i n e by plasma emission spectrometry i s described.
First,
p l a s m a e m i s s i o n s p e c t r o m e t r y and i t s p r e s e n t s t a t u s a r e
reviewed e s p e c i a l l y f o r t h e d e t e r m i n a t i o n o f n o n m e t a l l i c elements. Secondly atomic emission spectrometry u s i n g h e l i u m microwave-induced plasma ( M I P ) a t atmospheric pressure,
w h i c h i s an e f f i c i e n t e x c i t a t i o n s o u r c e f o r n o n m e t a l -
l i c elements as w e l l as m e t a l l i c elements, s e l e c t i v e d e t e c t o r i n gas c h r o m a t o g r a p h y (GC).
i s d i s c u s s e d a s an e l e m e n t Finally,
the determination
o f f l u o r i n e i n w a t e r s a m p l e s b y t h e GC/helium M I P s y s t e m i s shown as an e x p e r i m e n t a l a p p l i c a t i o n o f h e 1 i u m M I P t o e n v i r o n m e n t a l and g e o c h e m i c a l sampl es.
INTRODUCTION S p e c t r o c h e m i c a l a n a l y s i s u s i n g h i g h t e m p e r a t u r e media has e v o l v e d s i n c e t h e w o r k o f B u n s e n a n d K i r c h h o f f (1).
They used a
c h e m i c a l f l a m e as an
e x c i t a t i o n s o u r c e i n f l a m e e m i s s i o n s p e c t r o m e t r y . Flames, a r c s , sparits, d i s c h a r g e t u b e s and s o f o r t h h a v e b e e n u s e d as a t o m i z a -
g r a p h i t e furnaces,
t i o n / e x c i t a t i o n s o u r c e s i n a n a l y t i c a l a t o m i c s p e c t r o s c o p y such as a t o m i c emission,
absorption,
and f l u o r e s c e n c e s p e c t r o m e t r y .
A t o m i c a b s o r p t i o n s p e c t r o m e t r y (AAS) w a s d e v e l o p e d a b o u t 30 y e a r s ago, and i t h a s b e e n u s e d a s a p o w e r f u l a n a l y t i c a l t o o l i n t r a c e e l e m e n t a n a l y s i s (2).
I n AAS,
f l a m e s and e l e c t r o t h e r m a l g r a p h i t e f u r n a c e s a r e c o m m o n l y
employed as e f f i c i e n t a t o m i z e r s f o r most elements.
As i s w e l l k n o w n ,
however, AAS i s used f o r t h e d e t e r m i n a t i o n o f m e t a l l i c e l e m e n t s and n o t n o n m e t a l l i c elements.
T h e r e a s o n why a t o m i c a b s o r p t i o n o r e v e n a t o m i c
e m i s s i o n s p e c t r o m e t r y cannot be a p p l i e d t o t h e d e t e r m i n a t i o n o f non-metall i c e l e m e n t s i s t h a t t h e i r a t o m i c r e s o n a n c e l i n e s e x i s t i n t h e vacuum u l t r a v i o l e t region,
a s shown i n T a b l e I. T h e r e f o r e ,
molecular absorption
o r e m i s s i o n s p e c t r o m e t r y h a s been e x a m i n e d f o r n o n m e t a l l i c e l e m e n t d e t e r m i nation. W i t h f l u o r i n e , t h e w a v e l e n g t h o f t h e a t o m i c r e s o n a n c e l i n e i s 95.5 nm. w h i C h r e q u i r e s an e x c i t a t i o n e n e r g y o f 12.92 eV.
The e x p e r i m e n t a l i n c o n v e -
32
TABLE I ATOMIC RESONANCE LINES OF NONMETALLIC ELEMENTS Element
Wavelength (nm)
H
nience
E x c i t a t io n E n e r g y (eV)
121.6
Ionization E n e r g y (eV)
10.21
13.60
C
165.7
7.45
11.26
N
113.5
70.87
14.53
0
130.2
9.48
13.62
F
95.5
12.92
17.42
P
177.5
6.95
10.49
S
180.7
6.83
10.36
c1
134.7
9.16
12.97
Br
148.8
8.29
11.81
I
183.0
6.78
10.45
of
vacuum UV s p e c t r o s c o p y and l a c k o f e f f i c i e n t e x c i t a t i o n s o u r c e s
have hampered t h e d e v e l o p m e n t o f a n a l y t i c a l a t o m i c s p e c t r o s c o p i c t e c h n i q u e s f o r f l u o r i n e determination.
However,
i f an e x c i t a t i o n s o u r c e w h i c h a l l o w s
t h e e x c i t a t i o n o f f l u o r i n e a t o m s much h i g h e r t h a n t h e l e v e l a t 12.91 eV i s invented, sible.
t h e n a t o m i c e m i s s i o n s p e c t r o m e t r y o f f l u o r i n e w i 11 become posR e c e n t d e v e l o p m e n t o f r a r e gas p l a s m a s u s i n g h e l i u m and a r g o n
p r o v i d e s such a p o t e n t i a l i t y .
Hence p l a s m a e m i s s i o n s p e c t r o m e t r y w i l l be
b r i e f l y discussed i n t h e n e x t section. Plasma e m i s s i o n s p e c t r o m e t r y f o r s p e c t r o c h e m i c a l a n a l y s i s P l a s m a e x c i t a t i o n s o u r c e s u s i n g r a r e gases s u c h as a r g o n and h e l i u m have been d e v e l o p e d f o r e m i s s i o n s p e c t r o c h e m i c a l a n a l y s i s . c a l l e d plasma emission spectrometry.
The method i s now
As d e s c r i b e d p r e v i o u s l y , AAS has been
a commonly u s e d s p e c t r o m e t r i c m e t h o d f o r t r a c e a n a l y s i s .
However,
plasma
e m i s s i o n s p e c t r o m e t r y has s u b s t a n t i a l l y r e p l a c e d AAS i n t r a c e a n a l y s i s because i t has many a n a l y t i c a l advantages,
i n c l u d i n g h i g h s e n s i t i v i t y and
p r e c i s i o n , w i d e d y n a m i c ranges, a n d s i m u l t a n e o u s m u l t i - e l e m e n t a n a l y s i s (3). The p l a s m a e x c i t a t i o n s o u r c e s w h i c h a r e now commonly used and commerc i a l l y a v a i l a b l e a r e as f o l l o w s : a r g o n gas; and,
( 1 ) d i r e c t c u r r e n t p l a s m a (DCP).
(2) i n d u c t i v e l y c o u p l e d p l a s m a ( I C P ) ,
rarely,
n i t r o g e n gas; (3) m i c r o w a v e - i n d u c e d
o r h e l i u m gas.
u s i n g .commonly, p l a s m a (MIP),
using
a r g o n gas u s i n g argon
33
ICP is excited by a radio frequency of 27.12 MHz. OCP and ICP using argon gas have been established as excitation sources for atomic emission spectrometry, and are extensively used for the determination of elements including P, S. and I (4). Despite their excellent excitation capability, they cannot be applied to the determination of most nonmetallic elements, especially halogens, for reasons mentioned previously. In 1976, Beenakker designed a new microwave cavity, making it possible to sustain helium plasma at atmospheric pressure (5). A schematic drawing of the Beenakker-type microwave cavity is shown i n Figure 1. The cavity is
T-@ c
observe
Figure 1. A simple drawing o f TM type cavity. (I), cylindrical wall; fixed bottom; (3), removajO\id; (4) quartz discharge tube: (5). holder; (6) coupling loop; (7) connector: (8) Teflon insulator: (9, 10) tuning screw.
(2).
made of copper metal with a diameter of about 10 cm. At the center, the cavity has a hole of 8 mm. i n which a quartz discharge tube of 6 mm in 0.d. is set. Helium plasma is sustained inside a discharge tube of 1-3 m m i n 1.d. by applying microwave power of 75-100 W with the helium gas flow rate of 80-500 ml/min. The helium M I P at atmospheric pressure is operated at the lower power with a relatively small gas flow rate, and thus it is a small string-like plasma.
Therefore, only gaseous volatile compounds can be directly intro-
ducsd into the plasma. Some electrothermal vaporization devices are required when nonvolatile or metallic compounds are analyzed (6). Despite this limitation in sample introduction, the large excitation energy of helium plasma offers an advantage over argon plasma. That is, the excitation erlergy o f helium plasma is about 19.5 eV, while that of
34
a r g o n p l a s m a i s a b o u t 11.7 eV.
The e x c i t a t i o n p o w e r i n h e l i u m o r a r g o n
plasma i s determined by t h e e x c i t a t i o n energy o f h e l i u m o r argon metastable a t o m s (Hem o r A r m ) .
I n T a b l e 11, e m i s s i o n w a v e l e n g t h s i n t h e r e g u l a r U V
and v i s i b l e r e g i o n s f o r s e v e r a l n o n m e t a l l i c e l e m e n t s a r e shown. f o r example,
Fluorine,
when e x c i t e d up t o 14.53 eV p r o v i d e s a n a t o m i c e m i s s i o n l i n e
a t 690.3 nm as
the
t r a n s i t i o n t o t h e e n e r g y l e v e l a t 12.92 eV i n s t e a d o f
t h e v a c u u m e m i s s i o n l i n e a t 95.5 nm. r e a l i z e d w i t h h e l i u m plasma.
Such an e x c i t a t i o n can o n l y be
Generally i f excitation a t a level higher
t h a n t h e f i r s t e x c i t a t i o n l e v e l i s performed,
emission l i n e s i n the regular
UV a n d v i s i b l e r e g i o n s a r e o b s e r v e d a s t h e t r a n s i t i o n s f r o m t h e h i g h e r level t o the lower level. TABLE I 1 WAVELENGTHS AND EXCITATION ENERGIES OF NONMETALLIC ELEMENTS I N THE REGULAR UV AND VISIBLE REGIONS Element
H
Wavelength” (nm)
Excitation E n e r g y (eV)
656.3
12.09
486.1
2.75
C
247.9
7.68
N
746.8
1.99
0
777.2
0.74
F
690.3
4.53
P
253.6
7.18
S
190.0
6.50
c1
479.5 I 1
5.89
Br
470.5 I 1
4.28
I
206.2
6.92
516.1 I 1
12.45
_I<
“ I 1 i n d i c a t e s i o n i c l i n e s , and o t h e r l i n e s a r e a t o m i c l i n e s .
____ P l a s m a e m i s s i o n s p e c t r o m e t r y o f f l u o r i n e and i t s a p p l i c a t i o n t o an s e l e c t i v e d e t e c t o r i n gas c h r o m a t o g r a p h y
I n T a b l e 111,
e l e m s
t h e wavelength t a b l e s o f f l u o r i n e emission l i n e s e x c i t e d
w i t h h e l i u m MIP a r e shown a l o n g w i t h t r a n s i t i o n a s s i g n m e n t s and r e l a t i v e i n t e n s i t i e s (7).
These e m i s s i o n l i n e s w e r e i d e n t i f i e d f r o m t h e e m i s s i o n
s p e c t r u m w h i c h was o b s e r v e d b y i n t r o d u c i n g gaseous c6F6 i n t o t h e h e l i u m plasma.
The
w a v e l e n g t h t a b l e s o f n o n m e t a l l i c e l e m e n t s (H,
C,
N, 0, F, P,
35
S.
C1,
Br,
I ) e x c i t e d w i t h h e l i u m M I P were proposed i n t h i s l a b o r a t o r y (7).
As can be seen i n T a b l e 111, t i o n p o t e n t i a l (cm-’
many a t o m i c e m i s s i o n l i n e s a r e o b s e r v e d i n
I n t h e t a b l e , I.P..
the v i s i b l e region.
o r eV).
atomic lines,
I, IR. and
IMIT
indicate ioniza-
o b s e r v e d r e l a t i v e i n t e n s i t y , and
r e p o r t e d r e l a t i v e i n t e n s i t y i n t h e MIT Wavelength T a b l e s (8).
respectively.
The i n t e r f e r i n g band means t h a t some m o l e c u l a r e m i s s i o n bands o v e r l a p w i t h f l u o r i n e atomic lines.
Such a t o m i c e m i s s i o n l i n e s a r e n o t a v a i l a b l e f o r
a n a l y s i s because o f band i n t e r f e r e n c e s .
I t c a n be n o t e d f r o m T a b l e I 1 1
t h a t t h e e m i s s i o n l i n e a t 685.602 nm i s t h e b e s t one f o r f l u o r i n e d e t e r m i n a t i o n because i t i s m o s t i n t e n s e and has no i n t e r f e r i n g band. TABLE 111
OBSERVED E M I S S I O N LINES
OF FLUORINE I N ATMOSPHERIC PRESSURE HELIUM-MIP
F l u o r i n e I.P. Wavelength (nm)
I
775.470
I I I I
748.272 742.564
I I
739.868
I I
140,554 cm-’
Intensity Transition IR IMIT
(17.42 eV) Energy (cm-’ )
Interfering Band
4
60
104,732-1 17,624
757.341
3
40
102.841-1 16,042
755.224
4
40
102.681 -1 1 5.91 9
2
80
102,681-1 16,042
8
150
102,681-1 16.144
32
400
102,407-1 1 5.91 9
733.195
15
200
102.407-116.042
731.102
5
125
104,732-118,406
720.237
3
125
105,057-1 18.938
I I
712.799
11
150
105.057-119.083
703.745
17
200
104,732-1 18.938
I
690.982
15
150
102,841-1 17.309
I I I 1
690.246
44
500
102,681-1 17.165
687.022
15
150
102,841-1 17.393
685.602
100
1000
102,407-1 16,988
683.426
21
300
102,681-1 17,309
I
677.397
15
100
132,407-117.165
I I
641.366
17
150
102.841-1 18.429
634.850
32
200
102,681-118,429
OH 2nd(w)
I
623.964
32
300
102,407-118.429
OH 2nd(w)
CN 2nd
N$,
N2 2nd
NH 2nd
36 As m e n t i o n e d p r e v i o u s l y , a r g o n I C P i s n o t a s u i t a b l e e x c i t a t i o n s o u r c e f o r n o n m e t a l l i c e l e m e n t s because o f i t s l i m i t a t i o n i n e x c i t a t i o n e n e r g y . Recently,
however,
r e p o r t e d (9).
f l u o r i n e e m i s s i o n l i n e s e x c i t e d w i t h a r g o n ICP w e r e
According t o t h i s
report,
f l u o r i n e emission l i n e s were
o b s e r v e d a t a r e l a t i v e l y l o w e r p o s i t i o n o f t h e p l a s m a o n l y when v o l a t i l e o r g a n i c compounds w i t h o u t h u m i d i t y w e r e i n t r o d u c e d i n t o t h e I C P . w e r e m o s t l y weak o r i n s e n s i t i v e l i n e s .
and t h e y
A r g o n ICP, t h e r e f o r e , i s n o t y e t
available f o r the determination o f fluorine i n trace quantities. As a G C d e t e c t o r f o r h a l o g e n s , t h e E C D ( e l e c t r o n c a p t u r e d e t e c t o r ) i s commonly used because o f i t s h i g h s e n s i t i v i t y . t o a l l halogens,
i.e.,
However,
t o f l u o r i n e w h i l e i t i s t o o t h e r halogens.
On t h e o t h e r h a n d , h e l i u m M I P
can d e t e c t each element by wavelength s e l e c t i o n . used as a
t h e ECD r e s p o n d s
i t has n o e l e m e n t s e l e c t i v i t y and i s n o t s e n s i t i v e
Thus, i f h e l i u m M I P i s
GC d e t e c t o r , i t can be s e n s i t i v e and e l e m e n t - s e l e c t i v e .
GC/helium M I P system.
i n w h i c h h e l i u m M I P i s u s e d as t h e GC d e t e c t o r .
The has
been d e v e l o p e d and s u c c e s s f u l l y a p p l i e d t o a n a l y s i s o f v a r i o u s samples e s p e c i a l l y f o r c h e m i c a l s p e c i a t i o n (10-16).
A s c h e m a t i c d i a g r a m o f t h e G C / M I P s y s t e m u s e d i n t h i s l a b o r a t o r y i s shown i n F i g u r e 2.
The i n s t r u m e n t a l c o m p o n e n t s a n d some o p e r a t i n g c o n d i t i o n s a r e
summarized i n Table IV.
GC
r---------
A S h i m a d z u GC-6A d u a l c o l u m n g a s c h r o m a t o g r a p h
t
F i g u r e 2. S c h e m a t i c d i a g r a m o f t h e G C - M I P s y s t e m . ( S ) helium tank, (T) t h e r m a l c o n d u c t i v i t y d e t e c t o r , (H) h e a t e d t r a n s f e r t u b e , (C) m i c r o w a v e c a v i t y , ( G ) m i c r o w a v e g e n e r a t o r , (L) l e n s , ( M ) m o n o c h r o m a t o r . ( P ) p h o t o m u l t i p l i e r , ( V ) h i g h v o l t a g e s u p p l y , (A) p i c o a m m e t e r , ( R ) c h a r t r e c o r d e r .
37 e q u i p p e d w i t h a t h e r m a l c o n d u c t i v i t y d e t e c t o r (TCD) i s employed. i s used f o r m o n i t o r i n g t h e s o l v e n t s i g n a l s .
The TCD
The i n t e r f a c e b e t w e e n t h e g a s
chromatograph and t h e d i s c h a r g e t u b e o f t h e M I P i s c o n s t r u c t e d f r o m a h i g h t e m p e r a t u r e e l e c t r o m a g n e t i c f o u r - w a y v a l v e and a h e a t e d t r a n s f e r tube. four-way
The
v a l v e i s a t t a c h e d t o t h e TCD o v e n and c a n be o p e r a t e d b y a s w i t c h
l o c a t e d on t h e f r o n t panel o f t h e gas chromatograph.
The v a l v e i s u s e d f o r
s o l v e n t v e n t i l a t i o n when a l a r g e a m o u n t o f s o l v e n t v a p o r i s e l u t e d o u t f r o m t h e GC column.
The t r a n s f e r t u b e ( 5 0 cm l o n g ) i s c o n s t r u c t e d f r o m an i n n e r
n i c k e l t u b e ( 2 mm i.d.), i n g g l a s s tape,
a copper p r o t e c t i o n tube,
nichrome wire,
a thermocoupler,
shield-
and o u t e r g l a s s t a p e i n s u l a t i o n .
TABLE IV INSTRUMENTAL COMPONENTS AND OPERATING CONDITIONS
I tern
Model o r C o n d i t i o n
Gas c h r o m a t o g r a p h
S h i m a d z u GC-6A
Detector
T h e r m a l c o n d u c t i v i t y (TCD)
C a r r i e r gas ( f l o w r a t e )
H e l i u m (80 m l / m i n )
Microwave generator
2.45 GHz, 20-200 W ( I t o h Chotampa, Ltd.)
Microwave c a v i t y
B e e n a k k e r ' s TMol0
cavity
Discharge tube
Quartz tube
Plasma gas ( f l o w r a t e )
H e l i u m (80 m l / m i n )
Monochromator
50-cm f o c a l l e n g t h ( N i p p o n J a r r e l l - A s h Co.)
1200 g r o o v e s / m m
Grating Photomult i p 1i e r tube
R955 (Hamamatsu P h o t o n i c s ,
Amp1 i f i e r
DC a m p l i f i e r ( p a r t o f ICAP-500) ( N i p p o n J a r r e l l - A s h Co.)
Ltd.)
D e t e r m i n a t i o n o f u l t r a t r a c e l e v e l s o f f l u o r i d e i n w a t e r and u r i n e s a m p l e s I n r e c e n t years, t h e d e t e r m i n a t i o n o f f l u o r i n e i n v a r i o u s samples has been e x t e n s i v e l y i n v e s t i g a t e d because o f c l i n i c a l interest.
and e n v i r o n m e n t a l
An i o n - s e l e c t i v e e l e c t r o d e f o r f l u o r i d e i o n ( F - I S E )
has been
w i d e l y used as a c o n v e n t i o n a l a n a l y t i c a l method because o f i t s convenience a n d e c o n o m y (17-20). f l u o r i d e ions only,
As i s w e l l known,
t h e F-ISE
i s sensitive t o free
and i t i s i n f l u e n c e d b y c o e x i s t i n g c a t i o n s .
Other
a n a l y t i c a l m e t h o d s f o r f l u o r i n e d e t e r m i n a t i o n a r e c o l o r i m e t r y (21) and g a s
38
chromatography (22).
Recently, a molecular absorption s p e c t r o m e t r i c method
f o r f l u o r i n e d e t e r m i n a t i o n was d e v e l o p e d , i n which t o t a l f l u o r i n e was d i r e c t l y d e t e r m i n e d by m e a s u r i n g aluminum m o n o f l u o r i d e (AlF) m o l e c u l a r absorption a t 227.45 nm (23-25).
I n t h i s s e c t i o n , t h e f e a s i b i l i t y of t h e GC-MIP
system i s evaluated a s a
conventional a n a l y t i c a l method f o r f l u o r i n e determination,
and applied t o
t h e a n a l y s i s o f s e a w a t e r , t a p w a t e r , pond w a t e r , and u r i n e (26). r e l i a b i l i t y of t h e a n a l y t i c a l values obtained by
GC-MIP
The
i s compared with
t h a t obtained by t h e F-ISE.
MATERIALS AND METHODS The GC-MIP
system used i s s i m i l a r t o t h e instrument shown in Figure 2.
A
Pyrex g l a s s chromatographic column packed w i t h 15% DC-200 on 80/100 mesh U n i p o r t 8 a n d a n o t h e r Pyrex g l a s s column packed w i t h 3% OV-17 on 80/100 mesh Uniport HP were used.
The operating c o n d i t i o n s of t h e gas chromato-
g r a p h a r e summarized i n T a b l e V.
The plasma o p e r a t i n g c o n d i t i o n s a n d
d e t e c t i o n systems a r e t h e same a s those shown i n Table IV. The f l u o r i d e ions in water and u r i n e samples were e x t r a c t e d by t h e f o l lowing procedure:
Four ml of s a m p l e s o l u t i o n were t a k e n i n a 15 ml t e s t
t u b e w i t h a ground g l a s s s t o p p e r ,
and 1 ml of 9.5 M H C 1 and 1 ml of
e x t r a c t a n t s o l u t i o n (0.5 g of t r i r n e t h y l c h l o r o s i l a n e (TMCS) dissolved i n 1 1 of t o l u e n e ) were t h e n added.
The t e s t t u b e was shaken v i g o r o u s l y f o r 30
TABLE V
OPERATING CONDITIONS OF GAS CHROMATOGRAPH Experimental Conditions Column Column packing
Pyrex g l a s s 1 m x 3 mm i.d. 15% OC-200 on 80/100 mesh Uniport B
Pyrex g l a s s 3 m x 3 mm i . d . 3% OV-17 on 80/100 mesh Uniport HP
40
80
I n j e c t i o n temp. ("C)
160
150
Detector oven temp. ("C)
160
150
Transfer tube temp. ("C)
170
160
Helium
Helium
80
80
Column temp. ("C)
C a r r i e r gas C a r r i e r g a s flow r a t e (ml/min)
39 min.
A p a r t o f TMCS i n t h e o r g a n i c p h a s e r e a c t e d w i t h f l u o r i d e i o n s i n t h e
sample s o l u t i o n ,
p r o d u c i n g TMFS (trimethylfluorosilane).
Therefore,
fluo-
r i d e i o n s i n t h e aqueous phase w e r e e x t r a c t e d I n t o t h e o r g a n i c phase as
TMFS. phase,
F o l l o w i n g t h e s e p a r a t i o n o f t h e o r g a n i c phase f r o m t h e aqueous t h e o r g a n i c p h a s e was t r a n s f e r r e d i n t o a s a m p l i n g t u b e .
I n t h e measurements o f plasma e m i s s i o n spectrometry, a t o m i c l i n e o f f l u o r i n e a t 685.6
nm was used.
s o l u t i o n was i n j e c t e d i n t o t h e GC column.
the analytical
One u l o f s a m p l e e x t r a c t a n t
The TMFS f l u o r i n e p e a k a p p e a r e d
f i r s t and was d e t e c t e d b y t h e M I P d e t e c t o r a t i t s c h a r a c t e r i s t i c r e t e n t i o n t i m e (24 s).
I m m e d i a t e l y a f t e r t h e TMFS peak appeared,
t h e four-way
valve
was s w i t c h e d t o v e n t t h e s o l v e n t w i t h m o n i t o r i n g of t h e s o l v e n t p e a k s b y t h e TCD. T h i s p r o c e d u r e was n e c e s s a r y t o a v o i d e x t i n g u i s h i n g t h e p l a s m a and t o m a i n t a i n good p l a s m a s t a b i l i t y .
A t y p i c a l chromatogram o f f l u o r i n e w i t h
t h e GC-MIP s y s t e m i s shown i n F i g u r e 3. t o 1 vg/ml
Only
a TMFS s i g n a l c o r r e s p o n d i n g
f l u o r i n e was s e l e c t i v e l y o b s e r v e d i n t h e c h r o m a t o g r a m u s i n g M I P
d e t e c t i o n (lower curve),
w h i l e i t w a s n o t d e t e c t e d u s i n g TCD d e t e c t i o n
( u p p e r curve).
d
S 1 2 3 4 5 6 7 8 9
RetentionTime (min) F i g u r e 3. T y p i c a l c h r o m a t o g r a m s o f f l u o r i n e w i t h t h e GC-MIP system. (a) TMFS, ( b ) b e n z e n e , ( c ) TMCS. ( d ) t o l u e n e . U p p e r : TCD d e t e c t i o n . Lower: M I P detection.
40 RESULTS The d e t e c t i o n l i m i t s , e x t r a c t i o n e f f i c i e n c i e s , r e p r o d u c i b i l i t i e s . and l i n e a r d y n a m i c r a n g e s o b t a i n e d f o r f l u o r i n e ( p r e s e n t a s TMFS) w i t h t h e DC-
200
column are summarized i n Table VI.
Preconcentration o f the extract
4 t i m e s g a v e 3% r e l a t i v e s t a n d a r d d e v i a t i o n , w i t h a 1 v1 sample.
a n d 95% e x t r a c t i o n e f f i c i e n c y
T h i s i s a v e r y r e l i a b l e method o f f l u o r i n e analysis.
A l t h o u g h p r e c o n c e n t r a t i n g t h e e x t r a c t 10
u s e o f 4 u1 s a m p l e s
t i m e s and
r e s u l t e d i n a somewhat p o o r e r e x t r a c t i o n e f f i c i e n c y ,
t h i s procedure allowed
f l u o r i n e d e t e c t i o n a t a v e r y l o w c o n c e n t r a t i o n w i t h good r e p r o d u c i b i l i t y . A l u m i n u m i o n s i n t e r f e r e d w i t h t h e d e t e r m i n a t i o n o n l y when t h e i r c o n c e n t r a t i o n was 100 t i m e s g r e a t e r t h a n t h a t o f f l u o r i d e i o n s . The G C - M I P
s y s t e m was a p p l i e d t o t h e d e t e r m i n a t i o n o f f l u o r i n e i n sea-
water, t a p water, pond water, a l s o used f o r analysis.
and u r i n e .
The c o n v e n t i o n a l F - I S E
m e t h o d was
The r e s u l t s a r e p r e s e n t e d i n T a b l e V I I .
The
a n a l y t i c a l d a t a o b t a i n e d f r o m t h e s e t w o methods a r e c o n s i s t e n t w i t h each The r e c o v e r i e s o f t h e G C - M I P s y s t e m w e r e i n t h e r a n g e o f 95% t o
other.
110%. and t h e r e p r o d u c i b i l i t y o f a n a l y s i s i n s e a w a t e r was a b o u t 6%. CONCLUSION Element-selective
detection a t the ultratrace level u t i l i z i n g atomic
spectroscopic methods i s a n e w l y developed, characterization.
useful technique f o r trace
Among t h o s e a n a l y t i c a l a t o m i c s p e c t r o m e t r i c methods, t h e
G C / h e l i u m M I P m e t h o d f o r f l u o r i n e i s a s a c c u r a t e and s e n s i t i v e a s t h e F-ISE m e t h o d w i t h t h e a d v a n t a g e s o f a g r e a t e r s e l e c t i v i t y and w i d e r a p p l i c a b i l ity.
We hope t h a t i n t h e n e a r f u t u r e p l a s m a e m i s s i o n s p e c t r o m e t r y u s i n g
h e l i u m M I P and a r g o n I C P c o m b i n e d w i t h g a s o r l i q u i d c h r o m a t o g r a p h y w i l l be f u r t h e r d e v e l o p e d a s a u s e f u l a n a l y t i c a l method. TABLE V I
ANALYTICAL
FIGURES OF MERIT
FOR THE GC-MIP Sample Size, vl
SYSTEM WITH A DC-ZOO
COLUMN^
DL vg/ml
EE %
RSD
%
LDR Decades
Preconcentrated i n extraction, 4 times
1
0.02
95
3.0
3.3
Preconcentrated i n e x t r a c t i o n , 10 t i m e s
4
0.004
75
6.5
4.1
'DL=detection l i m i t ; EE=extrattion e f f i c i e n c y : RSD=relative standard d e v i a t i o n : LDR=linear dynamic range.
41 TABLE V I I APPLICATION OF THE G C - M I P SYSTEM TO DETERMINATION OF FLUORINE I N V A R I O U S SAMPLES ( c o n c . o f F; u g / m l )
F-ISE i n sample'
GC-MIP i n sample'
Recovery added t o sample
total
added
%
Seawater
1.30f0.06
1.30f0.03
1.25
2.48
1.18
94
Tap w a t e r
0.11~0.01
0.12+0.01
0.10
0.23
0.11
110
Pond w a t e r
0.13tO. 01
0.11*0.01
0.10
0.21
0.10
100
Urine
0.98t0.03
0.93+0.03
1.00
1.66
0.95
95
+
'The v a l u e s are estimated concentration d e v i a t i o n s corresponding t o the e t e r r e a d i n g s (mV). 'The v a l u e s f a r e e s t i m a t e d s t a n d a r d d e v i a t i o n s f o r t h r e e s e p a r a t e measurements.
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26. C h i b a K, Y o s h i d a K, Tanabe K. O z a k i M, H a r a g u c h i H, W i n e f o r d n e r , JD, Fuwa K (1982) A n a l Chem 54:761-764
43
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 43-46 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands
SEPARATION A N D DETERMINATION
OF
FLUORIDE I O N B Y HIGHLY SELECTIVE I O N -
EXCHANGE R E S I N S H I S A S H I TANAKA,
YOSHITO OKABAYASHI, TERUMICHI NAKAGAWA, A I K O INOUE,
R I E OH,
AND MASAHIKO C H I K U M A F a c u l t y o f Pharmaceutical Sciences. K y o t o 606, J a p a n
K y o t o U n i v e r s i t y , Y o s h i d a , Sakyo-ku,
ABSTRACT New f u n c t i o n a l r e s i n s f o r t h e s e l e c t i v e c o l l e c t i o n o f f l u o r i d e i o n s w e r e p r e p a r e d b y t h e m o d i f i c a t i o n o f an a n i o n - e x c h a n g e r e s i n w i t h l a n t h a n u m c o m p l e x e s o f a1 i r a r i n - c o m p l e x o n e ALC-La
(ALC) o r i t s s u l f o n a t e d compound (ALCS).
i s adsorbed by p h y s i c a l adsorption,
b o t h ion-exchange
and p h y s i c a l
w h e r e a s ALCS-La
i s adsorbed by
a d s o r p t i o n o n t o anion-exchange
A d s o r p t i o n o f f l u o r i d e i o n s b y t h e r e s i n l o a d e d w i t h ALC-La
resin.
o r ALCS-La
and
t h e d e s o r p t i o n o f f l u o r i d e i o n s w i t h sodium hydroxide s o l u t i o n were a l m o s t complete.
These r e s i n s m a i n t a i n e d t h e c a p a b i l i t y o f f l u o r i d e i o n a d s o r p -
t i o n even a f t e r b e i n g used t e n times.
Interferences by
w e r e n e g l i g i b l e e x c e p t f o r Fe3+ a n d AT3+
ions.
c o u l d be masked by t h e a d d i t i o n o f t a r t a r i c
c a t i o n s and a n i o n s
I n t e r f e r e n c e by these ions Satisfactory results
acid.
were o b t a i n e d by t h e use o f t h e s e r e s i n s i n t h e p r e c o n c e n t r a t i o n o f f l u o r i d e i o n s i n t a p water. INTRODUCTION F l u o r i n e i s an e l e m e n t t h a t commands a t t e n t i o n b o t h as a h a z a r d o u s e l e ment and a n u t r i e n t .
The s e n s i t i v i t y o f t h e
ion-selective electrode
method i s n o t always s a t i s f a c t o r i l y h i g h f o r t h e d e t e r m i n a t i o n o f f l u o r i d e i o n s i n b i o l o g i c a l o r e n v i r o n m e n t a l samples. i s o f t e n necessary.
However,
Preconcentration o f the ions
a h i g h l y s e l e c t i v e and e f f e c t i v e s o r b e n t f o r
t h e c o l l e c t i o n o f t h e f l u o r i d e i o n has n o t been deals w i t h t h e development o f f u n c t i o n a l t i o n o f f l u o r i d e ion.
developed.
This paper
resins f o r the selective collec-
A t t e m p t s w e r e made t o d e v e l o p f u n c t i o n a l r e s i n s
t h r o u g h m o d i f i c a t i o n o f a n i o n - e x c h a n g e r e s i n w i t h some r e a g e n t s w h i c h react with
fluoride
ion specifically.
c o m p l e x o n e - l a n t h a n u m c o m p l e x (ALC-La)
(1) w e r e examined.
ALCS-La
For t h i s
purpose,
alizarin-
and i t s s u l f o n a t e d compound (ALCS-La)
has t w o c h a r a c t e r i s t i c s ,
i.e.,
i t s high selec-
t i v i t y and s e n s i t i v i t y t o w a r d s f l u o r i d e i o n s . and i t s a b i l i t y t o a d s o r b onto t h e ion-exchange
resin.
On t h e o t h e r h a n d ,
ALCS-La,
due t o t h e
44 presence o f a s u l f o n a t e group, anion-exchange r e s i n ,
has an i o n - e x c h a n g e
c a p a b i l i t y w i t h an
i n a d d i t i o n t o t h e t w o c h a r a c t e r i s t i c s mentioned
A r e a g e n t s u c h a s ALCS-La i s r e f e r r e d t o a s a t e r f u n c t i o n a l r e a -
above.
F i g u r e l s h o w s t h e s t r u c t u r e o f ALC a n d ALCS s y n t h e s i z e d f r o m
g e n t (2).
alizarine-5-sulfonic
a c i d and i m i n o d i a c e t i c acid.
Lanthanum complexes o f
t h e s e c h e l a t i n g a g e n t s f o r m deep c o l o r e d t e r n a r y c o m p l e x e s w i t h t h e f l u o r i d e ion. MATERIALS AND METHODS Preparation o f resin An aqueous s o l u t i o n o f ALC o r ALCS ( 4 0 u m o l e ) was m i x e d w i t h 0.1 M n i t r i c a c i d s o l u t i o n o f La(N03)3 (40 umole), d i l u t e d t o 100 m l ,
and t h e r e s u l t i n g s o l u t i o n was
f o l l o w i n g a d j u s t m e n t o f t h e pH t o 4.5
-
5.0.
e x c h a n g e r e s i n , A m b e r l i t e - C G 4 0 0 ( 1 0 0 - 2 0 0 mesh; n i t r a t e f o r m ;
An a n i o n 1 9) w a s
a d d e d t o t h e s o l u t i o n o f ALC o r ALCS, a n d t h e m i x t u r e w a s s t i r r e d f o r 2 4 hours.
The
r e s i n (40 u m o l e / g anion-exchange
w i t h water,
r e s i n ) was c o l l e c t e d , washed
and a i r d r i e d .
A d s o r p t i o n and d e s o r p t i o n o f f l u o r i d e i o n T h e r e a c t i o n c o n d i t i o n s a r e d e s c r i b e d i n t h e l e g e n d o f F i g u r e 2.
The
f l u o r i d e i o n was d e t e r m i n e d b y i o n s e l e c t i v e e l e c t r o d e method. RESULTS AND D I S C U S S I O N The a d s o r p t i o n o f f l u o r i d e i o n s d e c r e a s e d w i t h an i n c r e a s e i n t h e pH o f the solution.
d u e t o c o m p e t i t i o n b e t w e e n f l u o r i d e i o n s and h y d r o x i d e i o n s .
T h i s suggests t h e p o s s i b l e d e s o r p t i o n o f f l u o r i d e i o n s b y t h e sodium hydroxide solution.
The a d s o r p t i o n and t h e d e s o r p t i o n o f f l u o r i d e i o n s
w e r e f o u n d t o be a l m o s t c o m p l e t e .
I n addition, the capability o f the resin
f o r f l u o r i d e i o n a d s o r p t i o n was m a i n t a i n e d e v e n a f t e r b e i n g u s e d t e n t i i n e s (Fig.
2).
The a d s o r p t i o n o f f l u o r i d e i o n s b y t h e a n i o n - e x c h a n g e
resin
shown i n F i g u r e 2 i n d i c a t e s t h a t u n s e l e c t i v e a d s o r p t i o n o f t h e f l u o r i d e i o n a s an a n i o n t o t h e a n i o n - e x c h a n g e extent.
r e s i n m a y h a v e t a k e n p l a c e t o some
S i m i l a r s a t i s f a c t o r y r e s u l t s w e r e o b t a i n e d b y t h e u s e o f ALCS-La.
The a m o u n t o f ALCS-La o f ALC-La,
l o a d e d on t h e r e s i n was f o u n d t o be a l m o s t t w i c e t h a t
because o f t h e ion-exchange c a p a c i t y e x h i b i t e d by t h e s u l f o n a t e
group.
T h i s i n d i c a t e s t h a t ALCS-La
ALC-La,
b e c a u s e ALCS-La
i o n s t h a n ALC-La
loaded resin.
decreases f l u o r i d e i o n adsorption. s i n c e an
i s a more advantageous reagent than
loaded r e s i n can adsorb g r e a t e r amounts o f f l u o r i d e The p r e s e n c e o f common a n i o n s s l i g h t l y T h i s may be c o n s i d e r e d a d i s t u r b a n c e ,
u n s e l e c t i v e a d s o r p t i o n o f i o n s o n t h e a n i o n e x c h a n g e r e s i n may
take place together w i t h a s e l e c t i v e adsorption.
However,
the selective
'
H* \
Hz-N(
miz
CHzCOOH CHZCOOH
K+
o;
ALC
F i g u r e 1.
/
-N
c ~ C O O H HzCOOH
ALCS
S t r u c t u r e o f ALC and ALCS
i / p
100
I I 8
0
I
2
I
1
I
,
3
4
5
6
--
1
'
8
7
8
'
910
T i m e s of use
F i g u r e 2.
Reuse o f t h e r e s i n ALC r e s i n , ----- CG-400
ALC R e s i n :
40 u m o l e / g - r e s i n
R e s i n column:
10 cm x 10 mm
Adsorption Conditions: f l o w r a t e : 0.23 ml/rnin F-: 38 ppm i n 0.1 M NaC1, pH 4.2 ( a c e t a t e b u f f e r ) Desorption Conditions: eluent: 10 m l o f 1N f l o w r a t e : 1 ml/min
NaOH
45
46 adsorption o f fluoride ions by the functional s i t e o f the resin i s not i n t e r f e r e d w i t h i n t h e presence o f o t h e r anions.
With the exception o f
A13+ a n d Fe3+, m o s t o f t h e m e t a l i o n s d i d n o t s h o w a n y i n t e r f e r e n c e . a d d i t i o n o f t a r t a r i c a c i d c o u l d mask t h e s e m e t a l i o n s . r e s u l t s w e r e o b t a i n e d when ALCS-La
The
Satisfactory
l o a d e d r e s i n was a p p l i e d t o t h e p r e c o n -
c e n t r a t i o n o f f l u o r i d e i o n s i n t a p water.
The r e s i n s p r e s e n t e d h e r e a r e
u s e f u l sorbents f o r t h e s e l e c t i v e c o l l e c t i o n o f f l u o r i d e ions. REFERENCES
1.
L e o n a l d MA,
2.
N a k a y a m a M, 31 :269-274
M u r r a y GT ( 1 9 7 4 ) A n a l y s t 99:645-651 I t o h K.
C h i k u m a M,
S a k u r a i H.
Tanaka H (1984) T a l a n t a
H. Tsunoda a n d M.-H. Y u (Editors)
47
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 47-58 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands
G A S CHROMATOGRAPHICAL MICRO-ANALYSIS O F FLUORIDE U S I N G TRIMETHYLCHLOROS I LANE Y O S H I H I R O ZAIMA AND S H I G E R U GOT0 D e p a r t m e n t o f E n v i r o n m e n t a l H e a l t h and Hygiene, M e d i c a l School, University
Osaka
ABSTRACT An a c c u r a t e and r a p i d m e t h o d f o r f l u o r i d e d e t e r m i n a t i o n b y g a s c h r o m a t o g r a p h y combined w i t h s o l v e n t e x t r a c t i o n has been e s t a b l i s h e d . extracted w i t h trimethylchlorosilane
fTMCSl f r o m
acidified
n-hexane as t r i m e t h y l f l u o r o s i l a n e (TMFS) i n a s e a l e d v e s s e l .
Fluoride i s solution into T r a c e amounts
o f f l u o r i d e i n t h e r a n g e o f 1 t o 50 n g c a n b e e x t r a c t e d w i t h i n o n e m i n u t e o f s h a k i n g and g l a s s v e s s e l s a r e a v a i l a b l e f o r t h e e x t r a c t i o n .
T h i s method
has been s u c c e s s f u l l y a p p l i e d t o t h e d e t e r m i n a t i o n o f f l u o r i d e d i s t r i b u t i o n i n t h e u p p e r i n c i s o r enamel o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l rat. INTRODUCTION For t h e d e t e r m i n a t i o n o f s m a l l amounts o f f l u o r i d e , t h e f l u o r i d e ions p e c i f i c e l e c t r o d e h a s b e e n w i d e l y used.
T h i s method i s based on t h e
d e t e r m i n a t i o n o f t h e i o n i c a c t i v i t y o f f l u o r i n e i n s o l u t i o n s w i t h a pH r a n g e o f 5 t o 8,
and t h u s r e q u i r e s d i s s o l v i n g s a m p l e s b e f o r e t h e a n a l y s i s .
I n t h e a n a l y s i s o f f l u o r i d e i n b i o l o g i c a l and a i r samples,
a s h i n g and
f l u o r i d e s e p a r a t i o n p r o c e d u r e s s u c h a s t h e a1 k a l i n e f u s i o n m e t h o d ( l , Z ) , and s t e a m d i s t i l l a t i o n method ( 5 ) a r e a v a i l a b l e .
d i f f u s i o n m e t h o d (3,4),
These d i s s o l u t i o n and s e p a r a t i o n t e c h n i q u e s c a u s e e r r o n e o u s r e s u l t s , are,
therefore,
and
n o t s u i t a b l e f o r t h e d e t e r m i n a t i o n o f t r a c e amounts o f
fluoride. Gas c h r o m a t o g r a p h y c o m b i n e d w i t h e x t r a c t i o n o f f l u o r i d e f r o m an a c i d i f i e d s o l u t i o n i n t o an o r g a n i c s o l v e n t i s c o n s i d e r e d u s e f u l f o r t h e a n a l y s i s o f t r a c e amounts o f f l u o r i d e . fluoride determination.
Bock and Semmler ( 6 ) a p p l i e d t h i s method t o They used t r i e t h y l s i l a n o l
and
triethyl-
c h l o r o s i l a n e a s t h e s i l i l a t i o n r e a g e n t s , t e t r a c h l o r e t h y l e n e and m - x y l e n e a s the organic solvents,
and
cyclohexane
as an i n t e r n a l s t a n d a r d .
They
obtained a l i n e a r c a l i b r a t i o n curve f o r f l u o r i d e i n t h e c o n c e n t r a t i o n range o f 3.8 t o 3 8 u g p e r m l .
The s h a k i n g t i m e f o r f l u o r i d e e x t r a c t i o n was 40 t o
50 m i n u t e s . Fresen,
Cox a n d W i t t e r ( 7 ) u s e d
t r i r n e t h y l c h l o r o s i l a n e (TMCS) a s t h e
48 sililation
reagent,
2-methylbutane
t h e o r g a n i c s o l v e n t . They
as t h e i n n e r standard,
and benzene as
d e t e r m i n e d t h e f l u o r i d e c o n t e n t s i n u r i n e and
s e r u m s a m p l e s a f t e r a 30 m i n u t e e x t r a c t i o n p e r i o d .
M u n k s g a a r d and B r u n n
(8), a n d G i b b s e t a l . ( 9 )
a p p l i e d t h i s method t o d e t e r m i n e t h e f l u o r i d e
c o n t e n t i n t o o t h enamel.
Although t h i s method is a p p l i c a b l e f o r v a r i o u s
samples,
r e s u l t s a r e erroneous because o f t h e
involved.
U s i n g TMCS,
long extraction time
Yamaya and Y o s h i d a ( 1 0 ) e x t r a c t e d f l u o r i d e b y d i f f u -
s i o n and d e t e r m i n e d i t b y gas chromatography.
They e x a m i n e d a n a l y t i c a l
c o n d i t i o n s s u c h as s t a n d i n g t i m e o r d i f f u s i o n p e r i o d and t h e q u a n t i t i e s o f TMCS.
solvent,
and s a m p l e s o l u t i o n s .
But they d i d not consider t h e vola-
t i l i t y o f t r i m e t h y l f l u o r o s i l a n e (TMFS) f o r m e d f r o m TMCS and f l u o r i d e .
I n o r d e r t o i m p r o v e t h e gas c h r o m a t o g r a p h i c method combined w i t h s o l v e n t extraction,
the authors studied the extracting conditions t h a t could a f f e c t
f l u o r i d e determination,
i n c l u d i n g t h e a c i d i t y o f t h e aqueous l a y e r ,
and t e m p e r a t u r e f o r t h e e x t r a c t i o n , chromatographic analysis,
time
s t a n d i n g t i m e f r o m e x t r a c t i o n t o gas
and t h e v e s s e l s u s e d f o r f l u o r i d e e x t r a c t i o n .
In
t h i s s t u d y TMCS w a s u s e d a s t h e f l u o r i d e e x t r a c t a n t , a n d n - h e x a n e a n d 2 m e t h y l b u t a n e were used as t h e s o l v e n t , tively.
and t h e i n t e r n a l
I t becomes c l e a r f r o m t h e r e s u l t s t h a t ,
s h a k i n g f o r one m i n u t e i s s u f f i c i e n t
standard,
respec-
t o extract fluoride,
and g l a s s v i a l s
are available.
E x t r a c t i o n w i t h a s e a l e d v e s s e l h a v i n g a s c r e w cap and a s i l i c o n e r u b b e r septum l a m i n a t e d w i t h a Teflonm sheet i s e f f e c t i v e i n p r e v e n t i n g t h e evaporation o f v o l a t i l e
TMFS (b.p..
16.4"C).
The s t a n d a r d d e v i a t i o n o f
t h e d e t e r m i n a t i o n o f 1 u g o f f l u o r i d e c a n be r e d u c e d t o b e l o w 1%. i s s e t a t 0.7 u g / m l ,
fluoride i n
t h e r a n g e o f 1 t o 50 n g c a n b e d e t e r m i n e d u s i n g t h e V - v i a l
w i t h 50 u l
When t h e c o n c e n t r a t i o n o f 2 - m e t h y l b u t a n e extractant.
By u s e o f t h i s method, t h e f l u o r i d e d i s t r i b u t i o n i n t h e enamel
o f t h e u p p e r i n c i s o r o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l r a t was s u c c e s s f u l l y determined. MATERIALS AND METHODS Apparatus Gas c h r o m a t o g r a p h :
Y a n a g i m o t o , model G-180 w i t h F I D
Column: S t a i n l e s s s t e e l ( 4 6 X 4m), ABS 6 9 0 / 1 0 0 Oven t e m p e r a t u r e :
70°C
I n j e c t o r temperature: C a r r i e r gas:
1 0 % s i l i c o n e o i l DC
150°C
nitrogen,
f l o w r a t e 30 m l / m i n
Atomic absorption spectroscopy:
H i t a c h i , model 708
200 o n
Anakrom
49 Reagents n-Hexane: Water:
Wako p u r e chem..
f o r d e t e r m i n a t i o n o f p e s t i c i d e r e s i d u e 300
d e m i n e r a l i z e d and d o u b l e d i s t i l l e d
A s o l u t i o n c o n t a i n i n g 0.9 mg t r i m e t h y l c h l o r o s i l a n e (TMCS) Extractant: and 1.7 v g 2 - m e t h y l b u t a n e p e r m l o f n-hexane was used. The s o l u t i o n was s t o r e d i n a sealed g l a s s b o t t l e i n a deep-freezer. S t o c k s o l u t i o n o f f l u o r i d e : An a q u e o u s s o l u t i o n c o n t a i n i n g 2 0 0 0 ppm F ( 2 . 2 1 0 0 g o f d r i e d NaF p e r 5 0 0 m l ) w a s p r e p a r e d . F r o m t h i s s o l u t i o n , t h e r e q u i r e d standard s o l u t i o n s were prepared by d i l u t i o n w i t h p i p e t s and b e a k e r s o f p o l y e t h y l e n e . Procedure
A s a m p l e s o l u t i o n was t a k e n i n a v e s s e l and p l a c e d i n a c o l d r o o m (5°C) f o r one hour.
F o l l o w i n g a d d i t i o n o f t h e e x t r a c t a n t t h e v e s s e l was s e a l e d
w i t h a screw cap and a s i l i c o n e r u b b e r septum l a m i n a t e d w i t h a T e f l o n @ sheet.
The a m o u n t o f aqueous l a y e r i n a p o l y c a r b o n a t e t u b e ,
V-vial
were 5 m l .
2.5 m l , a n d 0.25 m l ,
e x t r a c t a n t were 1 ml,
respectively,
0.5 m l , a n d 50 v l , r e s p e c t i v e l y .
shaken v i g o r o u s l y b y hand a t a r a t e
m i n i v i a l and
w h i l e those o f the The v e s s e l was
of a b o u t 200 r e c i p r o c a t i o n s p e r m i n u t e .
I n c a s e s w h e r e t h e e x t r a c t i o n was done a t d i f f e r e n t t e m p e r a t u r e s ,
the
v e s s e l s p r e p a r e d were a l l o w e d t o s t a n d more t h a n one h o u r i n t h e room a d j u s t e d t o t h e d e s i r e d t e m p e r a t u r e b e f o r e shaking. Two v l o f t h e e x t r a c t w e r e p i p e t t e d w i t h a m i c r o s y r i n g e p i e r c i n g t h r o u g h t h e s e p t u m and i n j e c t e d i n t o t h e g a s c h r o m a t o g r a p h column.
The f l u o r i d e
c o n t e n t w a s d e t e r m i n e d f r o m t h e p e a k h e i g h t r a t i o o f TMFS t o 2 - m e t h y l butane. RESULTS AND D I S C U S S I O N Blank c o r r e c t i o n The TMFS and 2 - m e t h y l b u t a n e (Fig.
1).
p e a k s w e r e s y m m e t r i c a l and s e p a r a t e d n i c e l y
R e l a t i v e peak h e i g h t s o f t h e b l a n k TMFS w i t h s t a n d a r d d e v i a t i o n s
a t a c o n c e n t r a t i o n l e v e l o f 0.2 ppm F - a r e l i s t e d i n T a b l e I.
Extraction
o f f l u o r i d e was c a r r i e d o u t i n a p o l y c a r b o n a t e t u b e w i t h t h e aqueous l a y e r acidified with perchloric
a c i d (0.47
mol/l).
R e s u l t s o f s i x t e e n measure-
m e n t s w e r e i n g o o d a g r e e m e n t w i t h e a c h o t h e r and t h e c o e f f i c i e n t o f v a r i a n c e (C.V.)
w a s 0.67%.
T h e r e l a t i v e p e a k h e i g h t o f t h e b l a n k w a s 0.6% o f
t h a t o f t h e s t a n d a r d s o l u t i o n and was w i t h i n e x p e r i m e n t a l m e a s u r i n g e r r o r . I n t h i s study,
then,
b l a n k c o r r e c t i o n was n o t made.
A c i d i t y o f aqueous l a y e r H y d r o c h l o r i c a c i d and p e r c h l o r i c a c i d w e r e used and 1 vg o f F-
was
e x t r a c t e d i n t h e p o l y c a r b o n a t e t u b e . Gas c h r o m a t o g r a p h i c a n a l y s i s was p e r f o r m e d a f t e r one m i n u t e s h a k i n g f o l l o w e d b y one m i n u t e s t a n d i n g .
Figure 2
50
F i g u r e 1. D e t e r m i n a t i o n o f f l u o r i d e b y gas c h r o m a t o g r a p h y . t r i m e t h y l f l u o r o s i l a n e ; B = 2 - m e t h y l b u t a n e ; C = n-hexane.
S
= start;
A =
a
c
I c
0 .c U
I
c
w
A c i d i t y of aqueous layer
( rnol I1 )
F i g u r e 2. R e l a t i o n s h i p b e t w e e n e x t r a c t i o n r a t e and m o l a r c o n c e n t r a t i o n o f a c i d i n t h e a q u e o u s l a y e r . 1 v g F (0.2 ppm F ) w a s e x t r a c t e d i n a p o l y c a r b o n a t e c e n t r i f u g e t u b e following 1 m i n s h a k i n g . 0. HC1; e. HC104.
51 TABLE I R E L A T I V E PEAK HEIGHT (TMFS/2-METHYL-BUTANE) STANDARD SOLUTION AND BLANK
F- Conc.
N
Mean k S.D.
(ppm) 0.2
C.V. (%)
16
0.8300 f 0.0056
0.67
5
0.0050 f 0 . 0 0 1 4
27.00
0
e x t r a c t i o n vessel: aqueous l a y e r : organic layer: s h a k i ng t i me:
OF
polycarbonate
5 ml
1 ml 1 min
shows t h e r e l a t i o n s h i p b e t w e e n e x t r a c t i o n r a t e and m o l a r c o n c e n t r a t i o n o f each a c i d i n t h e aqueous l a y e r .
E x t r a c t l o n r a t e was e x p r e s s e d a s a p e r -
c e n t a g e o f t h e r e l a t i v e peak h e i g h t o f TMFS t o t h a t i n t h e r a n g e o f a c i d i t y higher than 5 mol/l,
C.V.
where t h e r e l a t i v e peak h e i g h t became c o n s t a n t and
w a s l e s s t h a n 1%.
H y d r o c h l o r i c a c i d and p e r c h l o r i c a c i d showed t h e
same e x t r a c t i o n r a t e f o r t h e same m o l a r i t y .
The e x t r a c t i o n r a t e was
r e d u c e d b y 2% w h e n t h e a c i d i t y w a s r e d u c e d f r o m 5 m o l / l o r m o r e t o 0.5
mol/l.
I n t h i s study,
p e r c h l o r i c a c i d was used.
S ha k inq t ime F i g u r e 3 shows t h e r e l a t i o n s h i p b e t w e e n s h a k i n g t i m e and t h e e x t r a c t i o n r a t e a t t h r e e c o n c e n t r a t i o n l e v e l s o f p e r c h l o r i c a c i d (0.13, One u g o f F-
mol/l).
0.47,
and 2.35
was e x t r a c t e d i n a p o l y c a r b o n a t e t u b e and d e t e r m i n e d
b y g a s c h r o m a t o g r a p h y f o l l o w i n g s h a k i n g a n d o n e m i n u t e s t a n d i n g a t 5°C. The s h a k i n g t i m e i s e x h i b i t e d i n t h e l o g a r i t h m i c s c a l e o n t h e t r a n s v e r s a l axis.
The r a t e o f f l u o r i d e e x t r a c t i o n r o s e s t e e p l y as s h a k i n g t i m e
i n c r e a s e d and r e a c h e d a p l a t e a u w h i c h corresponded t o t h e a c i d i t y o f t h e aqueous l a y e r .
M i n i m u m t i m e r e q u i r e d t o a c c o m p l i s h maximum e x t r a c t i o n
became l o n g e r as t h e a c i d i t y o f t h e aqueous l a y e r was l o w e r e d . r e q u i r e d was o n l y 5 s e c o n d s when t h e a c i d i t y was 2.35 m o l / l .
The t i m e
b u t was 2 m i n
when t h e a c i d i t y was 0.19 mol/l. I n a p r e v i o u s s t u d y on t h e d e t e r m i n a t i o n o f f l u o r i d e b y gas chromatography, min. leak.
s h a k i n g was c a r r i e d o u t w i t h a m e c h a n i c a l s h a k e r f o r m o r e t h a n 30
Such a l o n g s h a k i n g i s d i s a d v a n t a g e o u s when t h e e x t r a c t i o n v e s s e l s I n t h i s study,
a s e a l e d v e s s e l a n d TMCS w e r e u s e d a n d a r a p i d
e x t r a c t i o n o f f l u o r i d e was a c c o m p l i s h e d . Standing time a f t e r shaking F i g u r e 4 shows t h e r e l a t i o n s h i p b e t w e e n s h a k i n g t i m e and r a t e o f f l u o r i d e
52
( %)
100
w
c
I C
? ! 50
"
-I c
x W
0 1 oo
1 o2
lo' Shaking
time
1
o3
10
( sec
Figure 3. Effect of shaking time on the extraction rate of fluoride (1 pg F). The aqueous layer was acidified with HC104. 0 , 2.35 mol/l; a, 0.47 rnol/l; 0, 0.19 mol/l.
5haklng
time
(sec)
Figure 4. Effect o f 2 4 hours standing on the extraction rate of fluoride (1 p g F). Concentrations of HC104: 0 . 2.35 rnol/l; a, 0.47 mol/l; 0 ,0.19 mol/l. extraction after standing 24 hours following shaking. The experimental conditions were the same as described previously.
The extraction vessels
were allowed to stand in the cold (5°C) for 24 hours after shaking. It i s clear from Figures 3 and 4 that fluoride unextracted after shaking was extracted at the rate of 90% a day by diffusion. Maximum or ceiling value o f the extraction rate rose only 0.6% after 24 hours for each acidity and the increase was not significant statistically.
53 When t h e a c i d i t y o f t h e aqueous l a y e r was 0.47 i n g t i m e w a s m o r e t h a n 20 s e c ( F i g . 3).
mol/l,
t h e r e q u i r e d shak-
A s h a k i n g t i m e o f one m i n u t e was
c h o s e n and t h e e f f e c t o f t h e s t a n d i n g t i m e a f t e r s h a k i n g was s t u d i e d .
For
t h e e x t r a c t i o n vessel,
The
p o l y c a r b o n a t e t u b e s and m i n i v i a l s w e r e used.
a n d 0.2
c o n c e n t r a t i o n s o f p e r c h l o r i c a c i d a n d f l u o r i d e w e r e 0.47 m o l / l , ppm,
respectively.
F i g u r e 5 shows t h e change i n e x t r a c t i o n r a t e f o r a 50-hour period.
standing
I t i s c l e a r t h a t t h e m a t e r i a l o f t h e vessel used f o r f l u o r i d e
e x t r a c t i o n . p o l y c a r b o n a t e and g l a s s , d i d n o t a f f e c t t h e e x t r a c t i o n r a t e . Also,
t h e standing t i m e f o l l o w i n g shaking increased t h e e x t r a c t i o n r a t e a t
t h e r a t e o f 0.02% a n h o u r .
Assuming t h a t t h e e x t r a c t i o n r a t e i s c o n s t a n t
f o r 50 h o u r s f o l l o w i n g s h a k i n g , i n F i g u r e 5 i s o n l y 1.09%. (0.67%).
t h e c o e f f i c i e n t o f v a r i a n c e o f a l l measures
Combined w i t h t h e c o e f f i c i e n t f r o m T a b l e I
t h e e r r o r o f d e t e r m i n a t i o n w i l l be w i t h i n 2% b y t h e gas c h r o m a t o -
g r a p h i c method. Temperature The c o n c e n t r a t i o n s o f f l u o r i d e and p e r c h l o r i c a c i d u s e d w e r e 0.2 ppm and 0.47 m o l / l .
respectively.
The a q u e o u s s o l u t i o n a n d t h e e x t r a c t i o n w e r e
p o u r e d i n t o a p o l y c a r b o n a t e c e n t r i f u g e t u b e a t 5°C and s e a l e d i m m e d i a t e l y w i t h a screw cap and a s i l i c o n e r u b b e r septum l a m i n a t e d w i t h a T e f l o n @ sheet.
The t u b e was p l a c e d i n t h e r o o m a d j u s t e d t o t h e p r e s c r i b e d t e m p e r a -
t u r e ( 0 . 1 3 and 20°C) f o r one h o u r f o l l o w e d b y one m i n u t e ' s s h a k i n g . F i g u r e 6 shows t h e r e l a t i o n s h i p b e t w e e n e x t r a c t i o n r a t e and
temperature.
The e x t r a c t i o n r a t e i s e x p r e s s e d a s t h e p e r c e n t a g e o f t h e r e l a t i v e p e a k
0
10 20 30 LO 50 Standing time after extraction ( hr)
F i g u r e 5. E f f e c t o f s t a n d i n g t i m e on t h e e x t r a c t i o n r a t e o f f l u o r i d e . F l u o r i d e (0.2 ppm F) was e x t r a c t e d i n a p o l y c a r b o n a t e c e n t r i f u g e t u b e and glass minivial. The c o n c e n t r a t i o n o f HC104 was 0.47 m o l / l .
54
0
5
10 15 Temperature
20
('C
)
F i g u r e 6. E f f e c t o f t e m p e r a t u r e on t h e e x t r a c t i o n r a t e o f f l u o r i d e . o f f l u o r i d e was e x t r a c t e d i n a p o l y c a r b o n a t e c e n t r i f u g e t u b e .
5°C.
h e i g h t o f TMFS t o t h a t a t t u r e rose,
A t t e m p e r a t u r e s above
5°C.
1 ug
a s t h e ternpera-
t h e e x t r a c t i o n r a t e d e c r e a s e d and t h e s t a n d a r d d e v i a t i o n o f t h e
d e t e r m i n e d v a l u e became l a r g e . and 20°C w e r e 0.67%.
0.68%,
T h e c o e f f i c i e n t s o f v a r i a n c e a t 0, 5, 1 3
1.36% and 1.71%.
respectively.
T h i s means t h a t
t h e v o l a t i l e TMFS e v a p o r a t e d m o r e as t h e t e m p e r a t u r e r o s e .
Accordingly,
f o r an a c c u r a t e d e t e r m i n a t i o n , t h e e x t r a c t i o n s h o u l d be c a r r i e d o u t a t a t e m p e r a t u r e l o w e r t h a n 5°C. Vessels used f o r f l u o r i d e e x t r a c t i o n
As s h o w n i n F i g u r e 5,
p o l y c a r b o n a t e c e n t r i f u g e t u b e s and g l a s s v i a l s
e x h i b i t e d t h e same e x t r a c t i o n r a t e s . fluoride extraction,
To s t u d y t h e e f f e c t o f v e s s e l s o n
t h e c a l i b r a t i o n c u r v e s made w i t h p o l y c a r b o n a t e t u b e s ,
m i n i v i a l s , and V - v i a l s w e r e compared.
The c o n c e n t r a t i o n o f p e r c h l o r i c
a c i d i n t h e aqueous l a y e r was a d j u s t e d t o 0.47 r i d e w e r e i n t h e r a n g e o f 0 t o 50 ppm.
mol/l
w h i l e those of f l u o -
S h a k i n g t i m e was one m i n u t e and t h e
t e m p e r a t u r e was 5°C. The c a l i b r a t i o n c u r v e s a r e shown i n F i g u r e s 7-9. linearity.
They show a v e r y good
The c o r r e l a t i o n c o e f f i c i e n t s f o r p o l y c a r b o n a t e t u b e s a n d f o r
b o t h g l a s s v i a l s w e r e 0.9989
and 0.9999,
respectively.
I r r e s p e c t i v e o f t h e k i n d o f v e s s e l used, 1 m l o f e x t r a c t a n t c o n t a i n i n g 0.9
mg TMCS c o u l d e x t r a c t u p t o 50 u g o f f l u o r i d e .
e x t r a c t e d w i t h a p o l y c a r b o n a t e tube,
The a m o u n t o f f l u o r i d e
m i n i v i a l and V - v i a l
corresponding t o
t h e r e l a t i v e peak h e i g h t o f TMFS t o t h e i n t e r n a l s t a n d a r d o f 1.0 w e r e 1.786 ug,
1.186 ug,
and 1.175 ug,
respectively.
The d i f f e r e n c e i n r e l a t i v e peak
h e i g h t s among d i f f e r e n t k i n d s o f v e s s e l s was o n l y 1% and was a l m o s t e q u i v a lent t o the dispersion o f the determinations
(Table
I).
I t can be
55
/ 50
1
2
3
4
Fluorine content
5
(
pg )
F i g u r e 7. C a l i b r a t i o n c u r v e o f f l u o r i d e u s i n g p o l y c a r b o n a t e c e n t r i f u g e tubes. The a m o u n t s o f aqueous l a y e r and e x t r a c t a n t a r e 5 m l and 1 m l . r e s p e c t i v e l y . 0 . d e n o t e s t h e r a n g e o f f l u o r i d e 0-5 ppm; 0.d e n o t e s t h e r a n g e o f f l u o r i d e 10-50 ppm.
/ I
3130
1
t!
1
~
10
a Fluorlne content
(yg)
F i g u r e 8. Calibration curve o f f l u o r i d e using glass minivials. The a m o u n t s o f a q u e o u s l a y e r a n d e x t r a c t a n t a r e 2.5 m l a n d 0.5 m l , r e s p e c 0 , f l u o r i d e 0-5 ppm; 0 , f l u o r i d e 10-50 ppm. tively. concluded,
therefore,
t h a t t h e r a t e o f f l u o r i d e e x t r a c t i o n was n o t a f f e c t e d
by t h e k i n d o f v e s s e l s used f o r e x t r a c t i o n . A n a l y s i s o f nanogram f l u o r i d e To d e t e r m i n e n a n o g r a m q u a n t i t i e s o f f l u o r i d e , i n g o f 0.9 mg TMCS and 0.7 p g 2 - m e t h y l b u t a n e prepared.
another e x t r a c t a n t consist-
p e r m i l l i l i t e r o f n-hexane
By u s i n g t h i s e x t r a c t a n t and a V - v i a l ,
was e x t r a c t e d and d e t e r m i n e d . c u r v e was o b t a i n e d .
0 t o 50 ng o f f l u o r i d e
As shown i n F i g u r e 10,
a linear calibration
The c o e f f i c i e n t o f c o r r e l a t i o n was 0.9990.
posed method makes t h e t r a c e a n y a l y s i s o f f l u o r i d e p o s s i b l e , v o l u m e o f s a m p l e s o l u t i o n a s s m a l l as 250 ul.
was
The p r o even i n a
56
( pg)
Fluorine content
Figure 9. Calibration curve of fluoride using glass V-vials. The amounts 50 111. respectively. 0 , fluoride 0-5 ppm; 0, fluoride 10-50 ppm.
o f aqueous layer and extractant are 250 111 and
0
10
20 Fluorine
40
50
content
( ng )
30
Figure 10. Calibration curve of fluoride using glass V-vials. The amount of 2-methylbutane, internal standard, in n-hexane was reduced to 0.7 ug/ml. The other conditions are the same as shown i n Figure 9. Fluoride in the tooth enamel of rats By using the proposed method, the fluoride distribution in the enamel of the upper incisor of an acutely fluorosed experimental rat could be determined. In experiment, a 2.5% NaF solution was injected subcutaneously into a male rat (SD strain, 160 f 59) at a dose of 0.5 mg per 100 g body Four days after the injection, the rat was sacrificed and the
weight.
upper incisors were extracted.
They were fixed with 10% formaldehyde
57
solution,
d e h y d r a t e d and embedded i n a p o l y e s t e r r e s i n .
200 urn t h i c k t r a n s v e r s a l l y , a n d c o a t e d w i t h
They w e r e s l i c e d .
n a i l enamel t o a v o i d a c i d
The e n a m e l o f t h e s u r f a c e was e t c h e d w i t h p e r c h l o r i c a c i d . and
etching.
t h e f l u o r i d e c o n t e n t was d e t e r m i n e d b y gas c h r o m a t o g r a p h y .
The c a l c i u m
c o n t e n t ? n t h e aqueous l a y e r was d e t e r m i n e d b y a t o m i c a b s o r p t i o n spec-
As shown i n F i g u r e 11, b o t h f l u o r i d e and c a l c i u m w e r e r e d u c e d a t
troscopy.
t h e h y p o m i n e r a l i z e d zone and i n c r e a s e d a t t h e h y p e r m i n e r a l i z e d zone. r a t i o s o f f l u o r i d e t o c a l c i u m a l s o showed a s i m i l a r t r e n d .
The
These r e s u l t s
indicate that, through f l u o r i d e t o x i c i t y , ameloblasts a r r e s t the deposition o f not only calcium but also fluoride.
And i t may s u g g e s t t h a t f l u o r i d e i s
d e p o s i t e d t o g e t h e r w i t h enamel m a t r i x . The d i s t r i b u t i o n p a t t e r n o f c a l c i u m a g r e e s w i t h t h e r e s u l t o b t a i n e d f r o m e l e c t r o n m i c r o p r o b e a n a l y s i s b y Suga (11).
CONCLUSION The a u t h o r s h a v e e s t a b l i s h e d an i m p r o v e d gas c h r o m a t o g r a p h i c m e t h o d combined w i t h e x t r a c t i o n o f f l u o r i d e as t r i m e t h y l f l u o r o s i l a n e .
,
S
Various
I
HPOl DEJ HPR
FlGu
F I Cd ( x ~ O )- ~
2i 0
10 20 30 40 50 Depth
(prn)
F i g u r e 11. D i s t r i b u t i o n o f f l u o r i d e and c a l c i u m i n t h e e n a m e l o f t h e u p p e r i n c i s o r o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l r a t . U p p e r p i c t u r e shows t h e contact microradiograph: S, s u r f a c e o f t h e e n a m e l : HPO, h y p o m i n e r a l i z e d zone: HPR, h y p e r m i n e r a l i z e d zone: DEJ, d e n t i n e - e n a m e l j u n c t i o n .
58
f a c t o r s a f f e c t i n g t h e e x t r a c t i o n have been s t u d i e d i n d e t a i l and s u i t a b l e c o n d i t i o n s have been e s t a b l i s h e d .
The i m p r o v e d m e t h o d p r o v e d e f f e c t i v e f o r
t h e d e t e r m i n a t i o n o f f l u o r i d e d i s t r i b u t i o n i n t h e enamel o f u p p e r i n c i s o r o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l r a t .
REFERENCES 1. Remmert LF, P a r k s AM, McBurney EH (1953) A n a l Chew 25:450-453
2. N i o s h Manual o f A n a l y t i c a l M e t h o d s (1977) I n : c a l Methods, 2nd ed. v o l 1:212-1--212-4
N I O S H Manual o f A n a l y t i -
3. S i n g e r L, A r m s t r o n g WD (1954) A n a l Chem 26:904-046 4. W i l l a r d HH,
W i n t e r OB (1933) I n d Eng Chem A n a l Ed 5:7-10
5. I n t e r s o c i e t y C o m m i t t e e o n M e t h o d f o r A m b i e n t A i r S a m p l i n g and A n a l y s i s (1969) H l t h Lab S c i 6:84-101 6. Bock R.
Semmler HJ (1967) F r e s e n i u s ' Z A n a l Chem 230:161-184
7. F r e s e n JA, Cox FH, W i t t e r MJ (1968) Pharm Weekblad 103:909-914
8. Munksgaard EC,
B r u n n C (1973) A r c h s O r a l B i o l 18:735-744
9. G i b b s M, R e t i e f DH. B r a d l e y EL, T a y l o r RE, W a l k e r A R ( 1 9 8 1 ) J D e n t Res 60:770-775
10. Yamaya K,
Y o s h i d a M (1984) J Chem SOC, J p n 4:563-568
11. S u g a S ( 1 9 8 3 ) I n : S u g a S ( e d ) M e c h a n i s m s o f T o o t h E n a m e l F o r m a t i o n . Q u i n t e s s e n c e P u b l i s h i n g Co, I n c . Tokyo, pp 157-303
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 59-71 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
DETERMINATION
OF
59
FLUORIDE IN DRINKING WATER BY COLORIMETRY WITH A TOTAL-
REFLECTION LONG CAPILLARY CELL WE1 L E I , KITAO FUJIWARA, AND K E I I C H I R O FUWA Department of Chemistry, F a c u l t y o f Science. Bunkyo-ku, T o k y o 113, J a p a n
The U n i v e r s l t y o f Tokyo,
ABSTRACT C o l o r i m e t r i c d e t e r m i n a t i o n o f f l u o r i d e i q n s i n d r i n k i n g w a t e r was c a r r i e d o u t i n a l o n g c a p i l l a r y c e l l (LCC).
C o l o r d e v e l o p m e n t was b a s e d o n t h e
Lanthanum-a1 i z a r i n complexone (La-ALL) method: s a m p l e and t h e fluoro-lanthanum-alizarin
La-ALC w a s a d d e d t o t h e
c o m p l e x o n e f o r m e d was e x t r a c t e d
w i t h isoamyl alcohol containing N,N-diethylaniline
and was m i x e d w i t h a
m i x t u r e o f c a r b o n d i s u l f i d e and a c e t o n e ( f i n a l r e f r a c t i v e i n d e x o f a n a l y t e solution
=
1.57).
Extension o f c e l l length longer than 1 m provided h i g h l y
sensitive colorimetry.
Enhancement o f a b s o r p t i o n was 0.5-2.5
g r e a t e r u s i n g a 4 m LCC.
x lo3 times
D e t e c t i o n l i m i t and r e c o v e r y of t h i s m e t h o d w e r e
1 0 n g / L a n d 93-104% ( f o r 50 u g / 1 ) ,
respectively.
The p r e s e n t m e t h o d was
a p p l i e d t o t h e d e t e r m i n a t i o n o f f l u o r i d e i n d r i n k i n g w a t e r samples c o l l e c t e d f r o m v a r i o u s s i t e s i n Tokyo c i t y .
INTRODUCTION Long c a p i l l a r y c e l l I t i s known t h a t t h e c o n c e n t r a t i o n o f f l u o r i d e i n d r i n k i n g w a t e r c o r r e l a t e s d i r e c t l y w i t h t h e o c c u r r e n c e o f d e n t a l d i s e a s e (1,Z). n a t i o n o f f l u o r i d e i n d r i n k i n g water, t h e environmental f i e l d .
therefore,
The d e t e r m i -
i s an i m p o r t a n t s u b j e c t i n
Although c o l o r i m e t r y using lanthanum a l i z a r i n
(3-6)
i s one o f t h e most s e n s i t i v e methods f o r t h e
determination o f fluoride,
i t cannot d e t e c t f l u o r i d e concentrations of l e s s
c o m p l e x o n e (La-ALC) t h a n 20 ng/ml, Recently, cells.
w h e n a n o r d i n a r y c o l o r i m e t r i c c e l l ( 1 cm c e l l ) i s u s e d .
l o n g c a p i l l a r i e s ( l o n g e r t h a n 1 m) w e r e used as c o l o r i m e t r i c
Because o f t h e e x t e n s i o n o f o p t i c a l p a t h l e n g t h i n s i d e t h e c e l l ,
the
u s e o f a l o n g c a p i l l a r y c e l l (LCC) p r o v i d e s e n h a n c e d d e t e c t i o n p o w e r i n o r d i n a r y c o l o r i m e t r y (7).
F i g u r e 1 s h o w s t h e o r d i n a r y c e l l s ( 1 cm a n d 5
cm) and v a r i o u s t y p e s o f LCCs e m p l o y e d i n t h e p r e s e n t s t u d y . cells,
t h e s e n s i t i v i t y o f absorption spectrometry,
I n ordinary
w h i c h obeys t h e Lambert-
60
Ordinary Cells
Capillary Cells
I
t
(5) --I
1.-
U
t
L=25,50m,
L = 2 , 4 , 1 0 m,....
1.
F i g u r e 1. O r d i n a r y and l o n g c a p i l l a r y c e l l s f o r s p e c t r o p h o t o m e t r y . (l'), 1 cm c e l l ; (2'), . 5 cm c e l l ; ( 1 ) . l i n e a r t y p e ; (2), c u r v e d t y p e ; ( 3 ) , l o o p e d t y p e ; (4), s p i r a l t y p e , ( 5 ) , " o p t i c a l f i b e r " t y p e .
Beer law, i s determined by t h e l i g h t pathlength
of
the
sample
cell.
w i t h t h e source l i g h t p a r a l l e l w i t h t h e c e l l wall.
I n t h i s system,
t h e d i a m e t e r o f t h e a p e r t u r e o f t h e LCC was s m a l l e r t h a n
t h a t o f t h e s o u r c e l i g h t beam, inside the capillary.
r e s u l t i n g i n m u l t i r e f l e c t i o n o f source l i g h t
T h i s c a u s e d t h e o p t i c a l p a t h l e n g t h t o become l o n g e r
t h a n t h e a p p a r e n t l e n g t h o f t h e LCC. m LCC,
Thus,
when u s i n g a l i n e a r l y shaped 1
t h e a b s o r b a n c e o f t h e s o l u t i o n was enhanced b y m o r e t h a n a f e w
hundred t i m e s t h a t o f o r d i n a r y
spectrometry (Fig. l(1)).
However, i t
1s
r a t h e r i n c o n v e n i e n t t o h a n d l e a l i n e a r LCC whose l e n g t h i s m o r e t h a n 1 m i n an o r d i n a r y l a b o r a t o r y . looped (Fig.
1(3)),
For c e l l s
more t h a n 1 m long,
o r s p i r a l (Fig. l ( 4 ) ) forms a r e preferable.
when t h e aqueous s o l u t i o n i s a p p l i e d t o t h e LCC, shape f r o m l i n e a r i t y c a u s e s a s u b s t a n t i a l c a p a c i t y o f t h e LCC.
curved (fig.
1(2)),
However,
the transformation o f c e l l
loss i n the l i g h t transmission
I t was f o u n d t h a t o n l y 0.001% o f t h e i n c i d e n t l i g h t
c o u l d reach t h e e x i t o f t h e s i n g l e looped c e l l . Condition f o r t o t a l r e f l e c t i o n For m a i n t a i n i n g source l i g h t t r a n s m i s s i o n i n s i d e t h e long c a p i l l a r y c e l l , successive t o t a l r e f l e c t i o n s must continue a t t h e i n t e r n a l w a l l o f t h e capillary.
This i s o n l y possible by applying a solvent,
r e f r a c t i v e index higher than t h a t o f t h e c e l l material, law.
Namely,
w h i c h has a
w h i c h obeys S n e l l ' s
the required condition f o r a t o t a l r e f l e c t i o n c e l l is:
61
"2 sin (90"-0) 2 n1 where 0 is the angle of the incident light, and n1 and n2 are the refractive indexes of the solvent and cell material (Pyrex), respectively. Figure 2 shows the patterns of source light transmission i n an ordinary 1 crn cell, L C C with specular reflection, and L C C with total reflection, where 10 and
I o ' are the intensities of source light before and after passing through the cell containing sample solutions, respectively. When n1 is smaller than n2, a s is the case when water is used as the solvent: n 1 = 1.33 (water), n2 = 1.474 (Pyrex), the light transmits through the internal cell wall with specular reflection (middle of Fig. 2). In this case the internal cell wall must be coated with a light reflective material. Since light reflectivities of most coating materials are smaller than loo%, the source light suffers strong attenuation during transmission i n this type of cell due to multiple reflection. On the other hand, when n1 is greater than n2, i.e., when refractive index of the solution exceeds that of the cell rnateria1,light transmission efficiency of the L C C is greatly improved. In other words, the source light is propagated through the inside cell wall via total reflections without the loss of intensity.
Io-c
(D
--
-I0f
ordinary cell
I0
I0
-
specular reflection LCC ( "1<"2 )
-1
t o t a l reflection LCC ( n1>n2 1
I o = I0 ' I o f I. >>> I
0
'
I.
2
0
Iof
nl: refractive index of solvent n2: refractive index of c e l l wall Figure 2. Patterns of source light transmissions ( 1 0 ) i n the ordinary cell, L C C with specular reflection, and L C C with total reflection.
For total reflection long capillary cell spectrometry, carbon disulfide is mainly used as the solvent, as its refractive index is 1.62, which is It is noted that the structure of
much higher than that of the Pyrex cell.
optical fiber is in principle the same as that of a long capillary cell
62
(8,9). F i g u r e l ( 5 ) s h o w s a l i q u i d c o r e o p t i c a l f i b e r c e l l w h o s e i n t e r n a l d i a m e t e r i s 250 pm. The s a m p l e v o l u m e r e q u i r e d i s o n l y a f e w m l f o r a 50 m When a 100 m o p t i c a l f i b e r c e l l i s used,
long cell.
a b s o r b a n c e i s enhanced
m o r e t h a n 6x104 t i m e s t h a t w i t h o r d i n a r y s p e c t r o m e t r y . I n t h i s paper,
t h e a p p l i c a t i o n o f t o t a l r e f l e c t i o n LCC t o t h e d e t e r m i n a -
t i o n o f f l u o r i d e i n t h e d r i n k i n g w a t e r samples o f Tokyo c i t y i s reported. MATERIALS AND METHODS Reagents S u b b o i l e d w a t e r d i s t i l l e d b y a q u a r t z d i s t i l l e r was u s e d t h r o u g h o u t t h e experiment. opment f o r (La-ALC)
A l l t h e r e a g e n t s u s e d w e r e o f a n a l y t i c a l grade.
Color deuel-
f l u o r i d e i o n s was b a s e d o n t h e l a n t h a n u m - a l i z a r i n e
m e t h o d (3-6).
d e v e l o p e d c o l o r (La-ALC-F)
m u s t be d i s s o l v e d i n c a r b o n d i s u l f i d e .
shows t h e m o l e c u l a r s t r u c t u r e s o f t h e l a n t h a n u m - a l i z a r i n (La-ALC),
complexone
F o r a t t a i n i n g t o t a l r e f l e c t i o n i n t h e LCC,
fluoro-lanthanum
a l i z a r i n c o m p l e x o n e (La-ALC-F)
the
Figure 3
comoplexone
and fluoro-N,N-
diethylanilino-lanthanum a l i z a r i n c o m p l e x o n e (La-ALC-DA-F).
The c o l o r
developing reagent,
La-ALC
amine-N,N-diacetic
acid) contains two molecules o f water attached t o
lanthanum (Fig.
3(A)).
(Lanthanum-1.2-dihydroxanthraquinonyl-3-methyl-
I n t h e p r e s e n c e o f b o t h La-ALC
and f l u o r i d e ,
f l u o r i d e i o n s u b s t i t u t e s one o f t h e w a t e r m o l e c u l e s i n La-ALC. ALC-F ( F i g . 3 ( B ) ) . tion o f fluoride.
f o r m i n g La-
T h i s p r i n c i p l e i s w i d e l y used f o r c o l o r i m e t r i c d e t e c However,
La-ALC-F
is insoluble i n organic solvents
because one m o l e c u l e o f w a t e r i s s t i l l a t t a c h e d t o lanthanum. ALC-F
a
Thus, La-
i s n o t s o l u b l e i n c a r b o n d i s u l f i d e a n d m u s t b e s u b s t i t u t e d b y some
o t h e r ligand.
I n t h e presence experiment,
N.N-diethylaniline
was used as a
A ( La-ALC )
€3
C
( La-ALC-F)
( La-ALC-DA-F )
(red color)
(purple color)
(blue c o l o r )
F i g u r e 3. M o l e c u l a r s t r u c t u r e s o f l a n t h a n u m a l i z a r i n c o m p l e x o n e and f l u o (A), La-ALC. m a j o r c o n s t i t u e n t o f " D o t i t e A l f u s o n e " ; r i d e derivatives. soluble i n ( B ) . La-ALC-F. i n s o l u b l e i n o r g a n i c s o l v e n t ; (C). La-ALC-DA-F, organic solvent.
63 s u b s t i t u t e f o r water,
r e s u l t i n g i n LaALC-DA-F
(Fig.
3(C)),
which i s soluble
i n o r g a n i c s o l v e n t s (10). C o l o r development. To a 25 m l s a m p l e s o l u t i o n , ALC),
10 m l a c e t o n e ,
t h e c o l o r development, and i r o n .
5 m l o f 5% l a n t h a n u m - a l i z a r i n c o m p l e x o n e (La-
and 2 m l a c e t y l a c e t o n e w e r e added.
Acetone improves
and a c e t y l a c e t o n e was t h e m a s k i n g a g e n t f o r a l u m i n u m
A p o r t i o n o f t h i s s o l u t i o n was t a k e n and i t s a b s o r p t i o n was
m e a s u r e d b y an o r d i n a r y c o l o r i m e t e r . i s o a m y l a l c o h o l s o l u t i o n was added. i n t o t h e isoamyl alcohol.
F o u r m l o f 5% N , N - d i e t h y l a n i l i n e The La-ALC-DA-F
in
f o r m e d was e x t r a c t e d
One m l o f t h e e x t r a c t a n t was m i x e d w i t h 99 m l o f
a m i x t u r e c o n s i s t i n g o f 80% c a r b o n d i s u l f i d e and 20% a c e t o n e .
This mixture
h a s a r e f r a c t i v e i n d e x o f 1.57 a n d i s s u i t a b l e a s a s o l v e n t f o r t h e t o t a l reflection cell.
La-ALC-DA-F
i s n o t s o l u b l e i n pure carbon d i s u l f i d e .
F i g u r e 4 i s a p h o t o g r a p h w h i c h s h o w s t h e a c t u a l c o l o r s o f La-ALC,
F i n aqueous p h a s e and La-ALC-DA-F
e x t r a c t e d i n t o isoarnyl alcohol.
La-ALCThe t w o
t e s t t u b e s o n t h e l e f t s i d e s h o w t h e c o l o r o f La-ALC w h i c h i s r e d a t pH 7. When La-ALC-F
forms, t h e c o l o r changes t o p u r p l e .
The t w o t e s t t u b e s o n
t h e r i g h t s i d e show t h e o r g a n i c and aqueous phases a f t e r s o l v e n t e x t r a c tion.
The u p p e r l a y e r i s i s o a m y l a l c o h o l .
a blue color. color.
On t h e o t h e r hand,
The e x t r a c t e d La-ALC-DA-F
t h e b l a n k (La-ALC-DA)
shows
gives a pale yellow
The aqueous p h a s e i s i n t h e l o w e r l a y e r and m a i n t a i n s t h e r e d c o l o r
o f La-ALC.
F i g u r e 4. C o l o r d e v e l o p m e n t o f l a n t h a n u m a l i z a r i n c o m p l e x o n e a n d f l u o r i d e ( A ) ( l ) . a q u e o u s s o l u t i o n (La-ALC): ( A ) ( 2 ) , a q u e o u s s o l u t i o n derivatives. 1o w e r (La-ALC-F): ( B ) ( 3 ) , u p p e r 1 a y e r : is o a m y l a 1 c o h o 1 p h a s e (La-ALC-DA); l a y e r : a q u e o u s p h a s e (La-ALC): ( B ) ( 4 ) , u p p e r l a y e r : i s o a m y l a l c o h o l p h a s e (La-ALC-DA-F): l o w e r l a y e r : aqueous phase (La-ALC).
64
Apparatus F i g u r e 5 shows t h e
LCC a b s o r p t i o n s p e c t r o m e t r y s y s t e m .
A tungsten
300 W ) was u s e d as t h e l i g h t source.
i n c a n d e s c e n t l a m p (Kondo S i l v a n i a ,
The i r r a d i a t i o n i n t e n s i t y o f t h e l i g h t s o u r c e was a d j u s t e d b y u s i n g a n e u t r a l d e n s i t y (N.D)
filter.
p e r s o n a l c o m p u t e r (NEC,
PC8001),
p l i e r was a m p l i f i e d b y a 3121).
The m o n o c h r o m a t o r was u n d e r t h e c o n t r o l o f a
DC
and t h e o u t p u t s i g n a l f r o m t h e p h o t o m u l t i -
a m p l i f i e r ( Y o k o k a w a E l e c t r i c W o r k : t y p e YEW
The a m p l i f i e d o u t p u t f r o m t h e DC a m p l i f i e r was r e c o r d e d b y a s t r i p
chart recorder (Hitachi;
t y p e 056).
a n d / o r was d i g i t i z e d w i t h a 1 2 - b i t
A/D
c o n v e r t e r ( D a t e 1 t y p e ADC HX 12BGC) w h i c h was s e t i n t o t h e same u n i v e r s a l b o a r d i n t h e 1/0 u n i t (NEC PC 8 0 1 2 ) f r o m w h i c h t h e d a t a w e r e b e i n g a c q u i r e d b y t h e same p e r s o n a l c o m p u t e r . c e l l s were employed.
I n t h i s experiment,
two types o f c a p i l l a r y
One w a s l i n e a r l y s h a p e d a n d t h e o t h e r s p i r a l .
o r d e r t o keep t h e s a m p l e v o l u m e down,
In
a c e l l equipped w i t h a c a p i l l a r y w i t h
a n i n s i d e d i a m e t e r o f l e s s t h a n 1 m m was used.
The v o l u m e o f s a m p l e
r e q u i r e d w a s a p p r o x i m a t e l y 1 m l f o r a 1 m LCC ( 1 m m i.d.).
A UV-210A w a s
used f o r c o n v e n t i o n a l c o l o r i m e t r y .
ILCC I I
1
SPAPLE
MONOCHROMATOR
Computer-control l e d absorption spectrophotometer w i t h t o t a l F i g u r e 5. r e f l e c t i o n long c a p i l l a r y cell.
65 RESULTS AND DISCUSSION T r a n s m i s s i o n and a b s o r p t i o n s p e c t r a o f a n a l y t e F i g u r e 6 shows t h e t r a n s m i s s i o n s p e c t r a o f La-ALC-DA carbon d i s u l f i d e i n a 2 m l o n g c a p i l l a r y c e l l . a n a q u e o u s s o l u t i o n o f La-ALC-F
and La-ALC-DA-F
in
The a b s o r p t i o n s p e c t r a o f
a n d a n La-ALC-DA-F
s o l u t i o n i n carbon
d i s u l f i d e o b t a i n e d b y a n o r d i n a r y s p e c t r o p h o t o m e t e r ( 1 cm c e l l ) a r e shown i n F i g u r e 7.
B o t h La-ALC-F
i n a q u e o u s p h a s e a n d La-ALC-DA-F
d i s u l f i d e p r o v i d e a n a b s o r p t i o n m a x i m u m a t a b o u t 6 2 0 nm.
i n carbon
So, t h e w a v e -
l e n g t h a t 620 nm f o r b o t h w a t e r and c a r b o n d i s u l f i d e was used.
1 k
:.arcar
v)
i_
# J ,
: .2 3 0 0 400 500 600 700 800 J.J
2 3 0 0 400 500 600 700 800
600 700 Wavelength (nm)
400
500
300 400 500 600 700 800 Wavelength (nrn)
800
F i g u r e 6. La-ALC-DA
T r a n s m i s s i o n s p e c t r a w i t h a 2 m l o n g t o t a l r e f l e c t i o n LCC. i n CS2; (B), La-ALC-DA-F i n CS2.
F i g u r e 7. H20; (B).
A b s o r p t i o n s p e c t r a w i t h a n o r d i n a r y 1 cm c e l l . ( A ) La-ALC-F La-ALC-DA-F i n CS2.
(A), in
P a t h w a y o f l i g h t w i t h s p e c u l a r and t o t a l r e f l e c t i o n i n LCC F i g u r e 8 shows t h e c a l i b r a t i o n c u r v e s o f f l u o r i d e when a p p l y i n g aqueous a n d c a r b o n d i s u l f i d e s o l u t i o n s t o 70 cm LCC.
When c a r b o n d i s u l f i d e w a s
u s e d a s t h e s o l v e n t , a b s o r b a n c e was t w i c e a s h i g h as t h a t when w a t e r was u s e d a s t h e medium.
T h i s means t h a t t h e o p t i c a l
pathlength o f t h e source
l i g h t i n t h e c e l l f o r specular r e f l e c t i o n i s d i f f e r e n t from t h a t f o r t o t a l reflection. The p a t h w a y s o f l i g h t i n t h e c e l l c a n be c l a s s i f i e d i n t o t w o p a t t e r n s (11).
One i s t h e m e r i d i o n a l r a y w h i c h i s t h e m a j o r l i g h t t r a n s m i s s i o n
p a t t e r n i n a specular r e f l e c t i o n c e l l ,
and t h e
o t h e r i s h e l i c a l ray, which
i s t h e m a j o r o p t i c a l p a t h w a y i n t h e t o t a l r e f l e c t i o n LCC ( F i g .
9). H e l i c a l
p r o g r e s s i o n c a n b e c o n s i d e r e d t h e c a s e i n s p e c u l a r r e f l e c t i o n LCC.
However
i t i s much a t t e n u a t e d due t o a number o f r e f l e c t i o n s whose c o e f f i c i e n t i s
66 below
100%.
Since t h e frequencies o f source l i g h t r e f l e c t i o n i n m e r i d i o n a l
progression are lower than those i n o f source
h e l i c a l progression,
the attenuation
l i g h t suffered v i a meridional progression i s s m a l l e r than t h a t
v i a h e l i c a l progression.
I n t h e specular r e f l e c t i o n c e l l ,
t r a n s m i t t e d l i g h t v i a m e r i d i o n a l pathways,
mainly,
therefore,
the
contributes t o the l i g h t
d e t e c t o r a t t h e e x i t o f t h e LCC, w h e r e t h e o p t i c a l p a t h l e n g t h i s g i v e n b y b x sec 8 (Fig.
9(1)).
On t h e o t h e r h a n d ,
h e l i c a l r a y i s g i v e n b y b,
the effective pathlength of
x c o s e c @ ( F i g . 9(2)).
total reflection
1.5V V
rl 1u
0
1.0-
specular
5
0
10
1s
20
Concentration of Fluoride ( 11;: in1
F i g u r e 8. cm).
C a l i b r a t i o n c u r v e s w i t h s p e c u l a r and t o t a l r e f l e c t i o n c e l l s
meridional ray
--
(70
helical ray
b
F i g u r e 9. P a t h w a y s o f l i g h t s o u r c e p r o g r e s s i o n i n LCC. (I), r e f l e c t i o n : m e r i d i o n a l ray; ( Z ) , t o t a l r e f l e c t i o n : h e l i c a l ray.
specular
67 T h e o r e t i c a l e q u a t i o n s o f a b s o r b a n c e f o r LCC Based on t h e a b o v e c o n s i d e r a t i o n o n o p t i c a l p a t h l e n g t h ,
the theoretical
s e n s i t i v i t y o f s p e c u l a r r e f l e c t i o n LCC ( m e r i d i o n a l r a y ) c a n be shown a s :
A(absorbance) = l o g
I 0 I
'
P(X) exp[-a(h)b
sec 8 c k ( c ) ] dXd0
And t h e t h e o r e t i c a l s e n s i t i v i t y o f t o t a l r e f l e c t i o n LCC c a n be g i v e n as:
A(absorbance)
=
log
I
2 I
w h e r e 10 i s t h e i n t e n s i t y o f i n c i d e n t l i g h t . I i s t h e i n t e n s i t y o f t r a n s mitted light,
h i s t h e w a v e l e n g t h o f t h e l i g h t source,
d i s t r i b u t i o n o f t h e l i g h t source, f u n c t i o n o f w a v e l e n g t h (A), absorbent,
P(A)
i s the spectral
a(X) i s t h e a b s o r p t i o n c o e f f i c i e n t as a
b i s t h e c e l l length,
c i s the concentration o f
k ( c ) i s a l m o s t e q u a l t o one i n t h e a b s o r p t i o n r a n g e o b s e r v e d i n
t h e experiment,
8 i s t h e a n g l e of i n c i d e n c e o f s o u r c e m e r i d i o n a l l i g h t , and
@ i s t h e angle o f incidence o f source h e l i c a l l i g h t .
Dependence o f s e n s i t i v i t y o f LCC on 8 and @ F i g u r e 1 0 s h o w s t h e d e p e n d e n c e s o f c a l c u l a t e d s e n s i t i v i t y o f LCC o n a n g l e s o f s o u r c e l i g h t i n c i d e n c e ( 0 , @(go"-@)) f o r m e r i d i o n a l and rays.
These a r e o b t a i n e d a c c o r d i n g t o E q u a t i o n s (1) and
I n t h e case o f t o t a l r e f l e c t i o n ,
t h e i n t e n s i t y o f I.
(2)
helical
shown above.
d o e s n o t depend o n t h e
a n g l e s o f 0 and I$ b u t t h e i n t e n s i t y o f I i s much d e p e n d e n t o n t h e s e a n g l e s . When t h e a n g l e s 8 a n d (go"-@) i n c r e a s e ,
1 decreases.
these angles increase, t h e absorbance a l s o increases. and (90"-I$) a r e c l o s e t o go", infinite.
However,
Therefore,
when
When t h e a n g l e s 8
t h e a b s o r b a n c e i n c r e a s e s and f i n a l l y becomes
the i n t e n s i t y o f I ( l i g h t transmitted through the
s a m p l e s o l u t i o n ) a l s o d e c r e a s e s w i t h i n c r e a s i n g 0 t o go",
and c a n n o t con-
t r i b u t e t o t h e d e t e c t i o n signal. Cell lenqth The c a l i b r a t i o n c u r v e s o b t a i n e d f r o m t h e t o t a l r e f l e c t i o n c e l l s o f v a r y i n g l e n g t h s a r e s h o w n i n F i g u r e 11.
Clearly, the sensitivity i n the
68
90r
Figure 10. The dependence of calculated sensitivity of LCC on angles 8 and of incidence of source light. 8, angle of incidence of meridional ray: @, angle of incidence of helical ray.
@
Figure 11. cell s.
CalibratTon curves obtained from various total reflection
total reflection LCC was dependent on cell length. It i s noted that the calibration curves showed strong curvature. This is due to the changes i n optical pathlength with variation in absorption of the solution: the optical path becomes shorter for solutions of higher concentrations. Thus the enhancement of smaller absorbance is more obvious than that of higher
A n absorbance o f 0.0002 and 0.002 given by an ordinary 1 cm cell can be amplified 2000 and 700 times, respectively, with a 4 m cell. The amplification o f absorbance by LCC is t w o to six times absorbances for the LCC.
greater than that of cell length.
69
C a l i b r a t i o n s i g n a l s and d e t e c t i o n l i m i t The a c t u a l c a l i b r a t i o n s i g n a l s o b t a i n e d b y a 4 m l o n g c e l l a r e shown i n F i g u r e 12. Aqueous s o l u t i o n s o f d i f f e r e n t f l u o r i d e c o n c e n t r a t i o n s w e r e t r e a t e d and e x t r a c t e d i n t o a carbon d i s u l f i d e - a c e t o n e m i x t u r e d e s c r i b e d previously.
I n t h e p r e s e n t system,
t h e d e t e c t i o n l i m i t was found t o be 10
n g / l w i t h a 4 m t o t a l r e f l e c t i o n LCC.
F i g u r e 12. A c t u a l c a l i b r a t i o n s i g n a l s ( t r a n s m i s s i o n s i g n a l s ) f o r f l u o r i d e o b t a i n e d f r o m t o t a l r e f l e c t i o n LCC (4 m) w i t h LaALC-DA-F.
TABLE I
R E C O V E R I E S OF FLUORIDE ADDED TO D R I N K I N G WATER ( n g / m l )
Sampl e l
A B
Fluoride Concentration Found Added Result (ng/ml) (ng/ml) (ng/ml)
107 92.8
C
120
D
153
E
90.0
'Samples were d i l u t e d 50 t i m e s
Recovery ( % )
50
151
50
149
104
50
173
102
96.2
50
196
96.6
50
130
92.9
70 Recoveries T a b l e I shows t h e r e c o v e r i e s o f t h e f l u o r i d e added t o v a r i o u s k i n d s o f d r i n k i n g water, 50 ng/ml
c o l l e c t e d o n t h e U n i v e r s i t y o f T o k y o campus.
fluoride
i o n s w e r e 1 n t h e r a n g e o f 93-104%.
Recoveries o f lndicatlng the
v a l i d i t y o f t h i s method i n t h e a n a l y s i s o f f l u o r i d e i n d r i n k i n g water. Application t o drinking water analysis T a b l e I 1 shows t h e a n a l y t i c a l r e s u l t s o f t h e f l u o r i d e i n d r i n k i n g w a t e r c o l l e c t e d f r o m v a r i o u s areas o f Tokyo c i t y ,
Japan,
using the present
c o l o r i m e t r i c t e c h n i q u e w i t h a t o t a l r e f l e c t i o n LCC (0.7 m ) a n d c o l o r i m e t r y w i t h a 1 cm c e l l .
ordinary
It i s c l e a r t h a t t h e values obtained from
t h e t o t a l r e f l e c t i o n LCC m e t h o d a g r e e d w e l l w i t h t h o s e r e s u l t i n g f r o m ordinary colorimetry.
Furthermore,
t h e p r e s e n t method i s more e f f e c t i v e i n
d e t e r m i n i n g t r a c e amounts o f f l u o r i d e , ng/ml,
e s p e c i a l l y those l o w e r than 10
which cannot be d e t e c t e d by o r d i n a r y c o l o r i m e t r y .
TABLE I 1 ANALYTICAL RESULTS
OF
FLUORIDE I N DRINKING WATER
OF
TOKYO CITY
Conc. o f F, Sampling s t a t i o n
Ordinary colorimetry ( 1 cm c e l l )
ng/ml Presenf method (LCC. 0 . 7 m )
110
107
2 ) I b i d , D e p t . o f Chem.
120
124
3) I b i d .
170
175
1 ) U n i v . o f Tokyo,
D e p t . o f Chem.
f o r L a b o r a t o r y Use
4 ) I b i d , T r e a t e d b y I o n Exchange R e s i n
ND3
4.352
5) I b i d , Student Union
95
93.8
6) I b i d , Student Union
80
83.1
Student Union
ND
8) I b i d . S t u d e n t U n i o n
165
166
9 ) I b i d , Dept. o f P h y s i c s
125
126
10) I b i d . Dept. o f P h y s i c s
ND
10.5
11) R e s i d e n t i a l Area,
90
88.4
7) I b i d .
Bunkyo-ku
' S a m p l e was d i l u t e d 20 t i m e s 'After 3ND,
deionization n o t detected
10.7
71 REFERENCES 1.
Fuwa K (1978) D e n t a l R e v i e w 4 2 7 : l l l - 1 1 6
2.
Underwood EJ (1975) T r a c e E l e m e n t s i n Human and A n i m a l N u t r i t i o n . Maruzen Co. Academic P r e s s , New York, p p 369-400
3.
F r e r e FJ (1961) A n a l Chem 33:644-645
4.
Yamamura SS, Wade MA, S i k e s JH (1962) A n a l Chem 34:1308-1312
5.
H a s h i t a n i H.
Y o s h i d a H,
Muto H (1967) B u n s e k i Kagaku 16:44-46
6.
H a s h i t a n i H,
Y o s h i d a H,
A d a c h i T (1979) B u n s e k i Kagaku 28:680-684
7.
We1 L, F u j i w a r a K.
8.
Fuwa K,
9.
F u j i w a r a K,
Wei
L,
Fuwa K (1983) A n a l Chem 55:951-955
F u j i w a r a K (1984) A n a l Chem 56:1640-1644
Fuwa K (1985) A n a l Chem 57:1012-1016
10. I n s t i t u t e o f E n v i r o n m e n t a l Science, B e i j i n g (1976) P r i n c i p a l Knowledge a b o u t P h y s i c a l and C h e m i c a l A n a l y s i s o f Water. C o n s t r u c t i o n I n d u s t r i a l Press. B e i j i n g , pp 194-197 11. We1 L. F u j i w a r a K,
Fuwa K (1985) J S p e c t r o s c SOC Japan 29:173-176
73
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 73-80 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
DETERMINATION OF TOTAL FLUORINE AND IONIZABLE FLUORINE LEVELS I N BLOOD SERA -
A COMPARATIVE STUDY
YOKO BESSHO,
OF V A R I O U S
ANALYTICAL METHODS
M I S A K O TOMITA. Y O S H I H I R O KANEKO
Department o f Hygiene and O r a l H e a l t h , School o f D e n t i s t r y , U n i v e r s i t y , 1-5-8, H a t a n o d a i , Shinagawa-ku. T o k y o 142, J a p a n
Showa
ABSTRACT
A c o m p a r a t i v e s t u d y was c a r r i e d o u t i n w h i c h f o u r a n a l y t i c a l m e t h o t i s w e r e t e s t e d f o r t h e d e t e r m i n a t i o n o f i o n i z a b l e and t o t a l n e w b o r n c a l f serum:
f l u o r i n e contents i n
(1) d i r e c t measurement o f i o n i z a b l e f l u o r i n e
i n the
serum u s i n g t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e ; ( 2 ) p r e t r e a t m e n t w i t h l o w t e m p e r a t u r e oxygen p l a s m a a s h i n g f o l l o w e d by t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e method o r b y gas c h r o m a t o g r a p h y ; ( 3 ) s e p a r a t i o n o f f l u o r i n e by and ( 4 )
m i c r o d i f f u s i o n f o l l o w e d by i t s d e t e r m i n a t i o n by gas chromatography;
d i r e c t e x t r a c t i o n w i t h 4% TMCS n-hexane f o l l o w e d b y gas c h r o m a t o g r a n h y . The f l u o r i d e e l e c t r o d e m e t h o d w i t h o u t a n y p r e t r e a t m e n t p r o v e d e f f i c i e n t i n determining the ionizable fluorine.
B o t h t h e m i c r o d i f f u s i o n m e t i o d and
t h e d i r e c t e x t r a c t i o n method proved d e f i c i e n t f o r t h e d e t e r m i n a t i o n o f t o t a l f l u o r i n e i n t h e serum. The b e s t r e s u l t s w e r e o b t a i n e d f r o m t h e l o w t e m p e r a t u r e o x y g e n o l a s m a a s h i n g method f o l l o w e d by gas chromatography.
The v a l u e s r e s u l t i n g f r o m
t h i s p r o c e d u r e p r e s u m a b l y i n d i c a t e d an a c t u a l t o t a l f l u o r i n e c o n t e n t
111 t h e
sample.
INTRODUCTION Numerous
analytical
methods and p r e t r e a t m e n t p r o c e d u r e s havc been
r e p o r t e d f o r t h e d e t e r m i n a t i o n o f t h e f l u o r i d e i n serum samples. on t h e l e v e l o f f l u o r i d e r e p o r t e d b y d i f f e r e n t r e s e a r c h e r s ,
Findings
however,
have
been c o n t r a d i c t o r y . Ashed s a m p l e s have been used t o s e p a r a t e f l u o r i n e b y m i c r o d i f f u s i o n . R e c e n t l y , however,
i t has been r e p o r t e d t h a t s a t i s f a c t o r y r e s u l t s w e r e
o b t a i n e d b y u s i n g s t r o n g p e r c h l o r i c a c i d t o s i m u l t a n e o u s l y decompose and d i f f u s e samples r i c h i n o r g a n i c m a t t e r ( 1 , Z ) . I n t h i s study,
we t e s t e d v a r i o u s a n a l y t i c a l methods,
e i t h e r alone o r i n
c o m b i n a t i o n w i t h some p r e t r e a t m e n t p r o c e d u r e s , i n an a t t e m p t t o f i n d an
74 adequate method f o r t h e d e t e r m i n a t i o n of
t h e i o n i z a b l e and t o t a l f l u o r i n e
c o n t e n t s i n c a l f serum.
MATERIALS AND METHODS
(1) l o w
The f o l l o w i n g a p p a r a t u s was u s e d t o c o n d u c t t h e s e e x p e r i m e n t s :
(2) gas chromatograph (Shimazu
t e m p e r a t u r e a s h e r ( B r a n s o n / I P C 1005-248 AN); GC-7AG,
FID);
(3) M i c r o p r o c e s s o r i o n a n a l y z e r ( O r i o n R e s e a r c h M o d e l 901);
(4) combination f l u o r i d e
e l e c t r o d e ( O r i o n Research Model
96-09);
(5)
s h a k i n g a p p a r a t u s (Thomas S c i e n t i f i c T - 2 2 s ) . I r v i n e S c i e n t i f i c ' s n e w b o r n c a l f s e r u m was u s e d a s t h e t e s t s a m p l e s e r u m t h r o u g h o u t t h i s work.
The i o n i z a b l e f l u o r i d e i n t h e s e r u m was d e t e r m i n e d
d i r e c t l y by a f l u o r i d e i o n - s e l e c t i v e electrode. Three p r o c e d u r e s were used t o d e t e r m i n e t h e t o t a l f l u o r i n e , b o t h i o n i z a b l e a n d combined, o f one
i n t h e serum.
including
Each o f t h e s e was a c o m b i n a t i o n
of t h e t h r e e p r e t r e a t m e n t m e t h o d s w i t h o n e o r b o t h o f t h e t w o
a n a l y t i c a l methods.
The p r e t r e a t m e n t m e t h o d s u s e d w e r e l o w t e m p e r a t u r e
oxygen plasma ashing (3)'
microdiffusion.
and d i r e c t e x t r a c t i o n .
a n a l y t i c a l methods used were t h e f l u o r i d e i o n - s e l e c t i v e
The
e l e c t r o d e method,
and g a s c h r o m a t o g r a p h y . The f o l l o w i n g t h r e e p r o c e d u r e s w e r e used:
( 1 ) I n t h i s p r o c e d u r e t h e s e r u m f l u o r i n e c o n t e n t was d e t e r m i n e d f i r s t b y t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e method f o l l o w i n g p r e t r e a t m e n t w i t h l o w t e m p e r a t u r e oxygen plasma ashing. a g a i n b y gas chromatography (Fig.
F l u o r i d e i n t h e s a m p l e was d e t e r m i n e d 1).
( 2 ) T h i i was a c o m b i n a t i o n o f m i c r o d i f f u s i o n and g a s c h r o m a t o g r a p h y ( F i g . 2).
A T u p p e r w a r e m i n i c u p was u s e d as a d i f f u s i o n b o t t l e .
A polyethylene
t u b e c o n t a i n i n g a p i e c e o f f i l t e r p a p e r w i t h s o d i u m h y d r o x i d e was p l a c e d i n t h e minicup.
The s a m p l e was p l a c e d i n t o t h e m i n i c u p ,
f o l l o w e d by t h e addi-
t i o n o f 60% p e r c h l o r i c a c i d s o l u t i o n s a t u r a t e d w i t h h e x a m e t h y l d i s i l o x a n e
(HMDS) as a r e l e a s i n g s o l u t i o n . chromatography. attempted.
The f l u o r i d e r e l e a s e d was a n a l y z e d b y gas
The e l e c t r o d e m e t h o d f o r a n a l y z i n g t h e f l u o r i d e was
However,
t h e sodium h y d r o x i d e p r e s e n t i n t h e sample s o l u t i o n
i n t e r f e r e d w i t h f l u o r i n e m e a s u r e m e n t b y t h e e l e c t r o d e and g a v e a f a l s e l y high value.
(3) I n t h i s p r o c e d u r e t h e s e r u m s a m p l e was e x t r a c t e d d i r e c t l y w i t h o u t p r i o r a s h i n g o r d e c o m p o s i t i o n t r e a t m e n t ( F i g . 3).
A 60% p e r c h l o r i c a c i d
s o l u t i o n and a hexane s o l u t i o n c o n t a i n i n g 4% t r i m e t h y l c h l o r o s i l a n e
(TMCS)
w e r e u s e d f o r t h e e x t r a c t i o n . The f l u o r i n e c o n t a i n e d i n t r i m e t h y l f l u o r o s i l a n e (TMFS) i n t h e h e x a n e l a y e r was t h e n d e t e r m i n e d b y g a s c h r o m a t o g r a p h y .
75 F i g u r e 1. S c h e m a t i c d i a gram showing d e t e r m i n a t i o n o f t o t a l F i n serum by F i o n e l e c t r o d e and b y g a s chromatography.
Sercim I .5Illl IJW
11111
I)W
1.5IIII
Ash
I---
I lexanc layer
II.0 l a y e r
I
GC
1-1?0l a y e r
I
Icxnllc?
I
GC
(1-11))
laycr (I 11 1)
76
Serum 2mi 4% TMCS-hexane Irnl 609; HClOs 2-4ml
H20
layer
Hexane layer
1
GC (FID) F i g u r e 3. Schematic diagram showing d e t e r m i n a t i o n o f t o t a l F i n serum by d i r e c t e x t r a c t i o n f o l l o w e d by gas chromatography.
RESULTS
AND DISCUSSION
W i t h t h e m i c r o d i f f u s i o n method (Fig.
4). a 100% r e c o v e r y o f t h e f l u o r i n e
added t o t h e serum as sodium f l u o r i d e was observed when t h e d i f f u s i o n t i m e was
3 hours o r longer.
I n contrast,
the experimental
value f o r the
f l u o r i n e i n t h e serum i t s e l f was i n c r e a s e d o n l y g r a d u a l l y w i t h t h e d i f f u sion time.
Moreover,
even a f t e r a d i f f u s i o n t i m e o f
74
hours, t h e f l u o r i n e
l e v e l observed never exceeded t h e v a l u e o f i o n i z a b l e f l u o r i n e measured by t h e d i r e c t e l e c t r o d e method.
L
J
O ' i
3
G
12
Diffusion time, h r
21
F i g u r e 4. E f f e c t o f d i f f u s i o n t i m e on t h e F v a l u e d e t e r m i n e d and t h e A 2 m l s e r u m s a m p l e w i t h o r w i t h o u t added r e c o v e r y r a t e o f added F. f l u o r i d e was t r e a t e d w i t h t h e m i c r o d i f f u s i o n method u s i n g 4 m l 60% HC104 s a t u r a t e d w i t h HMDS f o r v a r i o u s t i m e p e r i o d s . The r e l e a s e d F was t h e n d e t e r m i n e d b y gas chromatography.
77
The r e s u l t s o b t a i n e d w i t h t h e d i r e c t e x t r a c t i o n m e t h o d w e r e a l r i l o s t t h e same as w i t h m i c r o d i f f u s i o n .
The f l u o r i n e l e v e l o b t a i n e d n e v e r e x c e e d e d
t h e i o n i z a b l e one i n t h e serum, e v e n t h o u g h t h e e x t r a c t i o n t i m e was p r o longed t o 8 hours (Fig.
5).
I
O i 2 3 1 6 Extraction t i m e s hP
1
3
F i g u r e 5. E f f e c t o f e x t r a c t i o n t i m e o n t h e F v a l u e d e t e r m i n e d i n serum. A 2 m l s e r u m s a m p l e was e x t r a c t e d w i t h 2 m l 60% HC104 and 1 m l 4% TMCS i n nhexane.
The e x t r a c t e d
F was t h e n d e t e r m i n e d b y g a s c h r o m a t o g r a p h y .
F i g u r e s 5 and 7 show t h e r e l a t i o n s h i p b e t w e e n t h e s a m p l e v o l u m e and t h e a m o u n t o f f l u o r i d e o b t a i n e d b y m i c r o d i f f u s i o n and t h e a m o u n t o b t a i n e d b y d i r e c t extraction,
respectively.
gas c h r o m a t o g r a p h y .
I n b o t h cases,
f l u o r i n e was d e t e r m i n e d b y
A l i n e a r r e l a t i o n s h i p e x i s t e d between t h e t w o v a r i -
a b l e s i n b o t h cases. i o n i z a b l e f l u o r i n e only,
The v a l u e s shown i n t h e t w o f i g u r e s r e p r e s e n t t h e as mentioned p r e v i o u s l y .
F i g u r e 8 shows t h e r e l a t i o n s h i p b e t w e e n t h e f l u o r i n e c o n t e n t s d e t e r m i n e d b y t h e e l e c t r o d e m e t h o d and t h o s e d e t e r m i n e d b y g a s c h r o m a t o g r a p h y ,
subse-
q u e n t t o p r e t r e a t m e n t o f t h e serum sample b y t h e l o w t e m p e r a t u r e a s h i n g method.
An e x c e l l e n t a g r e e m e n t w a s f o u n d b e t w e e n t h e d e t e r m i n e d v a l u e s
f r o m b o t h methods,
even though t h e v a l u e s o b t a i n e d f r o m gas Chromatography
were s l i g h t l y h i g h e r t h a n t h o s e f r o m t h e e l e c t r o d e method i n most o f t h e samp 1es. The f l u o r i d e c o n t e n t s i n t h e c a l f s e r u m s a m p l e d e t e r m i n e d b y d i f f e r e n t a n a l y t i c a l m e t h o d s a r e s u m m a r i z e d i n T a b l e I.
The mean v a l u e o f t h e i o n i z -
a b l e f l u o r i d e l e v e l d e t e r m i n e d b y t h e d i r e c t e l e c t r o d e m e t h o d w a s 0.169 ug/ml.
Samples t h a t were p r e t r e a t e d w i t h t h e l o w t e m p e r a t u r e a s h i n g method
and t h e n d e t e r m i n e d f o r f l u o r i d e b y t h e e l e c t r o d e method o r b y gas c h r o -
78
F i g u r e 6. The r e l a t i o n s h i p b e t w e e n s a m p l e v o l u m e a n d t h e F v a l u e s d e t e r m i n e d b y t h e m i c r o d i f f u s i o n method. The v o l u m e o f 60% HC104 u s e d was t h r e e t i m e s t h a t o f t h e s e r u m sample. The r e l a t i o n s h i p b e t w e e n s a m p l e v o l u m e and t h e F v a l u e s d e t e r F i g u r e 7. m i n e d b y d i r e c t e x t r a c t i o n method. The v o l u m e o f 60% HC104 u s e d was t h r e e t i m e s t h e v o l u m e o f t h e s e r u m sample.
E .& 1.5-
2 E!
5
1.2.
0.9.
c ._
-um *
0.6.
L
0
0.304 0
-
0.3
U.6
0.9
1.2
1.5
T o t a l F in s e r u m ( G C ) ( r J q / m l )
F i g u r e 8. The r e l a t i o n s h i p b e t w e e n t o t a l F v a l u e s o b t a i n e d b y g a s chromat o g r a p h y (GC) and b y F i o n e l e c t r o d e m e t h o d ( I E ) .
I9 matography gave h i g h e r v a l u e s :
0.199 p g / m l
0.232 p g / m l b y g a s c h r o m a t o g r a p h y .
by t h e e l e c t r o d e method,
and
M o r e o v e r , when a s t a n d a r d a d d i t i o n
method was a p p l i e d t o t h e above methods,
even h i g h e r v a l u e s were o b t a i n e d ,
0.257 p g / m l b y t h e e l e c t r o d e m e t h o d , and 0.264 p g / m l b y g a s c h r o m a -
i.e.,
tography.
An e x a m p l e o f t h e c a l i b r a t i o n c u r v e f o r t h e s t a n d a r d a d d i t i o n
method i s shown i n F i g u r e 9. Contrary t o this,
w i t h t h e gas c h r o m a t o g r a p h i c method f o l l o w i n g t h e
pretreatment, e i t h e r w i t h m i c r o d i f f u s i o n o r w i t h d i r e c t e x t r a c t i o n , t h e v a l u e s o b t a i n e d n e v e r exceeded t h e l e v e l of t h e i o n i z a b l e f l u o r i n e . indicates
This
t h a t t h e c o m b i n e d f l u o r i n e i n b l o o d serum c a n n o t be d e t e c t e r '
w i t h t h e s e procedures. C o n c e r n i n g m i c r o d i f f u s i o n , V e n k a t e s w a r l u (4) and Paez
fi g .
(5) p o i n t e d
o u t a s i m i l a r f a c t i n t h e i r c o m p a r a t i v e s t u d i e s on methods f o r e v a l u a t i n g f l u o r i d e i n b l o o d serum.
They f o u n d m i c r o d i f f u s i o n t o be i n e f f e c t i v e as a
p r e t r e a t m e n t p r o c e d u r e f o r d e t e r m i n i n g t h e t o t a l f l u o r i n e c o n t e n t when i t was d i r e c t l y a p p l i e d t o unashed samples.
TABLE I FLUORIDE CONTENTS I N CALF SERUM DETERMINED BY DIFFERENT ANALYTICAL METHODS
n
Method
F i n Serum (w/m1) Mean ? SD
Ratio t o Ionizable F
~~~
IE' (ionizable) IE-sL2
15
0.169
0.003
1.00
9
0.199
0.019
1.18
36
0.257
-----
1.52
8
0.232
0.011
1.37
31
0.264
-----
1.56
0.165
0.008
0.98
_____
0.92
Ashing IE-st.
add3
GC-st. Ashing GC-st.
.
add3
D if f 4-
GC
4
E x t r . 5-
GC
2
'F ion electrode 'Standard c u r v e method 3 S t a n d a r d a d d i t i o n method
0.1 55
4 M i c r o d i f f us i o n method 5 D i r e c t e x t r a c t i o n method
80
-c,5
0
c.5
1.3
F added
1.5
2.0
(ug)
F i g u r e 9. C a l i b r a t i o n c u r v e o f t h e s t a n d a r d a d d i t i o n m e t h o d f o r F d e t e r m i n a t i o n b y gas chromatography.
CONCLUSION The u s e o f t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e w i t h o u t a p r i o r s e p a r a t i o n p r o c e d u r e was f o u n d t o b e e f f i c i e n t f o r t h e d e t e r m i n a t i o n o f t h e i o n i z a b l e f l u o r i n e i n b l o o d serum.
For the determination o f the t o t a l
f l u o r i n e i n b l o o d serum,
pretreatment w i t h microdiffusion o r d i r e c t extrac-
t i o n proved deficient.
The b e s t r e s u l t s w e r e o b t a i n e d b y u s e o f t h e l o w
t e m p e r a t u r e ashing method f o l l o w e d b y gas c h r o m a t o g r a p h y o r b y f l u o r i d e e l e c t r o d e measurement, a l t h o u g h t h e l a t t e r method gave a s l i g h t l y l o w e r value.
The v a l u e s o b t a i n e d f r o m t h e s e p r o c e d u r e s p r e s u m a b l y i n d i c a t e an
a c t u a l t o t a l f l u o r i n e c o n t e n t i n t h e sample.
REFERENCES Hayashi
H,
1.
H a r a K,
2.
D a b e k a RD, M c K e n z i e AD, 62: 1065-1069 S u z u k i S,
H i r o k a w a K.
I i z u k a K (1983) J D e n t H l t h 33:296-297
C o n a c h e r HBS ( 1 9 7 9 ) J A s s o c O f f A n a l Chem
K u r o i w a S, Kaneko Y (1983) J D e n t H l t h 33:183-196
3.
T o m i t a M,
4.
V e n k a t e s w a r l u P (1975) Biochem Med 14:368-371
5.
Paez DM, 13: 65-70
deB1anchi LP, G i l BA,
Dapas J r 0, C o r o n a t o
RG (1980) F l u o r i d e
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 81-88 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands
81
SAMPLING OF HYDROGEN FLUO2IDE BY DIFFUSIONAL TRANSPORT I N A CIRCULAR GLASS TUBE COATED WITH SODIUM CARBONATE TAKAAKI YANAKA
N i i g a t a P r e f e c t u r a l Research L a b o r a t o r y Sowa, N i i g a t a C i t y , J a p a n 950-21
f G r
H e a l t h and E n v i r o n m e n t ,
314-1
ABSTRACT Hydrogen f l u o r i d e o f t h r o u g h a t u b e whose
l o w c o n c e n t r a t i o n s (0.004 inside wall
was c o a t e d
-
2 ppm) was p a s s e d
w i t h sodium
R e y n o l d s n u m b e r s o f t h e f l o w w e r e b e t w e e n 2 5 0 a n d 10,000.
carbonate. For laminar
flow, t h e d i s t r i b u t i o n o f f l u o r i d e on t h e i n s i d e w a l l corresponded w e l l with the theoretical
f o r m u l a o f G o r m l e y and Kennedy.
For t u r b u l e n t flow,
t h e d i f f u s i o n c o e f f i c i e n t was e x p r e s s e d a s a f u n c t i o n of R e y n o l d s number. S a m p l i n g e f f i c i e n c i e s o f t h e t u b e a r e shown i n r e l a t i o n from t h e tube entrance,
t o the distance
t h e f l o w r a t e and t h e R e y n o l d s number.
From t l i e s e
r e s u l t s , i t a p p e a r s t h a t h y d r o g e n f l u o r i d e c a n be e f f e c t i v e l y c o l l e c t e d w i t h t h i s t y p e o f tube,
s e v e r a l d e c a c e n t i m e t e r s l o n g and w i t h a d i a m e t e r
o f 0.4 t o 1 cm.
INTRODUCTION I n the f i e l d o f aerosol science,
a method o f d i f f u s i o n a l t r a n s p o r t o f
p a r t i c l e s b y B r o w n i a n movement o n t o t h e i n s i d e w a l l o f a t u b e h a s been used f o r t h e m e a s u r e m e n t o f p a r t i c l e s i z e and c o n c e n t r a t i o n . t h e t u b e f o r t h i s m e t h o d i s c a l l e d a d i f f u s i o n tube.
The m a i n p a r t o f
The c r o s s s e c t i o n o f
the tube is usually c i r c u l a r o r f l a t l y rectangular. For P o i s e u i l l e f l o w
through a c i r c u l a r
tube,
t h e d i s t r i b u t i o n of
d i f f u s i o n a l t r a n s p o r t o n t o t h e i n s i d e w a l l was s o l v e d t h e o r e t i c a l l y b y G o r m l e y and Kennedy ( 1 ) u n d e r t h e c o n d i t i o n o f n e g l e c t i n g a x i a l d i f f u s i o n . The d i f f u s i o n e q u a t i o n i n a s t e a d y s t a t e i s
and b o u n d a r y c o n d i t i o n s a r e x =
0
C
=
r = R , C = O
Co ( c o n s t )
82 I n these equations, x i s t h e distance from the tube entrance, r i s r a d i a l
C
coordinate,
i s t h e c o n c e n t r a t i o n o f a e r o s o l a t (X,r),
particles,
and R i s t h e r a d i u s o f t h e t u b e .
-
c(x) =
0.8191 exp(-14.63A)
+
LO
-
V
i s t h e mean v e l o -
D i s t h e Brownian d i f f u s i o n c o e f f i c i e n t o f aerosol
c i t y t h r o u g h t h e tube,
+
The s o l u t i o n i s
0.0976 exp(-89.2A)
+ ...
0 . 0 3 2 5 exp(-228A)
,
(3)
W h e r e C ( x ) i s t h e m e a n c o n c e n t r a t i o n o f a e r o s o l a t a d i s t a n c e x.
A i s the
d i f f u s i o n parameter f o r a c i r c u l a r tube
A
r
D x 4VR
= X / P e , d = 2 R d
and Pe i s t h e d i f f u s i v e P e c l e t number ( v d / D ) . F o r t u r b u l e n t f l o w t h r o u g h a t u b e , M o n t g o m e r y and C o r n (2) s u m m a r i z e d t h e theories o f deposition o f p a r t i c l e s claimed by Friedlander
&
21. ( 3 ) .
D a v i e s (4). and B e a l (5). and p o i n t e d o u t t h a t t h e i r e x p e r i m e n t a l r e s u l t s were f a r h i g h e r t h a n t h e t h e o r e t i c a l values.
B u t L i u and Agarwal (6)
c l a i m e d t h a t t h e i r e x p e r i m e n t a l values were i n agreement w i t h t h e t h e o r i e s o f Friedlander gt
21. a n d
Beal.
It i s presumed t h a t t h e t h e o r y o f d i f -
f u s i o n a l deposition o f p a r t i c l e s i n a t u r b u l e n t f l o w i n a tube i s s t i l l not c o m p l e t e l y solved. The d i f f u s i o n c o e f f i c i e n t o f t h e m o l e c u l a r m o t i o n o f g a s i s b y f a r h i g h e r t h a n t h a t o f p a r t i c l e s b y Brownian motion.
F o r t h i s reason,
i f the inside
w a l l o f a tube i s coated w i t h a m a t e r i a l which reacts r a p i d l y w i t h a s p e c i f i c gas, and t h e n a g a s c o n t a i n i n g t h a t s p e c i f i c g a s f l o w s t h r o u g h t h e tube,
i t i s e x p e c t e d f r o m Boundary c o n d i t i o n s 2 and S o l u t i o n 3 t h a t t h e
s p e c i f i c gas can be e f f e c t i v e l y c o l l e c t e d on t h e i n s i d e w a l l i n a s h o r t l e n g t h o f tube.
O k i t a e t al.
(7) showed t h a t a t u b e c o a t e d w i t h sodium
carbonate c o u l d c a p t u r e hydrogen f l u o r i d e .
B u t s a m p l i n g e f f i c i e n c y has n o t
been i n v e s t i g a t e d u n d e r d i f f e r e n t c o n d i t i o n s . The p u r p o s e o f t h i s p a p e r i s t o show t h e d i s t r i b u t i o n o f d i f f u s i o n a l t r a n s p o r t o f h y d r o g e n f l u o r i d e i n a c i r c u l a r g l a s s t u b e whose i n s i d e w a l l i s c o a t e d w i t h sodium c a r b o n a t e f o r b o t h l a m i n a r and t u r b u l e n t f l o w s . MATERIALS AND METHODS
A i r c o n t a i n i n g 0.004 - 2 ppm h y d r o g e n f l u o r i d e g e n e r a t e d b y a P e r m e a c a l Permeator (PD
-
10, G a s t e c Co.)
was
passed through Pyrex g l a s s tubes
83 ( d i f f u s i o n t u b e ) o f 0.4.
0.6,
and 1.0 cin i.d.,
whose i n s i d e w a l l s w e r e
c o a t e d w i t h s o d i u m c a r b o n a t e ( d r i e d a f t e r b e i n g c o a t e d w i t h 1M Na2C03). F i g u r e 1 i s a s c h e m a t i c d i a g r a m o f t h e e x p e r i m e n t a l apparatus.
The appara-
t u s c o n s i s t s o f a g a s g e n e r a t i o n s y s t e m and a c o l l e c t i n g s y s t e m f o r t h e
A T e f l o n t u b e E,
d i l u t e d hydrogen f l u o r i d e . o f t h e d i f f u s i o n tube,
whose r a d i u s was equal t o t h a t
was u s e d f o r t h e f u l l d e v e l o p m e n t o f t h e f l o w
p r o f i l e . The l e n g t h o f t h e t u b e was 1 6 0 cm (8). A t t h e d o w n s t r e a m end o f an a l k a l i impregnated f i l t e r was s e t i n a T e f l o n h o l d e r
t h e d i f f u s i o n tube,
t o c o l l e c t hydrogen f l u o r i d e t h a t passed t h r o u g h t h e d i f f u s i o n tube.
The
a l k a l i impregnated f i l t e r was prepared by s o a k i n g a c e l l u l o s e f i l t e r (No. 51-A,
Toyo R o s h i Co.,
s o l u t i o n o f ca.
O.lM,
4 cm i n e f f e c t i v e d i a m e t e r ) i n a s o d i u m c a r b o n a t e f o l l o w e d by a i r - d r y i n g a t 60°C.
I n t h i s experiment,
t h e t e m p e r a t u r e o f t h e gas f l o w i n g t h r o u g h t h e t u b e was m a i n t a i n e d a t
201rZ"C,
and t h e p r e s s u r e was k e p t a t 760 - 773 T o r r f o r l a m i n a r f l o w ,
and
760 -860 T o r r f o r t u r b u l e n t f l o w . A f t e r sampling,
t h e d i f f u s i o n t u b e was c u t i n t o s e v e r a l s e c t i o n s and each
p i e c e was washed w i t h 25 m l o f water.
The f l u o r i d e i n t h e a l k a l i impreg-
n a t e d f i l t e r was e x t r a c t e d w i t h 2 5 m l o f warm w a t e r .
, T h e amount o f
f l u o r i d e i n t h e s e samples was d e t e r m i n e d b y a f l u o r i d e i o n s e l e c t i v e e l e c trode.
I
-v
-
I
F i g u r e 1. Schematic diagram o f t h e e x p e r i m e n t a l apparatus. ( C ) c o m p r e s s o r , ( V ) v a l v e , ( G ) r e g u l a t o r , ( A ) a i r f i l t e r , (P) P e r m e a c a l P e r m e a t o r , ( R ) f l o w m e t e r , ( M ) gas m i x e r , ( E ) T e f l o n Tube, (D) d i f f u s i o n tube, ( F ) a l k a l i impregnated f i l t e r , ( T ) thermometer.
RESULTS AND
DISCUSSION
F u l l y developed f l o w The d i s t r i b u t i o n o f d i f f u s i o n a l d e p o s i t i o n o f hydrogen f l u o r i d e i n t h e d i f f u s i o n t u b e was measured by s e t t i n g t h e e n t r a n c e r e g i o n a t i t s upstream. The e x p e r i m e n t a l r e s u l t s f o r P o i s e u i l l e f l o w and f u l l y developed t u r b u l e n t f l o w a r e i l l u s t r a t e d i n F i g u r e s 2 and 3, r e s p e c t i v e l y .
The c o - o r d i n a t e s
1
I
I
1
200
3oa
0.1
> 0
0
v
1 0
0.01
R e = 707 R e = 283
0.001
100
x /d
F i g u r E 2. D i f f u s i o n a l t r a n s p o r t o f h y d r o g e n f l u o r i d e i n a d i f f u s i o n t u b e f o r P o i s e u i l l e f l o w . Re, R e y n o l d s n u m b e r ; C(x), mean c o n c . o f a e r o s o l a t a d i s t a n c e x; Co, c o n c . a t e n t r a n c e ; x 1 d i s t a n c e f r o m t h e t u b e e n t r a n c e ; d, i n s i d e : d = 1.0 cm. d i a m e t e r o f tube. o : d = 0.4 cm; + : d = 0.6 cm; D i f f u s j o n a l t r a n s p o r t o f hydrogen f l u o r i d e i n a d i f f u s i o n tube i n f u l l y developed t u r b u l e n t flow. F i g u r e 3. Re, R e y n o l d s number; C(x), mean conc. o f a e r o s o l a t a d i s t a n c e x; Co. conc. a t e n t r a n c e ; x, d i s t a n c e f r o m t u b e e n t r a n c e ; d, i n s i d e d i a m e t e r o f t u b e . o : d = 0.4 cm.
85
a r e i n d i c a t e d i n d i m e n s i o n l e s s v a l u e s as t h e r a t i o o f t h e d i s t a n c e f r o m t h e t u b e i n l e t t o t h e d i a m e t e r o f t h e t u b e and t h e f r a c t i o n o f h y d r o g e n f l u o r i d e a r r i v i n g a t t h e cross-section.
4 r e p l i c a t e s o f t h e experiment,
a n d t h e r e p r o d u c i b i l i t y w a s v e r y good.
When t h e R e y n o l d s number was l e s s t h a n 1.d.
Each p l o t i s t h e mean v a l u e f o r 2-
2,000, t u b e s w i t h 0.4.
w e r e u s e d , w h i l e b e y o n d 2,000. t h o s e w i t h 0.4 cm 1.d.
e v e r y R e y n o l d s number,
0.6 o r 1.0 cm
w e r e used.
For
t h e a r r i v i n g f r a c t i o n o f hydrogen f l u o r i d e is l i n e a r
on a s e m i - l o g a r i t h m i c g r a p h w i t h t h e e x c e p t i o n o f a s h o r t r e g i o n n e a r t h e e n t r a n c e o f t h e tube.
I f D i s a s s u m e d t o b e 0.26 c m 2 / s , a n d t h e d i s t a n c e
f r o m t h e t u b e e n t r a n c e i s expressed b y t h e d i m e n s i o n l e s s v a l u e (x/d)/Pe. a l l e x p e r i m e n t a l r e s u l t s i n P o i s e u i l l e f l o w (Re<2.300) a r e i n a g r e e m e n t w i t h t h e t h e o r e t i c a l v a l u e s o f G o r m l e y and Kennedy ( 1 ) as i l l u s t r a t e d i n F i g u r e 4.
S i n c e t h e v a l u e o f 0.26 c m 2 / s i s n e a r l y e q u a l t o t h e m o l e c u l a r
d i f f u s i o n c o e f f i c i e n t o f w a t e r i n a i r (0.25 c r n 2 / s a t 2O"C, 7 6 0 T o r r ) . a n d t h e m o l e c u l a r w e i g h t of hydrogen f l u o r i d e i s n e a r l y equal t o t h a t o f water, i t seems r e a s o n a b l e t o c o n s i d e r t h a t t h e v a l u e o f
0.26 c m 2 / s i s t h e
m o l e c u l a r d i f f u s i o n c o e f f i c i e n t o f hydrogen f l u o r i d e i n a i r .
Comparison o f e x p e r i m e n t a l r e s u l t s i n P o i s e u i l l e f l o w w i t h theoF i g u r e 4. r e t i c a l v a l u e s s o l e d by-Gormley and Kennedy. D, B r o w n i a n d i f f u s i o n c o e f f i c i e n t (=0.26 cm /s); C(x), m e a n c o n c . o f a e r o s o l a t a d i s t a n c e X ; Co, c o n c . a t e n t r a n c e ; X, d i s t a n c e from t u b e e n t r a n c e : d, i n s i d e d i a m e t e r o f t u b e : Pe, d i f f u s i v e P e c l e t number (=Vd/D)
5
86
F i g u r e 5. Diffusion coefficient o f hydrogen f l u o r i d e o f f u l l y developed f l o w i n d i f f u s i o n tube. Re, R e y n o l d s numb r; D, B r o w n i a n d i f f u s i o n c o e f f i c i e n t o f a e r s o l p a r t i c l e s . D = 0.26 cm / s ( 2 8 3 < R e < 2 , 3 0 0 ) ; D = 2.04 l o g ( R e 1 1 5 0 0 ) cm / s (2.300iRe<10,000)
5
9
I f t h e f l o w i n t h e t u b e e x c e e d s t h e c r i t i c a l R e y n o l d s number (ca. 2,300). diffusional
transport onto the inside wall
laminar conditions.
becomes m o r e r a p i d t h a n u n d e r
T h i s phenomenon c a n b e e x p l a i n e d b y a d d i n g t h e e f f e c t s
o f eddy d i f f u s i o n t o m o l e c u l a r d i f f u s i o n .
The r e s u l t s o f t h e c a l c u l a t i o n
o f D f o r t u r b u l e n t f l o w a r e shown i n F i g u r e 5 a c c o r d i n g t o R e y n o l d s numbers.
The e x p e r i m e n t a l f o r m u l a f o r D w h i c h c a n be a p p l i e d t o R e y n o l d s
n u m b e r s f r o m 2,300 t o 10,000 i s
D
=
Re
2.04 l o g __ 1500
Therefore,
cm2/s
(4)
s u b s t i t u t i n g F o r m u l a ( 4 ) i n t o t h e f i r s t t e r m o f S o l u t i o n (3).
the distribution o f diffusional
transport for turbulent flow
can be
e x p r e s s e d as f o l l o w s : -
c(x) =
0.819[
-29.8
CO
vd
log Re 1500
3
(5)
(2,300~Re~10,000) Developing f l o w T h e t u b e E.
i l l u s t r a t e d i n F i g u r e 1, w a s r e p l a c e d w i t h t h e a p p a r a t u s
i l l u s t r a t e d i n F i g u r e 6. T e f l o n f i l t e r (PF-1,
The c y l i n d e r was made o f a c r y l i c r e s i n ,
and a
T o y o R o s h i Co.) w a s u s e d f o r a r r a n g i n g s t r e a m l i n e s .
T h e i n s i d e d i a m e t e r w a s 4 cm,
and t h e d i s t a n c e from t h e f i l t e r t o t h e
e n t r a n c e o f t h e d i f f u s i o n t u b e was a b o u t 5 cm.
F i g u r e 7 shows t h e d i f f u s i o n
c o e f f i c i e n t s o f hydrogen f l u o r i d e c o r r e s p o n d i n g t o Reynolds numbers i n l a m i n a r and t u r b u l e n t f l o w .
There were no s i g n i f i c a n t d i f f e r e n c e s between
t h e s e e x p e r i m e n t a l r e s u l t s and t h o s e o f e x p e r i m e n t s i n v o l v i n g f u l l y d e v e l oped f l o w .
I -+
a
t
a
out
F i g u r e 6. Apparatus f o r developing f l o w i n t h e entrance r e g i o n o f d i f f u ( F ) T e f l o n f i l t e r , (D) d i f f u s i o n tube, s c a l e u n i t i n m i l l i s i o n tube. meters.
W
1.0 0
0.5 -
OL 100
1
I
too0
Re
. . . .I loo00
F i g u r e 7. D i f f u s i o n c o e f f i c i e n t o f hydrogen f l u o r i d e o f d e v e l o p i n g f l o w i n tube. D i s t a n c e f r o m T e f l o n f i l t e r and e n t r a n c e o f d i f f u s i o n t u b e i s a p p r o x i m a t e l y 5 cm. Re, R e y n o l d s n u y e r ; D, B r o w n i a n d i f f u s i o n c o e f f i c i e n t o f aerosol particles. D = 0.26 cm / s ( 2 5 0 < R e < 2 , 3 0 0 ) , D = 2.04 l o g ( R e / 1 5 0 0 ) c m / s ( 2,300 < Re< 10.000)
88
Q and Re).
R e l a t i o n b e t w e e n s a m p l i n g e f f i c i e n c y and p a r a m e t e r s ( x .
F i g u r e 8 shows t h e r e l a t i o n b e t w e e n s a m p l i n g e f f i c i e n c i e s (90, 95 and 99%) a n d
f l o w r a t e Q a n d R e y n o l d s n u m b e r Re.
d i s t a n c e x,
i s constant i n the laminar condition,
t h e s a m p l i n g e f f i c i e n c y i s dependent
on t h e t u b e l e n g t h r e g a r d l e s s o f t h e r a d i u s o f t h e t u b e . i s constant i n turbulent flow,
If t h e f l o w r a t e If the flow rate
t h e h i g h e r t h e Reynolds number ( t h e s m a l l e r
t h e tube diameter) t h e higher t h e sampling e f f i c i e n c y . From t h e s e r e s u l t s ,
i t a p p e a r s t h a t h y d r o g e n f l u o r i d e c a n be e f f e c t i v e l y
c o l l e c t e d b y a d i f f u s i o n t u b e s e v e r a l deca c e n t i m e t e r s l o n g and w i t h a d i a m e t e r f r o m 0.4 t o 1 cm.
0 =90%
’
95%
99%
150r
150 -
‘7
5
” 100 100 -
50
0
~
50 -
t
OC
I
0
,
,
.
,
I
.
.
.
.
/
20
10
30 CLhinI
Q
F i g u r e 8. R e l a t i o n between s a m p l i n g e f f i c i e n c y o f d i f f u s i o n tube, d i s t a n c e , f l o w r a t e , a n d R e y n o l d s n u m b e r . Re, R e y n o l d s n u m b e r ; Q. f l o w r a t e ; x, d i s t a n c e f r o m tube entrance.
REFERENCES Kennedy M (1949) P r o c Roy I r i s h Acad 52A:163-169
1.
G o r m l e y PG,
2.
M o n t g o m e r y TL,
C o r n M (1970) J A e r o s o l S c i 1:185-213
3.
F r i e d l a n d e r SK,
4.
D a v i e s CN (1966) A e r o s o l S c i e n c e . A c a d e m i c P r e s s ,
5.
B e a l SK (1968) 765
J o h n s t o n e HF (1957) I n d Engng Chem 49:1151 New York,
p 436
W e s t i n g h o u s e E l e c t r i c C o r p o r a t i o n P u b l i c a t i o n WAPD-TM-
A g a r w a l JK (1974) J A e r o s o l S c i 5:145-155
6.
L i u BYH.
7.
O k i t a T, S u g a i R, K a n e d a K ( 1 9 7 0 ) J J a p a n SOC A i r P o l l 5 : 8 8
8.
S c h l i c h t i n g H (1968) Boundary-Layer
Theory.
McGraw-Hill.
pp
282 -
561
H. Tsunoda and M.-H. Yu (Editors)
91
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 91-97 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
FLUORIDE TRANSPORT I N TERRESTRIAL ECOSYSTEMS AROUND INDUSTRIAL AREAS
FRANK M U R R A Y ~ Department of B i o l o g i c a l Sciences, Wales, A u s t r a l i a
U n i v e r s i t y o f Newcastle,
New S o u t h
School o f E n v i r o n m e n t a l and L i f e Sciences, Murdoch 'Present address: U n i v e r s i t y , Murdoch. W e s t e r n A u s t r a l i a 6150, A u s t r a l i a ABSTRACT
A E u c a l y p t u s - d o m i n a t e d f o r e s t e c o s y s t e m and an A v i c e n n i a m a r i n a - d o m i n a t e d m a n g r o v e f o r e s t e c o s y s t e m a r o u n d f l u o r i d e e m i s s i o n s o u r c e s i n New S o u t h Wales.
A u s t r a l i a , have been s u b j e c t t o c o n t i n u o u s f l u o r i d e i n p u t s i n c e
a b o u t 1970.
S t u d i e s o f t h e e f f e c t s o f f l u o r i d e e m i s s i o n s on t h e e c o l o q y o f
t h e s e e c o s y s t e m s h a v e shown t h a t t r e e s b e 1 i e v e d t o be f l u o r i d e - s e n s i t i v e demonstrated changes i n metabolism,
and f l u o r i d e - s e n s i t i v e
t r e e s and s h r u b s
i n areas w i t h h i g h f l u o r i d e i n p u t died.
F l u o r i d e concentrations increased
i n a number o f ecosystem components.
Important t r a n s f e r processes o f
t h r o u g h f a l l and s t e m f l o w , w e r e r e s p o n s i b l e f o r s i g n i f i c a n t f l u o r i d e f l u x e s . T h r e e zones c o u l d be d e f i n e d i n a r e a s s u b j e c t t o h i g h f l u o r i d e i n p u t and are characterized by the following:
1.
M a j o r e f f e c t s r e s u l t i n g i n v i s i b l e i n j u r y and l o s s o f s e n s i t i v e e x p o s e d s p e c i e s f r o m t h e ecosystem.
2.
Subtle
e f f e c t s d e m o n s t r a t e d b y changes i n p l a n t m e t a b o l i s m w i t h p o t e n -
t i a l e f f e c t s on g r o w t h , t i v e species.
species composition,
3.
Innocuous
r e p r o d u c t i o n and d i s e a s e r e s i s t a n c e o f s e n s i -
A t an e c o s y s t e m l e v e l , t h e r e may be l o n g - t e r m changes i n diversity,
b i o m a s s and n u t r i e n t c y c l i n g .
e f f e c t s c h a r a c t e r i z e d by t h e ecosystem a c t i n g as a s i n k f o r
f 1u o r i d e . INTRODUCTION F l u o r i d e i s w i d e l y d i s t r i b u t e d i n t h e n a t u r a l environment,
ranking 13th
among t h e e l e m e n t s i n o r d e r o f a b u n d a n c e i n t h e e a r t h ' s c r u s t . i n rocks,
s o i l s , groundwater,
surface waters,
air,
It occurs
p l a n t s and a n i m a l s .
F l u o r i d e i s a l s o an i m p o r t a n t i n d u s t r i a l e m i s s i o n w h i c h i s r e l e a s e d when fluoride-containing
m a t e r i a l s a r e heated t o h i g h temperatures.
i n d u s t r i a l processes involved are c o a l - f i r e d power stations, smelters, works,
steel plants,
phosphate f e r t i l i z e r works,
brick.
The m a i n aluminum
t i l e and g l a s s
o i l r e f i n e r i e s and some o r e - r e f i n i n g and c h e m i c a l p l a n t s .
92 E f f e c t s on P l a n t s F l u o r i d e i s one o f t h e m o s t p h y t o t o x i c o f t h e common a i r p o l l u t a n t s .
In
t e r m s o f i t s e f f e c t on p l a n t s i t i s r e g a r d e d a s t h e s e c o n d m o s t i m p o r t a n t a i r p o l l u t a n t i n E u r o p e ( 1 ) a n d t h e f o u r t h i n t h e U S A (2).
I t may c a u s e
i n j u r y a t c o n c e n t r a t i o n s i n a i r a b o u t 100 t i m e s l o w e r t h a n ozone,
sulphur
I t i s a l s o a c c u m u l a t i v e i n p l a n t s and may be
d i o x i d e o r n i t r o g e n oxides.
p a s s e d on t o a n i m a l s w h i c h consume f l u o r i d e - c o n t a m i n a t e d
vegetation.
For
t h e s e r e a s o n s f l u o r i d e h a s t h e c a p a c i t y t o h a v e l o n g - t e r m e f f e c t s o n ecosystems. The e f f e c t s o f f l u o r i d e o n f o r e s t e c o s y s t e m s a r e d e t e r m i n e d by:
(a)
f a c t o r s r e l a t i n g t o f l u o r i d e e x p o s u r e such as t h e c h e m i c a l and p h y s i c a l form o f the fluoride,
t h e concentration,
and o t h e r c l i m a t i c c o n d i t i o n s :
d u r a t i o n o f exoosure.
windspeed
and ( b ) f a c t o r s r e l a t i n g t o t h e r e s p o n s e o f
t h e e c o s y s t e m s u c h as t h e r e s i s t a n c e o f t h e e c o s y s t e m c o m p o n e n t s o r p r o cesses,
d u r i n g t h e p e r i o d o f exposure.
A l t h o u g h t h e r e i s a s u b s t a n t i a l amount o f i n f o r m a t i o n r e l a t i n g t o t h e responses of
agricultural
plants t o fluoride,
a b o u t t h e responses o f f o r e s t e d ecosystems. the r a t e o f uptake of
r e l a t i v e l y l i t t l e i s known I n a d d i t i o n some k n o w l e d g e o f
f l u o r i d e by ecosystems i s r e q u i r e d i n order t o
i m p r o v e models o f f l u o r i d e d i s p e r s i o n around e m i s s i o n sources.
The accu-
r a c y o f p r e d i c t i o n s r e s u l t i n g f r o m t h e s e models depends i n p a r t on t h e a s s u m p t i o n s t h e y make a b o u t r a t e s o f r e m o v a l o f a i r b o r n e f l u o r i d e b y veget a t i o n and s o i l ,
b u t l i t t l e f i e l d d a t a i s a v a i l a b l e f o r most ecosystems.
I n t h i s p a p e r some c h a r a c t e r i s t i c s o f t h e i n p u t o f f l u o r i d e t o e c o s y s t e m s a r e examined w i t h emphasis on t h e t r a n s p o r t o f f l u o r i d e w i t h i n forested ecosystems. C h a r a c t e r i s t i c s o f F l u o r i d e E n t r y t o F o r e s t e d Ecosystems F l u o r i d e e n t e r s f o r e s t e d e c o s y s t e m s a r o u n d e m i s s i o n s o u r c e s b y means o f a e r i a l d e p o s i t i o n o n t o v e g e t a t i o n and s o i l s u r f a c e s ,
o r by transport i n
s u r f a c e w a t e r o r g r o u n d w a t e r s f r o m o t h e r a r e a s w i t h h i g h f l u o r i d e concent r a t i o n s due t o n a t u r a l o r a n t h r o p o g e n i c c o n t a m i n a t i o n . F l u o r i d e d e p o s i t e d o n l e a v e s may be i n a g a s e o u s o r p a r t i c u l a t e f o r m , i n solution associated w i t h precipitation.
or
P a r t i c u l a t e f l u o r i d e s may
adhere t o t h e l e a f surface b u t they o n l y s l o w l y cross t h e c u t i c l e .
The
r a t e o f t r a n s c u t i c u l a r m i g r a t i o n i n c r e a s e s w i t h h u m i d i t y (3). Gaseous f l u o r i d e s may be a d s o r b e d o n t h e s u r f a c e o f t h e l e a f o r may e n t e r t h e l e a f through t h e stomata where t h e m a j o r i t y remains i n a f r e e i o n i c f o r m and i s t r a n s l o c a t e d t o p e r i p h e r a l a r e a s o f t h e l e a f i n t h e t r a n s p i r a t i o n s t r e a m (4).
Solutions o f fluoride i n rain,
mist,
f o g o r snow p r o b a b l y
c r o s s t h e c u t i c l e more q u i c k l y t h a n p a r t i c u l a t e s under most c o n d i t i o n s ,
but
93 appear u n l i k e l y t o g a i n e n t r y t o t h e l e a f t h r o u g h t h e stomata.
Fluorides
deposited on o t h e r p l a n t organs such as branches a r e l e s s l i k e l y t o g a i n e n t r y t o t h e p l a n t a l t h o u g h some u p t a k e o f g a s e o u s f l u o r i d e s b y l e n t i c e l s p r o b a b l y o c c u r s (4).
Organs w i t h l a r g e s u r f a c e area:volume r a t i o s have
g r e a t e r f l u o r i d e c o n c e n t r a t i o n s t h a n those w i t h s m a l l s u r f a c e area:volume ratios. F l u o r i d e I n p u t Processes I n o r d e r t o q u a n t i f y some o f t h e p r o c e s s e s b y w h i c h f l u o r i d e e n t e r s and i s t r a n s p o r t e d w i t h i n f o r e s t e d ecosystems,
t w o n a t u r a l f o r e s t e d ecosystems
were studied. F l u o r i d e e n t r y i n t o a E u c a l y p t u s f o r e s t n e a r an a l u m i n u m s m e l t e r a t K u r r i Kurri.
A u s t r a l i a was e s t i m a t e d u s i n g r a i n f a l l c o l l e c t o r s .
The s o u r c e was
e m i t t i n g a b o u t 200 t o n n e s o f g a s e o u s and p a r t i c u l a t e f l u o r i d e s p e r y e a r , l a r g e l y f r o m r o o f vents.
The r a i n f a l l c o l l e c t o r s w e r e n o t e v e n t - a c t u a t e d .
They m e a s u r e d c l e a r f a l l ( p r e c i p i t a t i o n g r a v i t a t i o n a l l y t r a n s f e r r e d t o t h e e a r t h ' s surface), canopy),
throughfall
( p r e c i p i t a t i o n t h a t has p a s s e d t h r o u g h a l e a f
a n d s t e m f l o w ( p r e c i p i t a t i o n d i v e r t e d down t h e sterns o f p l a n t s ) ,
b u t were a l s o c o n t i n u o u s l y exposed t o d r y d e p o s i t i o n . used t o measure each parameter, source,
F i v e c o l l e c t o r s were
a t d i s t a n c e s o f 0.5 a n d 9.9 k m f r o m t h e
f o r a p e r i o d o f 1037 d a y s (5).
The r e s u l t s show t h a t f l u o r i d e c o n c e n t r a t i o n s i n s t e m f l o w w e r e m o r e t h a n one and a h a l f t i m e s t h o s e i n t h r o u g h f a l l and a l m o s t f i v e t i m e s t h o s e i n c l e a r f a l l i n t h e p o l l u t e d a r e a s ( F i g u r e 1).
The f l u o r i d e c o n c e n t r a t i o n s i n
c l e a r f a l l , t h r o u g h f a l l and s t e m f l o w i n t h e p o l l u t e d a r e a w e r e a l m o s t 10, and 27 t i m e s t h e r e s p e c t i v e v a l u e s f o r t h e c o n t r o l area. i n p u t t o t h e ecosystem,
15
The f l u o r i d e
a l l o w i n g f o r t h e percentage t r e e cover a t t h e study
sites,
was c o n s i d e r a b l y g r e a t e r as t h r o u g h f a l l t h a n c l e a r f a l l i n b o t h
areas.
The n e t t h r o u g h f a l l ( t h r o u g h f a l l m i n u s c l e a r f a l l )
i n t h e p o l l u t e d ecosystem, other studies (6,7),
b u t o n l y 0.016 g / m 2 / y r
was 0.39 g / m 2 / y r
i n t h e c o n t r o l area.
In
estimates indicate t h a t stemflow f l u x represents less
t h a n 5% o f n u t r i e n t i n p u t i n o p e n f o r e s t s .
However, t h i s n u t r i e n t i n p u t
i s i m p o r t a n t because i t i s d e p o s i t e d i n a s m a l l a r e a around t h e base o f t h e tree. Fluoride i n Vegetation The a m o u n t o f f l u o r i d e i n v e g e t a t i o n c a n be e x p r e s s e d i n t e r m s o f concent r a t i o n s i n one o r g a n such as p l a n t leaves, o r i n a number o f organs, b u t
i n r e l a t i o n t o e c o s y s t e m s i t i s b e s t e x p r e s s e d a s t h e mass o f f l u o r i d e p e r u n i t area,
t h a t i s b y summing t h e s t a n d i n g c r o p o f f l u o r i d e ( t h e p r o d u c t o f
d r y mass p e r u n i t a r e a and f l u o r i d e c o n c e n t r a t i o n ) f o r a l l o f t h e l a r g e s t e c o s y s t e m components.
The f l u o r i d e s t a n d i n g c r o p i n u n d e r s t o r y and g r o u n d -
94
0.5km
-
DISTANCE FROM A SOURCE
~
--
~
O.Okm
FLUORIDE FLUX ( 0 avi2yr-’1
F i g u r e 1: forest.
Some f l u o r i d e t r a n s p o r t p r o c e s s e s a n d f l u x e s i n a E u c a l y p t u s
c o v e r s p e c i e s i n t h e E u c a l y p t u s f o r e s t s was e s t i m a t e d b y c l e a r i n g a l l biomass f r o m f o u r r e p l i c a t e d p l o t s f o r each o f t w o years,
and m e a s u r i n g d r y
w e i g h t s and f l u o r i d e c o n c e n t r a t i o n f o r each c o m p o n e n t f o r e a c h o f a b o u t 30 p l a n t species p r e s e n t i n each p l o t .
The t o t a l f l u o r i d e s t a n d i n g c r o p i n
u n d e r s t o r y a n d g r o u n d c o v e r s p e c i e s w a s f o u n d t o b e 10.2 mg F / m 2 a t 0.5 k m a n d 1.4 mg F / m 2 a t 9.9 k m ( T a b l e I ) . The s t a n d i n g c r o p o f f l u o r i d e i n p l a n t l i t t e r was m e a s u r e d q u a r t e r l y f o r t w o y e a r s a t each o f f i v e s i t e s . nents:
leaves,
b r a n c h e s ( > 5 mm),
components such as f l o w e r s , was d r i e d ,
L i t t e r was d i v i d e d i n t o d i f f e r e n t compot w i g s (< 5 mm),
bark,
and m i s c e l l a n e o u s
f r u i t and p a r t l y decomposed p l a n t l i t t e r .
w e i g h e d and a n a l y z e d f o r f l u o r i d e s e p a r a t e l y .
Each
The r e s u l t s show
t h a t t h e t o t a l f l u o r i d e s t a n d i n g c r o p i n p l a n t l i t t e r w a s 1 5 7 mg F / m 2 a t
0.5 km a n d 1 3 mg F / m 2 a t 9.9 kni. As a c o m p a r i s o n ,
a s t u d y o f a mangrove f o r e s t g r o w i n g n e a r a hydrogen
f l u o r i d e m a n u f a c t u r i n g f a c i l i t y on Kooragang I s l a n d ,
N.S.W.,
Australia,
and
a s i m i l a r m a n g r o v e f o r e s t i n a r e l a t i v e l y u n p o l l u t e d a r e a ( G o s f o r d ) showed t h a t f l u o r i d e i n p u t t o t h e Kooragang I s l a n d ecosystem as b u l k d e p o s i t i o n o f
95 TABLE I FLUORIDE FLUX AND STANDING CROP I N FORESTED ECOSYSTEMS Eucalyptus f o r e s t ecosystem
Mangrove f o r e s t ecosystem
1.
D i s t a n c e f r o m s o u r c e (km)
0.5
9.9
0.5
2.
Bulk deposition o f f l u o r i d e (g/m2/yr)
0.93
0.21
0.31
0.04
3.
Fluori e standing crop (mgF/m ) i n 1.4
NA
NA
NA
9
a ) u n d e r s t o r y and groundcover species
10.2
b) p l a n t l i t t e r c ) t o t a l above g r o u n d standing crop 4.
157
13
NA
ND
ND
270
4.0
176
1.4
F l u o r i d e t r a n s f e r (mgF/m2/yr)
54
a ) 1 it t e r f a l l NA
50
-
ND - n o t d e t e r m i n e d
samples n o t a v a i l a b l e
f l u o r i d e was 0.31 g / m 2 / y r
2.0
and t h i s v a r i e d w i t h t h e d i s t a n c e and d i r e c t i o n
o f e a c h s i t e i n r e l a t i o n t o f l u o r i d e e m i s s i o n s o u r c e s (8).
Most o f t h e
i n p u t a t Kooragang I s l a n d was gaseous f l u o r i d e , b u t a s m a l l p a r t i c u l a t e f l u o r i d e i n p u t occurred.
Bulk deposition o f f l u o r i d e a t a r e l a t i v e l y
u n c o n t a m i n a t e d s i t e a t F u l l e r t o n Cove was a b o u t 0.04 g/m2/year.
virtually
a l l gaseous f l u o r i d e . The a b o v e - g r o u n d and 21.7 d r y t / h a ,
b i o m a s s a t b o t h s i t e s w a s p r a c t i c a l l y i d e n t i c a l (21.8 respectively) b u t t h e f l u o r i d e concentrations i n leaves
and branches were a l m o s t t w o o r d e r s o f m a g n i t u d e g r e a t e r a t Kooragang I s l a n d than a t Gosford.
Consequently t h e above-ground
fluoride i n the
s t a n d i n g c r o p w a s m u c h g r e a t e r a t K o o r a g a n g I s l a n d (2.7 k g F / h a ) t h a n a t G o s f o r d (0.04 k g F / h a ) . I n t h e mangrove f o r e s t ,
a comparison o f p l a n t l i t t e r f a l l .
f l u o r i d e con-
c e n t r a t i o n and f l u o r i d e d e p o s i t i o n i n l i t t e r f a l l a t t h e K o o r a g a n g I s l a n d s i t e and a t F u l l e r t o n Cove showed t h a t a l t h o u g h l i t t e r f a l l r a t e s w e r e s i m i l a r (562 and 514 g/m2/yr,
r e s p e c t i v e l y ) t h e r a t e s o f f l u o r i d e deposi-
t i o n i n l i t t e r f a l l w e r e a l s o t w o o r d e r s o f m a g n i t u d e d i f f e r e n t (176 and 1.42 mg F/m2/yr,
r e s p e c t i v e l y ) due t o t h e d i f f e r e n c e s i n f l u o r i d e concen-
t r a t i o n i n a l l components o f p l a n t l i t t e r a t t h e t w o s i t e s .
These l i t t e r -
f a l l r a t e s a r e s i m i l a r t o o t h e r d a t a r e p o r t e d f o r mangrove l i t t e r p r o d u c t i o n a t t h e same l a t i t u d e (8).
96 As a consequence o f f l u o r i d e i n p u t by l i t t e r f a l l ,
deposition,
buffer-extractable
w e t d e p o s i t i o n and d r y
f l u o r i d e concentrations
i n s o i l s a t Koora-
gang I s l a n d were v a r i a b l e , b u t were g e n e r a l l y w i t h i n t h e range 4-15 u g F/g.
A t F u l l e r t o n Cove and G o s f o r d t h e b u f f e r - e x t r a c t a b l e f l u o r i d e concent r a t i o n s i n s o i l were r e l a t i v e l y c o n s t a n t w i t h mean f l u o r i d e c o n c e n t r a t i o n s o f 2.0 and 1.9 u g F / g . r e s p e c t i v e l y . S o i 1s
F l u o r i d e i s r e l e a s e d f r o m p l a n t l i t t e r by f i r e and i t has been e s t i m a t e d t h a t about 68% i s e x p o r t e d f r o m t h e ecosystem as gaseous and p a r t i c u l a t e fluoride,
w i t h 32% r e m a i n i n g as ash.
High s o l u b l e f l u o r i d e c o n c e n t r a t i o n s
o c c u r i n t h e ash and have been found t o measure up t o about 1600 u g / g a f t e r a b u s h f i r e around an aluminum s m e l t e r ( 9 )
These c o n c e n t r a t i o n s c o u l d a l s o
impede g e r m i n a t i o n and s e e d l i n g g r o w t h f o r a p e r i o d o f t i m e b u t r a i n f a l l w i l l u l t i m a t e l y leach t h e water-soluble
a c t as a s i n k f o r f l u o r i d e .
fluoride i n t o the soil.
Most s o i l s
They have a h i g h c a p a c i t y t o b i n d f l u o r i d e i n
an i n s o l u b l e and u n a v a i l a b l e form, and t h e f a c t o r s r e s p o n s i b l e f o r t h i s a r e i r o n , a l u m i n u m , c a l c i u m and o t h e r a g e n t s ( 1 0 , l l ) .
Some s a n d y and s a l i n e
s o i l s l a c k t h e s e f a c t o r s and may n o t have t h i s c a p a c i t y ,
and consequently
t h e r e may be some p o t e n t i a l i n s o i l s w i t h t h e s e c h a r a c t e r i s t i c s f o r f l u o r i d e t o be t a k e n up by r o o t s ,
o r leached t o groundwaters.
DISCUSSION I n t h e a s s e s s m e n t o f t h e e f f e c t s o f f l u o r i d e s on f o r e s t e d a r e a s a r o u n d e m i s s i o n sources i t i s n e c e s s a r y t o c o n s i d e r t h a t ecosystems c o n s i s t o f o r g a n i s m s w h i c h a r e o r g a n i z e d i n s t r u c t u r a l and f u n c t i o n a l c o m p o n e n t s u n i t e d by t r a n s f e r p r o c e s s e s (12).
Consequently,
evaluations o f the
e f f e c t s o f f l u o r i d e on ecosystem processes d i f f e r c o n s i d e r a b l y f r o m evaluat i o n s o f t h e e f f e c t s on i n d i v i d u a l species.
even t h e dominant species,
as
t h e s e n s i t i v e p o i n t s w i t h i n t h e e c o s y s t e m s may be f u n c t i o n a l p r o c e s s e s . N a t u r a l f o r e s t ecosystems a r e u s u a l l y complex and i n o r d e r t o understand t h e e f f e c t s o f f l u o r i d e on t h e s e ecosystems,
t h e most s e n s i t i v e organisms
and ecosystem processes need t o be i d e n t i f i e d and t h e c h a r a c t e r i s t i c s o f t h e i r r e a c t i o n s need t o be u n d e r s t o o d .
I n f o r e s t e d ecosystems t h e most
s e n s i t i v e organisms t o t h e e f f e c t s o f a i r p o l l u t a n t s a r e t h e producers and decomposers (12).
w h i c h a r e a l s o t h e t w o most i m p o r t a n t groups o f organisms
w i t h i n f o r e s t e d e c o s y s t e m s , as t h e y c o n t r o l t h e p r o c e s s e s o f u p t a k e and t r a n s f e r o f energy and m i n e r a l s which may be r e a d i l y d i s t u r b e d (13).
It i s
b y means o f i m p a c t s a t t h e s e l e v e l s t h a t p o l l u t a n t s s u c h a s f l u o r i d e may a d v e r s e l y a f f e c t ecosystems i n a s u b t l e manner i n i t i a l l y , more obvious i m p a c t s w i t h t i m e i f t h e s t r e s s continues.
developing i n t o
97 E c o s y s t e m s a r o u n d m a j o r e m i s s i o n s o u r c e s may b e r e c o g n i z e d a s c a p a b l e of b e i n g c l a s s i f i e d i n t o d i s t i n c t zones.
The a r e a n e a r e s t t o t h e e m i s s i o n
s o u r c e may be s u b j e c t t o m a j o r i m p a c t s a s s o c i a t e d w i t h s i g n i f i c a n t e c o l o g i c a l d e g r a d a t i o n o f f u n c t i o n a l and a e s t h e t i c a t t r i b u t e s .
S e n s i t i v e species
a r e r e m o v e d and v i s i b l e i n j u r y t o f o l i a g e i s o b v i o u s i n t h i s zone.
Further
f r o m t h e e m i s s i o n s o u r c e o r i n l e s s exposed l o c a t i o n s i s f o u n d a zone i n w h i c h s u b t l e e f f e c t s o f f l u o r i d e may be r e c o g n i z e d .
I n t h i s zone t h e r e a r e
changes o r s u b t l e e f f e c t s d e m o n s t r a t e d by a l t e r a t i o n s i n p l a n t m e t a b o l i s m w i t h p o t e n t i a l e f f e c t s on growth, s e n s i t i v e species.
r e p r o d u c t i o n and d i s e a s e r e s i s t a n c e o f
A t an ecosystem l e v e l ,
t h e r e may b e l o n g - t e r m
changes
i n s p e c i e s c o m p o s i t i o n , d i v e r s i t y , b i o m a s s and n u t r i e n t c y c l i n g . Beyond t h i s zone i s a zone o f innocuous e f f e c t s c h a r a c t e r i z e d b y t h e ecosystem a c t i n g as a s i n k f o r f l u o r i d e .
The f l u o r i d e c o n c e n t r a t i o n s i n some e c o s y s -
tem components, and f l u o r i d e f l u x i n c y c l i n g processes a r e i n c r e a s e d i n t h i s zone. ACKNOWLEDGEMENTS The a s s i s t a n c e o f A l c a n A u s t r a l i a Ltd, A u s t r a l i a n F l u o r i n e C h e m i c a l s , R o s l y n Avery,
John Clancy.
Rosemary H u t c h i s o n ,
M a r i n a McGlinn,
Elizabeth
R a t c l i f f e and D a v i d R o s h i e r i s g r a t e f u l l y a c k n o w l e d g e d . REFERENCES 1. K n a b e W ( 1 9 7 8 ) I n : O l s c h o w y G ( e d ) U n w e l t s c h u t z i n d. B u n d e s r e p u b l i k D e u t s c h l a n d . P a u l Parey, Hamburg, p p 697-709
2. Heck W W (1982)
I n : U n s w o r t h MH, O r m r o d DP ( e d s ) E f f e c t s o f Gaseous A i r P o l l u t i o n i n A g r i c u l t u r e and H o r t i c u l t u r e . B u t t e r w o r t h S c i e n t i f i c , London, pp 411-435
3.
In: Proc I n t Clean A i r M c C u n e DC. S i l b e r m a n DH, W e i n s t e i n LH ( 1 9 7 7 ) C o n g r 4 t h . Tokyo, J a p a n e s e U n i o n o f A i r P o l l u t i o n P r e v e n t i o n A s s o c i a t i o n s , Tokyo, p p 116-118
4.
W e i n s t e i n LH (1977) J Occup Med 19:49-78
5.
M u r r a y F (1985a) P e r s p e c t i v e s E n v i r o n B o t 1 ( i n p r e s s )
6. O v i n g t o n JD (1954) F o r e s t r y 27:41-53 7. G u t h r i e HB,
A t t i w i l l PM.
L e u n i g R (1978) A u s t J B o t 26:189-201
8. M u r r a y F (1985b) J A p p l E c o l 22:277-285 9.
M u r r a y F (1981) I n : Webb KA. S m i t h AJ ( e d s ) S e v e n t h I n t e r n a t i o n a l C l e a n A i r C o n f e r e n c e : Ann A r b o r S c i e n c e . Ann A r b o r , Mich.. p p 451-462
10. M u r r a y F ( 1 9 8 3 ) Water,
Air,
S o i l P o l l u t 20:361-367
11. M u r r a y F ( 1 9 8 4 ) E n v i r o n P o l l u t (Ser.
B) 7:83-95
12. S m i t h WH ( 1 9 8 4 ) F o r e s t E c o l Manage 9:193-219 13. E h r l i c h PR.
Mooney HA ( 1 9 8 3 ) B i o S c i e n c e 33:248-254
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 99-106 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
99
PROSPECTIVE SURVEY ON FLUORIDE CONTENTS OF R I C E
S H I R O SAKURAI, KAZUYOSHI I T A I , AND HUM10 TSUNODA Department o f Hygiene and P u b l i c Health, U n i v e r s i t y , Morioka, I w a t e 020 Japan
School o f Medicine,
I w a t e Medical
ABSTRACT
A p r o s p e c t i v e 17-year s u r v e y was conducted on changes i n f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e f r o m an a r e a n e a r an a l u m i n u m r e f i n e r y p l a n t .
Rice
samples were a l s o taken f o r a n a l y s i s from areas around v a r i o u s types o f i n d u s t r i e s known t o e m i t f l u o r i d e , and t h e r e s u l t s were compared w i t h t h o s e o b t a i n e d 10 y e a r s before.
It was shown t h a t u n p o l i s h e d r i c e r e a d i l y accu-
m u l a t e d a i r b o r n e f l u o r i d e and t h a t i t c o u l d s e r v e as an i n d i c a t o r f o r longt e r m and l o w l e v e l exposure t o a t m o s p h e r i c f l u o r i d e .
The f l u o r i d e c o n t e n t s
o f unpolished r i c e grown i n areas around ceramics works were g e n e r a l l y h i g h e r t h a n t h o s e found i n samples c o l l e c t e d f r o m o t h e r i n d u s t r i a l areas. A l t h o u g h areas p o l l u t e d by a i r b o r n e f l u o r i d e e m i t t e d f r o m c e r a m i c s works are l i m i t e d t o those surrounding t h e plants, t e r e d a c r o s s Japan.
It i s possible,
amounts o f f l u o r i d e - c o n t a m i n a t e d problem.
many c e r a m i c s works l i e s c a t -
therefore,
t h a t i n g e s t i o n o f excessive
r i c e c o u l d become a s e r i o u s p u b l i c h e a l t h
I t i s s t r e s s e d t h a t c o n t r o l m e a s u r e s s h o u l d be t a k e n t o c u r b
f l u o r i d e e m i s s i o n a t c e r a m i c s works.
INTRODUCTION A c c u m u l a t i o n o f h i g h l e v e l s o f f l u o r i d e i n v e g e t a t i o n i s commonly observed i n areas p o l l u t e d by a i r b o r n e f l u o r i d e .
The most i m p o r t a n t prob-
lem i n v o l v e d i n human h e a l t h i s t h e i n t a k e o f e x c e s s i v e amounts o f f l u o r i d e through i n g e s t i o n o f foods contaminated by t h e p o l l u t a n t . t h e main s t a p l e f o r t h e Japanese,
Since r i c e is
ingestion o f fluoride-contaminated
rice
c o u l d a c c o u n t f o r a s i g n i f i c a n t p a r t o f t o t a l f l u o r i d e i n t a k e s i n Japan
(1).
The p r e s e n t s t u d y was c a r r i e d o u t i n an a t t e m p t t o d e t e r m i n e
fluo-
r i d e c o n t a m i n a t i o n i n r i c e caused by s e v e r a l t y p e s o f i n d u s t r i a l a c t i v i ties.
C u r r e n t c o n c e r n w i t h a i r b o r n e f l u o r i d e p o l l u t i o n was a l s o
considered.
100 MATERIALS AND METHODS A p r o s p e c t i v e 17-year
s u r v e y was c o n d u c t e d t o s t u d y t h e f l u o r i d e c o n t e n t
i n u n p o l i s h e d r i c e t a k e n f r o m an a r e a n e a r an aluminum r e f i n e r y p l a n t . T o t a l p r o d u c t i o n o f a l u m i n u m b y t h e p l a n t was 40.000 t o n s p e r y e a r . R i c e s a m p l e s w e r e c o l l e c t e d a n n u a l l y b e g i n n i n g i n 1967. R i c e s a m p l e s w e r e a l s o t a k e n f r o m a r e a s a r o u n d 1 8 i n d u s t r i a l p l a n t s known t o e m i t f l u o r i d e i n t o t h e atmosphere. r e f i n e r y p l a n t s (A-F), smelters,
They c o n s i s t e d o f s i x aluminum
f o u r p h o s p h a t e f e r t i l i z e r p l a n t s (A-D),
three steel
and f i v e c e r a m i c s w o r k s i n c l u d i n g t h o s e p r o d u c i n g c h i n a ,
tile,
r o o f t i l e and g l a s s f i b e r . D e t e r m i n a t i o n o f f l u o r i d e f o r t h e r i c e s a m p l e s t a k e n p r i o r t o 1984 was m e t h o d (2).
p e r f o r m e d a c c o r d i n g t o t h e A.O.A.C.
F l u o r i d e ions i n s o l u t i o n
w e r e t h e n d e t e r m i n e d b y t h e i o n - s e l e c t i v e e l e c t r o d e method.
Samples t a k e n
d u r i n g 1 9 8 4 w e r e d e t e r m i n e d b y I t a i ' s m e t h o d (3). RESULTS F i g u r e 1 shows t h e l o n g - t e r m
changes i n f l u o r i d e c o n t e n t s o f u n p o l i s h e d
r i c e f r o m a n a r e a n e a r a n a l u m i n u m r e f i n e r y p l a n t d e s i g n a t e d a s P l a n t B. I n 1968,
when v i s i b l e i n j u r y o f v e g e t a t i o n was v e r y s e v e r e t h e e i g h t - h o u r
mean f l u o r i d e c o n c e n t r a t i o n i n t h e a t m o s p h e r e was f o u n d t o be 3.3 ppb, a maximum o f 14.8 ppb,
w i t h i n a 0.5
1.0 km r a d i u s o f t h e p l a n t ,
m
::I
with
Even w i t h i n a
t h e v a l u e s w e r e 1.6 p p b a n d 4.8 ppb,
respec-
-+ --+-
Y i t h i n 0 . 5 km
1st Electrolvsas closed
S
km r a d i u s o f t h e p l a n t .
12.0-
- Beyond 0.5 km
.!? I0.0.0
Q
5
-.
0
2 0
md E l e c t r o l y s i s closed
8.0..
P l s n f ctosed
2.0
:
:
:
:
:
:
:
:
:
F i g u r e 1. Changes i n f l u o r i d e c o n t e n t s o f u n p o l i s h e d r i c e f r o m a r e a s n e a r an a l u m i n u m r e f i n e r y p l a n t ( B ) .
101 tively.
T h e m e a n f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e w a s 1 2 ppm ( 4 ) .
1972 t h e f i r s t e l e c t r o l y s i s p l a n t , was c l o s e d .
Subsequently,
In
t h e main source o f f l u o r i d e emission,
a marked r e d u c t i o n o f f l u o r i d e c o n t e n t s i n t h e
r i c e s a m p l e s was o b s e r v e d .
F l u o r i d e l e v e l s i n t h e samples have f u r t h e r
d e c r e a s e d t o 1.5 pprn s i n c e 1 9 8 1 when t h e p l a n t was c l o s e d c o m p l e t e l y .
E
a Q
Ic
0
c
K 0 0
L
LL
0 3
cunl"
CERnlCs
PH(1BcRTE
87EEL
F i g u r e 2-1. F l u o r i d e c o n t e n t s (mean) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s o f 1 8 i n d u s t r i a l p l a n t s (1975).
0
0
L
0 3
A B C D E F A P C D E A I C D A B C
ii
F i g u r e 2-2. F l u o r i d e c o n t e n t s (mean) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s o f 1 8 i n d u s t r i a l p l a n t s (1984).
102 The d a t a on t h e f l u o r i d e c o n t e n t s o f u n p o l i s h e d r i c e t a k e n f r o m a r e a s around i n d u s t r i a l f l u o r i d e sources a r e shown i n F i g u r e 2 w h i l e t h e i r maximum v a l u e s a r e shown i n F i g u r e 3.
I t i s o b v i o u s t h a t t h e maximum v a l u e s
appear t o be independent o f t h e d i s t a n c e f r o m e m i s s i o n s o u r c e (Fig.
3).
E n n 6 0
I
L
I? .c .v)
0
n
C
3
.c
0 v)
c
C 0)
+. C
0
0 Q,
E
a"
-3 LI F i g u r e 3-1. F l u o r i d e c o n t e n t s (max) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s o f 18 i n d u s t r i a l p l a n t s (1975).
Ea
P
v) *
C 0) c C
0 0
Y
F l u o r i d e c o n t e n t s (max) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s F i g u r e 3-2. o f 18 i n d u s t r i a l p l a n t s (1984).
103 The d a t a o b t a i n e d f r o m t h i s s t u d y were compared w i t h t h o s e o b t a i n e d f r o m a s i m i l a r s u r v e y p e r f o r m e d 10 y e a r s before.
I t was o b s e r v e d t h a t t h e
f l u o r i d e c o n t e n t s o f r i c e f r o m t h e v i c i n i t y o f many phosphate f e r t i l i z e r p l a n t s had been r e d u c e d t o a n o r m a l l e v e l .
R i c e samples f r o m a r e a s near
aluminum p l a n t s showed a s i m i l a r r e d u c t i o n .
On t h e o t h e r hand,
r i c e sam-
p l e s o b t a i n e d f r o m areas around s e v e r a l c e r a m i c s works w h i c h showed a h i g h f l u o r i d e l e v e l o f 19 ppm i n 1975 s t i l l had f l u o r i d e l e v e l s i n excess o f 10 ppm i n 1984,
a l t h o u g h t h e p o l l u t e d areas were l i m i t e d t o t h o s e a d j a c e n t t o
t h e e m i s s i o n sources.
A f l u o r i d e c o n t e n t as h i g h as 37.8 ppm was found i n
r i c e samples o b t a i n e d f r o m an a r e a near a r o o f t i l e m a n u f a c t u r i n g p l a n t .
DISCUSSION R e l a t i o n s h i p between a i r b o r n e f l u o r i d e and f l u o r i d e c o n t e n t o f r i c e The r e s u l t s o f t h e p r o s p e c t i v e 17-year
s u r v e y conducted i n an area near
aluminum r e f i n e r y p l a n t B have c l e a r l y shown t h a t f l u o r i d e c o n t e n t s o f t h e r i c e samples decreased,
as e x p e c t e d ,
f l u o r i d e i n t h e atmosphere.
w i t h decreasing concentration o f
I n actuality,
i s h e d r i c e had been reduced t o a
t h e f l u o r i d e c o n t e n t s i n unpol-
normal l e v e l f o l l o w i n g t h e removal o f t h e
f l u o r i d e e m i s s i o n source. The f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e i s a good i n d i c a t o r o f
long-term
and s u c c e s s i v e e x p o s u r e t o a t m o s p h e r i c f l u o r i d e s i n c e t h e e a r o f a r i c e p l a n t r a r e l y a b s c i s e s as l e a v e s do, u n l e s s t h e c o n c e n t r a t i o n o f a i r b o r n e f l u o r i d e i s so h i g h as t o cause n e c r o s i s and d e a t h o f t h e p l a n t . Concerning t h e d o s e - r e l a t i o n s h i p
between t h e l e v e l o f a t m o s p h e r i c f l u o -
r i d e and t h e f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e ,
i t was f o u n d i n t h i s
f i e l d study (1) t h a t the f l u o r i d e content i n unpolished r i c e increased t o more t h a n 4.0
ppm when t h e a t m o s p h e r i c f l u o r i d e c o n c e n t r a t i o n exceeded 1
ppb d u r i n g t h e r i c e g r o w i n g period. However,
T o m a t s u r i (5) exposed r i c e p l a n t s t o 2-5
ppb hydrogen f l u o r i d e
f o r 40 days and concluded t h a t t h e c o r r e l a t i o n between t h e c o n c e n t r a t i o n o f a t m o s p h e r i c f l u o r i d e and t h e f l u o r i d e c o n t e n t i n t h e l e a v e s o f r i c e p l a n t s was s i g n i f i c a n t ,
b u t t h a t t h e c o r r e l a t i o n between t h e c o n c e n t r a t i o n o f
a t m o s p h e r i c f l u o r i d e and t h e f l u o r i d e c o n t e n t i n t h e u n p o l i s h e d r i c e was not.
I n t h i s e x p e r i m e n t , t h e c o n t r o l p l a n t s w e r e a l s o e x p o s e d t o 0.1 ppb
hydrogen f l u o r i d e . control.
The c o n c e n t r a t i o n u s e d may h a v e been t o o h i g h f o r a
R i c e can a c c u m u l a t e f l u o r i d e when t h e exposure p e r i o d i s pro-
l o n g e d , e v e n when t h e l e v e l o f a i r b o r n e f l u o r i d e may b e v e r y l o w .
This
c o u l d account f o r t h e f a c t t h a t t h e d i f f e r e n c e i n f l u o r i d e l e v e l s between t h e e x p o s u r e g r o u p and t h e c o n t r o l g r o u p becomes i n s i g n i f i c a n t . h i s conclusion,
Despite
Tomatsuri’s study (5) i m p l i e s t h a t t h e f l u o r i d e content i n
104 u n p o l i s h e d r i c e s e r v e s as a good i n d i c a t o r f o r l o w l e v e l e x p o s u r e t o a i r borne f l u o r i d e . Nakao
g
a.( 6 )
measured t h e c o n c e n t r a t i o n s o f gaseous f l u o r i d e i n t h e
s u r r o u n d i n g s o f c e r a m i c s w o r k s (D).
They r e p o r t e d t h a t t h e eight-hour
m a x i m u m f l u o r i d e c o n c e n t r a t i o n i n t h e a t m o s p h e r e w a s 3 - 4 ppb, w h i l e t h e f l u o r i d e c o n t e n t o f t h e u n p o l i s h e d r i c e was 8.5 ppm. R e l a t i o n s h i p between y i e l d o f r i c e and f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e T o m a t s u r i ( 5 ) f u m i g a t e d r i c e w i t h 5 p p b HF a n d c o n c l u d e d t h a t HF h a d n o e f f e c t on t h e y i e l d o f r i c e , even though i t s f l u o r i d e c o n t e n t exceeded 8 ppm.
A s i m i l a r f i n d i n g has been r e p o r t e d b y Katsumi
t d.(7).
On t h e
o t h e r hand, Nakao @, d.(6), based on t h e i r f i e l d o b s e r v a t i o n s , s u g g e s t e d t h a t an i n c r e a s e d f l u o r i d e c o n c e n t r a t i o n i n t h e atmosphere a f f e c t e d t h e g r o w t h and y i e l d o f r i c e .
Tsunoda
eC, d.(8) a l s o r e p o r t e d i n t h e i r f i e l d
s t u d y t h a t up t o 20% r e d u c t i o n i n y i e l d o c c u r r e d when t h e f l u o r i d e c o n t e n t o f p o l i s h e d r i c e exceeded 10 ppm. ppm
T h i s i s e q u i v a l e n t t o a p p r o x i m a t e l y 20
f l u o r i d e i n u n p o l i s h e d r i c e (4,9).
Cho g t
21. ( 1 0 )
a l s o presented
evidence i n d i c a t i n g a s i g n i f i c a n t y i e l d r e d u c t i o n i n r i c e fumigated w i t h
HF.
I n t h e p r e s e n t s t u d y , a 90% r e d u c t i o n i n y i e l d was o b s e r v e d i n r i c e
c o l l e c t e d f r o m a n a r e a 1 5 0 m f r o m t h e c e r a m i c s w o r k s (C).
The f l u o r i d e
c o n t e n t i n t h e u n p o l i s h e d r i c e was f o u n d t o be 37.8 ppm. K a t s u m i @,
d.(11)
exposed r i c e p l a n t s t o 5 ppb HF w h i l e h a v i n g t h e husk
wrapped o r t h e l e a v e s o f t h e p l a n t s c u t o f f .
Judging f r o m t h e observation
t h a t t h e r e was no i n c r e a s e i n f l u o r i d e c o n t e n t i n t h e u n p o l i s h e d r i c e i n e i t h e r treatment,
t h e y showed c l e a r l y t h a t
unpolished r i c e accumulated
a t m o s p h e r i c f l u o r i d e o n l y t h r o u g h t h e husk and t h a t t h e r e was no t r a n s l o c a t i o n o f f l u o r i d e from t h e leaves t o t h e unpolished rice. The f o r e g o i n g d e s c r i p t i o n l e a d s t o t h e c o n c l u s i o n t h a t f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e i s a good i n d i c a t o r o f l o n g - t e r m
and l o w l e v e l e x p o s u r e
t o atmospheric fluoride. R e l a t i o n between f l u o r i d e c o n t e n t o f r i c e and d i s t a n c e f r o m e m i s s i o n s o u r c e The f l u o r i d e c o n t e n t i n r i c e a p p e a r s t o be i n d e p e n d e n t o f t h e d i s t a n c e f r o m e m i s s i o n source.
Concerning t h i s ,
Nakao
&a.(6)
concluded t h a t t h e
r i c e p l a n t absorbed atmospheric f l u o r i d e t h r o u g h stomata, c o u l d b e c l a s s i f i e d as " s t o m a t a type",
as a r i c e p l a n t
and t h e y f e l t t h a t t h e r e was a c l o s e
r e l a t i o n s h i p b e t w e e n t h e c o n c e n t r a t i o n o f a i r b o r n e f l u o r i d e and t h e f l u o r i d e content o f unpolished rice.
As t h e c o n c e n t r a t i o n s o f a t m o s p h e r i c
f l u o r i d e w e r e n o t measured i n t h e p r e s e n t s t u d y , b e made,
no d e t a i l e d comment c o u l d
but nevertheless i t i s possible t h a t the f a c t o r a f f e c t i n g the
f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e may be t h e c o n c e n t r a t i o n o f f l u o r i d e i n t h e atmosphere,
w h i c h i n t u r n i s dependent on w i n d d i r e c t i o n ,
t h e d i s t a n c e f r o m t h e e m i s s i o n source.
r a t h e r than
105 R e c e n t p r o b l e m s a s s o c i a t e d w i t h a i r b o r n e f l u o r i d e p o l l u t i o n i n Japan I n Japan, from
i t was r e c o g n i z e d t h a t f l u o r i d e c o n t e n t s i n r i c e c r o p s o b t a i n e d
a r e a s a r o u n d many p h o s p h a t e f e r t i l i z e r p l a n t s and a l u m i n u m r e f i n e r y
p l a n t s had b e e n r e d u c e d t o n o r m a l l e v e l s .
Presumably,
t o i n s t a l l a t i o n o f e m i s s i o n c o n t r o l systems,
o r c l o s u r e o f p l a n t s as a r e s u l t o f depression. samples
obtained from
areas
t h i s was due e i t h e r
o r t o reduction i n operation Several unpolished r i c e
a r o u n d some c e r a m i c s w o r k s ,
however,
still
showed h i g h l e v e l s o f f l u o r i d e . Japanese c e r a m i c s w o r k s a r e o f s m a l l s c a l e and t h e t o t a l volume o f e x h a u s t g a s f r o m t h e m i s a b o u t 1-3 N k m 3 / h r .
The o b s e r v a t i o n t h a t t h e
f l u o r i d e c o n t e n t o f unpolished r i c e obtained i n t h e v i c i n i t y o f these works was s t i l l h i g h may b e due t o t h e c h e m i c a l f o r m o f f l u o r i d e i n t h e e f f l u e n t .
d.(12)
Itai
r e p o r t e d t h a t 70 t o 80% o f t o t a l a t m o s p h e r i c f l u o r i d e came
from ceramics manufacturing plants. emissions from tunnel
Nakao
g d.( 1 3 )
studied fluoride
k i l n s o f t i l e w o r k s and c o n c l u d e d t h a t t h e f l u o r i d e
i n t h e e f f l u e n t was l a r g e l y i n g a s e o u s f o r m .
The e x p o s e d r i c e p l a n t s may
a c c u m u l a t e a h i g h l e v e l o f f l u o r i d e r e l e a s e d f r o m c e r a m i c s works. I n c r e a s e i n body burden o f f l u o r i d e I n c r e a s e i n t h e body burden o f f l u o r i d e a s s o c i a t e d w i t h i n c r e a s e i n t h e f l u o r i d e c o n t e n t o f r i c e may come i n t o q u e s t i o n as r e p o r t e d p r e v i o u s l y (1). F o r example,
r i c e has been g r o w n d e s p i t e q u a l i t a t i v e and q u a n t i t a t i v e
r e d u c t i o n s i n r i c e c r o p s o r a r e d u c t i o n i n t h e v a l u e o f goods. quently,
t h e f a r m e r s may be f o r c e d t o consume p o l l u t e d r i c e w i t h o u t p u t t i n g
i t on t h e market.
market,
Conse-
When s u c h c o n t a m i n a t e d r i c e i s s o l d a n d f o u n d i n t h e
t h e p u b l i c w i l l be e x p o s e d t o it, r e s u l t i n g i n e x c e s s i v e i n t a k e o f Since ceramics works l i e scattered across t h e country, such a
fluoride.
p r o b l e m as t h i s c o u l d become a m a t t e r o f concern. CONCLUSION
A p r o s p e c t i v e 1 7 - y e a r s u r v e y was p e r f o r m e d t o t r a c e t h e changes i n f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e f r o m an a r e a n e a r an a l u m i n u m r e f i n e r y plant.
Additionally,
f l u o r i d e l e v e l s o f unpolished r i c e from areas
a d j a c e n t t o v a r i o u s t y p e s o f i n d u s t r i e s c o n s i d e r e d t o be f l u o r i d e e m i s s i o n s o u r c e s w e r e s t u d i e d and c o m p a r e d w i t h t h e r e s u l t s o b t a i n e d f r o m a s i m i l a r i n v e s t i g a t i o n c o n d u c t e d 10 y e a r s b e f o r e .
Based o n r e s u l t i n g o b s e r v a t i o n s ,
t h e f o l l o w i n g c o n c l u s i o n s were reached: 1.
P o l l u t i o n b y a i r b o r n e f l u o r i d e d i s a p p e a r e d when t h e e m i s s i o n s o u r c e was removed.
2.
F l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e was f o u n d t o s e r v e as a good i n d i c a t o r o f l o n g - t e r m and l o w l e v e l e x p o s u r e t o a t m o s p h e r i c f l u o r i d e .
106 3.
The f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e grown i n a r e a s around cerami c s w o r k s was g e n e r a l l y h i g h and a l e v e l as h i g h as 37.8 ppm was found i n samples o b t a i n e d f r o m t h e v i c i n i t y o f one o f t h e s e p l a n t s .
4.
A l t h o u g h areas p o l l u t e d by a i r b o r n e f l u o r i d e e m i t t e d f r o m ceramics works a r e l i m i t e d t o those areas surrounding t h e p l a n t s , t h e r e are many c e r a m i c s works i n Japan.
The p o s s i b i l i t y e x i s t s t h a t i n g e s t i o n
o f f l u o r i d e - c o n t a m i n a t e d r i c e i n l a r g e q u a n t i t i e s c o u l d become a s e r i o u s p u b l i c h e a l t h p r o b l e m . I t i s s t r e s s e d t h a t t h e r e i s a need f o r c o n t r o l l i n g t h e f l u o r i d e e m i s s i o n a t c e r a m i c s works.
REFERENCES 1. Sakurai S,
2. H o r w i t z t o n , D.C..
W
I t a i K. Tsunoda H (1983) F l u o r i d e 16:175-180 (ed)(1970) O f f i c i a l Methods o f A n a l y s i s o f t h e AOAC, p p 405-411
Washing-
3. I t a i K, Tsunoda H ( 1 9 8 5 ) I n : Tsunoda H and Yu MH ( e d s ) P r o c e e d i n g s o f t h e 1 4 t h Conference o f t h e I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e Research. E l s e v i e r , Tokyo 4. Tsunoda H,
K u n i t a H (1973) Kougai t o T a i s a k u 9:69-75
5. T o m a t s u r i M (1971) J Japan
SOC of A i r P o l l u t i o n 6:336-342
6. Nakao M, Tanaka F, T a c h i b a n a C. Zenkokukougaikenkaishi 2:45-55
Fukuda K,
H a y a s h i K.
S a i t o K (1980)
7. Katsumi H (1984) B u l l Fukui P r e f A g r i Exp S t a 8:12-14 8. Tsunoda H,
Haneda
M
(1968) Sangyou Kankyou Kougaku 59:2-11
9. Hara S (1983) B u l l J o s a i Den U n i v 12:71-82 10. Cho JK, K i m BY,
Jeh SY (1984)
Res Rept ORD26-1(SPMU),
pp 29-38
11. Katsumi H (1978) B u l l Fukui P r e f A g r i Exp S t a 9:34-37 12. I t a i K,
Sakurai S , Tsunoda H (1983)
13. Nakao M, Tachibana C. 8: 81 -86
Tanaka F,
F l u o r i d e 16:229-234
S a i t o K (1983)
Zenkokukougaikenkaishi
107
H. Tsunoda a n d M.-H. Y u (Editors)
Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 107-112 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
FLUORIDE ABSORPTION AND EXCRETION I N HUMAN SUBJECTS FOLLOWING INGESTION OF F-CONTAMINATED AGRICULTURAL PRODUCTS
H U M I O TSUNODA AND N O R I K O TSUNODA D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , U n i v e r s i t y , M o r i o k a 020, J a p a n
School o f Medicine,
Iwate Medical
ABSTRACT F i v e v o l u n t e e r s p a r t i c i p a t e d i n a s t u d y i n w h i c h f l u o r i d e u p t a k e and e x c r e t i o n were i n v e s t i g a t e d f o l l o w i n g t h e i n g e s t i o n o f r i c e and g r e e n l e a f y v e g e t a b l e s p r o d u c e d i n an a r e a known t o be p o l l u t e d b y a i r b o r n e f l u o r i d e . The r e s u l t s w e r e c o m p a r e d w i t h t h o s e o b t a i n e d f r o m e x p e r i m e n t s i n w h i c h t h e s u b j e c t s were f e d d i e t s c o n s i s t i n g o f s i m i l a r b u t non-contaminated foods. The a v e r a g e d a i l y i n t a k e o f f l u o r i d e b y e a c h p a r t i c i p a n t was 7.84 mg when t h e s u b j e c t s w e r e on t h e e x p e r i m e n t a l menu,
w h i l e i t was 1.89 mg when t h e y
w e r e o n t h e c o n t r o l menu. A h i g h r a t e ( 9 2 % ) o f f l u o r i d e a b s o r p t i o n w a s o b s e r v e d when t h e s u b j e c t s w e r e on t h e e x p e r i m e n t a l menu. i m a t e d t h a t o f NaF.
The r a t e a p p r o x -
I n addition, t h e r e t e n t i o n t i m e o f f l u o r i d e s contained
i n t h e e x p e r i m e n t a l m e a l s was f o u n d t o b e l o n g e r t h a n t h a t o f NaF. The i m p o r t a n c e o f f l u o r i d e a c c u m u l a t i o n i n a g r i c u l t u r a l p r o d u c t s i n r e l a t i o n t o p u b l i c h e a l t h i n J a p a n was d i s c u s s e d .
INTRODUCTION One o f t h e c h a r a c t e r i s t i c s o f f l u o r i d e - i n d u c e d
a i r pollution i s the
a c c u m u l a t i o n o f f l u o r i d e s i n v e g e t a t i o n g r o w n i n t h e p o l l u t e d a r e a (1.2). F e e d i n g d o m e s t i c a n i m a l s a g r i c u l t u r a l products contaminated by f l u o r i d e s has been shown t o r e s u l t i n f l u o r o s i s ,
posing a serious problem i n the
l i v e s t o c k i n d u s t r y (3,4). I n Japan,
a c c u m u l a t i o n o f f l u o r i d e i n r i c e and l e a f y v e g e t a b l e s grown i n
a r e a s p o l l u t e d b y a i r b o r n e f l u o r i d e h a s been r e c o g n i z e d (5,6), of
public
h e a l t h c o n c e r n (7.8).
When h u m a n s
and h a s been
i n g e s t such f l u o r i d e -
contaminated a g r i c u l t u r a l products, t h e r e s u l t i n g t o x i c i t y may be d e t e r mined by t h e e x t e n t t o w h i c h t h e f l u o r i d e i s absorbed t h r o u g h t h e gastrointestinal tract.
108 T h i s p a p e r d e a l s w i t h a b a l a n c e s t u d y i n human s u b j e c t s i n w h i c h t h e i n t a k e and e x c r e t i o n o f f l u o r i d e t h r o u g h i n g e s t i o n o f f l u o r i d e - c o n t a m i n a t e d a g r i c u l t u r a l p r o d u c t s w e r e examined. MATERIALS AND METHODS F i v e h e a l t h y male a d u l t s p a r t i c i p a t e d i n t h i s study.
They w e r e a s k e d t o
b o a r d t o g e t h e r and p e r f o r m d e s k w o r k i n a d e s i g n a t e d h o u s e f o r f i v e days. The s u b j e c t s w e r e f e d d i e t s c o n s i d e r e d a d e q u a t e f o r e a c h i n d i v i d u a l r e s p e c t t o t h e i r c a l o r i c and p r o t e i n a l l o w a n c e s . days,
with
On t h e s e c o n d and t h i r d
t h e s u b j e c t s w e r e f e d m e a l s c o n t a i n i n g r i c e and g r e e n l e a f y vege-
t a b l e s p r o d u c e d i n an a r e a p o l l u t e d b y a i r b o r n e f l u o r i d e , o t h e r t h r e e days,
while during the
t h e m e a l s i n c l u d e d r i c e and v e g e t a b l e s g r o w n i n an a r e a
w i t h no known f l u o r i d e p o l l u t i o n . Throughout t h e e x p e r i m e n t a l period,
t h e f o o d and d r i n k i n g e s t e d b y each
p a r t i c i p a n t was w e i g h e d and t h e f l u o r i d e c o n t e n t d e t e r m i n e d . the total
d a i l y excretion o f fluoride,
w e r e t a k e n , b e g i n n i n g a t 8 a.m., was d e t e r m i n e d .
24-hour
To d e t e r m i n e
u r i n a r y and f e c a l s a m p l e s
and t h e f l u o r i d e c o n t e n t i n each sample
D e t e r m i n a t i o n o f f l u o r i d e c o n t e n t s i n f o o d and f e c e s was
b a s e d o n t h e A.O.A.C.
m e t h o d (11).
subjected t o d i s t i l l a t i o n .
S a m p l e s w e r e a s h e d a n d t h e a s h was
The r e l e a s e d f l u o r i d e was t h e n d e t e r m i n e d by
t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e (12,13),
w h i l e u r i n a r y f l u o r i d e was
d e t e r m i n e d d i r e c t l y b y t h e same m e t h o d (14). The e x p e r i m e n t s w e r e c o n d u c t e d d u r i n g t h e w i n t e r ,
s o a s t o p r e v e n t an
e x t e n s i v e l o s s o f f l u o r i d e t h r o u g h p e r s p i r a t i o n (9,lO). RESULTS The f l u o r i d e c o n t e n t s o f t h e a g r i c u l t u r a l p r o d u c t s f r o m b o t h p o l l u t e d and c o n t r o l areas d i f f e r e d f r o m each o t h e r .
Average f l u o r i d e c o n t e n t s o f r i c e
and g r e e n l e a f y v e g e t a b l e s f r o m t h e p o l l u t e d a r e a w e r e 5.2 ppm and 7.4-10.2 ppm,
respectively,
tively,
w h i l e t h e y w e r e 0.57
ppm a n d 0.44-0.94
ppm,
respec-
i n t h e c o n t r o l samples.
The d a i l y i n t a k e o f f l u o r i d e f r o m f o o d and t h e f l u o r i d e e x c r e t i o n i n u r i n e a n d f e c e s f o r e a c h i n d i v i d u a l a r e s h o w n i n T a b l e I. quantities
Because t h e
o f f o o d i n g e s t e d and c o m p o s i t i o n o f t h e m e a l s v a r i e d s l i g h t l y
a m o n g t h e p a r t i c i p a n t s , i n t a k e s o f f l u o r i d e d i f f e r e d f r o m d a y t o d a y and from individual t o individual.
The a v e r a g e d a i l y i n t a k e o f f l u o r i d e b y each
p a r t i c i p a n t w a s 7.84 mg f r o m t h e e x p e r i m e n t a l m e a l , a n d 1.89 mg f r o m t h e c o n t r o l meal. T a b l e I 1 shows t h e r a t e o f d a i l y u r i n a r y and f e c a l f l u o r i d e e x c r e t i o n i n r e l a t i o n t o corresponding f l u o r i d e i n t a k e .
The a v e r a g e r a t e o f f l u o r i d e
e x c r e t i o n was 69% when t h e s u b j e c t s w e r e f e d n o n - c o n t a m i n a t e d food,
while
109 TABLE I FIVE-DAY INTAKE AND EXCRETION OF FLUORIDE BY VOLUNTEERS Subject
Day 1
D a i l y F l u o r i d e I n t a k e o r O u t p u t , mg 2 3 4
5
S.S.
Intake' Output Urine Feces
1.53 0.99 0.54
7.58 3.45 0.57
7.24 2.69 0.36
2.12 1.72 0.11
1.93 1.30 0.38
H.T.
Intake Output
Urine Feces
1.66 1.15 0.33
8.37 3.61 0.48
7.26 2.55 0.39
2.12 1.99 0.16
1.93 1.39 0.27
M.K.
Intake Output
Urine Feces
1.72 0.81 0.52
8.12 2.35 0.25
7.33 3.15 0.09
1.98 0.77 0.12
2.02
1.22 0.09
Y.K.
Intake Output
Urine Feces
1.60 0.26 0.91
8.50 2.04 0.99
8.36 3.37 0.74
2.24 0.93 0.27
2.09 0.77 0.17
K.S.
Intake Output
Urine Feces
1.52 0.59 0.16
8.33 2.73 0.81
7.35 1.88 1.27
2.04 1.19 0.17
1.85 0.94 0.13
Average' Intake Output
Urine Feces
1.61 +O. 09 0.76kO.35 0.49+0.28
8.18k0.36 2.84+0.68 0.62+0.29
7.51k0.48 2.73+0. 58 0.57k0.45
2.1 OkO. 10 1.32k0.52 0.17+0.06
1.96+0.09 1.12k0.26 0.21 +o. 12
' I n t a k e i n c l u d e s f l u o r i d e s i n f o o d and beverage 'Values a r e means and s t a n d a r d d e v i a t i o n
i t was 43% when t h e y were f e d e x p e r i m e n t a l food.
These r e s u l t s e x c l u d e t h e
f i r s t dzy's d a t a as t h e y were a f f e c t e d by t h e f o o d i n g e s t e d p r i o r t o t h e i n i t i a t i o n o f t h e experiment.
On t h e o t h e r hand,
t h e average e x c r e t i o n o f
f e c a l f l u o r i d e was 8% w i t h l i t t l e v a r i a t i o n between days, w h i l e t h e u r i n a r y e x c r e t i o n r a t e was l o w e r on t h o s e days when t h e s u b j e c t s were f e d c o n t a m i n a t e d f o o d t h a n when t h e y were f e d t h e non-contaminated
food.
The r a t e o f f l u o r i d e a b s o r p t i o n t h r o u g h i n g e s t i o n o f t h e e x p e r i m e n t a l f o o d can be e s t i m a t e d f r o m t h e f l u o r i d e e x c r e t e d i n t h e feces.
Since t h e
p e r c e n t a g e o f f e c a l e x c r e t i o n o f f l u o r i d e i s a b o u t 8%. t h e r a t e o f f l u o r i d e a b s o r p t i o n f o r t h e a g r i c u l t u r a l p r o d u c t s i s 92%. I n a d d i t i o n ,
the fluoride
r e t e n t i o n r a t e i n t h e body can a l s o be e s t i m a t e d f r o m t o t a l t h e f l u o r i d e e x c r e t i o n , i.e..
u r i n a r y and f e c a l f l u o r i d e e x c r e t i o n .
I t was o b s e r v e d
t h a t f l u o r i d e r e t e n t i o n r e s u l t i n g from i n g e s t i o n o f the contaminated a g r i c u l t u r a l p r o d u c t s was g r e a t e r t h a n t h a t o f t h e c o n t r o l s .
110
TRBLE 11 RATE OF FLUORIDE EXCRETION Siibject
Item
-
Day 1
s. s.
Total Urine Feces
H. T.
Rate o f D a i l y Excretion, %
2
3
4
5
100.0 64.7 35.3
53.0 45.5 7.5
42.2 37.2 5.0
86.3 81.1 5.2
87.1 67.4 19. *'
Total Urine Feces
89.2 69.3 19.9
48.8 43.1 5.7
40.5 35.1 5.4
101.4 93.9 7.5
86.0 72.0 14.0
M. K.
Total Urine Feces
77.3 47.1 30.2
32.0 28.9 3.1
44.2 43.0 1.2
45.0 38.9 6.1
64.9 60.4 4.5
Y.K.
Total Urine Feces
73.2 16.3 56.9
35.6 24.0 11.6
49.2 40.3 8.9
53.6 41.5 12.1
44.9 36.8 8.1
K. S.
Total Urine Feces
49.3 38.8 10.5
42.5 32.8 9.7
42.9 25.6 17.3
66.6 58.3 8.3
57.8 50.8 7.0
77.8217.1 47.2k19.1 30.6k15.7
42.427.8 34.9k8.2 7.5k3.0
43.8t3.0 36.226.0 7.625.4
70.6220.8 62.7k21.7 7.8k 2.4
68.1k16.3 57.5+12.6 10.7k 5.5
Average1 Total Urine Feces 'Values
a r e means and s t a n d a r d d e v i a t i o n
DISCUSSION As m e n t i o n e d p r e v i o u s l y ,
problems associated w i t h fluoride-induced
air
p o l l u t i o n l i e i n t h a t i t n o t o n l y caused i n j u r y t o p l a n t s b u t a l s o r e s u l t s i n f l u o r i d e a c c u m u l a t i o n i n v e g e t a t i o n exposed t o t h e p o l l u t a n t .
Numerous
r e p o r t s have been p u b l i s h e d d e s c r i b i n g f l u o r o s i s i n a n i m a l s f e d f l u o r i d e c o n t a r n i n a t e d v e g e t a t i o n (3.4.8). 'Several
types o f agricultural
known t o a c c u m u l a t e f l u o r i d e . green l e a f y vegetables,
p r o d u c t s c o m m o n l y consumed b y humans a r e
I n Japan,
i t h a s been o b s e r v e d t h a t r i c e and
main s t a p l e s i n t h i s country,
produced i n p o l l u t e d
areas c o u l d c o n t a i n f l u o r i d e t h a t i s s e v e r a l t o more t h a n t e n t i m e s h i g h e r t h a n t h a t f o u n d i n n o n - c o n t a m i n a t e d p r o d u c t s (5,6).
Problems associated
w i t h t h e c o n s u m p t i o n o f such c o n t a m i n a t e d p r o d u c t s have been r e c o g n i z e d
(8.15).
The t o x i c i t y o f f l u o r i d e i s d e p e n d e n t o n t h e s o l u b i l i t y o f v a r i o u s
t y p e s o f f l u o r i d e and t h e a m o u n t a b s o r b e d (16).
Results obtained from t h i s
s t u d y show t h a t t h e a b s o r p t i o n r a t e o f f l u o r i d e s c o n t a i n e d i n r i c e and green l e a f y vegetables produced i n f l u o r i d e - p o l l u t e d
90%, a p p r o x i m a t i n g t h a t of NaF (9.17).
a r e a s was g r e a t e r t h a n
111 I n a d d i t i o n , t h e r a t e o f u r i n a r y e x c r e t i o n o f f l u o r i d e as a r e s u l t o f i n g e s t i n g t h e c o n t a m i n a t e d f o o d was f o u n d t o be l o w e r t h a n t h a t o f i n g e s t i n g MaF ( 9 , 1 4 ) ,
s u g g e s t i n g a l o n g e r r e t e n t i o n t i m e i n t h e body.
For t h i s
r e a s o n , t h e f l u o r i d e s c o n t a i n e d i n r i c e and l e a f y v e g e t a b l e s a r e o f (nuch c o n c e r n i n t e r m s o f t c l e i r i n f l u e n c e on p u b l i c h e a l t h .
T h e r e i s a need f o r
c o n t r o l l i n g f l u o r i d e e m i s s i o n b y i n d u s t r i e s so t h a t r i c e and g r e e n l e a f y v e g e t a b l e s g r o w n i n t h e v i c i n i t y o f i n d u s t r i a l p l a n t s may b e f r e e f r o m accumulation o f the pollutant. CONCLUSION T h e a b s o p t i o n o f f l u o r i d e t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t h a s been s t u d i e d i n f i v e human v o l u n t e e r s . and g r e e n
They w e r e f e d d i e t s c o n s i s t i n g o f r i c e
e a f y v e g e t a b l e s p r o d u c e d i n an a r e a known t o be p o l l u t e d b y
a i r b o r n e f u o r i d e . The a v e r a g e r a t e o f f l u o r i d e a b s o r p t i o n b y t h e s e p a r t i c i p a n t s was f o u n d t o be a b o u t 92%.
I n addition,
i t was o b s e r v e d t h a t t h e
f l u o r i d e present i n these foods e x h i b i t e d a longer r e t e n t i o n t i m e i n the body t h a n t h a t o f NaF. The i m p o r t a n c e o f p r e v e n t i n g f l u o r i d e a c c u m u l a t i o n b y a g r i c u l t u r a l p r o d u c t s g r o w n i n f l u o r i d e p o l l u t e d a r e a s i n r e l a t i o n t o human h e a l t h i s stressed.
REFERENCES 1. Tsunoda H,
Haneda M (1968) I n d u s t r E n v i r o n Eng 59:2-11
2. C o m m i t t e e o n B i o l o g i c E f f e c t s o f A t m o s p h e r i c P o l l u t a n t s (1971) F l u o r i d e s . NAS. Washington, DC. p p 77-162 3. P h i l l i p s PH, Greenwood DA, Hobbs CS, H u f f m a n C F (1955) The F l u o r o s i s Problem i n L i v e s t o c k Production. A Report o f t h e Committee on Animal N u t r i t i o n . NAS-NCR P u b l i c a t i o n 381, Washington, DC, p p 1-20 4. S h u p e J L ( 1 9 6 9 ) F l u o r o s i s o f L i v e s t o c k . A i r Q u a l i t y M o n o g r a p h 69-4, A m e r i c a n P e t r o l e u m I n s t i t u t e , New York, p p 1-29 5. Tsunoda H,
K u n i t a H (1973) J E n v i r o n P o l l u t C o n t r o l 9:69-75
6. S a k u r a i S,
I t a i K,
Tsunoda H (1983) F l u o r i d e 16:175-180
7. Tsunoda H (1971) J Japan SOC S a f e t y Eng 10:75-85
8. Tsunoda H (1978) N i p p o n D e n t Rev 427:137-150 9. H o d g e HC, S m i t h FA ( 1 9 6 5 ) I n : S i m o n s JH ( e d ) F l u o r i n e C h e m i s t r y , V o l 4. Academic Press, New York, p p 137-176 10. C r e m e r HD. B i t t n e r W ( 1 9 7 0 ) Health. WHO, Geneva, p p 75-91
I n : E r i c s s o n Y ( e d ) F l u o r i d e a n d Human
11. H o r o w i t z W (ed) (1970) O f f i c i a l M e t h o d s Washington, DC, p p 405-411 12. F r a n t MS, 13. N e e f u s JD.
o f A n a l y s i s o f AOAC 1 1 t h ed.
Ross J W (1966) S c i e n c e 154:1553-1554 C h o l a k J,
S a l t z m a n BE (1970) Am I n d Hyg Assoc J 1:168
14. I s h i k a w a S (1976) I w a t e I g a k u Z a s s h i 28:207-222
112
15. T s u n o d a H. S a k u r a i S , I t a i K. (1984) F l u o r i d e 17:159-167
S a t 0 T. Nakaya S . M i t a M,
16. E a g e r s R Y ( 1 9 6 9 ) T o x i c P r o p e r t i e s E l s e v i e r , Essex, p 33
Tatsumi M
o f I n o r g a n i c F l u o r i n e Compounds.
17. Largent EJ (1961) Fluorosis-The Health Aspects of F l u o r i n e Compounds. Ohio S t a t e U n i v e r s i t y P r e s s , Columbus, p p 34-39
113
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 113-125 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
EFFECTS O F EFFLUENTS FROM A F O S S I L FUEL POWERED GENERATING PLANT O N THE HEALTH OF BEEF CATTLE
J A M E S L. SHUPE, ARLAND E. OLSON, JAY W. RAGHUBIR P.
BAGLEY.
SHARMA, AND GENE W.
CALL,
JOHN E. BUTCHER, CLELL V.
MILLER
D e p a r t m e n t o f V e t e r i n a r y S c i e n c e , UMC 56. U t a h S t a t e U n i v e r s i t y , L o g a n , U t a h 84322, U.S.A. ABSTRACT E f f l u e n t waters from a coal-fueled e l e c t r i c i t y generating p l a n t were s p r i n k l e - i r r i g a t e d on forage crops which were grazed o r harvested f o r w i n t e r feed f o r c a t t l e .
The w a t e r s w e r e h i g h e r t h a n n o r m a l i n t o t a l d i s -
s o l v e d s a l t s and f l u o r i d e s . f o r drinking. ogy,
The c a t t l e s o m e t i m e s had a c c e s s t o t h i s w a t e r
A n a l y s e s h a v e shown no c o n s i s t e n t a b n o r m a l i t i e s i n Ihematol-
c l i n i c a l chemistries.
o r heavy m e t a l c o n c e n t r a t i o n s i n blood,
selected
s o f t t i s s u e s o r bones.
F l u o r i n e c o n t e n t o f bone and some u r i n e s a m p l e s
were s l i g h t l y elevated.
T e e t h t h a t h a v e f o r m e d and m i n e r a l i z e d w h i l e t h e
c a t t l e w e r e on t h e f a r m h a v e m o d e r a t e t o m a r k e d l e s i o n s o f d e n t a l f l u o r o sis.
Independently,
n e i t h e r the e f f l u e n t water,
nor forage sprinkle-
i r r i g a t e d w i t h t h a t w a t e r h a v e a h i g h enough f l u o r i n e c o n t e n t t o i n d u c e t h e s e v e r i t y o f d e n t a l l e s i o n s observed,
b u t t h e combined sources have induccd
chronic fluoride toxicosis i n the cattle. INTRODUCTION F o r s e v e r a l decades,
s c i e n t i s t s a t U t a h S t a t e U n i v e r s i t y (USU) h a v e Seen
s t u d y i n g t h e e f f e c t s on t h e e n v i r o n m e n t o f e m i s s i o n s f r o m many d i f f e r e n t types o f industry.
The c u r r e n t 5 - y e a r
f i n d i n g s are from a planned t e n year
s t u d y t o m o n i t o r h e a l t h r e l a t e d e f f e c t s on c a t t l e a f t e r i n g e s t i o n o f f o r a g e c r o p s t h a t h a v e been s p r i n k l e - i r r i g a t e d
w i t h e f f l u e n t waters from a coal-
fueled e l e c t r i c i t y generating plant.
The e f f l u e n t w a t e r i s o r i g i n a l l y
i n t r o d u c e d i n t o t h e g e n e r a t i o n p l a n t f r o m r e l a t i v e l y p r i s t i n e sources. Regulations f o r b i d t h e discharge o f any w a t e r from t h e p l a n t back i n t o n a t u r a l s t r e a m channels.
The w a t e r i s r e c i r c u l a t e d t e n t i m e s f o r c o o l i n g
and p o l l u t i o n c o n t r o l p u r p o s e s u n t i l t h e w a t e r i s h e a v i l y l a d e n e d w i t h chemicals.
Most o f t h e p l a n t e f f l u e n t s a r e i n t h e d i s c h a r g e water.
stack emissions are negligible. i n t o a storage reservoir.
Some o f t h e w a t e r e v a p o r a t e s , b u t much
used t o s p r i n k l e i r r i g a t e crops.
Smoke
The h e a v i l y p o l l u t e d w a t e r i s d i s c h a r g e d is
also
The c r o p s a r e d i v e r s i f i e d b u t m o s t a r e
114 f o r a g e c r o p s f o r g r a z i n g by c a t t l e o r h a r v e s t f o r w i n t e r feed. MATERIALS AND METHODS V e g e t a t i o n g r o w t h and d e v e l o p m e n t i s e v a l u a t e d throughout t h e growing s e a s o n and c o m p a r e d t o c o n t r o l v e g e t a t i o n o n t h e USU f a r m .
Analyses are
made o n v e g e t a t i o n s a m p l e s f o r l e a d , c o p p e r , z i n c , cadmium,
b o r o n , and
fluorine. D e t a i l e d p h y s i c a l and c l i n i c a l e x a m i n a t i o n s a r e c o n d u c t e d on t h e c a t t l e periodically t o evaluate t h e i r h e a l t h status.
A n i m a l s o f d i f f e r e n t ages
a r e a n n u a l l y s e l e c t e d a n d s a c r i f i c e d and t i s s u e s c o l l e c t e d and a n a l y z e d f o r s p e c i f i c elemental contents. selected tissues.
B l o o d s a m p l e s f r o m a l l a n i m a l s a r e a n a l y z e d f o r a number
o f i n o r g a n i c elements. measured.
P a t h o l o g i c e v a l u a t i o n s a r e a l s o made o f
Analytical
B i o c h e m i c a l and p h y s i o l o g i c a l p a r a m e t e r s a r e a l s o r e s u l t s a r e compared w i t h e a r l i e r r e s u l t s o f a n i m a l s
on t h e r e s e a r c h f a r m .
C o m p a r a b l e n o r m a l c a t t l e a t t h e USU f a r m n e a r Logan,
U t a h s e r v e as c o n t r o l s . Heavy m e t a l s and o t h e r e l e m e n t s w e r e a n a l y z e d f o r b y u s i n g a p p r o p r i a t e flame/flameless atomic absorption spectrometry. and u r i n e w e r e c o n d u c t e d w i t h a s p e c i f i c
F l u o r i d e analyses o f blood
i o n electrode.
Routine d i g e s t i o n
o r o t h e r p r e p a r a t i o n o f a l l s a m p l e s was done p r i o r t o a n a l y s e s .
Values a r e
g r o u p e d a c c o r d i n g t o t h e t i m e o f y e a r o f s a m p l i n g and t h e age g r o u p o f t h e an ima1 s. RESULTS AND D I S C U S S I O N To d a t e ,
a l l analyses o f blood, l i v e r ,
kidney. s k e l e t a l muscle, lung,
s p l e e n a n d bone h a v e b e e n w i t h i n t h e n o r m a l r a n g e s f o r i r o n , s i u m , c o p p e r , cadmium,
chromium, m e r c u r y and lead.
zinc.
magne-
O f these elements,
v a l u e s h a v e shown t h e g r e a t e s t f l u c t u a t i o n s t o d a t e i n t h e s t u d y ,
lead
but are
s t i l l b e l o w l e v e l s w h e r e c l i n i c a l e f f e c t s w o u l d be a p p a r e n t .
A l l serum e l e c t r o l y t e ,
enzyme o r o t h e r b i o c h e m i c a l v a l u e s a r e w i t h i n t h e
normal ranges f o r c a t t l e .
The t e s t s i n d i c a t e n o a l t e r a t i o n o f t h e p h y s i o -
l o g i c a l s t a t e o r organ d y s f u n c t i o n i n these animals. t i m e v a r i a t i o n s observed i n s e l e c t e d parameters,
There were time-to-
b u t t h e s e a r e e i t h e r o f no
adverse p h y s i o l o g i c a l consequence o r r e f l e c t t h e b i o l o g i c a l ,
seasonal,
or
age v a r i a t i o n i n t h e a n i m a l s f r o m whom t h e s a m p l e s w e r e o b t a i n e d . Similarly,
t h e h e m a t o l o g i c a l parameters o f a l l a n i m a l s were n o t i n f l u -
enced by t h e e n v i r o n m e n t a l
factors.
A l l c u r r e n t values a r e comparable t o
t h o s e o b t a i n e d f r o m e a r l i e r s a m p l i n g s and a r e w i t h i n t h e n o r m a l r a n g e s reported f o r cattle. The e s s e n t i a l m e t a l s i n b l o o d o r s e r u m o f t h e s e a n i m a l s h a v e been w i t h i n
115
the normal ranges. Slight variations may be due to biological variation or environmental factors, but did not significantly alter the levels of these elements and none of the values posed any health risk. The fluoride content o f the urine o f normal cattle ingesting a normal diet fluctuates, but it is usually less than 6 ppm F. Few of the samples collected to date were above 6 ppm F (Table 1). The average urine fluoride content o f all animals has been below the level normally associated with detrimental lesions. The fluoride levels i n urine and bone do, however, reflect an elevated fluoride intake by these animals. Urinary fluoride is an index of the exposure to this element although wide variations can be expected. An increase in the bone and urine fluoride is also consistent with increased amounts of fluoride in the water and vegetation.
TABLE I FLUORIDE CONTENT
OF
URINE FROM RESEARCH FARM CATTLE
Age (years)
PPM F
3.7
8.9
3.7
3.3
7.6
2.3
3.7
1.2
8.7
5.3
1.7
2.6
1.6
2.7
2.7
7.6
1.6
0.8
5.7
4.6
1.7
1.2
1.6
0.8
3.7
5.4
3.6
6.3
6.7
2.8
1.6
1.7
0.5
0.4
2.7
0.8
2.7
2.7
7.5
9.0
116 Other evidence o f increased l e v e l s o f f l u o r i d e
i n t a k e are present.
F l u o r i d e d e n t a l l e s i o n s a r e i n d u c e d w h i l e t e e t h a r e f o r m i n g and m i n e r a l i z i n g p r i o r t o e r u p t i n g i n t o t h e o r a l c a v i t y ( F i g . 1). have formed,
mineralized,
and e r u p t e d ,
c h a r a c t e r i s t i c enamel m o t t l i n g .
Once t h e t e e t h
e x c e s s i v e f l u o r i d e does n o t induce
Dental l e s i o n s c h a r a c t e r i s t i c o f f l u o r i d e
t o x i c o s i s a r e p r e s e n t i n t h e c a t t l e t h a t had permanent i n c i s o r t e e t h form, m i n e r a l i z e and e r u p t w h i l e on t h e R e s e a r c h Farm.
Teeth t h a t were formed
b e f o r e t h e c a t t l e w e r e p l a c e d on t h e f a r m showed n o f l u o r i d e i n d u c e d l e s i o n s ( F i g . 2).
The f l u o r i d e i n d u c e d d e n t a l l e s i o n s a r e m o r e s e v e r e i n
t h e second p a i r o f permanent i n c i s o r t e e t h t h a n i n t h e f i r s t p a i r ,
indica-
t i n g an i n c r e a s e d l e v e l o f f l u o r i d e i n t a k e a s t h e c a t t l e g r e w and i n c r e a s e d t h e i r v e g e t a t i o n and w a t e r i n t a k e ( F i g s .
3-4).
The c l a s s i f i c a t i o n o f s l i g h t
d e n t a l f l u o r o s i s i n t h e f i r s t p a i r o f permanent i n c i s o r s ,
moderate e f f e c t s
i n t h e second p a i r , and marked t o s e v e r e e f f e c t s i n t h e t h i r d and f o u r t h p a i r s i n d i c a t e t h a t t h e a n i m a l s r e c e i v e d more than t h e t o l e r a n c e t h r e s h o l d l e v e l o f f l u o r i d e when t h e t e e t h w e r e f o r m i n g ( T a b l e 11).
F i g u r e 1. Photograph o f dissected a n t e r i o r mandible of a 16 month o l d h e i f e r . The e r u p t e d t e e t h a r e a l l d e c i d u o u s . The d i s s e c t i o n shows t h e f o r m i n g and m i n e r a l i z i n g f i r s t ( 1 ) a n d s e c o n d (2) p a i r s o f p e r m a n e n t i n c i sors. Number 1 shows t y p i c a l m o d e r a t e f l u o r i d e i n d u c e d l e s i o n s .
117
F i g u r e 2. P e r m a n e n t i n c i s o r t e 3 t h f r o m a 4-1/2 y e a r o l d H e r e f o r d . T h e s e t e e t h were formed p r i o r t o exposure t o h i g h f l u o r i d e i n t a k e and a r e essent i a l l y normal.
Photograph o f dissected a n t e r i o r mandible of a 2 year 8 month F i g u r e 3. old H e r e f o r d female. The f i r s t p a i r o f p e r m a n e n t i n c i s o r s (1) has erupted. The s e c o n d p a i r (2) a r e f o r m e d , m i n e r a l i z e d and show soiro f l u o r i d e induced mottling. The c r o w n o f t h e L h i r d p a i r o f i n c i s o r s ( 3 ) i s m o s t l y f o r m e d and p a r t i a l l y m i n e r a l i z e d .
118
P e r m a n e n t i n c i s o r t e e t h o f a 3 y e a r 8 m o n t h o l d cow t h a t was F i g u r e 4. r a i s e d o n t h e Research Farm and exposed t o e x c e s s i v e f l u o r i d e . Note t h e a b n o r m a l d i s c o l o r a t i o n and c h a l k y appearance o f t h e second, t h i r d and f o u r t h p a i r s o f teeth. TABLE I 1 PERMANENT I N C I S O R TOOTH CLASSIFICATIONS OF CATTLE R A I S E D ON RESEARCH FARM Age ( y e a r s )
Incisor Classifications
4-7-7-7
2-3
3-7-7-7
3-3-7-7 i
2-6-7-7
+++
f
2-3-3-7
3-4
2-3-4-7
+
1-3-7-7
2-3-4-6
1-3-4-7 4-5
2-3-4-6
2-3-4-7
5 +
2-3-4-4 2-3-3-3
3-3-4-4
++
i
f
F o r t h e purpose o f c l i n i c a l l y c l a s s i f y i n g t h e v a r i o u s degrees o f d e n t a l fluorosis,
0.
s t a n d a r d s have been developed. Normal:
a d o p t e d a n d a r e u s e d (1-4).
smooth, t r a n s l u c e n t , glossy
w h i t e appearance o f enamel;
t o o t h h a s n o r m a l shape and s i z e .
1.
Questionable effect: determine;
may
a b e r r a t i o n s w i t h e x a c t cause d i f f i c u l t t o
have
enamel
flecks;
cavities,
i f present,
may b e u n i l a t e r a l o r b i l a t e r a l ; n o m o t t l i n g .
2.
Slight
effect:
slight,
t i o n s ( m o t t l e d enamel); a b n o r m a l wear;
opaque,
dull,
chalky-white cross s t r i a -
may h a v e s l i g h t d i s c o l o r a t i o n b u t n o
t o o t h w i l l h a v e n o r m a l shape and s i z e .
119 3.
Moderate
effect:
diffuse,
(generalized mottling);
dull,
chalky-white
enamel
t o o t h may h a v e a s l i g h t l y i n c r e a s e d
r a t e o f wear and may be s t a i n e d .
4.
Marked e f f e c t :
definite mottling:
c o l o r e d and evidence
enamel
may b e c r e a m
h y p o p l a s i a and hypomineralization:
e n a m e l may b e p i t t e d and e r o d e d ;
d e f i n i t e abrasion o f tooth:
s t a i n i n g may o r may n o t be present.
5.
Severe e f f e c t :
e f f e c t s more severe t h a n d e s c r i b e d i n number f o u r
classification. T e e t h c l a s s i f i e d a s n u m b e r 2 ( s l i g h t ) o r 3 ( m o d e r a t e ) d o n o t w e a r away any f a s t e r t h a n normal t e e t h and have cosmetic, animals.
non-detrimental
The e c o n o m i c i m p a c t i s n e g l i g i b l e i n a n i m a l s .
e f f e c t s a r e i m p o r t a n t t o people,
effects i n
Such c o s m e t i c
however.
D e n t a l f l u o r o s i s i n a n i m a l s i s u s u a l l y diagnosed by e x a m i n i n g t h e i n c i s o r teeth.
The use o f b o t h d i r e c t and back l i g h t i n g o f t h e t e e t h enhances t h e
a c c u r a c y o f examination.
Cheek ( p r e m o l a r and m o l a r ) t e e t h a r e more d i f f i -
c u l t t o examine i n t h e l i v e a n i m a l because i t i s sometimes d i f f i c u l t t o r e s t r a i n the animal properly,
i l l u m i n a t i o n o f t h e t e e t h i s o f t e n poor,
the
tongue and u n s w a l l o w e d f o o d may i n t e r f e r e , and d i s c o l o r a t i o n r e s u l t i n g f r o m f a c t o r s o t h e r t h a n f l u o r i d e c o m p l i c a t e t h e examination.
The c r i t e r i a used
i n d i a g n o s i n g and e v a l u a t i n g whether d e n t a l f l u o r o s i s i s a f f e c t i n g premol a r s and m o l a r s a r e b a s e d o n t h e d e g r e e o f s e l e c t i v e a b r a s i o n a n d a r e c o r r e l a t e d w i t h t h e degree o f i n c i s o r f l u o r o s i s .
P r e m o l a r and m o l a r abra-
s i o n i s d e l a y e d somewhat beyond t h a t o f i n c i s o r t e e t h .
Abraded cheek t e e t h
can cause i m p r o p e r m a s t i c a t i o n and u t i l i z a t i o n o f roughage.
Damaged t e e t h
may be s o m e w h a t p r o t e c t e d b y a d j a c e n t sound t e e t h t h a t a r e n o t abraded. A b r a s i v e f e e d s s u c h as c o a r s e , f i b r o u s , t o u g h r o u g h a g e w i l l i n c r e a s e t h e r a t e o f d e n t a l abrasion. t o x i c o s i s i n animals,
I n d i a g n o s i n g and e v a l u a t i n g c h r o n i c f l u o r i d e
t h e cheek t e e t h s h o u l d be examined and t h e f i n d i n g s
c o r r e l a t e d w i t h o t h e r fluoride-induced
symptoms and l e s i o n s (5).
The degree o f d e n t a l f l u o r o s i s can u s u a l l y be c o r r e l a t e d w i t h t h e amount o f f l u o r i d e i n t h e bones, t h e d e g r e e o f o s t e o f l u o r o s i s . t h e d u r a t i o n o f exposure, ingestion,
age o f
the
animal
during the time of
t h e amount o f f l u o r i d e ingested,
o f t h e body.
The d e n t a l l e s i o n s ,
diagnosing fluoride
toxicosis.
excessive
fluoride
and o t h e r p h y s i o l o g i c processes
therefore,
are useful i n c l i n i c a l l y
To b e m o s t m e a n i n g f u l ,
however,
the
f l u o r o t i c d e n t a l changes must be c o r r e l a t e d w i t h o t h e r symptoms and l e s i o n s indicative o f fluoride toxicosis. n o t be used as t h e
I n o t h e r words, d e n t a l l e s i o n s s h o u l d
s o l e c r i t e r i o n when e v a l u a t i n g f l u o r i d e t o x i c o s i s .
I n g e s t e d f l u o r i d e s have g r e a t a f f i n i t y f o r bone as w e l l a s f o r d e v e l o p i n g
120 and m i n e r a l i z i n g t e e t h .
E x c e s s i v e f l u o r i d e s may a f f e c t bones a t a n y t i m e
d u r i n g an animal's l i f e ;
however,
s i v e t o exce.;sive
bones i n y o u n g a n i m a l s a r e m o r e r e s p o n -
f l u o r i d e l e v e l s t h a n a r e those o f mature animals.
There
has been an i n c r e a s e i n t h e f l u o r i d e c o n t e n t o f bone f r o m t h e s a c r i f i c e d a n i m a l s t h a t w a s a g e r e l a t e d ( T a b l e 111).
To d a t e ,
no c h a r a c t e r i s t i c
f l u o r i d e induced l e s i o n s o f bone h a v e b e e n o b s e r v e d g r o s s l y ,
radiograph-
i c a l l y o r m i c r o s c o p i c a l l y i n any o f t h e necropsied animals f r o m t h e R e s e a r c h Farm.
However,
an increased ingestion o f
fluorides
for
a
p r o l o n g e d p e r i o d o f t i m e c a n c a u s e d e f i n i t e c h a r a c t e r i s t i c bone changes. TABLE 111 FLUORINE CONTENT
OF BONES FROM
Age ( m o n t h s ) 18
SACRIFICED CATTLE
PPM
F -
Metacarpal
dried fat-free basis Metatarsal
Rib
343
352
455
32
595
670
1 1 50
91
954
1020
1925
106
1080
1110
1940
I f t h e amounts o f f l u o r i d e i n g e s t e d a r e s u f f i c i e n t l y h i g h e r t h a n normal over an appreciable l e n g t h o f time, evident.
Grossly,
s t r u c t u r a l bone changes w i l l become
bones t h a t a r e s e v e r e l y a f f e c t e d b y f l u o r i d e appear
c h a l k y w h i t e w i t h a roughened i r r e g u l a r p e r i o s t e a l surface.
They a r e
l a r g e r i n d i a m e t e r and h e a v i e r t h a n n o r m a l . The t y p e o f bone c h a n g e s seen depend o n ' f a c t o r s toxicosis.
that influence fluoride
One o r m o r e o f t h e f o l l o w i n g c o n d i t i o n s m a y o c c u r :
Osteo-
porosis, osteosclerosis. hyperostosis. osteophytosis. o r osteomalacia. C h a r a c t e r i s t i c h i s t o l o g i c a l changes a r e a s s o c i a t e d w i t h t h e v a r i o u s degrees o f osteofluorosis.
These h a v e b e e n e l u c i d a t e d ,
d e s c r i b e d and i l l u s t r a t e d
(1,2,4,6,7).
A s l i g h t i n c r e a s e i n t h e f l u o r i d e c o n t e n t o f bone above t h e normal c o n c e n t r a t i o n c a n o c c u r w i t h o u t d e t e c t a b l e chnnges i n bone s t r u c t u r e o r function.
T h i s a p p e a r s t o be t h e s t a t u s o f R e s e a r c h Farm a n i m a l s c u r r e n t l y
( T a b l e 111).
A s l i g h t t o moderate increase i n t h e f l u o r i d e content induces
o n l y s l i g h t m i c r o s c o p i c bone changes w i t h no d e t e c t a b l e a l t e r a t i o n o f function.
As a m o u n t s o f b o n e f l u o r i d e i n c r e a s e , p r o g r e s s i v e s t r u c t u r a l
changes o c c u r t h a t r e s u l t i n d e f i n i t e bone l e s i o n s .
It i s important to
d i s c e r n t h e d i f f e r e n c e b e t w e e n n o r m a l g r o w t h and r e m o d e l i n g o f bone, fluoride-induced
and
lesions.
The d e g r e e o f p r o g r e s s i v e s t r u c t u r a l bone c h a n g e s d u e t o e x c e s s i v e f l u o r i d e i n t a k e and t h e i r p a t t e r n s o f c o m b i n a t i o n s a r e g o v e r n e d b y t h e v a r i o u s factors t h a t influence the manifestations o f fluoride toxicosis.
Some o f
t h e g r o s s and h i s t o l o g i c a l c h a n g e s i n d u c e d b y f l u o r i d e t o x i c o s i s may resemb l e bone l e s i o n s and a l t e r a t i o n s t h a t a r e a s s o c i a t e d w i t h o t h e r bone diseases.
T h e r e f o r e , t h e l e s i o n s observed must be c o r r e l a t e d c a r e f u l l y
w i t h o t h e r l e s i o n s and s y m p t o m s i n m a k i n g a d e f i n i t i v e d i a g n o s i s o f f l u o r i d e t o x i c o s i s (5.8). Bone c h a n g e s i n t h e r e s e a r c h f a r m a n i m a l s h a v e n o t y e t a p p e a r e d and t h u s t h e r e has been none o f t h e n o n - s p e c i f i c
lameness o r s t i f f n e s s t h a t i s
s o m e t i m e s seen i n a d v a n c e d c a s e s o f o s t e o f l u o r o s i s . E x c e s s i v e f l u o r i d e s f o r c a t t l e may a r i s e f r o m s e v e r a l s o u r c e s w h i c h may include:
f o r a g e s c o n t a m i n a t e d e i t h e r d i r e c t l y o r i n d i r e c t l y w i t h indus-
t r i a l effluents;
w a t e r w h i c h has a h i g h f l u o r i d e c o n t e n t , e i t h e r n a t u r a l l y ,
as i n t h e c a s e o f m o s t g e o t h e r m a l w a t e r s , effluents.
(9).
H i g h f l u o r i d e c o n t e n t s o i l may b e c a r r i e d b y w i n d o r b e r a i n s p l a s h e d
o n t o f o r a g e s w h i c h a r e t h e n consumed. fore,
or polluted with industrial
M i n e r a l supplements may e x c e e d recommended f l u o r i d e c o n t e n t F l u o r i d e s a r e c u m u l a t i v e and, t h e r e -
c o m b i n a t i o n s o f moderate amounts o f f l u o r i d e s f r o m t h e above sources
may add u p t o an e x c e s s i v e t o t a l i n t a k e . The b e e f c a t t l e i n t h i s s t u d y have, t h e t i m e t o t w o f l u o r i d e sources,
t o date,
had a c c e s s a t l e a s t p a r t o f
n a m e l y c o n t a m i n a t e d f o r a g e s and w a t e r .
N e i t h e r o f these sources i s e x t r e m e l y h i g h i n f l u o r i n e content (Tables I V -
V I ) b u t t h e combined s o u r c e s can i n d u c e t h e t y p e s and s e v e r i t y o f d e n t a l l e s i o n s we h a v e seen t o d a t e . harvested,
power generating plant. i n g water,
A l l o f t h e f o r a g e s consumed,
h a v e been s p r i n k l e - i r r i g a t e d
b o t h g r a z e d and
w i t h the e f f l u e n t water from the
T h i s w a t e r i s n o t i n t e n t i o n a l l y provided as d r i n k -
b u t c a t t l e have had a c c e s s t o it, and have d r u n k i t a t t i m e s .
E f f o r t s a r e now b e i n g made t o p r e v e n t c a t t l e a c c e s s t o t h e h i g h F c o n t e n t water.
This should lessen t h e s e v e r i t y o f t h e f l u o r i d e toxicosis.
There a r e a p p a r e n t l y few f l u o r i d e c o n t a i n i n g emissions f r o m t h e stacks o f t h e g e n e r a t i n g p l a n t because f l u o r i d e c o n t e n t o f n a t i v e u n i r r i g a t e d p l a n t s i n t h e v i c i n i t y o f t h e R e s e a r c h Farm i s w e l l w i t h i n e x p e c t e d n o r m a l l i m i t s , e v e n a t t h e e n d o f t h e g r o w i n g season when F c o n t e n t w o u l d u s u a l l y be e x p e c t e d t o be a t i t s h i g h e s t l e v e l . We h a v e s e e n t h e e f f e c t o r a p p l i c a t i o n o f s e v e r a l o f t h e f a c t o r s t h a t influence the expression o f chronic f l u o r i d e toxicosis.
Those f a c t o r s are:
(1) amount o f f l u o r i d e i n g e s t e d ; (2) d u r a t i o n o f i n g e s t i o n : (3) b i o a v a i l -
122 TABLE I V FLUORIDE CONTENT OF ALFALFA AT RESEARCH FARM A n a l y s e s PPM F1 July August
June Area
I
Area I I B
4.0
10.8
13.0
7.7
October
8.6
36.9
59.2
23.3
' A n a l y s e s a r e o n d r y w t . b a s i s and a r e an a v e r a g e o f 3 samples.
TABLE V FLUORIDE CONTENT OF MIXED PASTURE AT RESEARCH FARM
June
A n a l y s e s PPM F1 July August
Area I A
40.4
28.
A r e a 111
40.1
19.9
54.5
Area I I I B
7.0
18.2
51.8
'Analyses a r e on d r y w t .
a
October 17.5 41.3
b a s i s and a r e an a v e r a g e o f 3 samples.
TABLE V I FLUORIDE ANALYSES (PPM) OF IRRIGATION SYSTEM WATER
July
Time August ~
Area I A r e a I1
5.5 5.6
Area I11
October ~~
4.7
5.8 5.8
a b i l i t y o f f l u o r i d e i n g e s t e d ; (4) s p e c i e s o f a n i m a l i n v o l v e d ; (5) age a t t i m e of excessive f l u o r i d e ingestion:
(6) l e v e l o f n u t r i t i o n ;
t i o n s i n ingestion levels o f f l u o r i d e ( i n t e r m i t t e n t ingestion);
(7) f l u c t u a -
(8) g e n e r a l
s t a t e o f h e a l t h o f t h e animal; (9) a d d i t i v e o r s y n e r g i s t i c e f f e c t s f r o m
123 exposure t o o t h e r substances: c a u s e d b y p o o r management:
(10) o t h e r s t r e s s f a c t o r s such as t h o s e
and ( 1 1 ) i n d i v i d u a l b i o l o g i c a l r e s p o n s e .
T h i s r e p o r t i n c l u d e s work conducted through t h e f i f t h y e a r o f a planned
10 y e a r p r o j e c t .
On t h e b a s i s o f r e s u l t s o b t a i n e d t h u s f a r ,
i t i s apparent
t h a t e f f e c t s on t h e h e a l t h s t a t u s o f a n i m a l s r a i s e d i n t h e v i c i n i t y o f t h e coal-fired
power-generation
p l a n t and f e d o n v e g e t a t i o n i r r i g a t e d w i t h t h e
e f f l u e n t s f r o m t h e p o w e r p l a n t has been l i m i t e d t o d e n t a l f l u o r o s i s and s l i g h t l y i n c r e a s e d f l u o r i d e c o n t e n t o f t h e bone and u r i n e . Dental l e s i o n s c h a r a c t e r i s t i c o f f l u o r i d e t o x i c o s i s were present i n t h e c a t t l e t h a t had p e r m a n e n t i n c i s o r t e e t h f o r m , t h e R e s e a r c h Farm.
m i n e r a l i z e and e r u p t w h i l e on
The f l u o r i d e i n d u c e d d e n t a l l e s i o n s a r e u s u a l l y m o r e
s e v e r e i n t h e s e c o n d and s u b s e q u e n t p a i r s o f p e r m a n e n t i n c i s o r t e e t h t h a n i n the f i r s t pair,
i n d i c a t i n g an i n c r e a s e d l e v e l o f f l u o r i d e i n t a k e a s t h e
c a t t l e g r e w and i n c r e a s e d t h e i r v e g e t a t i o n and w a t e r i n t a k e .
The c l a s -
s i f i c a t i o n o f s l i g h t d e n t a l f l u o r o s i s i n t h e f i r s t p a i r o f permanent i n c i sors,
moderate e f f e c t s i n t h e second p a i r ,
and m a r k e d t o s e v e r e e f f e c t s i n
t h e t h i r d and f o u r t h p a i r s i n d i c a t e t h a t t h e a n i m a l s r e c e i v e d m o r e t h a n t h e t h r e s h o l d l e v e l o f f l u o r i d e when t h e t e e t h w e r e f o r m i n g .
For reference,
e s t a b l i s h e d f l u o r i d e t o l e r a n c e l e v e l s i n b o t h f e e d and w a t e r f o r s e v e r a l animal species are given i n Table V I I .
Tolerance l e v e l s f o r o t h e r species
c a n be e x t r a p o l a t e d . When i n g e s t i o n o f e l e v a t e d l e v e l s o f f l u o r i d e i s s u s p e c t e d a n d s u p p o r t i v e c l i n i c a l evidence i s lacking, w i t h a d i a g n o s i s (10).
urinalysis,
i f p r o p e r l y evaluated,
can a s s i s t
S e v e r a l v a r i a b l e s m u s t be k e p t i n m i n d when e v a l u -
a t i n g u r i n e analyses f o r fluoride.
A t a given f l u o r i d e intake level,
older
animals w i l l v o i d more f l u o r i d e i n t h e u r i n e than w i l l younger animals. The f l u o r i d e c o n t e n t o f t h e u r i n e o f n o r m a l c a t t l e i n g e s t i n g a n o r m a l d i e t fluctuates,
b u t i t i s u s u a l l y l e s s t h a n 6 ppm F.
The a v e r a g e u r i n e f l u o r i d e c o n t e n t o f m o s t o f t h e a n i m a l s o n t h e R e s e a r c h F a r m was b e l o w t h e l e v e l o f h e a l t h c o n c e r n . and bone do,
however,
The f l u o r i d e l e v e l s i n u r i n e
r e f l e c t an e l e v a t e d f l u o r i d e i n t a k e b y these animals.
T h i s has been c o n f i r m e d b y t h e d e n t a l c l a s s i f i c a t i o n o f c a t t l e r a i s e d o n t h e f a r m and u r i n a r y f l u o r i d e l e v e l s i n some a n i m a l s .
Urinary fluoride i s
an i n d e x o f t h e e x p o s u r e t o t h i s e l e m e n t a l t h o u g h w i d e v a r i a t i o n s c a n be expected.
An i n c r e a s e i n t h e b o n e a n d u r i n e f l u o r i d e i s a l s o c o n s i s t e n t
w i t h t h e i n c r e a s e d a m o u n t o f f l u o r i d e i n t h e w a t e r and v e g e t a t i o n . M o n i t o r i n g o f a n i m a l h e a l t h w i l l c o n t i n u e o n t h i s R e s e a r c h Farm.
Samples
w i l l be a n a l y z e d f o r s e v e r a l e l e m e n t s t h a t may b e s y n e r g i s t i c o r a n t a g o n i s -
t i c t o b i o l o g i c a l e f f e c t s o f i n g e s t e d f l u o r i d e s on c a t t l e .
124 TABLE V I I A G U I D E TO FLUORIDE TOLERANCE' LEVELS I N FEED AN0 WATER FOR DOMESTIC
ANIMALS~
BASED ON CLINICAL
SIGNS
AND LESIONS
Species
2.5
Cattle,
D a i r y and B e e f H e i f e r s
30
Cattle,
Dairy, Mature
40
3 -
Cattle,
Beef, Mature
50
4 -
Cattle,
Finishing
Sheep,
Breeding
Lambs,
Feeder
12
100
60
4
-
-
6
a 15
5 12
150
-
Horses
60
4-
Swine, G r o w i n g
70
5 -
8 15
a a
Turkeys, Growing
100
10 - 12
Chickens, Growing
150
10
Dogs, G r o w i n g Mink, Growing
-
13
50
3 -
8
45
3 -
a
1 8 i o l o g i c a l e f f e c t s depend on t h e f a c t o r s m e n t i o n e d i n t h e t e x t . 'The v a l u e s s h o u l d b e r e d u c e d p r o p o r t i o n a l l y w h e n b o t h w a t e r a n d f e e d c o n t a i n a p p r e c i a b l e amounts o f f l u o r i d e s . 3 T h i s i s a s u g g e s t e d g u i d e when F i n t h e f e e d i s e s s e n t i a l l y t h e s o u r c e o f fluoride. T o l e r a n c e s b a s e d on s o d i u m f l u o r i d e o r o r o t h e r f l u o r i d e s o f s im i1a r t o x ic it y. 4The a v e r a g e a m b i e n t a i r t e m p e r a t u r e and t h e p h y s i c a l and b i o l o g i c a l a c t i v i t y o f t h e a n i m a l s i n f l u e n c e t h e a m o u n t o f w a t e r consumed and hence t h e w i d e range o f t o l e r a n c e l e v e l s suggested. For a c t i v e animals i n a warm c l i m a t e , t h e l o w e r v a l u e s s h o u l d be u s e d as c r i t i c a l l e v e l i n d i c a tors. REFERENCES
1. S h u p e JL ( 1 9 6 7 ) I n : I V t h I n t e r n M e e t i n g o f t h e W o r l d A s s o c f o r B u i atrics,
2.
P u b l No 4.
Zurich,
Shupe JL, M i n e r ML. V e t Res 24:964-984
S w i t z e r l a n d , p p 15-30
Greenwood DA,
H a r r i s LE,
S t o d d a r d GE (1953) Amer J
3. S h u p e JL, A l t h e r EW ( 1 9 6 6 ) I n : E i c h l e r 0, F o r a n A, H e r k e n H, W e l c h AD, S m i t h FA ( e d s ) Handbook o f E x p e r i m e n t a l P h a r m a c o l o g y . S p r i n g e r - V e r l a g , New York, 20:307-354 4.
S h u p e J L , O l s o n AE ( 1 9 8 4 ) I n : S h u p e J L . L e o n e NE, P e t e r s o n HB ( e d s ) F l u o r i d e s - E f f e c t s o n V e g e t a t i o n , A n i m a l s and Humans. P a r a g o n P r e s s , p p 319-338
5. Shupe J L (1970) Amer V e t P u b l , Wheaton,
Ill, pp 288-301
125 6. J o h n s o n LC ( 1 9 6 5 ) I n : S i m o n s JH ( e d ) F l u o r i n e C h e m i s t r y , V o l 4. Acad e m i c Press, New York, pp 424-441 7. M i l l e r GW,
Shupe JL (1962) Amer J V e t Res 23(92):24-31
8. Shupe JL (1961) Can V e t J 2 3 6 9 - 3 7 6 9. A A F C O ( 1 9 8 2 ) O f f i c i a l P u b l i c a t i o n . Assoc Amer Feed C o n t r o l O f f i c i a l s , I n c , C h a r l e s t o n , W Va 10. Shupe JL. H a r r i s LE, Greenwood DA, B u t c h e r JE, N i e l s o n HM (1963) Amer J V e t Res 24:300-30
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 127-134 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
127
DENTAL LESIONS I N CATTLE AND SHEEP DUE TO INDUSTRIAL POLLUTION CAUSED BY COAL COMBUSTION
FRANKLIN
RIET-CORREA~,
MARIA
c.
M ~ N D E Z ~ ANA , L. SCHILD~.
JOXO A.
OLIVEIRA~,
AND O D A I R ZENEBON' ' L a b o r a t b r i o R e g i o n a l De D i a g n 6 s t i c o . 96100 P e l o t a s , RS., 5 r a z i l
'Institute
Szo P a u l o , SP.,
A d o l f o Lutz,
F a c u l d a d e De V e t e r i n s r i a .
Ufpel,
Brazil
ABSTRACT D e n t a l l e s i o n s i n c a t t l e w e r e s t u d i e d i n 11 f a r m s l o c a t e d w i t h i n 1.2 t o 9.6 km f r o m a c o a l c o m b u s t i o n t h e r m o e l e c t r i c p l a n t .
I n c i s o r t e e t h o f some
c a t t l e showed e v i d e n c e o f c h r o n i c f l u o r i d e p o i s o n i n g . a l t e r a t i o n was d e n t a l a t t r i t i o n .
The m o s t i m p o r t a n t
C a t t l e f r o m f a r m s n e a r t h e p l a n t had
complete a t t r i t i o n o f i n c i s o r s a t 6 t o 7 years.
The d e g r e e o f wear was
r e l a t e d t o t h e d i s t a n c e between t h e f a r m s and t h e p l a n t as a l i n e a r f u n c t i o n y = 4.11
+
(-0.42~);
lesions t o those o f cattle. ppm.
r 2 = 0.75
(P<0.05).
Sheep had s i m i l a r
Bone f l u o r i d e l e v e l s w e r e f r o m 265 t o 5673
Two m e c h a n i s m s seem t o b e r e s p o n s i b l e f o r d e n t a l l e s i o n s :
sive ei'fect o f p a r t i c u l a t e matter, especially s i l i c a , atmosphere,
t h e abra-
emitted into the
and t h e e f f e c t o f f l u o r i d e d e c r e a s i n c t h e e n a m e l r e s i s t a n c e .
INTRODUCTION F l u o r i d e i n t o x i c a t i o n has been r e c e n t l y d e s c r i b 2 d i n B r a z i l a s a conseq u e n c e o f i n d u s t r i a l p o l l u t i o n c a u s e d b y p h o s p h a t e F r o c e s s i n g p l a n t s (1). I n a n a r e a w i t h i n t h e m u n i c i p a l i t y o f Bag&. s t a t e o f R i o G r a n d e d o S u l , where a c o a l combustion e l e c t r i c power p l a n t i s located,
many f a r m e r s
c l a i m e d t h a t a t m o s p h e r i c e m i s s i o n o f t h i s p l a n t caused d e n t a l a t t r i t i o n i n animals.
The o b j e c t i v e o f t h i s w o r k was t o s t u d y d e n t a l l e s i o n s i n c a t t l e
and sheep f r o m t h e area.
MATERIALS AND METHODS D e n t a l l e s i o n s were s t u d i e d i n 11 f a r m s l o c a t e d a t d i f f e r e n t d i s t a n c e s and d i r e c t i o n s f r o m t h e t h e r m o e l e c t r i c p l a n t .
A t each farm,
incisor teeth
o f a v a r i a b l e n u m b e r o f c a t t l e ( T a b l e I)w e r e o b s e r v e d a n d p h o t o g r a p h e d , making b l a c k and w h i t e p r i n t s .
The d e g r e e o f l e s i o n o f e a c h i n c i s o r was
TABLE I EFFECT OF INDUSTRIAL POLLUTION ON I N C I S O R LESION AND ATTRITION Farma
Number o f cattle observed
Distance from p l a n t (krn) min. max. X
OF
CATTLE
Number o f i n c i s o r t e e t h a f f e c t e d w i t h each d e g r e e o f l e s i o n 0 1 2 3 4
Mean d e g r e e o f lesion
Mean d e g r e e of a t t r i t i o n ( X ? SX)
0.55
3 . 4 8 f 0.27
0.36
2.24
0
6 0 0
EF
34
1.2
3.7
2.45
80
12
11
1
RM
37
4.8 5.4
3.1 3.1
171
59
13
1
45
14
1
0.21
TA
26
1.4 0.8
DX
30
3.3
8.0
5.65
120
47
3
0
0
0.31
EC
30
3.3
6.8
5.05
85
16
1
0
0.18
---
GL
38
4.5
6.8
5.65
216
34
5
0 0
0
0.17
224 f 0 . 1 8
ES
37
4.5
9.0
6.75
200
70
0
0
0
170 k 0.18
PF
21
5.5
9.6
7.55
140
18
1
JM
35
6.4
9.5
7.95
147
38
0 3
EL
28
7.6
9.4
8.5
179
32
9
0 0 0
0.26 0.13
a
The d e g r e e o f l e s i o n o f i n c i s o r t e e t h f r o m f a r m JE t h e Dhotos o b t a i n e d
0 2
f
_--
0.27
141 k 0.22
56
f
0.23 0.25
was n o t d e t e r m i n e d due t o t h e low q u a l i t y
Of
0.26
c l a s s i f i e d i n t o t h e f o l l o w i n g s c o r e s : 0) w i t h o u t l e s i o n s ; 1 ) s l i g h t m o t t l i n g o f e n a m e l ; 2) d e f i n i t e m o t t l i n g ; 3) d e f i n i t e m o t t l i n g . hypocalcification:
h y p o p l a s i a and
4) d e f i n i t e m o t t l i n g , hypoplasia o r hypocalcification
and i n c r e a s e d w e a r when c o m p a r e d w i t h a d j a c e n t i n c i s o r s . p l e t e wear were n o t considered.
T e e t h w i t h com-
The mean s c o r e o f a l l p e r m a n e n t i n c i s o r
t e e t h o b s e r v e d i n e a c h f a r m was d e s i g n e d as mean d e g r e e o f l e s i o n .
The
mean d e g r e e o f l e s i o n i n r e l a t i o n t o t h e d i s t a n c e f r o m t h e p l a n t was studied by regression analysis. Dental
a t t r i t i o n o f i n c i s o r t e e t h was s t u d i e d i n 6 f a r m s w i t h r e c o r d s of
a n i m a l ages.
The age c a l c u l a t e d b y i n c i s o r s w e a r was d e t e r m i n e d b y
c o m p a r i s o n w i t h b l a c k and w h i t e p r i n t s f r o m n o r m a l c a t t l e b e l o n g i n g t o t h e Pelotas U n i v e r s i t y farm. c a l c u l a t e d b y t h e wear, tion.
The d i f f e r e n c e ( i n y e a r s )
b e t w e e n t h e age
a n d t h e r e a l age was d e s i g n a t e d a s d e g r e e o f a t t r i -
The mean d e g r e e o f a t t r i t i o n i n e a c h f a r m ,
i n relation t o the
d i s t a n c e f r o m t h e p l a n t , was s t u d i e d b y r e g r e s s i o n a n a l y s i s . L e s i o n s i n m o l a r and p r e m o l a r t e e t h w e r e o b s e r v e d i n 20 s l a u g h t e r e d cows. I n c i s o r t e e t h f r o m t h e s e a n i m a l s were f i x e d i n lox f o r m a l i n e ,
decalcified
w i t h f o r m i c a c i d and s o d i u m c i t r a t e and embedded i n p a r a f f i n .
Sections
w e r e s t a i n e d w i t h h e m a t o x y l i n and e o s i n . Humerus and m a n d i b l e s w e r e c o l l e c t e d f r o m s l a u g h t e r h o u s e s o r f r o m a n i m a l s w h i c h d i e d f r o m d i f f e r e n t causes.
F l u o r i d e l e v e l s i n bone ashes w e r e
determined w i t h t h e f l u o r i d e i o n electrode. D e n t a l l e s i o n s i n sheep w e r e s t u d i e d i n 20 a n i m a l s o f d i f f e r e n t a g e s f r o m f a r m s RM,
E F a n d JE.
The f a r m s w e r e c o d e d u s i n g t h e i n i t i a l s o f t h e
o w n e r ' s names. RESULTS I n c i s o r t e e t h o f some c a t t l e showed c h a l k y w h i t e , c o l o r a t i o n and h y p o p l a s i a o f enamel.
y e l l o w o r brown d i s -
Such l e s i o n s w e r e c o n s i d e r a b l e i n
t h o s e f a r m s n e a r t h e p l a n t and d i s c r e t e i n t h e o t h e r s . c a t t l e and p e r m a n e n t i n c i s o r s o b s e r v e d i n e a c h f a r m , o f individual i n c i s o r teeth,
t h e degree o f l e s i o n
t h e mean d e g r e e o f l e s i o n a n d t h e d i s t a n c e
f r o m t h e p l a n t a r e s h o w n i n T a b l e I. s h o w n i n F i g u r e 1.
The number of
Farms and p l a n t l o c a l i z a t i o n a r e
R e g r e s s i o n a n a l y s i s o f t h e d e g r e e o f l e s i o n as a
f u n c t i o n o f t h e d i s t a n c e f r o m t h e p l a n t i s p r e s e n t e d i n F i g u r e 2. D e n t a l a t t r i t i o n was t h e m o s t i m p o r t a n t a l t e r a t i o n .
Regression a n a l y s i s
o f t h e d e g r e e o f wear as a f u n c t i o n o f t h e d i s t a n c e f r o m t h e p l a n t i s p r e s e n t e d i n F i g u r e 3.
T e e t h o f many a n i m a l s w i t h 8 p e r m a n e n t i n c i s o r s
w e r e w o r n t o gum l e v e l ( F i g . 4). incisors,
I n some a n i m a l s w i t h 4 o r 6 p e r m a n e n t
t h e 1, was s h o r t e r t h a n o t h e r s a s a c o n s e q u e n c e o f w e a r ( F i g .
5).
130
F i g u r e 1. D r a w i n g o f t h e a r e a s t u d i e d s h o w i n g t h e f a r m s and p l a n t (UTPM) l o c a l i z a t i o n . T h e t o t a l s u r f a c e o f t h e 1 1 f a r m s i s a p p r o x i m a t e l y 11.500 h e c t a r e s and t h e w h o l e a r e a 30.000 h e c t a r e s .
2
Mean d e g r e e o f t o o t h l e s i o n i n e a c h f a r m ( y a s a f u n c t i o n o f F i g u r e 2. y = 0 . 4 7 1 7 + ( - 0 . 0 3 6 ~ ) ; r 4 . 4 2 (PcO.05). t h e d i s t a n c e f r o m t h e p l a n t (x).
131
DISTANCE
, mm
f i g u r e 4. S e v e n - y e a r - o l d cow f r o m f a r m TA. I n c i s o r s a r e w o r n t o gum l e v e l showing c o n s i d e r a b l e amounts o f secondary d e n t i n e . Severe g i n g i v a l hyperplasia.
I n many a n i m a l s p e r m a n e n t i n c i s o r s s h o w e d v a r i a b l e a m o u n t s o f s e c o n d a r y d e n t i n w h i c h a p p e a r e d d a r k b r o w n o r b l a c k ( F i g . 4); c a v i t y was exposed.
i n others the pulp
The m a j o r i t y o f a n i m a l s showed c o n s i d e r a b l e g i n g i v a l
h y p e r p l a s i a ( F i g . 4-5).
D e c i d u o u s i n c i s o r s a l s o showed e x c e s s i v e wear.
E x c e s s i v e and uneven a t t r i t i o n was a l s o o b s e r v e d i n m o l a r and p r e m o l a r teeth. H i s t o l o g i c a l l e s i o n s o f p e r m a n e n t i n c i s o r t e e t h showed h y p e r p l a s i a o f t h e c e m e n t and p r o l i f e r a t i o n o f s e c o n d a r y d e n t i n .
I n many t e e t h ,
t h i s second-
132 a r y d e n t i n had m o r e w e a r t h a n p r i m a r y d e n t i n and e n a m e l , c a v i t y impacted w i t h d e t r i t u s .
l e a v i n g an e m p t y
I n o t h e r s t h e p u l p c a v i t y was exposed.
The
g i n g i v a showed m a r k e d h y p e r p l a s i a o f t h e s t r a t i f i e d squamous e p i t h e l i u m .
T h r e e - y e a r - o l d cow f r o m f a r m RM. Central incisors are shorter F i g u r e 5. Gingival hyperplasia. t h a n I 2 a s a c o n s e q u e n c e o f wear.
TABLE I 1 FLUORIDE LEVELS I N BOVINE BONE ASHES FROM DIFFERENT FARMS Age
Farm
F l u o r i d e (ppm) Humerus
DX
5.65
Mandible
559-491-475 41 3-21 Oa
adults
PF
7.55
3 years
265
ES
6.75
4 years
594
386
RM
3.10
3 years
GL
5.65
adults
681
702-533-508 490-461
JE
4.30
adults
832
954-772
5673-3966 3680-31 56 1526-1 502 11 72-1 091 1081
2931 -2580-2554 2244-2087-1 990 1852-1 770-1 476 1281-975-849 81 0-747-733 714-667-528 499-435-389
1937
1317
adult
EF
2.45
1-7 y e a r s
aEach v a l u e i s f r o m a d i f f e r e n t a n i m a l .
133 T e e t h o f n e a r l y a l l s h e e p w i t h 8 p e r m a n e n t i n c i s o r s w e r e w o r n t o gum level.
Wear was a l s o e x c e s s i v e i n a n i m a l s w i t h 2 p e r m a n e n t i n c i s o r s :
in
sheep w i t h 4 o r 6 p e r m a n e n t i n c i s o r s t h e I 1 was s h o r t e r t h a n t h e o t h e r s . R e s u l t s o f f l u o r i d e d e t e r m i n a t i o n s i n bone a s h e s a r e shown i n T a b l e 11.
DISCUSSION The r e s u l t s o f t h i s w o r k e s t a b l i s h e d t h e d i a g n o s i s o f f l u o r i d e p o i s o n i n g as a c o n s e q u e n c e o f c o a l c o m b u s t i o n ;
otherwise it i s clear t h a t fluoride i s
not the only f a c t o r responsible f o r the occurrence o f dental lesions i n c a t t l e and sheep i n t h e a r e a s t u d i e d .
Dental lesions o f t h e most severe
degree, s u c h as t h o s e s c o r i n g 3 o r 4 i n enamel h y p o p l a s i a , w e r e o b s e r v e d m a i n l y i n t h e farms l o c a t e d near t h e plant.
T o x i c l e v e l s o f f l u o r i d e were
d e t e c t e d i n bone a s h e s i n a n i m a l s f r o m t h e s e f a r m s .
M o s t i n c i s o r s showed
l e s i o n s w i t h s c o r e 1 , r e p r e s e n t e d b y s l i g h t m o t t l i n g o f enamel, be o b s e r v e d i n a n i m a l s n o t e x p o s e d t o f l u o r i d e .
which can
T h i s i s confirmed by t h e
l o w c o e f f i c i e n t o f i n d e t e r m i n a t i o n b e t w e e n t h e d e g r e e o f l e s i o n s and t h e distance from the plant. F l u o r i d e i n t o x i c a t i o n was d e s c r i b e d i n a p r e v i o u s p a p e r i n t h e m u n i c i p a l i t y o f R i o G r a n d e (1).
palities,
Comparing t h e a l t e r a t i o n s observed i n b o t h munici-
we c a n c o n c l u d e t h a t d e n t a l l e s i o n s c h a r a c t e r i s t i c o f f l u o r o s i s
w e r e l e s s m a r k e d a n d f l u o r i d e l e v e l s w e r e l o w e r i n Bag6 a s a c o n s e q u e n c e o f coal combustion, consequence o f
t h a n i n R i o Grande where t h e d i s e a s e o c c u r r e d as a
rock phosphate processing.
s i t u a t e d 5.5 km f r o m t h e f a c t o r i e s ,
I n R i o Grande,
i n a farm
t h e m e a n d e g r e e o f l e s i o n w a s 1.99,
w h i l e i n B a g ’ e i n a f a r m l o c a t e d 2.4 km f r o m t h e p l a n t t h e mean d e g r e e was
0.55. Grande.
O t h e r w i s e d e n t a l a t t r i t i o n was m o r e m a r k e d i n Bag& t h a n i n R i o i n d i c a t i n g t h a t f l u o r i d e p o i s o n i n g i s n o t t h e u n i q u e cause o f t e e t h
l e s i o n s due t o i n d u s t r i a l p o l l u t i o n caused by c o a l combustion. U n t i l O c t o b e r 1984 when t h i s w o r k was f i n i s h e d ,
t h e amounts o f p a r t i c u -
l a t e s e m i t t e d i n t o t h e atmosphere were very high,
as t h e p l a n t w i t h a
c a p a c i t y o f 126 m e g a v o l t s was e q u i p p e d w i t h o n l y 2 e l e c t r o s t a t i c t a t o r s o f v e r y l o w e f f i c i e n c y (0% t o 50%).
precipi-
Taking i n t o account a d a i l y
c o n s u m p t i o n o f a p p r o x i m a t e l y 1 8 6 7 t o n s o f c o a l , c o n t a i n i n g 52% t o 5 9 % o f a s h e s , t h e p r o d u c t i o n o f p a r t i c u l a t e s w a s a b o u t 855 t o n s d a i l y , o f w h i c h
40% t o 80% w e r e e l i m i n a t e d w i t h t h e f l u e g a s ( S e c r e t a r y o f H e a l t h , E n v i r o n mental Department,
R i o Grande do Sul.
1985.
Unpublished data).
Such
p a r t i c u l a t e s w e r e composed m a i n l y o f S i O 2 (70%), A1203 (21.4%) and Fez03
(4.1%) ( A n d r a d e A.
1985. U n i v e r s i d a d e F e d e r a l d o R i o G r a n d e d o Sul.
Unpub-
lished data). Considering t h e abrasive e f f e c t o f these elements deposited i n considera b l e amounts on pastures,
it i s evident that a t least two factors are
134 r e s p o n s i b l e f o r t o o t h wear:
a b r a s i o n caused b y t h e f l y a s h d u r i n g g r a z i n g
and c h e w i n g and t h e e f f e c t o f f l u o r i d e d e c r e a s i n g t h e enamel r e s i s t a n c e . A b r a s i o n b y p h y s i c a l a g e n t s i s a w e l l known mechanism o f t o o t h wear, i n sheep i n w h i c h t h e i n g e s t i o n o f s o i l , i n feces,
mainly
e s t i m a t e d b y t h e amount o f s i l i c a
i s r e l a t e d t o t o o t h a t t r i t i o n (2.3).
The p o s s i b i l i t y o f o t h e r
elements contained i n t h e p a r t i c u l a t e c o n t r i b u t i n g t o t h e e t i o l o g y o f t e e t h l e s i o n s c a n n o t be e x c l u d e d . D e n t a l l e s i o n s o f c a t t l e and sheep d e s c r i b e d i n t h i s p a p e r w e r e c e r t a i n l y an i m p o r t a n t c a u s e o f e c o n o m i c l o s s e s t o t h e b e e f and w o o l i n d u s t r y o f t h e area studied.
fortunately,
i n O c t o b e r 1 9 8 4 t h e p l a n t i n s t a l l e d t w o new
e l e c t r o s t a t i c p r e c i p i t a t o r s a b l e t o r e m o v e 99.4% o f t h e p a r t i c u l a t e s formed.
They a l s o remove 91.3% o f t h e f l u o r i d e ,
t h e f l u e g a s (4).
t h u s l e a v i n g o n l y 7.6% i n
For these reasons i t i s expected t h a t t h e seriousness o f
d e n t a l l e s i o n s w i l l decrease. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y EMBRAPA/UEPAE/Pelotas. REFERENCES 1.
R i e t - C o r r e a F, O l i v e i r a JA. M6ndez MC, S c h i l d AL ( 1 9 8 3 ) P e s q V e t B r a s 3: 107-114
2.
A r n o l d GW, McManus WR, 107
3.
H e a l y WB,
4.
Page AL,
Bush I G (1966) A u s t J Exp A g r i c Anim Husb 6 : l O l -
L u d w i g TG (1965) N Z J A g r i c Res 8:737-752 E l s e e w i AA.
S t r a u g h a n I R (1979) R e s i d u e R e v i e w s 71:83-120
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985,Studies in Environmental Science, Volume 27, pp. 135-141 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands
HEALTH EFFECTS
OF A H Y D R O G E N
135
FLUORIDE E M I T T I N G GLASS F I B R E PLANT I N T H E
NETHERLANDS KLAAS BIERSTEKER AND JAN S. M.
BOLEIJ
Department o f Environmental Health, Agricultural F o u l k e s w e g 43. 6703 BM Wageningen, The N e t h e r l a n d s
University,
Gen.
e m i t t i n g 12-15 k g HF/hr.
were
ABSTRACT The h e a l t h e f f e c t s o f a g l a s s f i b r e p l a n t ,
A v i l l a g e u p w i n d a t 1 5 km
studied i n a v i l l a g e close t o the plant. d i s t a n c e served as c o n t r o l i n t h e study.
The s o c i o e c o n o m i c d a t a o f b o t h
p o p u l a t i o n s i n d i c a t e d good c o m p a r a b i l i t y e x c e p t f o r a h i g h e r l e v e l o f environmental concern i n t h e p o l l u t e d v i l l a g e . The c o l l e c t e d d a t a showed t h a t l o c a l l y g r o w n v e g e t a b l e s had a h i g h e r f l u o r i d e c o n t e n t i n t h e exposed v i l l a g e . only slightly,
however,
Urine concentrations differed
p r o b a b l y as a r e s u l t o f l i m i t e d consumption o f t h e
l o c a l vegetables. Lung f u n c t i o n s f o r 40 d a y s ,
o f 20 s c h o o l c h i l d r e n i n b o t h v i l l a g e s w e r e t e s t e d d a i l y
a f t e r t h e c h i l d r e n had been i n t h e o u t d o o r a i r ,
p o t e n t i a l acute e f f e c t s o f a i r pollution.
increased p o l l u t i o n l e v e l i n t h e exposed v i l l a g e , f u n c t i o n was observed.
t o study
There were a few days w i t h b u t no e f f e c t on lung
The e x p o s e d v i l l a g e h a d m o r e p e o p l e w i t h a h i s t o r y
o f pulmonary symptoms t h a n t h a t o f t h e c o n t r o l s .
T h i s d i f f e r e n c e may be
due t o t h e h i g h e r degree o f environmental concern o f t h e people i n t h e e x p o s e d v i 1 1 age. The s t u d e n t s who p a r t i c i p a t e d i n t h e s t u d y consumed a m e a l o f l o c a l l y g r o w n k a l e and c o l l e c t e d t h e i r u r i n e f o l l o w i n g t h e meal. l e v e l s o f t h e k a l e r e a c h e d 100 ppm. rapid resorption,
The f l u o r i d e
Urinary content d i d not contradict the
b u t stayed below danger l e v e l .
INTRODUCTION T h e r e was c o n c e r n a b o u t r e s p i r a t o r y e f f e c t s and q u a l i t y o f l o c a l l y g r o w n vegetables i n a v i l l a g e located close t o a glass f i b r e p l a n t i n the north of
t h e Netherlands.
The g l a s s f i b r e p l a n t ( 8 0 0 employees,
28000 t o n s
p r o d u c t i o n a n n u a l l y ) u s e d CaF2 i n t h e g l a s s o v e n s t o i m p r o v e t h e q u a l i t y o f the fibres.
It was assumed t h a t ,
due t o t h e h i g h t e m p e r a t u r e ,
HF w a s
e m i t t e d as gas t o g e t h e r w i t h t h e e m i s s i o n s o f t h e oven b u r n e r s (SO2,
NO,).
136 T h e r e w e r e s i x c h i m n e y s , 30 m h i g h t o d i s p e r s e t h e e m i s s i o n s .
The p l a n t
w a s i n c o n t i n u o u s o p e r a t i o n a n d t h e HF e m i s s i o n v a r i e d , a c c o r d i n g t o t h e p l a n t d i r e c t o r , b e t w e e n 12-15 k g p e r h o u r .
Concern about h e a l t h e f f e c t s
was s t i m u l a t e d b y t h e p r o v i n c i a l h e a l t h i n s p e c t o r ,
who a d v i s e d p e o p l e n o t
t o consume m o r e t h a n t w i c e a week l o c a l l y g r o w n v e g e t a b l e s .
This advice
was b a s e d o n a s m a l l s a m p l e o f v e g e t a b l e s t h a t had been a n a l y z e d f o r f l u o r i d e content. I n an e a r l i e r s t u d y i n t h e s o u t h o f t h e N e t h e r l a n d s (1,Z)
u r i n e had been
used f o r b i o l o g i c a l m o n i t o r i n g o f t h e f l u o r i d e exposure o f school c h i l d r e n l i v i n g c l o s e t o an a l u m i n u m s m e l t e r .
The f l u o r i d e e m i s s i o n s t h e r e w e r e i n
d u s t f o r m and i t was d e m o n s t r a t e d t h a t p r o p e r w a s h i n g o f t h e v e g e t a b l e s b e f o r e c o o k i n g removed most o f t h e f l u o r i d e s . Compared w i t h o t h e r f l u o r i d e compounds, t o t h e s k i n and mucosa.
HF i s known t o be v e r y a b r a s i v e
A s e c o n d p o i n t o f i n t e r e s t was t h e r e f o r e w h e t h e r
an e f f e c t on t h e r e s p i r a t o r y t r a c t o f s c h o o l c h i l d r e n c o u l d be d e m o n s t r a t e d i n t h e e x p o s e d v i 1 1age.
A group o f environmental
h y g i e n e s t u d e n t s d e v e l o p e d a s t u d y d e s i g n and
c a r r i e d o u t t h e f i e l d work.
T h i s paper i s a condensed v e r s i o n o f t h e f i n a l
r e p o r t t h a t was s u b m i t t e d t o t h e l o c a l and p r o v i n c i a l h e a l t h a u t h o r i t i e s , t h e p l a n t d i r e c t o r and t h e p o p u l a t i o n o f t h e v i l l a g e W e s t e r b r o e k i n 1978. MATERIALS AND METHODS Design o f t h e study The s t u d y a t t e m p t e d t o answer t w o q u e s t i o n s : a.
Is t h e r e a demonstrable e f f e c t on pulmonary h e a l t h i n t h e v i l l a g e ?
b.
Is t h e r e a d e n t a l h e a l t h r i s k i n c o n s u m i n g l o c a l v e g e t a b l e s ?
To a n s w e r t h e f i r s t q u e s t i o n ,
l u n g f u n c t i o n t e s t s o f 20 c h i l d r e n w e r e
s t u d i e d d a i l y on s c h o o l g o i n g d a y s i n t h e m o r n i n g a f t e r p l a y t i m e o u t d o o r s i n W e s t e r b r o e k a n d i n a c o n t r o l v i l l a g e l o c a t e d 1 5 km f r o m t h e p l a n t a n d s o c i o e c o n o m i c a l l y comparable.
I n addition the parents o f schoolchildren
were asked t o r e g i s t e r on s p e c i a l forms, schools,
d i s t r i b u t e d weekly by the two
w h e t h e r t h e r e had b e e n r e s p i r a t o r y s y m p t o m s i n t h e f a m i l y i n t h e
p a s t week. To a n s w e r t h e s e c o n d q u e s t i o n v e g e t a b l e s w e r e c o l l e c t e d t h r e e t i m e s and analyzed
f o r f l u o r i d e content.
lyzed f o r f l u o r i d e content. l o o k i n g meals,
A l s o t h e u r i n e o f s c h o o l c h i l d r e n was ana-
Finally,
t h e students organized two s i m i l a r
o n e w i t h p o l l u t e d k a l e and one w i t h c l e a n k a l e ,
t o register
t h e e f f e c t on e x c r e t i o n o f f l u o r i d e s w i t h urine. The i m m i s s i o n F l u o r i d e concentrations c l o s e t o t h e p l a n t were determined several t i m e s and i t was d e m o n s t r a t e d
by sampling separately f o r
particulate fluorides
t h a t p r a c t i c a l l y a l l o f t h e f l u o r i d e was p r e s e n t a s g a s ( T a b l e I).
137 TABLE I PRESENCE OF FLUORIDES I N DOUBLE FILTER SAMPLES I N EXPOSED VILLAGE (4 HOUR AVERAGES )
particJes
time
21 21-22 25 31 36 37 38 38
w/mJ
16.3 3.5 3.4 3.0 4.3 16.7 6.0 13.6
13.00-1 7.00 22.00- 2.00 5.00- 9.00 5.00- 9.00 13.00-1 7.00 5.00- 9.00 5.00- 9.00 13.00-17.00
0 0.5 0 0 0 0 0 0.1
TABLE I 1 FREQUENCY OF FLUORIDE AN0 SO2 CONCENTRATIONS AT THE PLAYGROUNDS OF THE SCHOOLS F Iuorjde ( i d m )
Exposed village A
Control village B
<1 1-2 2-3 >3
37 0 1 (+) 1 (”)
37 1 0 0
Total
39
38
Exposed village A
Control village B
90
26 6 4 1 0 3
22 9 8 1 1 0
Total
40
SO2 (w/m3)
(“1
41
average A: average B: (+’)A-day 21: (+)A-day 24:
0.40 ug/m3 0.12ug/rn3 8.50 ugF-/m3 2.30 vgF-/m3
average A:
18.35
average B: 16.13 (“)A-day 7: 107.00 day 21: 96.00 day 29: 232.00
ug/m33 vg/m pgS02/m3 vgSO2/m; ugS02/m
138
SO2
and HF w e r e m e a s u r e d a s 1 h o u r a v e r a g e s a t t h e p l a y g r o u n d o f t h e
s c h o o l e a c h m o r n i n g when t h e c h i l d r e n p e r f o r m e d l u n g f u n c t i o n t e s t s a f t e r p l a y t i m e outdoors.
As s h o w n i n T a b l e I 1 t h e r e w e r e o n l y t w o d a y s w i t h
e l e v a t e d HF c o n c e n t r a t i o n s and t h r e e d a y s w i t h e l e v a t e d
SO2
concentrations
a t t h e exposed school. RESULTS AND D I S C U S S I O N E f f e c t s on l u n q f u n c t i o n Both schools provided
20
c h i l d r e n who w e r e w i l l i n g t o p a r t i c i p a t e i n t h e
l u n g f u n c t i o n t e s t i n g t h a t t o o k p l a c e e a c h d a y a t a b o u t 1O:OO a.m.
The
t e s t s w e r e c o n d u c t e d w i t h a w e t Lode s p i r o m e t e r and a pneumotachograph. The d a t a w e r e f e d i n t o a c o m p u t e r and p r i n t o u t s f o r e a c h c h i l d and each g r o u p o f c h i l d r e n w e r e a v a i l a b l e a t t h e end o f t h e s t u d y . F o r a n a l y s i s o f an e f f e c t on l u n g f u n c t i o n s ,
d a y s 21 and 24 w e r e c o m p a r e d
w i t h s u r r o u n d i n g d a y s and a l s o w i t h d a y s w i t h s i m i l a r w e a t h e r c o n d i t i o n s , b u t w i t h o u t HF i m m i s s i o n s . B o t h t y p e s o f s t a t i s t i c a l a n a l y s i s f a i l e d t o d e m o n s t r a t e a l o w e r i n g o f l u n g f u n c t i o n s o n t h e p o l l u t e d days. d a y w i t h an i n c r e a s e d SO2 c o n c e n t r a t i o n o f ZOO+ p g S02/m3
The s i n g l e
also failed t o
d e m o n s t r a t e an e f f e c t o n l u n g f u n c t i o n p a r a m e t e r s o f t h e s c h o o l c h i l d r e n . A m u l t i p l e r e g r e s s i o n a n a l y s i s f o r a l l days, temperature,
r e l a t i v e humidity,
v a r i a b l e s demonstrated e f f e c t s o f time, villages,
u s i n g number o f t h e day,
r u n n i n g nose, SO2 and HF as i n d e p e n d e n t t e m p e r a t u r e and common c o l d i n b o t h
b u t no e f f e c t o f t h e t w o a i r p o l l u t a n t s i n t h e e x p o s e d v i l l a g e .
R e s p i r a t o r y symptoms The p a r e n t s of a l l s c h o o l c h i l d r e n w e r e a s k e d w e e k l y t o l i s t r e s p i r a t o r y s y m p t o m s t h a t h a d o c c u r r e d i n t h e f a m i l y o n a f o r m w i t h q u e s t i o n s s u c h as: d u r i n g t h e l a s t week, w h i c h members o f t h e f a m i l y had a f e v e r , a r u n n i n g nose,
a sore throat,
etc.
T a b l e 111 shows t h e p e r c e n t a g e o f p a r t i c i p a n t s
i n b o t h v i l l a g e s who had s y m p t o m s d u r i n g a 12-week
p e r i o d . T h e r e w e r e 316
r e s p o n d e n t s i n W e s t e r b r o e k and 2 2 6 i n t h e c o n t r o l v i l l a g e .
Westerbroek
showed a h i g h e r p e r c e n t a g e w i t h symptoms and d o c t o r v i s i t s .
Whether t h i s
i s due t o g r e a t e r c o n c e r n a b o u t h e a l t h i n W e s t e r b r o e k t h a n i n t h e c o n t r o l population o r t o a i r p o l l u t i o n i s hard t o
prove.
An a t t e m p t t o c o r r e l a t e
w e e k l y a v e r a g e s o f HF p o l l u t i o n l e v e l s w i t h w e e k l y p r e v a l e n c e s o f symptoms d i d n o t s h o w s i g n i f i c a n t r e l a t i o n s . When t h e s c h o o l c h i l d r e n u n d e r w e n t a p h y s i c a l examination a t t h e beginning o f t h e study,
i t was e v i d e n t t h a t
W e s t e r b r o e k a l s o had m o r e c h i l d r e n w i t h a h i s t o r y o f r e s p i r a t o r y d i s e a s e . The r e a s o n f o r t h e d i f f e r e n c e s i s n o t c l e a r .
T h e r e was m o r e e n v i r o n -
m e n t a l c o n c e r n i n t h e e x p o s e d v i l l a g e and t h i s may h a v e i n f l u e n c e d t h e r e p o r t i n g o f symptoms.
139 TABLE 111 REPORTED RESPIRATORY SYMPTOMS I N THE TWO VILLAGES (AVERAGE RESPONDENTS) Exposed
PERCENTAGE
Control
a. 3
Running nose Fever Cough Sore t h r o a t Doctors v i s i t Absent from school o r from work
OF
9.1
1.2 8.6
0.5
7.9 2.9 1.2 0.8
4.5
1.3 1.5
Fluoride i n urine o f schoolchildren The p e r c e n t a g e o f s c h o o l c h i l d r e n c o n s u m i n g v e g e t a b l e s f r o m t h e i r own g a r d e n was l o w e r i n W e s t e r b r o e k t h a n i n t h e c o n t r o l v i l l a g e ( T a b l e I V ) . The u s e o f f l u o r i d e t a b l e t s b y t h e c h i l d r e n was l e s s t h a n 1 0 p e r c e n t . Compared w i t h t h e c h i l d r e n u s i n g f l u o r i d a t e d a r i n k i n g w a t e r i n t h e N e t h e r lands,
the l e v e l s w e r e low, and t h e r e were no s i g n i f i c a n t d i f f e r e n c e s
between t h e t w o v i l l a g e s .
The absence o f d i f f e r e n c e s i s p r o b a b l y due t o
t h e i r r e g u l a r consumption of
l o c a l vegetables containing elevated f l u o r i d e
levels. V e g e t a b l e s w e r e c o l l e c t e d t h r e e t i m e s i n t h e e x p o s e d v i l l a g e and i n t h e control village.
K a l e as a w i n t e r v e g e t a b l e was c h o s e n a s a good c o l l e c t o r
TABLE I V FLUORIDE CONCENTRATION ( P P M )
I N URINE
OF
THE SCHOOLCHILDREN
Use o f t o o t h p a s t e o r t a b l e t s w i t h FYes Period Exposed: Nov. March June Oct.
Number
1976 1977 1977 1977
Control : Nov. 1976 M a r c h 1977 J u n e 1977 Oct. 1977
23 20 34
No Average
0.41 0.40 0.45
za
0.41
15
0.45
34
0.53
17 20
0.44 0.32
Number
Average
37 39 36 39
0.38 0.37 0.31 0.31
19 18 19 15
0.31
0.30 0.31
0.28
140 TABLE V FLUORIDE CONTENT
SOURCE
OF
KALE I N WESTERBROEK WITH INCREASING DISTANCE FROM THE
Rank O r d e r distance
November unwashed washed
1
81 20 21 20 17 81
2 3 4 5 6 7 8 9 10 11 12 13 14 15
March unwashed washed
58
83
16
14 14 10 54
-
-
39 37 53 56 27 40 12
39 30 43 32 21 34 8
6 6 7 6 7
6 4
8
8
66
-
-
28 53 32 93 61 58 86 60
17 43 23 71 48 42 49
-
-
44 52 38 31
41 31 34 28
5 6 4 4 8
6
50
Control v i l l a g e 1 2 3 4
5
6
4 7
o f f l u o r i d e s i n e a r l i e r studies. g i v e n i n T a b l e V. s m e l t e r (2),
6 4 4
6
The d a t a f o r t w o o f t h e k a l e s a m p l e s a r e
I n c o n t r a s t w i t h e a r l i e r s t u d i e s around an aluminum
washing d i d n o t remove most o f t h e f l u o r i d e .
The k a l e e x p e r i m e n t During t h e i r stay, kale,
t h e s t u d e n t s consumed a k a l e meal t w i c e ( a m i x o f
p o t a t o e s and meat),
once p r e p a r e d w i t h u n p o l l u t e d k a l e and once
prepared w i t h l o c a l l y grown p o l l u t e d kale.
T h e i r u r i n e was c o l l e c t e d a t
r e g u l a r i n t e r v a l s f o r 24 h o u r s a f t e r t h e meals. r i d e c o n c e n t r a t i o n s f o r b o t h occasions.
Table V I gives t h e f l u o -
It i s e v i d e n t t h a t t h e k a l e i n t h e
exposed v i l l a g e s contained b i o l o g i c a l l y a v a i l a b l e f l u o r i d e s . c o n t e n t was 1 6 ppm i n f r e s h k a l e ( 9 4 ppm i n d r y r e s t ) , c o n t a i n e d 0.6 p p m ( 4 pm i n d r y r e s t ) .
The k a l e
the nonpolluted kale
The l e v e l o f t h e p o l l u t e d k a l e was
t y p i c a l f o r k a l e grown c l o s e t o t h e factory. D a i l y consumption o f l a r g e q u a n t i t i e s o f such k a l e m i g h t l e a d t o u n d e s i r a b l e u p t a k e o f f l u o r i d e s (3),
b u t i t i s i m p r o b a b l e t h a t t h i s occurs.
141 TABLE V I FLUORIDE CONTENT O F U R I N E I N P P M AFTER CONSUMPTION OF NONPOLLUTED A N D POLLUTED KALE H o u r s A f t e r N o n p o l l u t e d and P o l l u t e d M e a l Student
0-3
3-6
1 2 3 4 5 6 7 8
0.45 0.37 0.46 0.28 0.32 0.35 0.34 0.55
0.35 0.32 0.56 0.36 0.21 0.24 0.15 0.39
Day 1 6-10
0.64 0.61
0.50 0.59 0.32 0.88 0.21 0.55
10-24
0-3
3-6
0.41 0.57 0.54 0.36 0.46 0.64 0.23 0.47
1.43 1.19 1.72 1.16 1.28 1.23 1.70 1.67
2.66 2.14 3.61 3.33 ----
3.13 3.10 3.09
Day 2
6-10
10-24
0.86 1.56 1.12 2.55 0.63 1.82 2.68 2.03
0.89 0.98 0.89 0.90 0.92 1.12 0.92 0.88
CONCLUSIONS An a c u t e e f f e c t o f h y d r o g e n f l u o r i d e i m m i s s i o n on c h i l d r e n ' s l u n g f u n c t i o n s c o u l d n o t be demonstrated.
T h e r e was a s o m e w h a t h i g h e r r a t e o f s e l f -
r e p o r t e d r e s p i r a t o r y symptoms i n t h e exposed v i l l a g e .
W h e t h e r t h i s was due
more t o environmental concern than t o environmental d i f f e r e n c e s i n a i r q u a l i t y c o u l d n o t be e s t a b l i s h e d . As f o r t h e u p t a k e o f f l u o r i d e s w i t h l o c a l l y g r o w n v e g e t a b l e s ,
even an
u n u s u a l l y h i g h e x p e r i m e n t a l r e s u l t among t h e s t u d e n t s r e m a i n e d b e l o w t h e l e v e l t h a t c a u s e s e n a m e l damage.
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1. 2. 3.
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236
B i e r s t e k e r K. Z i e l h u i s RL, B a c k e r D i r k s 0, Van L e e u w e n P. Van R a a y A
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E n v i r o n m e n t a l P r o t e c t i o n Agency (1985) N a t i o n a l P r i m a r y R e g u l a t i o n s . F l u o r i d e . Fed Reg 50(93):20164-20175
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FLUORIDE
EMISSION FROM TUNNEL KILNS OF TILE WORKS
MAKOTO NAKAO AND F U M I O TANAKA S h i m a n e P r e f e c t u r a l I n s t i t u t e o f P u b l i c H e a l t h and E n v i r o n m e n t a l S c i e n c e , Nishi-Hamasada, Matsue, Shimane, 690-01, J a p a n ABSTRACT F l u o r i d e e m i s s i o n f r o m t u n n e l k i l n s has been assessed based on d a t a o b t a i n e d a t s e v e r a l t i l e works i n t h e Iwarni D i s t r i c t , P r e f e c t u r e , Japan. t h e f l u e gas.
w e s t e r n Shimane
The r e s u l t s showed an a v e r a g e e m i s s i o n o f 24 mgF/m3N i n
F o l l o w i n g an a l k a l i c l e a n i n g p r o c e s s t h e c o n c e n t r a t i o n
d e c r e a s e d t o 1 mgF/m3N.
The b u l k o f t h e f l u o r i d e i n t h e f l u e gas was i n
g a s e o u s f o r m , a n d m o s t o f t h e f l u o r i d e e m i t t e d may h a v e o r i g i n a t e d f r o m m a t e r i a l clay.
F l u o r i d e r e l e a s e r a t e i n t h e f i r i n g p r o c e s s was 40-60% i n
general. The q u a n t i t y o f f l u o r i d e e m i t t e d f r o m a t u n n e l k i l n was a p p r o x i m a t e l y i n p r o p o r t i o n t o t h e w e i g h t o f t h e product.
The w e i g h t o f t h e p r o d u c t c a n be
e s t i m a t e d f r o m t h e number o f p r o d u c t s o r f r o m t h e q u a n t i t i e s o f m a t e r i a l used.
Based on t h e s e d a t a ,
the t o t a l fluoride emission from tunnel k i l n s
t h r o u g h o u t t h e P r e f e c t u r e was
estimated.
The f l u o r i d e e m i s s i o n f a c t o r f o r
t u n n e l k i l n s was 1 2 0 g F / t o n o f m a t e r i a l used. INTRODUCTION Sekishugawara, a t i l e w i t h a 360-year h i s t o r y , i s manufactured i n t h e I w a m i D i s t r i c t . w h i c h i s l o c a t e d i n t h e w e s t e r n p a r t o f Shimane P r e f e c t u r e . Because o f t h e i n t r o d u c t i o n o f t u n n e l k i l n s and t h e housing.boom. as t h e h i g h q u a l i t y o f t h e t i l e , 1965,
r e a c h i n g 6 0 m i l l i o n p i e c e s a n n u a l l y 1 0 y e a r s ago,
5 y e a r s ago,
and now 1 9 0 m i l l i o n p i e c e s .
a t a t e m p e r a t u r e o f 1200-13OO0C,
as w e l l
p r o d u c t i o n has r a p i d l y grown s i n c e about Since t h e Sekishu-tile
20-100°C
o t h e r t i l e p r o d u c t i o n a r e a s i n Japan,
138 m i l l i o n p i e c e s i s fired
h i g h e r than t h a t employed i n
the r a t e a t which f l u o r i d e is
r e l e a s e d f r o m r a w m a t e r i a l i s c o n s i d e r e d t o be h i g h e r t h a n i n o t h e r p r o d u c t i o n areas.
F u r t h e r m o r e , due t o t h e i n c r e a s e i n t h e number o f k i l n s . t h e
larger kiln-size.
and r e g i o n a l c o n c e n t r a t i o n o f k i l n s i n r e c e n t y e a r s ,
the
p o s s i b i l i t y e x i s t s t h a t s e r i o u s l o c a l a i r p o l l u t i o n m i g h t r e a d i l y occur. I n fact,
damages t o v e g e t a t i o n and t h e s i l k w o r m i n d u s t r y h a v e been r e c o g -
n i z e d s i n c e around 1972 i n areas n e a r t i l e m a n u f a c t u r i n g works.
The
a u t h o r s h a v e been i n v o l v e d i n i n v e s t i g a t i n g t h e s t a t e o f a i r p o l l u t i o n b y
144 f l u o r i d e s (1-7).
I n t h i s paper,
t h e s t a t e o f f l u o r i d e e m i s s i o n from tunnel
k i l n s p r e s e n t i n most o f t h e t i l e works i s r e p o r t e d . MATERIALS AND METHODS Fifty-seven
tunnel k i l n s f o r Sekishu-tile
f i r i n g methods (8) ( s i d e - f i r i n g ,
manufacture were c l a s s i f i e d by
sandwich-firing,
and t o p - f i r i n g ) ,
produc-
t i o n , and o p e r a t i n g c o n d i t i o n s ( f i r i n g t e m p e r a t u r e , c a r r i a g e f e e d r a t e , etc.) o f t h e i n d i v i d u a l k i l n . f o r the investigation. f l u e gas m o n i t o r i n g ,
T h i r t e e n t y p i c a l k i l n s were then selected
Factors investigated included operating conditions,
f l u o r i n e c o n t e n t i n r a w m a t e r i a l s and p r o d u c t s ,
and so
on. Gaseous and p a r t i c u l a t e f o r m s o f f l u o r i d e s w e r e c o l l e c t e d s e p a r a t e l y w i t h
M i 1 1 i p o r e F i 1t e r s ( T y p e AAWP). M a t e r i a l c l a y and g l a z e w e r e t a k e n f r o m t h e c l a y y a r d and g l a z e t a n k , A b o u t t e n p i e c e s o f u n f i r e d t i l e s o f Type I o r Type I V p e r
respectively.
k i l n were t a k e n b e f o r e t h e y were loaded on t h e
carriage.
Three t o t e n
p i e c e s f r o m each t y p e o f f i r e d t i l e s were t a k e n i m m e d i a t e l y a f t e r t h e f i r i n g process.
Samples w e r e p u l v e r i z e d i n t o 100 mesh o r s m a l l e r f o r
a n a l y s i s b y t h e t h e r m a l d e c o m p o s i t i o n m e t h o d - i o n e l e c t r o d e m e t h o d (5). RESULTS AND D I S C U S S I O N C o n c e n t r a t i o n and c o m p o s i t i o n o f f l u o r i d e s i n f l u e qas The c o n c e n t r a t i o n o f g a s e o u s f l u o r i d e s e m i t t e d f r o m t u n n e l k i l n s was 9.23 0 mgF/m3N ( m e a n o f 1 3 k i l n s :
2 3 mgF/m3N).
Based o n f i r i n g methods,
gaseous f l u o r i d e c o n c e n t r a t i o n o f t h e s i d e - f i r i n g - t y p e mgF/m3N ( m e a n o f 6 k i l n s : mgF/m3N (mean o f 4 k i l n s : 2 8 mgF/m3N),
2 0 mgF/m3N). 24 mgF/m3N)
k i I n s w a s 9.2-27
The c o n c e n t r a t i o n s w e r e 21-27
and 25-30
f o r t h e s a n d w i c h - and t o p - f i r i n g
mgF/m3N (mean o f 3 k i l n s :
methods,
r e s p e c t i v e l y . There
seemed t o be l i t t l e d i f f e r e n c e i n g a s e o u s f l u o r i d e c o n c e n t r a t i o n s b e t w e e n d i f f e r e n t f i r i n g methods,
though t h e t o p - f i r i n g - t y p e
somewhat h i g h e r c o n c e n t r a t i o n t h a n t h e o t h e r s .
Also,
k i l n s seemed t o h a v e as seen i n F i g u r e 1,
gaseous f l u o r i d e c o n c e n t r a t i o n i n t h e f l u e gas d i d n o t f l u c t u a t e g r e a t l y . On t h e o t h e r h a n d ,
f o l l o w i n g the a l k a l i cleaning process the f l u o r i d e
c o n c e n t r a t i o n i n t h e f l u e g a s d e c r e a s e d t o 0.78-1.4 k i l n s : 1.0 mgF/m3N).
mgF/m3N ( m e a n o f 3
T h e p e r c e n t a g e o f f l u o r i d e r e m o v a l was. t h e r e f o r e ,
e s t i m a t e d t o be m o r e t h a n 90%. The e f f e c t s o f f l u o r i d e s o n a n i m a l s and p l a n t s depend o n t h e p h y s i c a l and chemical p r o p e r t i e s o f f l u o r i d e s ,
and t h e i r c o n c e n t r a t i o n s .
I n general,
t h e t o x i c i t y o f f l u o r i d e i s h i g h e r i n gaseous form, s o l u b l e p a r t i c u l a t e , and i n s o l u b l e p a r t i c u l a t e form,
i n t h a t order.
F o r example, i t has been
145
Y
-I- 1 1
Fluorine concentration ( P P ~ F i g u r e 1. E x a m p l e o f c o n t i n u o u s a u t o m a t i c m e a s u r e m e n t r e c o r d o f g a s e o u s f l u o r i n e c o n c e n t r a t i o n i n f l u e gas. r e p o r t e d t h a t gaseous f l u o r i d e s a r e e i g h t t o t e n t i m e s more t o x i c t h a n p a r t i c u l a t e f l u o r i d e s (9). fore,
To e v a l u a t e f l u o r i d e t o x i c i t y .
i t is, there-
i m p o r t a n t t o know t h e p r o p e r t i e s o f f l u o r i d e s i n t h e f l u e gas and i n
t h e atmosphere. T a b l e I shows t h e c o n c e n t r a t i o n s o f v a r i o u s f o r m s o f f l u o r i d e s i n f l u e gas.
S i n c e a p a r t i c u l a t e f o r m o f f l u o r i d e was d e t e c t e d i n o n l y one sample,
i t a p p e a r s t h a t f l u o r i d e s e m i t t e d f r o m t u n n e l k i l n s w e r e m o s t l y i n gaseous form.
Gaseous f l u o r i d e s a r e h i g h l y p o i s o n o u s t o a n i m a l s and p l a n t s .
thermore,
Fur-
s i n c e 70-80% o f t h e a t m o s p h e r i c f l u o r i d e s a r o u n d t i l e w o r k s a r e
i n gaseous form,
and m o s t p a r t i c u l a t e - f o r m f l u o r i d e s a r e s o l u b l e ,
emphasis
s h o u l d be focused on measures t o c o n t r o l f l u o r i d e p o l l u t i o n f r o m t i l e works. F l u o r i n e c o n t e n t s i n m a t e r i a l c l a y and g l a z e F l u o r i d e s c o n t a i n e d i n t h e f l u e gas o r i g i n a t e f r o m m a t e r i a l c l a y and glaze.
To s e c u r e t h e r e f r a c t o r i n e s s t o w i t h s t a n d h i g h - t e m p e r a t u r e
and t o s e c u r e t h e q u a l i t y o f t h e f i n i s h e d S e k i s h u - t i l e , material
firing
several kinds o f
c l a y s a r e g e n e r a l l y m i x e d i n t o t h e compound m a t e r i a l .
Material
c l a y s c a n b e c a t e g o r i z e d i n t o c l a y , sand, a n d s i l i c a sand.
The f l u o r i n e
c o n t e n t i n t h e m a t e r i a l s was 195-438 ppm ( s a m p l e number: 40;
mean: 311 ppm)
f o r clay,
45-217
( s a m p l e number:
ppm ( s a m p l e number: 11; mean: 1 3 8 ppm) f o r sand, 2;
mean:
18-21 ppm
20 ppm) f o r s i l i c a sand,
and 227-330 ppm ( s a m p l e
n u m b e r 1 1 ; mean: 2 7 5 p p m ) f o r c o m p o u n d m a t e r i a l .
On t h e o t h e r h a n d , t h e
f l u o r i n e c o n t e n t i n g l a z e was 80-268
ppm ( s a m p l e n u m b e r : 2 2 ; mean: 1 7 4
146 TABLE I
GASEOUS AND PARTICULATE FORMS OF FLUORIDE I N THE FLUE GAS Gaseous Form
Kiln
P a r t i c u l a t e Form Soluble Insoluble
Total
ND
ND
23
ND
ND
23
9.2
9.2
19
ND
ND
19
27
ND
ND
27
21
ND
ND
21
23
0.4
ND
23.4
4
ND < 0.1 mgF m3N ( s o l u b l e ) ND < 1 mgF/m N ( i n s o l u b l e ) ppm).
No m a t e r i a l w i t h an e x t r e m e l y h i g h c o n c e n t r a t i o n was o b s e r v e d ,
and
t h e r e were no marked d i f f e r e n c e s between d i f f e r e n t k i n d s o f m a t e r i a l s . Thus,
since t h e f l u c r i d e content i n glaze i s considerably lower than t h a t
o f m a t e r i a l c l a y o r compound m a t e r i a l , and,
furthermore,
since the quantity
o f g l a z e u s e d i s m u c h s m a l l e r t h a n t h a t o f m a t e r i a l c l a y (e.g. tile,
a few percent i n weight),
f o r Type I
most o f t h e f l u o r i d e s e m i t t e d from tunnel
k i l n s may o r i g i n a t e f r o m m a t e r i a l c l a y ,
although t h e r e i s a tendency t h a t
t h e f l u o r i n e c o n t a i n e d c l o s e r t o t h e s u r f a c e o f a t i l e i s more e a s i l y r e l e a s e d i n t h e f i r i n g process. Release r a t e o f f l u o r i d e i n f i r i n g process F l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s i s c o n s i d e r e d dependent on t h e f l u o r i n e c o n t e n t o f t h e r a w m a t e r i a l and t h e f l u o r i n e r e l e a s e r a t e f r o m t i l e s i n t h e f i r i n g process.
And,
t h e r e l e a s e r a t e i s supposed t o be
i n f l u e n c e d b y t h e c o m p o s i t i o n o f m a t e r i a l c l a y and g l a z e , fluorides,
f i r i n g c o n d i t i o n , t i l e shape,
the form o f
and so f o r t h .
To s t u d y t h e d i f f e r e n c e s b e t w e e n t h e r e l e a s e r a t e and t i l e shape,
ten
samples f r o m each o f t h e f o u r k i n d s o f t i l e s f i r e d s i m u l t a n e o u s l y i n a k i l n were c o l l e c t e d f o r measurement o f f l u o r i n e content.
As shown i n T a b l e 11, t h e r e l e a s e r a t e was 48-62%, d e p e n d e n t o n t h e shape of the tile.
of tiles, weight. examined. rates.
Though t h e shape, s i z e , and t h i c k n e s s v a r i e d w i t h t h e k i n d s
l a r g e r and t h i c k e r t i l e s a r e c o n s i d e r e d t o h a v e a h e a v i e r p r o d u c t The r e l a t i o n s h i p b e t w e e n r e l e a s e r a t e and p r o d u c t w e i g h t was t h u s The r e s u l t s showed t h a t t h e h e a v i e r t i l e s had l o w e r r e l e a s e
147 TABLE I 1 FLUORINE RELEASE RATE ACCORDING TO TYPES
Release Rate (%) Product Weight (kg)
47.4
OF
TILE
48.8
58.7
3.55
2.90
II
I
61.6
2.84
m
2.06
N
TABLE I 1 1 FLUORINE RELEASE RATE OF TYPE I T I L E
Kiln
Firing Method
A
1
S i d e - f i r i ng
F l u o r i n e C o n t e n t (ppm) Fired U n f ir e d
Release Rate (%)
Product Weight (kg)
X
Y
21
264
126
52.3
2.95
C
S i d e - f i r ing
300
159
D
S i d e - f ir i n g
255
104
47.0 59.2
2.84
G
Side-f i r ing
228
42
81.6
3.31
E
Sandwich-firing
248
103
58.5
3.06
F
Sandwich-firing
255
138
45.9
3.07
H
Sandwich-firing
268
102
61.9
2.96
I J
Sandwich-firing
231
100
56.7
2.96
Top-firing
320
181
43.4
3.01
K
Top-firing
284
159
44.0
3.19
z=-
x - y
X
x100
3.07
148 S i n c e l i t t l e d i f f e r e n c e was f o u n d i n s i z e a n d w e i g h t b e t w e e n d i f f e r e n t t i l e w o r k s , and s i n c e t h e f o r m o f f l u o r i d e s and t h e c o m p o s i t i o n o f compound m a t e r i a l and g l a z e w e r e c o n s i d e r e d t o b e s i m i l a r among t i l e w o r k s ,
i t was
supposed t h a t t h e d i f f e r e n c e i n r e l e a s e r a t e o f Type I t i l e s between t i l e
So,
w o r k s depended o n d i f f e r e n t f i r i n g c o n d i t i o n s .
the influence o f f i r i n g
c o n d i t i o n s o n t h e r e l e a s e r a t e was s t u d i e d b y c o m p a r i n g t h e r e l e a s e r a t e f r o m Type I t i l e s o f d i f f e r e n t t i l e works. r e l e a s e r a t e r a n g e d f r o m 43.4-81.6% E x c e p t f o r k i l n G,
A s s h o w n i n T a b l e 111, t h e
w i t h a mean o f 55.1%.
w h i c h had a h i g h e r f i r i n g temperature than t h a t o f
m o s t S e k i s h u - t i l e k i l n s b y a b o u t 50°C b e c a u s e o f t h e u s e o f a h i g h m e l t i n g g l a z e a n d a s l o w e r f e e d r a t e o f t h e c a r r i a g e , t h e r e l e a s e r a t e w a s 43.461.9% w i t h a mean o f 52.1%.
Concerning t h e
o f the f a s t e r carriage feed-rate,
f i r i n g method,
perhaps because
t o p - f i r i n g k i l n s had a s l i g h t l y l o w e r
r e l e a s e r a t e t h a n o t h e r f i r i n g methods.
B u t , t h e i r d i f f e r e n c e was con-
sidered small. I n conclusion,
t h e r e l e a s e r a t e i n c r e a s e d as t h e t i l e s i z e o r t h e f i r i n g
temperature decreased,
o r as t h e c a r r i a g e f e e d - r a t e
regular tunnel k i l n f o r the Sekishu-tile
firing,
increased.
for a
t h e r e l e a s e r a t e was
40-
60%. E s t i m a t e d and measured f l u o r i n e e m i s s i o n F l u o r i n e e m i s s i o n f r o m a t u n n e l k i l n c a n be e s t i m a t e d b y u s i n g E q u a t i o n
( 1 ) f r o m t h e m a t e r i a l balance. Ec where:
=
Ec
Ci(FIi
x
WI,
x (1-~1,/100)
- Fol x Wgl]
(1)
= F l u o r i n e emission (gF/day)
F I ~ = F l u o r i n e c o n t e n t i n u n f i r e d t i l e s o f Type
i
Fo1 = F l u o r i n e c o n t e n t i n f i r e d t i l e s o f Type
(ppm)
W I ~ = W e i g h t o f u n f i r e d t i l e s o f Type
i
i
(ppm)
(ton/day)
Woi
= W e i g h t o f f i r e d t i l e s o f Type i ( t o n / d a y )
mi
= Q u a n t i t y o f w a t e r a t t a c h e d t o u n f i r e d t i l e s o f Type
i
(%)
I n t h e c a l c u l a t i o n o f f l u o r i n e emission from tunnel k i l n s using Equation
(I),t h e v a l u e s c a l c u l a t e d f r o m t h e p r o d u c t i o n and t h e m e a s u r e d u n i t w e i g h t o f e a c h t i l e t y p e w e r e used f o r W I ~and
FI,
a n d Fol,
and a l s o ,
Wol,
and t h e measured v a l u e s f o r
measured v a l u e s o f Type
I
t i l e w e r e u s e d f o r mi
w h i c h was c o n s i d e r e d t o show l i t t l e d i f f e r e n c e w i t h t i l e t y p e .
On t h e
o t h e r hand, m e a s u r e d f l u o r i n e e m i s s i o n was c a l c u l a t e d f r o m t h e f l u o r i n e c o n c e n t r a t i o n i n t h e f l u e gas and t h e d r y - g a s
volume.
T h e e s t i m a t e d a n d m e a s u r e d v a l u e s a r e p l o t t e d i n F i g u r e 2.
While the
149
-
-
o Side-f i r i n g
- i r ing Top-firing o
A w i ch f 0 Sand Top-firin;/ 0
A
A
0 0
w
c1
4
m 3 09'
a
a
I
1
t
z
4
6
I
ia
8
Measured v a l u e (KgF/day) F i g u r e 2.
R e l a t i o n b e t w e e n e s t i m a t e d and measured f l u o r i n e e m i s s i o n .
e s t i m a t e d values tended t o be l a r g e r t h a n t!ie measured values f o r k i l n s w i t h h i g h f l u o r i n e emission, s l o p e o f 45".
Consequently,
p l o t t e d p o i n t s gathered near t h e l i n e w i t h t h e since t h e estimated values almost coincided
w i t h t h e measured v a l u e s f o r t u n n e l k i l n s , seemed t o e x i s t i n t u n n e l k i l n s . w i l l
a m a t e r i a l balance f o r f l u o r i n e
The m e a s u r e d v a l u e s o f f l u o r i n e e m i s s i o n
be u s e d i n t h e d i s c u s s i o n h e r e a f t e r .
R e l a t i o n s h i p b e t w e e n f l u o r i n e e m i s s i o n and w e i q h t o f f i r e d p r o d u c t s Since f l u o r i d e s e m i t t e d from tunnel k i l n s o r i g i n a t e from raw materials, i t i s supposed t h a t t h e e m i s s i o n i s r e l a t e d t o t h e q u a n t i t y o f m a t e r i a l s o r
t h e weight o f products.
The r e l a t i o n b e t w e e n f l u o r i n e e m i s s i o n and t h e
w e i g h t o f p r o d u c t s i s p l o t t e d i n F i g u r e 3.
The r e l a t i o n s h i p i s e x p r e s s e d
b y E q u a t i o n (2).
Em
=
0.13 x W
w h e r e Em r e p r e s e n t s f l u o r i n e e m i s s i o n (kgF/day). (ton/day).
(2) and W,
weight o f products
S i n c e t h e c o e f f i c i e n t o f c o r r e l a t i o n i s 0.97,
a high correla-
t i o n i s recognized. The v i r t u a l l y l i n e a r r e l a t i o n s h i p b e t w e e n f l u o r i n e e m i s s i o n and w e i g h t o f t h e product suggests t h a t f l u o r i n e content i n raw m a t e r i a l s , f i r i n g method,
etc.
are not significant factors,
t i l e shape,
though they s l i g h t l y
i n f l u e n c e t h e e m i s s i o n . Thus, t o e s t i m a t e f l u o r i n e e m i s s i o n f r o m t u n n e l k i Ins,
k i I n p r o d u c t i o n c a p a c i t y s h o u l d be i d e n t i f i e d .
r a t h e r than f i r i n g
150
t
Side-firing
0
0
a 4 0
60
Product w e i g h t (ton/day) F i g u r e 3.
R e l a t i o n b e t w e e n f l u o r i n e e m i s s i o n and p r o d u c t w e i g h t .
method o r o t h e r f i r i n g c o n d i t i o n s . or top-firing
methods,
Therefore,
k i l n s using
sandwich-firing
which are being b u i l t i n c r e a s i n g l y recently,
be examined s i n c e t h e y have a c a p a c i t y t w o t o t h r e e
should
times larger than
c o n v e n t i o n a l k i l n s and n a t u r a l l y h a v e h i g h e r f l u o r i d e e m i s s i o n s . Method o f e s t i m a t i o n o f f l u o r i n e e m i s s i o n E s t i m a t i o n o f f l u o r i d e emission f r o m tunnel k i l n s i s e s s e n t i a l f o r promoting environmental p r o t e c t i o n against f l u o r i d e p o l l u t i o n . as e n v i r o n m e n t a l i m p a c t assessment,
e v a l u a t i o n o f data,
Such m e a s u r e s
and f o r m u l a t i o n o f
a r e g i o n a l p o l l u t i o n c o n t r o l program a r e a l l important. E q u a t i o n ( 2 ) c a n be used.
To e s t i m a t e t h e f l u o r i n e e m i s s i o n ,
Although
t h e method f o r e v a l u a t i n g p r o d u c t w e i g h t f r o m t h e u n i t w e i g h t o f a p r o d u c t and t h e number o f p r o d u c t s i s h i g h l y a c c u r a t e , cated.
Therefore,
every kiln.
t h e measurement i s c o m p l i -
i t i s d i f f i c u l t t o obtain a real product weight f o r
I n practice,
s u c h m e t h o d s as u s i n g t h e p r o d u c t i o n o r t h e quan-
t i t i e s o f r a w m a t e r i a l s may be adopted. The m e t h o d o f u s i n g t h e p r o d u c t i o n ,
i n which t h e product w e i g h t (product-
n u m b e r b a s i s ) i s a p r o d u c t o f t h e p r o d u c t i o n c a l c u l a t e d i n t e r m s o f Type I t i l e ( t h e q u o t i e n t o f t h e t o t a l o u t p u t d i v i d e d b y t h e u n i t p r i c e o f Type I t i l e i s t r a d i t i o n a l l y c a l l e d t h e p r o d u c t i o n a t t i l e w o r k s ) and t h e u n i t w e i g h t o f t h e ' T y p e I t i l e ( a s s h o w n i n T a b l e 111. u n i t w e i g h t o f T y p e I t i l e i s a b o u t 3 Kg w i t h l i t t l e d i f f e r e n c e among t i l e w o r k s : o f 3.0
therefore,
use
Kg a s a u n i t w e i g h t m a y h a v e l i t t l e i n f l u e n c e o n t h e a c c u r a c y o f
t h e e s t i m a t i o n ) i s expressed a t f o l l o w s :
151 W,
=
3 x P/30
(3)
where U p i s p r o d u c t w e i g h t c a l c u l a t e d from p r o d u c t number (ton/day),
and P
i s p r o d u c t number c a l c u l a t e d i n t e r m s o f Type I t i l e ( l o 3 pieces/month). The method o f u s i n g t h e q u a n t i t i e s o f raw m a t e r i a l s ,
i n which the product
weight ( m a t e r i a l basis) i s evaluated from t h e q u a n t i t y of m a t e r i a l c l a y used,
w i t h c o r r e c t i o n s f o r t h e q u a n t i t y o f water attached t o t h e m a t e r i a l
c l a y ( 1 8 % was used,
w h i c h was t h e mean o f m e a s u r e d v a l u e s ,
i g n i t i o n l o s s (5% was used,
16-20%) and
w h i c h was t h e mean of measured values,
446%).
i s expressed i n E q u a t i o n (4).
wr where Wr
=
R x (1
--
100
)
X
(
X
100
(4)
)
i s t h e p r o d u c t w e i g h t c a l c u l a t e d from m a t e r i a l q u a n t i t y (ton/day)
and R t h e q u a n t i t y o f m a t e r i a l c l a y used (ton/day).
Here,
the quantity of
t h e g l a z e used i s n e g l e c t e d f o r t h e reason d e s c r i b e d p r e v i o u s l y . The c a l c u l a t e d v a l u e s f r o m E q u a t i o n ( 3 ) and E q u a t i o n ( 4 ) a r e p l o t t e d t o t h e measured v a l u e s i n F i g u r e 4.
I n b o t h cases,
number b a s i s and on t h e m a t e r i a l basis, w i t h a s l o p e o f 45".
c a l c u l a t e d on t h e p r o d u c t -
p l o t t e d p o i n t s g a t h e r e d near a l i n e
Thus, i t was f o u n d t h a t t h e p r o d u c t w e i g h t c o u l d be
e s t i m a t e d f r o m e i t h e r E q u a t i o n (3) o r E q u a t i o n (4) w i t h c o n s i d e r a b l e accuracy.
t
0
0
Product-number bas i s
o Material basis
20
40
60
Reasured value ( t o d d a y )
F i g u r e 4.
R e l a t i o n between c a l c u l a t e d and measured p r o d u c t w e i g h t .
152 Emission f a c t o r s tunnel k i l n s Emission f a c t o r i s g e n e r a l l y used t o o b t a i n t h e amount o f discharged environmental pollutant. i n Table IV.
Emission f a c t o r s f o r t u n n e l k i l n s a r e summarized
Because o f t h e
o f the side-firing
i n s u f f i c i e n t m e a s u r e s f o r f u e l economy,
lower fluorine emission factors, t h e o t h e r kiln-types, o f products,
kilns
t y p e have h i g h e r r a t e s o f f u e l consumption and t h e r e f o r e based on f u e l consumption,
compared w i t h
However, b a s e d on t h e w e i g h t o f p r o d u c t s ,
o r the quantities o f materials,
l i t t l e difference i n fluorine
e m i s s i o n f a c t o r i s r e c o g n i z e d b e t w e e n d i f f e r e n t f i r i n g methods. emission f a c t o r s a r e 157 gF/ton on a product-weight p i e c e s on a product-number basis,
t h e number
basis.
The mean
443 gF/103
and 1 2 0 g F / t o n on a m a t e r i a l b a s i s .
Method o f r e d u c t i o n o f f l u o r i n e e m i s s i o n To r e d u c e t h e f l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s , considered. content,
These i n c l u d e
selection o f
s e v e r a l m e t h o d s may b e
raw m a t e r i a l s w i t h l o w e r f l u o r i n e
c o n t r o l o f t h e f l u o r i n e release r a t e from raw materials,
and
t r e a t m e n t o f t h e f l u e gas. I n the Sekishu-tile
industry,
however,
since selection o f the material
c l a y i s d i f f i c u l t i n view o f t h e possible exhaustion o f e x c e l l e n t m a t e r i a l clay,
and a l s o ,
s i n c e f l u o r i d e compounds s u c h a s f l u o r i t e o r c r y o l i t e a r e
n o t u s e d as t h e s o l v e n t o f t h e g l a z e ,
reduction o f f l u o r i n e emission by
s e l e c t i o n o f r a w m a t e r i a l s c a n n o t be e x p e c t e d . control o f the f l u o r i n e release rate,
improvement o f
t h e o p e r a t i o n o f k i l n s or a d d i t i o n o f a r e l e a s e i n h i b i t o r .
On t h e o t h e r hand,
s u c h as s l a k e d
l i m e , t o t h e m a t e r i a l c l a y may b e c o n s i d e r e d . I n the Sekishu-tile
i n d u s t r y , however, s i n c e improvement i n t h e o p e r a t i o n
o f k i l n s h a s been a c h i e v e d f r o m t h e v i e w p o i n t o f f u e l economy,
by increas-
i n g the feed r a t e o f the carriage o r by lowering the f i r i n g temperature w i t h i n t h e r a n g e so t h a t t h e q u a l i t y o f p r o d u c t s m i g h t s t i l l be m a i n t a i n e d , f u r t h e r d e c r e a s e o f t h e f l u o r i n e r e l e a s e r a t e c a n n o t be e x p e c t e d f o r m o s t o f t h e t i l e works.
Fluorine release inhibitor i s said t o exhibit the
c o n t r o l e f f e c t o n l y when i t i s added i n s u c h abundance a s t o a d v e r s e l y a f f e c t t h e q u a l i t y o f t h e p r o d u c t (10).
Therefore,
development o f inexpen-
s i v e and m o r e e f f e c t i v e i n h i b i t o r s i s needed. Thus,
w h i l e keeping t h e q u a l i t y o f t h e Sekishu-tile a t i t s present level,
t h e r e seems t o be n o o t h e r way t o e f f e c t i v e l y r e d u c e f l u o r i n e e m i s s i o n t h a n t o t r e a t f l u e gas.
A f l u o r i n e r e m o v a l r a t e o f m o r e t h a n 90% c a n b e
a c h i e v e d b y t h e a 1 k a l i c l e a n i n g method.
CONCLUSIONS The a c t u a l s t a t e o f f l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s was i n v e s t i g a t e d
TABLE I V
E M I S S I O N FACTORS OF TUNNEL KILNS Kiln
Fuel Consumpti on
F l u e gas Volume pry)
(l/ton-product)
Total
S id e - f ir ing
S a n d w i c h - f i r i ng
Top-firing
(lo3,
/ton-product)
Fuel Basis (kgF/kl)
F l u o r i n e Emission Product-weight Product-number M a t e r i a1 Basis Basis Basis (gF/ton-product) (gF/103pieces) ( g F / t o n - m a t e r i a l )
61-237 ( n = l l . 123)
3.9-1 4.3 ( n = l l , 7.4)
0.78-0.88 ( n = l l , 1.30)
11 7-256 ( n = l l , 157)
281 -658 ( n = l l , 443)
91 -1 44 (n=9, 120)
122-237 (n=6. 167)
6.1-14.3 (n=6, 9.1)
0.78-1.61 (n=6, 1.08)
11 7-256
( n = 6 , 174)
281 -658 (n=6, 471)
91-1 41 (n=4, 117)
61-113 (n=4, 8 4 )
4.8-7.6 (n=3, 5.8)
1.07-1.88 (n=3, 1.55)
(n=3, 138)
323-51 1 (n=3, d02)
105-1 56 (n=3, 123)
83-89 (n=3, 8 6 )
(n=2.
3.9-5.1 4.5)
1.39-1.73 (n=2, 1.56)
119-154 (n=2. 137)
380-463 (n=2, 422)
96-1 44 (n=2, 120)
121-167
154
among t h e S e k i s h u - t i l e m a n u f a c t u r i n g i n d u s t r i e s s p e c i f i c t o t h e Iwami D i s t r i c t , Shi mane P r e f e c t u r e . T h e mean c o n c e n t r a t i o n o f g a s e o u s f l u o r i d e s i n t h e f l u e g a s was 2 4 mgF/m3N.
T h e bulk of t h e f l u o r i d e s i n t h e f l u e g a s was i n g a s e o u s form,
and t h e c o n t r i b u t i o n of p a r t i c u l a t e - f o r m nificant.
f l u o r i d e s was found t o be i n s i g -
F l u o r i d e o r i g i n a t i n g from t h e g l a z e i s s c a r c e i n t h e f l u e gas.
a n d m o s t o f i t may o r i g i n a t e f r o m m a t e r i a l c l a y .
Fluorine release r a t e
from raw m a t e r i a l s i n t h e f i r i n g p r o c e s s was 40-60% i n g e n e r a l . T h e q u a n t i t y o f f l u o r i d e s e m i t t e d from a t u n n e l k i l n i s a p p r o x i m a t e l y i n p r o p o r t i o n t o t h e w e i g h t o f t h e p r o d u c t , w h i c h i n turn c a n be e s t i m a t e d e i t h e r from t h e number of p r o d u c t s o r from t h e q u a n t i t i e s of m a t e r i a l c l a y used.
By t h i s m e t h o d , t h e t o t a l f l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s
t h r o u g h o u t t h e p r e f e c t u r e was e s t i m a t e d .
The f l u o r i n e e m i s s i o n f a c t o r f o r
t u n n e l k i l n s was 120 gF/ton o f m a t e r i a l used. T h e r e s e e m s t o b e no o t h e r way f o r an e f f e c t i v e r e d u c t i o n o f f l u o r i n e e m i s s i o n from t u n n e l k i l n s t h a n t h e t r e a t m e n t of f l u e gas.
Fluorine
r e m o v a l r a t e o f m o r e t h a n 90% c a n be a c h i e v e d by t h e a l k a l i c l e a n i n g method. REFERENCES 1. Nakao M, Kimura T, S a i t o K (1973) Report o f Shimane P r e f I n s t Pub Hlth Environ S c i e n c e 15:52-53
2. Nakao M, Tanaka F (1977) Shimane PIOPHAES Report 19:96-97 3. T a n a k a F, Fukada K , Nakao M, Kimura T ( 1 9 7 7 ) S h i m a n e PIOPHAES R e p o r t 19 :65-72 4. Shimane P r e f e c t u r e (1976) Report of Survey f o r A d d i t i o n a l D e s i g n a t i o n of Toxic S u b s t a n c e s (on commission of t h e Environ Agency) 5. Shimane P r e f e c t u r e (1979) Report on F l u o r i d e E m i t t i n g Source Survey (on commission of t h e Environ Agency)
6. Nakao M, Tanaka F, Tachibana C, S a i t o K (1979) Shimane PIOPHAES Report 21 :75-76 7. Nakao M, T a n a k a F, T a c h i b a n a C. Fukada K , H a y a s h i K , S a i t o K ( 1 9 8 0 ) J Environ Lab Assoc 5:105-115
8. Tanaka M (1980) Handbook of Clay-Tile.
Gihodo, Tokyo
9. Tsunoda F, S a k u r a i S, I t a i K and Nakaya S (1978) C r i t e r i a on F l u o r i d e s (on commission of t h e Environ Agency) 10. Gunma P r e f e c t u r e (1975) Report of Gunma P r e f I n s t I n d u s t S c i Tech 28
H. Tsunoda and M.-H. Yu (Editors)
155
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 155-163 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands
D I S T R I B U l I O N OF FLUORIDE I N H U M A N B O N E S COLLECTED FOR THE EVALUATION O F INDUSTRIAL AIR POLLUTION
M A R C A. V A N D E N H E E D E ~ , J A C Q U E S 1.1. TIMPERMAN*, JAN A.
AUBIN
M. H E Y N D R I C K X ~ .
AND
CORDONNIER~
'Department o f Toxicology, B-9000 Ghent. B e l g i u m
S t a t e U n i v e r s i t y o f Ghent,
H o s p i t a a l s t r a a t 13,
'Department o f Legal Medicine, State U n i v e r s i t y o f K l u y s k e n s s t r a a t 29, B-9000 Ghent. B e l g i u m
Ghent.
P r o f . J.
ABSTRACT F l u o r i d e c o n c e n t r a t i o n s w e r e d e t e r m i n e d i n human bones i n o r d e r t o e s t a b l i s h w h e t h e r bone f l u o r i d e l e v e l s c o u l d be i n d i c a t i v e o f a c h r o n i c e x p o s u r e t o a f l u o r i d e contaminated environment.
The bone s a m p l e s s t u d i e d w e r e
m a i n l y o b t a i n e d f r o m t h i r t e e n p o s t mortem examinations. cases,
t h e p e r s o n s i n v o l v e d had l i v e d i n r u r a l a r e a s ,
p o l l u t i o n ( r e f e r e n c e cases).
I n t h e o t h e r t h r e e cases,
i n v o l v e d had l i v e d f o r many ,;ears t i n g plant.
I n ten o f these
f r e e o f any f l u o r i d e the individuals
i n t h e neighborhood o f a f l u o r i d e e m i t -
I n a d d i t i o n , an a n a l y s i s was a l s o p e r f o r m e d o n a bone f r a g m e n t
p r o c u r e d by a 71-year-old t i o n o f t h e hip.
woman who u n d e r w e n t a n a r t h r o p l a s t i c r e c o n s t r u c -
T h i s p a t i e n t a l s o had a p r e v i o u s h i s t o r y o f c h r o n i c
f l u o r i d e intoxication. The f l u o r i d e c o n c e n t r a t i o n s measured i n t h e f o u r s u s p e c t e d cases w e r e w i t h i n t h e l i m i t s i n d i c a t e d b y t h e r e f e r e n c e cases.
As s u c h t h e y w e r e n o t
d i r e c t l y i n d i c a t i v e o f a c h r o n i c exposure t o i n d u s t r i a l f l u o r i d e p o l l u t i o n .
INTRODUCTION With t h e c u r r e n t increased a t t e n t i o n t o environmental protection.
i t has
been n o t e d t h a t t h e r i s i n g i n d u s t r i a l f l u o r i d e e m i s s i o n s a s an e l e m e n t of a i r p o l l u t i o n s h o u l d i n d e e d t r i g g e r c o n c e r n among t h o s e i n v o l v e d w i t h public health protection.
However,
i t i s known t h a t t h e d i a g n o s i s of
c h r o n i c f l u o r i d e i n t o x i c a t i o n i n man i s v e r y d i f f i c u l t . s e r i e s o f n o n s p e c i f i c symptoms,
Apart from a
the only practical c r i t e r i a available f o r
e s t a b l i s h i n g a d i a g n o s i s a r e X - r a y e v i d e n c e and f l u o r i d e d e t e r m i n a t i o n s i n b l o o d and u r i n e samples.
The p u r p o s e o f t h i s s t u d y was t o e s t a b l i s h
w h e t h e r a g i v e n f l u o r i d e c o n c e n t r a t i o n f o u n d i n t h e bone t i s s u e o f an i n d i v i d u a l s u s p e c t e d o f h a v i n g been e x p o s e d t o t o x i c l e v e l s o f f l u o r i d e i n
156 t h e a i r c o u l d be i n d i c a t i v e o f a c h r o n i c f l u o r i d e i n t o x i c a t i o n .
The accum-
u l a t i o n o f f l u o r i d e i n human bone t i s s u e was s t u d i e d b y a n a l y z i n g bone ma-lerial obtained from t h i r t e e n r o u t i n e f o r e n s i c autopsies. cases,
I n three
t h e p o s t m o r t e m bone s a m p l e s o r i g i n a t e d f r o m p e r s o n s who had been
l i v i n g f o r a number o f years i n t h e neighborhood o f a f l u o r i d e - e m i t t i n g factory.
The p o s t m o r t e m bone s a m p l e s c o l l e c t e d i n t h e o t h e r t e n cases
b e l o n g e d t o p e r s o n s who h a d b e e n l i v i n g i n a r e a s f r e e o f a n y known f l u o r i d e pollution.
The f l u o r i d e c o n c e n t r a t i o n s f o u n d i n t h e l a t t e r cases c o u l d
t h u s be c o n s i d e r e d as r e f e r e n c e v a l u e s o r blanks. and r e p r o d u c i b i l i t y ,
I n view o f u n i f o r m i t y
bone s a m p l e s w e r e t a k e n f r o m t h e same a n a t o m i c s i t e s
i n a l l t h e p o s t mortem cases studied.
T h e s e s i t e s , a s s h o w n i n F i g u r e 1.
a r e t h e " P o s t e r i o r Calvarium" o f t h e s k u l l ,
t h e "Manubrium" o f t h e sternum,
t h e "Caput" o f t h e femur, and a p a r t o f t h e r i b . F r o m t w o i n d i v i d u a l s who h a d b e e n l i v i n g i n a n a r e a o f h e a v y f l u o r i d e c o n t a m i n a t i o n ( p o s t m o r t e m c a s e s 1 2 and 13), femur
was a l s o a n a l y z e d .
bone s a m p l e s ,
w h i c h were c o l l e c t e d p o s t mortem,
s u r g i c a l l y removed f r o m a 71-year-old struction of the hi?
F i g u r e 1.
t h e "Trochanter
iii
Major" o f t h e
A p a r t f r o m t h e d e t e r m i n a t i o n s p e r f o r m e d on t h e
t h e '!i?jv?rsi':;
N a t u r e o f t h e bone samples.
t h e "Caput"
o f t h e femur
woman d u r i n g an a r t h r o p l a s t i c r e c o n : ! ? z . > i t s l z i n J a n u a r y 1977.
was a l s o
157 analyzed.
T h i s c a s e was c h o s e n a f t e r i t was shown d u r i n g a p r e v i o u s s t a y
i n t h e U n i v e r s i t y H o s p i t a l s i n 1975,
w h e r e t h e woman was u n d e r g o i n g t r e a t -
m e n t f o r a r t h r i t i s and p o l y n e u r i t i s .
t h a t t h e p a t i e n t had i n c r e a s e d f l u o -
r i d e c o n c e n t r a t i o n s i n t h e u r i n e ( u p t o 3.88 ppm).
as compared w i t h t h e
t o l e r a n c e v a l u e s d e s c r i b e d b y V i g l i a n i (1) and Hodge
gt G.
A t this
(2).
t i m e t h e woman d r a n k b o t t l e d V i c h y m i n e r a l w a t e r a t an a m o u n t o f a b o u t 1.5
L a day.
D u r i n g h e r s t a y i n t h e h o s p i t a l t h e u r i n a r y f l u o r i d e concen-
t r a t i o n r e m a i n e d h i g h so an a n a l y s i s o f s e v e r a l b o t t l e s o f V i c h y w a t e r was performed.
The a n a l y s i s r e v e a l e d t h a t t h e V i c h y w a t e r c o n t a i n e d u p t o 9 L a t e r i t was c o n f i r m e d b y V e r g o t e gt
ppm o f f l u o r i d e .
~ 1 (.3 )
that the
f l u o r i d e c o n c e n t r a t i o n s i n b o t t l e d V i c h y w a t e r s were h i g h as opoosed t o those found i n o t h e r m i n e r a l waters. o f Vichy water,
A f t e r d i s c o n t i n u i n g t h e consumption
A
t h e u r i n a r y f l u o r i d e c o n c e n t r a t i o n decreased.
m o n t h s a f t e r t h e p a t i e n t had l e f t t h e h o s p i t a l . f l u o r i d e c o n c e n t r a t i o n was n o t e d again.
few
an i n c r e a s e o f t h e u r i n a r y
Further investigation revealed
t h a t t h i s woman a l s o l i v e d i n t h e n e a r v i c i n i t y o f a f l u o r i d e e m i t t i n g plant,
and t h a t a c c o r d i n g t o t h e p r e v a i l i n g d i r e c t i o n
of
the
wind,
a t m o s p h e r i c f l u o r i d e l e v e l s o f u p t o 57 pg/m3 w e r e m e a s u r e d i n h e r g a r d e n a t a d i s t a n c e o f a b o u t 700 m f r o m t h e p l a n t .
Measurements performed a f t e r -
w a r d s on p l a n t m a t e r i a l g r o w i n g i n h e r g a r d e n r e v e a l e d t h a t t h e l e a v e s o f t h e b r u s h w o o d and some v e g e t a b l e s c o n t a i n e d f l u o r i d e i n e x c e s s i v e l y h i g h c o n c e n t r a t i o n s (bush rose, a d r y w e i g h t basis).
37.2
ppm;
parsley,
29.1 ppm;
b o t h c a l c u l a t e d on
The r i s e i n u r i n a r y f l u o r i d e c o n c e n t r a t i o n c o u l d t h u s
be b r o u g h t i n r e l a t i o n w i t h a r e n e w e d e x p o s u r e t o e n v i r o n m e n t a l f l u o r i d e pollution. MATERIALS AND METHODS Reaqents A l l c h e m i c a l s used w e r e r e a g e n t g r a d e and w e r e u s e d w i t h o u t f u r t h e r purification,
except f o r sodium f l u o r i d e ,
a Merck suprapure product,
which
was p r e v i o u s l y d r i e d f o r 6 h o u r s a t 110°C and s u b s e q u e n t l y s t o r e d i n a desiccator. F l u o r i d e s t o c k s o l u t i o n ( 1 , 9 0 0 p p m ) w a s p r e p a r e d b y d i s s o l v i n g 4.199 g s o d i u m f l u o r i d e i n d o u b l e d i s t i l l e d w a t e r a n d d i l u t i n g t o 1 L.
Fluoride
standard s o l u t i o n s were prepared from t h e stock s o l u t i o n by d i l u t i n g w i t h double d i s t i l l e d water. T o t a l i o n i c s t r e n g t h a d j u s t m e n t b u f f e r (TISAB) was p r e p a r e d by a d d i n g 57 mL g l a c i a l a c e t i c a c i d , 5 8 g s o d i u m c h l o r i d e a n d 0.3 g s o d i u m c i t r a t e t o 500 mL o f d o u b l e d i s t i l l e d w a t e r . t o pH 5.0
-
The s o l u t i o n was t i t r a t e d w i t h 5 M NaOtl
5.5 and f u r t h e r d i l u t e d t o 1 L w i t h d o u b l e d i s t i l l e d w a t e r .
158
Instrumentation The f o l l o w i n g i n s t r u m e n t a t i o n was u t i l i z e d : t y p e K R 1 7 0 (W.C.
H e r a e u s G.M.B.H.,
e l e c t r o d e (Beckman Model 39600): t r o d e (Beckman M o d e l 39402).
Hanau);
(1) m u f f l e f u r n a c e ,
Heraeus
(2) f l u o r i d e i o n s e l e c t i v e
s i n g l e j u n c t i o n reference calomel elec-
R e a d i n g s w e r e made on a Beckman E x p a n d o m a t i c
SS-2 pH m e t e r w i t h expanded s c a l e c a p a b i l i t y . Sample P r e p a r a t i o n Each bone s a m p l e was chopped i n s m a l l p i e c e s , w h i c h w e r e b o i l e d t w i c e i n d o u b l e d i s t i l l e d w a t e r f o r 10 m i n u t e s and d r i e d .
According t o t h e proce-
d u r e s o f S i n g e r and A r m s t r o n g (4) and Zober and
S c h e l l m a n n (5) a w e l l
d e f i n e d a m o u n t o f d r y bone t i s s u e ( 1 t o 1 0 g r a m s ) was w e i g h e d i n a t a r e d q u a r t z c r u c i b l e w i t h c o v e r ( c a p a c i t y : 5 0 mL) a n d a s h e d f o r 1 5 h o u r s i n a m u f f l e f u r n a c e a t 550°C.
After cooling i n
2
desiccator,
t h e corresponding
amount o f a s h was d e t e r m i n e d and homogenized. Fluoride Determination The a p p l i e d m e t h o d was a s l i g h t m o d i f i c a t i o n o f a t e c h n i q u e f o r t h e d e t e r m i n a t i o n o f f l u o r i d e i n v e g e t a t i o n p r e v i o u s l y d e s c r i b e d b y Van d e n tieede
2 d.(6).
An a c c u r a t e l y w e i g h e d a m o u n t o f a s h ( 1 5 t o 2 0 mg) w a s
d i s s o l v e d i n 3.5 mL o f 0.25 M HC1.
The o b t a i n e d s o l u t i o n w a s p a r t i a l l y
n e u t r a l i z e d b y a d d i n g 4 mL o f 0.125 M NaOH a n d f u r t h e r a d j u s t e d t o pH 4.7 b y t i t r a t i n g w i t h a 0.05
M sodium a c e t a t e s o l u t i o n .
was d i l u t e d t o 20 mL w i t h d o u b l e d i s t i l l e d w a t e r .
Finally the solution
After
o f a t o t a l i o n i c s t r e n g t h a d j u s t m e n t b u f f e r (TISAB),
a d d i t i o n o f 2 0 mL
a f l u o r i d e i o n selec-
t i v e e l e c t r o d e and a s i n g l e j u n c t i o n r e f e r e n c e c a l o m e l e l e c t r o d e were i m m e r s e d i n t o t h e s o l u t i o n w h i c h was s t i r r e d u n t i l a c o n s t a n t m i l l i v o l t a g e r e a d i n g was o b t a i n e d .
D i f f e r e n t f l u o r i d e standard solutions, containing
f l u o r i d e i o n c o n c e n t r a t i o n s b e t w e e n 0.38 and 38 ppm, w e r e p r e p a r e d f r o m t h e standard stock solution. o f 0.25
To 5 mL o f each f l u o r i d e s t a n d a r d s o l u t i o n 3.5
M HC1 a n d 6 mL o f 0.125 M NaOH w e r e s u b s e q u e n t l y added.
mL The
s o l u t i o n w a s a d j u s t e d t o pH 4.7 b y t i t r a t i n g w i t h a 0.05 M s o d i u m a c e t a t e s o l u t i o n and f u r t h e r d i l u t e d t o 20 m l w i t h d o u b l e d i s t i l l e d w a t e r .
After
a d d i t i o n o f 2 0 mL o f TISAB, a f l u o r i d e i o n a c t i v i t y m e a s u r e m e n t w a s p e r f o r m e d as a l r e a d y d e s c r i b e d .
The f l u o r i d e c o n t e n t s o f t h e bone a s h s a m p l e s
were c a l c u l a t e d from a c a l i b r a t i o n curve, drawn by p l o t t i n g t h e m i l l i voltage readings w i t h t h e standard solutions on the rectangular a x i s against t h e i r fluoride
i o n a c t i v i t i e s ( c o n c e n t r a t i o n s ) on t h e l o g a r i t h m i c
a x i s o f s e m i l o g a r i t h m i c paper. o f 58 m V p e r decade.
T h i s g r a p h was a s t r a i g h t l i n e w i t h a s l o p e
159
RESULTS AND DISCUSSION D a t a e x h i b i t i n g t h e r e p r o d u c i b i l i t y o f t h e proposed method f o r t h e d e t e r m i n a t i o n o f f l u o r i d e i n bone t i s s u e ,
obtained by determining f l u o r i d e i n
f i v e f r a g m e n t s o f b o t h t h e " P o s t e r i o r C a l v a r i u m " o f t h e s k u l l and t h e a r e s u m m a r i z e d i n T a b l e I. A l l b o n e
"Trochanter Major" o f t h e femur,
f r a g m e n t s o r i g i n a t e d f r o m t h e same body,
o f w h i c h no s p e c i f i c a t i o n s were
a v a i l a b l e , s i n c e t h e y w e r e c o l l e c t e d t o p e r f o r m some p r e l i m i n a r y t e s t s before the actual study started.
Some i m p o r t a n t s p e c i f i c a t i o n s r e g a r d i n g
t h e t h i r t e e n p o s t m o r t e r n c a s e s s t u d i e d a r e s h o w n i n T a b l e 11. TABLE
A l l data
I
REPRODUCIBILITY OF THE PROPOSED METHOO FOR THE DETERMINATION OF FLUORIDE I N BONE TISSUE ( F I V E FRAGMENTS O F BOTH THE POSTERIOR CALVARIUM OF THE SKULL AN0 THE TROCHANTER MAJOR OF THE FEMUR ARE TAKEN FROM THE SAME BODY) Nature o f the bone sample Skull
Femur
' d r y bone/ash
Weight (9) d r y bone ash
ratio'
F l u o r i d e C o n c e n t r a t i o n (ppm) d r y bone ash
1
1.502
1.012
1.5
1,112
1.650
2
0.854
0.567
1.5
863
1,300
3
1.272
0.793
1.6
71 1
1.140
4
1.153
0.707
1.6
705
1.150
5
0.884
0.666
1.5
795
1,175
x
1.54
837.20
1,283
SD
0.05
166.86
214.93
sx
0.02
74.62
96.12
CV%
3.56
19.93
16.75
1
1.527
2
0.382
0.241
3
0.990
0.637
0.992
844
1,300
1.6
931
1,475
1.6
836
1,300 1.125
1.5
4
0.808
0.503
1.6
700
5
0.524
0.319
1.6
852
1,400
832.6
1.320
jT
1.50
SD
0.04
83.34
131.58
sx
0.02
37.27
58.84
CV%
2.83
10.01
9.97
160 TABLE I 1 SPECIFICATIONS ABOUT THE CASES STUDIED Case
Sex
Age
Cause o f Death
N
Profession
a
b
1
M
74
Drowning
Retired
4 days
2
M
53
Drowning
Dealer i n c a t t l e fodder
1 day
3
F
20
T r a f f i c accident
Housekeeper
3 days
4
F
50
F a l l from a b u i l d i n g
Factory-worker
3 days
5
M
70
Strangulation
Old servant
1 day
6
M
63
Drowning
Baker
3 days
7
M
73
Drowning
Reti red
3 days
8
M
49
B a r b i t u r a t e overdose
Metal-worker
3 days
9
F
79
E x h a u s t i o n and f a l l
Housekeeper
1 day
10
F
74
T r a f f i c accident
Housekeeper
11
M
53
B r o n c h i a l carcinoma
So 1d ie r
1 day 70 days
12
F
76
Cerebrovascular accident
Housekeeper
18 months
13
M
72
Cardiovascular s c l e r o s i s
Farmer
2 months
a: t i m e i n t e r v a l between d e a t h and autopsy o r exhumation b: l i v i n g o r w o r k i n g i n an a r e a suspected o f c o n t a m i n a t i o n
TABLE 111 DATA CONCERNING THE ASHING AND THE FLUORIDE DETERMINATIONS AS PERFORMED ON THE BONE T I S S U E S COLLECTED I N THIRTEEN POST MORTEM CASES AND A SURGICALLY REMOVED BONE FRAGMENT Case N
Nature o f t h e bone sample
Weight ( 9 ) d r y bone ash ratio’
F l u o r i d e Conc. (ppm) ash d r y bone
1
Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium)
1.953 3.187 1.273 5.213
0.633 0.819 0.708 3.126
3.1 3.9 1.8 1.6
3.653 3,523 3.132 3,266
1,184 905 1,742 1,958
2
Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior C a l v a r i urn)
1.806 2.978 2.722 2.330
0.125 1.040 1.449 1.543
14.4 2.9 1.9 1.5
833 1,247 91 1 642
58 435 485 425
3
Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium)
1.730 2.570 2.566 4.156
0.235 0.684 1.275 2.631
7.3 3.7 2.0 1.6
2,525 3.083 1.822 1,913
343 821 905 1,211
161
4
Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium)
2.632 3.305 2.185 4.141
0.081 1.648 1.319 2.685
32.5 2.0 1.7 1.5
223 452 369 31 1
7 225 223 201
5
Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium)
4.094 12.130 3.361 5.991
0.819 3.806 1.848 3.61 5
5.0 3.2 1.8 1.6
1,636 1,655 1,288 1,532
327 51 9 708 925
6
Sternum (Manu b r ium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium)
3.792 6.197 3.533 7.442
0.637 2.335 1.355 4.585
6.0 2.7 2.6 1.6
1.199 1,380 1.131 869
201 520 434 535
7
Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium)
3.132 7.110 2.584 5.314
0.558 2.527 1.231 3.306
5.6 2.8 2.1 1.6
1,529 2.134 1,636 1,665
21 2 7 58 779 1,036
8
Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Cal v a r i urn)
5.447 6.242 2.489 7.810
1.526 2.045 1.006 4.778
3.6 3.0 2.5 1.6
1,016 883 908 767
285 289 367 469
9
Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium)
3.574 4.463 2.646 3.895
0.858 1.053 0.816 2.378
4.2 4.2 3.2 1.6
1,162 1.475 1.023 955
266 348 31 5 583
10
Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium)
5.426 5.132 3.471 4.077
1.392 1.724 0.997 2.667
3.9 3.0 3.5 1.5
798 850 787 749
205 286 226 490
11
Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium)
3.017 6.041 1.860 4.606
0.582 1.402 0.987 2.899
5.2 4.3 1.9 1.6
877 825 71 1 700
169 191 377 441
12
Sternum (Manubrium) Femur ( C a p u t ) Femur ( T r o c h a n t e r ) Rib Skull (Posterior Calvarium)
1.139 2.295 1.276 0.746 1.955
1,300 1,403 1,061 1,157 1.033
433 528 467 432 668
Sternum (Manubrium) Femur ( C a p u t ) Femur ( T r o c h a n t e r ) Rib Skull (Posterior Calvarium)
2.818 2.497 2.289 2.320 3.803
-
-
1,414 1,598 1,483 1,357 1,109
576 644 508 581 709
Femur ( C a p u t )
5.392
-
-
2,177
963
13
14
' d r y bone/ash
-
-
-
-
-
162 c o n c e r n i n g t h e a s h i n g and t h e f l u o r i d e d e t e r m i n a t i o n s as p e r f o r m e d on t h e bone t i s s u e s c o l l e c t e d i n t h o s e t h i r t e e n cases, ("Caput"
and t h e bone f r a g m e n t
o f t h e f e m u r ) w h i c h was s u r g i c a l l y removed f r o m t h e 71-year-old
woman, w e r e l i s t e d i n T a b l e 111.
As c o u l d b e e x p e c t e d , t h e r a t i o o f t h e
w e i g h t o f t h e d r y bone t o t h e w e i g h t o f t h e c o r r e s p o n d i n g amount o f ash r e m a i n e d v e r y c o n s t a n t f o r c o m p a c t b o n e t i s s u e s s u c h as t h e s k u l l .
How-
ever, f o r h i g h l y v a s c u l a r i z e d bone t i s s u e s t h i s v a l u e v a r i e d o v e r a w i d e As i l l u s t r a t e d i n T a b l e s I V and V t h e f l u o r i d e l e v e l s f o u n d i n t h e
range.
bone samples o r i g i n a t i n g f r o m t h e t e n r e f e r e n c e cases showed h i g h l y i n d i v i d u a l d i f f e r e n c e s (CV%:
58.90
-
68.21).
However, t h o s e f o u n d i n t h e bone
s a m p l e s w h i c h o r i g i n a t e d f r o m a s i n g l e p e r s o n u s u a l l y w e r e i n t h e same range.
T h i s i n d i c a t e d a good c o r r e l a t i o n (r
=
0.94 - 0.99)
without a sig-
n i f i c a n t d i f f e r e n c e (pO.05).
TABLE I V
MEAN VALUES OF THE FLUORIDE CONCENTRATIONS FOUND I N THE TEN REFERENCE CASES Sternum
X
1,457.10
Femur 1,668.20
Rib 1,300.70
Sku1 1 1,266.90
SD
982.87
984.44
766.08
864.14
SX
310.81
311.31
242.86
273.26
CVZ
67.45
59.01
58.90
68.21
X: mean; SD: s t a n d a r d d e v i a t i o n : SX: s t a n d a r d e r r o r : CV%: c o e f f i c i e n t o f variation % TABLE V CORRELATION DATA CALCULATED FOR THE TEN REFERENCE CASES DeterminatioQ Intercept constant, r a
Slope b
Correlation coefficient, r
X
Y
femur
skull
0.89
-114.20
0.33
0.94
femur
rib
0.89
72.97
0.74
0.95
femur
sternum
0.93
-1 48.40
0.96
0.96
skul 1
rib
0.97
192.54
0.87
0.99
skul 1
sternum
0.96
46.94
1.11
0.98
rib
sternum
0.96
-1 75.38
1.26
0.98
(y=a+bx;
iJ=lO;
p=NS)
163 It was a l s o i n t e r e s t i n g t o n o t e t h a t t h e maximum ( c a s e 1) a s w e l l a s t h e m i n i m u m f l u o r i d e l e v e l s ( c a s e 4) w e r e f o u n d i n t h e bone s a m p l e s t a k e n f r o r r p e r s o n s who h a d b e e n l i v i n g i n r u r a l a r e a s , fluoride pollution.
supposedly f r e e f r o m i n d u s t r i a l
T h i s suggests t h a t t h e f l u o r i d e concentrations
found
i n t h e f o u r suspected cases were w i t h i n t h e l i m i t s o f t h e s o - c a l l e d r e f e r ence v a l u e s o r blanks.
I t c o u l d be c o n c l u d e d t h a t u p t a k e and m i n e r a l i z a -
t i o n o f f l u o r i d e i n t h e bones o f t h e s e persons were t h o s e o f t h e random population.
F r o m t h e s e f i g u r e s h o w e v e r i t was i m p o s s i b l e t o d e t e r m i n e
which were t h e fluoride-exposed
persons.
CONCLUSION Cur i n v e s t i g a t i o n s
p e r f o r m e d o n a s e r i e s o f human bones i n d i c a t e d t h a t
the f l u o r i d e concentrations i.e.,
found indeed v a r i e d f r o m l o w t o h i g h levels,
f r o m a b o u t 300 t o 3 , 0 0 0 p p m ( i n b o n e ash).
H o w e v e r , i t w a s n o t e d t h a t f l u o r i d e c o n c e n t r a t i o n s u p t o 2,000 ppm ( i n b o n e a s h ) w e r e f o u n d i n a b o n e f r a g m e n t p r o c u r e d b y a n o l d woman, e x p o s u r e t o i n d u s t r i a l f l u o r i d e e m i s s i o n s was u n d e n i a b l e . p e r c e n t a g e o f t h e r e f e r e n c e c a s e s l o w e r l e v e l s w e r e found. h i g h amounts,
however,
were a l s o detected.
whose
I n a large Exceptionally
The r e a s o n f o r t h e s e e m e r g i n g
concentrations remained obscure since the available information did not s p e c i f y t h e source o f p o s s i b l e contamination. REFERENCES 1.
V i g l i a n i EC (1959) Med d L a v o r o 50:323-327
2.
F l u o r i d e s and iluman H e a l t h . H o d g e HC, S m i t h FA. G e d a l i a 1 ( 1 9 7 0 ) I n : W o r l d H e a l t h O r g a n i z a t i o n , Monograph S e r i e s No.59, Geneva, p p 141-161
3.
V e r g o t e G, Van Z e l e W, B e l g 56: 130-1 34
De C l e r c k F,
H e y n d r i c k x A (1979) F a r m T i j d s c h r
4.
S i n g e r L, A r m s t r o n g WD (1968) A n a l Chem 40:613-614
5.
Z o b e r A,
6.
S c h e l l m a n n B (1975) Z K l i n Chem K l i n B i o c h e m 13:197-201
Van d e n Heede MA,
H e y n d r i c k x AM,
J A s s o c O f f A n a l Chem 58:1135-1137
Van Peteghem CH,
Van Z e l e WA (1975)
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 165-172 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
165
DENTAL FLUOROSIS I N D U C E D BY WATER-BORNE FLUORIDE I N THE EFFLUENT O F A N ALUMINUM PLANT
TORU YAMAUCHI~,
YASUO H O N M A ~ , AND HUMIO
TSUNODA~
' D e p a r t m e n t o f P u b l i c H e a l t h , F u k u s h i m a M e d i c a l C o l l e g e , F u k u s h i m a 960, Japan 'Department
o f H y g i e n e and P u b l i c H e a l t h ,
I w a t e M e d i c a l U n i v e r s i t y , Japan
ABSTRACT Twenty-six
c a s e s o f d e n t a l f l u o r o s i s (DF) i n c l u d i n g p r e s u m a b l e c a s e s w e r e
o b s e r v e d among y o u n g p e o p l e ( l e s s t h a n 24 y e a r s o l d ) i n a v i l l a g e w h e r e t h e a i r was s l i g h t l y p o l l u t e d b y a i r - b o r n e f l u o r i d e ( F ) f r o m an a l u m i n u m p l a n t .
I t was shown b y an e p i d e m i o l o g i c a l s t u d y t h a t s t r e a m s i n t h e v i l l a g e w e r e c o n t a m i n a t e d b y h i g h l e v e l s o f F f r o m an e f f l u e n t o f t h e a l u m i n u m p l a n t and t h e r e s i d e n t s u s u a l l y consumed t h e s e s t r e a m - w a t e r s purposes,
w i t h o u t any knowledge o f F p o l l u t i o n .
daily,
even f o r d r i n k i n g
The h i g h l e v e l o f F i n t h e
e f f l u e n t r e s u l t e d f r o m a wet scrubber system i n t h e plant.
INTRODUCTION T h e r e h a v e been many r e p o r t s on e n v i r o n m e n t a l p o l l u t i o n b y a i r - b o r n e f l u o r i d e f r o m i n d u s t r i a l s o u r c e s (1-4). i n g water-borne o f water-borne
However,
few papers e x i s t concern-
f l u o r i d e f r o m i n d u s t r i a l sources. fluoride,
As f o r t h e h e a l t h e f f e c t
a l m o s t a l l t h e cases r e p o r t e d e a r l i e r were caused
by consumption o f water containing high concentrations o f f l u o r i d e from s u c h n a t u r a l s o u r c e s as s p r i n g s o r w e l l - w a t e r s
(5,6).
I n t h i s paper, t h e o u t b r e a k o f d e n t a l f l u o r o s i s i n d u c e d b y w a t e r - b o r n e f l u o r i d e f r o m an i n d u s t r i a l s o u r c e i s r e p o r t e d .
15 y e a r s ago,
Although i t occurred about
t h e r e s u l t s o f t h e study are s t i l l considered t o be valuable
today i n terms o f c o n t r o l l i n g environmental p o l l u t i o n . MATERIALS AND METHODS The s u b j e c t a r e a o f t h e e p i d e m i o l o g i c a l s u r v e y was a s m a l l v i l l a g e named Nagao o f K i t a k a t a C i t y , Japan (Fig.
1).
i n t h e n o r t h w e s t e r n p a r t o f Fukushima Prefecture,
T h e r e was an a l u m i n u m p l a n t l o c a t e d a b o u t 2 km f r o m Nagao.
F l u o r i d e l e v e l s i n t h e a i r around t h e p l a n t , demiological
survey,
determined p r i o r t o t h e epi-
a r e a l s o shown i n F i g u r e 1
surrounded m a i n l y by r i c e f i e l d s .
(2,4).
The p l a n t was
166
F i g u r e 1. S u r v e y e d a r e a a n d f l u o r i d e l e v e l s Tn t h e a i r (2). s e n t a t m o s p h e r i c f l u o r i d e l e v e l s (ugF/dm2/mon.).
Figures repre-
The s u b j e c t s o f t h e e p i d e m i o l o g i c a l s u r v e y w e r e a l l r e s i d e n t s o f Nagao and w e r e 7 t o 6 9 y e a r s o f age.
The s u r v e y i n c l u d e d t h e f o l l o w i n g :
1. D e n t a l e x a m i n a t i o n o f a l l s u b j e c t s b y d i r e c t e x a m i n a t i o n a n d p h o t o graphs.
2. G e n e r a l h e a l t h e x a m i n a t i o n o f t h e i n c l u d i n g p h y s i c a l examination, (protein,
3.
s u b j e c t s t e n y e a r s o l d and above,
blood analysis (hematocrit),
and u r i n a l y s i s
g l u c o s e and pH).
M e a s u r e m e n t o f f l u o r i d e c o n t e n t i n u r i n e ( s p o t and 24 h o u r u r i n e ) .
4. E n v i r o n m e n t a l s u r v e y i n c l u d i n g a q u e s t i o n n a i r e a b o u t t h e s o u r c e s o f w a t e r f o r d a i l y c o n s u m p t i o n i n t h e p a s t and p r e s e n t , o f f l u o r i d e l e v e l s i n w e l l - w a t e r and s t r e a m - w a t e r
and t h e d e t e r m i n a t i o n s
i n the village.
RESULTS The s u r v e y was c o n d u c t e d i n 1971. i n t h e e x a m i n a t i o n (85% c o v e r a g e ) ,
O u t o f 220 s u b j e c t s , and i n p a r t i c u l a r ,
185 p a r t i c i p a t e d
a l l c h i l d r e n 7 t o 15
years o l d received t h e examination. Dental examination The r e s u l t s o f t h e d e n t a l e x a m i n a t i o n a r e shown i n F i g u r e 2. the prevalence o f dental f l u o r o s i s (mottled teeth)
A s t u d y on
indicated that the
167
DMF Rate 20
- 24 (9)
1p
0
?,S
0.0
-
16 18 (1 0 13 15 (1 7)
-
10 12 I 2 1) 1 - $
(1 5)
30 20 10 0 PREVALENCE OF MOTTLED TEETH”
SO\
48
F i g u r e 2.
Ap. (n)
0
1
2
3
Results o f dental examination
highest occurrence t h e s u b j e c t s were,
was i n t h e 10 t o 15 y e a r age g r o u p , t h e lower t h e prevalence.
o b s e r v e d i n t h e g r o u p aged 25 y e a r s o r o l d e r . 24,
6
S
b
DMFTlperson
and t h a t t h e o l d e r
No m o t t l e d t e e t h w e r e
O u t o f 78 SubJeCtS aged 7 t o
1 3 c a s e s (17%) w e r e d i a g n o s e d a s p r e s u m a b l e d e n t a l f l u o r o s i s (MQ),
a n o t h e r 1 3 c a s e s a s h a v i n g m o t t l e d t e e t h (MI
+
M2).
and
The d i s t r i b u t i o n o f
t h e s e c a s e s i s i l l u s t r a t e d i n F i g u r e 3.
‘,
F i g u r e 3.
Wet scrubber in Aluminum Plant
I
D i s t r i b u t i o n o f d e n t a l f l u o r o s i s and F - l e v e l s (ppm). 0 ,c a s e o f d e n t a l f l u o r o s i s ; Q , case o f presumable d.f.
i n stream-waters
168 I n contrast,
t h e DMF (decayed,
missing,
and f i l l e d ) r a t e i n d i c a t e d t h a t
t h e l o w e s t l e v e l was i n t h e g r o u p aged 1 0 t o 1 5 prevalence o f dental fluorosis.
who a l s o h a d t h e h i g h e s t
DMFT/person was t h e l o w e s t i n t h e g r o u p
aged 7 t o 9 y e a r s and i n c r e a s e d w i t h age o f t h e s u b j e c t s . Fluoride content i n urine F l u o r i d e c o n c e n t r a t i o n s f o u n d i n t h e 24 h o u r u r i n e a r e shown i n F i g u r e 4. The mean
l e v e l o f f l u o r i d e i n u r i n e i n c r e a s e d w i t h i n c r e a s e i n age u n t i l
t h e age g r o u p o f 50 y e a r s i n m a l e and 4 0 y e a r s i n f e m a l e .
I n n e i t h e r spot
n o r 24 h o u r u r i n e s a m p l e s w e r e a b n o r m a l l y h i g h l e v e l s o f f l u o r i d e found.
1.5 -Male
i O T 10
2Ll
19
29
r
F i g u r e 4.
-
7
30
F-contents
5:0 59
49
3 9
6,0 AGE GROUP
n 24 hour u r i n e samples o f t h e r e s i d e n t s .
Other h e a l t h examination F i g u r e 5 shows t h e mean l e v e l o f h e m a t o c r i t ( H t ) b y d i f f e r e n t age groups. No a b n o r m a l H t l e v e l s n o r p a t h o l o g i c a l c o n d i t i o n s w e r e o b s e r v e d i n t h e examination. F l u o r i d e l e v e l i n w a t e r samples The f l u o r i d e c o n c e n t r a t i o n s i n t h e w e l l - w a t e r s a m p l e s a r e shown i n T a b l e
I. E x c e p t f o r o n e s a m p l e
w h i c h s h o w e d m o r e t h a n 0.1 ppm.
37 o u t o f 46
s a m p l e s (80%) g a v e l e v e l s w i t h i n 0.050 t o 0 . 0 9 9 ppm. a n d t h e mean F l e v e l was 0.057
ppm.
The f l u o r i d e l e v e l s i n 20 w a t e r s a m p l e s f r o m t h e s t r e a m s i n Nagao v i l l a g e a r e shown i n F i g u r e 3. i n t o t h e s e streams. 8.20 ppm.
F
The e f f l u e n t f r o m t h e a l u m i n u m p l a n t was d i s c h a r g e d The mean F l e v e l was 3.34 ppm w i t h a r e r g e o f 2.10 t o
l e v e l s o f t h e samples taken f r o m s i t e s near t h e p l a n t were
h i g h e r than those o f samples taken f r o m t h e streams d i s t a n t f r o m t h e plant.
169
I Male
V'
0
I
,
,
I
I
12 1519 2 9
F i g u r e 5.
39
49
59
A0g e
69
Group
D i s t r i b u t i o n o f h e m a t o c r i t b y age g r o u p s .
TABLE I FLUORIDE LEVEL I N WELL-WATERS Fluoride level (PPm)
Number o f w e l l s
L e s s t h a n 0.049
0.050
-
0.099
0.100 o r more
8
(17.4%)
37
(80.4%)
l a ( 2.2%) 46
Total
~~
aMean l e v e l was 0.68 ppm (0.057 ppm i f t h e h i g h e s t one was e x c l u d e d ) .
On e x a m i n a t i o n o f t h e d i s t r i b u t i o n o f d e n t a l f l u o r o s i s c a s e s , m a n y w e r e d i s t r i b u t e d i n t h e west p a r t o f t h e v i l l a g e where t h e f l u o r i d e l e v e l s i n t h e stream-water
w e r e h i g h e r t h a n 3.0 ppm.
Results o f questionnaire F i g u r e 6 shows t h e a n n u a l t r e n d o f t h e s o u r c e s o f w a t e r f o r d a i l y cons u m p t i o n i n Nagao, b a s e d o n r e s u l t s o f t h e q u e s t i o n n a i r e .
I n t h e 1950s,
n e a r l y 80% o f t h e r e s i d e n t s c o n s u m e d t h e s t r e a m - w a t e r n e a r t h e i r h o u s e s daily,
and h a l f
3f
them used t h e w a t e r f o r d r i n k i n g purposes. These propor-
t i o n s b e g a n t o d e c r e a s e i n t h e b e g i n n i n g o f t h e 1960s w h e n t h e a l u m i n u m p l a n t began t o t r e a t i t s e m i s s i o n w i t h wet-scrubbers.
170
H i s t o r y o f w a t e r s u p p l y and consumption 1 Aluminum p l a n t began t o t r e a t i t s e f f l u e n t . 2 Many c a r p i n t h e streams d i e d f r o m an unknown cause. 3 Aluminum p l a n t began t o o p e r a t e i t s wet-scrubber system.
F i g u r e 6.
DISCUSSION S e v e r a l c a s e s o f p r e s u m e d d e n t a l f l u o r o s i s w e r e o b s e r v e d among s c h o o l c h i l d r e n i n Nagao v i l l a g e ,
Fukushima P r e f e c t u r e ,
Japan,
i n 1968
and 1969.
There was an aluminum p l a n t near Nagao and i t was known t h a t t h e a i r i n t h e s u r r o u n d i n g a r e a was p o l l u t e d b y a i r - b o r n e Although the f l u o r i d e l e v e l s i n the those i n non-polluted
air
fluoride
(Figs. 1 , 2 ) , t h e y
were
n o t c o n s i d e r e d t o be
s e r i o u s enough t o a f f e c t
the health o f the residents.
several
fluorosis
cases o f d e n t a l
( T a r o m a r u v. and T a k a y o s h i v.,
from the plant.
a i r i n 1968 were s l i g h t l y h i g h e r t h a n I n fact,
were found i n o t h e r
villages
only
(3)
F i g . 1 ) w h i c h w e r e c l o s e r t h a n Nagao t o t h e
a l u m i n u m p l a n t , and w h e r e t h e f l u o r i d e l e v e l i n t h e a i r was h i g h e r t h a n t h a t i n Nagao as w e l l .
An e p i d e m i o l o g i c a l survey,
therefore,
was planned
t o i n v e s t i g a t e t h e cause o f t h e o u t b r e a k o f d e n t a l f l u o r o s i s i n Nagao. The aluminum p l a n t s t a r t e d i t s o p e r a t i o n i n 1943,
and i n 1965 t h e annual
p r o d u c t i o n was mo.re t h a n 50,000 tons. The p r e v a l e n c e o f d e n t a l f l u o r o s i s among y o u n g p e o p l e l i v i n g i n l o c a l areas where f l u o r i d e c o n t a m i n a t e d w a t e r was consumed had been e s t i m a t e d t o b e 1 0 t o 1 3 % (7). hand,
T h i s s u r v e y showed a p r e v a l e n c e o f 33.3%.
On t h e o t h e r
t h e p r e v a l e n c e of d e n t a l c a r i e s i n Nagao was compared w i t h t h o s e i n
Y a m a s h i n a c i t y and S h u g a k u i n t o w n ( 6 ) , b o t h i n K y o t o . I n Y a m a s h i n a c i t y ,
171 TABLE I 1
COMPARISON OF DMF RATE AND DMFT/PERSON I N NAGAO V. OTHER AREAS
WITH
Area
Nagao V.
Yamashina C.
Shugakuin T.
6.9
10-1 2
5.1
1 .oo
13-1 5
5.8
1.59
(6)
( 1 964)
0.32-1.48
7- 9
4.9-1 1.3
10-12
10.6-1 1 .4
1.73-3.81
13-1 5
12.7-14.1
3.34-3.81
7- 9
1 1 .8-20.0
0.84-2.28
10-1 2
13.5-17.3
2.90-4.17
13-1 5
16.6-23.2
4.43-6.46
(6)
( 1 964)
0.73
7- 9
f l u o r i d a t e d w a t e r was s u p p l i e d .
B o t h D M F r a t e and DMFT/person i n Nagao
w e r e much l o w e r t h a n i n t h o s e t w o a r e a s ( T a b l e 11).
A high prevalence o f
d e n t a l f l u o r o s i s and a l o w p r e v a l e n c e o f d e n t a l c a r i e s were observed among young people aged 10 t o 15.
R e s u l t s f r o m t h e d e n t a l e x a m i n a t i o n suggested
t h e p o s s i b i l i t y o f exposure t o r e l a t i v e l y h i g h l e v e l s o f f l u o r i d e by t h e r e s i d e n t s i n Nagao.
However,
the low content o f f l u o r i d e i n the u r i n e o f
t h e r e s i d e n t s and t h e l o w l e v e l o f f l u o r i d e i n w e l l - w a t e r s
consumed a t t h e
t i m e o f survey suggested t h a t t h e f l u o r i d e p o l l u t i o n m i g h t have o c c u r r e d i n t h e p a s t and n o t a t t h e t i m e o f t h e survey. A n a l y s i s o f t h e q u e s t i o n n a i r e showed t h a t n e a r l y 8 0 % o f t h e r e s i d e n t s consumed t h e s t r e a m - w a t e r n e a r t h e i r h o u s e s d a i l y ,
and,
i n particular,
a b o u t h a l f o f t h e m consumed t h e w a t e r f o r d r i n k i n g p u r p o s e s i n t h e 1 9 5 0 s ( F i g . 6).
T h i s c o u l d e x p l a i n why c a s e s o f d e n t a l f l u o r o s i s w e r e d i s t r i b -
u t e d a l o n g t h e streams where t h e f l u o r i d e c o n c e n t r a t i o n s o f t h e w a t e r were high. The f i n d i n g s o f t h e e p i d e m i o l o g i c a l survey d e s c r i b e d above have l e a d t o t h e f o l l o w i n g conclusion:
The e f f l u e n t f r o m t h e a l u m i n u m p l a n t c a u s e d
f l u o r i d e p o l l u t i o n o f t h e streams i n t h e v i l l a g e .
T h i s had o c c u r r e d s i n c e
t h e b e g i n n i n g o f t h e 1960s, when t h e p l a n t i n s t a l l e d a w e t - s c r u b b e r
to
c o l l e c t gas c o n t a i n i n g h i g h l e v e l s o f f l u o r i d e and d i s c h a r g e d t h e e f f l u e n t i n t o t h e streams.
The r e s i d e n t s a l o n g t h e s t r e a m s had consumed t h e stream-
172 w a t e r w i t h o u t any k n o w l e d g e o f t h e p o l l u t i o n .
The h i g h l e v e l s o f f l u o r i d e
i n t h e i r d r i n k i n g w a t e r c a u s e d d e n t a l f l u o r o s i s i n y o u n g p e o p l e whose t e e t h were j u s t growing a t t h a t time. I n t h e 1960s,
h a r d l y anybody,
n a t u r a l l y n o t t h e residents o r t h e workers
o f t h e p l a n t , o r even t h e s c i e n t i s t s s p e c i a l i z i n g i n f l u o r i d e p o l l u t i o n , c o n s i d e r e d t h e hazardous e f f e c t o f water-borne f l u o r i d e discharged from i n d u s t r i a l s o u r c e s (5.6).
While air-borne
f l u o r i d e i s known t o a f f e c t
v e g e t a t i o n and a n i m a l s s u c h a s l i v e s t o c k g r a z i n g o n g r a s s , fluoride,
i r r e s p e c t i v e o f i t s source,
water-borne
may d i r e c t l y a f f e c t human h e a l t h .
The e p i s o d e r e p o r t e d h e r e s u g g e s t s t h a t a m e a s u r e t a k e n a g a i n s t one t y p e o f e n v i r o n m e n t a l p o l l u t i o n m i g h t have caused a n o t h e r t y p e o f p o l l u t i o n w h i c h a d v e r s e l y had a f f e c t e d t h e h e a l t h o f t h e r e s i d e n t s . REFERENCES
1.
A g a t e JN, B e l l GH, B o d d i e GF, B o w l e r RG. B u c k e l l M, C h e e s e m a n EA, D o u g l a s THJ. D r u e t t HA, G a r r a d J , H u n t e r 0, P e r r y K M A . R i c h a r d s o n JD, d e V.Weir JB ( 1 9 6 8 ) I n d u s t r i a l f l u o r o s i s : a s t u d y o f t h e h a z a r d t o man and a n i m a l n e a r F o r t W i l l i a m , S c o t l a n d . Med Res C o u n c i l Mem No 22, 47. HM S t a t i o n a r y O f f i c e , London
2.
T s u n o d a H, 59: 2-1 1
Haneda M (1968) Sangyou-Kougaku
( I n d u s t r i a l Engineering)
3.
Tsunoda H (1968) J J p n SOC A i r P o l l 2:135-137
4.
Tsunoda H (1970) J E n v i r o n P o l l C o n t r o l 6(7):16-20
5.
Cornmi t t e e o n B i o l o g i c a l E f f e c t o f A t m o s p h e r i c P o l l u t i o n : F l u o r i d e s ( 1 9 7 1 ) N a t Acad S c i e n c e s , W a s h i n g t o n , DC, pp 5-11
6.
L e o n e NC, M a r t i n AE. M i n o g u c h i G, S c h l e s i n g e r ER, S i d d i q u i A H ( 1 9 7 0 ) WHO (Geneva): 2 7 3 - 3 2 1
7.
T s u j i Y (1973) J E n v i r o n P o l l C o n t r o l 9(4):4-10
and 6(8):3-8
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 173-18C 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
173
COMMUNITY DENTAL FLUOROSIS AND THE FLUORIDE CONTENT OF SALIVA AND U R I N E I N THAI CHILDREN PRATHIP PHANTUMVANIT172, Y U P I N SONGPAISAN2,
AND R U D I G. SCHAMSCHULA3
'Department o f Biochemistry, F a c u l t y o f D e n t i s t r y , U n i v e r s i t y , H e n r i - D u n a n t Road, Bangkok 10500, T h a i l a n d 'Intercountry Thai land
Centre f o r Oral Health,
3 1 n s t i t u t e o f D e n t a l Research,
Chulalongkorn
M i n i s t r y o f Pub1 i c H e a l t h , C h i a n g m a i ,
Sydney,
Australia
ABSTRACT C o m m u n i t y i n d i c e s o f f l u o r o s i s ( F c i ) and t h e f l u o r i d e ( F ) c o n c e n t r a t i o n s i n u r i n e and s a l i v a w e r e assessed i n 184 c h i l d r e n , f r o m Chiangmai Province,
Thailand,
aged 11 t o 13 y e a r s ,
who h a d been c o n s u m i n g w a t e r c o n t a i n i n g
0.05 ppm, 0 . 3 8 ppm, 0.76 ppm, 1.76 ppm. a n d 3.09 ppm F, r e s p e c t i v e l y .
The
mean F c i v a l u e s a n d t h e u r i n a r y a n d s a l i v a r y F l e v e l s i n c r e a s e d c o n s i s t e n t l y w i t h increase i n F c o n t e n t o f t h e water supply, r e l a t i o n s h i p i n each instance.
An F c i l e v e l o f 0.4,
was r e a c h e d when t h e F c o n t e n t i n
considered i n s i g n i f i c a n t i n populations, t h e w a t e r was 0.44
ppm;
exhibiting a sigmoid a t which fluorosis i s
c o r r e s p o n d i n g mean u r i n a r y and s a l i v a r y F concen-
t r a t i o n s w e r e 0.84 ppm and 33.2 ppb.
respectively.
These r e s u l t s c o n f i r m e d
t h e usefulness o f u r i n a r y and s a l i v a r y F as short-term exposure i n small groups o f children.
indicators o f F
The f i n d i n g s a l s o c a u t i o n a g a i n s t
applying e x i s t i n g formulas f o r determining optimal F l e v e l s o f water supply i n d e v e l o p i n g and t r o p i c a l c o u n t r i e s w i t h o u t e x p l o r i n g t h e d i e t a r y and n u t r i t i o n a l c h a r a c t e r i s t i c s o f t h e community concerned.
INTR3DUCTION T h a i l a n d i s one o f t h e m a i n f l u o r s p a r p r o d u c i n g c o u n t r i e s i n t h e w o r l d . Much o f t h e m i n e r a l i s m i n e d f r o m numerous d e p o s i t s s i t u a t e d i n t h e n o r t h e r n p a r t o f t h e c o u n t r y w h e r e L e a t h e r w o o d and a s s o c i a t e s f o u n d h i g h p r e v a l e n c e o f d e n t a l f l u o r o s i s (1).
E n d e m i c f l u o r o s i s h a s been c o n f i r m e d
i n t h e n o r t h e r n p r o v i n c e o f Chiangmai i n a r e c e n t study, mapping o f d r i n k i n g w e l l w a t e r s d i s c l o s e d v a r i a t i o n s 4.38 ppm ( 2 ) .
where f l u o r i d e ( F )
r a n g i n g f r o m 0.01
to
The e x i s t e n c e o f s u b s t a n t i a l r u r a l p o p u l a t i o n s exposed t o
highly contrasting levels o f F
17
f u r t h e r i n v e s t i g a t i o n s of f l u c r ? ; ' e
n;Ltilr I Y ,G-
r a t e r p r o v i d e d an o p p o r t u n i t y f o r
r
7nd u t i l i z a t i o n .
174 The a s s o c i a t i o n b e t w e e n F c o n c e n t r a t i o n s i n d r i n k i n g w a t e r and t h e p r e v a l e n c e and s e v e r i t y o f d e n t a l f l u o r o s i s h a s b e e n w e l l e s t a b l i s h e d u n d e r t e m p e r a t e c l i m a t i c c o n d i t i o n s (3,4).
Under t h e s e c o n d i t i o n s ,
the F content
o f u r i n e has been w i d e l y used as an i n d i c a t o r o f F u t i l i z a t i o n and f o r q u a n t i t a t i n g c u r r e n t F i n t a k e (5).
I n contrast,
s a l i v a r y F h a s r a r e l y been
u t i l i z e d f o r corresponding purposes i n f i e l d studies. The p u r p o s e o f t h e p r e s e n t s t u d y was t o e x a m i n e t h e i n t e r r e l a t i o n s between a wide range o f n a t u r a l l y o c c u r r i n g F i n t h e water, indices o f
dental
fluorosis
and t h e F
(Fci)(6)
community
levels of
u r i n e and
a c t i v a t e d whole saliva, under t h e t r o p i c a l c o n d i t i o n s p r e v a i l i n g i n t h e C h i a n g m a i area. MATERIALS AND METHODS P r i o r t o f i n a l d e f i n i t i o n o f t h e sample, t o 13 years,
1,687 s c h o o l c h i l d r e n ,
aged 11
who w e r e c o n s t a n t r e s i d e n t s o f o n e o f t h e 71 r u r a l c o m m u n i t i e s
o f t h r e e n e i g h b o u r i n g d i s t r i c t s o f Chiangmai, samples f r o m t h e i r d r i n k i n g w e l l s .
Those c h i l d r e n whose combined w a t e r
supply,
i n c l u d i n g h o u s e h o l d and s c h o o l w a t e r ,
sions,
i.e.,
<0.1:
0.3-0.5:
were asked t o c o l l e c t w a t e r
0.7-0.9;
f e l l within the f i v e F divi-
1.5-2.0,
a n d 2.4-4.8
ppm.
r e g a r d e d as e l i g i b l e and were asked t o p a r t i c i p a t e i n t h e study. sample comprised 184 o f t h e consenting c h i l d r e n , f u l f i l l q u o t a s o f 30 t o 40 p e r F d i v i s i o n , t i o n s o f b o t h sexes.
were The
randomly selected t o
w i t h approximately equal propor-
Dental examinations were c a r r i e d o u t a t t h e schools
i n n a t u r a l d a y l i g h t u s i n g v i s u a l and t a c t i l e means.
F l u o r o s i s was r e c o r d e d
a c c o r d i n g t o Dean's i n d e x (6) as t h e i n d i v i d u a l s c o r e f o r e a c h p e r m a n e n t tooth,
and averaged f o r each s u b j e c t .
The F c i means w e r e c a l c u l a t e d sepa-
r a t e l y f o r each o f t h e f i v e F d i v i s i o n s . Two s a m p l e s e a c h o f u r i n e and s t i m u l a t e d w h o l e s a l i v a w e r e c o l l e c t e d f r o m e a c h s u b j e c t i n t h e m o r n i n g w i t h an i n t e r v a l o f t w o weeks b e t w e e n t h e duplicates.
F i e l d b l a n k s w e r e made u p w i t h e a c h b a t c h o f s a m p l e s .
s a m p l e s and b l a n k s w e r e a n a l y z e d f o r F i n a 1O:l buffer,
The
s u s p e n s i o n o f TISAB
u s i n g a s p e c i f i c i o n e l e c t r o d e ( O r i o n 940911) and s t a n d a r d p r o c e -
d u r e s d e s c r i b e d p r e v i o u s l y (7). The r e l a t i o n s h i p s
between t h e F c o n c e n t r a t i o n s
t h o s e i n t h e b i o l o g i c a l samples,
i n t h e w a t e r s u p p l y and
and t h e F c i o b t a i n e d f r o m e a c h F d i v i s i o n
were examined u s i n g l i n e a r r e g r e s s i o n analyses. RESULTS T a b l e I shows t h e m a i n c h a r a c t e r i s t i c s o f t h e s a m p l e s s t u d i e d ,
including
t h e F c o n c e n t r a t i o n s i n t h e h o u s e h o l d and s c h o o l w a t e r s and t h e d i s t r i b u t i o n o f children according t o F divisions.
175 TABLE I SAMPLE CHARACTERISTICS
F division
No. o f Children M F Total
Range ( PPm)
F i n w a t e r (ppm)
Household
School
Combined
1
15
24
39
0.05k0.04a
0.04k0.02
0.05k0.03
2
0.3-0.5
16
22
38
0.39k0.14
0.38k0.06
0.38k0.09
3
0.7-0.9
24
15
39
0.74k0.15
0.78+0.20
0.76k0.08
4
1.5-2.0
24
16
40
1.82+0. 34
1.69kO. 11
1.76kO. 17
5
2.4-4.8
15
13
28
3.50k1.20
2.67k0.84
3.09kO. 57
aMeans k S.D.
The mean u r i n a r y and s a l i v a r y F c o n c e n t r a t i o n s and t h e F c i v a l u e s i n e a c h d i v i s i o n a r e shown i n T a b l e 11.
V a r i a t i o n s o f t h e u r i n a r y and s a l i v a r y F
l e v e l s i n each o f t h e F d i v i s i o n s a r e i n d i c a t e d by standard deviations. Several o f these a r e high, b u t a r e n o t excessive i n t h e c o n t e x t o f i n d i v i d ual physiological differences. v a r i a t i o n (CV),
30-502,
The g e n e r a l r a n g e o f t h e c o e f f i c i e n t s o f
i n d i c a t e s moderate o v e r l a p o f values between neigh-
b o u r i n g d i v i s i o n s f o r m o s t samples.
I n contrast,
the coefficients f o r Fci
values are h i g h l y variable i n the f i r s t three F divisions, 129%, and 952,
1.e..
2672,
respectively, r e f l e c t i n g t h e occasional incidence o f s i g n i f -
i c a n t degree o f f l u o r o s i s a t r e l a t i v e l y l o w F l e v e l s i n t h e water. I n absolute terms, o f water,
t h e mean u r i n a r y F c o n c e n t r a t i o n a l w a y s exceeds t h a t
i n d i c a t i n g c o n t r i b u t i o n f r o m o t h e r sources.
T h i s e f f e c t i s most
TABLE I 1 FLUORIDE CONCENTRATIONS I N D R I N K I N G WELL WATER, U R I N A R Y A N D SALIVARY SAMPLES, AND COMMUNITY FLUOROSIS I N D E X ( F c i ) ACCORDING TO F D I V I S I O N
F Division
Water-F (PPm)
Urinary F ( PPm)
Salivary F (PPb)
Fci
1
0.05+0. 04a
0.33kO. 1 4
28.2k12.5
0.06k0.16
2
0.39kO. 1 4
0.86k0.32
32.4k14.0
0.31 k0. 40
3
0.74kO. 1 5
1.03k0.49
34.4k14.1
0.64k0.61
4
1.82kO. 34
2.57k1.08
58.0k25.6
1.52kO. 72
5
3.50k1.20
3.79k1.84
77.1k36.7
2.30kO. 73
aMeans k S.D.
176
”-
T
q
.-C
T I
I
1
T 3 Well-water
fluoride, ppn1
The relationship b e t w e e n f l u o r i d e c o n c e n t r a t i o n i n t h e d r i n k i n g F i g u r e 1. w e l l w a t e r i n C h i a n g m a i and community f l u o r o s i s i n d e x ( F c i ) .
1 I I
I
0
1
Well-water
2
3
4
fluoride, ppm
F i g u r e 2. The r e l a t i o n s h i p b e t w e e n w e l l - w a t e r r i d e i n c h i l d r e n o f Chiangmai.
f l u o r i d e and u r i n a r y f l u o -
177 a p p a r e n t i n t h e l o w F d i v i s i o n s and i s i n c r e a s i n g l y m a r k e d w i t h i n c r e a s e i n
F c o n c e n t r a t i o n i n t h e water. t o F i n u r i n e i s 1:6.6,
F,
Thus,
i n Division 1 the r a t i o o f F i n water
w h e r e a s i n D i v i s i o n 5 i t i s 1:1.08.
With s a l i v a r y
t h e a b s o l u t e c o n c e n t r a t i o n i s a l w a y s much l o w e r t h a n t h a t i n w a t e r and
u r i n e , b u t s t i l l shows a c o n s i s t e n t i n c r e a s e w i t h b o t h p a r a m e t e r s . The a s s o c i a t i o n b e t w e e n t h e F c o n t e n t o f w e l l w a t e r and t h e F c i ( F i g . u r i n a r y F (Fig.
Z ) , and s a l i v a r y F (Fig.
1).
3) shows a r i s i n g t r e n d i n t h e
lneans w i t h i n c r e a s e d f l u o r i d e e x p o s u r e . The c o r r e l a t i o n i s n e a r l y a b s o l u t e (r=0.989-0.990).
However,
t h e u s e o f means,
i t s h o u l d be n o t e d t h a t t h i s i s due,
which eliminates within-group
variability.
i n part,
to
The r e g r e s -
s i o n e q u a t i o n s and t h e s l o p e s o f t h e l i n e s o f b e s t f i t i n d i c a t e t h a t t h e u n i t change i n each dependent v a r i a b l e i s s u b s t a n t i a l w i t h change i n F concentration i n t h e water.
A closer examination o f Figures 1 - 3 dis-
c l o s e s a d e f i n i t e t r e n d o f s i g m o i d shape between each p a i r o f v a r i a b l e s , indicating t h a t the cause-effect r e l a t i o n s h i p i s c o n s i d e r a b l y reduced a t t h e l o w and h i g h e n d s o f t h e s c a l e s .
1 I
I
0
1
Well-water
2
3
fluoride, ppm
F i g u r e 3. The r e l a t i o n s h i p b e t w e e n w e l l - w a t e r r i d e i n c h i l d r e n o f Chiangmai.
f l u o r i d e and s a l i v a r y f l u o -
178 DISCUSSION The h i g h c o r r e l a t i o n b e t w e e n F c o n t e n t o f w a t e r and F c i f o u n d i n t h i s s t u d y i s i n a c c o r d w i t h p r e v i o u s o b s e r v a t i o n s i n t e m p e r a t e c l i m a t e s (3,4). However,
t h e 0.4 l i m i t o f F c i ,
w h e r e Dean (8) c o n s i d e r e d t h e p r e v a l e n c e and
severity o f fluorosis insignificant,
was r e a c h e d i n t h e p r e s e n t s t u d y a t a
v e r y l o w w a t e r F c o n c e n t r a t i o n o f 0.44 c o n t r a s t w i t h t h e 0.8-1.2 t e m p e r a t e areas.
ppm ( F i g .
1).
This i s i n sharp
ppm F r a n g e u s u a l l y c o n s i d e r e d a p p r o p r i a t e f o r
Similarly,
t h e F c i r a n g e o f 0.4-0.6,
w h i c h was r e p o r t e d
b y Dean ( 8 ) t o b e o f b o r d e r l i n e p u b l i c h e a l t h s i g n i f i c a n c e , 0.44-0.74
corresponded t o
ppm F i n t h e w a t e r , a s o p p o s e d t o r a n g e s r e p o r t e d b y i n v e s t i g a -
t o r s i n t h e U.S.A.
(1.0-1.5
p p m ) ( 3 ) a n d i n D e n m a r k (1.5-2.0
ppm)(4).
One
obvious reason f o r t h e d i f f e r e n c e s between these f i n d i n g s i s t h e higher w a t e r i n t a k e under t r o p i c a l c o n d i t i o n s .
However, a c o n s i s t e n t and s u b s t a n -
t i a l e l e v a t i o n o f u r i n a r y F o v e r t h e F c o n t e n t o f w a t e r shown i n t h i s s t u d y such as F i n foods,
( T a b l e 11) s u g g e s t s t h a t o t h e r f a c t o r s ,
s i g n i f i c a n t l y t o F exposure and t h e h i g h Fci.
may c o n t r i b u t e
A n o t h e r a s p e c t t o be con-
s i d e r e d i s t h e l i k e l y c o n t r i b u t i o n o f p o o r n u t r i t i o n a l s t a t u s o f t h e subj e c t s t o F u t i l i z a t i o n and d e n t a l f l u o r o s i s ,
as s u g g e s t e d b y an e a r l y s t u d y
i n I t a l y (9) and b y more r e c e n t o b s e r v a t i o n s i n A u s t r a l i a n A b o r i g i n a l c h i l d r e n (10).
I n t h e same c o n t e x t ,
s t u d y i n Hungary,
it i s o f interest that,
w h e r e n o m a l n u t r i t i o n was found,
i n a parallel
f l u o r o s i s posed no
c l i n i c a l o r a e s t h e t i c problems a t t h e community l e v e l where F concentrat i o n s o f 1.6-3.1
ppm w e r e f o u n d i n t h e w a t e r (7).
The f o r e g o i n g a s p e c t s d e s e r v e f u r t h e r i n v e s t i g a t i o n s i n y o u n g e r age groups where permanent t o o t h f o r m a t i o n i s s t i l l i n progress,
and i n
sam-
p l e s s e l e c t e d b a s e d on c o n t r a s t i n g n u t r i t i o n a l s t a t u s . I n children,
u r i n a r y F excretion i s n o t always expected t o p a r a l l e l
F
i n t a k e f r o m d r i n k i n g w a t e r t o t h e same e x t e n t a s i n a d u l t s , because o f d e p o s i t i o n o f F i n d e v e l o p i n g and m i n e r a l i z i n g h a r d t i s s u e s .
However,
C o l l i n s and S e g r e t o ( 1 1 ) f o u n d a s t r o n g d i r e c t a s s o c i a t i o n i n t h i s r e s p e c t i n 7 t o 12 and 1 4 t o 1 8 - y e a r - o l d
children.
t i o n s h i p was d e m o n s t r a t e d i n t h i s s t u d y . a l s o f o u n d i n H u n g a r i a n c h i l d r e n (7).
A s i m i l a r dose-response
rela-
A c o r r e s p o n d i n g a s s o c i a t i o n was
although the urinary
F
concentration
was t w i c e a s h i g h i n t h e T h a i s a m p l e a t a l l l e v e l s o f F i n t h e w a t e r . As i n t h e H u n g a r i a n s t u d y (7).
a p o s i t i v e a s s o c i a t i o n b e t w e e n w a t e r and
s a l i v a r y F l e v e l s was c o n f i r m e d i n t h e p r e s e n t s t u d y . that,
It s h o u l d be n o t e d
e v e n t h o u g h i d e n t i c a l c o l l e c t i o n and a n a l y t i c a l m e t h o d s w e r e used,
t h e s a l i v a r y F c o n t e n t s o f t h e Thai samples were t h r e e t o f o u r t i m e s h i g h e r than those i n t h e Hungarian study.
Urinary F l e v e l s were a l s o t w i c e as
h i g h i n t h i s s t u d y as i n t h e Hungarian study.
I t may be assumed t h a t
179 differences i n climatic conditions,
d i e t a r y patterns,
and n u t r i t i o n a l
s t a t u s c o u l d have c o n t r i b u t e d t o t h e s e v a r i a t i o n s . D e s p i t e t h e d i f f e r e n c e s i n u r i n a r y and s a l i v a r y F c o n c e n t r a t i o n s f o u n d i n t h i s s t u d y and i n H u n g a r y (7).
i n c r e a s e i n t h e mean F v a l u e s o f t h e b i o l o g -
i c a l s a m p l e s w i t h i n c r e a s e d F c o n c e n t r a t i o n i n t h e w a t e r was c o n s i s t e n t i n both instances.
Thus,
t h e present f i n d i n g s c o n f i r m t h a t both parameters
are useful indicators o f short-term systemic F turnover i n r e l a t i v e l y small g r o u p s o f s u b j e c t s c o n s i s t i n g o f 20 t o 40. The s i g m o i d r e l a t i o n s h i p b e t w e e n t h e F c o n c e n t r a t i o n i n t h e w a t e r and a l l t h r e e dependent v a r i a b l e s examined supports t h e a s s e r t i o n t h a t t h e f o r m o f a s s o c i a t i o n b e t w e e n d e n t a l c o n d i t i o n s and t h e i r d e t e r m i n a n t s i s o f i m p o r t a n c e (12).
T a k i n g F i g u r e 1 as an example,
the i n f l e x i o n s o f the curve
s u g g e s t t h a t i n t h e s e samples, t h e preva:ence and s e v e r i t y o f f l u o r o s i s i n c r e a s e d m o s t s t e e p l y w h e n F l e v e l i n t h e w a t e r w a s b e t w e e n 0.4 ppm a n d 2.0
ppm.
I n t h e p r e s e n c e o f a h i g h i n f l e x i o n t h e i m p a c t i s so s t r o n g t h a t
l i t t l e a d d i t i o n a l damage i s p o s s i b l e , a l r e a d y been a f f e c t e d . H u n g a r i a n s t u d y (7).
as t h e g r e a t m a j o r i t y o f t e e t h have
Whereas f l u o r o s i s was o f no consequence i n t h e
i t i s o f i n t e r e s t i n t e r m s o f F e x p o s u r e and u t i l i z a -
t i o n t h a t t h e a s s o c i a t i o n b e t w e e n t h e F c o n c e n t r a t i o n i n w a t e r and t h o s e i n u r i n e and s a l i v a (as w e l l as t h o s e i n p l a q u e , curvilinear,
A
n a i l s and h a i r ) was a l s o
r e s e m b l i n g t h e s i g m o i d r e l a t i o n s h i p found i n t h i s study.
m a j o r i t y o f t h e samples used i n t h i s study were w e l l s u p p l i e d o r
oversupplied w i t h water-borne
F and s t u d i e s o n d e f l u o r i d a t i o n a r e a l r e a d y
i n p r o g r e s s w i t h WHO s u p p o r t . areas i n Thailand,
where,
However,
t h e r e a r e numerous F - d e f i c i e n t
i n v i e w o f i n c r e a s i n g c a r i e s i n c i d e n c e (13),
w a t e r f l u o r i d a t i o n o r o t h e r f l u o r i d e s u p p l e m e n t s w o u l d be b e n e f i c i a l justified.
and
Such m e a s u r e s r e q u i r e p r i o r d e t e r m i n a t i o n o f o p t i m a l F concen-
t r a t i o n i n t h e w a t e r , f o r w h i c h f o r m u l a s based on temperature-dependent w a t e r c o n s u m p t i o n h a v e been d e v e l o p e d (3.14.15). G a l a g a n - V e r m i l l i o n f o r m u l a (14),
Application o f the
intended f o r unusual c l i m a t i c c o n d i t i o n s .
w o u l d i n d i c a t e an o p t i m a l l e v e l o f 0.7 ppm f o r m o s t a r e a s o f T h a i l a n d , e v e n t h e m o r e c o n s e r v a t i v e M i n o g u s h i (15) a p p r o a c h o f 0.55 be e x c e s s i v e .
These f i n d i n g s i n d i c a t e t h a t ,
developing countries,
pre-fluoridation
and
ppm a p p e a r s t o
p a r t i c u l a r l y i n t r o p i c a l and
t r i a l s should include determination
o f F i n t a k e f r o m sources o t h e r t h a n water,
exploration o f the nutritional
s t a t u s o f t h e community w i t h emphasis on c h i l d r e n ,
and t h e u s e o f r e l i a b l e
b i o l o g i c a l i n d i c a t o r s o f a c t u a l F e x p o s u r e and u t i l i z a t i o n . ACKNOWLEDGEMENT T h i s s t u d y was s u p p o r t e d t h r o u g h WHO A g r e e m e n t No D2/181/52. t h a n k Ms. J. S a r d o - I n f i r r i
The a u t h o r s
and D r . T h a w o r n A n u m a r n r a j a d h o n f o r l o g i s t i c s
180 arrangements,
M i s s P. S. H. Un f o r a d v i c e ,
c a l assistance, schools,
and t h e p r i n c i p a l s ,
M i s s M. T a n t a n a d i l o k f o r t e c h n i -
teachers,
and c h i l d r e n a t v a r i o u s
f o r t h e i r cooperation.
REFERENCES 1. L e a t h e r w o o d EC. B u r n e t t GW. Pub H1t h 55: 1792-1 799
C h a n d r a v e j j s m a r n R.
S i r i k a y a P (1965) Am J
2. P h a n t u m v a n i t P, S o n g p a i s a n Y. V i s a r u r a t h S . P a t i m a n u k a s e m P ( 1 9 8 2 ) O s t e r r Z S t o m a t o l 79:303-304 3. G a l a g a n DJ. 4.
Lamson GG ( 1 9 5 3 ) P u b l H l t h Rep 68:497-508
D e n t a l F l u o r o s i s and C a r i e s . M d l l e r IJ (1965) S c i e n c e P u b l i s h e r s , Copenhagen, p p 146-147
Rhodes I n t e r n a t i o n a l
5. E r i c s o n Y (1971) A c t a O d o n t Scand 29:43-51 6.
Dean HT ( 1 9 3 4 ) J Am D e n t Assoc 21:1321-1426
7.
S c h a m s c h u l a RG. S u g a r E, Un PSH, T o t h K. C o m m u n i t y D e n t O r a l E p i d e m i o l 13:104-107
B a r m e s DE. A d k i n s BL ( 1 9 8 5 )
F l u o r i n e and D e n t a l Health. Am 8. D e a n HT ( 1 9 4 2 ) I n : M o u l t o n F R ( e d ) Assoc Adv S c i , W a s h i n g t o n DC, p p 23-31 9.
M a s s l e r M,
S c h o u r I (1952) J Am D e n t Assoc 44:156-169
10. S c h a m s c h u l a RG. C o o p e r MH (1982) I n : B i e n n i a l Report, t u t e o f D e n t a l R e s e a r c h , Sydney, p p 29-31 11. C o l l i n s EM,
1980-82,
Insti-
S e g r e t o VA ( 1 9 8 4 ) J D e n t c h i l d 51:352-355
12. S c h a m s c h u l a R G , A d k l n s BL, Agus HM. B a r m e s DE, C h a r l t o n G ( 1 9 7 8 ) I n : B i b b y BG, S h e a r n RJ ( e d s ) P r o c e e d i n g s , M e t h o d s o f C a r i e s P r e d i c t i o n . Spec S u p p l t o M i c r o b i o l A b s t r a c t s ,
13. P a r n - y a - g h a r m Bangkok 2002. 14. G a l a g a n DJ,
p 289-297
P h a n t u m v a n i t P. S o n g p a i s a n Y ( 1 9 8 2 ) I n : S y m p o s i u m C h u l a l o n g k o r n U n i v e r s i t y . Bangkok, p p 15.1-15.21
Y,
V e r m i l l i o n JR ( 1 9 5 7 ) Pub H l t h Rep 72:191-193
15. M i n o g u c h i G ( 1 9 7 0 ) I n : F l u o r i d e s and Human H e a l t h . z a t i o n , Geneva, p p 294-304
World Health Organi-
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 181-186 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
181
SKELETAL FLUOROSIS A S SEEN I N A H I G H FLUORIDE ZONE
SHIV
CHANDRA~
D e p a r t m e n t o f P r e v e n t i v e and S o c i a l M e d i c i n e , J a i p u r : 302004, I n d i a
S.M.S.
Medical College,
P r e s e n t Address: D e p a r t m e n t o f P r e v e n t i v e and S o c i a l M e d i c i n e , M e d i c a l C o l l e g e , A j r n e r : 305001, I n d i a .
J.L.N.
ABSTRACT An e p i d e m i o l o g i c a l s t u d y o f e n d e m i c s k e l e t a l f l u o r o s i s h a s b e e n c a r r i e d o u t among p e r s o n s 1 5 y e a r s o l d and a b o v e i n a n a r e a w h e r e t h e d r i n k i n g w a t e r c o n t a i n e d 5 ppm F and above.
I n a s a m p l e o f 1 2 6 p e r s o n s t h e sequence
o f c o m p l a i n t s n o t e d w a s : b a c k a c h e , 31.74%; j o i n t p a i n s , lower limbs,
17.46%;
and p a r e s t h e s i a ,
a n y s u g g e s t i v e symptoms r e v e a l e d : r i g i d i t y o f spine,
17.96% e a c h ;
12.69%.
26.98%;
Examination o f persons w i t h
b r i s k r e f l e x e s , 46.51%:
and bony e x o s t o s i s ,
symptoms and t h r e e s i g n s were p r e s e n t t o g e t h e r , case o f c l i n i c a l s k e l e t a l f l u o r o s i s .
pain i n
k y p h o s i s and
3.46%.
When t h r e e
a p e r s o n was d i a g n o s e d a s a
Thus 1 5 p e r s o n s w e r e f o u n d t o be
s u f f e r i n g f r o m s k e l e t a l f l u o r o s i s g i v i n g an o v e r a l l p r e v a l e n c e o f 1.13% i n t h e s t u d y area.
U r i n a r y f l u o r i d e e x c r e t i o n i n t h e s e i n d i v i d u a l s was h i g h e r
t h a n t h e f l u o r i d e l e v e l i n t h e i r d r i n k i n g water.
T h i s d i f f e r e n c e was f o u n d
s t a t i s t i c a l l y s i g n i f i c a n t when c o m p a r e d a g a i n s t a c o n t r o l group.
Measure-
m e n t o f t h e I n d e x o f I n d e p e n d e n c e i n t h e A c t i v i t i e s o f D a i l y L i v i n g among t h e s e i n d i v i d u a l s d i s c l o s e d some l i m i t a t i o n i n p e r f o r m i n g d a i l y a c t i v i t y .
INTRODUCTION I n t h e f l u o r o t i c b e l t s s p r e a d t h r o u g h o u t w e s t and s o u t h I n d i a , f l u o r o s i s c o n t i n u e s t o be a p r o b l e m o f p u b l i c h e a l t h magnitude.
skeletal Epidemio-
l o g i c a l s t u d i e s o f endemic f l u o r o s i s c a r r i e d o u t i n v a r i o u s areas i n d i c a t e t h a t p u b l i c h e a l t h e x p e r t s m u s t d e v e l o p a s i m p l e and f e a s i b l e method o f i d e n t i f y i n g t h e m e d i c a l g e o g r a p h y o f f l u o r o s i s i n s u c h zones.
The o b j e c -
t i v e s o f t h e p r e s e n t work are: (1) measuring t h e prevalence o f s k e l e t a l f l u o r o s i s i n a f l u o r o t i c b e l t , (2) developing c l i n i c a l diagnosis o f skeletal fluorosis i n the t h e absence of t e c h n i c a l i n v e s t i g a t i v e aids,
and
( 3 ) m e a s u r i n g t h e i n t e n s i t y o f d i s e a s e a t an i n d i v i d u a l l e v e l .
field in
182 MATERIALS AND METHODS T h i s s t u d y was c a r r i e d o u t i n a r u r a l a r e a i n w e s t e r n I n d i a w h e r e p u b l i s h e d (1,Z).
I n t h e present paper observations r e l a t e d t o s k e l e t a l
involvement are presented.
The a r e a was d i v i d e d i n t o t w o p a r t s b a s e d on
the F concentrations i n d r i n k i n g water:
A r e a I, 5.1 t o 1 0 ppm; A r e a 11.
10.1 ppm a n d a b o v e . RESULTS S i n c e s k e l e t a l f l u o r o s i s i s a d i s e a s e i n a d u l t s i n a n e n d e m i c zone, p e r s o n s 1 5 y e a r s o l d and above w e r e i n t e r v i e w e d and e x a m i n e d f o r t h e p r e s ence o f s k e l e t a l f l u o r o s i s .
S i n c e n o symptoms r e l a t e d t o s k e l e t a l i n v o l v e -
m e n t w e r e seen i n t h e 1 5 t o 19 y e a r s o l d group, i n p e r s o n s 20 y e a r s o l d and above.
p r e v a l e n c e was i n v e s t i g a t e d
The m o s t common symptom was backache
s e e n i n 28.57% o f t h e s a m p l e i n A r e a I a n d 35.71% i n A r e a 11, r e s p e c t i v e l y ( T a b l e I). W i t h t h e i n c r e a s e i n F c o n t e n t o f d r i n k i n g w a t e r , i n t h e number o f s y m p t o m a t i c s
was
found
t h e increase
statistically significant
(p<0.005). I n symptomatics found d u r i n g i n t e r v i e w s .
c l i n i c a l e x a m i n a t i o n was made
f o r t h e r i g i d i t y o f s p i n e , k y p h o s i s , b r i s k r e f l e x e s and bony e x o s t o s i s . B r i s k r e f l e x e s was t h e m o s t f r e q u e n t l y e n c o u n t e r e d s i g n w h i l e bony e x o s t o s i s was seen i n o n l y t h r e e p e r s o n s ( T a b l e 11).
The s y m p t o m a t i c s h a v i n g one
o r m o r e s i g n s w e r e 72.72% i n A r e a 11, c o m p a r e d t o 28.57% i n A r e a I. W i t h t h e increase i n F concentrations i n t h e d r i n k i n g water, t h i s increase i n
TABLE I OBSERVED SYMPTOMS OF SKELETAL FLUOROSIS
SYMPTOMS
N=70
I
SAMPLE A R E A ' Percent
N=56
I1
Percent
P a i n i n Lower Limbs
10
14.28
12
Backache
20
28.57
20
35.71
Pain i n J o i n t s
17
24.28
17
30.35
Number o f P a r a e s t h e s i a
10
14.28
6
10.71
T o t a l Symptomatics
42
60.00
44
78.57
X2=4.94;
d.f.=l;
21.42
~ ~ 0 . 0 5
' F l u o r i d e i n d r i n k i n g w a t e r : A r e a I, 5.1-10.0
ppm; A r e a 11. 210.1 ppm
183 TABLE I 1 OBSERVED SIGNS OF SKELETAL FLUOROSIS ~
SIGNS
N=42
I
~
SAMPLE
~
AREA^
Percent
~
11
N=44
Percent
R i g i d i t y o f Spine
3
7.15
14
31.81
Kyphosis
7
16.66
10
22.72
B r i s k Reflexes
10
23.80
30
68.18
Bony E x o s t o s e s
0
0.00
3
6.81
12
28.57
32
72.72
S y m p t o m a t i c s h a v i n g one o r more s i g n s X’z16.75:
d.f.=l;
p
’ F l u o r i d e i n d r i n k i n g w a t e r : A r e a I . 5.1-10.0
ppm: A r e a 11.
10.1 ppm
t h e number o f s y m p t o m a t i c s h a v i n g one o r m o r e p o s i t i v e s i g n s was s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (p
I and I 1 w e r e c l a s s i f i e d as c a s e s o f s k e l e t a l f l u o r o s i s .
Thus,
1 5 c a s e s o f s k e l e t a l f l u o r o s i s w e r e d e t e c t e d ( T a b l e 111). c a s e was a 2 8 - y e a r - o l d
a total of
The y o u n g e s t
m a l e w h i l e t h e o l d e s t c a s e was a 7 0 - y e a r - o l d
female,
b o t h o f whom consumed w a t e r c o n t a i n i n g 10.1 ppm F and above.
O f t h e s e 15
cases, 12 were bona f i d e r e s i d e n t s and t h r e e were m i g r a n t s .
A l l of the
m i g r a n t s were female. U r i n a r y F e x c r e t i o n o f t h e s e c a s e s was
compared t o t h a t o f a c o n t r o l
g r o u p w h o w e r e o f t h e s a m e s e x , h a d d r i n k i n g w a t e r f r o m t h e same s o u r c e , had s i m i l a r r e s i d e n t i a l s t a t u s , and w e r e w i t h i n t e n y e a r s o f t h e age c l a s s . O n l y 12 c o n t r o l cases c o u l d be obtained. fluorosis,
Among t h e c a s e s o f s k e l e t a l
u r i n a r y F was a l w a y s h i g h e r t h a n t h e F c o n t e n t i n d r i n k i n g w a t e r
except f o r a 48-year-old
m a l e who e x c r e t e d 11.2 ppm F i n u r i n e whereas h i s
d r i n k i n g w a t e r c o n t a i n e d 11.8 ppm F ( T a b l e I V ) . an e r r a t i c p a t t e r n o f u r i n a r y F e x c r e t i o n .
The c o n t r o l g r o u p r e v e a l e d However,
the relationship
b e t w e e n u r i n a r y F e x c r e t i o n and t h e F c o n t e n t i n d r i n k i n g w a t e r was f o u n d t o b e s t a t i s t i c a l l y s i g n i f i c a n t ( ~ ~ 0 . 0 5 i )n c a s e s c o m p a r e d t o c o n t r o l s . T h e p a t t e r n o f u r i n a r y F e x c r e t i o n s e e n among c h i l d r e n i n t h e s t u d y a r e a has been p u b l i s h e d e l s e w h e r e (3).
184
TABLE I 1 1 DISTRIBUTION OF SKELETAL FLUOROSIS ACCORDING TO FLUORIDE CONTENT IN DRINKING WATERa Cases of Skeletal Fluorosis Age Group (Years)
20 - 29
Male
Area I (N=38)
Female Area I Area I 1 (N=32) (N=26)
Area I 1 (N=30)
Total (N=126)
0
1
0
0
1
30 - 39
0
0
-
49
0
1 5
0
40
0
lb
1 6
50
-
59
0
1
0
lb
2
60
- above
2
1
Ib
1
5
9
1 (3.12%)
3 (11.54%)
15 (11.9%)
Total Grand Total
2
(5.26%)
(30.0%)
11 (16.17%)
4 (6.8%)
'Fluoride in drinking water: Area I , 5.1-10.0 pprn; Area 11. 2 10.1 ppm bMi grants
TABLE i V URINARY FLUORIDE EXCRETION AMONG CASES OF SKELETAL FLUOROSIS AND CONTROLS Fluoride i n Drinking Water
Cases of Skeletal Fluorosis Age Sex Urinary F
Age
Controls Sex Urinary F
( PPm)
(ppm)
( PPm)
6.8
70 60
M F
a. a
7.8
64 52
M F
4.2
10.7
48 48 56 45 48 28
M
70.8
48
M
16.4
56 40
M
9.8 30.0
M
15.6 9.2 7.6 5.3 5. a 6.9 15.2
11.8
50 48 45 60
t=2.16; d.f.=2; ~ ~ 0 . 0 5
M
M M M M F M F
F
38.0 38.8 28.0 18.8 15.6 19.2 11.2 14.2 14.8
25 27 45 45 40 50 55
M M
F M
M F
F
4.
a
185 The I n d e x o f I n d e p e n d e n c e i n t h e A c t i v i t i e s o f D a i l y L i v i n g ( 4 ) was measured i n t h e 15 cases o f s k e l e t a l f l u o r o s i s and was compared w i t h t h e r e s t o f t h e p o p u l a t i o n ( 6 2 7 p e r s o n s ) 20 y e a r s o l d and a b o v e l i v i n g i n t h e same a r e a .
O n l y 40% o f t h e s k e l e t a l f l u o r o s i s c a s e s w e r e i n d e p e n d e n t i n
c a r r y i n g o u t a l l t h e s i x f u n c t i o n s c o n s i d e r e d i n measuring t h e ADL index, w h i l e i n t h e g e n e r a l p o p u l a t i o n 96.4% c o u l d p e r f o r m t h e same f u n c t i o n i n d e p e n d e n t l y (Table V).
TABLE V
I N D E X OF INDEPENDENCE I N A C T I V I T I E S OF DAILY LIVING I N PERSONS 20 YEARS OLD AND ABOVE Skeletal Fluorosis Index
Number (N=l5)
Others
Percent
Number (N=612)
Percent
A
40.00
590
96.40
B
33.33
17
2.77
C
6.66
0
0.00
D
0.00
0
0.00
E
0.00
0
0.00
F
6.66
3
0.50
G
0.00 13.33
0 2
0.00 0.33
Others
DISCUSSION D u r i n g an e p i d e m i o l o g i c a l s t u d y o f endemic f l u o r o s i s i n a remote r u r a l area i n I n d i a ,
t h e need t o c l i n i c a l l y diagnose t h e cases o f s k e l e t a l f l u o -
r o s i s was f e l t .
On t h e b a s i s o f t h r e e s y m p t o m s and t h r e e s i g n s , c a s e s
w h i c h met i n v e s t i g a t i v e c r i t e r i a were l a b e l e d as s u f f e r i n g f r o m s k e l e t a l fluorosis.
S k e l e t a l f l u o r o s i s was observed more i n males t h a n i n f e m a l e s as
reported e a r l i e r by J o l l y
d.(5).
T h i s o c c u r s l a r g e l y because males
p e r f o r m more s t r e n u o u s work w h i c h r e s u l t s i n a h i g h e r w a t e r i n t a k e . Fewer cases a r e observed i n f e m a l e s i n t h e s t u d y area a l s o due t o t h e i r m i g r a t i o n f r o m n o n - f l u o r o t i c areas when t h e y marry.
Singh and J o l l y (6) n o t e d back-
ache i n persons t h e y examined f o r s k e l e t a l i n v o l v e m e n t i n a f l u o r o t i c b e l t . Mathur e t al.
( 7 ) a l s o r e p o r t e d b a c k a c h e i n 78.2% o f t h e p e r s o n s i n a
f l u o r o t i c b e l t i n t h e same region.
H i g h e r amounts o f c a l c i u m i n t h e d r i n k -
i n g w a t e r i n t h e s t u d y a r e a was r e s p o n s i b l e f o r l o w e r p r e v a l e n c e o f s k e l e t a l f l u o r o s i s i n t h e s t u d y area.
186 REFERENCES
SK ( 1 9 8 0 ) Comm D e n t -
1.
C h a n d r a S, S h a r m a R, T h e r g a o n k a r VP, C h a t u r v e d i O r a l E p i d e m i o l 8:92-96
2.
Chandra S (1983) E p i d e m i o l o g i c a l s t u d i e s o f endemic f l u o r o s i s i n n o r t h e r n I n d i a . P a p e r p r e s e n t e d a t 1 3 t h C o n f e r e n c e o f I.S.F.R., New D e l h i . Nov. 13-17
3.
Chandra S,
4.
K a t z S, F o r d AB. M a s k o w i t z RW, J a c k s o n BA, M a l j o r d WJ. JAMA 185: 91 4-91 5
T h e r g a o n k a r VP (1984) F l u o r i d e 17:155-159 M a l j o r d C (1963)
5.
J o l l y SS, S i n g h BM, M a t h u r OC. M a l h o t r a KC (1968) BMJ 4:427-429
6.
S i n g h A.
7.
M a t h u r GM. T a m b o l i BL, M a t h u r RM, Ray AK, M a t h u r GL, G o y a l OP ( 1 9 7 6 ) I n d J PSM 7:90-96
J o l l y SS (1961) Q u a r t J Med 30:357-360
187
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 187-191 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
LONG-TERM RETENTION OF FLUORIDE I N THE BONES OF FORMER ALUMINUM WORKERS CHARLES A. JEAN M.
BAUD, SEONG BANG, CHRISTIANE DEMEURISSE, H E N R I J. TOCHON-DANGUY,
VERY
I n s t i t u t e o f Morphology, Centre M i d i c a l U n i v e r s i t a i r e , S e r v e t , 121 1 Gengve 4, S w i t z e r l a n d
1, r u e M i c h e l -
ABSTRACT F l u o r i d e d e p o s i t e d i n t h e s k e l e t o n i s p r o g r e s s i v e l y removed b y osteoclast i c r e s o r p t i o n process.
A small p a r t o f the released f l u o r i d e i s recycled
i n n e w l y f o r m i n g bone.
A t e v e r y step,
t h e bone m i n e r a l ( c r y s t a l l i n i t y index,
the crystallographic properties o f c r y s t a l s i z e and/or p e r f e c t i o n ,
unit
c e l l p a r a m e t e r s ) a r e c l o s e l y c o r r e l a t e d t o t h e l o c a l f l u o r i d e concentration. E l i m i n a t i o n o f f l u o r i d e f r o m t h e skeleton,
and a c c o m p a n y i n g c h a n g e s i n
t h e bone m i n e r a l s u b s t a n c e , w e r e s t u d i e d o n 1 0 2 i l i a c b i o p s i e s f r o m
68 m a l e
a l u m i n u m w o r k e r s 0-32 y e a r s a f t e r e x p o s u r e t o f l u o r i d e ceased. F l u o r i d e i n m i n e r a l i z e d t i s s u e was d e t e r m i n e d w i t h a s p e c i f i c e l e c t r o d e
(1).
The f l u o r i d e c o n t e n t o f c o m p a c t bone was f o u n d t o d e c r e a s e p r o -
g r e s s i v e l y w i t h t i m e a f t e r e x p o s u r e h a s ended, e s t i m a t i o n s (2),
confirming earlier indirect
b u t i n o p p o s i t i o n t o t h e o p i n i o n o f Dominok gt
The e l i m i n a t i o n r a t e was a b o u t 6.5%/year
(Fig.
1).
21. (3).
I n comparison, t h e
t u r n o v e r r a t e e s t i m a t e d by h i s t o m o r p h o m e t r i c methods amounts t o 8-l0%/year
(4).
The d i f f e r e n c e i n d i c a t e s t h a t f l u o r i d e r e l e a s e d d u r i n g bone r e s o r p -
t i o n i s i n p a r t r e c y c l e d d u r i n g bone f o r m a t i o n , p r o v e d b y Guo g t
a process e x p e r i m e n t a l l y
d.(5).
Topographic d i s t r i b u t i o n o f f l u o r i d e , r a y microanalysis (6.7),
as d i s p l a y e d b y e l e c t r o n p r o b e X-
i s characterized by an i r r e g u l a r r e p a r t i t i o n o f
t h e f i e l d s w i t h a h i g h f l u o r i d e l e v e l d u r i n g t h e p e r i o d o f exposure (Fig. 2) and b y an e v o l u t i o n t o w a r d s l o w l e v e l s and homogeneity a f t e r t h e end o f c o n t a m i n a t i o n ( F i g . 3). The c r y s t a l l i n i t y o f t h e bone m i n e r a l s u b s t a n c e was e v a l u a t e d b y i n f r a r e d s p e c t r o m e t r y (8).
The c r y s t a l l i n i t y i n d e x d e c r e a s e s as a f u n c t i o n o f
d e c r e a s i n g f l u o r i d e c o n t e n t (Fig.
4).
C r y s t a l s i z e and/or p e r f e c t i o n i s i n v e r s e l y c o r r e l a t e d w i t h B values, w i d t h o f X-ray d i f f r a c t i o n
lines a t half
the
i n t e n s i t y ( 9 ) . The B v a l u e s
i n c r e a s e when t h e f l u o r i d e c o n c e n t r a t i o n i s f a l l i n g ( F i g .
5).
The d i m e n s i o n s o f t h e u n i t c e l l o f t h e a p a t i t e w e r e d e t e r m i n e d b y means
188
*I* F Duration of Exposure to F= 30'- 3 years = 5 5 % 6 years Age at withdrawal
.8 .6
.4 .2
5
10 15 20 Years after withdrawal
25
F i g u r e 1. F l u o r i d e c o n t e n t o f t h e i l i a c c o m p a c t bone t i s s u e m i n e r a l o f a l u m i n u m w o r k e r s , 0 t o 2 5 y e a r s a f t e r e x p o s u r e t o f l u o r i d e ceased.
I
:I E
4o
0
.2
it
Duration of Exposure l o F - 4 3 years Duration of Post-exposure = 0
1700 3400 5100 6800 8500 10200 11900 13600 15300 p p rn F
Control
40
20
1 I
0
,
,
,
,
,
,
,
,
1700 3400 5100 6800 8500 10200119001360015300 p p m F
F i g u r e 2. H i s t o g r a m s showing t h e d i s t r i b u t i o n o f f l u o r i d e i n t h e i l i u m o f an aluminum w o r k e r exposed t o f l u o r i d e f o r 43 y e a r s ( t o p ) and o f a c o n t r o l s u b j e c t o f t h e same age group (bottom).
:i #
189 Duration of Expowre l o F = I 4 years Duration of P-I-exposun
= 20 years
20
1tW 2200 Ooo 4400 3500 6640 vpm F
Control
60
4
p
10
:
m
it
20
1 1 0 0 2 1 0 0 u o O 4 4 0 0 5 5 0 0 ~p p m F
F i g u r e 3. H i s t o g r a m s showing t h e d i s t r i b u t i o n o f f l u o r i d e c o n c e n t r a t i o n s , i n p e r c e n t a g e volume o f compact bone t i s s u e , i n t h e i l i u m o f an aluminum w o r k e r 20 y e a r s a f t e r e x p o s u r e t o f l u o r i d e c e a s e d ( t o p ) and o f a c o n t r o l s u b j e c t o f t h e same age group (bottom).
0 Controls
75
X
8
C
@ Exposed
0
0 After
withdrawal
70
I
1
E-.-
-
65
6
60
a
n
2.
0
.5
1.0
*I. F F i g u r e 4. V a r i a t i o n o f t h e c r y s t a l l i n i t y i n d e x o f t h e i l i a c compact bone t i s s u e mineral asa f u n c t i o n o f f l u o r i d e concentration.
190
re')=?
' ( J I 0 )
0 Controls @Exposed
600
o After withdrawal r = -. 78
0
500
400
300
1.0
.5
0
O/o
F
F i g u r e 5. V a r i a t i o n o f t h e c r y s t a l s i z e a n d / o r p e r f e c t i o n ( B (31.0) v a l u e ) o f t h e i l i a c c o m p a c t bone t i s s u e m i n e r a l a s a f u n c t i o n o f f l u o r i d e concentration.
a
(All
0 Controls
Exposed
9.44 -
0After withdrawal
r = -. 94
I 0
.5
'lo F
1.0
.
F i g u r e 6. V a r i a t i o n o f the a p a t i t e u n i t c e l l a parameter o f the i l i a c c o m p a c t bone t i s s u e m i n e r a l as a f u n c t i o n o f f l u o r i d e c o n c e n t r a t i o n .
191 o f h i g h r e s o l u t i o n X-ray d i f f r a c t i o n (9).
The a p a r a m e t e r o f t h e u n i t c e l l
o f t h e bone a p a t i t e c r y s t a l s l e n g t h e n s p r o g r e s s i v e l y as a f u n c t i o n o f decreasing f l u o r i d e c o n c e n t r a t i o n (Fig.
6).
The c o r r e l a t i o n b e t w e e n f l u o r i d e c o n t e n t and t h e o t h e r p a r a m e t e r s demons t r a t e s t h a t t h e f l u o r i d e i s t h e most i m p o r t a n t f a c t o r f o r t h e progressive a l t e r a t i o n o f t h e bone m i n e r a l d u r i n g t h e p o s t - c o n t a m i n a t i o n p e r i o d as w e l l as d u r i n g t h e p e r i o d o f e x p o s u r e t o f l u o r i d e . REFERENCES 1.
McCann HG (1968) A r c h O r a l B i o l 13:475-477
2.
L a r g e n t EJ (1952) A r c h I n d u s t r H y g i e n e
3.
Dominok G,
4.
P a r f i t t A M (1983) I n : R e c k e r RR (ed) Bone H i s t o m o r p h o m e t r y : and I n t e r p r e t a t i o n . CRC Press, I n c , Boca Raton, p p 143-223
5.
Guo MK,
6.
Baud CA, Bang S (1972) I n : S h i n o d a G, K o h r a K, I c h i n o k a w a T (eds) Proc e e d i n g s o f t h e S i x t h I n t e r n a t i o n a l C o n f e r e n c e o n X-ray O p t i c s and M i c r o a n a l y s i s . U n i v e r s i t y o f Tokyo Press, p p 835-840
7.
B a n g S , B a u d CA, B o i v i n G, D e m e u r i s s e C, G o s s i M, T o c h o n - D a n g u y HJ. V e r y JM (1978) I n : F l u o r i d e and Bone, P r o c 2nd Symp CEMO. Huber, Bern, p p 168-175
8.
B a u d CA, P o u e z a t JA, s u p p l : 452-456
9.
V e r y JM, Baud CA (1984) I n : D i c k s o n GR (ed) Methods o f C a l c i f i e d T i s s u e P r e p a r a t i o n . E l s e v i e r , Amsterdam, pp 369-387
Siefert
K,
- Occup
Med 6:37-42
F r e g e J, Dominok B (1984) F l u o r i d e 17:23-26
M e s s e r HH, Ophaug R,
Techniques
S i n g e r L (1985) J D e n t Res 64:357
Tochon-Danguy
HJ ( 1 9 7 6 )
C a l c i f T i s s u e Res 2 1
193
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 193-199 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
SYMPTOMATOLOGY OF WORKERS I N THE FLUORIDE INDUSTRY AND FLUORSPAR PROCESSING PLANTS
V I K A S K.
BHARAT S.
DESAI.
BHAVSAR,
NATVARLAL R.
MEHTA,
DINESH KUMAR SAXENA
AND SHANTILAL L. KANTHARIA Community M e d i c i n e Department, Government M e d i c a l College, Gujarat, India
Surat.
395 001,
ABSTRACT R a p i d i n d u s t r i a l d e v e l o p m e n t w i t h i n c r e a s i n g u s e o f f l u o r i d e compounds i n I n d i a n e c e s s i t a t e s b a s e l i n e s t u d i e s on i n d u s t r i a l were undertaken i n G u j a r a t State. HF,
f r e e z i n g gas,
cryolite,
etc.,
fluorosis.
Two s t u d i e s
One on w o r k e r s i n i n d u s t r i e s p r o d u c i n g was c o n d u c t e d f r o m 1976 t o 1979.
Another
o n w o r k e r s i n f l u o r s p a r p r o c e s s i n g p l a n t s was p e r f o r m e d f r o m 1981 t o 1984.
O f 4 3 8 i n d u s t r i a l w o r k e r s c o n s u m i n g d r i n k i n g w a t e r c o n t a i n i n g <0.8 ppm F. 34% h a d c o m p l a i n t s .
M a j o r c o m p l a i n t s w e r e p a i n and s t i f f n e s s o f t h e l u m b a r
s p i n e (14.1%) a n d p a i n and s t i f f n e s s o f j o i n t s (12.1%).
T h e i r mean u r i n a r y
F l e v e l w a s 1.96 ppm.
O f 3 6 6 p l a n t w o r k e r s w h e r e d r i n k i n g w a t e r c o n t a i n e d 0.8 61.48% h a d c o m p l a i n t s . l u m b a r s p i n e (42.6%). p a i n (34.9%).
3.0 ppm F,
-
M a j o r c o m p l a i n t s w e r e p a i n and s t i f f n e s s o f t h e p a i n a n d s t i f f n e s s o f j o i n t s (23.2%) and e p i g a s t r i c
T h e i r mean u r i n a r y F l e v e l was 3.17
ppm.
I n b o t h groups o f w o r k e r s symptoms were more severe i n employees o f h i g h r i s k departments.
The s y m p t o m s w e r e r e l a t e d t o mode o f exposure.
Symptoms
w e r e u n r e l a t e d t o d e n t a l f l u o r o t i c c h a n g e s o r r a d i o l o g i c a l changes.
INTRODUCTION R a p i d i n d u s t r i a l d e v e l o p m e n t w i t h i n c r e a s i n g u s e o f F compounds i n I n d i a n e c e s s i t a t e s b a s e l i n e s t u d i e s on t h e i n d u s t r i a l f l u o r o s i s problem. studies,
f r e e z i n g gas, plants,
Two
t h e f i r s t o f t h e i r k i n d i n I n d i a o n i n d u s t r i e s p r o d u c i n g HF, cryolite,
etc.,
and on w o r k e r s i n f l u o r s p a r
processing
were undertaken i n G u j a r a t State.
D e n t a l and s k e l e t a l f l u o r o s i s a r e c o n s p i c u o u s and t h o r o u g h l y s t u d i e d m a n i f e s t a t i o n s o f t h e i n d u s t r i a l and e n d e m i c f l u o r o s i s p r o b l e m . been a c c e p t e d t h a t t h e y a r e n o t t h e o n l y f e a t u r e s o f t h e d i s e a s e .
I t h a s now
194 Roholm ( 1 ) i n h i s c l a s s i c book h a s d e s c r i b e d a w i d e s p e c t r u m o f m a n i f e s t a t i o n s o t h e r t h a n i n v o l v e m e n t o f bones and t e e t h . were a l s o d e s c r i b e d b y M u r r a y and W i l s o n (4).
Such m a n i f e s t a t i o n s
(2). W a l d b o t t ( 3 ) . F r a d a g t
21.
C z e r w i n s k i and L a n k o s z ( 5 ) i n t h e i r s t u d i e s on human f l u o r o s i s .
T h i s study a t t e m p t s t o analyze b a s e l i n e i n f o r m a t i o n o f symptomatology i n i n d u s t r i a l and p l a n t w o r k e r s who h a v e d i f f e r e n t b a c k g r o u n d s and d i f f e r e n t environments. MATERIALS AND METHODS
A t o t a l o f 4 3 8 i n d u s t r i a l w o r k e r s , i n c l u d i n g 286 m a n u a l l a b o r e r s , w h o c o n s u m e d d r i n k i n g w a t e r c o n t a i n i n g 0.8
ppm F w e r e s t u d i e d .
They w e r e
r e s i d e n t s o f nearby c i t i e s o r v i l l a g e s . In addition,
366 p l a n t w o r k e r s ,
sumed w a t e r c o n t a i n i n g 0.8
-
3.0
i n c l u d i n g 247 t r i b a l l a b o r e r s , ppm F w e r e s t u d i e d .
who con-
They w e r e r e s i d e n t s
o f a workers' colony o r nearby t r i b a l v i l l a g e s . W o r k e r s w e r e i n t e r v l e w e d a n d c l i n i c a l l y examined.
Spot u r i n e samples
w e r e c o l l e c t e d f r o m 210 i n d u s t r i a l w o r k e r s and f i r s t m o r n i n g u r i n e s a m p l e s were c o l l e c t e d from
( R t . f o r e a r m , A.P.
319 p l a n t w o r k e r s . X-rays
view)
w e r e t a k e n o f 183 i n d u s t r i a l w o r k e r s and 82 p l a n t w o r k e r s who w e r e r a n d o m l y selected.
F levels o f water
Water samples were c o l l e c t e d f r o m t h e source.
and u r i n e s a m p l e s w e r e d e t e r m i n e d u s i n g an O r i o n i o n s p e c i f i c e l e c t r o d e . RESULTS AND D I S C U S S I O N E n d e m i c f l u o r o s i s h a s been e x t e n s i v e l y s t u d i e d and r e p o r t e d f r o m I n d i a , w h i l e i n d u s t r i a l f l u o r o s i s i s a new f i e l d .
Inter-individual
known i n v a r i o u s t y p e s o f m a n i f e s t a t i o n s o f f l u o r o s i s . p r e v i o u s and c u r r e n t F i n t a k e s , individual susceptibility,
extent,
variations are
They a r e based on
mode and d u r a t i o n o f F i n t a k e ,
and n u t r i t i o n a l s t a t u s ,
etc.
M a n i f e s t a t i o n s o t h e r t h a n s k e l e t a l and d e n t a l f l u o r o s i s p r e c e d e o r accompany c l a s s i c a l
changes
i n c h r o n i c f l u o r i d e exposure.
Roholm (1) has
d e s c r i b e d a w i d e s p e c t r u m o f m a n i f e s t a t i o n s o t h e r t h a n i n v o l v e m e n t o f bones and teeth.
s u c h as n e u r o m u s c u l a r symptoms, t a c h y c a r d i a , p o l y d i p s i a , and
a l l e r g i c s k i n lesions.
W a l d b o t t and C e c i l i o n i ( 6 ) have a l s o d e s c r i b e d
symptomatology r e l a t e d t o musculoskeletal,
gastro-intestinal,
system and c h i z z o l a macula i n neighborhood f l u o r o s i s .
respiratory
A medical research
c o u n c i l s t u d y ( 7 ) r e v e a l e d m i l d d y s p e p t i c symptoms. S y m p t o m a t o l o g y o f i n d u s t r i a l and p l a n t w o r k e r s i s shown i n T a b l e I. over-all
The
p r e v a l e n c e r a t e was 34% and 61.5% i n i n d u s t r i a l a n d p l a n t w o r k e r s ,
respectively.
P a i n and s t i f f n e s s o f t h e spine,
predominantly t h e lumbar
s p i n e , and p a i n and s t i f f n e s s o f j o i n t s , p r e d o m i n a n t l y knee j o i n t s , w e r e
195
TABLE I SYMPTOMATOLOGY
OF WORKERS Percent
Symptoms
Industry
Plant
P a i n and s t i f f n e s s o f s p i n e
14.1
42.6
P a i n and s t i f f n e s s o f j o i n t
12.1
20.8
2.7
34.9
1.8
9.6
Nausea,
vomiting,
epigastric pain
Headache Muscle s t i f f n e s s ,
3.3
5.2
Paresthesia
twitching
3.8
0.3 5.5
L o s s o f w e i g h t and weakness
2.5
Emphysema, asthma, c h e s t p a i n
3.4
3.3
Hearing disturbances
1.3
3.3
Nasal c o n j u n c t i v a l i r r i t a t i o n
4.7
3.3
U r i n a r y complaints
1.5
3.3
Overall prevalence
34.0
61.5
Specific
18.0
48.4
t h e m a i n symptoms i n b o t h groups. 34.9% o f t h e p l a n t w o r k e r s ,
B u r n i n g e p i g a s t r i c p a i n was r e p o r t e d b y
compared t o 2.7% b y t h e i n d u s t r i a l w o r k e r s .
T o i d e n t i f y symptoms r e l a t e d t o i n d u s t r i a l exposure,
t h e symptoms can b e
compared w i t h those found i n i d e n t i c a l p o p u l a t i o n s n o t employed i n fluoride-related
industry.
I n t h e absence o f s u c h r e c o r d s ,
t h i s study can
s e r v e as a b a s e l i n e . S p e c i f i c symptoms r e l a t e d t o bones and j o i n t s w h i c h can be s t r o n g l y a t t r i b u t e d t o F e x p o s u r e w e r e h i g h e r (48.4%) i n p l a n t w o r k e r s t h a n i n i n d u s t r i a l w o r k e r s (18.0%) I n d u s t r i a l w o r k e r s who consumed w a t e r w i t h n o r m a l F l e v e l s w e r e exposed t o t h e same e n v i r o n m e n t t h r o u g h o u t t h e y e a r s o f t h e i r e m p l o y m e n t i n t h e absence o f i n t e r d e p a r t m e n t a l r o t a t i o n a t t h e t i m e o f study.
Industrial
w o r k e r s w e r e a t r i s k o f e x p o s u r e t o F - c o n t a i n i n g gas and d u s t . The m a j o r i t y o f t h e p l a n t w o r k e r s ( l a b o r e r s ) w e r e r e s i d e n t s o f s u r r o u n d i n g t r i b a l v i l l a g e s w i t h d i f f e r e n t w a t e r F l e v e l s b e t w e e n 0.8 Compared t o o t h e r d e p a r t m e n t s ,
-
3.0 ppm.
h i g h r i s k d e p a r t m e n t s had a s i g n i f i c a n t l y
h i g h p r o p o r t i o n o f w o r k e r s who w e r e e m p l o y e d f o r l e s s t h a n t e n y e a r s , w e r e f r o m a r e a s w h e r e t h e r e was m o r e t h a n 1 ppm F i n t h e w a t e r , and w e r e a t r i s k o f b e i n g exposed t o F - c o n t a i n i n g dust.
196 Symptomatology i n t h i s s t u d y appears t o r e l a t e s t r o n g l y w i t h t h e mode o f exposure.
H y d r o f l u o r o s i s and i n g e s t i o n o f F - c o n t a i n i n g d u s t r e s u l t e d i n
h i g h e r p r e v a l e n c e o f s p e c i f i c symptoms and b u r n i n g e p i g a s t r i c p a i n i n p l a n t workers. (7).
I n t h e s t u d i e s b y R o h o l m ( 1 ) and t h e M e d i c a l R e s e a r c h C o u n c i l
r a d i o l o g i c a l c h a n g e s w e r e r e l a t e d t o d u s t i n h a l a t i o n and d e g r e e o f
I n t h e M e d i c a l Research C o u n c i l s t u d y ( 7 ) u r i n a r y F e x c r e t i o n
exposure.
was r e l a t e d t o s e v e r i t y o f exposure. As shown i n Table 11,
s p e c i f i c symptoms i n t h i s s t u d y show a s i g n i f i c a n t
a s s o c i a t i o n t o r i s k o f exposure.
S p e c i f i c symptoms were s i g n i f i c a n t l y
h i g h e r i n w o r k e r s i n h i g h r i s k d e p a r t m e n t s i n i n d u s t r y (29.2%) and i n p l a n t s (70.9%) t h a n i n w o r k e r s o f l o w r i s k d e p a r t m e n t s i n i n d u s t r y (9.9%) and i n p l a n t s (26.9%).
respectively.
S p e c i f i c symptoms w e r e h i g h e r i n i n d u s t r i a l w o r k e r s w i t h m o r e t h a n 10 y e a r s employment (19.8%) t h a n i n t h o s e w i t h l e s s t h a n 10 y e a r s employment (12.0%). health.
T h i s suggests a c u m u l a t i v e e f f e c t I n p l a n t workers,
o f t h e e n v i r o n m e n t on workers‘
s p e c i f i c symptoms were s i g n i f i c a n t l y h i g h e r i n
t h o s e employed f o r l e s s t h a n 10 y e a r s (52.3%) t h a n o t h e r s (33.3%).
This i s
because a m a j o r i t y o f h i g h r i s k department w o r k e r s a r e employed f o r l e s s t h a n f i v e y e a r s a f t e r w h i c h t h e y e i t h e r s h i f t t o s a f e r departments o r l e a v e t h e job. S p e c i f i c symptoms w e r e s i g n i f i c a n t l y a s s o c i a t e d w i t h d r i n k i n g w a t e r F level.
These s y m p t o m s w e r e p r e s e n t i n
w o r k e r s e x p o s e d t o <1, 1-2 7.63,
d.f.=
2,
40.5%,
58.3% and 61.5% o f t h e
and 2-3 ppm F i n t h e w a t e r , r e s p e c t i v e l y (X’:
~~0.05).
TABLE I 1 SPECIFIC SYMPTOMS I N RELATION TO R I S K OF EXPOSURE TO INDUSTRY AND PLANT
WORKERS
R i s k o f Exposure High Moderate
Low
S p e c i f i c Symptoms I n d u s t r y workers P l a n t workers Present Absent Present Absent
(29.2%)
50
121 (70.8%)
61 (70.9%)
25 (29.1%)
14 (12.2%)
101 (87.8%)
84 (52.2%)
17 (47.8%)
15
137 (89.1%)
32 (26.9%)
a7 (73.1%)
(9.9%) X2 d.f. P
25.48 2
<0.05
40.43 2
197 F l u o r o t i c d e n t a l changes a r e a r e s u l t o f c h i l d h o o d exposure t o h i g h F levels.
I t s s i g n i f i c a n t a s s o c i a t i o n w i t h s p e c i f i c symptoms i n p l a n t work-
e r s i n d i c a t e s c o n t r i b u t i o n o f water F l e v e l t o which workers are continuo u s l y exposed t o f r o m b i r t h i n a d d i t i o n t o F - c o n t a i n i n g d u s t exposure a t t h e plants.
I n t h e absence o f h y d r o f l u o r o s i s i n i n d u s t r i a l w o r k e r s t h e r e
was n o a s s o c i a t i o n b e t w e e n d e n t a l c h a n g e s and s p e c i f i c s y m p t o m s ( T a b l e 111).
TABLE I 1 1
S P E C I F I C SYMPTOMS I N RELATION TO FLUOROTIC DENTAL CHANGES I N INDUSTRY AND PLANT WORKERS
S p e c i f i c Symptoms Present Absent
Total
F l u o r o t i c D e n t a l Changes I n d u s t r y workers P l a n t workers Present Absent Present Absent
7 (8.9%)
35 (9.7%)
102 (57.6%)
80 (42.3%)
72 (91.1%)
324 (90.3%)
75 (42.4%)
109 (57.7%)
79
359
177
189
0.05 1 >O. 05
X2 d.f. P
8.56 1
U r i n a r y F e x c r e t i o n i n t h e p r e s e n c e o f h y d r o f l u o r o s i s and F - c o n t a i n i n g d u s t exposure a t p l a n t s showed a s i g n i f i c a n t r e l a t i o n t o s p e c i f i c symptoms, w h e r e t h e mean u r i n a r y F l e v e l was s i g n i f i c a n t l y h i g h e r i n w o r k e r s w i t h s p e c i f i c symptoms t h a n t h o s e n o t e x h i b i t i n g any symptoms.
Such a r e l a t i o n -
s h i p was n o t observed i n i n d u s t r i a l w o r k e r s (Table I V ) .
O f t h e t o t a l w o r k e r s X-rayed, 30.5% o f p l a n t w o r k e r s
o n l y 11.4% o f t h e i n d u s t r i a l w o r k e r s and
had b o t h s p e c i f i c symptoms and q u a l i t a t i v e r a d i o -
l o g i c a l changes. D y s p e p t i c symptoms a r e d e s c r i b e d i n a M e d i c a l Research C o u n c i l Study (7). I n t h e aluminum i n d u s t r y s t u d y a t F o r t W i l l i a m ,
a s i g n i f i c a n t l y h i g h preva-
l e n c e o f b u r n i n g e p i g a s t r i c p a i n (34.9%) i s n o t e w o r t h y i n p l a n t w o r k e r s i n t h i s study.
P r e v a l e n c e o f b u r n i n g e p i g a s t r i c p a i n was s i g n i f i c a n t l y h i g h e r
(52.3%) i n h i g h r i s k departments t h a n i n l o w r i s k departments (30.3%; Table V).
The M e d i c a l R e s e a r c h C o u n c i l S t u d y ( 7 ) a l s o r e p o r t e d an a s s o c i a t i o n
between d y s p e p t i c symptoms and s e v e r i t y o f exposure.
198 TABLE I V SPECIFIC SYMPTOMS I N RELATION TO URINARY F EXCRETION I N INDUSTRY AND PLANT
WORKERS
Mean U r i n a r y Industry
S p e c i f i c Symptoms
F
1.95
Present
% o f Workers w i t h >4 ppm L e v e l
L e v e l (ppm) Plant
Industry
3.24
t1.90
Plant
18.4
28.9
20.2
17.3
Q.59
2.10
2.78
t1.76
G'. 41
X*
0.5
2.29
2.26
2.49
P
>O. 05
>O. 05
<0.05
Absent
H y d r o f l u o r o s i s i s a l s o p r e s e n t i n h i g h r i s k departments.
However,
the
f a c t that burning epigastric pain i s not related t o water F levels indicates t h a t d u s t i n g e s t i o n p l a y s a r o l e i n h i g h r i s k departments.
That
b u r n i n g e p i g a s t r i c p a i n i s n o t r e l a t e d t o f l u o r o t i c d e n t a l changes suggests t h e same c o n c l u s i o n . T h e m e a n u r i n a r y F l e v e l (3.55
ppm) a n d w o r k e r s w i t h m o r e t h a n 4 ppm
u r i n a r y F l e v e l s (30.3%) w e r e s i g n i f i c a n t l y h i g h e r i n w o r k e r s w i t h b u r n i n g e p i g a s t r i c p a i n t h a n i n w o r k e r s w i t h o u t b u r n i n g e p i g a s t r i c p a i n (2.85
ppm
TABLE V BURNING EPIGASTRIC P A I N I N RELATION EXPOSURE I N PLANT WORKERS Burning Epigastric Pain
High
R i s k o f Exposure Moderate
TO R I S K OF E X P O S U R E AND
Low
DURATION O F
D u r a t i o n o f Exposure (years) L e s s t h a n 10 M o r e t h a n 10
Present
45 (52.3%)
62 (38.5%)
36 (30.3%)
123 (41.0%)
19 (28.8%)
Absent
41 (47.7%)
99 (61.5%)
83 (69.7%)
177 (59.0%)
47 (71.2%)
161
119
300
Total
X2
d.f. P
86 10.25 2
3.39 1 >0.05
66
199 and
19.3%. r e s p e c t i v e l y ) .
non-water-soluble
T h i s suggests absorption o f F from r e l a t i v e l y
f l u o r s p a r d u s t i n g e s t e d by workers.
CONCLUSION T h i s b a s e l i n e s t u d y o f i n d u s t r i a l and p l a n t w o r k e r s u n d e r p r e v a i l i n g c o n d i t i o n s r e v e a l s t h a t s y m p t o m s r e l a t e d t o bones and j o i n t s w e r e t h e m a j o r ones. S p e c i f i c s y m p t o m s w e r e r e l a t e d t o mode o f F i n j e s t i o n , s e v e r i t y o f exposure,
d u r a t i o n o f e x p o s u r e and w a t e r F l e v e l .
Only i n t h e presence o f
h y d r o f l u o r o s i s were s p e c i f i c symptoms r e l a t e d t o f l u o r o t i c d e n t a l changes and u r i n a r y F e x c r e t i o n .
Ingestion o f fluorspar dust resulting i n burning
e p i g a s t r i c p a i n was r e l a t e d t o s e v e r i t y and d u r a t i o n o f e x p o s u r e . baseline study w i l l help i n further follow-up
Such a
s t u d i e s o f workers t o under-
s t a n d t h e e f f e c t o f e x p o s u r e i n t h e w o r k i n g e n v i r o n m e n t on t h e i r h e a l t h and p l a n n i n g o f p r e v e n t i v e measures. REFERENCES
A C l i n i c a l H y g e n i c Study. H. K.
1.
Roholrn K (1937) F l u o r i n e I n t o x i c a t i o n , L e w i s and Co. Ltd., London
2.
(1946) L a n c e t 2:821-824 (1962) I n t A r c h A v e r g 20(Suppl):1-60 F r a d a G, M e n t e s a n a G, N a l b o n e G (1963) M i n e r v a Med 54:451-459 C z e r w i n s k i E, L a n k o s z W (1977) F l u o r i d e 10:125-136 W a l d b o t t GL, C e c i l i o n i VA (1969) C l i n T o x i c o l 2:387-396 M e d i c a l R e s e a r c h C o u n c i l Memorandum (1949) No 22 I n d u s t r i a l F l u o r o s i s
3. 4. 5. 6. 7.
M u r r a y MM.
W i l s o n DC
W a l d b o t t GL
HMSO. London
H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 203-210 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
203
INFLUENCE OF PROTEIN AND ASCORBIC A C I D ON FLUORIDE-INDUCED CHANGES I N BLOOD COMPOSITION AND SKELETAL FLUORIDE DEPOSITION I N M I C E
MING-HO
YU AND HUEY-LIY S. HWANG
Huxley College o f Environmental Studies, B e l l i n g h a m , W a s h i n g t o n 98225, U.S.A.
I d s s t e r n Washington U n i v e r s i t y .
ABSTRACT F e m a l e m i c e ( N u s m u s c u l u s . S w i s s W e b s t e r s ) f e d a d i e t c o n t a i n i n g low (4%) p r o t e i n s were t e s t e d f o r t h e e f f e c t s o f supplemental a c i d on body weight, tion.
f l u o r i d e and a s c o r b i c
fluoride deposition i n the tibia.
and b l o o d composi-
The r e s u l t s w e r e c o m p a r e d w i t h t h o s e o b t a i n e d f r o m c o n t r o l m i c e f e d
a d i e t c o n t a i n i n g n o r m a l (27%) p r o t e i n .
A t t h e end o f 12 weeks,
the
a n i m a l s m a i n t a i n e d o n t h e l o w p r o t e i n d i e t showed a 40% r e d u c t i o n i n b o d y weight,
w h i c h was u n a f f e c t e d b y s u p p l e m e n t a l f l u o r i d e a t 1 0 ppm.
Fluoride
a d m i n i s t r a t i o n e n h a n c e d t h e t i b i a f l u o r i d e l e v e l b y 550% i n t h e l o w p r o t e i n g r o u p a n d b y 160% i n t h e n o r m a l p r o t e i n group.
These i n c r e a s e s were mar-
k e d l y d i m i n i s h e d i n t h e presence o f supplemental ascorbic acid.
The e f f e c t
o f f l u o r i d e a n d a s c o r b i c a c i d o n s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y was s i m i l a r t o t h a t on b o n e f l u o r i d e d e p o s i t i o n .
A pronounced i n c r e a s e i n t h e
enzyme a c t i v i t y was o b s e r v e d e s p e c i a l l y i n t h e l o w p r o t e i n group.
INTRODUCTION M a n y e p i d e m i o l o g i c a l a n d e x p e r i m e n t a l s t u d i e s h a v e shown t h a t d i e t a r y f a c t o r s s u c h a s p r o t e i n s , c a l c i u m , v i t a m i n C, effects o f fluoride. i n d u c e d t o x i c i t y (1,Z).
etc..
can m o d i f y t h e t o x i c
G e n e r a l u n d e r n u t r i t i o n a p p e a r s t o enhance f l u o r i d z The a s s o c i a t i o n o f a s c o r b i c a c i d w i t h f l u o r i d e
t o x i c i t y a p p e a r s t o be f i r s t r e p o r t e d b y P h i l l i p s i n 1933 (3). t h e ensuing s t u d i e s have demonstrated
A number O F
mitigation of fluorosis
m e n t a l a n i m a l s b y t h e a d m i n i s t r a t i o n o f v i t a m i n C (4-7).
i n experi-
while others
s h o w e d t h a t t h e a d d i t i o n o f t h e v i t a m i n h a d n o b e n e f i c i a l e f f e c t s (8). Many s t u d i e s h a v e shown d i s t u r b a n c e s i n s k e l e t a l d e v e l o p m e n t i n humans and e x p e r i m e n t a l a n i m a l s f e d a d i e t d e f i c i e n t i n p r o t e i n s (9-11). study w i t h guinea pigs, d i e t a r y v i t a m i n C,
Parker
fi d.( 1 2 )
In their
investigated the interaction o f
p r o t e i n , and c a l c i u m w i t h f l u o r i d e ,
and showed i n c r e e s e d
a c c u m u l a t i o n o f f l u o r i d e i n t h e g r o u p n a i n t a i n e d on a l o w p r o t e i n d i e t .
204
Reports on the interaction of dietary factors with fluoride, however, have been largely concerned with epidemiological studies on humans o r experimental studies on domestic animals. For studies dealing with the effect o f vitamin C, guinea pigs or monkeys have been widely used. Little information is available on the effects of fluoride on wildlife or the effect of ascorbic acid on animals that do not require this vitamin because of their ability to synthesize it. Newman and Yu (13) observed marked dental disfigurement and abnormal tooth wear patterns in black-tailed deer taken from an area near an industrial fluoride source.
More recently, Yu
and Driver (14) reported that supplementation o f fluoride in the diet of growing cockerels resulted in significantly altered tissue ascorbic acid and dehydroascorbic acid levels. Thrs suggests that in animals that do not require vitamin C, supplemental fluoride and ascorbic acid may induce a response similar to that in those animals that require the vitamin. The present work was initiated to further test this possibility. In particular, the interaction of protein and supplemental ascorbic acid with fluoride in laboratory mice was studied.
MATERIALS AND METHODS One-week-old female mice (Mbl musculus. Swiss Websters) were fed diets containing either normal (27%) protein o r low (4%) protein for three weeks. The diets were obtained from Nutritional Biochemicals, Cleveland, Ohio,
U.S.A.
The
normal protein diet consisted of vitamin free casein, 27%;
starch, 32%; sucrose, 27%; vegetable oil, 10%: and salt mixture and vitamins, 4%. The low protein diet differed from the normal protein diet only in the casein and starch contents, which were 4% and 55%, respectively. At the end of the three-week period, the mice in both diet groups were divided into four sub-groups varying only in drinking water: the control group continued to receive water with no supplements.while the others received water supplemented with 10 ppm F as NaF, 20 ppm ascorbic acid, and 10 pprn F plus 20 ppm ascorbic acid, respectlvely. In all cases the animals received the food and drinking water ad libitum. These regimens were continued for 12 weeks. At the end o f the experimental period, the mice were sacrificed and the blood samples and the tibias were collected. The tibias were dried, defatted. and ashed. The fluoride content of the bone ash was then determined by a fluoride specific ion electrode by the method of Singer and Armstrong (15).
The serum samples were analyzed for albumin, glucose,
calcium, and cholesterol contents, and the alkaline phosphatase activity, respectively.
The analyses were carried out in an ACCU-STAT blood chemis-
try analyzer (Clay-Adams, Inc.. New Jersey, U.S.A.).
205 RESULTS The e x p e r i m e n t a l m i c e m a i n t a i n e d on t h e l o w p r o t e i n d i e t e x h i b i t e d a general poor growth,
a n d t h e r e was a m o r e t h a n 40% (p<0.05)
reduction i n
body w e i g h t i n comparison w i t h t h e c o n t r o l a n i m a l s f e d t h e normal p r o t e i n (Table I).
F l u o r i d e o r a s c o r b i c a c i d a l o n e c a u s e d l i t t l e change.
While
b o t h NaF and a s c o r b i c a c i d were supplemented a t t h e same t i m e , t h e r e was a s l i g h t r e d u c t i o n (6%) i n t h e body w e i g h t o f t h e normal p r o t e i n group, w h i l e a 19% i n c r e a s e i n t h e l o w p r o t e i n g r o u p was o b s e r v e d .
These d i f f e r e n c e s
were n o t found t o be s t a t i s t i c a l l y s i g n i f i c a n t a t t h e 95% l e v e l o f c o n f i dence,
however.
As shown i n Table 11. an average o f 123 ppm F was observed 1n t h e t i b i a s o f t h e c o n t r o l m i c e r e c e i v i n g n o n f l u o r i d a t e d water. ppm
F
i n the drinking water resulted i n
d
A d m i n i s t r a t i o n o f 10
s i g n i f i c a n t i n c r e a s e i n bone
f l u o r i d e i n b o t h n o r m a l and l o w p r o t e i n g r o u p s .
However,
t h e r e was a
s t r i k i n g d i f f e r e n c e i n bone f l u o r i d e r e t e n t i o n b e t w e e n t h e t w o d i e t a r y groups.
The i n c r e a s e i n f l u o r i d e d e p o s i t i o n i n t h e n o r m a l p r o t e i n g r o u p
was 160% (p
These i n c r e a s e s w e r e d i m i n i s h e d when
a s c o r b i c a c i d was supplemented s i m u i t a n e o u s l y . c i a l l y marked f o r t h e l o w p r o t e i n group: and a s c o r b i c a c i d used,
whereas i n t h e l o w proThe d i m i n u t i o n was espe-
a t t h e concentrations o f f l u o r i d e
a 47% decrease i n bone d e p o s i t i o n was observed.
For t h e mice fed normal protein,
s u p p l e m e n t a l f l u o r i d e caused l i t t l e
change i n serum a l k a l i n e phosphatase a c t i v i t y w h i l e i n t h e presence o f
TABLE I
EFFECTS OF FLUORIDE AND ASCORBIC A C I D ON BODY WEIGHT OF M I C E FED NORMAL AND LOW PROTEIN DIETS Treatment
Normal P r o t e i n Body Weight (9)
Control
32.5k5.4'
N
(4)
Low P r o t e i n
Percent o f Control
Body Weight (9)
-
20.0k2.8
N
Percent o f Control
(3)
-
32.7k5.8
(3)
100
20.5k3.3
(3)
102
A A ~
33.1k9.6
(3)
101
20.7k1.7
(3)
103
+
30.4k3.2
(4)
94
23.8k0.8
(2)
119
NaF2 NaF
AA
' V a l u e s a r e means f S.D.
' F l u o r i d e and a s c o r b i c a c i d (AA) were supplemented i n t h e d r i n k i n g w a t e r a t 10 ppm and 20 ppm. r e s p e c t i v e l y .
206 a s c o r b i c a c i d a l o n e , a 14% r e d u c t i o n was o b s e r v e d ( T a b l e 111).
Far the
a n i m a l s f e d a l o w - p r o t e i n d i e t , h o w e v e r , m a r k e d i n c r e a s e s i n t h e enzyme a c t i v i t y w e r e shown i n a l l s u b - g r o u p s .
The s u b - g r o u p t r e a t e d w i t h NaF
showed a 54% e l e v a t i o n o v e r t h e w a t e r c o n t r o l s .
I n t h e presence o f
a s c o r b i c a c i d t h i s i n c r e a s e was d i m i n i s h e d t o 21%.
TABLE I 1
EFFECTS OF FLUORIDE AND ASCORBIC A C I D Old FLUORIDE DEPOSITION I N T I B I A S M I C E FED NORMAL AND LOW PROTEIN D I E T S Treatment
Normal P r o t e i n
F conc.
OF
Low P r o t e i n
N
Percent o f Control
F conc.
P Pm
PPm
N
Percent o f Control
Con t r o 1
123+46'
(4)
-
227k122
(3)
-
NaF2
320k147
(3)
260
1810k743
(3)
653
A A ~
105+29
(3)
85
289283
(3)
104
NaFtAA
280k137
(4)
227
964+348
(2)
348
'Values a r e
means
_C
S.D.
' F l u o r i d e and a s c o r b i c a c i d (AA) were supplemented i n t h e drinl:ir,g 10 ppm and 20 ppm, r e s p e c t i v e l y .
water a t
TABLE I 1 1 EFFECTS OF FLUORIDE AND A S C O R B I C A C I D ON SERUM ALKALINE PHOSPHATASE ACTIVITY OF M I C E FED NORMAL AND LOW PROTEIN DIETS Treatment
Normal P r o t e i n Percent
Enzyme
activity
N
of C o n t r o l
activity
N
o f Control
32k4.9l
(4)
-
82k8.5
(3)
153
Enzyme
Control
Low P r o t e i n Percent
NaF2
33k3.5
(3)
103
125kl. 1
(3)
A A ~
2721.4
(3)
86
7625.6
(3)
93
Na F+AA
32k0.6
(4)
100
99+2.1
(2)
121
'Values a r e means k S.D.
Babson u n i t .
' F l u o r i d e and a s c o r b i c a c i d (AA) were supplemented i n t h e d r i n k i n g w a t e r a t 10 ppm and 20 ppm, r e s p e c t i v e l y .
TABLE IV EFFECTS OF FLUORIDE AND ASCORBIC ACID ON SERUM COMPOSITION OF MICE FED NORMAL AND LOW PROTEIN DIETS Treatment
Normal Protein Albumin g/dl
Glucose mg/dl
Cholesterol mg/dl
Ca mg/dl
A1 bumi n g/dl
G1 ucose mg/dl
Cholesterol mg/dl
Ca mg/dl
~~
Control NaF2
A A ~ Na F+AA
2.341 (0.05) 2.30 (0.14) 2.53 (0.25) 2.49 (0.20)
21 2 (31.7) 205 (5.0) 225 (21.8) 207 (24.7)
147 (16.9) 139 (12.7) 126 (15.3) 134 (7.9)
9.6
(0.6)
10.0 (2.3) 9.4 (0.6) 9.1 (0.3)
'Values are means and S.D.. in parenthesis. 'Fluoride and ascorbic acid (AA) w e r e supplemented in the respectively.
1.93 (0.27) 1.86 (0.08) 1.96 (0.29) 2.73 (0.53)
drinking
153 (28.4) 176 (17.6) 218 (46) 175 (20.5)
124 (19.1) 135 (35.3) 158 (26.3) 168 (24.7)
water a t 10 ppm and 20 ppm,
8.7 (0.3) 9.9 (1.5) 8.4 (0.5) 9.3 (0.5)
208 Whereas b o t h s e r u m g l u c o s e and a l b u m i n l e v e l s i n m i c e m a i n t a i n e d o n t h e low-protein d l e t were g e n e r a l l y l o w e r than those i n t h e normal p r o t e i n group,
f l u o r i d e o r a s c o r b i c a c i d induced no s i g n i f i c a n t changes ( T a b l e I V ) .
Serum c a l c i u m and c h o l e s t e r o l l e v e l s w e r e no-t shown t o be s i g n i f i c a n t l y a l t e r e d b y t h e a d m i n i s t r a t i o n o f e i t h e r f l u o r i d e o r a s c o r b i c acid.
For t h e
m i c e f e d a l o w - p r o t e i n d i e t , b o t h f l u o r i d e and a s c o r b i c a c i d r e s u l t e d i n some i n c r e a s e s i n s e r u m c h o l e s t e r o l c o n t e n t ,
b u t t h e d i f f e r e n c e s between
t h e e x p e r i m e n t a l and t h e w a t e r c o n t r o l s w e r e f o u n d n o t s i g n i f i c a n t a t t h e 95% l e v e l o f c o n f i d e n c e .
DISCUSSION The e x p e r i m e n t a l d a t a p r e s e n t e d h e r e c l e a r l y d e m o n s t r a t e t h e i m p o r t a n c e o f d i e t a r y p r o t e i n i n t h e r e t e n t i o n o f f l u c r i d e i n t h e bone o f m i c e , low dietary protein resulted i n higher fluoride
accumulation.
o b s e r v a t i o n w i t h g u i n e a p i g s h a s b e e n r e p o r t e d (12). e x p e r i m e n t a l c o n d i t i o n s used,
i.e.,
A similar Under t h e
s u p p l e m e n t a t i o n o f f l u o r i d e a t 10 ppm i n t h e
d r i n k i n g w a t e r d e p o s i t e d n e a r l y f i v e t i m e s more f l u o r i d e i n t h e t i b i a of mice maintained on a low p r o t e i n d i e t than i n t h e normal p r o t e i n group r e c e i v i n g t h e s a m e a m o u n t o f f l u o r i d e i n t h e d r i n k i n g w a t e r ( T a b l e 11). Even w i t h o u t f l u o r i d e s u p p l e m e n t a t i o n ,
t h e l o w p r o t e i n group accumulated
125% m o r e f l u o r i d e t h a n t h e n o r m a l p r o t e i n group.
The f l u o r i d e i n t h e bone
It
o f t h e c o n t r o l a n i m a l s p r e s u m a b l y was d e r i v e d f r o m t h e d i e t i t s e l f .
appears t h a t d i e t a r y p r o t e i n i s an independent f a c t o r a f f e c t i n g f l u o r i d e d e p o s i t i o n i n t h e bone o f m i c e . T h a t s u p p l e m e n t a l a s c o r b i c a c i d m a r k e d l y reduced f l u o r i d e r e t e n t i o n i n t h e bones o f t h e e x p e r i m e n t a l a n i m a l s i s c l e a r l y shown i n T a b l e 11.
It i s
i m p o r t a n t t o n o t e t h a t t h e d i m i n i s h i n g e f f e c t o f a s c o r b i c a c i d i s more p r o n o u n c e d i n t h e l o w p r o t e i n g r o u p (46%) t h a n i n t h e n o r m a l p r o t e i n g r o u p (12%).
W h i l e t h e r e a s o n f o r s u c h d i f f e r e n c e i s n o t known,
i n t e r m s o f h e a l t h e f f e c t may be i m p o r t a n t .
i t s implication
The e a r l i e r o b s e r v a t i o n s t h a t
v i t a m i n C a d m i n i s t r a t i o n p r e v e n t e d t h e d e v e l o p m e n t o f s e v e r e bone f l u o r o s i s
(7).
o r d i m i n i s h e d t h e t o x i c e f f e c t s o f f l u o r i d e i n bones o f monkeys (ll),
may be e x p l a i n e d i n t e r m s o f t h e r e d u c t i o n
0-:
bone f l u o r i d e a c c u m u l a t i o n
i n f l u e n c e d by t h e vitamin.
As shown i n T a b l e 111, s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y was m a r k e d l y e n h a n c e d i n a l l t r e a t m e n t g r o u p s m a i n t a i n e d on a l o w p r o t e i r ! d i e t .
A
s t r i k i n g s i m i l a r i t y e x i s t s b e t w e e n s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y and fluoride retention
i n t h e bone w i t h r e s p e c t t o t h e i n f l u e n c e o f f l u o r i d e
and a s c o r b i c a c i d s u p p l e m e n t a t i o n :
exposure t o supplemental f l u o r i d e
enhanced f l u o r i d e d e p o s i t i o n i n t h e bone and serum a1 k a l i n e p h o s p h a t a s e activity,
and s u p p l e m e n t a t i o n o f a s c o r b i c a c i d d i m i n i s h e d t h e i n c r e a s e
209 ( T a b l e s I 1 and 111).
The d i f f e r e n c e s i n enzyme a c t i v i t y e x h i b i t e d b y t h e
t w o d i e t a r y p r o t e i n groups a r e considerable.
Since t h e p r e c i s e metabolic
f u n c t i o n s o f t h e enzyme a r e n o t y e t f u l l y understood, a s s o c i a t e d w i t h t h e s e d i f f e r e n c e s i s n o t known.
the implication
However,
because t h e
enzyme may f a c i l i t a t e t r a n s f e r o f m e t a b o l i t e s a c r o s s c e l l m e m b r a n e s , b e associated w i t h l i p i d transport,
and be i n v o l v e d i n t h e c a l c i f i c a t i o n
p r o c e s s o f b o n e s y n t h e s i s (16),
t h e p o s s i b i l i t y e x i s t s t h a t one o r m o r e o f
t h e s e f u n c t i o n s m y be a l t e r e d .
A p a t h o l o g i c a l l e g i o n may a l s o be p r e s e n t
i n any o f t h e v a r i o u s organs.
I t i s n o t i c e a b l e t h a t r e p o r t s on c h a n g e s i n s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y as a r e s u l t o f exposure t o f l u o r i d e have been c o n t r a d i c t o r y . W h i l e o u r o b s e r v a t i o n s a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d b y Reddy and S r i k a n t i a (11).
and tlough and Freeman (1~/), t h e y c o n t r a s t w i t h o t h e r s
s h o w i n g a l o w e r e d enzyme a c t i v i t y a s s o c i a t e d w i t h p r o t e i n m a l n u t r i t i o n (1821).
These d i s c r e p a n c i e s p r e s u m a b l y a r e caused b y such f a c t o r s as t h e
s p e c i e s o f a n i m a l s and e x p e r i m e n t a l c o n d i t i o n s used, i n a d d i t i o n t o t h e c o m p l e x n a t u r e o f t h e enzyme i t s e l f . ACKNOWLEDGEMENT We w i s h t o t h a n k t h e B u r e a u o f F a c u l t y R e s e a r c h , University,
Western Washington
f o r s u p p o r t i n t h e c o u r s e o f t h i s work.
REFERENCES K r i s h n a s w a m y K (1974) I n d J Med Res 62:1415-1423
1.
K r i s h n a m a c h a r i KAVR,
2.
S u t t i e JW (1983) I n : Shupe JL, P e t e r s o n HB. Leone NC ( e d s ) F l u o r i d e s E f f e c t s o n V e g e t a t i o n , A n i m a l s , a n d Humans. P a r a g o n P r e s s , S a l t L a k e C i t y , p p 291-304
3.
P h i l l i p s PH (1933) J B i o l Chem 100:79
4.
Evans RJ,
5.
Wadhwani TK (1952) I n d Med Gaz 87:5-7
P h i l l i p s PH (1939) J N u t r 18:353-360
6. Wadhwani TK ( 1 9 5 4 ) J I n d I n s t S c i 36:64-69 7.
Wadhwani TK ( 1 9 5 5 ) I n d J Med Res 45:377-386
8.
V e n k a t e s w a r l u P,
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J h a GJ, Deo MG,
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J o n e s PRM,
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9eddy GS,
Rao ND ( 1 9 5 7 ) I n d J Med Res 45:377-386 R a m a l i n g a s w a m y V ( 1 9 6 8 ) Amer J P a t h 53:1111-1123
Dean RFA (1959) J P e d i a t 54:176 S r i k a n t i a SG ( 1 9 7 1 ) M e t a b o l i s m 20:642-656
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P a r k e r CM,
S h a r n a RP,
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Newman JR.
Yu MH ( 1 9 7 6 ) J W i l d l i f e D i s 12:39-41
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Yu MH,
Shupe J L ( 1 9 7 9 ) C l i n T o x i c o l 15:301-311
D r i v e r CJ (1982) F l u o r i d e 15:97-104
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S i n g e r LD, A r m s t r o n g WD (1968) A n a l Chem 40:613-614
16.
T i e t z NW ( 1 9 7 9 ) I n : G r i f f i t h s JC ( e d ) C l i n i c a l E n z y m o l o g y . M a s s o n P u b l i s h i n g , New York, p p 69-79
210
17.
Hough VH,
Freeman S (1942) Amer J P h y s i o l 138:184-189
18.
D a v i e s JEW, E l l e r y PM, H e y w o r t h PG,
19.
S c h w a r t z R (1956) J C l i n P a t h 9:33
Hughes RE (1978) S p e c i a l i a 15:429
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W a t e r l o w JC, S t e p h e n JML (1969) P r o c N u t r SOC 28:234-242
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E d o z i e n JC (1961) P e d i a t r i c s 27:325-333
H.Tsunoda and M.-H. Y u (Editors)
211
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 211-224 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
CHANGES I N THE METABOLISM
OF
GLUCOSE AND CALCIUM FOLLOWING A SINGLE LARGE
DOSE OF FLUORIDE TO RATS YASUNOBU SUKETA, Y U K A R I KANAMOTO, YUKO SATOH, AND SHOJI OKADA S h i z u o k a C o l l e g e o f P h a r m a c y , D e p t o f E n v i r o n m e n t a l B i o c h e m i s t r y , 2-2-1 O s h i k a , S h i z u o k a , S h i z u o k a 422, J a p a n
ABSTRACT Serum g l u c o s e l e v e l was e l e v a t e d i m m e d i a t e l y a f t e r i p a d m i n i s t r a t i o n o f a s i n g l e l a r g e d o s e o f f l u o r i d e (NaF 3 5 m g / k g ) t o r a t s .
The e l e v a t i o n o f
serum g l u c o s e was m a r k e d l y s u p p r e s s e d b y a d r e n a l e c t o m y , propranolol,
b u t n o t b y thyroid-parathyroidectomy.
glucose
found
was
t o be a s s o c i a t e d
d i benamine o r
E l e v a t i o n o f seruni
w i t h enhancement o f
glucose-6-
phosphatase a c t i v i t i e s i n t h e l i v e r and k i d n e y o f f l u o r i d e - t r e a t e d On t h e o t h e r hand,
rats.
r e n a l c a l c i u m was e l e v a t e d g r a d u a l l y as c o m p a r e d t o t h e
a u g m e n t o f s e r u m g l u c o s e and c y c l i c AMP f o l l o w i n g t h e f l u o r i d e a d m i n i s t r a tion.
The e l e v a t i o n o f r e n a l c a l c i u m was m a r k e d l y s u p p r e s s e d b y t h r y r o i d -
parathyroidectomy.
INTRODUCTION Taylor
gt
4. ( 1 )
f l u o r i d e (NaF 20-30
r e p o r t e d t h a t a d m i n i s t r a t i o n o f n e a r - l e t h a l doses o f mg/kg,
i v ) markedly increased u r i n a r y sugar excretion.
T h e i r f i n d i n g s w e r e c o n f i r m e d and i t was shown t h a t a s i n g l e l a r g e d o s e o f f l u o r i d e (NaF 3 5 m g / k g , i p ) c a u s e d t h e e l e v a t i o n o f n o t o n l y u r i n a r y g l u c o s e e x c r e t i o n b u t a l s o serum g l u c o s e c o n c e n t r a t i o n (2a).
Suketa gt
.a!
( 2 b ) f o u n d t h a t t h e c a l c i u m c o n t e n t i n t h e k i d n e y s was a u g m e n t e d b y f l u o r i d e administration. cium i n f l u x .
G l u c o s e p r o d u c t i o n i s known t o he r e g u l a t e d b y c a l -
I n t h e p r e s e n t s t u d y , a p o s s i b l e m e c h a n i s m f o r changes i n t h e
m e t a b o l i s m o f g l u c o s e and c a l c i u m i n r a t s f o l l o w i n g a s i n g l e l a r g e d o s e o f f l u o r i d e was i n v e s t i g a t e d .
MATERIALS AND METHODS Materials Glucose-6-phosphate
(disodiurn salt),
D-fructose-1.6-diphosphate ( s o d i u m
salt), phosphoenolpyruvate (nonopotassium s a l t ) , i n o s i n e 5'-diphosphate (sodium s a l t ) ,
g l u t a t h i o n e ( r e d u c e d form),
6-nicotinamide
adenine dinucleo-
212 t i d e , r e d u c e d f o r m (NADH).
ma1 i c d e h y d r o g e n a s e a n d ( - ) - e p i n e p h r i n e
o b t a i n e d f r o m S i g m a C h e m i c a l Company ( S t . c h l o r i d e ) and D L - p r o p r a n o l o l (Kyoto,
Japan);
Louis.
(hydrochloride),
were
dibenamine (hydro-
from Nakarai Chemical Ltd.
17-hydroxycorticosterone and g l y c o g e n , f r o m Wako P u r e Chem-
i c a l Industries,
L t d . (Osaka, Japan);
and s o d i u m b i ~ a r b o n a t e - [ C ' ~ ] ( O . 2 5
mCi/mg) and c a l c i u m ~ h l o r i d e - [ C a ~ ~ ] ( 2 3 . 1uCi/mg), 8 (Boston,
Mo);
f r o m New E n g l a n d N u c l e a r
Mass).
Treatment M a l e W i s t a r a l b i n o r a t s w e i g h i n g 95-105 g w e r e u s e d i n t h i s s t u d y . r a t s w e r e m a i n t a i n e d i n an a i r - c o n d i t i o n e d r o o m a t d i e t M F ( O r i e n t a l Y e a s t Co. L t d . ,
22"C,
The
and w e r e f e d b a s a l
Tokyo, J a p a n ) and w a t e r ad l l b i t u m .
At
t h e end o f s i x days, t h e r a t s w e r e f a s t e d 24 h o u r s b e f o r e and a f t e r each s e t of
experiments t o
m i n i m i z e g l u c o s e 3 b s o r p t i o n f r o m t h e g u t and
s t a b i l i z e u r i n a r y e x c r e t i o n o f glucose.
Adrenalectomy
and t h y r o i d -
p a r a t h y r o i d e c t o m y w e r e c a r r i e d o u t on r a t s m a i n t a i n e d o n b a s a l d i e t M F and w a t e r f o r 5 days.
F o l l o w i n g a s i n g l e l p a d m i n i s t r a t i o n o f f l u o r i d e (NaF.
3 5 m g / k g ) o r c h l o r i d e (NaC1 48.1 r n g / k g ) ,
used as c o n t r o l s , t h e r a t s were
a n e s t h e t i z e d w i t h e t h e r and k i l l e d b y c a r d i a c p u n c t u r e . I n j e c t i o n o f epinephrine, 0101 Epinephrine and/or
17-hydroxycorticosterone, d i b e n a m i n e and pro=
17-hydroxycorticosterone ( 3 ) w e r e i n j e c t e d i p 1 0 n i i n
a f t e r the fluoride administration.
D i benami ne (4) a n d / o r p r o p r a n o l o l (5)
w e r e i n j e c t e d s c 10 m i n a f t e r t h e f l u o r i d e a d m i n i s t r a t i o n . D e t e r m i n a t i o n o f g l u c o s e and 17-hydroxycorticosterone. Serum and u r i n a r y g l u c o s e and 17-hydroxycorticosterone w e r e d e t e r m i n e d b y t h e m e t h o d s of H u l t m a n
(6). and P o r t e r and S i l v e r (7).
respectively.
Enzyme a s s a y The a c t i v i t i e s o f g l u c o s e - 6 - p h o s p h a t a s e , phosphoenolpyruvate carboxykinase,
fructose-1.6-diphosphatase.
and g l y c o g e n p h o s p h o r y l a s e w e r e d e t e r -
m i n e d b y t h e m e t h o d s o f S w a n s o n (8),
M c G i l v e r y (9).
B a l l a r d and Hanson
( 1 0 ) . a n d M e y e r g t g l . (ll), respectively. Assay o f c a l c i u m f l u x D e t e r m i n a t i o n o f r e n a l and h e p a t i c c a l c i u m - i n f l u x and - e f f l u x was c a r r i e d o u t w i t h k i d n e y and l i v e r s l i c e s a c c o r d i n g t o t h e m e t h o d o f W a l l a c e (12) u s i n g 4 5 ~ a . D e t e r m i n a t i o n o f magnesium and c a l c i u m C a l c i u m and magnesium w e r e m e a s u r e d b y t h e m e t h o d o f W i l l i s ( 1 3 ) . Determination o f protein P r o t e i n was d e t e r m i n e d b y t h e method o f L o w r y
aJ.(l4).
g G.
213 P r e p a r a t i o n s o f m i c r o s o m e s , c y t o s o l and g l y c o g e n g r a n u l e s M i c r o s o m e s and c y t o s o l w e r e p r e p a r e d a c c o r d i n g t o t h e m e t h o d o f J d r g e n s e n (15).
At the time o f sacrifice,
k i l l e d by cardiac puncture.
t h e r a t s w e r e a n e s t h e t i z e d w i t h e t h e r and
The k i d n e y s and l i v e r s w e r e r e m o v e d and t h e
t i s s u e s (1 g) were i m m e d i a t e l y homogenized i n a P o t t e r - E l v e h j e m t e f l o n g l a s s h o m o g e n i z e r w i t h 5 m l o f i c e - c o l d 0.25M b u f f e r (pH 7.2).
sucrose-0.03M
histidine
Reproducible p r e p a r a t i o n s o f t h e heavy microsomal f r a c t i o n
w e r e o b t a i n e d b y c e n t r i f u g a t i o n ( 2 5 3 0 0 x g.
30 m i n ) o f t h e s u p e r n a t a n t
a f t e r s e d i m e n t a t i o n o f t h e m i t o c h o n d r i a a t 1 0 8 0 0 x g f o r 3 0 min.
Cytosol
f r a c t i o n s were o b t a i n e d by s e d i m e n t a t i o n o f t h e l i g h t m i c r o s o m a l f r a c t i o n ( 1 0 5 0 0 0 x g. 6 0 m i n ) f r o m t h e s u p e r n a t a n t ( 2 5 3 0 0 x g, 3 0 m i n ) .
Glycogen
9d.(ll).These
g r a n u l e s were p r e p a r e d a c c o r d i n g t o t h e method o f Meyer p r e p a r a t i o n s ( 1 mg o f p r o t e i n / m l o f t h e r e s p e c t i v e b u f f e r )
were s t o r e d
at
-20°C.
RESULTS AND DISCUSSION E f f e c t o f f l u o r i d e a d m i n i s t r a t i o n on serum g l u c o s e Serum g l u c o s e was e l e v a t e d t o a maximum 3 h o u r s f o l l o w i n g a s i n g l e i p a d m i n i s t r a t i o n o f f l u o r i d e (NaF 3 5 m g / k g ) ( F i g .
SERUM
la).
E l e v a t i o n o f serum
a
W
z
U 3
U
0
5
0
U w
m
3
12
5
b
URINE
z
24
W
ln
0 0
3
d
. z
2 M
-6
0
6
12
24
HOURS AFTER ADMINISTRATION
F i g u r e 1. Changes i n s e r u m and u r i n a r y g l u c o s e c o n t e n t s o f r a t s . The r a t s were k i l l e d a t v a r i o u s t i m e s f o l l o w i n g a s i n g l e i p a d m i n i s t r a t i o n o f f l u o r i d e (NaF, 3 5 rng/kg). T h e v a l u e s a r e a v e r a g e a n d S.E. (n=8). Fluoridei n) Figure t r e a t e d (.)in F i g u r e l a . Control(=) a n d f l u o r i d e - t r e a t e d (0 lb. " S i g n i f i c a n t d i f f e r e n c e f r o m t h e c o n t r o l . p
214 g l u c o s e b y f l u o r i d e was s u g g e s t e d t o b e p o s s i b l y d u e t o d e c r e a s e d u r i n a r y glucose excretion,
o r t o enhancement o f g l y c o l y s i s o r gluconeogenesis.
t e s t these p o s s i b i l i t i e s ,
f l u o r i d e w e r e f i r s t examined. v a l u e s 6-12
To
changes i n u r i n a r y g l u c o s e e x c r e t i o n induced by U r i n a r y g l u c o s e e x c r e t i o n r e a c h e d maximum
hours f o l l o w i n g t h e f l u o r i d e a d m i n i s t r a t i o n (Fig.
lb).
These
r e s u l t s suggest t h a t t h e e l e v a t i o n o f serum glucose i n f l u o r i d e - t r e a t e d r a t s i s n o t l i k e l y due t o d e c r e a s e d u r i n a r y g l u c o s e e x c r e t i o n . To t e s t w h e t h e r o r n o t e l e v a t i o n o f s e r u m g l u c o s e was a s s o c i a t e d w i t h a c t i v a t i o n o f g l y c o l y s i s o r gluconeogenesis-related fluoride
enzymes, t h e e f f e c t s o f
a d m i n i s t r a t i o n on t h e a c t i v i t i e s o f glucose-6-phosphatase.
fructose-1.6-diphosphatase.
p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e and g l y c o g e n
p h o s p h o r y l a s e ( a a n d b) i n t h e l i v e r w e r e e x a m i n e d .
As shown i n T a b l e I ,
t h e a c t i v i t y o f g l u c o s e - ~ 6 - p h o s p h a t a s e was m a r k e d l y enhanced b y f l u o r i d e .
TABLE I FLUORIDE-INDUCED C H A N G E S I N THE A C T I V I T I E S OF HEPATIC ENZYMES RELATED TO GLUCONEOGENESIS AND GLYCOLYSIS. Enzyme
Hour a f t e r adrninistration
Glucose-6-phosphatase
Fructose-l.6d i phosphatase
Phosphoenol p y r u v a t e c a r b o x y k i nase
a
Enzyme a c t i v i t y nmol/min/mg
protein
R e l a t i v e value
0
168.0 f 9 . 8
3
294.5 f 12.5
0
1320 f 47
1
1
1345 f 41
1.02
3
1530
D
60.7 f 3.3
3
30.7 f 4.5
0
540 f 9
3
270 f 8
0
1 6 0 0 t 24
+
71
1 1.75a
1. 16b 1
0. 50a 1
0. 50a
Glycogen phosphorylase b
3
600 f 180
1
0. 38a
T h e r a t s w e r e k i l l e d 1 o r 3 h o u r s a f t e r f l u o r i d e a d m i n i s t r a t i o n (NaF 3 5 m g / k g . i p ) o r c h l o r i d e a d m i n i s t r a t i o n (NaC1 48.7 m g / k g . i p ) a s c o n t r o l t o rats. V a l u e s a r e a v e r a g e s o b t a i n e d f r o m f o u r t o s i x r a t s f SE. a v b S i g n i f i c a n t c h a n g e f r o m z e r o t i m e : ap
215 E f f e c t o f a d r e n a l e c t o m y on e l e v a t i o n of s e r u m g l u c o s e c a u s e d administration
by
fluoride
The p r o d u c t i o n o f g l u c o s e i s known t o be s t i m u l a t e d b y e p i n e p h r i n e a n d / o r a d r e n a l g l u c o c o r t i c o i d s (3). n a l f u n c t i o n by f l u o r i d e ,
I n o r d e r t o examine t h e enhancement o f adre-
a d r e n a l e c t o m y was c a r r i e d o u t o n r a t s .
As shown
i n T a b l e 11. a d r e n a l e c t o m y m a r k e d l y s u p p r e s s e d t h e e l e v a t i o n o f s e r u m glucose induced by f l u o r i d e . r e n a l glucose-6-phosphatase by adrenalectomy.
I n addition,
t h e e n h a n c e m e n t o f h e p a t i c and
a c t i v i t y was f o u n d t o be c o m p l e t e l y s u p p r e s s e d
These r e s u l t s ,
s u g g e s t t h a t e l e v a t i o n o f serum g l u c o s e
may b e due t o t h e e n h a n c e m e n t o f g l u c o s e - 6 - p h o s p h a t a s e l i v e r and k i d n e y s i n f l u o r i d e - t r e a t e d
a c t i v i t y i n the
rats.
TABLE I 1 EFFECT OF ADRENALECTOMY ON ELEVATIONS OF SERUM GLUCOSE, AND OF HEPATIC AND RENAL GLUCOSE-6-PHOSPHATASE A C T I V I T I E S BY A SINGLE LARGE DOSE OF FLUORIDE Treatment
Serum g l u c o s e c o n c e n t r a t i o n ( m g / d l ) Intact
Adrenalectomized
(AX)
Control
104.8 f 8.2
56.6 f 10.5
F 1u o r i d e
187.3
55.8 f 4.1
f
11.8
Treatment
Suppression by AX (%)
46.0 100.6
H e p a t i c gl ucose-6-phosphatasea Intact
Adrenalectomized
(AX)
Suppression by AX (%)
Control
168.6 f 9.8
138.9 f 0.9
17.6
F1 u o r i d e
294.4 f 12.5
139.7 f 2.7
99.5
Treatment
Renal glucose-6-phosphatasea Intact
Control
F 1u o r i d e
Adrenalectomized
(AX)
70.0 f 1.9 195.9
?
2.8
54.1 i 1.4
50.4
f
4.8
Suppression b y AX ( X )
22.7 102.6
The r a t s w e r e k i l l e d 3 h o u r s a f t e r f l u o r i d e a d m i n i s t r a t i o n (NaF 35 mg/kg. i p ) o r c h l o r i d e a d m i n i s t r a t i o n (NaC1 48.7 m g / k g . i p ) a s c o n t r o l t o r a t s . V a l u e s a r e a v e r a g e s o b t a i n e d f r o m f o u r t o s i x r a t s i SE. a E n z y m e a c t i v i t y i s e x p r e s s e d a s n m o l e s P i l i b e r a t e d p e r mg p r o t e i n p e r min.
TABLE 111 SUPPRESSIVE EFFECTS OF PROPRANOLOL AND/OR DIBENAMINE ON ELEVATIONS OF HEPATIC AND RENAL GLUCOSE-6PHOSPHATASE ACTIVITIES, AND OF SERUM GLUCOSE BY A SINGLE LARGE DOSE OF FLUORIDE Glucose-6-phosphatase activity Treatment
Enzyme acti v i tya
Fluoride None Pr
Control
Liver Suppression by antagonist ( % )
Di Pr+Di
475.5 f 42.3 257.6 k 4.0 218.4 rf 15.5 125.4 f 6.8
None
139.0
k
17.4
0 64.7b 76.4b 104b -
Serum glucose
Kidney Enzyme Suppression acti vi tya by antagonist ( % ) 300.4 f 171.3 f 189.9 ?I 121.2 f
18.2 9.2 5.9 9.9
114.0 k 8.0
Concentration (mg/dl)
Suppression by antagonist (Z)
0 69.3b 59.3b 96.1b
155.0
k
6.2
D
104.3 112.2 95.1
k
k
6.7 4.3 7.3
63.qb 53.5b 73.Zb
-
15.5
k
4.1
k
Propranolol (Pr) (1.0 mg/kg) or dibenamine (Di) (2.0 mg/kg) was subcutaneously two times immediately and 30 min after fluoride administration (NaF 35 mg/kg, ip) or chloride administration (NaC1 48.7 mg/kg, ip) as control to rats. The rats were killed 3 hours after the fluoride dose. Values are averages obtained from four rats k SE. aEnzyme activity is expressed as nmoles Pi liberated per mg protein per min. bSuppression by antagonist: p
217
Suppressive effects of propranolol and/or dibenamine on elevation of qlucose-6-phosphatase activity and serum qlucose after a single larqe dose of fluoride The contribution of adrenomedullary function to elevation o f serum glucose i n fluoride-treated rats was examined by use of dibenamine (4) and/or propranolol (5) as an 01- or &blocker, respectively. The elevation of serum glucose was significantly suppressed by dibenamine and/or propranHowever, the suppression of serum glucose level by concomitant
0101.
treatment with dibenamine and propranolol was not significantly higher than that by dibenamine or propranolol. alone (Table 111). Increase in glucose-6-phosphatase activities in the liver and kidneys was suppressed by dibenamine and/or propranolol (Table 111). These results suggest that elevation of serum glucose in fluoride-treated rats may be due to the stimulation of adrenomedullary function by fluoride. Changes in adrenocortical function and serum glucose caused by fluoride Production of glucose i s known to be stimulated by glucocorticoids secreted by the adrenal cortex (3).
Changes i n 1 7 - h y d r o x y c o r t i c o s t e r o n e in
ul
z _ d
p :
9-
CC
2
6 -
V
*
I - P
0
URINE
b
Figure 2 . Changes in serum and urinary 1 7 - h y d r o x y c o r t i c o s t e r o n e contents o f rats. The rats were killed at various times following a single ip administration of fluoride (NaF, 35 mg/kg). Values are average and S.E. (n=8). Fluoride-treated ( 0 ) . Figure 2a. Control (I and) fluoride-treated (a), Figure 2b. Significant difference from the control, +‘piO.O2, +%p
N
P
W
TABLE I V EFFECTS OF E P I N E P H R I N E AND 17-HYDROXYCORTICOSTERONE ON HEPATIC AND RENAL GLUCOSE-6-PHOSPHATASE AND ON SERUM GLUCOSE OF RATS TREATED WITH FLUORIDE
ACTIVITIES,
~~
Glucose-6-phosphatase Treatment
Fluoride
Control
Enzyme activitya
Liver Stimulation b y Ep o r Hc
(Z)
Serum g l u c o s e
activity Enzyme activitya
Kidney Stimulation b y Ep o r Hc
Concentration (mg/dl)
(Z)
None
294.5 f 1 2 . 5
0
195.9
*
2.8
0
Hc
349.1 f 10.2
32. qb
216.1
f
5.4
28.gb
229.3
187.3 f 11.8 k
6.4
Stimulation b y Ep o r Hc
(Z)
0 40.1
EP
350.2 ?r 13.5
33. Ob
214.6 f 6.1
26.8b
296.0 f 35.8
103.7b
HctEp
346.5 f 14.1
30. gb
210.5
24.8b
365.9 f 45.2
170.4b
None
168.6 f 9.8
-
f
5.7
70.0 f 1.9
-
104.8 f 8.2
-
E p i n e p h r i n e (56.6 v g / k g ) a n d / o r 17-hydroxycorticosterone ( 1 0 0 u g / k g ) w e r e a d m i n i s t e r e d i p 1 0 m i n a f t e r a s i n g l e d o s e o f f l u o r i d e (NaF 3 5 m g / k g , i p ) t o r a t s . T h e r a t s w e r e k i l l e d 3 h o u r s a f t e r a s i n g l e d o s e o f f l u o r i d e o r c h l o r i d e (NaC1 48.7 rng/kg, i p ) as c o n t r o l t o r a t s . V a l u e s a r e a v e r a g e s o b t a i n e d f r o m f o u r r a t s f SE. aEnzyrne a c t i v i t y i s e x p r e s s e d as n m o l e s P i l i b e r a t e d p e r rng p r o t e i n p e r min. b S t i r n u l a t i o n b y Ep a n d / o r Hc:p
219 t h e s e r u m and u r i n e w e r e e x a m i n e d 0-24 h o u r s f o l l o w i n g a s i n g l e i p a d m i n i s S e r u m 17-hydroxycorticosterone w a s f o u n d t o b e
tration o f fluoride.
i n c r e a s e d p r i o r t o an e l e v a t i o n o f s e r u m g l u c o s e f o l l o w i n g f l u o r i d e a d m i n i s t r a t i o n (Fig.
2a).
Moreover,
u r i n a r y 17-hydroxycorticosterone
was i n c r e a s e d b y f l u o r i d e ( F i g .
2b).
excretion
These d a t a i n d i c a t e t h a t a d r e n o c o r -
t i c a l f u n c t i o n was s t i m u l a t e d b y f l u o r i d e . Moreover,
r e g u l a t i o n o f serum g l u c o s e l e v e l b y g l u c o c o r t i c o i d s has been
r e p o r t e d t o b e a s s o c i a t e d w i t h e p i n e p h r i n e (4). hydroxycorticosterone fluoride-treated
and/or
epinephrine
Thus, t h e e f f e c t s o f 17serum glucose
level
in
r a t s w e r e e x a m i n e d a s a c o m p a r i s o n w i t h t h e e f f e c t s on t h e
c o r r e s p o n d i n g changes i n g l u c o s e - 6 - p h o s p h a t a s e kidney.
on
a c t i v i t i e s i n t h e l i v e r and
E p i n e p h r i n e and 17-hydroxycorticosterone c o u l d be r e c o n f i r m e d t o
i n c r e a s e s e r u m g l u c o s e l e v e l a s shown i n T a b l e I V . Glucose-6-phosphatase
a c t i v i t i e s i n t h e l i v e r and k i d n e y were m a r k e d l y
e n h a n c e d b y t h e i n j e c t i o n o f e p i n e p h r i n e a n d 17-hydroxycorticosterone (Table IV).
However,
i n c r e a s e i n enzyme a c t i v i t y b y c o n c o m i t a n t
injection
was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t b y i n j e c t i o n o f e i t h e r d r u g alone. On t h e o t h e r h a n d ,
a n i n c r e a s e i n p l a s m a PTH l e v e l s i s k n o w n t o b e a
w i t h Genu Valgum (16).
c h a r a c t e r i s t i c symptom o f human e n d e m i c f l u o r o s i s Goiter-like
s w e l l i n g o f t h e t h y r o i d o f a d o g was r e p o r t e d t o be i n d u c e d b y
t h y r o x i n e a n d PTH ( 1 8 ) .
Thus,
i n order t o understand t h e r e l a t i o n s h i p
between changes i n t h y r o i d - p a r a t h y r o i d glucose i n f l u o r i d e - t r e a t e d on t h e animals.
rats,
f u n c t i o n and e l e v a t i o n o f serum
thyroid-parathyroidectomy was p e r f o r m e d
T h e r e s u l t s a r e s h o w n i n T a b l e V.
E l e v a t i o n o f serum
g l u c o s e b y f l u o r i d e was n o t s u p p r e s s e d b y thyroid-parathyroidectomy. Changes i n c a l c i u m and cAMP i n t h e administration
livers
and
kidneys a f t e r f l u o r i d e
The mode o f t r a n s m i s s i o n o f t h e membrane-bound a d e n y l a t e c y c l a s e a c t i v i t y i s n o t y e t c l e a r a n d i n some t a r g e t c e l l s t h e a c t i v i t y i s k n o w n t o be m a r k e d l y i n f l u e n c e d b y La2+ i o n s (19).
On t h e o t h e r h a n d ,
known t o s t i m u l a t e a d e n y l a t e c y c l a s e i n v i t r o (20).
Thus,
fluoride i s
t h e changes i n
c a l c i u m and cAMP i n t h e l i v e r and k i d n e y f o l l o w i n g a s i n g l e l a r g e d o s e o f f l u o r i d e were examined.
A s s h o w n i n F i g u r e s 3 a a n d b, r e n a l a n d h e p a t i c
cAMP c o n c e n t r a t i o n s w e r e i n c r e a s e d a p p a r e n t l y p r i o r t o e l e v a t i o n o f t h e c a l c i u m c o n t e n t s i n t h e s e organs.
The e l e v a t i o n o f c a l c i u m c o n c e n t r a t i o n s
i n t h e k i d n e y and l i v e r i n d u c e d b y f l u o r i d e was s i g n i f i c a n t l y s u p p r e s s e d b y
thyroid-parathyroidectomy ( 2 1 )( Ta b l e V I ) .
220
TABLE V EFFECT OF THYROID-PARATHYROIOECTOMY O N ELEVATIONS O F S E R U M AN0 U R I N A R Y GLUCOSE BY A SINGLE LARGE DOSE OF FLUORIDE
Treatment
Control F1 u o r i d e
Treatment
Serum g l u c o s e c o n c e n t r a t i o n ( m g / d l ) a Intact TPTX
71.5 f 13.1 133.6 f 1 1 . 6
Suppression b y TPTX ( % )
85.8 f 9.0 131.4
?
-10.7
14.9
3.9
U r i n a r y g l u c o s e e x c r e t i o n (mg/day/lOOg)b Intact TPTX
Suppression b y TPTX ( % )
Control
0.334 k 0.042
0.423 f 0.040
-26.7
F1u o r i d e
5.359 f 1.711
7.048 f 2.715
-33.6
The r a t s w e r e k i l l e d 3a o r 24b h o u r s a f t e r f l u o r i d e a d m i n i s t r a t i o n (NaF 35 mg/kg, i p ) o r c h l o r i d e a d m i n i s t r a t i o n (NaC1 48.7 mg/kg. i p ) as c o n t r o l t o rats. V a l u e s a r e a v e r a g e s o b t a i n e d f r o m e i g h t r a t s t SE. S u p p r e s s i o n b y TPTX: n o s i g n i f i c a n c e (p>O.O5)
Ca
lL==++dw 0
3
6
12
2.
HOURS AFTER ADMINISTRATION
F i g u r e 3. E f f e c t o f f l u o r i d e a d m i n i s t r a t i o n o n c a l c i u m and cAMP c o n t e n t s i n t h e k i d n e y a n d l i v e r . C h a n g e s i n t h e c a l c i u m a n d cAMP c o n t e n t s w e r e d e t e r m i n e d a t v a r i o u s t i m e s a f t e r a s i n g l e f l u o r i d e a d m i n i s t r a t i o n (NaF, 35 T h e v a l u e s a r e a v e r a g e a n d S.E. (n=6). 0 . c a l c i u m ; 0,CAMP. mg/kg).
. . >
+
z
0
L
C O
L
w o
L O
w v -J w w m L L Q
~
. . .
m m o c u m m o m
w e d
n
n
-
0
n
6
. o. d.
m
0
m o r -
- 7 0 0
. . .
N 0
. o. o.
m
w m m m - c u 0 +I
0
U
0
0
N
0
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. . .
0
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v
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.
-
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0
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+I
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E
40
L 4a, c
u m
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0
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LA a,
m L
>
a,
m a,
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m 3
LA a, 7
m
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a; LA LA
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c, L
LA
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rn
41 LA a,
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7
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m -0
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v
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7
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a, L E
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a, LA LA a,
a
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. -. 7 .
m
0 0 . . 0 0 +I +I N L n
.
I
0
W
. . .
r - - m
+I
0
C
m ? - . . .
r - m e
0 +I
0 +I
6
9
+I +I +I - O N
-
o . m ." m N
. . .
r - 6 L n
m c u e
I
+I
0 ~ 0
.
- 0 6 +I +I +I
7 - r -
c u m -
.
0
rm.
d & G m
I
.
0 +I
* c u e 0 . 0 +I
. . .
a - m N m r f 0
0
0
0
-
-
0
a,
0
LA
c, 8
C
a, c,
Ln
0 +I
. . . 0
N
0
m
0
0 +I
m
0 +I
+I
. . . 0
0 2
b 0 0 +I
I
0
. r. - . m
r m c u
0
0
V
x
. . .
0
-
mcu c u m 6
LA
7
L
r
- a , U E v 0 L A O a,oLA L 0 0 V Q
J
x a
C
.- 0 3 x 0 Y
221
222
~
In vitro 1 iv e r
effects
o f f l u o r i d e and PTH o n c a l c i u m - f l u x i n t h e
kidney
and
C o n t r a r y t o e x p e c t a t i o n , t h e h e p a t i c and r e n a l c a l c i u m e f f l u x was n o t s u p p r e s s e d b y e i t h e r f l u o r i d e o r PTH ( T a b l e V I I ) .
Moreover, t h e c a l c i u m
i n f l u x i n r e n a l and h e p a t i c m i t o c h o n d r i a and c y t o s o l s was s i g n i f i c a n t l y d e c r e a s e d b y b o t h f l u o r i d e and PTH, I n t h i s experiment,
as shown i n T a b l e V I I I .
i t w a s f o u n d t h a t e l e v a t i o n o f s e r u m g l u c o s e was
a s s o c i a t e d w i t h enhancement o f g l u c o s e - 6 - p h o s p h a t a s e t h e l i v e r and k i d n e y o f f l u o r i d e - t r e a t e d
a c t i v i t i e s (22-24)
in
r a t s . R e n a l a n d h e p a t i c CAMP
concentrations were increased markedly p r i o r t o t h e fluoride-induced elevat i o n o f serum glucose.
However, e l e v a t i o n o f r e n a l and h e p a t i c c a l c i u m
c o n t e n t s c o u l d n o t b e d e m o n s t r a t e d i n a s s o c i a t i o n w i t h t h e augment o f serum glucose by t h e f l u o r i d e administration.
TABLE V I I EFFECTS OF FLUORIDE AND PARATHORMONE ON CALCIUM-WASHOUT I N RAT KIDNEY 45Ca E f f l u x ( n m o l / g / m i n ) Kidney Control
F-
2TH
Liver
18.75 f 0.53 ( 4 )
16.43 f 0.60 ( 4 )
10-3 M
20.25 k 1.28 ( 4 )
17.40 f 1.28 ( 4 )
lo-‘
M
30.50 f 2 . 7 0 a ( 4 )
20.70 f 1 . 5 0 a ( 4 )
1 pg m l - I
18.75 f 1.35 ( 4 )
19.28 f 0.75 ( 4 )
10 pg m l - I
16.50 f 1.50b(4)
22.35 f 1 . 0 5 a ( 4 )
For experimental details, ee i n t h e t e x t . The r a t e o f c a l c i u m w a s h o u t i s g i v e n as t h e f r a c t i o n o f 4eCa a c t i v i t y l o s t f r o m t h e t i s s u e p e r min. a ’ b S i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l : ap<0.02, bp<0.05
e
0
m 0
e
0
m 0
0 7
X
+I
0
m
L
0
.r
0
. .
0
. .
0 +I
3
Cr-
+I
0
w
0 %
0 +I
0
r - w *
.
w
e
5
0
. w.
N
+I
0
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CO
-V
ue, .0P1a
z
r - m
0
m m m m 0
N
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-
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X 3 F-
+I
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m -
. w.
m
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m r -
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m
mu3
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0 -
m +I
0
m 0
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0
0 7
0
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0
c
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-
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m
0
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m c o 0
c
7
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0
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7
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0
7
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0
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0
7
0 0 - 0.
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0
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0
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e
0
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F
0
m
z z
d d
r-r-
w
W o o 0 +I
0
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0
m r0
. .
+I
0
W c
0
c
W
+I
. .
m
C
c
uw
I
0 C
ar
m -
N
W
I%
m
L
m m
'r
x c
E
0
-
\
U E c \ o m I
u
0
+ 'r
-
X
u
m
I
r
% C
?--
X 3
V
m
I
aJ
%
+
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0
m
7
0 +
x
i,
3
7
Lc Lc
m I 0
c
L 0
+ C 0 U
223
224
REFERENCES 1.
T a y l o r JM, S c o t t JK. M a y n a r d EA. S m i t h FA, Hodge H D ( 1 9 6 1 ) T o x i c o l A p p l Pharmac 3:278-289
2a. S u k e t a Y, Asao Y, Kanamoto Y, Pharmac 80:199-205 2b. S u k e t a Y,
S a k a s h i t a T, Okada S (1985) T o x i c o l A p p l
M i k a m i E, H a y a s h i M (1977) T o x i c o l A p p l Pharmac 39:313-319
3.
E x t o n JH, FrTedmann N, Wong EH, B r i n e a u JP. C o r b i n J B i o l Chem 247:3579-3588
4.
T o l b e r t MEM,
JD,
P a r k CR (1972)
B u t c h e r FR, F a i t JN (1973) J B i o l Chem 248:5686-5692
U i M (1976) Biochem P h a r m a c o l 25:841-845
5.
S a i t o h Y,
6.
H u l t m a n E (1959) N a t u r e 183:108-109
7.
P o r t e r CC, S i l b e r RC ( 1 9 5 0 ) J B i o l Chem 1 8 4 : 2 0 1 - 2 0 7
8.
Swanson M A (1955) I n : C o l o w i c k SP, l o g y . Acad Press, New York, V o l 2,
9.
M c G i l b e r t RW ( 1 9 5 5 ) I n : C o l o w i c k SP, K a p l a n NO ( e d s ) , Enzymology. Acad Press, New York. V o l 2, p p 543-546
K a p l a n NO (eds) Methods i n EnzymoFP 541-543
10.
B a l l a r d FJ, Hanson RW (1969) J B i o l Chem 244:5625-5630
11.
M e y e r F, H e i l m e y e r J r LMG, H a s c h k e RJ, 245: 6642-6648
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W a l l a c e J.
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W i l l i s JB (1960) S p e c t r o c h i m A c t a 16:259-272
14.
L o w r y OH, R o s e b r o u g h NJ, 193: 265-275
15.
J d r g e n s e n PL (1968) B i o c h i m B l o p h y s A c t a 151:212-224
16.
K r i s h n a m a c h a r i KAVR,
17.
McLaren JR (1976) F l u o r i d e 9:105-116
18.
Ashmore J, Weber G (1959) V a t a m i n e s Hormones 17:91-132
19.
Bradshaw RA,
20.
R a l l TW. S u t h e r l a n d EM (1958) J B i o l Chem 232:1065-1076
P i n t a d o E,
Methods i n
F i s c h e r E H ( 1 9 7 0 ) J B l o l Chem
S c a r p a A (1983) F E B S L e t t e r s 151:83-88 F a r r AF.
R a n d a l l RJ ( 1 9 5 1 ) J B i o l Chem
S i v k u m a r B (1976) F l u o r i d e 9:185-200
F r a z i e r WA (1977) C u r r Top C e l l Regul 1 2 : l - 3 7
21.
S u k e t a Y,
Kanamoto Y (1983) T o x ~ c o l o g y26:335-345
22.
S u k e t a Y,
S a t o M (1980) T o x i c o l A p p l Pharmac 52:386-390
23.
S u k e t a Y,
S a t o M.
24.
S u k e t a Y,
Yamada M, Hasegawa J, Asao Y (1982) M o l Pharmac 22:116-120
K u r a M (1980) E x p e r i e n t i a 36:438-439
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 225-229 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
225
FLUORIDE AND CALCIUM INTERRELATIONSHIP I N MATERNAL AND CORD BLOOD SERA MOT00 NIWA, JUN TAKIMOTO, AND TATSUHIKO TSUJI D e p a r t m e n t o f P r e v e n t i v e and C o m m u n i t y D e n t i s t r y , N i p p o n D e n t a l U n i v e r s i t y , F u j i m i , Chiyoda-ku,
T o k y o 102, J a p a n
ABSTRACT M a t e r n a l and c o r d s e r u m p a i r s w e r e c o l l e c t e d f r o m 121 d e l i v e r i e s . m a t e r n a l t o t a l s e r u m F was f o u n d t o b e 0.072+0.051 t o t a l s e r u m F was 0.055+0.028
ug/ml.
ug/ml.
The
while the fetal
T o t a l F l e v e l s i n m a t e r n a l serum were
f o u n d t o be s i g n i f i c a n t l y h i g h e r t h a n t h o s e i n c o r d serum,
but the differ-
e n c e i n i o n i c F l e v e l s b e t w e e n m a t e r n a l and c o r d s e r a was n o t s t a t i s t i c a l l y significant.
On t h e o t h e r hand,
t h e t o t a l and i o n i c Ca l e v e l s i n m a t e r n a l
serum w e r e f o u n d t o be s i g n i f i c a n t l y l o w e r t h a n t h o s e i n c o r d serum.
A
p o s i t i v e c o r r e l a t i o n was f o u n d b e t w e e n t h e i o n i c F l e v e l s i n m a t e r n a l and c o r d sera.
M a t e r n a l i o n i c Ca l e v e l s c o r r e l a t e d s i g n i f i c a n t l y a n d p o s i -
t i v e l y w i t h t h o s e i n c o r d serum.
No s i g n i f i c a n t c o r r e l a t i o n w a s f o u n d
b e t w e e n Ca and F i n m a t e r n a l and c o r d sera. t h e concept o f a placental barrier.
These f i n d i n g s d o n o t s u p p o r t
I o n i c F found i n t h e serum i n d i c a t e s
t h a t t h e r e i s no d i s e q u i l i b r a t i o n o f i o n i c F i n m a t e r n a l a n d f e t a l c i r c u l a tions.
INTRODUCTION I n o r d e r t o i n c r e a s e t h e knowledge o f t h e pharmacokinetics o f f l u o r i d e
( F ) i n t h e body, f e t u s (1). determined
i t i s i m p o r t a n t t o s t u d y t h e t r a n s f e r o f F from mother t o
However, because o f l a c k o f samples, o n l y a few a u t h o r s have
F l e v e l s i n human m a t e r n a l and c o r d b l o o d s e r a .
The e x a c t
q u a n t i t a t i v e r e l a t i o n s h i p b e t w e e n m a t e r n a l and c o r d b l o o d F l e v e l s r e m a i n s unknown
(2. 3).
S i n c e il number m a t e r n a l and c o r d b l o o d s a m p l e s w e r e s i m u l -
taneously obtained.
t h e i r i o n i c and t o t a l F l e v e l s were studied.
t i o n , t h e l e v e l s o f c a l c i u m (Ca),
I n addi-
w h i c h h a s a h i g h a f f i n i t y f o r F, w e r e
a l s o determined.
MATERIALS AND METHODS I n c l u d e d i n t h e p r e s e n t s t u d y w e r e p r e g n a n t wcmon who d e l i v e r e d a t an OB-
G Y N h o s p i t a l i n Tokyo, Japan, b e t w e e n 1 9 8 2 a n d 1984. lected durlng parturition;
C o r d b l o o d was c o l -
m a t e r n a l b l o o d was w i t h d r a w n a s soon a s p o s s i b l e
226 after parturition.
B o t h b l o o d s a m p l e s w e r e o b t a i n e d f r o m 92 women.
Ionic
and t o t a l F l e v e l s i n b l o o d s e r u m w e r e d e t e r m i n e d b y t h e F e l e c t r o d e m e t h o d and t h e A1F m o l e c u l a r a b s o r p t i o n m e t h o d (4),
respectively.
I o n i c and t o t a l
Ca l e v e l s i n b l o o d s e r a w e r e d e t e r m i n e d b y t h e Ca e l e c t r o d e m e t h o d and b y molecular absorption analysis,
respectively.
RESULTS AND D I S C U S S I O N I o n i c and t o t a l F l e v e l s i n b o t h m a t e r n a l and c o r d b l o o d p r o d u c e d a l m o s t r e g u l a r frequency d i s t r i b u t i o n patterns.
Mean i o n i c F l e v e l s w e r e compar-
a b l e i n m a t e r n a l and c o r d b l o o d s e r a , w h i l e t o t a l F l e v e l s i n c o r d b l o o d s e r u m w e r e s i g n i f i c a n t l y l o w e r t h a n t h o s e i n m a t e r n a l b l o o d serum.
TABLE I
AND CALCIUM
FLUORIDE
LEVELS IN MATERNAL AND CORD SERA (n=92)' M a t e r n a l Serum
0.036
I o n i c f l u o r i d e (ppm)
f
0.004
P
C o r d Serum
0.035
f
0.004
NS
0.028
~0.01
T o t a l f l u o r i d e (ppm)
0.070
*
0.040
0.055
I o n i c c a l c i u m (mM)
0.944
k
0.062
0.965
*
0.088
NS
T o t a l c a l c i u m (mM)
2.585 f 0.177
2.939
f
0.148
<0.001
' V a l u e s a r e means f
S.D.
f
NS: ~ 0 . 0 5
TABLE I 1 CONCENTRATIONS Feto-maternal
OF I O N I C
FLUORIDE I N 1,lATERNAL AND CORD SERA
rate'
7
Frequency (%)
-
85
85
-
90
9
9.8
90
-
95
15
7.6
14
16.3 15.2
-
47
51.0
Total
92
99.9
95 - 1 0 0 100
1
n
I o n i c F i n c o r d serum I o n i c F i n m a t e r n a l serum
100 ( % )
48.9
Ca
227 l e v e l s i n c o r d b l o o d serum were s i g n i f i c a n t l y h i g h e r t h a n t h o s e i n m a t e r n a l blood serum (Table I). I o n i c F l e v e l s i n c o r d b l o o d serum were l o w e r than t h o s e i n m a t e r n a l b l o o d s e r u m i n 48.9% o f t h e s a m p l e s ,
and t h e y w e r e e q u a l
t o o r h i g h e r t h a n t h o s e i n m a t e r n a l b l o o d s e r u m i n t h e r e m a i n i n g 51.1%. T o t a l F l e v e l s were l o w e r i n c o r d b l o o d serum t h a n i n m a t e r n a l b l o o d serum i n 64.2% o f t h e s a m p l e s a n d h i g h e r i n t h e r e m a i n i n g 35.8%. the rates o f transfer, therefore, f e t u s more e a s i l y than t o t a l
F
Judging from
i o n i c F seems t o b e t r a n s f e r r e d t o t h e
( T a b l e s I 1 a n d 111). I n o r d e r t o e v a l u a t e
t h e c o r r e l a t i o n b e t w e e n F a n d Ca l e v e l s i n m a t e r n a l a n d c o r d b l o o d s e r a , t h e c o r r e l a t i o n c o e f f i c i e n t w a s d e t e r m i n e d f o r i o n i c a n d t o t a l F a n d Ca levels. (Figs.
A s i g n i f i c a n t c o r r e l a t i o n was f o u n d b e t w e e n i o n i c F and Ca l e v e l s 1 a n d 2).
/ . . ... .
-t ga 0011
r=0.59 ( p < O . O l ) Y=O.49X+O.O17
. . -.. .
. - . . .-..
v
. ..
2
003
V
1/o 0 2
('0
0 03
0 04
0 05
M a t e r n a l serum (ppm) F i g u r e 1. S c a t t e r d i a g r a m and r e g r e s s i o n l i n e o f i o n i c f l u o r i d e c o n c e n t r a t i o n s i n c o r d and m a t e r n a l s e r a .
Newborns were grouped by sex i n o r d e r t o d e t e r m i n e t h e c o r r e l a t i o n c o e f f i c i e n t b e t w e e n s e r u m F l e v e l s and b i r t h w e i g h t . t i o n was found.
However,
No s i g n i f i c a n t c o r r e l a -
a s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n ( r = -0.3,
p4I.01)
was p r e s e n t b e t w e e n m a t e r n a l age and t o t a l F l e v e l s i n c o r d b l o o d
serum,
suggesting a v e r y c l o s e c o r r e l a t i o n between t h e t w o parameters.
228 TABLE 111 CONCENTRATIONS OF TOTAL FLUORIDE I N MATERNAL AND CORD SERA Feto-maternal
r a t e1
-
25
5
5.4
25
-
50
ia
19.6
50
-
75
25
27.2
75 - 1 0 0
11
12.0
-
33
35.
Total
92
100.0
100
1
Frequency (%)
n
T o t a l F i n c o r d serum T o t a l F i n m a t e r n a l serum
. .
a
(z)
100
..
Y=0.64X+0.36
. . : ::: :/
..
64.2
..
Maternal serum (ri
'
F i g u r e 2. S c a t t e r d i a g r a m and r e g r e s s i o n l i n e o f i o n i c c a l c i u m c o n c e n t r a t i o n s i n c o r d and m a t e r n a l sera.
CONCLUSION R e s u l t s f r o m t h e p r e s e n t s t u d y s u g g e s t t h a t w h i l e Ca, w h i c h i s needed f o r f e t a l g r o w t h , was t r a n s f e r r e d t o t h e f e t u s t o a c o n s i d e r a b l e e x t e n t , t h e t r a n s f e r o f F was r a t h e r r e s t r i c t e d .
Furthermore,
organic F levels (which
are probably equal t o t o t a l F l e v e l s minus i o n i c F l e v e l s ) decreased i n c o r d b l o o d s e r u m as c o m p a r e d t o t h o s e i n m a t e r n a l b l o o d serum.
This also
229 suggests a p a r t i a l b l o c k i n g a t t h e p l a c e n t a l b a r r i e r .
I n order t o deter-
mine t h e e x t e n t t o which F i s t r a n s f e r r e d i n t o t h e fetus,
however,
studies
i n v o l v i n g a g r e a t e r number o f s a m p l e s ( i n c l u d i n g d e t e r m i n a t i o n o f p l a c e n t a l
F l e v e l s ) a s w e l l a s a n i m a l s t u d i e s a r e needed. REFERENCES 1.
G e d a l i a I (1979) I n :
2.
A r m s t r o n g WD. 434
3.
Shen YW.
4.
N i s h i d a A.
F l u o r i d e s and Human H e a l t h .
S i n g e r L,
WHO,
Geneva,
p 103
M a k o w s k i E ( 1 9 7 0 ) Am J O b s t e t G y n e c o l 107:432-
T a v e s DR (1974) Am J O b s t e t G y n e c o l 119:205-207 N i w a M (1984) O d o n t o l o g y 72:871-881
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 231-239 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
FLUORIDE T O X I C I T Y :
231
ERYTHROCYTE MEMBRANE ABNORMALITY AND "ECHINOCYTE"
FORMATI O N A.
K.
SUSHEELA AND SURESH K.
JAIN
F l u o r o s i s and F l u o r i d e Research L a b o r a t o r y , D e p a r t m e n t o f Anatomy, I n d i a I n s t i t u t e o f M e d i c a l S c i e n c e s , New D e l h i - 1 1 0 029, I n d i a
A1 1
ABSTRACT I n g e s t i o n o f f l u o r i d e i n excess leads t o i t s accumulation i n t h e e r y t h r o c y t e membrane.
The b i o c h e m i c a l and s t r u c t u r a l a b n o r m a l i t i e s d e t e c t e d a r e
r e p o r t e d i n t h i s communication. B l o o d s a m p l e s o f r a b b i t s a d m i n i s t e r e d w i t h 1 0 mg NaF/kg b o d y w e i g h t a t 24 hour i n t e r v a l s f o r v a r y i n g d u r a t i o n s were investigated.
Human s u b j e c t s
a f f l i c t e d w i t h h y d r o f l u o r o s i s were a l s o investigated. F l u o r i d e and c a l c i u m c o n t e n t s o f serum and e r y t h r o c y t e membrane w e r e e s t i m a t e d u s i n g an i o n s e l e c t i v e e l e c t r o d e and a n a t o m i c a b s o r p t i o n spectrometer respectively.
The e r y t h r o c y t e m o r p h o l o g y was s t u d i e d u s i n g a
scanning e l e c t r o n microscope. The r e s u l t s i n d i c a t e t h a t f l u o r i d e a c c u m u l a t i o n i n t h e e r y t h r o c y t e memb r a n e l e d t o d e p l e t i o n o f c a l c i u m i n t h e membrane.
Erythrocytes w i t h
s p i n o u s p r o j e c t i o n s w e r e a t a maximum o f 7% i n human s u b j e c t s .
However,
t h e number i n t h e r a b b i t r o s e f r o m 1% t o 9% a s t h e d u r a t i o n o f e x p o s u r e t o f l u o r i d e was i n c r e a s e d f r o m 1-20
months.
I t was a l s o e v i d e n t t h a t o l d e r
e r y t h r o c y t e s were more s e r i o u s l y a f f e c t e d t h a n t h e n e w l y formed e r y t h r o c y t e s as f l u o r i d e a c c u m u l a t i o n increased. T h i s s t u d y has c o n f i r m e d t h a t f l u o r i d e i s one o f t h e c h e m i c a l a g e n t s responsible f o r inducing echinocyte formation. INTRODUCTION Susheela e t al.
(1) have r e p o r t e d an a c c u m u l a t i o n o f f l u o r i d e i n t h e
e r y t h r o c y t e membrane i n r a b b i t s t h a t had i n g e s t e d e x c e s s f l u o r i d e .
Changes
h a v e been r e p o r t e d i n t h e h e m a t o l o g i c a l p r o f i l e o f r a b b i t s a s a r e s u l t o f excess f l u o r i d e ingestion. ingestion.
Anemia a l s o occurred as a r e s u l t o f f l u o r i d e
The c o n c l u s i o n s d r a w n w e r e b a s e d o n s t u d i e s c o n d u c t e d u s i n g 10,
30 and 50 mg d o s e s o f f l u o r i d e a d m i n i s t e r e d t o r a b b i t s f o r f i v e m o n t h s (2). I n f o r m a t i o n o n b l o o d c e l l p r o f i l e was f u r t h e r augmented b y s t u d i e s cond u c t e d b y S u s h e e l a a n d J a i n (3).
According t o these i n v e s t i g a t o r s t h e
e r y t h r o c y t e and l e u c o c y t e p o p u l a t i o n r e v e a l e d a s i g n i f i c a n t r e d u c t i o n a f t e r
6 and 1 2 m o n t h s o f f l u o r i d e i n g e s t i o n . However,
t h o s e a n i m a l s t h a t w e r e on
232 NaF f o r 7 , m o n t h s p r o d u c e d o f f s p r i n g w h i c h d i d n o t r e v e a l a n y d e v i a t i o n e i t h e r i n e r y t h r o c y t e o r leucocyte population.
Basophils.
lymphocytes and
hemoglobin c o n t e n t were a l s o s i g n i f i c a n t l y reduced i n b o t h t r e a t e d a n i m a l s and t h e i r o f f s p r i n g .
The r e d u c t i o n i n b l o o d c e l l c o u n t and h e m o g l o b i n
c o n t e n t was c o n s i d e r e d as a r e s u l t o f a d r e n a l c o r t i c a l m a l f u n c t i o n .
Rao
and S u s h e e l a ( 4 ) h a v e r e p o r t e d e a r l i e r t h a t h y p e r t r o p h y o f t h e a d r e n a l g l a n d and r e d u c t i o n i n d e l t a 5,3
b e t a s t e r o i d dehydrogenase a c t i v i t y was
due t o e x c e s s i v e f l u o r i d e i n g e s t i o n . The p r e s e n t communication r e p o r t s t h a t e r y t h r o c y t e membrane a b n o r m a l i t y o c c u r s i n human s u b j e c t s a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s and i n r a b b i t s t h a t i n g e s t e x c e s s i v e amounts o f f l u o r i d e . MATERIALS AN0 METHODS Human S u b j e c t s A f f l i c t e d w i t h S k e l e t a l F l u o r o s i s B l o o d a n d u r i n a r y s a m p l e s w e r e c o l l e c t e d f r o m t w o p a t i e n t s ( A and B) a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s diagnosed r a d i o l o g i c a l l y and t h e b l o o d samples were used f o r s t u d i e s on e r y t h r o c y t e morphology u s i n g SEM. R a b b i t Model Male a l b i n o r a b b i t s w e i g h i n g 750-1000 g each were k e p t under l a b o r a t o r y conditions,
segregated a t random i n t o t w o groups,
and f e d a balanced d i e t
o b t a i n e d f r o m H i n d u s t a n Lever (Bombay) and w a t e r ad l i b i t u m .
The a n i m a l s
i n one group were g i v e n o r a l l y 10 mg o f NaF/kg body w e i g h t one dose d a i l y f o r a p e r i o d o f 20 months. sex-matched c o n t r o l s .
A n i m a l s i n t h e s e c o n d g r o u p s e r v e d as age and
B l o o d was d r a w n a t i n t e r v a l s o f 1,
months a f t e r f l u o r i d e t r e a t m e n t .
3,
9 and 20
Blood samples f r o m c o n t r o l a n i m a l s were
a l s o c o l l e c t e d a t t h e same i n t e r v a l s .
The samples were used f o r scanning
e l e c t r o n m i c r o s c o p i c s t u d i e s o f e r y t h r o c y t e s , serum f l u o r i d e c o n t e n t and plasma c a l c i u m c o n t e n t d e t e r m i n a t i o n s . Scanning E l e c t r o n M i c r o s c o p i c S t u d i e s o f E r y t h r o c y t e s Blood samples f r o m humans and r a b b i t s were t r a n s f e r r e d i n t o c o l d b u f f e r e d (pH 7.4)
g l u t e r a l d e h y d e (2%) f o r 45 minutes.
washed t h r e e t i m e s i n c o l d p h o s p h a t e
After fixation,
b u f f e r (pH 7.4) (5). A t h i n f i l m o f
t h e m a t e r i a l t h u s o b t a i n e d was p l a c e d o n a s m a l l s t u b , s p u t t e r c o a t e d w i t h gold.
samples were a i r d r i e d and
The c e l l s were scanned a t 30 K V u s i n g a P h i l i p s
Scanning E l e c t r o n Microscope 501. C o u n t i n g o f Abnormal E r y t h r o c y t e s
A m i n i m u m o f 1 1 and a maximum o f 300 c e l l s f r o m a n u m b e r o f s p e c i m e n s o b t a i n e d f r o m t h e 2 p a t i e n t s i n v e s t i g a t e d were counted on t h e t e l e v i s i o n s c r e e n o f t h e s c a n n i n g e l e c t r o n m i c r o s c o p e a t a m a g n i f i c a t i o n o f 640. F u r t h e r , a minimum o f 78 and a maximum o f 310 c e l l s f r o m a number o f s t u b s
233 obtained from t h e experimental animals ( f o r d e t a i l s r e f e r Table I ) were a l s o counted.
A b n o r m a l c e l l s among t h e t o t a l p o p u l a t i o n o f c e l l s w e r e
s c o r e d and t h e p e r c e n t a g e o f a b n o r m a l c e l l s was c a l c u l a t e d s i m u l t a n e o u s l y .
A s e r i e s o f p h o t o g r a p h s w e r e t a k e n f r o m t h e s a m e f i e l d s a t t h e s a m e magn i f i c a t i o n ( 6 4 0 X),
and c e l l s w e r e c o u n t e d f r o m t h e p h o t o g r a p h s t o c o n f i r m
t h e o b s e r v a t i o n s made on t h e s c r e e n . Serum F l u o r i d e D e t e r m i n a t i o n (Human and R a b b i t ) Serum f l u o r i d e was d e t e r m i n e d b y u s i n g a pH m e t e r m o d e l PHM 8 4 ( R a d i o meter,
Copenhagen) w i t h F- s p e c i f i c e l e c t r o d e s a s d e s c r i b e d b y H a l l
&
d.
(6). Plasma C a l c i u m E s t i m a t i o n ( R a b b i t ) T o t a l c a l c i u m f r o m t h e p l a s m a o f t h e r a b b i t was d e t e r m i n e d w i t h a Pye Unicam A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t e r B a r k e r gt
d.(7).
M o d e l SP-900
as described by
The p r e c i s i o n was c h e c k e d b y i n t e r n a l s t a n d a r d s .
The
p l a s m a c a l c i u m i o n c o n c e n t r a t i o n o f r a b b i t w a s e s t i m a t e d b y u s i n g a pH meter
model
PHM 8 4
(Radiometer,
Copenhagen)
with
calcium-specific
e l e c t r o d e s b y a p p l y i n g t h e m e t h o d o f H u l a n i c k i a n d T r o j a n o w i c z (8).
The
g l a s s w a r e and s a m p l i n g b o t t l e s w e r e c l e a n e d i n h o t n i t r i c a c i d t o r e m o v e a l l possible contamination. RESULTS E r y t h r o c y t e Morphology E r y t h r o c y t e s f r o m human s u b j e c t s a f f l i c t e d w i t h f l u o r o s i s and s o d i u m f l u o r i d e t r e a t e d r a b b i t s revealed c e r t a i n s p e c i f i c m o r p h o l o g i c a l changes w h i c h a r e d e p i c t e d i n F i g u r e s la,b,c
and 2a,b,c,
respectively.
It i s c l e a r
f r o m t h e s e e l e c t r o n m i c r o g r a p h s t h a t t h e e a r l i e s t change o b s e r v e d was t h a t t h e o u t e r membrane h a s a w a v y o u t l i n e ( F i g s .
la,
2a).
The c r e n a t i o n o f t h e
c e l l s enhanced t o t h e e x t e n t t h a t p r o j e c t i o n s f r o m t h e s u r f a c e o f t h e e r y t h r o c y t e i n a l l d i r e c t i o n s appeared as l a r g e spinous processes (Figs. 1 b . c a n d 2b,c).
B l o o d s a m p l e s o b t a i n e d f r o m n o r m a l human s u b j e c t s and
c o n t r o l r a b b i t s d i d n o t r e v e a l any such a b n o r m a l i t y . The human s u b j e c t s i n v e s t i g a t e d had s e r u m f l u o r i d e c o n t e n t s r a n g i n g f r o m 0.12
t o 0.2
ppm ( n o r m a l v a l u e :
0.02
ppm) and u r i n a r y f l u o r i d e r a n g i n g f r o m
1.7 t o 2.8 ppm a n d t h e n u m b e r o f a b n o r m a l e r y t h r o c y t e s w e r e 4.4% a n d 6.7% i n the two subjects investigated. I n rabbits,
t h e p e r c e n t a g e o f e r y t h r o c y t e s w h i c h had undergone such
m o r p h o l o g i c a l changes i n c r e a s e d w i t h d u r a t i o n o f exposure t o f l u o r i d e .
In
T a b l e I i t i s r e v e a l e d t h a t t h e p e r c e n t a g e o f a b n o r m a l e r y t h r o c y t e s was 1.28% i n r a b b i t s t r e a t e d f o r one month,
whereas i n a n i m a l s t r e a t e d f o r 9
a n d 2 0 m o n t h s t h e a b n o r m a l a p p e a r i n g e r y t h r o c y t e s i n c r e a s e d f r o m 6.25% t o
234
F i g u r e la. Scanning e l e c t r o n micrograph o f e r y t h r o c y t e of human p a t i e n t A showing normal e r y t h r o c y t e s ( a ) and t h o s e w i t h c r e n a t e d margin ( X 6300). F i g u r e Ib. Scanning e l e c t r o n micrograph o f e r y t h r o c y t e o f human p a t i e n t B s h o w i n g a n o r m a l e r y t h r o c y t e and a n o t h e r w i t h wavy o u t l i n e and s p i n o u s membrane o u t g r o w t h s ( X 13125). F i g u r e Ic. Scanning e l e c t r o n micrograph o f e r y t h r o c y t e o f human p a t i e n t B showing a l a r g e number o f s p i n o u s membrane o u t g r o w t h s ( X 9800).
235
F i g u r e 2a. S c a n n i n g e l e c t r o n m i c r o g r a p h o f e r y t h r o c y t e o f r a b b i t t r e a t e d w i t h NaF f o r 9 m o n t h s ( X 15750). N o t e t h e wavy m a r g i n o f e r y t h r o c y t e . F i g u r e s 2b,c. Scanning e l e c t r o n micrographs o f e r y t h r o c y t e s o f r a b b i t t r e a t e d w i t h NaF f o r 1 9 m o n t h s ( X 17500). N o t e t h e m e m b r a n e a b n o r m a l i t y l e a d i n g t o spinous p r o j e c t i o n .
236
9.0%. I t i s a l s o e v i d e n t t h a t t h e s e r u m f l u o r i d e c o n t e n t i n c r e a s e d f r o m 0.09 ppm i n n o r m a l a n i m a l s t o 0.33 ppm and 0.78 ppm o v e r a p e r i o d o f 1-20 months o f NaF t r e a t m e n t .
The number o f abnormal e r y t h r o c y t e s i n c r e a s e d as
c i r c u l a t i n g l e v e l s o f f l u o r i d e increased. The t o t a l and i o n i c c a l c i u m c o n t e n t s o f t h e plasma i n r a b b i t s i n c r e a s e d s i g n i f i c a n t l y (P<0.05) ( T a b l e 11) a f t e r f l u o r i d e i n g e s t i o n f o r 9 months.
TABLE I
SERUM FLUORIDE CONTENT AND PERCENTAGE OF ABNORMAL ERYTHROCYTE POPULATION I N HUMAN SUBJECT AFFLICTED WITH FLUOROSIS AND I N R A B B I T S EXPOSED TO NaF FOR VARIOUS TIME INTERVALS Serum Flevels ( PPm)
Duration o f exposure t o NaF
Percentage o f erythrocytes with altered morphology
Number o f s t u b s examined w i t h r e f e r e n c e t o number o f human/animals s t u d i e d
Human S u b j e c t s P a t i e n t A (35 y r )
0.2
6.7%
3 stubs/patient A
Patient 6 (51 y r )
0.12
4.4%
3 stubs/patient B
Normal
0.09
-_-_-
4 stubs/2 animals
1 month
0.33
I . 28%
4 stubs/2 animals
9 months
0.63
6.25%
6 stubs/3 animals
20 months
0.78
9.0%
6 stubs/2 animals
Rabbits
TABLE I 1
TOTAL
AND I O N I C CALCIUM CONCENTRATIONS (PPM) I N PLASMA T o t a l Calcium PPm (n=5)
OF
RABBITS
I o n i c Calcium PPm (n=4)
Normal R a b b i t s
61.6
f
6.7'
16.35
?
1.48
Rabbits t r e a t e d w i t h NaF f o r 9 months
76.3
f
5.3
18.52
?
1.22 (p<0.05)
'Values a r e means
*
S.D.
237 DISCUSSION The m o r p h o l o g i c a l changes o f e r y t h r o c y t e s i n human s u b j e c t s a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s and t h e e x p e r i m e n t a l f l u o r o s i s i n r a b b i t s r e p o r t e d i n t h e p r e s e n t s t u d y b e a r c o n s i d e r a b l e s i m i l a r i t y t o r e d c e l l membrane pathological stages I
-
I 1 r e f e r r e d t o as e c h i n o c y t e I
-
I 1 (9).
I t has
been r e p o r t e d t h a t e c h i n o c y t e f o r m a t i o n can be i n d u c e d by (a) washing o f r e d c e l l s f r e e o f p l a s m a and e x a m i n i n g b e t w e e n g l a s s s l i d e s , factors,
and ( c ) i n t r i n s i c f a c t o r s .
I n t h e p r e s e n t study,
(b) e x t r i n s i c
washing erythro-
c y t e s t o remove p l a s m a and e x a m i n i n g them between g l a s s s l i d e s d i d n o t occur s i n c e t h e experimental procedure i n v o l v e d an e n t i r e l y d i f f e r e n t method.
The e f f e c t o f g l a s s c a n a l s o b e r u l e d o u t a s s u c h c h a n g e s w e r e n o t
o b s e r v e d i n b l o o d s a m p l e s o b t a i n e d f r o m n o r m a l m a t e r i a l and p r o c e s s e d s i m u l t a n e o u s l y i n i d e n t i c a l manner. B e c a u s e o f t h e s c a r c i t y o f human m a t e r i a l ,
only the erythrocytes from
f l u o r o s e d r a b b i t s were f u r t h e r i n v e s t i g a t e d f o r t h e p o s s i b l e involvement o f e x t r i n s i c and i n t r i n s i c f a c t o r s r e s p o n s i b l e f o r p r o d u c i n g e r y t h r o c y t e membrane a b n o r m a l i t y s i m i l a r t o echinocyte I
-
11.
I t i s known t h a t c e r t a i n c h e m i c a l s u b s t a n c e s s u c h a s f a t t y a c i d s , lecithin,
b i l e acids,
barbiturates,
dipyridamole,
lyso-
phenylbutazone,
s a l i c y l a t e a n d r o s e b e n g a l c a n i n d u c e e c h i n o c y t e t r a n s f o r m a t i o n (9.10). According t o Hoffman (lo), a l t e r a t i o n s i n t h e morphology o f t h e e r y t h r o c y t e depend upon a t r a n s m e m b r a n e c o n c e n t r a t i o n g r a d i e n t o f r e s p e c t i v e c h e m i c a l agents.
R e c e n t s t u d i e s h a v e shown t h a t f l u o r i d e p e n e t r a t e s b i o l o g i c a l
membranes a s t h e u n d i s s o c i a t e d a c i d , r a t e i s pH-dependent
stances such as s a l i c y l i c acid,
(11).
hydrogen f l u o r i d e ,
i n b o t h humans and r a t s ,
whose a b s o r p t i o n
as i s t h e c a s e w i t h sub-
a c e t y l s a l i c y l i c a c i d and some b a r b i t u r a t e s
T h i s pH d e p e n d e n t a b s o r p t i o n o f f l u o r i d e t h r o u g h t h e membranes l e a d s
t o t h e f o r m a t i o n o f a t r a n s m e m b r a n e g r a d i e n t (12).
Hence, f l u o r i d e c a n
a l s o b e g r o u p e d w i t h t h o s e a g e n t s w h i c h a r e known t o c a u s e e c h i n o c y t e formation.
I t may a l s o be p o s s i b l e t h a t c e r t a i n i n t r i n s i c f a c t o r s a r e r e s p o n s i b l e f o r t h e o b s e r v e d t r a n s f o r m a t i o n o f e r y t h r o c y t e s r e p o r t e d i n t h i s communication.
Weed a n d C h a i l l e y (13) h a v e s u g g e s t e d t h a t m o r p h o l o g i c a l t r a n s f o r -
mation i s associated with a generalized increase i n permeability t o calcium.
The i n c r e a s e i n p e r m e a b i l i t y t o c a l c i u m i s a t t r i b u t e d t o e n e r g y
depletion o f the erythrocytes.
Susheela and J a i n (14) have r e p o r t e d a
s i g n i f i c a n t decrease i n t h e a c t i v i t i e s o f glucose-6-phosphate
dehydrogenase
and p y r u v a t e k i n a s e i n e r y t h r o c y t e s f r o m r a b b i t s f o l l o w i n g e x c e s s i v e i n t a k e s o f sodium f l u o r i d e .
Reduced e r y t h r o c y t e membrane c a l c i u m c o n t e n t ,
and enhanced plasma and h e m o l y s a t e c a l c i u m l e v e l s have been r e p o r t e d by
238 J a i n a n d S u s h e e l a (15), fluoride toxicity.
s u g g e s t i n g enhanced p e r m e a b i l i t y o f c a l c i u m i o n s i n
The o b s e r v e d e r y t h r o c y t e membrane a b n o r m a l i t y i n human
s u b j e c t s a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s and i n e x p e r i m e n t a l r a b b i t s e x p o s e d t o f l u o r i d e may be a s s o c i a t e d w i t h an i n d i r e c t e f f e c t o f
fluoride
o n i n t r i n s i c f a c t o r s s u c h a s i n c r e a s e d membrane p e r m e a b i l i t y t o c a l c i u m and energy d e p l e t i o n o f erythrocytes.
Therefore,
the present study points out
t h e p o s s i b i l i t y t h a t t h e o b s e r v e d e r y t h r o c y t e membrane a b n o r m a l i t y may h a v e been c a u s e d b y f l u o r i d e and o t h e r c a u s a t i v e f a c t o r s .
However,
i t s h o u l d be
n o t e d t h a t t h e e n t i r e e r y t h r o c y t e p o p u l a t i o n d o e s n o t show t h e a b n o r m a l i t y . I n t h e t w o human s u b j e c t s s t u d i e d t h e p e r c e n t a g e o f a b n o r m a l e r y t h r o c y t e
r a n g e d f r o m 4.4
t o 6.7% w h i l e i n t h e r a b b i t s t h e number r a n g e d f r o m 1.28 t o
9.0% when e x p o s e d t o f l u o r i d e f r o m 1 t o 20 months.
T h i s i s p o s s i b l y due t o
t h e f a c t t h a t e r y t h r o c y t e s a r e known t o h a v e a d e f i n i t e l i f e span.
In
humans e r y t h r o c y t e s , t h i s r a n g e s f r o m 120 t o 130 days, w h e r e a s i n r a b b i t erythrocytes
It t h e r e f o r e emerges t h a t i n o l d e r
i t i s 6 8 days.
e r y t h r o c y t e s t h e f l u o r i d e c o n t e n t i n t h e membrane may b e h i g h e r and m o r e severely affected than t h a t i n newly formed cells.
This explains the
r e a s o n why o n l y a c e r t a i n p e r c e n t a g e o f c e l l s h a v e s h o w n " e c h i n o c y t e " f o r m a t ion. I n conclusion,
i t may be s t a t e d t h a t f l u o r i d e i s one o f t h e c h e m i c a l
substances r e s p o n s i b l e f o r "echinocyte"
formation.
ACKNOWLLDGEMENTS We a r e g r a t e f u l
t o Dr.
T.
Research i n Reproduction (IRR),
C.
Anand Kumar,
Bombay,
e l e c t r o n microscope f o r p r e l i m i n a r y studies, and Dr.
Director,
Institute for
f o r a l l o w i n g us t o use t h e s c a n n i n g and t o
Dr. K a m l a G o p a l k r i s h n a
Ashok Kumar o f I R R f o r t h e i r h e l p d u r i n g t h e SEM s t u d i e s .
thank Mr.
S.
C.
P.
I n d i a I n s t i t u t e o f Medical Sciences (AIIMS), assistance.
We a l s o
Sharma o f t h e e l e c t r o n m i c r o s c o p e l a b o r a t o r y o f t h e A l l New D e l h i .
f o r h i s technical
We a c k n o w l e d g e t h e u s e o f t h e R e g i o n a l S o p h i s t i c a t e d I n s t r u -
mentation Centre (RSIC) f a c i l i t y ,
AIIMS,
w h e r e t h i s s t u d y was c o m p l e t e d .
T h i s i n v e s t i g a t i o n was s u p p o r t e d b y g r a n t s made a v a i l a b l e t o one o f u s
( A K S ) b y t h e D e p a r t m e n t o f E n v i r o n m e n t , Government o f I n d i a , New D e l h i . REFERENCES 1. S u s h e e l a AK. Sharma YD, F l u o r i d e 15(4):173-177
Mohan J,
S i n g h M,
Jagannath
B,
J a i n SK
(1982)
2. H i r a o M (1972) F l u o r i d e 5(1):33-37 3. S u s h e e l a AK,
4. Rao K,
J a i n SK (1983a) B u l l E n v i r o n Contam T o x i c o l 30:388-393
S u s h e e l a A K (1979) F l u o r i d e 12:67-71
239
5. C l a r k e JA, S a l s b u r y AJ (1967) N a t u r e 215:402-404 6. H a l l LL, S m i t h PA, De L o p e z OH, 1458
G a r d n e r DE ( 1 9 7 2 ) C l i n Chem 1 8 : 1 4 5 5 -
7. B a r k e r DH, R e n d e r AC. M i t t a l BM, S h a n b h a g SU, S h a r m a VN, S h a r m a L S (1976) I n : H e m p h i l DD ( e d ) T r a c e S u b s t a n c e s i n E n v i r o n m e n t a l H e a l t h . V o l X, U n i v o f M i s s o u r i , C o l u m b i a 8. H u l a n i c k i A.
T r o j a n o w i c z M (1974) A n a l y t i c a C h i m i c a A c t a 68:155-160
9. M o h a n d a s N. Weed R I , B e s s i s M ( 1 9 8 0 ) I n : T r u m p BF, A r s t i l a A U ( e d s ) P a t h o b i o l o g y o f C e l l Membranes. Academic Press, p p 41-91 10. H o f f m a n
JF
(1972) c f : P a t h o b i o l o g y o f C e l l Membranes,
11. W h i t f o r d CM.
1980
P a s h l e y DH (1984) C a l c i f T i s s u e I n t 36(3):302-307
12. M i l l e r GW. P u s h n i k JC, G i a n n i n i J, B r i s k i n OD (1985) 1 4 t h C o n f e r e n c e o f I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e Research, M o r i o k a 13. Weed R I , C h a i l l e y B ( 1 9 7 3 ) I n : B e s s i s M. Week R I . L e p l o n g PF ( e d s ) Red C e l l Shape, P h y s i o l o g y P a t h o l o g y , U l t r a s t r u c t u r e . S p r i n g e r V e r l a g , p 55 14. S u s h e e l a AK, 15. J a i n SK,
J a i n SK (1983b) I R C S 11:14-15
S u s h e e l a AK B u l l E n v i r o n Contam T o x i c o l ( i n p r e s s )
241
H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 241-251 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
A MODEL FOR SUBCELLULAR
DISTRIBUTION OF
FLUORIDE
I N H I G H E R PLANTS:
RELATION TO I N VITRO CONCENTRATIONS R E Q U I R E D FOR ENZYME I N H I B I T I O N
GENE W.
MILLER, JAMES C.
B i o l o g y Department,
PUSHNIK, AND JOHN G I A N N I N I
Utah State University,
Logan,
UT 84322,
U.S.A.
ABSTRACT P h y s i o l o g i c d a t a o f whole p l a n t response t o e l e v a t e d ambient a i r l e v e l s o f f l u o r i d e w o u l d seem i n c o n s i s t e n t w i t h c o n c e n t r a t i o n s r e q u i r e d t o a f f e c t b i o c h e m i c a l mechanisms i n v i t r o .
Reconciliation o f the apparently higher
-~ i n v i t r o c o n c e n t r a t i o n s and t h e a n t i c i p a t e d l o w e r i n v i v o o b s e r v a t i o n s has been o b t a i n e d by s t e a d y - s t a t e
modeling o f t h e s u b c e l l u l a r p a r t i t i o n i n g o f
f l u o r i d e s i n t o c e l l u l a r f r a c t i o n s d r i v e n by a non-ionic
d i f f u s i o n gradient.
A p p l i c a t i o n o f t h i s p r e d i c t i v e model t o r e p o r t e d l i t e r a t u r e v a l u e s and p h y s i o l o g i c o b s e r v a t i o n s h a s r e s u l t e d i n an u n d e r s t a n d i n g o f t h e sequence o f biochemical a l t e r a t i o n s o f metabolism associated w i t h elevated ambient atmospheric concentrations o f f l u o r i d e .
T h i s m o d e l c a n be a p p l i e d t o o t h e r
e n v i r o n m e n t a l p o l l u t a n t s w h i c h a c t a s weak a c i d - b a s e c o u p l e s and may be u s e d t o p r e d i c t t h e c o n c e n t r a t i o n s o f t h e s e t o x i c compounds a t s u b c e l l u l a r sites.
Such a m o d e l w o u l d p r o v i d e u s e f u l i n f o r m a t i o n i n s t u d y i n g c o n t e m -
p o r a r y problems such as a c i d r a i n .
I n f o r m a t i o n on t h e s u b c e l l u l a r p a r t i -
tioning o f pollutants w i l l help elucidate the i n i t i a l s i t e s o f t o x i c i t y i n t h e o r g a n i s m and a i d i n t h e d e v e l o p m e n t o f r e a l i s t i c a l l o w a b l e
levels of
t h e s e compounds i n t h e e n v i r o n m e n t . FLUORIDE I N THE ENVIRONMENT The p h y t o t o x i c e f f e c t s o f f l u o r i d e on p l a n t s and t h e i r c h e m i s t r y h a s been w e l l documented.
V e g e t a t i o n damage has been a t t r i b u t e d t o i n d u s t r i a l
atmospheric f l u o r i d e e m i s s i o n s f r o m copper smelters, generating plants, w o r k s and g l a s s ,
superphosphate p l a n t s , and c e r a m i c f a c t o r i e s .
coal-fired
electric
aluminum r e f i n i n g plants,
brick-
F o l i a r damage i s c h a r a c t e r i z e d b y
i n i t i a l g e n e r a l l e a f c h l o r o s i s and e v e n t u a l m a r g i n a l n e c r o s i s w i t h p r o l o n g e d exposure.
The a v a i l a b l e d a t a s u g g e s t t h a t f o l i a r n e c r o s i s i n
s u s c e p t i b l e p l a n t s o c c u r s a t a t h r e s h o l d r e s p o n s e o f 40 ppm f l u o r i d e i n t h e tissue,
w h i l e l e s s s e n s i t i v e s p e c i e s do n o t e x h i b i t v i s i b l e symptoms u n t i l
h i g h e r l e v e l s o f a c c u m u l a t i o n o c c u r . A c c o r d i n g t o a N a t i o n a l Academy o f S c i e n c e R e p o r t (NAS),
40 pprn w o u l d r e s u l t f r o m e x p o s u r e t o a v e r a g e a m b i e n t
a i r c o n c e n t r a t i o n s o f g a s e o u s f l u o r i d e s o f 0.5 u g / m 3 f o r a p e r i o d o f 3 0
242 days:
m o r e r a p i d a c c u m u l a t i o n h a s been d e m o n s t r a t e d w i t h h i g h e r e x p o s u r e
l e v e l s (1). Environmental Factors A f f e c t i n q P l a n t Uptake The p r i n c i p a l mode o f f l u o r i d e e n t r y i n t o p l a n t s i s t h r o u g h t h e s t o m a t e s d u r i n g a c t i v e g a s exchange. enter the leaf, opening. tal
S o l u b l e p a r t i c u l a t e forms o f f l u o r i d e can a l s o
b o t h b y d i f f u s i o n t h r o u g h t h e c u t i c l e and v i a t h e s t o m a t a 1
These p a s s i v e a c c u m u l a t i o n s a r e r e s p o n s i v e t o v a r i o u s e n v i r o n m e n -
factors:
1i g h t ,
and p l a n t s p e c i e s .
temperature,
w a t e r balance,
nutritional
status,
age
A l l t h e s e i n t e r a c t i n g f a c t o r s m o d i f y f o l i a r response.
Dark exposure o f Jerusalem c h e r r y l e a v e s t o gaseous f l u o r i d e r e s u l t e d i n l o w e r a c c u m u l a t i o n l e v e l s t h a n l i g h t t r e a t e d c o n t r o l s (2). t r e a t e d p l a n t s d i s p l a y e d no v i s i b l e f o l i a r damage:
however,
The d a r k
when t h e y w e r e
t r a n s f e r r e d t o t h e l i g h t s e v e r e damage r e s u l t e d . Sunflowers accumulated h i g h e r l e v e l s o f f l u o r i d e a t elevated temperature, w h i l e g l a d i o l u s d i s p l a y e d t h e opposite response b u t w i t h g r e a t e r v i s i b l e damage (3).
P o o v a i a h and W i e b e ( 4 ) d e m o n s t r a t e d t h a t s o y b e a n s f u m i g a t e d
w i t h f l u o r i d e d i s p l a y e d i n c r e a s e d v i s i b l e damage a t e l e v a t e d t e m p e r a t u r e even a f t e r t h e t e r m i n a t i o n o f t h e f u m i g a t i o n . P l a n t s g r o w i n g u n d e r w a t e r s t r e s s c o n d i t i o n s a p p e a r t o be m o r e r e s i s t a n t t o f l u o r i d e d a m a g e (5).
b u t t h i s does n o t appear t o be t h e case i f t h e
i m p o s i t i o n o f w a t e r s t r e s s o c c u r s a f t e r f u m i g a t i o n i s t e r m i n a t e d (4). Accumulation o f F l u o r i d e by t h e F o l i a q e o f Higher P l a n t s Once f l u o r i d e h a s p e n e t r a t e d l e a f e n t r y r e s i s t a n c e s ,
it dissolves i n the
s u b s t o m a t a l l i q u i d p h a s e s and moves w i t h t h e t r a n s p i r a t i o n a l s t r e a m t h r o u g h t h e a p o p l a s t t o w a r d t h e l e a f t i p s and margins, accumulation.
the principal sites o f
F l u o r i d e does n o t d i s t r i b u t e e v e n l y a c r o s s t h e l e a f b u t
a p p e a r s t o a f f e c t t h e c e l l s c l o s e s t t o t h e s i t e e n t r y and a l o n g t h e t r a n s p i r a t i o n a l p a t h w a y (6).
G a r r e c gJ
d.( 7 )
employed e l e c t r o n microprobe
a n a l y s i s of f l u o r i d e exposed f i r n e e d l e s and d e m o n s t r a t e d t h a t f l u o r i d e d i s t r i b u t e d i n a steep g r a d i e n t from t h e l e a f t i p d i m i n i s h i n g toward t h e l e a f base.
These i n v e s t i g a t o r s a l s o e s t a b l i s h e d t h a t t h e g r a d i e n t d i s -
p l a y e d t w o peaks,
t h e f i r s t i n t h e l e a f t i p and a s e c o n d a t t h e zone w h i c h
separated t h e n e c r o t i c region from t h e uninjured tissue. C e l l u l a r Location o f F l u o r i d e by F r a c t i o n a t i o n Studies F r a c t i o n a t i o n s t u d i e s on f l u o r i d e exposed t o m a t o p l a n t s r e v e a l e d t h a t f l u o r i d e was f o u n d i n d e c r e a s i n g c o n c e n t r a t i o n s i n t h e c e l l w a l l s , plasts,
soluble proteins,
mitochondria,
and m i c r o s o m e s (8).
chloro-
The h i g h f l u o -
r i d e c o n c e n t r a t i o n a s s o c i a t e d w i t h t h e c e l l w a l l i s i n accordance w i t h t h e known h i g h c a l c i u m c o n c e n t r a t i o n s o f t h a t f r a c t i o n . The a c c u m u l a t i o n i n t h e c h l o r o p l a s t was s u b s t a n t i a t e d b y Chang and Thompson (9) and i s c o n s i s t e n t
243 w i t h known u l t r a s t r u c t u r a l (10.11)
and p h y s i o l o g i c r e s p o n s e s (12).
P h y s i o l o g i c Response t o E l e v a t e d F l u o r i d e L e v e l s The e a r l i e s t m a n i f e s t a t i o n s o f f l u o r i d e e x p o s u r e a r e a l t e r a t i o n s i n t h e r a t e s o f p h o t o s y n t h e s i s and r e s p i r a t i o n .
I n v i v o s t u d i e s h a v e shown t h a t
p h o t o s y n t h e t i c and r e s p i r a t o r y a c t i v i t i e s r e s p o n d m o s t r a p i d l y t o f l u o r i d e e x p o s u r e a s q u i c k l y a s 2 h o u r s a f t e r f u m i g a t i o n w i t h 4 0 p p b HF. p r o c e s s e s a r e s i g n i f i c a n t l y a l t e r e d (13).
These
R e c e n t s t u d i e s on t h e e f f e c t s o f
f l u o r i d e on t h e b i o c h e m i s t r y o f c h l o r o p l a s t s h a v e c l e a r l y d e m o n s t r a t e d t h a t t h e i n h i b i t i o n i s a s s o c i a t e d w i t h t h e a c t i o n o f t h e F i o n on t h e photosynthetic
c o u p l i n g f a c t o r (ATPase)
(14;
Table
I).
I n contrast t o the
observed f l u o r i d e i n h i b i t i o n o f photosynthetic activities, mitochondrial r e s p i r a t i o n i s i n i t i a l l y enhanced b u t w i t h c o n t i n u e d e x p o s u r e i n h i b i t i o n o f o x y g e n c o n s u m p t i o n i s s e e n (15,16).
While decreased t i s s u e . r e s p i r a t i o n i s
g e n e r a l l y a t t r i b u t e d t o i n h i b i t i o n o f r e s p i r a t o r y enzymes,
t h e reason f o r
t h e i n i t i a l s t i m u l a t i o n i s somewhat obscure. M e t a b o l i c s t u d i e s show v a r y i n g d e g r e e s o f f l u o r i d e s u s c e p t i b i l i t y o f enzymes.
Cytoplasmic
enzymes,
such
as
enolase
(17)
and
phospho-
g l u c o m u t a s e (18) d i s p l a y s e n s i t i v i t i e s t o l o w l e v e l s o f s o l u b l e f l u o r i d e ( 5
mM).
B i o e n e r g e t i c w o r k h a s shown t h a t t h e m i t o c h o n d r i a l ( 1 5 ) and c h l o r o -
p l a s t l o c a l i z e d ATPases ( 1 9 ) a r e i n h i b i t e d a t a p p a r e n t l y h i g h e r c o n c e n t r a t i o n s o f f l u o r i d e (30 mM; T a b l e I ) . enzymes (dehydrogenases),
Some o f t h e m i t o c h o n d r i a l b o u n d
however, a r e i n h i b i t e d s i g n i f i c a n t l y a t l o w e r
f l u o r i d e c o n c e n t r a t i o n s ( T a b l e I). T h e t o n o p l a s t a s s o c i a t e d ATPase d i s p l a y s a s e n s i t i v i t y t o f l u o r i d e s i m i l a r t o t h e m i t o c h o n d r i a l ATPase (20). b u t t h e m e m b r a n e pH g r a d i e n t f o r m a t i o n i s i n h i b i t e d a t 1 0 m M
(Table I).
The p l a s m a l e m m a l ATPase a p p e a r s t o be s e n s i t i v e t o f l u o r i d e i n h i b i t i o n a t 5
mM ( T a b l e I ) and i s i n h i b i t e d a t t h e l o w e s t c o n c e n t r a t i o n o f a l l membrane bound A T P a s e s ( 2 1 ) . M c N u l t y and L o r d s (22) r e p o r t e d t h a t h i g h e r c o n c e n t r a t i o n s o f p h o s p h o r y l a t e d n u c l e o t i d e s were detected i n h i g h e r p l a n t s t r e a t e d w i t h f l u o r i d e than i n comparable c o n t r o l
plants.
T h e s e a u t h o r s s u g g e s t e d t h a t f l u o r i d e may
d i s r u p t basic c e l l u l a r energetics,
t h u s i n c r e a s i n g t h e oxygen consumption
b y i n c r e a s i n g t h e a m o u n t s o f p h o s p h a t e a c c e p t o r s ( A D P ) o r d o n o r s (ATP). R e v i e w o f t h e r e l e v a n t l i t e r a t u r e shows t h a t b o t h s i t u a t i o n s a r e o b s e r v e d . Yang a n d M i l l e r ( 1 8 ) p r o p o s e d t h a t i n c r e a s e d ADP c o n c e n t r a t i o n s w e r e responsible for
f l u o r i d e s t i m u l a t i o n o f r e s p i r a t i o n i n n e c r o t i c tissues.
M i l l e r and M i l l e r (16)
i n v e s t i g a t e d t h e r e s p i r a t i o n o f f l u o r i d e fumigated
t i s s u e and t h e ATPase a c t i v i t y o f m i t o c h o n d r i a i s o l a t e d f r o m t h i s t i s s u e . T h e s e a u t h o r s f o u n d a good c o r r e l a t i o n among t i s s u e r e s p i r a t i o n , d r i a l respiration,
a n d ATPase a c t i v i t y .
mitochon-
These o b s e r v a t i o n s l e d t o t h e
244 p o s t u l a t i o n t h a t r e s p i r a t o r y s t i m u l a t i o n was c o n t r o l l e d b y t h e r e l a t i v e r a t i o o f c e l l u l a r ADP/ATP.
A l t e r n a t e l y i t was s u g g e s t e d t h a t HF was a c t i n g
a s a c l a s s i c weak u n c o u p l e r o f t h e r e s p i r a t o r y c h a i n (15).
These s t u d i e s
w e r e c o n s i s t e n t w i t h e a r l i e r r e s u l t s o f t h e c o m p a r i s o n o f DNP a n d H F o n r e s p i r a t o r y a c t i v i t y (23).
Ballentyne
(24).
on t h e o t h e r h a n d , r e p o r t e d
i n c r e a s e s i n t h e l e v e l s o f ATP e x t r a c t e d f r o m f l u o r i d e f u m i g a t e d t i s s u e s . Model
o f Fluoride Subcellular Distribution:
Physical Factors
I n an a t t e m p t t o e q u a t e c o n c e n t r a t i o n s o f t i s s u e f l u o r i d e i n v o l v e d i n t h e -~ i n vivo
o b s e r v a t i o n s o f a t m o s p h e r i c f l u o r i d e damage w i t h i n v i t r o e x p e r -
imental levels associated w i t h biochemical alterations, m o d e l o f s u b c e l l u l a r d i s t r i b u t i o n i s proposed. physical properties o f fluoride i n solution:
The m o d e l i s b a s e d o n t h e
HF i s a
and e x i s t s i n t w o f o r m s a t p h y s i o l o g i c a l pH:
a steady-state
weak a c i d (pKa 3.45)
F- and HF.
R e l a t i v e concen-
t r a t i o n s - o f these i o n i c species are mathematically predictable by the Henderson-Hasselbalch e a u a t i o n as f o l l o w s : pH=pKa
+
log [F-]/[HF]
The t w o i o n i c f o r m s e x h i b i t e x t r e m e l y d i f f e r e n t p e r m e a b i l i t y c o e f f i c i e n t s t h r o u g h l i p i d b i l a y e r membranes (25).
The c h a r g e d i o n c a n b e d e m o n s t r a t e d
t o c r o s s a m o d e l membrane ( l e c i t h i n and c h o l e s t e r o l ) b i l a y e r a t 4.9 f 2.3 x IO-”cm/sec,
w h i l e t h e u n d i s s o c i a t e d m o l e c u l e moves a p p r o x i m a t e l y 6 o r d e r s
o f m a g n i t u d e m o r e r a p i d l y (1.4
*
0.3 x 1 0 - 4 c m / s e c ) .
A p r e d i c t a b l e con-
sequence o f t h e s e p r o p e r t i e s i s t h a t a d i f f e r e n t i a l s u b c e l l u l a r d i s t r i b u t i o n w i l l o c c u r b a s e d p r i m a r i l y o n l o c a l i z e d pH g r a d i e n t s w i t h i n t h e c e l l . T h i s d i s t r i b u t i o n w i l l occur as t h e f l u o r i d e t r a n s p i r a t i o n a l route,
moves f r o m t h e a p o p l a s t i c
a c r o s s t h e plasmalemma, and e v e n t u a l l y i n t o t h e
s u b c e l l u l a r compartments.
The movement i s d e p e n d e n t on t h e a t m o s p h e r i c and
t i s s u e f l u o r i d e c o n c e n t r a t i o n s and t h e m a g n i t u d e and d i r e c t i o n o f t h e pH d i f f e r e n c e on t w o s i d e s o f a membrane. Mechanics
o f Fluoride Partitioninq Within the Cell
R e g r e s s i o n m o d e l s have been p r e s e n t e d w h i c h r e l a t e t h e a m b i e n t atmospheric f l u o r i d e concentrations w i t h the r a t e o f f o l i a r accumulation
(1.26).
The d o s e - r a t e
response f o r accumulation o f atmospheric f l u o r i d e i s
described by t h e equation:
AF
= KCT
where K i s t h e accumulation c o e f f i c i e n t ,
C represents t h e concentration of
h y d r o g e n f l u o r i d e i n ug/m3 and T i s t h e t i m e o f e x p o s u r e i n days. The v a l u e
245 o f K h a s b e e n d i f f i c u l t t o e s t a b l i s h f o r i n d i v i d u a l s p e c i e s and w i t h v a r y ing environmental conditions.
R e g r e s s i o n m o d e l v a l u e s h a v e been p r i m a r i l y
d e t e r m i n e d i n e n c l o s e d e n v i r o n m e n t a l chambers i n w h i c h t h e r e d u c t i o n o f a i r movement e s t a b l i s h e s a l a r g e r e s i s t a n c e b o u n d a r y l a y e r and r e s u l t s i n l o w e r a c c u m u l a t i o n t h a n w o u l d b e e x p e c t e d u n d e r f i e l d c o n d i t i o n s (26). result, lower,
As a
t h e K v a l u e s f o r m o s t p l a n t s d e t e r m i n e d i n t h e s e c h a m b e r s w o u l d be t h a n m i g h t be e x p e c t e d ,
under f i e l d conditions.
These l i m i t a t i o n s
i n p r e d i c t i n g a c c u m u l a t i o n r a t e s o f a m b i e n t gaseous hydrogen f l u o r i d e a r e recognized,
b u t a c c u m u l a t i o n s o f f l u o r i d e i n t h e v a r i o u s s u b c e l l u l a r com-
p a r t m e n t s r e s u l t f r o m t h e r e s i s t a n c e s e n c o u n t e r e d d u r i n g t h e p a s s i v e movement o f f l u o r i d e i n t h e t r a n s p i r a t i o n a l f l o w t o w a r d t h e l e a f margin. The f i r s t m a j o r r e s i s t a n c e b a r r i e r t o c e l l u l a r f l u o r i d e a c c u m u l a t i o n imposed by t h e l e a f i s t h e c e l l wall. g u i s h a b l e spaces; I n t h e WFS,
Cell walls consist o f two distin-
a w a t e r f r e e s p a c e (WFS)
and t h e Donnan f r e e s p a c e (DFS).
c a t i o n s and a n i o n s a r e i n r o u g h l y e q u a l c o n c e n t r a t i o n s ,
while
t h e DFS c o n t a i n s f i x e d n e g a t i v e c h a r g e s and t e n d s t o p r o m o t e a n i o n e x c l u sion. (27).
C e l l w a l l s a r e v i e w e d as c o n s i s t i n g o f m a c r o p o r e and m i c r o p o r e s p a c e s The f o r m e r i s u n c h a r g e d a n d c o r r e s p o n d s t o t h e WFS.
while the latter
r e p r e s e n t s t h e DFS w h i c h i s h i g h l y c h a r g e d due t o i o n i z a b l e u r o n i c a c i d and p e c t i c substances.
The DFS d i s p l a y s p r e f e r e n t i a l b i n d i n g o f d i v a l e n t o v e r
monovalent ions.
Sequestration o f ions by t h e c e l l w a l l i s p r i m a r i l y
e l e c t r o s t a t i c i n nature.
C a l c i u m i s o r d i n a r i l y t h e m a j o r c a t i o n bound i n
the c e l l w a l l i n t h e form o f calcium-pectate.
Concentration o f t h i s ion
v a r i e s w i t h p l a n t s p e c i e s and may be a c o n t r i b u t i n g f a c t o r
i n the relative
degree o f s u s c e p t i b i l i t y t o f l u o r i d e t o x i c i t y .
As a t m o s p h e r i c f l u o r i d e f o l l o w s t h e t r a n s p i r a t i o n a l
movement t o w a r d t h e
l e a f m a r g i n t h r o u g h t h e macropore space i n t h e c e l l w a l l ,
i t may b e g i n t o
p a r t i t i o n i n t o t h e m i c r o p o r e s p a c e s w h i c h h a v e become c h a r g e c o m p e n s a t e d
As t h e f l u o r i d e
concentration
i n c r e a s e s i n t h e m i c r o p o r e space, f l u o r i d e w i l l d i s p l a c e Ca”
from the c e l l
due t o accumulated c a l c i u m (Fig. wall.
This displacement
s t r e n g t h s o f t h e CaF2 vs. s o l u b i l i t y products. bound.
1).
i s due t o t h e
Ca p e c t a t e
large difference i n binding
as i l l u s t r a t e d
by t h e i r respective
The c e l l w a l l w i l l a c c u m u l a t e F u n t i l a l l t h e Ca”
is
T h i s phenomenon i s a n a l o g o u s t o a c a t i o n e x c h a n g e c o l u m n b i n d i n g
a n i o n s u n t i l t h e a c t i v e s i t e s a r e c o m p l e t e l y exhausted.
T h i s phenomenon
might account f o r the d i f f e r e n c e i n the tolerance o f d i f f e r e n t p l a n t s p e c i e s t o t h r e s h o l d l e v e l s of a c c u m u l a t e d f l u o r i d e a s t h e c e l l w a l l c a l c i u m w o u l d be a c t i n g as a b u f f e r a g a i n s t c e l l u l a r f l u o r i d e a c c u m u l a t i o n . Once t h e b u f f e r i n g c a p a c i t y o f t h e c e l l w a l l i s d e p l e t e d b y p r e c i p i t a t i o n a s f l u o r o c o m p l e x e s o f t h e p o l y a n i o n b o u n d d i v a l e n t c a t i o n s ( F i g . 1).
the
246
Pectates
o=c
o= c
o=c
0
0
0
I
I
.Ca
....
o,.-cs
Ca
I
....
Ca
I
c=o
c=o
c
0
I
I
I
c =o
01
I
c=o
F
o= c
o= c
1
no
n ca 0 c
Hb
Ca
0-c
OIC
F-;a
0 I
Ca
Ca
eoe0eo* F
0
P
0
i
F i g u r e 1. T h e i n t e r a c t i o n o f f l u o r i d e w i t h c a l c i u m p e c t a t e . A m o d e l d e p i c t i n g t h e b u f f e r i n g a c t i o n o f t h e c e l l w a l l on f l u o r i d e , t h e d i s p l a c e m e n t o f c a l c i u m f r o m t h e p e c t a t e f r a c t i o n and p r e c i p i t a t i o n o f CaF2. f l u o r i d e i o n encounters t h e second b a r r i e r t o t h e i r accumulation,
the
plasmalemma. The p l a s m a l e m m a i s t h e l i m i t i n g membrane o f t h e c e l l i n s o m u c h a s i t d i s p l a y s s e l e c t i v i t y i n p e r m e a b i l i t y t o substances d i f f u s i n g from t h e exterior.
The p l a s m a l e m m a i s a p r o t e i n and l i p i d c o m p l e x i n w h i c h many o f
t h e p r o t e i n s f u n c t i o n a s t r a n s p o r t m o l e c u l e s f o r t r a n s m e m b r a n e i o n and m e t a b o l i t e movement.
The d r i v i n g f o r c e f o r t h e t r a n s p o r t o f t h e s e m o l e -
c u l e s i s a n a s y m m e t r i c p r o t o n p u m p i n g , ApH, i n w h i c h t h e e x t e r i o r h a s a h i g h e r H+ c o n c e n t r a t i o n .
The movement o f H+ t o t h e e x t e r i o r r e s u l t s i n t h e
l o w e r i n g o f t h e pH o f t h e a p o p l a s t i c s p a c e a d j a c e n t t o t h e plasmalemma. a p p r o x i m a t e l y 5.8.
pH
R e d u c t i o n i n pH i n t h i s r e g i o n o f t h e c e l l w o u l d r e s u l t
i n a m a j o r s h i f t i n t h e f l u o r i d e Henderson-Hasselbalch e q u i l i b r i u m d i s t r i b u t i o n i n f a v o r o f t h e protonated form,
HF.
S i n c e t h e HF f o r m h a s b e e n
demonstrated t o be a p p r o x i m a t e l y 6 o r d e r s o f magnitude more permeable than
241
t h e n o n p r o t o n a t e d c o u n t e r p a r t (28).
a h i g h e r r a t e o f d i f f u s i o n w o u l d be
e x p e c t e d a c r o s s t h e p l a s m a l e m m a ( F i g . 2).
D i f f u s i o n t o w a r d s an a r e a o f
l o w e r c o n c e n t r a t i o n would continue u n t i l e q u i l i b r i u m i s established.
How-
e v e r , t h e pH o f t h e c y t o p l a s m i s m a i n t a i n e d a t 7.2 w h i c h a g a i n s h i f t s t h e H e n d e r s o n - H a s s e l b a l c h e q u i l i b r i u m t o w a r d t h e n o n p r o t o n a t e d f o r m , F-.
Since
t h e HF f o r m i s t h e d i f f u s i n g s p e c i e s and t h e membrane i s r e l a t i v e l y i m p e r m e a b l e t o t h e F- f o r m ,
a n e t a c c u m u l a t i o n i n t h e c e l l w i l l o c c u r d r i v e n by
t h e m a g n i t u d e o f t h e n o n i o n i c d i f f u s i o n g r a d i e n t o f t h e HF f o r m . The m a g n i t u d e o f t h i s g r a d i e n t i s d i r e c t l y d e p e n d e n t o n t h e t r a n s m e m b r a n e pH gradient.
EXTERIOR Space
INTERIOR Space
Membrane F-
HF HF
F
F-
”+
Hi F
HF
H
F-
-
+
H
HF
HF
HF HF
HF
+
F
-
HF
<:+
HF HF
HF
L o w PH
High
pH
F i g u r e 2. H y p o t h e t i c a l model o f f l u o r i d e a c c u m u l a t i o n b y a n o n i o n i c d i f f u s i o n gradient. I n c r e a s e o f p r o t o n s a t t h e e x t e r i o r o f t h e membrane i n d u c e s HF f o r m a t i o n e n h a n c i n g f l u o r i d e t r a n s p o r t a c r o s s t h e membrane. I n t h e cytoplasm t h e accumulated f l u o r i d e would randomly d i s t r i b u t e by free diffusion.
This free diffusion within the c e l l w i l l result i n the
movement o f t h e f l u o r i d e i n t o t h e v a r i o u s s u b c e l l u l a r c o m p a r t m e n t s v i a t h e same p r i n c i p l e s as t h e t r a n s p l a s m a l e m m a movement.
Based o n t h e Henderson-
Hasselbalch c a l c u l a t i o n s t h e two p r i n c i p a l s i t e s of accumulation would appear t o be t h e c h l o r o p l a s t and t h e m i t o c h o n d r i a : e f f e c t s w o u l d a l s o be e x p e c t e d i n t h e t o n o p l a s t and m i c r o s o m a l v e s i c l e s ( F i g .
3).
A c c u m u l a t i o n w i t h i n t h e m i t o c h o n d r i a w o u l d r e s u l t f r o m pH d e p e n d e n t m e c h a n i s m s s i m i l a r t o t h o s e o p e r a t i n g i n t h e plasmalemma. elle,
I n t h i s organ-
a c t i v e r e s p i r a t i o n r e s u l t s i n g e n e r a t i o n o f a t r a n s m e m b r a n e pH g r a -
d i e n t v i a a c t i v e extrusion o f proton,
H+ i o n s , w h i c h a r e u s e d f o r t h e
t r a n s p o r t o f m e t a b o l i t e s a c r o s s t h e membrane and a l s o d r i v e r e s p i r a t o r y
248
Ir
cell wall
5.8
cytoplasm
PH
7 2
l e a l margan
F i g u r e 3. T h e d i f f u s i o n o f f l u o r i d e ( H F ) a c r o s s t h e p l a s m a accumulation i n various organelles o f t h e c e l l . phosphorylation.
membrane and
C y t o p l a s m i c f l u o r i d e ( l i p i d s o l u b l e HF) n e a r t h e o r g a n e l l e
membrane s u r f a c e w o u l d r e a d i l y move i n t o t h e membrane.
Since t h e m a t r i x of
t h e m i t o c h o n d r i a h a s a pH o f a b o u t 7.8 w h e r e F- w o u l d a c c u m u l a t e , a n o n i o n i c d i f f u s i o n g r a d i e n t w o u l d r e s u l t i n t h e one-way movement o f c y t o p l a s m i c HF i n t o t h e m i t o c h o n d r i a 1 m a t r i x r e s u l t i n g i n a c c u m u l a t i o n . The c h l o r o p l a s t r e s p o n s e w o u l d be s i m i l a r i n p r i n c i p l e b u t d i f f e r e n t i n d i r e c t i o n o f t h e p r o t o n movement.
Under l i g h t c o n d i t i o n s photosynthetic
e l e c t r o n t r a n s p o r t moves p r o t o n s f r o m t h e s t r o m a i n t o t h e l u m e n o f t h e t h y l a k o i d r e s u l t i n g i n t h e r e d u c t i o n o f t h e s t r o m a l pH t o a v a l u e g r e a t e r t h a n 8.0.
T h i s r e d u c t i o n i n t h e pH o f t h e c h l o r o p l a s t s t r o m a c r e a t e s a
s i n k f o r t h e p o r t i o n o f t h e c y t o p l a s m i c f l u o r i d e w h i c h i s p r e s e n t i n t h e HF form.
The s t e e p n e s s o f t h i s g r a d i e n t makes t h e c h l o r o p l a s t t h e p r i m a r y
s i t e o f fluoride accumulation w i t h i n the cell. O t h e r c e l l u l a r c o m p o n e n t s w o u l d a l s o be a f f e c t e d b y f l u o r i d e ,
b u t more
l i k e l y f r o m i n h i b i t i o n o f e n z y m e s a s s o c i a t e d w i t h membrane o r m e t a b o l i c functions,
t h a n f r o m a c c u m u l a t i o n (Table I).
example o f t h i s response.
The t o n o p l a s t i s a p r i m e
T o n o p l a s t ATPases a r e s u s p e c t e d t o be l o c a l i z e d
on t h e c y t o p l a s m i c s i d e o f t h e membrane and a r e s e n s i t i v e t o f l u o r i d e . Inhibition o f tonoplast transport
ATPases
or
the
membrane
pH
gradient
f o r m a t i o n w o u l d r e s u l t i n d i s f u n c t i o n ( c h a n g e i n p e r m e a b i l i t y ) and e v e n t u a l b r e a k d o w n o f t h e membrane. The f o r e g o i n g d i s c u s s i o n p r o v i d e s a m o d e l f o r t h e s u b c e l l u l a r p a r t i t i o n o f f l u o r i d e s i n t o t h e v a r i o u s o r g a n e l l e s based o n s t e a d y s t a t e m a t h e m a t i c a l
249
TABLE I FLUORIDE EFFECTS ON ENZYMATIC REACTIONS
Enzymes o r System
Inhibition %
Concentration mM
Reference
Glycolysis Phosphoglucomutase
29
UOP glucose-fructose transglucosylase Enolase
29 94
Respiration Succinic dehydrogenase Malate dehydrogenase Peroxidase Ascorbic acid oxidase Polyphenol oxidase
29 8 60 86 16
10
10
31 31 31 32 32
40
10
14
30
10
14
Photosynthesis PGA-Dependent 0 evolution (chloroplasts? Photosynthetic Phosphorylation, Membrane-Associated
5 1 1 mM: Poi3: 5 mM)
30
(Mg”
10
10
10
ATPases Mitochondria Chloroplast Tonoplast Plasma membrane
30 54 42 40
Other Membrane (Tonoplast) pH gradient formations
24
30 30 30 5 (Mg/ATP. 1 : 1 )
10
15 19 20 21
20
equation. This is summarized in Table I 1 and Figure 3, assuming a pKa of 3.45. The calculated values (Table 11) are based on the equilibrium distribution of fluoride in adjacent cellular compartments induced by pH gradients and calculated using the Henderson-Hasselbalch, equation (e.g., a 25 fold accumulation could be expected between the apoplast and the cyto-
plasm, a 6.3 and 5.0 fold increase i s expected in the chloroplast and the mitochondria, respectively, relative to the cytoplasmic fluoride level).
25 0
TABLE I 1 HENDERSON-HASSELBALCH DISTRIBUTION OF SUBCELLULAR FLUORIDE
Cellular Fraction
PH
Apopl a s t
5.8
Fluoride Concentration (mM) (PPm) 0.1
1.9
Cytoplasm
7.2
2.5
47.5
Chloroplast
8.0
15.7
298.3
Mitochondria
7.8
10.0
190.0
C a l c u l a t i o n s determined u s i n g t h e Henderson-Hasselbalch e q u a t i o n assuming a pKa o f 3.45. F l u o r i d e accumulation process.
i n t h e l i v i n g t i s s u e i s a d y n a m i c and c o n t i n u o u s
Accumulation o f atmospheric f l u o r i d e i n the f o l i a g e o f higher
plants i s subjected t o various environmental conditions, earlier,
as d i s c u s s e d
a l l o f which p r i m a r i l y a f f e c t the concentration o f f l u o r i d e
p r e s e n t w i t h i n t h e t r a n s p i r a t i o n a l stream.
The s u b c e l l u l a r p a r t i t i o n i n g o f
f l u o r i d e would be determined by t h e f l u o r i d e c o n c e n t r a t i o n p r e s e n t i n t h e n o n i o n i c f o r m a t t h e e x t e r i o r s u r f a c e o f t h e plasmalemma.
The h y p o t h e t i c a l
model i l l u s t r a t e s t h a t even s l i g h t e l e v a t i o n i n t h e l e v e l o f f l u o r i d e s above t h e b u f f e r i n g c a p a c i t y o f t h e c e l l w a l l w i l l v e r y r a p i d l y r e s u l t i n high l e v e l o f organelle accumulation.
These s l i g h t i n c r e a s e s r e s u l t i n
h i g h a c c u m u l a t i o n l e v e l s i n t h e c h l o r o p l a s t and m i t o c h o n d r i a r e s u l t i n g f r o m a h i g h pH g r a d i e n t b e t w e e n t h e o r g a n e l l e a n d c y t o p l a s m .
Such a model
i n d i c a t e s t h a t t h e use o f h i g h e r f l u o r i d e l e v e l s i n i n v i t r o o r g a n e l l e o r enzyme s t u d i e s i s v a l i d and c o m p a r a b l e t o u s i n g much l o w e r c o n c e n t r a t i o n s i n i n v i v o whole organism studies. REFERENCES
7.
N a t i o n a l Academy o f S c i e n c e s (1971) F l u o r i d e s . Washington,
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G a r r e c JP, B l a n c h a r d B, B r u n JC, B i s c h A. B l i g n y R, F o u r c y A ( 1 9 7 3 ) C R Acad S c i / P a r i s 277:805-808
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Wei LL (1972) Ph D Thesis. U t a h S t a t e Univ.
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L h o s t e A M (1976) F l u o r i d e 9:63-70
H i l l AC (1973) J E n v i r o n Qua1 2:526-530
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G i a n n i n i JL,
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P u s h n i k JC. M i l l e r GW ( 1 9 8 3 ) I n : S u s h e e l a A K ( e d ) F l u o r i d e T o x i c i t y . I n t SOC F l u o r i d e Res, Kalpana P r i n t i n g House, New D e l h i
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M i l l e r JE, M i l l e r GW (1974) P h y s i o l P l a n t 32:115-121
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P u s h n i k JC (1985) F l u o r i d e 18:72-79
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20. G i a n n i n i JL, M i l l e r GW. P u s h n i k JC, B r i s k i n . D (1985) P l a n t P h y s i o l o g y (submitted)
21. G i a n n i n i J, P u s h n i k J, B r i s k i n D, M i l l e r G ( 1 9 8 5 ) P l a n t S c i e n c e ( i n press)
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H. Tsunoda and M.-H.Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 253-256 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
THE EFFECT
OF
FLUORIDE
ON DNA
AND RNA SYNTHESIS BY BARLEY MESOPHYLL
PROTOPLAST
YUMIN L I AND H U I X I A N MA Department o f I n d u s t r i a l Hygiene, Hunan I n s t i t u t e o f M e t a l l u r g i c a l Protect i o n , Shu Mu Ling, Changsha, Hunan, China
ABSTRACT T r e a t m e n t w i t h f l u o r i d e d e c r e a s e d t h e s y n t h e s i s r a t i o o f DNA and i n c r e a s e d t h a t o f RNA i n b a r l e y ( H o r d i u m d i s t i c h o n v a r nudum) m e s o p h y l l protoplasts. E x p e r i m e n t a l r e s u l t s showed t h a t NaF promoted 3H-UdR i n c o r p o r a t i o n i n t o
RNA.
The a v e r a g e p r o m o t l o n r a t i o s w e r e 18.3%. 39.5%, and 49.9% when NaF
c o n c e n t r a t i o n s w e r e 1.0,
10.0,
and 100 ppm, r e s p e c t i v e l y .
However NaF
i n h i b i t e d 3H-TdR i n c o r p o r a t i o n i n t o DNA. A v e r a g e i n h i b i t i o n r a t i o s w e r e 11.2%. 11.1%. 22.8%, and 64.3% when NaF c o n c e n t r a t i o n s w e r e 0.1,
1.0,
10.0,
and 100 ppm. r e s p e c t i v e l y
INTRODUCTION The e f f e c t s o f f l u o r i d e on p l a n t s c a n b e shown a t v a r i o u s l e v e l s o f b i o l o g i c a l organization.
Those e f f e c t s ,
as o c c u r r i n g i n t h e t i s s u e ,
l a r , o r g a n e l l e , and m o l e c u l a r l e v e l s , w e r e r e v i e w e d b y M i l l e r Mohamed 0.01
e G . demonstrated
cellu-
fit. (1).
t h a t g r o w i n g young o n i o n r o o t s t r e a t e d w i t h
M NaF showed c h r o m o s o m a l a b e r r a t i o n s (2).
H y d r o g e n f l u o r i d e was
t h o u g h t t o b l o c k t h e r e p l i c a t i o n o f DNA i n t o m a t o p l a n t s (3). I n o r d e r t o s t u d y t h e e f f e c t s o f f l u o r i d e on DNA and RNA s y n t h e s i s , b a r l e y (Hordium d i s t i c h o n v a r nudum) m e s o p h y l l p r o t o p l a s t s w e r e t r e a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f sodium f l u o r i d e and t h e i n c o r p o r a t i o n o f 3H-TdR ( t h y m i d i n e ) o r 3H-UdR ( u r i d i n e ) i n t o DNA o r R N A was d e t e r m i n e d . P r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t f l u o r i d e caused t h e s y n t h e s i s r a t i o s o f
DNA t o be decreased and RNA t o be increased.
MATERIALS AND METHODS L a b e l e d 3H-TdR
(4.5
C/mM)
and 3H-UdR (20 C / m M )
were produced by t h e
Shanghai Atomic I n s t i t u t e . B a r l e y (Hordium d i s t i c h o n v a r &)
seeds were g e r m i n a t e d on n y l o n net,
and t h e s e e d l i n g s were grown by w a t e r c u l t u r e .
A f t e r g r o w i n g f o r 6 days,
254 t h e l e a v e s w e r e c u t o f f and t h e f i b e r l a y e r was r e m o v e d f r o m t h e b o t t o m surface. 1.5
T h e l e a v e s w e r e i n c u b a t e d i n 4% p e c t i n a s e a n d 4 % c e l l u l a s e f o r
h o u r s a t 28°C t o c a u s e t h e c e l l w a l l t o p e e l o f f .
The i n c u b a t i o n
m i x t u r e was t h e n f i l t e r e d t h r o u g h a n y l o n s i e v e o f 300-400 mesh and c e n t r i f u g e d a t 1000 rpm t o o b t a i n t h e m e s o p h y l l p r o t o p l a s t s .
The m e s o p h y l l
p r o t o p l a s t s w e r e s u s p e n d e d i n M r a s h i g e and Skoog c u l t u r e m e d i a and t h e c e l l c o u n t was 1 x 106/m1.
M o r e t h a n 95% o f t h e p r o t o p l a s t s w e r e shown t o h a v e
v i t a l it y . S a m p l e s w e r e d i v i d e d i n t o f i v e g r o u p s based on t h e amount o f NaF used: 0 (control),
0.1.
1.0,
10.0,
a n d 1 0 0 ppm NaF.
The r e a c t i o n was a l l o w e d t o p r o c e e d i n t e s t t u b e s . c o n t a i n e d a r e a c t i o n m i x t u r e c o n s i s t i n g o f M S media, t i o n . NaF, a n d 3H-TdR o r 3H-UdR, d i d n o t c o n t a i n added NaF. i n c u b a t e d f o r 12 hours.
Each o f t h e t u b e s p r o t o p l a s t prepara-
i n a t o t a l v o l u m e o f 1.0 m l .
The c o n t r o l
The t u b e s c o n t a i n i n g t h e r e a c t i o n m i x t u r e w e r e A t t h e end o f t h e i n c u b a t i o n p e r i o d . t h e tubes
w e r e s u b m e r g e d i n i c e - w a t e r t o s t o p t h e r e a c t i o n . T h e m i x t u r e was t h e n f i l t e r e d t h r o u g h g l a s s f i b e r a n d washed w i t h 10 m l e a c h o f 0.7 M m a n n i t o l , 100% e t h a n o l ,
and 10% t r i c h l o r o a c e t a t e .
The d r i e d g l a s s f i b e r was p l a c e d
i n t o l i q u i d s c i n t i l l a t i o n v i a l s c o n t a i n i n g 1 0 m l s c i n t i l l a t i o n liquid. and t h e r a d i o a c t i v i t y was d e t e r m i n e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r . Promotion o r i n h i b i t i o n r a t i o s o f i n c o r p o r a t i o n were c a l c u l a t e d according t o t h e f o l l o w i n g formula:
Ratio =
CPMNaF - C P M c o n t r o l
x 100 (CPM, c o u n t s p e r m i n u t e )
RESULTS R e s u l t s f r o m p r e l i m i n a r y e x p e r i m e n t s showed t h a t b o t h 3H-TdR and 3H-UdR a t t a i n e d t h e i r peak o f i n c o r p o r a t i o n i n 1 0 h o u r s a n d r e m a i n e d t h e r e f o r 1 8 hours.
I n c u b a t i o n t i m e i n t h i s e x p e r i m e n t was.
therefore,
T h e i n c o r p o r a t i o n o f 3H-TdR w a s i n h i b i t e d b y NaF. r a t i o s w e r e 11.1%, 19.9%, 22.7%. w e r e 0.1.
1.0,
s e t a t 1 2 hours.
Average i n h i b i t i o n
a n d 64.3% w h e n t h e c o n c e n t r a t i o n s o f NaF
10.0, a n d 100 ppm. r e s p e c t i v e l y ( T a b l e I ) .
The i n c o r p o r a t i o n o f 3H-UdR,
on t h e o t h e r hand, was p r o m o t e d b y NaF.
a v e r a g e p r o m o t i o n r a t i o s w e r e 18.3%. t i o n s o f NaF w e r e 1.0.
10.0,
39.5%,
and 1 0 0 ppm,
The
and 49.9% when t h e c o n c e n t r a -
r e s p e c t i v e l y ( T a b l e 11).
255
TABLE I EFFECT OF NaF ON THE INCORPORATION OF 3H-TdR INTO DNA NaF ( PPm)
CPM o f each e x p e r i m e n t
Range o f Average i n h i b i t i o n in h i b i t i on r a t i o (%) r a t i o (%)
1
2
3
4
5
6
0
2.836
5,062
5,431
4,563
3,560
3,748
0.1
2.592
4.586
4,899
4,034
3.104
1.0
2,388
4,070
4,535
3,541
2,681
10.0
2,286
4,019
4,182
3,445
2,631
2.906
19.4-26.1
22.7k2.46
100.0
1,202
1,853
2,194
1,556
1,050
1,181
57.6-70.5
64.3t5.03
-
-
3.216
8.6-14.2
11.2k2.17
2,976
15.8-24.7
19.9k3.41
TABLE I 1 EFFECT
OF NaF ON THE INCORPORATION OF 3H-UdR INTO RNA
NaF
( PPm)
0
CPM o f each e x p e r i m e n t
1
2
3
4
5
6
Range o f promotion r a t i o (%)
5,869
7,602
6,973
2,831
2,674
4,556
-
2,663
4,553
Average promotion r a t i o (%)
-
-0.8-0.6
0.1
5,822
7,564
6,966
2,814
1.0
7,072
9,282
8,096
3,261
3,142
5,394
15.2-22.1
18.3k2.62
10.0
8,498
10.920
9,469
3,751
3,655
6,542
32.5-44.8
39.5k5.14
100.0
8.622
11,190
10,083
4,258
4,109
7,135
44.6-56.7
49.9f4.55
-
DISCUSSION
Mohamed
& d.r e p o r t e d t h a t v i g o r o u s young o n i o n r o o t s t r e a t e d w i t h 0.01
M NaF showed chromosomal a b e r r a t i o n (2).
Mohamed c o n c l u d e d
b l o c k DNA r e p l i c a t i o n d i r e c t l y o r i n d i r e c t l y (3).
t h a t HF w o u l d
B a l e and H a r t ( 4 )
t r e a t e d b a r l e y (Hordium v u l g a r e ) s e e d l i n g r o o t t i p s w i t h e i t h e r 0.01
M NaF
o r 0.01 M HF and n o t e d a m a r k e d l y s l o w e d r a t e o f s e e d l i n g g r o w t h .
It
a p p e a r s , t h e r e f o r e , t h a t DNA, t h e b a s i c m a t e r i a l o f c e l l n u c l e i . m u s t b e a f f e c t e d by fluoride. 3H-TdR
S i n c e t h y m i d i n e i s a component o f DNA. t h e r a t i o s o f
i n c o r p o r a t i o n i n t o DNA can r e p r e s e n t i t s r e p l i c a t i o n .
m e s o p h y l l p r o t o p l a s t s F- i n h i b i t e d DNA s y n t h e s i s .
With barley
The r e s u l t s o b t a i n e d i n
t h i s e x p e r i m e n t appear t o be c o n s i s t e n t w i t h t h o s e o f o t h e r e x p e r i m e n t s .
256
F l u o r i d e has been r e p o r t e d t o cause a decrease i n polysome l e v e l s and an i n c r e a s e i n t h e number and c o n c e n t r a t i o n o f ribosomes and s u b u n i t s combined (5).
Chang (6) compared t h e b i o c h e m i c a l mechanisms o f g r o w t h r e t a r d a t i o n
caused by f l u o r i d e and ozone.
The f l u o r i d e - i n d u c e d
biochemical a l t e r a t i o n
was t h o u g h t t o c o n s i s t o f t h e d i s i n t e g r a t i o n o f t h e polysome i n t o s m a l l e r particles.
I t i s p o s s i b l e t h a t 3H-UdR may come i n t o c o n t a c t w i t h ribosomes
more r e a d i l y .
T h i s c o u l d e x p l a i n why 3H-UdR i n c o r p o r a t i o n r a t i o s w e r e
promoted. I t s h o u l d be s t r e s s e d t h a t t h e s e r e s u l t s a r e p r e l i m i n a r y .
Further
s t u d i e s on t h e mechanisms i n v o l v e d i n t h e observed changes a r e necessary.
REFERENCES 1.
M i l l e r GW, Yu MH, Pushnik JC (1983) In: Shupe JL, P e t e r s o n HB, Leone NC ( e d s ) F l u o r i d e s - E f f e c t s on V e g e t a t i o n , A n i m a l s and Humans. P a r a g o n Press, Inc., S a l t Lake City, pp 83-104
2.
Mohamed AH,
Applegate HG, S m i t h
J D (1966) Can J Genet C y t o l 8:241-244
3.
Mohamed AH (1968) J A i r P o l l u t C o n t r o l Assoc 18:395-398
4.
B a l e SS,
5.
Chang CW (1970) Can J Biochem 48:450-454
6.
Chang CW (7978) F l u o r i d e 11:55-59
H a r t GE (1973) Can J G e n e t i c C y t o l 15:695-702
257
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 257-261 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
THE EFFECTS
OF
FLUORIDE ON THE A C T I V I T I E S
OF
SEVERAL ENZYMES A!!D
ON CYCLIC
AMP LEVELS I N HUMAN LYMPHOCYTES I N VITRO TSUTOMU SATO, MOT00 NIWA, YOSHIKAZU NISHIDA. AND TATSUHIKO TSUJI D e p a r t m e n t o f P r e v e n t i v e and C o m m u n i t y D e n t i s t r y , 1-19-20, F U j i m i . Chiyoda-ku. T o k y o 102. J a p a n
Nippon Dental U n i v e r s i t y .
ABSTRACT T h e a c t i v i t y o f g l y c o l y t i c e n z y m e s (ALD a n d LDH) a n d p h o s p h a t a s e s ( A l p and AcP).
and cAMP l e v e l s i n human l y m p h o c y t e s c u l t u r e d i n t h e p r e s e n c e o f
F (0.53 m M o r 1.05 mM) f o r 24 h o u r s w e r e d e t e r m i n e d . examined by t r a n s m i s s i o n e l e c t r o n microscopy. was n o t shown i n f l u o r i d e - t r e a t e d c e l l s . observed between f l u o r i d e - t r e a t e d
T h e i r m o r p h o l o g y was
Loss o f lymphocyte v i a b i l i t y
No m o r p h o l o g i c a l d i f f e r e n c e s w e r e
c e l l s and
the controls.
The g l y c o l y t i c
enzymes and p h o s p h a t a s e s w e r e n o t a f f e c t e d b u t cAMP l e v e l s w e r e enhanced b y
1.05 m M F.
INTRODUCTION A l t h o u g h t h e e f f e c t s o f f l u o r i d e ( F ) on m a m m a l i a n c e l l s h a v e b e e n s t u d i e d under v a r i o u s c o n d i t i o n s ,
f e w s t u d i e s h a v e been c a r r i e d o u t on i m m u n o c y t c s .
It i s w e l l k n o w n t h a t a l a r g e n u m b e r o f e n z y m a t i c r e a c t i o n s ( i n c l u d i n g g l y c o l y t i c e n z y m e s ) a r e i n h i b i t e d b y F.
On
t h e o t h e r hand,
i t has been
s h o w n t h a t F e n h a n c e s t h e p r o d u c t i o n o f a d e n o s i n e 3', 5 ' - m o n o p h o s p h a t e
(CAMP)
b y s t i m u l a t i n g t h e a c t i v i t y o f a d e n y l a t e c y c l a s e (1).
Phosphatases
h a v e a l s o b e e n shown t o b e a c t i v a t e d b y F (2,3).
Thus, i n t h i s s t u d y , t h e
a c t i v i t y o f g l y c o l y t i c enzymes and p h o s p h a t a s e s ,
and cAMP l e v e l s i n human
l y m p h o c y t e s c u l t u r e d i n t h e p r e s e n c e o f F w e r e examined. The m o r p h o l o g y was observed under a t r a n s m i s s i o n e l e c t r o n microscope.
MATERIALS AND METHODS Preparation o f lymphocytes L y m p h o c y t e s w e r e s e p a r a t e d f r o m f r e s h venous b l o o d s a m p l e s f r o m t e n human v o l u n t e e r s (age:
24-31)
b y c e n t r i f u g a t i o n o n F i c o 1-Paque
(Pharmacia.
Sweden). L y m p h o c y t e c u l t u r e s and F t r e a t m e n t . S e p a r a t e d l y m p h o c y t e s w e r e washed w i t h Ca2+/Mg2+s o l u t i o n (PBS-)
r e e phosphate b u f f e r
and s e e d e d i n RPMI 164.0 s u p p l e m e n t e d w i t h 20% ( v / v )
fetal
258 calf
serum
at
a density o f
2-5x106
cells
p r e i n c u b a t i o n f o r 2 4 h o u r s a t 37°C ( 0 t i m e ) ,
per plastic
tube.
After
F s o l u t i o n (NaF) was added t o
t h e p r e i n c u b a t e d l y m p h o c y t e s a t t h e f i n a l c o n c e n t r a t i o n o f 0.53 m M o r 1.05 nM.
T h e s e s a m p l e s w e r e f u r t h e r i n c u b a t e d a t 37°C f o r 2 4 h o u r s , and b i o -
c h e m i c a l and m o r p h o l o g i c a l s t u d i e s w e r e c a r r i e d o u t . Enzyme a s s a y F o u r c e l l u l a r enzymes, (LDH),
i n c l u d i n g a l d o l a s e (ALD),
l a c t i c dehydrogenase
a c i d p h o s p h a t a s e (AcP) and a1 k a l i n e p h o s p h a t a s e ( A l p ) .
p e n d e d i n t h e same b u f f e r s o l u t i o n . tured using a Teflon-glass An a u t o a n a l y z e r ,
JCA-N6R
were measured
L y m p h o c y t e s w e r e washed w i t h c o l d PBS- a n d sus-
i n c u l t u r e d lymphocytes.
Cells, maintained on ice, were rup-
h o m o g e n i z e r and t h e n u s e d f o r t h e enzyme assay.
(JEOL).
was u s e d f o r t h e assay.
Enzyme a c t i v i t y
was e x p r e s s e d a s s p e c i f i c a c t i v i t y . C y c l i c AMP a s s a y L y m p h o c y t e s w e r e washed a n d d i s r u p t e d b y t h e same p r o c e d u r e d e s c r i b e d a b o v e . A f t e r t h e a d d i t i o n o f c o l d TCA t o t h e h o m o g e n a t e . centrifuged.
t h e m i x t u r e was
The s u p e r n a t a n t was c o m b i n e d w i t h e t h e r s a t u r a t e d w i t h w a t e r .
A f t e r m i x i n g , CAMP i n t h e aqueous l a y e r was q u a n t i f i e d w i t h a r a d i o i m m u n o a s s a y k i t (YAMASA). T h e r e s u l t s w e r e e x p r e s s e d a s f e m t o m o l s p e r mg c e l l protein. Preparation f o r transmission e l e c t r o n microscopy L y m p h o c y t e s w e r e washed a s d e s c r i b e d above.
P r i m a r y f i x a t i o n was done i n
2.5% g l u t a r a l d e h y d e i n p h o s p h a t e b u f f e r s o l u t i o n c o n t a i n i n g 0.05 A f t e r t w o h o u r s p o s t f i x a t i o n i n 2% o s m i u m t e t r o x i d e ,
M sucrose.
t h e c e l l s were stained
f o r o n e h o u r w i t h 2% u r a n y l a c e t a t e a n d l e a d , d e h y d r a t e d i n e t h a n o l , and T h i n s e c t i o n s w e r e c u t o n LKB U l t o r o t o m e I V ( m o d e l
embedded i n EPON 812.
8800) and e x a m i n e d w i t h a JEOL l O O C e l e c t r o n m i c r o s c o p e a t 8 0 kV. RESULTS The a d d i t i o n o f F (0.53
rnM o r 1.05
mM) t o t h e c u l t u r e media d i d n o t
a f f e c t t h e v i a b i l i t y o f l y m p h o c y t e s w h i c h was
determined using t h e trypan
b l u e d y e e x c l u s i o n m e t h o d ( d a t a n o t shown).
The e f f e c t s o f f l u o r i d e on
enzyme a c t i v i t i e s i n l y m p h o c y t e s a r e shown i n
Figures
1-4.
The b a s a l ALD
a c t i v i t y m e a s u r e d i n t e n c o n t r o l s a m p l e s ( n o t t r e a t e d w i t h F) c o u l d be d i v i d e d i n t o t w o groups: However.
a h i g h a c t i v i t y group,
and a l o w a c t i v i t y group.
t h e i r a c t i v i t i e s w e r e n o t c o n s i s t e n t l y a f f e c t e d b y F ( F i g . 1).
T h e LDH a c t i v i t y w a s a l s o n o t a f f e c t e d b y F ( F i g .
2).
The,AcP a c t i v i t i e s
i n l y m p h o c y t e s c u l t u r e d w i t h F d i d n o t change f r o m c o n t r o l v a l u e s w i t h i n a 24 h o u r p e r i o d ( F i g .
3).
S i n c e t h e A1P a c t i v i t y i n l y m p h o c y t e s was t o o l o w
f o r an a c c u r a t e d e t e r m i n a t i o n , s o n a l s a m p l e s ( F i g . 4).
d a t a c o u l d be o b t a i n e d o n l y f r o m f i v e p e r -
Therefore,
t h e e f f e c t o f F o n A1P a c t i v i t y w a s
259
c
4
2
1000
0 L-
a
z
\
3
H
-E
500
4
-4
>4
u u irJ
I : 4 ci
0
F F i g u r e 1. hours.
0.53 ( nM
1.05
0
0
1.05
0.53 F ( mM
)
ALD a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t F f o r 24
F i g u r e 2. LDH a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t F f o r 24 hours. A c t i v i t i e s o f t w o samples were n o t detected.
0
0.53
1.05
F ( W ) A l p a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t F f o r 24 F i g u r e 3. hours. A c t i v i t i e s o f f i v e samples were n o t detected. F i g u r e 4. AcP a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t hours. A c t i v i t i e s o f t w o samples were n o t detected.
F
f o r 24
260
o b s c u r e i n t h i s study.
5).
C y c l i c AMP l e v e l s were enhanced by 1.05 m M F (Fig.
U l t r a s t r u c t u r a l d i f f e r e n c e s c o u l d n o t be found between c o n t r o l c e l l s
and f l u o r i d e - t r e a t e d c e l l s ( F i g . 6A-C).
0
0.53
1 .05
F ( d 1 )
F i g u r e 5. 2 4 Hours.
C y c l i c AMP l e v e l s i n lymphocytes c u l t u r e d w i t h o r w i t h o u t F f o r
261
Figure 6. Electron micrographs of lymphocytes c u l t u r e d i n t h e absence (A: c o n t r o l ) and p r e s e n c e (B: 0.53 mM, C: 1.05 m M ) o f f l u o r i d e f o r 2 4 hours. The s p a r s e cytoplasm and few o r g a n e l l e s were shown i n both c o n t r o l and Ft r e a t e d c e l l s . N: nucleus, M: mitochondrion, Ps: pseudopod, Bar=l.O urn.
REFERENCES 1 . Rall TW. Sutherland EW (1958) J Biol Chem 2321065-1076 2.
Lai CC, Singer L, Armstrong WD (1974) Clin Orthop Re1 Res 100:387-391
3.
Farley JR, Wergedal JE, Baylink DJ (1983) Science 222:330-332
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 263-266 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
263
INDUCTION OF PROTEIN I N HELA CELLS BY SODIUM FLUORIDE TOSHIO I M A I , K U M I GOJO,
TSUTOMU SATO, R Y O I C H I NISHIKAWA, MOT00 NIWA AND
TATSUHIKO TSUJI D e p a r t m e n t o f P r e v e n t i v e and C o m m u n i t y D e n t i s t r y N i p p o n D e n t a l U n i v e r s i t y , 1-9-20, F u j i m i Chiyoda-Ku. Tokyo, 102. J a p a n ABSTRACT When HeLa c e l l s w e r e t r e a t e d w i t h s o d i u m f l u o r i d e , synthesis
of
77000 d a l t o n s , inhibited. cells,
a
specific
protein
with
a
t h e r e was an i n c r e a s e d
molecular
weight
of
about
w h i l e t h e s y n t h e s i s o f m o s t o t h e r c e l l u l a r p r o t e i n s was
T h i s p h e n o m e n o n w a s c o m m o n t o HeLa-S3 c e l l s ,
human d i p l o i d f i b r o b l a s t c e l l s and HT-1080 c e l l s .
were t r e a t e d w i t h bromide o r iodide, sodium f l u o r i d e d i d n o t occur.
C l o n e 1-5c-4
When HeLa c e l l s
h o w e v e r , t h e phenomenon o b s e r v e d w i t h
M o r e o v e r , when s t r e s s e d b y t e m p e r a t u r e ,
i n d u c t i o n o f s p e c i f i c 77KD p r o t e i n s y n t h e s i s i n H e L a c e l l s
was
the not
observed. INTRODUCTION
I t i s w e l l known t h a t sodium f l u o r i d e i s c y t o t o x i c a t h i g h c o n c e n t r a t i o n s , c a u s i n g g r o w t h i n h i b i t i o n a n d c e l l d e a t h (1-4).
The m a i n c a u s e o f
t h i s e f f e c t i s p r o b a b l y i n h i b i t i o n o f c e l l u l a r p r o t e i n s y n t h e s i s (4,5). P r e v i o u s s t u d y h a s shown t h a t when HeLa c e l l s w e r e e x p o s e d t o s o d i u m f l u o ride,
the synthesis of a particular protein w i t h a molecular weight of
a b o u t 77.000 d a l t o n s (77KD) was i n d u c e d , c e l l u l a r p r o t e i n s was i n h i b i t e d (6).
w h i l e t h e synthesis o f most o t h e r
The p u r p o s e o f t h e p r e s e n t s t u d y was
t o i n v e s t i g a t e t h e m e c h a n i s m i n v o l v e d i n t h e s y n t h e s i s o f 77KD p r o t e i n i n cells. MATERIALS AND METHODS Cell culture HeLa c e l l s ,
HeLa S3 c e l l s ,
C l o n e 1-5c-4
cells,
human d i p l o i d f i b r o b l a s t
c e l l s and HT-1080 c e l l s w e r e c u l t i v a t e d i n m o n o l a y e r c u l t u r e s w i t h E a g l e ' s m i n i m a l e s s e n t i a l medium s u p p l e m e n t e d w i t h 20 p g / m l k a n a m y c i n e and 10% h e a t i n a c t i v a t e d f e t a l c a l f s e r u m a t 37°C i n a 5% C02 a t m o s p h e r e . Labeling condition Twenty-four
hours a f t e r seeding c e l l s ,
s o d i u m f l u o r i d e was added t o t h e
c u l t u r e m e d i u m a t a f i n a l c o n c e n t r a t i o n o f 1.5 mM. a n d t h e c u l t u r e s w e r e t h e n i n c u b a t e d f o r 2.5.
5.0 a n d 7.5 h o u r s , r e s p e c t i v e l y .
The m e d i u m was
264 r e m o v e d f r o m t h e c u l t u r e s and r e p l a c e d w i t h P B S ( - ) vCi/dish)
c o n t a i n i n g 1.5 m M NaF
and I 4 C - p r o t e i n
h y d r o l y s a t e (0.5
and l a b e l e d f o r 1 hour a t 37°C.
A f t e r labeling,
t h e c e l l s were washed w i t h PBS(-) and were scraped o f f t h e
p l a t e s and homogenized u s i n g a t e f l o n homogenizer. Gel e l e c t r o p h o r e s i s Samples o f 1 4 C - p r o t e i n h y d r o l y s a t e - l a b e l e d
p r o t e i n were analyzed i n 11-
13% g r a d i e n t p o l y a c r y l a m i d e g e l s a c c o r d i n g t o t h e procedure o f Laemmli (7). Gels were t r e a t e d w i t h sodium s a l i c y l a t e ( 8 ) , d r i e d .
and t h e n exposed t o X-
ray f i l m .
RESULTS AND D I S C U S S I O N P r e v i o u s s t u d i e s have r e v e a l e d a t r e n d o f i n c r e a s e d s y n t h e s i s o f t h e 77KD p r o t e i n i n c e l l s i n t h e presence o f sodium f l u o r i d e .
A q u e s t i o n arose as
t o whether t h i s 77KD p r o t e i n was unique t o HeLa c e l l s o r t o human c e l l s i n general.
To i n v e s t i g a t e o t h e r human c e l l s , H e l a S3, C l o n e 1-5c-4,
diploid fibroblasts
and
HT-1080
c e l l s were
human
t r e a t e d w i t h sodium f l u o r i d e
and l a b e l e d . The l a b e l e d p r o t e i n s were f r a c t i o n a t e d on SDS-polyacrylamide
gels.
The
s y n t h e s i s o f 77KD p r o t e i n i n HeLa S3 c e l l s was i n d u c e d b y t r e a t m e n t w i t h s o d i u m f l u o r i d e ( F i g . 1). other cells.
I n d u c t i o n o f 77KD p r o t e i n was a l s o o b s e r v e d i n
T h i s suggests t h a t i n d u c t i o n o f 77KD p r o t e i n m i g h t be common
t o human c e l l s . Two t y p e s o f e x p e r i m e n t s w e r e done t o i n v e s t i g a t e t h e s p e c i f i c i t y o f sodium f l u o r i d e i n r e l a t i o n t o t h e s y n t h e s i s o f 77KD p r o t e i n . c e l l s were t r e a t e d w i t h bromide o r i o d i d e f o r 5 hours,
First,
HeLa
l a b e l e d f o r 1 hour,
and t h e p a t t e r n o f t h e s y n t h e s i z e d p r o t e i n was examined by SDS-polyacrylamide g e l a n a l y s i s .
The c o n c e n t r a t i o n o f t h e h a l i d e s used i n t h e s e s t u d i e s
(80 m M ) was c y t o t o x i c .
The i n d u c t i o n o f 77KD p r o t e i n s y n t h e s i s was n o t
o b s e r v e d ( F i g . 2). Second, c e l l s w e r e e x p o s e d t o a t e m p e r a t u r e o f 29°C f o r 5 h o u r s i n t h e presence o r absence o f sodium f l u o r i d e . period,
A t t h e end o f t h e s h i f t e d - d o w n
c e l l s were r e t u r n e d t o 37°C and l a b e l e d f o r 1 hour.
o f sodium f l u o r i d e ,
I n t h e absence
t h e i n d u c t i o n o f 77KD p r o t e i n s y n t h e s i s i n s h i f t e d - d o w n
c e l l s was n o t observed.
I n t h e presence o f sodium f l u o r i d e ,
o f 77KD p r o t e i n was d i m i n i s h e d w i t h most c e l l u l a r p r o t e i n . a b i l i t y t o i n d u c e 77KD p r o t e i n s y n t h e s i s was n o t l o s t .
the synthesis However,
the
These r e s u l t s
suggest t h a t t h e i n d u c t i o n o f 77KD p r o t e i n s y n t h e s i s i s a s p e c i f i c phenomenon t o sodium f l u o r i d e t r e a t m e n t .
1
2
3
Figure 1. Induction o f 77KD protein in HeLa S3 cells by sodium fluoride. Cells were treated with 1.5 m M NaF for 0 (1). 2.5 hours (2) and 7.5 hours ( 3 ) . respectively. After treatment, the cellular proteins w e r e labeled w i t h protein hydrolysate for 1 hour. The labeled proteins were analyzed in SDS-polyacrylamide gel and visualized by fluorography. Black arrow indicates the 77KD protein. Figure 2. Fluorogram o f SDS-polyacrylamide slab gel of 14C-protein hydrolysate-labeled proteins from HeLa cells treated with fluoride, bromide and iodide. Cells were treated with drug at 37°C for 5 hours and then labeled for 1 hour. (1) control: (2) 1.5 m M NaF; (3) 80 m M bromide; (4) 80 m M iodide. Black arrow indicates the 77KD protein.
N
0)
01
266 ACKNOWLEDGEMENT We t h a n k Dr. M. U e d a f o r h e l p f u l d i s c u s s i o n a n d f o r r e v i e w i n g t h e manuscript. REFERENCES 1.
B e r r y RJ, T r i l l w o o d W (1963) B r i t Med J 23:1964
2.
A r m s t r o n g WC, B l o m q u i s t CH, S i n g e r B r i t Med J 1:486-488
L,
P o l l o c k ME, M c L a r e n LC ( 1 9 6 5 )
3.
B e r r y RJ (1969) F l u o r i d e 2:157-167
4.
I m a i T, N i w a M, Ueda M (1983) A c t a p h a r m a c o l e t t o x i c o l 52:8-11
5.
H o l l a n d R I (1979) A c t a p h a r m a c o l e t t o x i c o l 49:96-101
6.
I m a i T, N i s h i k a w a R. N i w a M (1983) J D e n t Res 6 2 : S p e c i a l
7.
L a e m m l i UK (1970) N a t u r e (London) 227-680-685
8.
C h a m b e r l a i n JP (1979) A n a l Biochem 98:132-135
I s s u e 692
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 267-276 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
267
EFFECTS OF FLUORIDES ON ORAL MICROORGANISMS MASATO I I D A ,
TSUGUO KAWASAKI, MASAHIRO FURUTANI, AND TAKUMI SAT0
D e p a r t m e n t o f O r a l and M a x i l l o f a c i a l S u r g e r y , S c i e n c e , S e t a O h t s u 520-21 Japan
Shiga U n i v e r s i t y o f Medical
ABSTRACT The p u r p o s e o f t h i s s t u d y was t o e x a m i n e t h e e f f e c t s o f f l u o r i d e s on v a r i o u s o r a l microorganisms i n c l u d i n g Streptococcus, Pseudomonus,
B a c t e r o i d e s and Fusobacterium,
Streptococcus mutans ( S .
mutans),
Actinomyces,
Candida.
on t h e a c i d production o f
and o n t h e s y n t h e s i s o f w a t e r i n s o l u b l e
g l u c a n c a t a l y z e d b y e x t r a c e l l u l a r enzyme ( g l u c o s y l t r a n s f e r a s e ) d e r i v e d f r o m S.
mutans.
T h e m i n i m u m i n h i b i t o r y c o n c e n t r a t i o n s o f f l u o r i d e p r o v i d e d a s NaF f o r Actinomyces,
C a n d i d a and Pseudomonus w e r e h i g h e r t h a n t h o s e f o r t h e o t h e r
microorganisms.
NaF a r r e s t e d b a c t e r i a l g r o w t h b a c t e r i o s t a t i c a l l y b u t d i d
not a f f e c t the synthesis o f water-insoluble
glucan.
G l u c a n s y n t h e s i s was
i n h i b i t e d o n l y b y CoF2 a t c o n c e n t r a t i o n s o f 600-1000 ppm F. L a c t i c a c i d p r o d u c t i o n was s u p p r e s s e d i n b o t h t h e c e l l r e s t i n g and c e l l g r o w i n g systems.
F l u o r i d e s o f b i v a l e n t c a t i o n s s u c h a s SnF2 a n d CoF2 i n h i b i t e d t h e
g r o w t h o f t h e m i c r o o r g a n i s m s m o r e m a r k e d l y t h a n NaF. INTRODUCTION F l u o r i d e s a r e w i d e l y used i n p r e v e n t i v e d e n t i s t r y s u c h a s t o p i c a l a p p l i c a t i o n and f l u o r i d a t i o n b e c a u s e o f t h e i r c a r i o s t a t i c e f f e c t s .
Such e f f e c t s
a r e p r o d u c e d a s a r e s u l t o f e n h a n c e d s t r e n g t h and a c i d t o l e r a n c e o f t o o t h m a t e r i a l through formation o f f l u o r a p a t i t e
and CaF2.
S t u d i e s on t h e e f -
f e c t s o f f l u o r i d e s have focused l a r g e l y on improvement of a c i d t o l e r a n c e o f t h e t o o t h (1-4).
Fluorides,
however,
e f f e c t s on d e n t a l p l a q u e - f o r m i n g
a r e a l s o known t o h a v e a n t i b a c t e r i a l
b a c t e r i a (5).
B i b b y a n d Van K e s t e r n (6)
r e p o r t e d i n 1940 t h a t f l u o r i d e s r e d u c e d t h e p r o d u c t i o n o f o r g a n i c a c i d s b y o r a l microbes i n culture,
and s u g g e s t e d t h a t t h e y a f f e c t e d b a c t e r i a l g r o w t h
and s a c c h a r o m e t a b o l i s m a t l o w c o n c e n t r a t i o n s . minimum i n h i b i t o r y c o n c e n t r a t i o n ( M I C )
They a l s o d e t e r m i n e d t h e
o f NaF f o r S t r e p t o c o c c u s t o be 250
ppm F, and r e p o r t e d s u p p r e s s i o n o f t h e p r o d u c t i o n o f o r g a n i c a c i d s a t l o w e r f l u o r i d e concentrations.
However,
much o f t h e e f f e c t o f f l u o r i d e on i n d i g -
enous o r a l m i c r o o r g a n i s m s r e m a i n s o b s c u r e . The p u r p o s e o f t h i s s t u d y was t o e x a m i n e t h e e f f e c t s o f f l u o r i d e s o n
268
various oral microorganisms including Streptococcus, Actinomyces, Candida. Pseudomonus, Bacteroides and Fusobacterium. on acid production of Strepto-
~coccus mutans (S. mutans).
and on the synthesis of water-insoluble glucan catalyzed by extracellular enzyme (glucosyltransferase, EC. 2.4.1.5)
derived from S_ mutans. In addition, the effects of NaF and other fluorides on cariogenic S. mutans at various concentrations were compared in_ vitro. _ _
MATERIALS AND METHODS Bacterial strains Oral microorganisms used in this study were provided by the Department of Bacteriology, Osaka Dental College and Shiga University of Medical Science. Bacteria were repeatedly stored once a month i n 10 ml brain-heart infusion agar media (BHI, Difco) at the Department of Oral Surgery, Shiga University of Medical Science. For preincubation, the culture (0.1 ml) was inoculated to a 10 ml broth and incubated at 37°C for 24 hours. The incubated
culture (0.1 ml) was reinoculated to the same media and incubated aerobically and anaerobically at 37°C for 18 hours. Table I shows the number and kind o f strains of microorganisms used in the present study. Chemi ca 1 s Sodium fluoride was obtained from the Hashimoto Chemical Co. while 1 1 other fluorides were obtained from the Nakarai Chemical Co., Japan.
TABLE I BACTERIAL STRAINS Streptococcus ( I 3a) S. mutans (13) Bacteroides (19) B. melaninogenicus (8) B. intermedius (3)
B. asaccharolyticus (3) B. gingivalis (2) B. fragilis (3) Candida (10)
C. albicans (5) C. tropicalis (4) C. krusei (1) anumber of strain
Fusobacteri um (9) F. nucleatum (5) F. mortiferum (2) F. vorium (2) Actinomyces (10) A. viscosus (5)
A. A. A. A.
islaelli (2) odontolyticus (1) meyeri (1)
naeslandi (1) Pseudomonus (10) P. aeruginosa (10)
269 C u l t u r e media
A B H I b r o t h was used as t h e l i q u i d medium, and agar p l a t e s were prepared A Gifu
b y a d d i n g B a c t o a g a r ( D i f c o ) t o BHI f o r a e r o b i c microorganisms. a n a e r o b i c medium (GAN,
N i s s u i ) was used f o r a n a e r o b i c microorganisms.
Measurement o f M I C and b a c t e r i a l growth The M I C v a l u e s w e r e d e t e r m i n e d w i t h t h e t h e a g a r d i l u t i o n t e c h n i q u e a c c o r d i n g t o t h e method o f t h e Japanese S o c i e t y f o r Chemotherapy (7).
One
p l a t i n u m l o o p o f t h e p r e - i n c u b a t e d sample s o l u t i o n ( c o n t a i n i n g approximatel y 6x106 c e l l s / m l
f o r aerobs and 8x108 c e l l s / m l
f o r anaerobs) was added t o
agar media w i t h f l u o r i n e c o n c e n t r a t i o n s o f 0-5960 f l u o r i d e i o n e l e c t r o d e ( O r i o n Research).
ppm F,
verified with a
A f t e r 24 hours o f i n c u b a t i o n a t
t h e m i n i m a l c o n c e n t r a t i o n a t w h i c h no b a c t e r i a l c o l o n y was g r o s s l y
37"C,
observed was d e t e r m i n e d t o be t h e M I C . W h i l e i n v e s t i g a t i n g t h e b a c t e r i a l growth,
t h e t u r b i d i t y absorbance was
measured e v e r y hour w i t h a s p e c t r o p h o t o m e t e r ( H i t a c h i , u n i t s (CFU)
Colony-forming a f t e r 0,
4,
8,
100-10)
a t 500 nm.
were s i m u l t a n e o u s l y measured on B H I agar p l a t e s
12. and 24 h o u r s o f i n c u b a t i o n .
added t o t h e l i q u i d and agar media
As a c o n t r o l ,
NaCl was
a t t h e same c o n c e n t r a t i o n s as NaF,
and
m e a s u r e m e n t o f t u r b i d i t y and C F U was made d u r i n g i n c u b a t i o n i n t h e same G r o w t h c u r v e s o f C F U w e r e p l o t t e d as l o g l o
m a n n e r as above.
CFU/ml o f
c u l t u r e over time. Measurement o f pH and l a c t i c a c i d The s a m p l e s o l u t i o n s w e r e c e n t r i f u g e d a t 1500 x g f o r 1 5 m i n and t h e pH o f t h e s u p e r n a t a n t was measured w i t h a pH m e t e r ( H i t a c h i - H o r i b a
F7).
A n a l y s i s o f l a c t i c a c i d was conducted w i t h a gas chromatograph (Shimazu GC-7A)
by an o r d i n a r y m e t h y l - e s t e r i f i c a t i o n method.
P r i o r t o t h e analysis.
l a c t i c a c i d was i d e n t i f i e d i n t h e s t a n d a r d and sample s o l u t i o n s by i t s mass number (105).
i n a mass s p e c t r o g r a p h (Shimazu AUTO GCMS 6020).
Preparation o f dextransucrase Dextransucrase was d e r i v e d f r o m S. (c).
OMZ-176 (d),
mutans AHT ( s e r o type,
OMZ-175 ( f ) and N I D R 6715 (9).
a),
NCTC 10449
A f t e r preculturing i n BHI
b r o t h f o r 24 and 18 hours t w i c e a t 37°C a e r o b i c a l l y ,
a 0.1 m l s o l u t i o n was
i n o c u l a t e d i n 30 m l B H I b r o t h f o r t h e purpose o f p r e p a r i n g d e x t r a n s u c r a s e as d e s c r i b e d below. The p r e c u l t u r a l f l u i d s o f S. m u t a n s w e r e i n o c u l a t e d i n t o 3 1 B H I b r o t h and i n c u b a t e d a t 37°C f o r 18 hours.
The c u l t u r e s were t h e n c e n t r i f u g e d a t
7,200 x g f o r 20 m i n and t h e s u p e r n a t a n t was t r e a t e d w i t h 60% s a t u r a t e d ammonium s u l f a t e a t 4°C f o r 18 hours.
Following centrifugation
c i p i t a t e was d i s s o l v e d i n 75 m l 0.05 M p h o s p h a t e b u f f e r (pH 6.8). m i x t u r e was d i a l y z e d a g a i n s t t h e same b u f f e r o v e r n i g h t .
t h e preand t h e
The d i a l y z a t e was
270 c e n t r i f u g e d t o r e m o v e i n s o l u b l e m a t e r i a l s and t h e s u p e r n a t a n t was used a s a crude p r e p a r a t i o n o f dextransucrase.
T h i s p r e p a r a t i o n was a d j u s t e d t o
convert sucrose t o w a t e r i n s o l u b l e glucan a t a r a t e o f
S.
The c r u d e enzyme p r e p a r a t i o n f r o m c o l u m n c h r o m a t o g r a p h y o n B i o G e l A-15m
0.6
umol/ml/min.
m u t a n s OMZ-176 w a s p u r i f i e d b y
and s u b s e q u e n t l y o n h y d r o x y l a p a t i t e .
T h i s enzyme p r e p a r a t i o n g a v e t h r e e bands o n a n a l y s i s b y SDS-PAGE
as
d e s c r i b e d p r e v i o u s l y (8). Enzyme a s s a y Enzyme a c t i v i t y was d e t e r m i n e d b y t h e b l u e d e x t r a n m e t h o d ( 9 ) a n d / o r I 4 C s u c r o s e m e t h o d (10).
NaCl was u s e d a s t h e c o n t r o l .
R e l a t i v e enzyme a c t i v i t y i n t h e r e a c t i o n m i x t u r e a t e a c h f l u o r i d e concent r a t i o n was e x p r e s s e d i n t e r m s o f t h e p e r c e n t a g e o f a c t i v i t y o b t a i n e d w i t h t h e s t a n d a r d m i x t u r e , as shown b e l o w :
Relative
enryme activity
A c t i v i t y a t each f l u o r i d e c o n c e n t r a t i o n A c t i v i t y w i t h t h e standard m i x t u r e
=
A l l t e s t s were performed i n t r i p l i c a t e . RESULTS T a b l e 11 shows t h e M I C d i s t r i b u t i o n of NaF f o r o r a l m i c r o o r g a n i s m s .
M I C f o r S. m u t a n s w e r e l o w e r t h a n 1 8 0 ppm
F.
The
The M I C f o r B a c t e r o i d e s and
F u s o b a c t e r i u m w e r e w i d e l y d i s t r i b u t e d w i t h i n t h e r a n g e o f 22.5 t o 1440 ppm
F.
On t h e o t h e r h a n d , t h e M I C v a l u e s f o r A c t i n o m y c e s ,
Pseudomonus and
Candida were h i g h e r t h a n those f o r o t h e r microorganisms. TABLE I 1 SENSITIVITY DISTRIBUTION OF ORAL MICROORGANISMS
M I C o f NaF
O r g a n i sms 22.5
45
90
180
360
720
1440
Bacteroides
2
2
4
1
8
1
1
Fusobacterium
1
1
2
3
2
1
4
1
A c t i nomyces
S. m u t a n s
2
5760 (ppm
7
8 6
Pseudomonus Candida
2880
6
4
4
F)
271 S. -~ mutans
g r o w t h a f t e r 24 h o u r s o f i n c u b a t i o n i n t h e p r e s e n c e o f NaF i s
p r e s e n t e d i n F i g u r e 1.
B a c t e r i a l growth,
expressed i n t e r m s o f absorbance
d e c r e a s e d w i t h i n c r e a s e i n NaF c o n c e n t r a t i o n f r o m 22.5
t o 360 ppm.
Fluo-
r i d e l e v e l s b e l o w 22.5 ppm had n o e f f e c t and t h e p r e s e n c e o f added NaCl had no i n h i b i t o r y e f f e c t w i t h i n t h e c o n c e n t r a t i o n r a n g e t e s t e d . T h e t i m e c o u r s e f o r t h e g r o w t h o f S.
mutans as a f f e c t e d b y d i f f e r e n t
c o n c e n t r a t i o n s o f f l u o r i d e i s s h o w n i n F i g u r e 2.
F l u o r i d e a t 22.5 ppm
showed n o i n h i b i t o r y e f f e c t , w h i l e a t 4 5 ppm F a s l i g h t r e d u c t i o n i n t h e g r o w t h o f t h e t e s t o r g a n i s m was o b s e r v e d .
A d d i t i o n o f 90 ppm F was c l e a r l y
i n h i b i t o r y t o g r o w t h . F l u o r i d e l e v e l s b e t w e e n 180 a n d 3 6 0 ppm a r r e s t e d b a c t e r i a l growth.
A t t h e s e f l u o r i d e l e v e l s t h e g r o w t h was r e d u c e d b y
a p p r o x i m a t e l y 1.5 l o g l o
u n i t s compared w i t h t h e c o n t r o l .
f i g u r e 3 shows t h e e f f e c t o f f l u o r i d e on pH v a l u e s and f i n a l l a c t i c a c i d c o n t e n t d u r i n g t h e g r o w t h o f S. m u t a n s Gs-5. values were i n v e r s e l y related.
L a c t i c a c i d c o n t e n t s a n d pH
A s i m i l a r o b s e r v a t i o n was a l s o made f r o m
e x p e r i m e n t s u s i n g r e s t i n g c e l l s ( d a t a n o t shown). T h e e f f e c t o f NaF o n t h e s y n t h e s i s o f w a t e r - i n s o l u b l e
dextran a t low
f l u o r i d e c o n c e n t r a t i o n s (0-1000 ppm) i s shown i n F i g u r e 4a.
The d i f f e r e n c e
i n a c t i v i t i e s a t d i f f e r e n t c o n c e n t r a t i o n s o f f l u o r i n e and c h l o r i n e , distinct, tions,
was n o t s i g n i f i c a n t .
while
The e f f e c t o f f l u o r i d e a t h i g h e r c o n c e n t r a -
c o m p a r a b l e t o t h o s e i n a 2% NaF s o l u t i o n u s e d f o r d i r e c t t o p i c a l
a p p l i c a t i o n , was t h e n s t u d i e d .
=;
46
90
F o r t h i s purpose, f l u o r i n e and c h l o r i n e
180
360
N.n
Concentration of F. pprn F i g u r e 1. E f f e c t s o f NaF o n t h e g r o w t h o f S. m u t a n s s t r a i n s . T u r b i d i t y a b s o r b a n c e was measured a f t e r i n c u b a t i o n f o r 24 h o u r s w i t h a s p e c t r o p h o t o m e t e r ( H i t a c h i , 100-10) a t 5 5 0 nrn. A s t h e c o n t r o l , N a C l w a s added t o t h e l i q u i d .
212
22.5 45 0
90
1
110
300 (pprnF)
I
L
1
4
8
I2
,I
'
I
24
Incubation Time. hr
F i g u r e 2. E f f e c t s o f NaF o n t h e g r o w t h o f S. m u t a n s s t r a i n s . C o l o n y - f o r m i n g u n i t s (CFU) w e r e m e a s u r e d on B H I a g a r p l a t e s a f t e r i n c u b a t i o n f o r 0, 4. 8, 12, a n d 2 4 h o u r s . G r o w t h c u r v e s o f C F U w e r e p l o t t e d a s l o g l o CFU/ml i n c u l t u r e o v e r t i m e .
I
4 Of
(Iacric acid mmoltml)
; 1
1
'
0
12
1 -
Incubation Time. hr
F i g u r e 3. E f f e c t o f f l u o r i d e on l a c t i c a c i d f o r m a t i o n d u r i n g g r o w t h o f S, m u t a n s Gs-5. c o n c e n t r a t i o n s o f 0, tested.
1000, 2000, 4000,
As shown i n F i g u r e 4b,
no c h a n g e s i n d e x t r a n s y n t h e s i s w e r e n o t e d
despite these h i g h concentrations. s u c r a s e f r o m S. m u t a n s 0142-176. different strains of results.
&
6000, 8000 a n d 10000 p p m w e r e
The enzyme u s e d was p u r i f i e d d e x t r a n Crude dextransucrase p r e p a r a t i o n s from
mutans were subsequently t e s t e d w i t h s i m i l a r
273
2'%
I 250
IOU0
500
wniF
NaF
I
0
1000
I
I
2000
I
1
1
8000
6000
4000
10000
Concentration of F. ppm F i g u r e 4. The e f f e c t o f NaF on t h e s y n t h e s i s o f w a t e r - i n s o l u b l e l o w ( a ) and h i g h ( b ) c o n c e n t r a t i o n s .
dextran a t
Experiments were c a r r i e d out t o t e s t the e f f e c t o f d i f f e r e n t f l u o r i d e compounds on t h e g r o w t h o f S. 111.
mutans.
The r e s u l t s a r e s u m m a r i z e d i n T a b l e
T h e M I C o f CoF2 a n d SnF2 w e r e l o w e r t h a n t h o s e o f o t h e r f l u o r i d e s .
B a c t e r i a l g r o w t h was i n h i b i t e d b a c t e r i c i d a l l y a t 2MIC and 3MIC o f b o t h CoF2 a n d SnF2.
The e f f e c t o f f l u o r i d e s o n t h e s y n t h e s i s o f w a t e r - i n s o l u b l e
g l u c a n was e x a m i n e d as i s shown i n F i g u r e 4.
G l u c a n s y n t h e s i s was i n h i b -
i t e d a p p r o x i m a t e l y 20% b y CoF2 o n l y a t c o n c e n t r a t i o n s o f 600-1000 ppm F.
DISCUSSION F l u o r i d e s a r e w i d e l y u s e d i n t h e f i e l d o f p r e v e n t i v e d e n t i s t r y b e c a u s e of t h e i r anticarious effects.
The p r e s e n t s t u d y shows t h a t NaF h a s d i f f e r e n t
M I C a g a i n s t o r a l m i c r o o r g a n i s r s and t h a t b a c t e r i a l s p e c i e s and s t r a i n s v a r y i n s e n s i t i v i t y t o NaF.
The M I C o f S. m u t a n s w e r e l e s s t h a n 180 ppm.
d . &
observations are consistent w i t h those o f M a l t z Candida,
On t h e o t h e r hand,
B a c t e r o i d e s and F u s o b a c t e r i u m t o t h e M I C
Maltz
fi fl
( 5 ) r e p o r t e d t h a t A c t i n o m y c e s was
m o r e s e n s i t i v e t o t h e b a c t e r i c i d a l a c t i v i t y o f NaF t h a n w e r e s a l i v a r i u s a n d S. known.
Actinomyces,
and Pseudomonus s t r a i n s w e r e shown i n t h e p r e s e n t s t u d y t o be l e s s
s e n s i t i v e than Streptococcus, results.
(5).
These
mutans.
S,
sanguis.
S,
The r e a s o n f o r t h e s e c o n t r a s t i n g r e s u l t s i s n o t
I t may i n d i c a t e t h a t d i f f e r e n t mechanisms a r e i n v o l v e d i n t h e
b a c t e r i c i d a l (MBC) and b a c t e r i o s t a t i c ( M I C ) a c t i o n s o f NaF.
The a n t i m i c r o -
214 TABLE I11 EFFECTS OF DIFFERENT FLUORIDE COMPOUNDS ON S. m u t a n s
F 1u o r i d e s
MIC
NaF
Inhibitory effect
GTA a c t i v i t y
1 8 0 ppm
bacteriostatic
not effective
1 8 0 ppm
bacteriostatic
not effective
360 ppm
bacteriostatic
not effective
KF CsF NH4F NaHF2
b
NH4F HF AlF3
CrF3 K 2 T i F6 L iF COF~
45 PPm
bactericidal
effective
SnF2
45 PPm
bactericidal
n o t determined
b i a l e f f e c t o f f l u o r i d e s o n B a c t e r o i d e s m e l a n i n o g e n i c u s s u b s p e c i e s and B a c t e r o i d e s a s a c c h a r o l y t i c u s was r e p o r t e d b y Yoon g&
d. (11).
When t h e y
e x a m i n e d t h e v i a b i l i t y o f e a c h o r g a n i s m a f t e r e x p o s u r e t o NaF f o r 8 hours. t h e r e s u l t s showed t h a t NaF was t h e l e a s t e f f e c t i v e a s c o n c e n t r a t i o n s o f 500 a n d 7 0 0 ppm F w e r e r e q u i r e d t o a f f e c t t h e v i a b i l i t y .
I n the MIC
e v a l u a t i o n i n t h e present study, t h e s u s c e p t i b i l i t y o f most Bacteroides s t r a i n s was shown t o b e h i g h .
F u s o b a c t e r i u m and B a c t e r o i d e s s t r a i n s showed
a s i m i l a r s e n s i t i v i t y t o NaF. T h e M I C v a l u e s f o r P s e u d o m o n u s a n d C a n d i d a were h i g h e r than t h o s e o f o t h e r s except f o r Actinomyces.
Thus,
the results
s u g g e s t t h a t NaF h a s s e l e c t i v e e f f e c t s o n o r a l m i c r o o r g a n i s m s . T h e a d d i t i o n o f 9 0 ppmF p r o v i d e d a s NaF
reduced
bacterial
growth
Changes i n CFU o f t h e b a c t e r i a l c u l t u r e i n d i c a t e d m a r k e d i n h i b i -
slightly.
t i o n o f b a c t e r i a l g r o w t h i n t h e p r e s e n c e o f 1 8 0 ppm F a s NaF, complete a r r e s t o f b a c t e r i a l 3 6 0 ppm F.
Brown
&
d.( 1 2 )
and s u g g e s t e d
g r o w t h and b a c t e r i o s t a t i c e f f e c t a t 1 8 0 and r e p o r t e d a s i g n i f i c a n t a r r e s t o f S.
t h e p r e s e n c e o f 3 0 0 ppm a n d 600 ppm F.
mutans i n
T h i s s t u d y a l s o showed d i s t i n c t
g r o w t h i n h i b i t i o n o f S. m u t a n s a t f l u o r i n e i o n c o n c e n t r a t i o n s c o m m o n l y used f o r m o u t h w a s h (100-500 ppm F). Bibby
gt .a!
r e p o r t e d i n h i b i t o r y e f f e c t s o f NaF o n t h e g r o w t h a n d a c i d
production o f Streptococcus,
L a c t o b a c i l l u s a n d o t h e r o r a l m i c r o b e s (5).
275 According t o these authors,
t h e a c i d p r o d u c t i o n o f S t r e p t o c o c c u s and L a c t o -
b a c i l l u s d e c r e a s e d b y a b o u t 50% i n i n c u b a t i o n m e d i a c o n t a i n i n g 1 0 0 ppm NaF. Their r e s u l t s are consistent w i t h these findings t h a t acid production d e c r e a s e d a b o u t 40% i n t h e p r e s e n c e o f 90 ppm F. B u n i c k and K a s h k e t (12),
I n addition,
s t u d y i n g t h e e f f e c t s o f f l u o r i n e on
p u r i f i e d e n o l a s e d e r i v e d f r o m S, s a l i v a r i e .
S. m u t a n s a n d S. s a n g u i s ,
suggested t h a t f l u o r i n e exerted a n t i b a c t e r i a l e f f e c t s through i t s a n t i enzymic a c t i o n on enolase. water-insoluble
The e f f e c t s o f f l u o r i n e on t h e s y n t h e s i s o f
d e x t r a n h a v e b e e n e x t e n s i v e l y examined.
S h i m u r a and O n i s h i
(14) r e p o r t e d t h a t p r o d u c t i o n o f d e x t r a n d e c r e a s e d i n t h e p r e s e n c e o f NaF, while Shklair
9 ~ 1(. 15)
o b s e r v e d no change i n p r o d u c t i o n a t a f l u o r i n e
c o n c e n t r a t i o n o f 100 ppm d e s p i t e a n o b v i o u s i n h i b i t i o n o f b a c t e r i a l g r o w t h . I n t h e w o r k o f P a t r i c k (16) w i t h v a r i o u s s t r a i n s o f S. m u t a n s , f l u o r i n e was shown t o e n h a n c e t h e s y n t h e s i s o f w a t e r - i n s o l u b l e d e x t r a n b y a c c e l e r a t i n g t h e dextransucrase a c t i v i t y . o f water-insoluble
These d i s c r e p a n c i e s i n d i c a t e d t h a t p r o d u c t i o n
d e x t r a n was i n h i b i t e d b y t h e p r e s e n c e o f NaF i n t h e c e l l
g r o w i n g s y s t e m b u t t h a t t h e p r o d u c t i o n p e r u n i t o f b a c t e r i a l p r o t e i n was enhanced.
A l t h o u g h t h e e f f e c t o f NaF i n t h e c e l l - f r e e
s y s t e m was e x a m i n e d
i n t h i s s t u d y a t h i g h e r c o n c e n t r a t i o n s t h a n t h o s e employed b y S h k l a i r , i n h i b i t i o n o f d e x t r a n s y n t h e s i s was o b s e r v e d .
no
These f i n d i n g s a r e c o n s i s t -
e n t w i t h t h o s e o f S h k l a i r ( 1 5 ) a n d C a r l s s o n (17). T h e f i n d i n g s t h a t Li',
Na',
K+,
NH4+,
Cr',
a n d Cs3+ h a d n o e f f e c t o n
d e x t r a n s y n t h e s i s a r e c o n s i s t e n t w i t h t h o s e o f C a r l s s o n ( 1 7 ) and W i l l i a m
(18).
T h e s e a u t h o r s , h o w e v e r , f o u n d t h a t b i v a l e n t c a t i o n s s u c h a s Zn2+,
Co2+,
Mg2+,
Ni2+,
and Cu2+ i n h i b i t e d g l u c a n s y n t h e s i s .
was shown t o i n h i b i t g l u c a n s y n t h e s i s ,
b u t AlF3.
ZnF2,
I n t h i s study, CaF2,
Co2+
MgF2 and SnF?
w e r e n o t o b s e r v e d t o f o r m p r e c i p i t a t e s i n t h e p r e s e n c e o f p o t a s s i u m phosphate,
a c e t i c a c i d b u f f e r and t r i s - m a l a t e
buffer.
S n F 2 a n d CoF2 g a v e l o w e r M I C f o r S. m u t a n s t h a n o t h e r f l u o r i d e s .
CoF2
seems t o h a v e a s p e c i f i c s y n e r g i s m w i t h f l u o r i n e and v a r i o u s c a t i o n s ,
as i n
t h e c a s e o f SnF2. a c t i o n o f CoF2.
F u r t h e r s t u d i e s a r e needed t o c l a r i f y t h e a n t i b a c t e r i a l B e c a u s e o f t h e s p e c i f i c c y t o t o x i c i t y o f Co2+,
caution
s h o u l d be e x e r c i s e d i n t h e c l i n i c a l a p p l i c a t i o n o f t h i s agent. I n t h e present study, antibacterial effects.
NaF a n d o t h e r f l u o r i d e s w e r e s c r e e n e d f o r t h e i r The g r e a t e r a n t i b a c t e r i a l e f f e c t o f CoF2 t h a n t h a t
o f NaF, s u g g e s t e d b y t h e c o m p a r i s o n o f M I C and i n s o l u b l e d e x t r a n f o r m a t i o n , w i l l s e r v e as a u s e f u l g u i d e l i n e i n t h e development o f t h i s study.
CONCLUSION The f o l l o w i n g r e s u l t s w e r e o b t a i n e d .
276
T h e M I C o f f l u o r i d e p r o v i d e d a s NaF f o r A c t i n o m y c e s .
_____ Pseudomonus
Candida and
were higher than those f o r t h e o t h e r microorganisms.
(Candida > A c t i n o m y c e s > Pseudomonus > > B a c t e r o i d e s
=
Fusobacterium >
S. _m_ u t_ ans) The i n h i b i t o r y e f f e c t o f NaF was r e l a t e d t o f l u o r i n e c o n c e n t r a t i o n and observed as a b a c t e r i o s t a t i c action. CoF2 and SnF2 i n h i b i t e d b a c t e r i a l g r o w t h a t c o n c e n t r a t i o n s o f 2MIC and 3MIC b a c t e r i c i d a l 1y. E x c e p t f o r CoF2,
n o e f f e c t s o f f l u o r i d e (0-10000
ppm F ) o n t h e
s y n t h e s i s o f w a t e r i n s o l u b l e g l u c a n w e r e observed. REFERENCES
1. Huysen GV.
M u h l e r JC (1948) J D e n t Res 27:46-51
2. H a t t o n WE, H e b e r g a l l WH,
M u h l e r JC (1955) J D e n t Res 34:350-357
3. C u r r e s s TM (1966) NZ D e n t J 62:26-31 4. Shannon I L (1970) J O r a l Med 25:12-17 5. M a l t z M,
E m i l s o n CG (1982) J D e n t Res 61:786-790
6. B i b b y BG, Van K e s t e r e n M (1940) J D e n t Res 19:391-402 7. Japanese C o m m i t t e e f o r Judgment o f M I C ( i n J a p a n e s e ) ( l 9 8 1 ) Chemotherapy 29: 76-81 8. F u r u t a n i M, 56: 793
Y a g i T,
I w a k i M,
9. F u r u t a n i M, Y a g i T, H e a l t h 35:173-179 10. R o b y t e JF,
I w a k i M,
I i d a M.
S a t 0 T,
N o z a k i K (1984) S e i k a g a k u
I i d a M, N o z a k i K, S a t 0 T ( 1 9 8 5 ) J D e n t
C o r r i n g a n AL (1977) A r c h Biochem B i o p h y s 83:726-781
11. Yoon A, Newman MG (1980) J C l i n P e r i o d o n t o l 7: 489-494 12. Brown LR, H a n d l e r SH, Res 50: 159-1 67
H o r t o n I M , S t r e c k f u s s JL,
Dreizen
S
(1980) J D e n t
13. B u n i c k FT, K a s h k e t SK (1981) I n f e c t Immun 34:856-863 14. S h i m u r a N, 15. S h k l a i r IL,
O n i s h i M (1978) J D e n t Res 57:928-931 W a l t e r RG,
L a m b e r t s BL (1977) AADR 56:147
16. P a t r i c k T (1981) J D e n t Res 51:1601-1610 17. C a r l s s o n J (1970) A L e v a n s u c r a s e f r o m S, mutans. 18. W i l l i a m R (197) C a r b o Res 42:325-331
C a r i e s Res 4:97-113
H.Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science,Volume 27, pp. 277-284 0 1986 Elsevier Science PublishersB.V., Amsterdam - Printed in The Netherlands
EXCITATION
OF
SKELETAL MUSCLE
277
BY FLUORIDE
TOSHIMI HATTORI AND HIROSHI MAEHASHI Department of Dental Pharmacology, Matsumoto Dental College, Shiojiri 39907. Japan
ABSTRACT The effects of NaF on the mechanical and electrical responses of sciatic nerve-sartorius muscle preparations from the bullfrog were investigated i n an attempt to elucidate the mechanism of excitation of skeletal muscle induced by fluoride. NaF at concentrations above 0.1 mM augmented the twitch and tetanus evoked by indirect stimulation of the partially fatigued muscle, and above 5.0 m M it induced fibrillation. Sodium oxalate at 2.5 sodium citrate at 1.7 m M , and calcium-deficient Ringer's solution neither induced fibrillation nor augmented the twitch. NaF increased the
mM.
amp1 itude of the miniature endplate potential (m.e.p.p.) even under pretreatment with 16 uM neostigmine. but the extent of the increment was only one half as much as that by NaF alone. NaF, 0.1 m M adrenaline, 1.0 m M theophylline, and 4.0 m M dibutyryl cyclic A M P , raised the m.e.p.p. frequency and increased the quanta1 content of the endplate potential.
These
findings suggest that fluoride may induce excitation of skeletal muscle in a manner unrelated to decalcification and that such excitation may be due to the activation of adenylate cyclase in the nerve ending, the inhibition of cholinesterase, and to increased sensitivity of the ZndPlate to acetylcholine.
INTRODUCTION Fluoride affects the l i v i n g cell i n various ways, for example, decalcification, inhibition o f enzymes, and activation of adenylate cyclase, etc. It also excites skeletal muscles. Fibrillation is observed in acute fluoride poisoning (1). and fluoride decurarizes muscles blocked by 4tubocurarine (2). Many investigators have ascribed acute fluoride poisoning to decalcification (3). However, there are some reports indicating that it is due rather to other properties of fluoride, e.g., its inhibitory effect on cholinesterase (4).
Most studies on fluoride toxicity have been
performed in vivo. The experimental data are complex, and the mechanism of
278 t h e poisoning i s s t i l l unclear.
An i n v i t r o s t u d y h a s b e e n m a d e o n t h e
e f f e c t s o f s o d i u m f l u o r i d e on m e c h a n i c a l a n d e l e c t r i c a l r e s p o n s e s o f t h e skeletal
muscle o f t h e frog,
t o d e t e r m i n e w h e t h e r o r n o t f l u o r i d e causes
muscle e x c i t a t i o n t h r o u g h a mechanism independent o f d e c a l c i f i c a t i o n . MATERIALS AND METHODS Sciatic nerve-sartorius
m u s c l e p r e p a r a t i o n s f r o m t h e b u l l f r o g Rana c a t e s The
b e i a n a ( w e i g h i n g b e t w e e n 1 0 0 and 2 0 0 g) w e r e u s e d a s t h e m a t e r i a l . p r e p a r a t i o n s h a v e been d e s c r i b e d p r e v i o u s l y (5). The p r e p a r a t i o n was (volume:
v e r t i c a l l y f i x e d o n t h e b o t t o m o f a Magnus t u b e
10 m l ) c o n t a i n i n g a e r a t e d R i n g e r ' s s o l u t i o n .
T w i t c h and t e t a n u s
were evoked b y e l e c t r i c a l s t i m u l a t i o n o f t h e n e r v e o r t h e muscle. n e r v e was s t i m u l a t e d w i t h a s u c t i o n e l e c t r o d e ( v o l t a g e :
supramaximum;
d u r a t i o n , 0.1 m s e c ; f r e q u e n c y , 0.1 a n d 50 Hz f o r t w i t c h a n d t e t a n u s , pectively).
res-
D i r e c t s t i m u l a t i o n o f t h e m u s c l e was done t r a n s v e r s e l y b y
f i e l d s t i m u l a t i o n w i t h p l a t i n u m p l a t e s ( 2 0 0 V, 0.1-1.0 l o w i n g t r e a t m e n t w i t h d - t u b o c u r a r i n e c h l o r i d e (6.0-30
m s e c , 0.1 H z ) f o l -
ugiml).
Tensions i n
t h e muscle were measured i s o m e t r i c a l l y w i t h a f o r c e - d i s p l a c e m e n t (SB-IT.
The
N i h o n Kohden).
transducer
The c o n c e n t r a t i o n o f c a l c i u m i n t h e R i n g e r ' s s o l u -
t i o n was d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y . E l e c t r i c a l r e s p o n s e s o f t h e m u s c l e f i b e r were i n t r a c e l l u l a r l y recorded w i t h a n o s c i l l o s c o p e (VC-10. f i l l e d w i t h 3M KC1. extracellularly. p o t e n t i a l (e.p.p.).
N i h o n Kohden)
a glass microelectrode
The compound a c t i o n p o t e n t i a l o f t h e n e r v e was d e r i v e d
For
d e t e r m i n a t i o n o f t h e quanta1 content o f t h e endplate
t h e m u s c l e was t r e a t e d w i t h a p e r f u s a t e c o n t a i n i n g 0.5
m M CaC12 a n d 6.0 m M MgC12 t h r o u g h o u t t h e e x p e r i m e n t a n d t h e e.p.p. m i n i a t u r e e n d p l a t e p o t e n t i a l (m.e.p.p.)
w e r e r e c o r d e d (6).
and
Quanta1 c o n t e n t
w a s o b t a i n e d b y b o t h t h e d i r e c t m e t h o d ( m e a n a m p l i t u d e o f e.p.p./mean a m p l i t u d e o f m.e.p.p.)
and t h e f a i l u r e s method,
i m p u l s e s / n u m b e r o f f a i l u r e s o f e.p.p.)
(6).
log,
(number o f nerve
The v a l u e s o b t a i n e d b y t h e
d i r e c t m e t h o d w e r e c o r r e c t e d f o r n o n l i n e a r s u m m a t i o n (7). The c o m p o s i t i o n o f n o r m a l R i n g e r ' s s o l u t i o n ( i n m M ) was a s f o l l o w s : NaC1, 5.6.
1 1 0 ; KC1,
1.9;
CaC12,
1.1;
T h e pH w a s a d j u s t e d t o 7.3.
s o d i u m f l u o r i d e (NaF),
NaH2P04,
0.4;
NaHC03.
2.4:
and g l u c o s e ,
The f o l l o w i n g c h e m i c a l s w e r e used:
sodium o x a l a t e , sodium c i t r a t e . and t h e o p h y l l i n e
(Na k a r a i ); n e o s t i g m i n e m e t h y l s u 1f a t e ( S h io n o g i); d - t u b o c u r a r in e c h 1 o r id e (d-Tc. (Sigma).
Yoshitomi);
adrenaline (Daiichi);
and d i b u t y r y l c y c l i c AMP (dbcAMP)
A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t r o o m t e m p e r a t u r e (20-25°C).
219 RESULTS NaF (0.1-10
mM) dose-dependently
a u g m e n t e d t h e t w i t c h and t e t a n u s e v o k e d
b y i n d i r e c t s t i m u l a t i o n o f t h e p a r t i a l l y f a t i g u e d m u s c l e whose t w l t c h t e n s i o n had d e c r e a s e d t o 60-70% o f t h e m a x i m a l v a l u e .
The e f f e c t o f NaF on
t h e t w i t c h i s i l l u s t r a t e d i n F i g u r e 1.
even a t a concentra-
t i o n a s h i g h a s 1 0 mM,
However,
NaF.
augmented n e i t h e r t h e maximal t w i t c h n o r t h e t w i t c h
induced by d i r e c t stimulation.
NaF a b o v e 5.0 m M i n d u c e d f i b r i l l a t i o n o f
t h e u n s t i m u l a t e d muscle. T h e Ca c o n c e n t r a t i o n i n t h e R i n g e r ' s s o l u t i o n w a s l o w e r e d f r o m 1.1 t o
0.94 mM b y 5 mM NaF.
To c o m p a r e NaF w i t h v a r i o u s c h e m i c a l a g e n t s f o r t h e i r
Ca-reducing e f f e c t ,
we i n v e s t i g a t e d t h e e f f e c t s o f t h e d e c a l c i f y i n g d r u g s ,
s o d i u m o x a l a t e (2.5
m M ) and s o d i u m c i t r a t e (1.7
t o 5.0 m M NaF i n t e r m s o f C a - b i n d i n g c a p a c i t y , c o n t a i n i n g 0.94
m M Ca.
mM),
which are equivalent
and C a - d e f i c i e n t R i n g e r
None o f t h e s e i n d u c e d f i b r i l l a t i o n o r a u g m e n t e d t h e
twitch.
180,
F i g u r e 1. L o g d o s e - r e s p o n s e c u r v e o f NaF o n t h e t w i t c h t e n s i o n o f t h e z a r t o r i u s muscle evoked b y s c i a t i c n e r v e s t i m u l a t i o n o f a p r e p a r a t i o n f r o m the bullfrog. NaF a u g m e n t e d t h e t w i t c h d o s e - d e p e n d e n t l y . Each p o i n t r e p r e s e n t s t h e mean o f r e l a t i v e v a l u e s (8-9 e x p e r i m e n t s ) o f t h e t w i t c h t e n s i o n s o b s e r v e d 3 m i n u t e s a f t e r a p p l i c a t i o n o f NaF c o m p a r e d w i t h t h a t before the application. The v e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d d e v i a t i o n . +$, ?yric: S i g n i f i c a n t l y d i f f e r e n t f r o m t h e v a l u e b e f o r e t h e a p p l i c a t i o n a t p<0.05 and p
280
200 r
-2
180
-
u
$e
-
ti d
f
140120-
looL0:5 1:O 210 5:O NgF concentration (mM) Figure 2. Rise of m.e.p.p. frequency produced by NaF. Each polnt represents the mean o f flve relatlve values compared with those before the application. )ep~0.05,i'"p
The effects of NaF on the nerve and muscle were studied to ascertain that they were not the sites of NaF action. As expected, NaF (5.0 and 10 mM) had no of the (r.p.), muscle
effect on their electrical responses, i.e., the alpha-wave amplitude compound action potential of the nerve and the resting potential membrane resistance, and wave form of the action potential of fiber remained unchanged by NaF treatment.
The influence of NaF on the electrical phenomena in the endplate was also NaF at 0.5-5.0 m M raised the m.e.p.p. frequency (i.e. the rate of spontaneous release of the transmitter) and increased the amplitude investigated.
of not only the m.e.p.p. but also the e.p.p. The increase of the m.e.p.p. frequency produced by NaF is shown in Figure 2. Since the elevation of m.e.p.p. frequency and e.p.p. amplitude seemed to be related to the improvement of transmitter release from the nerve ending, the effect of NaF on the quantal content of e.p.p. (i.e., the amount of transmitter released in response to a single nerve impulse) was studied. NaF increased the quantal contents calculated by both methods, as illustrated in Figure 3. The quantal content obtained by the direct method was corrected by Martin's method (7); namely, the value was multiplied by (r.p.-15)/(r.p.-15-e.p.p.) after r.p. was substituted by 89.4 or 92.0, obtained from measurement during the control or test period. In addition, the effects of adrenaline. an activator of adenylate cyclase. theophylline, an inhibitor of phosphodiesterase, and dbcAMP, a derivative o f cyclic AMP, on the transmitter release were studied to see how NaF affected the nerve ending. The quantal content and m.e.p.p.
frequency were increased by 0.1 mM adrenaline, 1.0 mM
theophylline, and 4.0 mM dbcAMP (Table I).
TABLE I
E F F E C T S O F ADRENALINE, THEOPHYLLINE. A N D dbcAMP
ON T H E
QUANTAL CONTENT O F e.p.p.
A N D O N THE
m.e. p. p. F R E Q U E N C Y . Treatment
Mean A m p l i t u d e o f e.p.p. ( m V )
23.7 30.0
1.50
0.31 0.36
4.53 5.15
29.3 51.0
0.13 0.87''
0.26 0.23
3.06 4.35
49.3 61.3
A d r e n a l i n e , 0.1 mM
0.85
Control
1.37
Control DbcAMP, "p
4.0 mM
Frequency o f m.e.p.p. (/min)
2.52 3.31
0.70
1.0 mM
Corrected Quanta1 Content
0.30 0.30
Control
Theophylline,
Mean A m p l i t u d e o f m.e.p.p. (mV)
282
-t140r c
C
d
130 -
5 120. -
10010:5 1:O 2:O 5:O NaF concentration (mM) Figure 3. Increment o f quanta1 content of e.p.p. produced by NaF. Each point represents the same as in Figure 2. 0 : direct method, A : failures m e t h od. 3c p < 0.0 5, 3*"p<0.0 1.
The effect of neostigmine, an inhibitor of cholinesterase (ChE), was investigated to find out whether the increase in the m.e.p.p.
amplitude
induced by NaF was related to C h E inhibition. Figure 4 shows that 5.0 m M NaF increased the m.e.p.p. amplitude about twice as much as neostigmine (16 uM). a concentration 8 times higher than that which inhibited ChE up to
80%. NaF increased the amplitude even after neostigmine application. but the increase was only one half as much as that by NaF alone (Fig. 4).
180r
T
"i
I d 120
100
NaF
Figure 4. Effects of NaF and neostigmine on m.e.p.p. amplitude. NaF increased the m.e.p.p. amplitude more markedly than neostigmine. The extent of the increment o f m.e.p.p. amplitude by N a f under pretreatment with neostigmine was only half as much as that by NaF alone. Each column represents the same as in Figure 2.
283
DISCUSSION NaF a t c o n c e n t r a t i o n s above 0.1 stimulation,
mM augmented t h e t w i t c h evoked by n e r v e
a n d a b o v e 5.0 mM i n d u c e d f i b r i l l a t i o n .
l a t e o r sodium c i t r a t e ,
However,
s o d i u m oxa-
b o t h a t a c o n c e n t r a t i o n e q u i v a l e n t t o 5.0 m M NaF,
or C a - d e f i c i e n t R i n g e r caused n e i t h e r f i b r i l l a t i o n n o r augmentation o f These r e s u l t s i n d i c a t e t h a t NaF a t c o n c e n t r a t i o n s l o w e r t h a n 5.0
twitch.
mM induces e x c i t a t i o n t h r o u g h a m e c h a n i s m u n r e l a t e d t o d e c a l c i f i c a t i o n . Moreover,
s i n c e NaF h a d no e f f e c t o n t h e t w i t c h e v o k e d b y d i r e c t s t i m u l a -
t i o n n o r on t h e e l e c t r i c a l responses o f t h e muscle, i t i s u n l i k e l y t o a c t on t h e muscle f i b e r d i r e c t l y .
S i n c e d e c r e a s i n g t h e e x t e r n a l Ca c o n c e n t r a -
t i o n raises the e x c i t a b i l i t y o f the nerve
(8). t h e e f f e c t o f NaF o n t h e
compound a c t i o n p o t e n t i a l o f t h e n e r v e was i n v e s t i g a t e d . wave f o r m r e m a i n e d unchanged.
Therefore,
I t s a m p l i t u d e and
t h e s i t e o f a c t i o n o f NaF seems
t o be n e i t h e r t h e n e r v e n o r m u s c l e b u t t h e n e u r o m u s c u l a r j u n c t i o n .
P.s t h e m.e.p.p.
frequency i s e n t i r e l y c o n t r o l l e d by t h e conditions o f t h e
p r e s y n a p t i c membrane (9),
i n c r e a s e i n f r e q u e n c y b y NaF s h o w e d t h a t NaF
a c t e d on t h e n e r v e e n d i n g and i n c r e a s e d t h e s p o n t a n e o u s r a t e o f t r a n s m i t t e r release.
I n addition,
i t a l s o s u g g e s t e d t h a t t h e i n c r e a s e i n t h e e.p.p.
a m p l i t u d e may b e d u e t o i m p r o v e m e n t o f t h e e v o k e d r e l e a s e o f t h e t r a n s mitter.
Thus,
t h e q u a n t a l c o n t e n t o f t h e e.p.p.
was measured.
5.0 m M i n c r e a s e d t h e q u a n t a l c o n t e n t , i n d i c a t i n g t h a t
NaF a t
0.5-
NaF i n c r e a s e d t h e
amount o f t r a n s m i t t e r r e l e a s e d b y a n e r v e impulse. S i n c e NaF a c t i v a t e s a d e n y l a t e c y c l a s e , various t i s s u e s (lo),
a l l o f w h l c h show a s i m i l a r a c t i o n , increased the
t h u s r a i s i n g t h e cAMP l e v e l i n
t h e e f f e c t s o f adrenaline,
theophylline,
were studied.
and dbcAMP,
Each o f t h e t e s t e d d r u g s
q u a n t a l c o n t e n t a n d r a i s e d t h e m.e.p.p.
frequency.
i m p l i e s t h a t cAMP may be i n v o l v e d i n t h e t r a n s m i t t e r r e l e a s e .
This
This idea i s
s u p p o r t e d b y m a n y r e p o r t s d e s c r i b i n g t h e r o l e o f cAMP i n n e u r o m u s c u l a r t r a n s m i s s i o n (11). dbcAMP,
F o r example, W i l s o n (12) i n v e s t i g a t e d t h e e f f e c t s o f
theophylline,
and a m i n o p h y l l i n e ( a n i n h i b i t o r o f p h o s p h o d i e s t e r a s e ) and r e p o r t e d t h a t a l l o f t h e t e s t e d
o n t h e t r a i n s o f r e p e t i t i v e e.p.p.s
d r u g s i n c r e a s e d t h e q u a n t a l c o n t e n t o f t h e f i r s t e.p.p.. and m o b i l i z a t i o n r a t e ,
releasable store,
s u g g e s t i n g t h a t cAMP was i n v o l v e d i n t h e s y n t h e s i s ,
m o b i l i z a t i o n , and s t o r a g e o f Ach.
We b e l i e v e , t h e r e f o r e ,
t h a t NaF i m p r o v e s
Ach r e l e a s e t h r o u g h e l e v a t i o n o f cAMP c o n c e n t r a t i o n s i n t h e n e r v e e n d i n g . B e c a u s e NaF r e d u c e s t h e a c t i v i t y o f ChE, m.e.p.p.
a m p l i t u d e was i n v e s t i g a t e d .
t h e i n f l u e n c e o f NaF o n t h e
T h e i n c r e a s e i n t h e m.e.p.p.
amp1 i-
t u d e b y NaF u n d e r p r e t r e a t m e n t w i t h n e o s t i g m i n e was o n l y o n e h a l f a s much as t h a t b y NaF a l o n e ;
therefore,
t h e p o t e n t i a t i o n o f t h e m.e.p.p.
may be p a r t i a l l y due t o t h e a n t i - C h E
a c t i o n o f NaF.
amplitude
284 NaF i n c r e a s e d t h e m.e.p.p. stigmine. t o Ach,
a m p l i t u d e e v e n u n d e r p r e t r e a t m e n t w i t h neo-
T h i s i n d i c a t e s t h a t NaF r a i s e d t h e s e n s i t i v i t y o f t h e e n d p l a t e
f o r t h e a m p l i t u d e o f t h e m.e.p.p.
t h e p o s t s y n a p t i c membrane (9). t h e i n c r e a s e i n t h e m.e.p.p.
i s c o n t r o l l e d by p r o p e r t i e s o f
Koketsu and Gerard ( 2 ) have d e s c r i b e d t h a t
a m p l i t u d e induced by NaF i s due t o t h e r i s e o f
Ach s e n s i t i v i t y r a t h e r t h a n t o ChE i n h i b i t i o n . F r o m t h e r e s u l t s d e s c r i b e d above,
t h e mechanism o f e x c i t a t i o n o f t h e
s k e l e t a l muscle by NaF i s t h o u g h t t o be as f o l l o w s : a t e cyclase,
spontaneous o r evoked r e l e a s e o f t h e t r a n s m i t t e r ; raised,
ChE i s i n h i b i t e d ,
increased;
NaF a c t i v a t e s adenyl-
t h u s r a i s i n g t h e cAMP l e v e l i n t h e n e r v e ending: a n d t h e Ach
t h e m.e.p.p.
cAMP improves frequency i s
s e n s i t i v i t y o f the endplate i s
d e p o l a r i z a t i o n o f t h e e n d p l a t e r e g i o n by Ach i s made so i n t e n -
s i v e t h a t t h e a c t i o n p o t e n t i a l i s generated;
f i n a l l y f i b r i l l a t i o n occurs o r
t h e t w i t c h i s a u g m e n t e d as a r e s u l t o f r e c r u i t m e n t o f t h e m u s c l e f i b e r s . It
IS
concluded t h a t f l u o r i d e may induce e x c i t a t i o n o f s k e l e t a l muscles i n
a fashion unrelated t o d e c a l c i f i c a t i o n ;
t h a t i s , e x c i t a t i o n may be due t o
t h e a c t i v a t i o n o f a d e n y l a t e c y c l a s e i n t h e nerve ending, ChE,
the i n h i b i t i o n o f
and t o t h e i n c r e a s e d s e n s i t i v i t y o f t h e e n d p l a t e t o Ach.
REFERENCES 1. Tappeiner H.
(1889) Arch Exper P a t h o l Pharmakol 25:203-224
2. Koketsu K, Gerard RW (1956) Am J P h y s i o l 186:278-282 3. Haynes RC. M u r a d F ( 1 9 8 0 ) I n : Goodman c o l o g i c a l B a s i s o f Therapeutics, 1550
4.
6 t h ed.
LS.
G i l m a n A ( e d s ) The PharmaM a c M i l l a n . New York, pp 1525-
Roholm K ( 1 9 3 8 ) I n : Handbuch d e r E x p e r i m e n t e l l e n P h a r m a k o l o g i e , Erganzungswerk, 7. J u l i u s S p r i n g e r , B e r l i n , pp 1-62
5. H a t t o r i T (1984) Matsumoto Shigaku 10:17-23
6. Del C a s t i l l o J, K a t z B (1954) J P h y s i o l 124:560-573 7. M a r t i n AR (1955) J P h y s i o l 130:114-122 8. Frankenheuser B,
Proc Roy SOC B i o l 155:455-477
9. K a t z B (1962) 10. S u t h e r l a n d EW, 11. Goldberg AL,
Hodgkin AL (1957) J P h y s i o l 137:218-244
R a l l TW,
Menon T ( 1 9 6 2 ) J B i o l Chen 237:1220-1227
S i n g e r J J (1969) Proc Nat Acad S c i 64: 134-141
12. Wilson DF (1974) J Pharmacol Exp Ther 188:447-452
H.Tsunoda and M.-H. Y u (Editors) Fiuoride Research 1985, Studies in Environmental Science,Volume 27,pp. 285-297 0 1986 Elsevier Science PublishersB.V., Amsterdam - Printed in The Netherlands
285
BIOLOGICAL SIGNIFICANCE OF FLUORIDE I N FISH TEETH SHOICHI SUGA'.
YASUHIKO TAK12, AND KOJI WADA3
'Address all correspondence to Dr. S. Suga, Department of Pathology, Nippon Dental University. 1-9-20, Fujimi, Chiyoda-ku, Tokyo, Japan 'Tokyo University o f Fisheries, Tokyo, Japan 3National Research Institute of Aquaculture, Mie. Japan ABSTRACT The teleost fishes may be classified into t w o groups
terms of the fluoride concentration in the enameloid of teeth: one group containing fluoride of more than about 1.4% i n the enameloid, and the other containing in
less than about 0.3%. There is no correlation between the fluoride concentration i n the enameloid or that i n the environmental water, the shape and size of teeth which show diverse morphological adaptation, and the feeding habits. The fluoride concentration in the enameloid i s related to the phylogeny of fishes. The enameloid is considered to provide a site where excess fluoride i n the body fluids is excreted i n order to detoxify the fishes. INTRODUCTION The fluoride concentration in the enamel of mammals is generally considered closely related to that i n their drinking water and diet.
However,
preliminary investigations of fluoride concentration in the teeth o f various teleosts and elasmobranchs from both marine and freshwater indicated that fish may be divided into t w o groups i n terms of fluoride concentrations i n the enameloid: one group containing more than about 2.5% fluoride in the enameloid, and the other containing less than about 0.17%. I n addition, it can be shown that the fluoride concentration in the enameloid might be related to fish phylogeny rather than to the environmental waters. (Marine water over all the oceans contains about 1.3 ppm fluoride (1) whereas freshwater contains less than 0.1 ppm) (2-4). I n order to study more precisely the relationship between the fluoride concentration in the enameloid, the fish phylogeny and the environmental water, quantitative fluoride analyses were performed on the teeth of many fishes collected systematically. Efforts were focused particularly on bony fishes (Osteichthyes) because there are more species of bony fishes in the world than cartilaginous fishes (Chondrichthyes) and they are distributed in
various geographical and environmental conditions.
286 The e n a m e l o i d o f f i s h h a s been c o n s i d e r e d a s a p r i m i t i v e f o r m o f enamel i n amphibians.
r e p t i l e s and mammals.
It i s h i g h l y m i n e r a l i z e d ,
although
i t s o r g a n i c m a t r i x c o m p r i s e s b o t h e c t o d e r m a l and m e s o d e r m a l p r o d u c t s w h i l e t h e enamel o f t e t r a p o d s c o n t a i n s o n l y e c t o d e r m a l products. T h i s paper i s a r e v i e w o f t h e r e s u l t s o f o u r r e c e n t i n v e s t i g a t i o n s i n t o t h e t e e t h o f f i s h e s b e l o n g i n g t o some i m p o r t a n t g r o u p s (5-7). MATERIALS AND METHODS The g r o u n d s e c t i o n s o f t e e t h and bone embedded i n p o l y e s t e r r e s i n w e r e f i r s t microradiographed u s i n g a s o f t X-ray mineralization pattern, b l o c k w i t h epoxy resin.
apparatus t o examine t h e
and t h e n m o u n t e d on a p o l i s h e d s u r f a c e o f a l u m i n u m A f t e r applying a t h i n coating o f carbon t o the
p o l i s h e d s u r f a c e o f t h e specimens,
quantitative
microprobe analyses were performed.
The e l e c t r o n m i c r o p r o b e a p p a r a t u s used
was a Shimadzu-ARL,
t y p e EMX-SM,
and l i n e scan e l e c t r o n
equipped w i t h a wavelength d i s p e r s i v e
m o n o c h r o m e t e r (WDX). Q u a n t i t a t i v e f l u o r i d e a n a l y s i s was c a r r i e d o u t b y m e a s u r i n g t h e i n t e n s i t y of
FKa e m i s s i o n o b t a i n e d b y t h e m o d e o f
point
analysis,
using the
c o n v e r g e n t m e t h o d w i t h f l u o r a p a t i t e ( D u r a n g o ) a s a s t a n d a r d sample. a n a l y s i s was made m a i n l y i n t h e s u r f a c e l a y e r o f e n a m e l o i d . l a y e r o f dentin,
Point
the middle
and t h e t o o t h s u p p o r t i n g bone.
RESULTS AND DISCUSSION Distribution o f fluoride i n the teeth A l i n e s c a n a n a l y s i s was c a r r i e d o u t a l o n g a l i n e c r o s s i n g b o t h e n a m e l o i d and d e n t i n l a y e r s o f t h e t e e t h .
I n t h e t e e t h o f some f i s h e s ,
the results
i n d i c a t e d t h a t f l u o r i d e c o n c e n t r a t i o n was much h i g h e r i n t h e e n a m e l o i d t h a n i n t h e dentin.
The c o n c e n t r a t i o n was t h e h i g h e s t i n t h e s u r f a c e l a y e r o f
t h e e n a m e l o i d and d e c r e a s e d g r a d u a l l y t o w a r d s t h e e n a m e l o i d - d e n t i n j u n c t i o n and t h e n showed a marked d e c r e a s e i n t h e d e n t i n . l e v e l was v e r y l o w i n t h e d e n t i n ,
Although the f l u o r i d e
i t tended t o increase s l i g h t l y from t h e
pulpal side towards the enameloid-dentin
j u n c t i o n i n the narrow dentin
F i g u r e 1. A c a n t h o p a g r u s s c h l e g e l i ( m a r i n e f i s h o f t h e P e r c i f o r m e s ) . a - M i c r o r a d i o g r a m o f g r o u n d s e c t i o n o f d e v e l o p i n g and e r u p t e d t e e t h . E l e c t r o n m i c r o p r o b e l i n e s c a n a n a l y s i s was p e r f o r m e d a l o n g t h e l i n e s d r a w n o n t h e m i c r o r a d i o g r a m ( x 80). b - L i n e s c a n a n a l y s i s o f CaKa, MgKa and FKa p e r f o r m e d a l o n g l i n e b o n t h e d e v e l o p i n g t o o t h germ a t t h e l a t e stage. c-Line scan a n a l y s i s performed a l o n g l i n e c on t h e e r u p t e d t o o t h . F l u o r i d e c o n c e n t r a t i o n i s much h i g h e r i n t h e e n a m e l o i d t h a n i n t h e Magnesium c o n c e n t r a t i o n i s l o w e r i n t h e e n a m e l o i d t h a n i n t h e dentin. d e n t i n . ES, e n a m e l o i d s u r f a c e ; EDJ, e n a m e l o i d - d e n t i n j u n c t i o n .
287
1b
Ic
Enamcsloidl
u
Enamsloid
Dbntin
288
l a y e r adjacent t o the enameloid-dentin
j u n c t i o n ( F i g s . 1 a n d 5).
A high
f l u o r i d e c o n c e n t r a t i o n was o b s e r v e d n o t o n l y i n t h e e r u p t e d e n a m e l o i d ,
but
a l s o i n t h e d e v e l o p i n g e n a m e l o i d a t a l a t e s t a g e o f m i n e r a l i z a t i o n (7.8) ( F i g . 1).
On t h e o t h e r h a n d , i n t h e t e e t h o f o t h e r f i s h e s , b o t h e n a m e l o i d
and d e n t i n a l s o showed a v e r y l o w f l u o r i d e l e v e l ( F i g .
6).
Fluoride concentration i n the teeth o f Perciform fishes (5) The P e r c i f o r m e s , fishes,
one o f t h e l a r g e s t o r d e r s o f t e l e o s t f i s h e s o f bony
i n c l u d e a c o n s i d e r a b l e number o f f r e s h w a t e r s p e c i e s i n a d d i t i o n t o
t h e predominant marine forms.
T h e s e f r e s h w a t e r s p e c i e s c a n be c l a s s i f i e d
i n t o s e v e r a l g r o u p s w h i c h d i f f e r i n t h e p r o c e s s o f e s t a b l i s h m e n t as freshwater
forms,
e.g.,
primary freshwater fishes which are s t r i c t l y
i n t o l e r a n t o f s a l i n e environments,
secondary f r e s h w a t e r
f i s h e s which are
r a t h e r s t r i c t l y c o n f i n e d t o f r e s h w a t e r b u t may be t o l e r a n t o f s e a w a t e r t o a c e r t a i n extent,
and v i c a r i o u s f r e s h w a t e r f i s h e s t h a t a r e s t r i c t l y
f r e s h w a t e r r e p r e s e n t a t i v e s o f p r i m a r i l y m a r i n e g r o u p s (9). The s p e c i m e n s used r e p r e s e n t 78 s p e c i e s f a l l i n g i n t o 40 f a m i l i e s i n 10 suborders.
The t e e t h o f f i s h e s o f t h e s e o r d e r s showed v a r i o u s shapes and
arrangements.
Some f i s h e s h a d s e p a r a t e a n d w e 1 1 - d e v e l o p e d ,
m o l a r - 1 ik e
(Fig. l a ) o r c h i s e l - l i k e (Fig. 2) t e e t h and o t h e r f i s h e s had sharp-edged, b e a k - l i k e t o o t h p l a t e s composed o f s u c c e s s i v e r o w s o f a r r o w h e a d - l i k e
teeth
w h i c h w e r e s u r r o u n d e d b y a l v e o l a r bone ( F i g . 3). The d i s t r i b u t i o n o f f l u o r i d e c o n c e n t r a t i o n s d e t e r m i n e d b y p o i n t a n a l y s i s i s p l o t t e d i n F i g u r e 4.
The e n a m e l o i d o f a l l t h e P e r c i f o r m s p e c i e s
examined c o n t a i n e d r a t h e r h i g h f l u o r i d e concentrations, t o 5.45%,
i n the surface layer,
t h a n 0.75% and 0.63% f l u o r i d e ,
r a n g i n g f r o m 1.44%
w h e r e a s t h e d e n t i n and bone c o n t a i n e d l o w e r respectively.
The f l u o r i d e c o n c e n t r a t i o n o f
t h i s o r d e r w a s m u c h h i g h e r t h a n t h a t i n h u m a n e n a m e l ( a b o u t 0.2% i n t h e s u r f a c e l a y e r ) and i n t h e e n a m e l o i d o f C y p r i n u s c a r p i o (common c a r p : 0.17%)(4)
and Fugu r u b r i p e s r u b r i p e s ( p u f f e r :
t h a t t h e r e was
0.17%)(6).
It was r e c o g n i z e d
n o s i g n i f i c a n t d i f f e r e n c e i n f l u o r i d e c o n c e n t r a t i o n among
m a r i n e and f r e s h w a t e r s p e c i e s o f d i f f e r e n t d i v i s i o n s . Fluoride concentration i n the teeth o f Tetraodontiform fishes (6) The T e t r a o d o n t i f o r m s a r e c o n s i d e r e d t o h a v e e v o l v e d f r o m P e r c i f o r m s d u r i n g t h e Eocene p e r i o d o f t h e C e n o z o i c Era. f i e d i n t o t w o suborders,
L i v i n g examples a r e c l a s s i -
t h e p r i m i t i v e B a l i s t o i d e i which contains s i x fami-
l i e s and t h e m o r e a d v a n c e d T e t r a o d o n t o i d e i
containing four
The B a l i s t o i d e i h a v e s e p a r a t e and well-developed, l o n g r o o t s (Fig.
5a).
On t h e o t h e r hand,
f a m i l i e s (10).
chisel-like teeth with
t h e T e t r a o d o n t o i d e i have a k n i f e -
shaped p l a t e f o r m e d b y f u s i o n o f s u c c e s s i v e r o w s o f d e v e l o p i n g p l a t e - l i k e teeth,
w h i c h a r e s u r r o u n d e d b y a l v e o l a r bone ( F i g .
6a).
289
F i g u r e 2. M i c r o r a d i o g r a m o f c h i s e l - l i k e t o o t h o f E p i n e p h e l u s l a t i f a s c i a t u s ( m a r i n e f i s h o f t h e P e r c i f o r m e s ) ( x 100)(5) F i g u r e 3. M i c r o r a d i o g r a m o f p l a t e - l i k e t o o t h o f S c a r u s g i b b u s ( m a r i n e f i s h o f t h e P e r c i f o r m e s ) ( x 80)(5)
290
A q u a n t i t a t i v e p o i n t analysis o f f l u o r i d e (Fig. 7 ) i n d i c a t e d t h a t t h e enameloid o f
all
the
Balistoidel
specimens
contained
r a n g i n g f r o m 2.31% t o 5.16%.
concentration o f fluoride.
a
very
hlgh
whereas i n t h e
T e t r a o d o n t o i d e i t h e c o n c e n t r a t i o n ranged o n l y f r o m 0.14% t o 0.28%. It s h o u l d a l s o be n o t e d t h a t t h e e n a m e l o i d o f t h e f a m i l y f r o m w h i c h t h i s o r d e r may have d e r i v e d ( t h e A c a n t h u r i d a e o f t h e P e r c i f o r m e s ) c o n t a i n e d v e r y h i g h amounts o f f l u o r i d e .
It i s speculated t h a t the f l u o r i d e concentrating
mechanism may have been l o s t d u r i n g t h e e v o l u t i o n o f t h e T e t r a o d o n t o i d e i . F l u o r i d e c o n c e n t r a t i o n s i n t h e d e n t i n r a n g e d f r o m 0.14% t o 0.82% i n a l l t h e f i s h e s o f t h i s o r d e r w i t h a tendency t o be s l i g h t l y h i g h e r i n t h e B a l i s t o i d e i than i n the Tetraodontoidei.
F l u o r i d e i n t h e bone showed a
s i m i l a r pattern. Role and s i g n i f i c a n c e o f h i g h d e p o s i t i o n o f f l u o r i d e i n t h e f i s h enameloid The f i n d i n g s d e s c r i b e d above s u p p o r t t h e p r e v i o u s s p e c u l a t i o n t h a t t h e f l u o r i d e concentration
i n the enameloid o f f i s h t e e t h i s r e l a t e d t o
phylogeny o f f i s h e s r a t h e r t h a n t h e f l u o r i d e c o n c e n t r a t i o n i n t h e e n v i r o n mental water.
R e s u l t s o f o t h e r r e c e n t i n v e s t i g a t i o n s p e r f o r m e d on t h e
t e e t h o f f i s h e s o f o t h e r formes,
s u c h as t h e O s t a r i o p h y s a n f i s h e s o f
t e l e o s t s (11) and t h e elasmobranchs (12),
a l s o support t h i s speculation.
The t e e t h o f f i s h e s show a r e m a r k a b l e r a n g e o f a d a p t a t i o n i n f o r m and size,
a s s o c i a t e d w i t h f e e d i n g h a b i t s and p h y s i c a l p r o p e r t i e s o f d i e t .
However,
i n these studies
(5,6.8).
t h e r e was no e v i d e n c e t h a t a
particular
c o r r e l a t i o n e x i s t e d between t h e f l u o r i d e c o n c e n t r a t i o n i n t h e enameloid and f o r m and s i z e o f t h e e n a m e l o i d .
I n o t h e r words,
the evolution o f the
c h e m i c a l c o m p o s i t i o n o f f i s h e n a m e l o i d i s c o m p l e t e l y independent o f t h e morphological adaptation o f teeth. Crystallographic investigation indicated that, enameloid,
fluoride
i s
apparently
present
i n the high fluoride i n
the
apatite
as
(13). C ~ ~ O ( P ~ ~ ) ~ ( Fc. rOCal0(P04)6F2 H) The f a c t t h a t t h e f l u o r i d e c o n c e n t r a t i o n i n t h e d e n t i n and b o n e o f a l l t h e f i s h e s e x a m i n e d was v e r y l o w , w h e r e a s t h e e n a m e l o i d o f some s p e c i e s contained high fluoride,
indicates t h a t the enameloid-forming
e s p e c i a l l y t h e ameloblasts.
cells,
have a p e c u l i a r a b i l i t y t o c o n c e n t r a t e t h e
f l u o r i d e i n t o d e v e l o p i n g enameloid. R e c e n t l y , i t has b e e n f o u n d t h a t i r o n i n t h e e n a m e l o i d shows a s i m i l a r phenomenon, a l t h o u g h t h e r e l a t i o n s h i p b e t w e e n i t s c o n c e n t r a t i o n and f i s h group i s n o t i d e n t i c a l w i t h t h a t o f f l u o r i d e (11,
14).
I n o r d e r t o u n d e r s t a n d t h e r o l e o f f l u o r i d e and i r o n i n e n a m e l o i d mineralization,
d e v e l o p i n g enameloids o f t w o f i s h e s ,
one c o n t a i n i n g h i g h
f l u o r i d e and i r o n (Pagrus m a j o r o f t h e P e r c i f o r m s ) and t h e o t h e r c o n t a i n i n g
3
1
2
2
Enamel o i d
Dentin
6
6
5
4
2
v
E
5 W
0
Bone
3
1 0.9 0.8 0.7 0.6
d 0.5
1 0.9
0.9
0,8
@,8
0.
0.7
0.
@,6
0.
0.5
0.
0,4
0.
0.3
0.
0,2
n,iJ
0,l
I
291
0 Marine f i s h
0 Primary freshwater f i s h
o
Vicarious freshwater f i s h Secondary freshwater f i s h
1
0 3
G
0.4 (J,
3
0.2
Human enamel \.(surface layer) rubripes. r u b r i pes
i-w 1
‘Cyprinus
0,l
carpio
F i g u r e 4. D i s t r i b u t i o n o f 78 p e r c i f o r m s p e c i e s e x a m i n e d i s p l o t t e d a c c o r d i n g t o t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e e n a m e l o i d , d e n t i n a n d b o n e (5). The f l u o r i d e c o n c e n t r a t i o n s i n human m a t u r e d enamel ( s u r f a c e l a y e r ) ( 4 ) . i n t h e e n a m e l o i d o f C y p r i n u s c a r p i o ( 4 ) and Fugu r u b r i p e s r u b r i p e s (6). and i n fluorapatite are also plotted.
292
v e r y l o w f l u o r i d e and i r o n ( C y p r i n u s c a r p i o o f t h e C y p r i n i f o r m e s ) were e x a m i n e d u s i n g m i c r o r a d i o g r a p h y and e l e c t r o n m i c r o p r o b e a n a l y s i s (7). The p r o g r e s s i v e m i n e r a l i z a t i o n p a t t e r n r e v e a l e d b y m i c r o r a d i o g r a p h y was almost
the
same
i n these
two
species.
The
process o f
progressive
m i n e r a l i z a t i o n i s c o n s i d e r e d t o be d i v i d e d i n t o a t l e a s t t h r e e s t e p s .
The
t h i r d step, w h i c h seems t o c o r r e s p o n d t o t h e m a t u r a t i o n s t a g e o f enamel formation,
i s composed o f t w o d i f f e r e n t p r o c e s s e s : one w h i c h p r o g r e s s e s
f r o m t h e i n n e r m o s t l a y e r towards t h e m i d d l e l a y e r a t t h e e a r l y stage,
and
t h e other which progresses from t h e surface l a y e r towards the inner l a y e r
b 5F :3.89
Dentin
ES
I
h
i ’”
.> /
c
-.
”
F
‘,.“*‘*
7 I .
F : OI 2 4
”\
,
F i g u r e 5. B a l i s t o i d e s c o n s p i c i l l u m ( m a r i n e f i s h o f t h e B a l i s t o i d e i o f Tetraodontiformes)(6). a - M i c r o r a d i o g r a m o f g r o u n d s e c t i o n ( x 100) b - L i n e s c a n a n a l y s i s o f CaKcr, MgKa a n d FKcr b y t h e e l e c t r o n m i c r o p r o b e , p e r f o r m e d a l o n g t h e l i n e c r o s s i n g t h e e n a m e l o i d and d e n t i n . Fluoride c o n c e n t r a t i o n i s much h i g h e r i n t h e e n a m e l o i d t h a n i n t h e d e n t i n . Magnesium c o n c e n t r a t i o n i s l o w e r i n t h e e n a m e l o i d t h a n i n t h e d e n t i n .
293
smeloid
Dentin
ES x
**
F i g u r e 6. T r i o d o n m a c r o p t e r u s ( m a r i n e f i s h o f t h e T e t r a o d o n t o i d e i o f T e t r a o d o n t i f o r m e s ) ( x 120)(6)-a - M i c r o r a d i o g r a m o f g r o u n d s e c t i o n ( x 100) b - L i n e s c a n a n a l y s i s o f CaKcc, MgKa a n d FKa b y t h e e l e c t r o n m i c r o p r o b e , p e r f o r m e d a l o n g t h e l i n e c r o s s i n g t h e m a t u r e d e n a m e l o i d and d e n t i n l a y e r s . F l u o r i d e c o n c e n t r a t i o n i s v e r y l o w i n b o t h t h e e n a m e l o i d and t h e d e n t i n . Magnesium i s l o w e r i n t h e e n a m e l o i d t h a n i n t h e d e n t i n .
294 a t t h e l a t e r stage, iron.
and i s a c c o m p a n i e d b y h e a v y d e p o s i t i o n o f f l u o r i d e and
S i n c e t h e t i m i n g o f d e p o s i t i o n and d i s t r i b u t i o n p a t t e r n s o f t h e s e
elements are n o t i d e n t i c a l
i n t h e developing enameloid.
t h a t t h e i r c o n c e n t r a t i n g mechanisms
are
i t i s considered
No p a r t i c u l a r
independent.
d i f f e r e n c e i n t h e p a t t e r n o f p r o g r e s s i v e m i n e r a l i z a t i o n was f o u n d b e t w e e n these two fishes.
I t i s t h u s c o n c l u d e d t h a t f l u o r i d e and i r o n d o n o t p l a y
any s p e c i a l r o l e i n t h e p r o c e s s o f e n a m e l o i d m i n e r a l i z a t i o n . The s i g n i f i c a n c e o f t h e d u a l d e p o s i t i o n o f f l u o r i d e and i r o n i n t h e developing
e n a m e l o i d o f some f i s h e s i s n o t u n d e r s t o o d .
A very similar
phenomenon has been p o i n t e d o u t i n t h e m i n e r a l i z e d t i s s u e s o f o t h e r b i o l o g i c a l systems,
s u c h a s t h e t e e t h o f r a d u l a o f some m o l l u s k s (15).
t h o u g h no r e a s o n a b l e i n t e r p r e t a t i o n has been g i v e n f o r such p e c u l i a r c o m b i n a t i o n s o f f l u o r i d e and i r o n d e p o s i t i o n i n t h e h e a v i l y m i n e r a l i z e d tissues. I n
certain
epithelial
tissues
(kidney,
digestive
glands
and
hepatopancreas) o f m o l l u s k s , h i g h l y i n s o l u b l e i n t r a c e l l u l a r m i n e r a l s appear i n a form o f e i t h e r crystallographically pure deposits containing w e l l
o r i e n t e d c r y s t a l s o r o f t e n amorphous m a t e r i a l s . c o n s i s t mainly o f calcium,
magnesium.
They h a v e been shown t o
phosphate and c a r b o n a t e i o n s and
contain also small
amounts o f v a r i o u s heavy metals,
aluminum.
lead,
cadmium,
iron, zinc,
evidence f o r t h e i r recycling.
such as s i l v e r ,
copper, and s i l i c o n .
There i s no
Such a b i o m i n e r a l i z a t i o n h a s been s u g g e s t e d
as a c e l l u l a r d e t o x i c a t i o n mechanism f o r c a l c i u m w h i c h i s one o f t h e p h a r m a c o l o g i c a l l y m o s t a c t i v e c a t i o n s and h e a v y m e t a l i o n s (16).
It i s speculated t h a t t h e h i g h l y m i n e r a l i z e d enameloid p r o v i d e s a s i t e w h e r e e x c e s s a m o u n t s o f f l u o r i d e and i r o n i n t h e b o d y f l u i d s a r e e x c r e t e d i n order t o d e t o x i f y t h e fishes. gland.
The a m e l o b l a s t s a c t a l s o a s an e x c r e t i n g
The e n a m e l o i d i s c o n s i d e r e d t o be a s u i t a b l e t i s s u e t o f i r m l y f i x
t h e e l e m e n t s e x c r e t e d , a s c o m p a r e d w i t h t h e d e n t i n a n d bone, b e c a u s e t h e f u l l y mineralized enameloid i s chemically very stable, t a c t with l i v i n g cells,
d o e s n o t h a v e con-
and i s n o t so p e r m e a b l e t o b o d y f l u i d s .
Further-
more, f i s h t e e t h a r e c o n t i n u o u s l y b e i n g r e p l a c e d t h r o u g h o u t t h e l i f e s p a n o f the fish.
It i s considered,
therefore,
that t h i s peculiar property o f
e n a m e l o i d e n a b l e s t h e f i s h e s t o r e m o v e e x c e s s f l u o r i d e and i r o n c o n t i n u o u s l y i n t o t h e i r s u r r o u n d i n g w a t e r s a s s t a b l e c h e m i c a l compounds w h i c h do F i g u r e 7. H i s t o g r a m s h o w i n g t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e e n a m e l o i d . The f l u o r i d e cond e n t i n and bone o f t h e f i s h e s o f T e t r a o d o n t i f o r m s (6). c e n t r a t i o n o f a l l t h e p r i m i t i v e B a l i s t o i d e i e x a m i n e d was h i g h e r t h a n 2.31%, w h e r e a s t h a t o f t h e a d v a n c e d J e t r a o d o n t o i d e i w a s l e s s t h a n 0.28%. The f l u o r i d e c o n c e n t r a t i o n s i n m a t u r e d and u n e r u p t e d human enamel and i t s t h e o r e t i c a l v a l u e i n t h e f l u o r a p a t i t e a r e a l s o shown.
295 Fluoride concentration (%) Triacanthodidae Triacanthodes nnamalus Triacanthidae Triacanthus breuirost r i s Ballst idae Canthidermis maculatus Balistoides conspicrllum Xanthichthys mento Monacanthdae S tephanolepi s cirrhifer S t ephanoleprs japonicus Rudarius ercodes Nauodon modes f us Alutera monoceros Pseudalutarrus nasicornis Aracandae Kenirocapros aculealus Ostracidae Ost r aci on cubicus Triodontdae Triodon macroplerus Tetraodontdae Fugu rubripes rubripes Fugu pardale Fugu nrphobles
0
0.25
"
LI
Om
.
I I1
w o . 1 9
0.49
a I
i
= r"cTJ
Enameloid
Fugu vermiculare porphyreum Fugu vermrculare uermiculare Lagocephalus lunaris spadiceus Tetraodon hrspidus Canthigaster rivulata Diodontdae Chilomycterus affznrs
Dentin
0Bone
D i odon ho lac ant hu s Diodon lrturosus Diodon h y s t r i r ~
Molidae Mola mola Fluorapatite Human tooth
~
Very thin enarneloid. could not be analysed
.:;:;.
- ....._. _.;_ . * :_. .:. ..: .....-_1 1 . 7 1 .......... ..,..,.: .. ..:. ...._ . .....::.:. .:_..._._
~~
'
296 n o t p o l l u t e t h e i r environment (7)(Table
I).
Thus, t h e c o n c e n t r a t i o n s o f
f l u o r i d e and i r o n i n t h e e n a m e l o i d seem t o b e r e l a t e d t o t h e e v o l u t i o n o f t h e s y s t e m i c system t o c o n t r o l body f l u i d s .
T h i s s p e c u l a t i o n may be sup-
p o r t e d b y t h e f a c t t h a t t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e d e n t i n a n d bone o f t h e f i s h e s o f B a l i s t o i d e i and P e r c i f o r m e s whose e n a m e l o i d c o n t a i n s h i g h f l u o r i d e a r e s l i g h t l y h i g h e r t h a n t h o s e o f f i s h e s o f T e t r a o d o n t o i d e i whose e n a m e l o i d c o n t a i n s l o w f l u o r i d e (5,6). TABLE
I
HISTOLOGICAL AND CHEMICAL CHARACTERISTICS OF THE ENAMELOID, DENTIN AND BONE O F F I S H A S A S I T E WHERE E X C E S S FLUORIDE AND I R O N I N BODY F L U I D S A R E EXCRETED.
M i n e r a l i z a t i o n degree Amount o f o r g a n i c substances
Enameloid
D e n t i n and bone
very high
1ow
+
+++ 1ow
high
Chemical s t a b i l i t y Permeability o f body f l u i d s Resorption by c l a s t i c cells F l u o r i d e and i r o n deposited
+
+++
-
t
a r e removed i n t o environment as s t a b l e c h e m i c a l compounds b y a t t r i t i o n and s h e d d i n g of teeth
are released p a r t l y t o body f l u i d s by r e s o r p t i o n and chemical exchange
REFERENCES 1. G r e e n h a l g h R,
R i l e y JP ( 1 9 6 3 ) N a t u r e 197:371-372
2. Suga S, Suga S.
Wada K.
3. Suga S,
Ogawa M (1978) J D e n t Res 57A:28D
4.
Wada K,
Ogawa M (1976) J D e n t Res 55D:117
S u g a S, Wada K. O g a w a M ( 1 9 8 0 ) I n : O m o r i M. W a t a b e N ( e d s ) T h e M e c h a n i s m s o f B i o m i n e r a l i z a t i o n i n A n i m a l s and P l a n t s . T o k a i U n i v P r e s s p p 229-240
5. Suga S,
T a k i Y,
Wada K ( 1 9 8 3 ) J a p a n J I c h t h y o l 30:81-93
6. Suga S,
Wada K.
Ogawa M (1981) J a p a n J I c h t h y o l 28:304-312
7. Suga S (1984) I n :
F e a r n h e a d RW. Suga S ( e d s ) T o o t h Enamel I V . S c i e n c e P u b l i s h e r s BV Amsterdam, pp 472-477
Elsevier
8. S u g a S, Wada K, O g a w a M ( 1 9 8 1 ) I n : B i n d e r K. H o h e n e g g e r M ( e d s ) F l u o r i d e M e t a b o l i s m . V e r l a g W i l h e l m M a u d r i c h , Wien,
9.
M y e r s GS ( 1 9 5 1 ) S t a n f o r d I c h t h o l B u l l 4 : l l - 2 1
pp 79-88
297
10. T y l e r JC (1980) O s t e o l o g y , Phylogeny, and H i g h e r C l a s s i f i c a t i o n o f t h e F i s h e s o f t h e O r d e r P l e c t o g n a t h i ( T e t r a o d o n t i f o r r n e s ) . US D e p t Commerce N a t O c e a n A t m o s A d m i n N a t M a r F i s h S e r v N O A A T e c h Rep NMFS C i r c 434: 422 11. S u g a S , T a k i Y ( 1 9 8 4 ) J D e n t Res 6 3 : 1 8 5 12. S u g a S ( 1 9 8 4 ) J D e n t Res 6 3 : 5 6 0 13. LeGeros RZ.
Suga S (1980) C a l c i f T i s s u e I n t 32:169-174
14. Suga S, Ogawa M (1980) J D e n t Res 59D31891 15. Lowenstam HA (1967) S c i e n c e 156:1373
16. S i r n k i s s K (1977) C a l c i f T i s s Res 24:199-200
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 299-305 0 1986 Elsevier Science Publishers B.V., Amsterdam -Printed in The Netherlands
299
EFFECTS OF FLUORIDE ON DEVELOPING ENAMEL AND DENTIN OF FAT I N C I S O R S TOSHIO ABE,
M I C H I O MASUOKA,
MASAJI
NOMURA, AND
HIROAKI
MIYAJIMA
D r u g S a f e t y E v a l u a t i o n L a b o r a t o r i e s , C e n t r a l Research D i v i s i o n , Takeda C h e m i c a l I n d u s t r i e s , Ltd., 569 6-10-1 H i m u r o - c h o T a k a t s u k i , Osaka, Japan
ABSTRACT The e f f e c t s o f f l u o r i d e on t h e d e v e l o p i n g e n a m e l and d e n t i n o f r a t i n c i sors were investigated. agents,
Rats were t r e a t e d o r a l l y w i t h t h e a n t i - n e o p l a s t i c
T e g a f u r a n d e t h y l t-6-butoxy-5-fluorohexahydro-2,
dine-r-5-carboxylate
(TAC-278),
These a n t i - n e o p l a s t i c
and NaF a t d i f f e r e n t d o s e s f o r e i g h t weeks.
agents c o n t a i n f l u o r i n e (F)
5-fluorouraci 1 a f t e r t h e treatment. enamel
ameloblasts,
d e g e n e r a t i o n and n e c r o s i s ,
and a p p e a r e d t o r e l e a s e
T e g a f u r a n d TAC-278
c h a r a c t e r i z e d by pigment-free
pathologically,
4-dioxopyrimi-
induced mottled
and c h a l k y w h i t e areas.
Histo-
a t t h e s e c r e t o r y t o m a t u r a t i o n stage,
showed
and f o r m e d a c y s t i c i r r e g u l a r a r r a y .
I n the
dentin, t h e r e were f o c a l o r d i f f u s e hypoplastic d e f e c t s r e s u l t i n g from d e g e n e r a t i o n and a t r o p h y o f o d o n t o b l a s t s .
Microradiographically,
t r a u m a t i c l i n e s r e s u l t i n g f r o m damage t o s e c r e t o r y a m e l o b l a s t s ,
calcio-
and c a l c i o -
t r a u m a t i c zones r e s u l t i n g f r o m damage t o a m e l o b l a s t s a t t h e m a t u r a t i o n s t a g e w e r e o b s e r v e d i n t h e enamel.
T h e s e c h a n g e s w e r e e s s e n t i a T l y t h e same
as t h e a b n o r m a l i t y r e s u l t i n g f r o m NaF t r e a t m e n t .
i n c i s o r s w a s h i g h e s t i n t h e NaF t r e a t e d r a t s , treated rats.
The
F
concentration i n the
a n d l o w e s t i n t h e TAC-278
The s e v e r i t y o f g r o s s l e s i o n o f t h e i n c i s o r s and t h e F
c o n c e n t r a t i o n i n t h e i n c i s o r s were w e l l correlated. i s suggested that,
From t h e s e r e s u l t s , i t
l i k e NaF, a n e x c e s s i v e d o s e o f T e g a f u r o r TAC-278 a f -
f e c t s t h e d e v e l o p i n g e n a m e l and d e n t i n o f r a t i n c i s o r s .
INTRODUCTION The e f f e c t s o f f l u o r i n e ( F ) on d e v e l o p i n g enamel and d e n t i n has been r e p o r t e d (1-7).
The t o x i c e f f e c t s o f e x c e s s i v e f l u o r i d e e x p o s u r e on t o o t h
f o r m a t i o n i s c a l l e d f l u o r o s i s o r m o t t l e d enamel (8-10).
The s e v e r i t y o f
f o r m a t i v e d e f e c t s i s d i r e c t l y r e l a t e d t o t h e amount o f f l u o r i d e i n g e s t e d ( 1 1,12). M o t t l e d enamel, c i f i e d areas,
c h a r a c t e r i z e d by pigment-free,
c h a l k y w h i t e and h y p o c a l -
was o b s e r v e d i n t h e i n c i s o r s o f r a t s t r e a t e d w i t h t h e a n t i -
300
n e o p l a s t i c agents,
appeared t o r e l e a s e 5 - f l u o r o u r a c i l I n t h i s study,
4-
T e g a f u r a n d e t h y l t-6-butoxy-5-fluorohexahydro-2.
dioxopyrimidine-r-5-carboxylate (TAC-278).
These d r u g s c o n t a i n F and
a f t e r t h e treatment.
these [mottled t e e t h were examined u s i n g h i s t o l o g i c
methods, c o n t a c t m i c r o r a d i o g r a p h y , and an a n a l y t i c method f o r i n c i s a l F concentration. series.
The s t u d y was c a r r i e d o u t as t w o i n d e p e n d e n t e x p e r i m e n t a l
I n t h e f i r s t e x p e r i m e n t , t h e h i s t o p a t h o l o g i c a l and m i c r o r a d i o -
g r a p h i c changes o f t h e i n c i s o r s were s t u d i e d .
I n t h e second e x p e r i m e n t ,
t h e r e l a t i o n o f t h e F c o n c e n t r a t i o n t o t h e s e v e r i t y o f t h e i n c i s o r damage was s t u d i e d . MATERIALS AND METHODS To s t u d y t h e h i s t o p a t h o l o g i c a l and m i c r o r a d i o g r a p h i c changes o f t h e incisors.
four-week-old
30 f e m a l e W i s t a r r a t s w e r e t r e a t e d o r a l l y w i t h 200
m g / k g / d a y o f T e g a f u r o r 500 m g / k g / d a y o f TAC-278 f o r e i g h t weeks.
When t h e
d o s i n g p e r i o d was c o m p l e t e d , e a c h a n i m a l was a n e s t h e t i z e d and p e r f u s e d w i t h 10% n e u t r a l b u f f e r e d f o r m a l i n t h r o u g h t h e a s c e n d i n g a o r t a .
One i n c i s o r
f r o m e a c h a n i m a l was r e m o v e d w i t h t h e s h e a t h o f a l v e o l a r bone and s o f t o e r i o d o n t a l t i s s u e , d e m i n e r a l i z e d w i t h 2 Na-EDTA,
embedded i n p a r a f f i n ,
s e c t i o n e d s a g i t t a l l y a t 5 Dm, and s t a i n e d w i t h h e m a t o x y l i n and e o s i n (H-E), B e r l i n b l u e , P A S a n d azan.
A s e c o n d i n c i s o r was r e m o v e d and embedded i n
o o l y e s t e r r e s i n ( R i g o l a c B ) a f t e r e t h a n o l d e h y d r a t i o n and s a g i t t a l s e c t i o n s , 5 0 t o 7 5 um t h i c k , w e r e p r e p a r e d b y g r i n d i n g .
Four microradiographs o f
undemineralized s e c t i o n were prepared a t d i f f e r e n t exposure t i m e s i n a c o n t a c t microradiographic apparatus.
The e x p o s u r e f a c t o r s w e r e s e l e c t e d
e m p i r i c a l l y t o p r o d u c e maximum c o n t r a s t b e t w e e n a r e a s w i t h s m a l l d i f f e r ences i n m i n e r a l content.
The m i c r o r a d i o g r a p h s w e r e p r e p a r e d o n Kodak
S p e c t r o s c o p i c F i l m 649-0 and d e v e l o p e d i n a Kodak X - r a y
Developer.
To s t u d y t h e r e l a t i o n s h i p b e t w e e n F c o n c e n t r a t i o n s and s e v e r i t y o f t h e i n c i s o r damage,
20 t h r e e - w e e k - o l d
i n t o f o u r groups:
female W i s t a r r a t s were d i v i d e d e q u a l l y
t h e c o n t r o l g r o u p and t h r e e e x p e r i m e n t a l g r o u p s .
Ani-
m a l s o f t h e e x p e r i m e n t a l g r o u p s w e r e t r e a t e d o r a l l y w i t h 21 m g / k g / d a y NaF,
100 mg/kg/day o f Tegafur,
T h e s e d o s e s w e r e e q u i v a l e n t t o 9.5 v g F / k g / d a y . added f l u o r i d e .
of
o r 1 4 0 m g / k g / d a y o f TAC-278 f o r e i g h t weeks. The c o n t r o l s r e c e i v e d no
When t h e d o s i n g p e r i o d was c o m p l e t e d ,
F concentration i n
t h e l o w e r i n c i s o r s was d e t e r m i n e d b y t h e L a n t h a n - A l i z a r i n C o m p l e x o n m e t h o d (13). Student's t - t e s t
was a p p l i e d t o e x a m i ne t h e s i g n i f i c a n c e o f d i f f e r e n c e
b e t w e e n t h e means o f measurements.
301 I?tSU LTS
A t t h e end o f t h e f i r s t e x p e r i m e n t a l p e r i o d ,
u p p e r and l o w e r i n c i s o r s o f
t h e c o n t r o l r a t s showed a d i s t i n c t , o r a n g e p i g m e n t a t i o n . The i n c i s o r s o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278. f r e e and c h a l k y w h i t e c o l o r . s o r s and i n T e g a f u r - t r e a t e d
on t h e o t h e r hand, showed p i g m e n t -
These c h a n g e s w e r e m a r k e d i n t h e l o w e r i n c i rats.
Some o f t h e T e g a f u r - t r e a t e d
r a t s showed
a l m o s t no i n c i s o r p i g m e n t a t i o n . B e f o r e e x p l a i n i n g t h e h i s t o p a t h o l o g i c a l f i n d i n g s o f t h e i n c i s o r s , we w i l l b r i e f l y describe t h e morphogenesis o f r a t incisors. calcify,
Rat i n c i s o r s grow,
and e r u p t c o n t i n u o u s l y t h r o u g h o u t t h e l i f e o f t h e a n i m a l ,
and
t h e r e f o r e each t o o t h shows t h e c o m p l e t e l i f e c y c l e o f t o o t h development f r o m i n c e p t i o n t o m a t u r i t y (10). o f r a t i n c i s o r s (2,4,10,14), sors
i n t o f o u r stages:
Based o n t h e m o r p h o l o g i c a l c l a s s i f i c a t i o n
we d i v i d e d t h e d e v e l o p i n g e n a m e l i n r a t i n c i proliferation,
m a t r i x formation,
transition,
and
maturation. I n t h e enamel o r g a n o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278, a t t h e t h e s e c r e t o r y t o m a t u r a t i o n s t a g e w e r e damaged. e r a t i o n and n e c r o s i s ,
ameloblasts
They showed degen-
and f o r m e d a c y s t i c i r r e g u l a r a r r a y ( F i g s .
t h e a r e a o f c y s t i c d e g e n e r a t i o n o f t h e enamel organ,
1-3).
In
d e f e c t i v e enamel
m a t r i x a n d d e p o s i t i o n o f amorphous o r g a n i c m a t e r i a l s w e r e o b s e r v e d .
Amelo-
b l a s t s a t t h e l a t e m a t u r a t i o n s t a g e w e r e i r r e g u l a r l y a r r a y e d and c o n t a i n e d a l i t t l e i r o n p i g m e n t (Fig. g r a n u l a r shape.
4).
No a b n o r m a l i t y
T h i s pigment e x h i b i t e d a g l o b u l a r o r round ::17s
o/>serv?:I
i , 1 .;;A,.
' . , - " l i f e r a t i o n stage o f
ameloblasts.
A section from the posterior t h i r d o f a lower i n c i s o r o f a r a t F i g u r e 1. A c y s t i c degeneration t r e a t e d w i t h 500 m g / k g o f TAC-278 f o r e i g h t weeks. o f t h e e n a m e l o r g a n ( 0 ) a n d a f o c a l h y p o p l a s t i c d e f e c t o f t h e d e n t i n (v) a r e observed. An a r r o w i n d i c a t e s t h e d i r e c t i o n o f i n c i s o r e r u p t i o n . H-E s t a i n , x 85. F i g u r e 2. A section from the posterior t h i r d o f a lower incisor o f a r a t t r e a t e d w i t h 200 mg/kg o f T e g a f u r f o r e i g h t weeks. Cystic degeneration o f t h e e n a m e l o r g a n and a b n o r m a l e n a m e l m a t r i x a r e o b s e r v e d . An a r r o w i n d i H--E s t a i n , x 170. cates t h e d i r e c t i o n o f i n c i s o r eruption.
ZOE:
303 I n t h e m i c r o r a d i o g r a p h s o f i n c i s o r s o f r a t s t r e a t e d w i t h T e g a f u r o r TAC278,
calcio-traumatic
l i n e s c o n s i s t i n g o f b o t h h y p e r - and h y p o m i n e r a l i z e d
c o m p o n e n t s c r o s s e d t h e enamel f r o m t h e d e n t i n o e n a m e l j u n c t i o n t o t h e s u r f a c e o f t h e e n a m e l ( F i g . 5).
T h i s p a t t e r n was e v i d e n c e d b y a r a d i o p a q u e
zone f o l l o w e d b y a zone o f d i s t i n c t r a d i o l u c e n c y . were a l s o observed i n t h e enamel o f t h e s e r a t s (Fig.
C a l c i o - t r a u m a t i c Tones 6).
The s u r f a c e l a y e r
o f t h e enamel was h y p e r m i n e r a l i z e d o n l y w i t h r e s p e c t t o t h e r e l a t i v e l y f o c a l l y h y p o m i n e r a l i z e d e n a m e l b e n e a t h it.
I n a d d i t i o n t o these lesions,
t h e p r e s e n c e o f c a l c i f i e d b o d i e s p e r i p h e r a l t o t h e d e f e c t s was o b s e r v e d (Fig.
7).
The r e s u l t s o f t h e second e x p e r i m e n t a r e g i v e n i n T a b l e I.
Fluorine
c o n c e n t r a t i o n i n t h e i n c i s o r s was t h e h i g h e s t i n t h e NaF t r e a t e d r a t s , t h e l o w e s t i;; c.': i n t h e iilci;c.:"s
-[.!. $:,-!/? -'-
'i7s
ti-ezL::l
c2rr:ilzt>d
rr?'is.
t i i t ?
and
The s e v z r i t y o f t h e d i s c o l o r a t i o n
t ! i F~1 ~ 0 ~ i i l -c72 n c e n t r a t i o n .
F i g u r e 7. A m i c r o r a d i o g r a p h o f a l o w e r i n c i s o r f r o m a r a t t r e a t e d w i t h 500 mg/kg o f TAC-278 f o r e i g h t weeks. C a l c i f i e d bodies are located outside o f t h e d e f e c t enamel (E). x 85. TABLE I
F CONCENTRATION I N THE I N C I S O R S AND DISCOLORATION OF THE I N C I S O R S
Compound
e
Control NaF
F concentration
Mol e c u 1a r Weight
(ppm)
--
60.0 f 1 3 . 3 a
42
604.6 f 37.gb
Tegafur
200
453.2
f 57.gb
TAC-278
276
146.6 f 1 7 . Z b
a V a l u e s a r e mean f SD. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l , p
-.
D iscolorationc -
-
++ +++ ++ +
304 DISCUSSION The e f f e c t s o f F on r a t i n c i s o r s h a v e been r e p o r t e d b y many w o r k e r s u s i n g d i f f e r e n t t e c h n i q u e s (1-7). follows.
T h e i r f i n d i n g s c o u l d be s u m m a r i z e d as
A c u t e f l u o r o s i s p r o d u c e s h o r i z o n t a l bands o f p i g m e n t - f r e e
i n r a t i n c i s o r enamel c o r r e s p o n d l n g t o t h e t i m i n g o f i n j e c t i o n s .
areas
Chronic
f l u o r o s i s i n t h e r a t i n c i s o r enamel v a r i e s f r o m l o s s o f p i g m e n t t o
severe
enamel hypoplasia.
demon-
The a m e l o b l a s t s a r e v e r y s e n s i t i v e t o f l u o r i d e ,
s t r a t e d by e a r l y reduction i n height.
Then f o l l o w s p r o l i f e r a t i o n o f t h e
enamel o r g a n l e a d i n g t o f o r m a t i o n o f c y s t s i n w h i c h amorphous c a l c i f i e d b o d i e s a r e found.
Microradiographic studies o f fluorosed r a t incisor
enairiel h a v e shown a h y p e r m i n e r a l i z e d s u r f a c e l a y e r s i m i l a r t o t h a t f o u n d i n human f l u o r o s e d t e e t h .
As a r e s u l t o f h i g h d o s a g e s o f f l u o r i d e ,
a b n o r m a l i t i e s may be seen i n t h e d e n t i n : h y p o i n i n e r a l i z e d i n t e r g l o b u l a r spaces,
striation,
four
hypoplastic defects,
and g r o s s d e f o r m a t i o n o f t h e e x t e r n a l
o u t l i n e o f t h e dentin. The h i s t o p a t h o l o g i c a l and m i c r o r a d i o g r a p h i c f i n d i n g s i n t h e p r e s e n t s t u d y o f r a t s . t r e a t e d w i t h T e g a f u r o r TAC-278 w e r e e s s e n t i a l l y t h e same as t h o s e observed i n f l u o r o s i s o f r a t i n c i s o r s . C y s t i c d e g e n e r a t i o n o f e n a m e l o r g a n and a b n o r m a l e n a m e l m a t r i x f o r m a t i o n w e r e o b s e r v e d i n t h e i n c i s o r s o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278. T h e s e c h a n g e s e x h i b i t e d t h e damage and p r o l i f e r a t i o n o f a m e l o b l a s t .
These
l e s i o n s d e v e l o p e d when l i m i t e d r e g i o n s o f a m e l o b l a s t a t r o p h i e d o r d i e d . surrounding ameloblasts continued t o form normal matrix.
a r e a s o f a m e l o b l a s t e p i t h e l i u m became s u r r o u n d e d b y e n a m e l m a t r i x . b l a s t s a t t h e l a t e m a t u r a t i o n stage were i r r e g u l a r l y arrayed, a l i t t l e i r o n pigment. o f pigment-free
and
I n t h i s way, Amelo-
and c o n t a i n e d
These c h a n g e s w e r e c o n s i d e r e d t o be r e l a t e d t o t h a t
enamel.
Microradiographically,
c a l c i o - t r a u m a t i c l e s i o n s were observed i n the
e n a m e l o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278.
These c a l c i o - t r a u m a t i c
l i n e s i n t h e e n a m e l w e r e c o n s i d e r e d t o r e s u l t f r o m damage t o a m e l o b l a s t s a t t h e m a t r i x f o r m a t i o n s t a g e (4.5).
The c a l c i o - t r a u m a t i c
zone was a l s o
c o n s i d e r e d t o r e s u l t f r o m damage t o a m e l o b l a s t s a t t h e m a t u r a t i o n s t a g e
(4.5).
Based on l i g h t and e l e c t r o n m i c r o s c o p i c a l
studies,
i t was assumed
t h a t t h e h y p o m i n e r a l i z e d zone i n d i c a t e d a d i s t u r b e d f u n c t i o n o f t h e s e c r e t o r y a m e l o b l a s t s d u e t o f l u o r i d e (11,15). Concerning t h e pathogenesis o f t h e enamel l e s i o n s i n t h e p r e s e n t study, h i s t o p a t h o l o g i c a l f i n d i n g s and m i c r o r a d i o g r a p h i c f i n d i n g s w e r e w e l l c o r r e lated.
305 Hypoplastic d e f e c t s i n t h e d e n t i n were observed i n r a t s t r e a t e d w i t h T e g a f u r o r TAC-278. excessive
These changes w e r e c o n s i d e r e d t o be t h e r e s u l t o f an
dosage o f F because t h e d e n t i n w a s n o t a s s e n s i t i v e t o F a s t h a t
o f t h e e n a m e l (8.10).
A l t h o u g h t h e r a t s w e r e t r e a t e d w i t h NaF. taining equivalent molecular weight o f F,
Tegafur,
o r TAC-278,
each con-
the F concentration i n the inci-
s o r s was f o u n d t o be t h e h i g h e s t a f t e r NaF t r e a t m e n t and t h e l o w e s t a f t e r TAC-278 t r e a t m e n t .
These d i f f e r e n c e s w e r e c o n s i d e r e d t o be r e l a t e d t o t h e
r a t e o f m e t a b o l i s m o f t h e compound.
S e v e r i t y o f g r o s s l e s i o n of t h e i n c i -
s o r s and t h e F c o n c e n t r a t i o n i n t h e i n c i s o r s w e r e w e l l c o r r e l a t e d .
The
e x t e n t o f damage i s d i r e c t l y r e l a t e d t o t h e amount o f f l u o r i d e i n g e s t e d (11,12). From t h e s e r e s u l t s ,
i t i s suggested t h a t ,
l i k e NaF.
an e x c e s s i v e dose o f
T e g a f u r o r TAC-278 a f f e c t s t h e d e v e l o p i n g e n a m e l a n d d e n t i n o f r a t i n c i sors. REFERENCES 1. Schour I , S m i t h MC (1934) P r o c SOC Exp B i o l Med 3 2 : l - 2
2. O t a k e T (1960) O d o n t o l o g y 48:l-54 3. Yaeger JA (1966) Am J A n a t 118:665-684 4. I s h i d a T (1970) J a p J O r a l B i o l 12:88-107
5. Hasegawa K (1971) J a p J O r a l B i o l 13:239-254 6.
I j u i n N (1971) J a p J O r a l B i o l 13:39-61
7. S h i n o d a H (1975) C a l c i f T i s s Res 18:91-100
8. P i n d b o r g J J ( 1 9 7 0 ) P a t h o l o g y o f t h e D e n t a l H a r d T i s s u e s . M u n k s g a a r d , Copenhagen. p 162 9. N i k i f o r u k G. G r a i n g e r RM (1964) I n : B e a t o n GH. McHenry EW (eds) N u t r i t i o n - A Comprehensive Treatise. Academic Press, New York, pp 417-461 10. S c h o u r I , M a s s l e r M ( 1 9 4 9 ) I n : F a r r i s EJ, G r i f f i t h JQ ( e d s ) The R a t i n L a b o r a t o r y I n v e s t i g a t i o n . J B L i p p i n c o t t Company, London, pp 104-165 11. K r u g e r BJ (1970) A r c h O r a l B i o l 15:109-114 12. Angmar-Mansson
B,
E r i c s s o n Y,
Ekberg, 0 (1976) C a l c i f T i s s Res 22:77-84
13. P h a r m a c e u t i c a l S o c i e t y o f J a p a n (1980). K i n b a r a P u b l i c a t i o n , Tokyo, P 60 14. Warshawsky H, S m i t h CE (1974) A n a t Rec 179:423-446 15. W a l t o n RE,
Eisenmann.
DR (1974) A r c h O r a l B i o l 19:171-182
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 307-312 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
EFFECTS OF LONG TERM,
LOW DOSE INGESTION
OF
307
FLUORIDE ON THE THYROID GLAND
I N RATS
MITSURU TANAKA’,
ISAO
TSUCHIDA~,
O K A Y A S U ~YUHKO K O H Y A M A ~ . HIROKO
FUMIYOSHI Y A N A G I S A W A ’ ,
KURIHARA~,
HEIZO
C H I G U S A DATE3, MASAYUKI HAYASH13. K A Y O
MU13, AND MASAHIRO ASADA3 ’ D e p a r t m e n t o f E p i d e m i o l o g y , M e d i c a l R e s e a r c h I n s t i t u t e . T o k y o M e d i c a l and D e n t a l U n i v e r s i t y , No. 3-10, 2-Chome, K a n d a - S u r u g a d a i C h i y o d a - k u , T o k y o 101, J a p a n ‘Pathology Center, U n i v e r s i t y H o s p i t a l . and D e n t a l U n i v e r s i t y 3Department o f P u b l i c Health,
School o f Medicine.
Tokyo M e d i c a l
Osaka C i t y U n i v e r s i t y M e d i c a l S c h o o l
ABSTRACT Four-week-old c o n t a i n i n g 0,
3.
W i s t a r m a l e r a t s w e r e g i v e n d i s t i l l e d w a t e r ad l i b i t u m
6,
12,
a n d 2 5 ppm F- a s NaF.
A t t h e end o f 20 months,
m o r p h o l o g i c a l changes and e n d o c r i n o l o g i c a l d a t a on t h e t h y r o i d g l a n d i n these animals were studied. H y p e r p l a s t i c nodules c o n s i s t i n g o f t h y r o i d p a r a f o l l i c u l a r c e l l s (C c e l l s ) w e r e o b s e r v e d i n t h e e x p e r i m e n t a l a n i m a l s r e c e i v i n g F-.
Both c a l c i t o n i n
and c h a r a c t e r i s t i c s e c r e t o r y g r a n u l e s w e r e d e t e c t e d i n t h e c y t o p l a s m s o f these hyperplastic cells.
The s t r i k i n g i n c r e a s e o f s e r u m c a l c i t o n i n l e v e l
seen i n r a t s w i t h h y p e r p l a s i a o f p a r a f o l l i c u l a r c e l l s s u b s t a n t i a t e d t h e m o r p h o l o g i c a l changes.
The t h y r o i d f o l l i c l e s o f t h e e x p e r i m e n t a l a n i m a l s
were l a r g e r than those o f t h e controls,
although t h e d i f f e r e n c e s were n o t
highly significant. INTRODUCTION Many e p i d e m i o l o g i c a l s t u d i e s (1,2)
and e x p e r i m e n t a l s t u d i e s (3.4)
e f f e c t s o f f l u o r i d e on t h e t h y r o i d h a v e been r e p o r t e d . consensus o f o p i n i o n on t h e i r r e l a t i o n s h i p .
on t h e
However t h e r e i s n o
Although there i s a r e p o r t
i n d i c a t i n g t h a t g o i t e r w o u l d b e i n d u c e d b y f l u o r i d e and a c c o m p a n y i n g i n s u f f i c i e n t i o d i n e i n t a k e ( 5 ) , i t h a s b e e n c o n c l u d e d b y WHO t h a t n o s p e c i f i c t o x i c i t i e s o f f l u o r i d e on t h e t h y r o i d e x i s t (6). T h i s e x p e r i m e n t a l w o r k was done t o s t u d y t h e e f f e c t o f a l o n g - t e r m , dose i n g e s t i o n o f sodium f l u o r i d e on t h e t h y r o i d g l a n d i n r a t s .
low
Emphasis
was p l a c e d on t h e m o r p h o l o g i c a l changes o f t h e t h y r o i d g l a n d as w e l l as s e r u m and e n d o c r i n o l o g i c a l o b s e r v a t i o n s .
308 MATERIALS AND METHODS Four-week-old group) and g i v e n 12,
(8-11
W i s t a r male r a t s were d i v i d e d i n t o f i v e groups
d i s t i l l e d d r i n k i n g w a t e r ad l i b i t u m c o n t a i n i n g 0. 3,
and 2 5 ppm F- (NaF),
were s a c r i f i c e d .
rats/
respectively.
6,
A t t h e end o f 20 m o n t h s t h e a n i m a l s
H i s t o l o g i c s e c t i o n s were obtained l o n g i t u d i n a l l y from t h e
middle portions o f b i l a t e r a l lobes o f the thyroid.
Hematoxylin-eosin
s t a i n i n g was done f o r r o u t i n e h i s t o l o g i c o b s e r v a t i o n s .
F o r measurement of
t h e s i z e s o f t h y r o i d f o l l i c l e s on t h e c u t surface,
microphotographs
taken f r o m t h e m i d d l e p o r t i o n o f each l o b e o f t h e t h y r o i d . Videoplan.
t h e a r e a s and d i a m e t e r s o f 232 t h y r o i d f o l l i c l e s w e r e measured
a t random i n each group. section,
were
U s i n g a MOP
I n order t o detect calcitonin i n the tissue
d e p a r a f f i n i r e d sections were stained by t h e immunoperoxidase
m e t h o d (7). Serum c a l c i t o n i n ,
t h y r o i d s t i m u l a t i n g h o r m o n e (TSH),
t h y r o x i n e (T4).
and
t r i i o d o t h y r o n i n e (T3) w e r e t e s t e d b y t h e s t a n d a r d r a d i o i m m u n o a s s a y method. RESULTS The t h y r o i d f o l l i c l e s o f t h e e x p e r i m e n t a l a n i m a l s w e r e l a r g e r t h a n t h o s e i n t h e c o n t r o l group,
a s s h o w n i n F i g u r e s 1 a n d 2.
e p i t h e l i a were a l s o flattened. i z e d i n T a b l e I.
Their follicular
The d a t a on t h y r o i d f o l l i c l e s a r e summar-
The mean v a l u e s o f b o t h t h e a r e a s a n d d i a m e t e r s o f
t h y r o i d f o l l i c l e s o f t h e e x p e r i m e n t a l g r o u p s w e r e much g r e a t e r t h a n t h o s e o f t h e c o n t r o l group. High incidence o f h y p e r p l a s t i c nodules c o n s i s t i n g o f p a r a f o l l i c u l a r c e l l s
( C c e l l s ) was o b s e r v e d i n t h e e x p e r i m e n t a l a n i m a l s ,
w h i l e no p r o l i f e r a t i o n
o f p a r a f o l l i c u l a r c e l l s as a mass was seen i n t h e c o n t r o l g r o u p ( T a b l e 11). Where m e d u l l a r y p r o l i f e r a t i o n o f p a r a f o l l i c u l a r c e l l s f o r m e d a mass i t . was d i a g n o s e d as a h y p e r p l a s t i c nodule.
M u l t i p l e h y p e r p l a s t i c nodules were
o c c a s i o n a l l y seen i n a s i n g l e t h y r o i d .
I n addition,
c a l c i t o n i n was
d e t e c t e d a s a d a r k d e p o s i t i o n i n t h e c y t o p l a s m o f p r o l i f e r a t i n g as w e l l as normal p a r a f o l l i c u l a r c e l l s i n t h e t h y r o i d , dase method (Fig.
3).
a s shown b y t h e i m m u n o p e r o x i -
Electron microscopic observation revealed character-
i s t i c s e c r e t o r y granules,
abundant rough endoplasmic r e t i c u l u m s ,
and p o l y -
ribosomes i n t h e cytoplasms o f h y p e r p l a s t i c p a r a f o l l i c u l a r c e l l s . As s h o w n i n T a b l e 1 1 1 , s e r u m c a l c i t o n i n l e v e l s w e r e e n h a n c e d i n t h e F t r e a t e d groups.
I n p a r t i c u l a r , t h e g r o u p r e c e i v i n g 2 5 ppm F- s h o w e d a
v a l u e s i x t i m e s h i g h e r t h a n t h e c o n t r o l group. where h y p e r p l a s t i c nodules were observed,
Furthermore,
i n most cases
t h e s e r u m c a l c i t o n i n l e v e l was
a l s o enhanced. The serum T j
v a l u e s o f t h e e x p e r i m e n t a l g r o u p s t e n d e d t o be h i g h e r t h a n
309
F i g u r e 1. H i s t o l o g y o f t h e t h y r o i d i n t h e c o n t r o l group. The f o l l i c l e s o f t h e t h y r o i d a r e u n i f o r m l y s m a l l i n s i z e . The p a r e n chymatous p a r a t h y r o i d g l a n d can be seen ir! t h 2 u p p e r l e f t p o r t i o n . 'Iir>stGxylin-eosin s'iiin. T'2 x.
H i s t o l o g y o f t h e t h y r o i d i n t h e g r o u p r e c e i v i n g 2 5 ppm F-. F i g u r e 2. The f o l l i c u l a r The f o l l i c l e s o f t h e t h y r o i d a r e l a r g e and v a r y i n s i z e . iIc!.i;.toxylin-eosin stain. 200 x. e p i t h e l i a (2t-2 r a t i i c r i'1at:;c;icd.
D e t e c t i o n o f c a l c i t o n i n i n h y p e r p l a s t i c p a r a f o l l i c u l a r c e l l s by F i g u r e 3. the immunoperoxidase technique. N o r m a l p a r a f o l l i c u l a r c e l l s a l s o show p o s i t i v e r e a c t i o n i n t h e surrounding t h y r o i d tissue. 400 x.
310 TABLE I S I Z E OF FOLLICLES I N THE T H Y R O I D GLAND
1999.1
f
1828.3l
46.7
t
3 ppm
5132.6
t
4850.8
73.1
f
34.6
6 ppm
f
4070.8
70.25e
f
29.5
12 ppm
4545.0'' 4810.0
69.7
f
35.6
25 ppm
4737.8
70.4
f
32.8
Control F-,
Diameter (wn)
Area (urn2)
Treatmen t
5297.1 i 4432.4 i
1L
" S i g n i f i c a n t l y d i f f e r e n t (piO.05) ' v a l u e s a r e means S.D.
19.0
f r o m t h e c o n t r o l group
TABLE I 1
I N C I D E N C E OF HYPERPLASTIC NODULE OF PARAFOLLICULAR CELLS I N THE THYROID GLAND F l u o r i n e (F-),
ppm
H y p e r p l a s t i c nodule' 'No.
0
0/8
3
6
12
25
3/11
3/9
7/10
7/10
o f case t h a t had h y p e r p l a s t i c nodules i n t h e t h y r o i d / t o t a l
TABLE 111
(F1
HORMONES I N SERUM
Control
F-,
3 ppm 6ppm
50.8 k 41.6 120.6
f
58.9*
80.3f30.2
1 2 ppm
182.5
25 ppm
319.1 f 209.2'ii:
f
102.8"
f SD)
34.1
f
10.8
2.2
f
0.2
15.5
4.9 f 0 . 8
47.6
f
24.8
1.6
f
0.7
29.8
2.7f0.6"
18.7
6 . 0 f 1.0" 6. o. 7Qit
2.1 f 0 . 7
24.0
6.9 k 1.2'"'
2.3
30.0
4.4 f 0.6
50.4f12.3 50.5
f
17.8"
62.0 f 25.2"
f
0.6
J,.t z - S i g n i f i c a n t l y d i f f e r e n t (~~0.05) from t h e c o n t r o l s S i g n i f i c a n t l y d i f f e r e n t (p
311 t h e c o n t r o l s a l t h o u g h no a p p r e c i a b l e d i f f e r e n c e s seen.
i n s e r u m T4 v a l u e s w e r e
S e r u m TSH v a l u e s w e r e h i g h e r i n a n i m a l s r e c e i v i n g 3.
6. a n d 1 2 ppm
F- t h a n i n t h e c o n t r o l s . No h i s t o l o g i c a l d i f f e r e n c e s w e r e f o u n d b e t w e e n t h e e x p e r i m e n t a l a n d c o n t r o l groups.
Parathyroid glands were parenchymatous i n a l l o f t h e
a n i ma1 s s t u d i e d .
DISCUSSION Histological
changes o f t h y r o i d glands,
h y p e r p l a s t i c nodules c o n s i s t i n g
o f p a r a f o l l i c u l a r c e l l s , c o n f i r m e d b y t h e i m m u n o p e r o x i d a s e method. f o u n d i n a n i m a l s r e c e i v i n g F-.
were
E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s showed
c h a r a c t e r i s t i c s e c r e t o r y g r a n u l e s and a b u n d a n t r o u g h e n d o p l a s m i c r e t i c u l u m s i n t h e cytoplasm, (8,9).
s u g g e s t i n g enhanced a c t i v i t y o f t h e s e h y p e r p l a s t i c c e l l s
I n c r e a s e d serum c a l c i t o n i n v a l u e s a l s o s u b s t a n t i a t e d
on h y p e r p l a s t i c nodules o f p a r a f o l l i c u l a r c e l l s .
observations
The r e s u l t s o f t h e h i s t o -
l o g i c a l i n v e s t i g a t i o n agree w e l l w i t h r e s u l t s o b t a i n e d f r o m b i o c h e m i c a l studies. These r e s u l t s r e v e a l e d t h a t l o n g - t e r m
i n g e s t i o n o f F- b y t h e r a t i n d u c e d
h y p e r p l a s t i c nodules i n t h e t h y r o i d gland.
It i s n o t known w h e t h e r t h e
changes were caused d i r e c t l y by f l u o r i d e o r i n d i r e c t l y t h r o u g h m e t a b o l i c a l t e r a t i o n s o f c a l c i u m i n bones ( 1 0 , l l )
o r i n t h e k i d n e y s (12,13).
I t i s s t i l l c o n t r o v e r s i a l a s t o w h a t e f f e c t s l o n g - t e r m i n g e s t i o n o f Fm i g h t have on t h y r o i d function.
The h i s t o l o g i c a l changes o f t h e t h y r o i d
i n c l u d i n g t h e appearance o f l a r g e t h y r o i d f o l l i c l e s l i n e d w i t h r a t h e r f l a t t e n e d e p i t h e l i a , a n d h i g h o r l o w s e r u m T3, suggest any u n i f o r m , Nevertheless,
T4 a n d TSH v a l u e s d o n o t
f u n c t i o n a l s t a t e o f t h e t h y r o i d gland.
t h e h i s t o l o g i c c h a n g e s d e s c r i b e d h e r e may s u g g e s t a s l i g h t
h y p o f u n c t i o n o f t h e t h y r o i d g l a n d i n r a t s r e c e i v i n g F-. T3 v a l u e s ,
however,
The s e r u m T 4 and
do n o t s u p p o r t h y p o f u n c t i o n o f t h e t h y r o i d g l a n d sug-
gested by t h e h i s t o l o g i c a l f i n d i n g s .
A
detailed examination o f thyroid
f u n c t i o n i n c o n j u n c t i o n w i t h f l u o r i d e a d m i n i s t r a t i o n i s t h u s necessary. REFERENCES 1. S i d d i q u i AH (1955) B r i t Med J 11:1408-1413
2. S i n g h
A,
J o l l y SS,
3. H a r r i s NO, 4. G a l l e t t i PM, 5. Day TK,
M a t h o u r CC (1963) M e d i c i n e 42:229-246
J o y e t G (1958) J C l i n E n d o c l i n o l 18:1102-1110
Powell-Jackson
6. WHO ( 1 9 7 0 ) Geneva
B a n s a l BC,
Hayes RL (1955) J D e n t R e s e a r c h 34:470-477
PR (1972) L a n c e t (May 27):1135-1138
F l u o r i d e a n d Human H e a l t h .
WHO M o n o g r a p h S e r i e s NO. 59,
312 7. Hsu SM,
R a i n e L, F a n g e r H (1981) J. H i s t o c h e m , Cytochem 29:577-580
8. De L e l l i s RA.
Nunnemacher G.
9. B l o o d w o r t h JMB (ed) (1982) B a l t i m o r e and London
W o l f e HJ (1977) Lab I n v e s t 36:237-248 Endocrine Pathology,
W i l l i a m s - W i l k i n s Co.
10. Sundstb’rm B (1972) A c t a P a t h M i c r o b i o l Scand Sec A 80:17-20
11. I s h i d a K (1981) O r a l H y g i e n e 31:2-16 12. K o u r K,
S i n g h J (1980) F l u o r i d e 13:163-167
13. A u s t i n LA,
H e a t h H (1981) N E n g l J Med 304:269-278
313
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 313-324 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
EXPERIMENTAL
ON
STUDIES
THE
EFFECTS O F
FLUORIDATION O N
RATS
WITH
EXPERIMENTALLY INDUCED GLOi~lERULONEPHRITIS TAKUMI SATO. KAZUSATA YOSHITAKE,
MASATO I I D A . AND TSUGUO KAWASAKI
D e p a r t m e n t o f O r a l and M a x i l l o f a c i a l S u r g e r y . Science, Japan
Shiga U n i v e r s i t y o f Medical
ABSTRACT The i n f l u e n c e o f f l u o r i d e i n g e s t i o n on r a t s w i t h g l o m e r u l o n e p h r i t i s h a s been i n v e s t i g a t e d . Thirty-day-old
W i s t e r r a t s were given nephrotoxin,
w e r e p r o v i d e d 1 0 ppm F ( a s NaF s o l u t i o n ) During t h e experimental period, i n the incisors, the
and m a i n t a i n e d f o r 100 days.
body w e i g h t ,
o c c u r r e n c e o f m o t t l e d enamel
and u r i n e and b l o o d c h e m i s t r y w e r e s t u d i e d .
experimental period,
t h e animals were s a c r i f i c e d ,
presence o f d e n t a l c a r i e s i n molars, molars,
and f i v e d a y s l a t e r ,
mandibles,
tibias.
A t t h e end o f
and s t u d i e d f o r t h e
and t h e f l u o r i d e c o n t e n t s o f i n c i s o r s ,
and s e v e r a l s o f t t i s s u e s w e r e d e t e r m i n e d .
His-
t o l o g i c a l e x a m i n a t i o n s w e r e p e r f o r m e d o n some o r g a n s . No s i g n i f i c a n t d i f f e r e n c e s i n b l o o d c h e m i s t r y o r i n h i s t o p a t h o l o g y o f s o f t tissues,
except t h e kidney,
were observed between t h e r a t s w i t h r e n a l
d i s e a s e and t h e c o n t r o l s .
A d e c r e a s e i n b o d y w e i g h t was n o t e d i n t h e e a r l y
stage o f t h e experiment.
Furthermore, n e p h r i t i c animals given d r i n k i n g
w a t e r s u p p l e m e n t e d w i t h 1 0 ppm F showed a m a r k e d l y e l e v a t e d f l u o r i d e l e v e l i n t h e hard tissues. INTRODUCTION E v e r s i n c e t h e p u b l i c a t i o n o f r e p o r t s i n t h e 1930's on e p i d e m i o l o g i c a l s t u d i e s o f t h e r e l a t i o n b e t w e e n f l u o r i d e i n g e s t i o n and d e n t a l
conditions,
w a t e r f l u o r i d a t i o n o r t o p i c a l a p p l i c a t i o n o f f l u o r i d e h a v e become p a r t o f d e n t a l c a r r i e s p r e v e n t i o n programs.
I t i s now w i d e l y a c c e p t e d t h a t ade-
q u a t e use o f f l u o r i d e c o u l d r e d u c e d e n t a l c a r i e s considerably. may a c t t o s t r e n g t h e n d e n t a l h a r d t i s s u e s , r i a l and a n t i - e n z y m a t i c
Fluoride
as w e l l as perform anti-bacte-
a c t i o n s on t o o t h s u r f a c e s .
It i s suggested t h a t
f l u o r i d e i s an e s s e n t i a l e l e m e n t as it i s an e f f e c t i v e f a c t o r i n t h e m i n e r a l i z a t i o n o f h a r d t i s s u e (1). n o t c l e a r l y understood. f l u o r i d e absorption,
On t h e o t h e r h a n d , i t s m e t a b o l i s m i s
There i s no agreement on t h e mechanism i n v o l v e d i n d i s t r i b u t i o n , and e x c r e t i o n .
Concerning absorption,
t h e e x i s t e n c e o f b o t h a c t i v e t r a n s p o r t and p a s s i v e d i f f u s i o n h a s been shown
314 i n t h e r a t (2.3). The a c t i o n and e f f e c t o f f l u o r i d e on a n i m a l s d e p e n d o n s e v e r a l f a c t o r s such as dose and t y p e o f f l u o r i d e compounds, cation,
m-thod and f r e q u e n c y o f a p p l i -
and p h y s i o l o g i c a l c o n d i t i o n s o r s p e c i f i c i t y i n r e a c t i o n s o f each
organ as w e l l as age and sex. healthy adults,
E x c r e t i o n o f f l u o r i d e i s m a r k e d l y high.
In
more t h a n 90% o f t h e i n g e s t e d f l u o r i d e i s c o n s i d e r e d t o be
e x c r e t e d i n t h e u r i n e (4). t r o i n t e s t i n a l secretion,
O t h e r r o u t e s w i l l be t h r o u g h s a l i v a and gas-
sweat,
lacrimal,
and mammary e x c r e t i o n s .
Adminis-
t r a t i o n o f a l a r g e d o s e o f f l u o r i d e has been r e p o r t e d t o c a u s e k i d n e y disturbances.
However,
r e p o r t s on t h e e x c r e t i o n and a c c u m u l a t i o n o f f l u o -
r i d e under t h e c o n d i t i o n o f k i d n e y d i s f u n c t i o n a r e r a r e i n t h e l i t e r a t u r e
(5). These e x p e r i m e n t s were designed t o i n v e s t i g a t e t h e i n f l u e n c e o f f l u o r i d e a d m i n i s t r a t i o n on a n i m a l s w i t h e x p e r i m e n t a l l y induced k i d n e y disease.
Rats
w i t h e x p e r i m e n t a l g l o m e r u l o n e p h r i t i s (Masugi's n e p h r i t i s ) were g i v e n f l u o r i d a t e d d r i n k i n g w a t e r and t h e i r t e e t h and b i o l o g i c a l c o n d i t i o n s w e r e studied. MATERIALS AND METHODS F o r t y - f o u r W i s t e r s t r a i n p u r e m a l e r a t s w e r e used.
The r a t s w e r e b r e d
and s u p p l i e d by t h e Pure S t r a i n Animal Center o f Kyoto U n i v e r s i t y , For preparing nephrotoxin (anti-rat
Japan.
n e p h r o c o r t e x r a b b i t serum),
mature r a t s and t h r e e h e a l t h y m a t u r e r a b b i t s were used.
ten
An e m u l s i o n con-
t a i n i n g 20% r a t k i d n e y c o r t e x was homogenized and 5 m l o f t h e homogenate was i n j e c t e d i n t o t h e abdominal c a v i t y o f t h e r a b b i t 16 t i m e s , t h r e e t o f o u r days. test.
once e v e r y
I m m u n o l o g i c a l v a l u e was checked by b l o o d s e d i m e n t a t i o n
Two weeks a f t e r t h e f i n a l i n j e c t i o n , t h e s e r u m was c o l l e c t e d and
i n a c t i v a t e d by h e a t i n g a t 65°C
f o r 30 min.
Anti-rat-nephrocortex
antigen
was o b t a i n e d by t h e a d d i t i o n o f 0.5% c a r b o l i c acid.
To produce Masugi's n e p h r i t i s ,
a volume o f t h e a n t i g e n was i n j e c t e d i n t o
t h e t a i l v e i n o f t h e experimental rats,
30 days o f age.
Dose o f s e n s i t i z a -
t i o n was 0.5 m l o r 0.25 m l o f t h e a n t i g e n p e r 100 g body w e i g h t o f t h e r a t . T h i s induced marked g l o m e r u l o n e p h r i t i s .
To s t u d y t h e e f f e c t o f f l u o r i d e on r a t s w i t h induced g l o m e r u l o n e p h r i t i s . t h e a n i m a l s were d i v i d e d i n t o 5 groups: water only (controls); nephrotoxin.
Group A a n i m a l s r e c e i v e d d i s t i l l e d
G r o u p N A r e c e i v e d i n t r a v e n o u s i n j e c t i o n o f 0.5ml
p l u s d i s t i l l e d w a t e r as d r i n k i n g w a t e r :
G r o u p nF r e c e i v e d
i n t r a v e n o u s i n j e c t i o n o f 0.25
m l o f t h e nephrotoxin,
c o n t a i n i n g 10 ppm F- (as NaF):
Group FIF r e c e i v e d 0.5 m l o f t h e n e p h r o t o x i n ,
p l u s d i s t i l l e d w a t e r c o n t a i n i n g 10 ppm F-;
plus d i s t i l l e d water
and Group F r e c e i v e d d i s t i l l e d
315 w a t e r c o n t a i n i n g 1 0 ppm F- as d r i n k i n g w a t e r and n o i n j e c t i o n . F l u o r i d e c o n t a i n i n g d r i n k i n g w a t e r was g i v e n s i n c e t h e f i f t h day a f t e r t h e i n j e c t i o n o f the nephrotoxin.
The a n i m a l s w e r e a l l f e d a b a s a l d i e t o f O r i e n t a l
pellet. The a n i m a l s were m a i n t a i n e d f o r a p e r i o d o f 100 days f o l l o w i n g a d m i n i s t r a t i o n o f t h e n e p h r o t o x i n . A t ten-day i n t h e i n c i s o r s were examined,
intervals,
body w e i g h t and changes
and b l o o d and u r i n e samples were analyzed.
A t t h e end o f t h e e x p e r i m e n t a l p e r i o d , t h e a n i m a l s were s a c r i f i c e d . C a r i o u s l e s i o n s o f t h e m o l a r s were examined and t h e f l u o r i d e c o n t e n t s o f s e v e r a l h a r d and s o f t t i s s u e s determined. on d i f f e r e n t t i s s u e s , pancreas,
H i s t o p a t h o l o g i c a l s t u d i e s were a l s o made
i n c l u d i n g t h e kidneys,
s a l i v a r y glands,
liver,
heart,
lungs,
spleen,
and t e s t e s .
RESULTS I n c r e a s e o f body w e i g h t and g e n e r a l f i n d i n g s The c h a n g e s i n t h e b o d y w e i g h t o f t h e a n i m a l s d u r i n g t h e e x p e r i m e n t a l p e r i o d a r e shown i n T a b l e I.
I n c r e a s e s i n b o d y w e i g h t i n g r o u p s NA. nF.
and NF w e r e l o w e r t h a n t h o s e i n g r o u p s A and F.
Rats t r e a t e d w i t h b o t h
n e p h r o t o x i n and f l u o r i d e showed l e s s i n c r e a s e i n w e i g h t t h a n t h o s e t r e a t e d w i t h F alone.
On t h e o t h e r hand,
n o s i g n i f i c a n t d i f f e r e n c e was f o u n d
between t h e c o n t r o l s and t h o s e a n i m a l s r e c e i v i n g f l u o r i d e alone. TABLE I
CHANGES I N THE BODY WEIGHT OF EXPERIMENTAL ANIMALS Group
N
Average body w e i g h t ( 4 ) initial final i n c r e a s e d f SDa
P (by t - t e s t )
10
92.3
341.5
249.2 f 24.7
NA
5
85.5
274.4
191.2 f 30.2
nF
7
81.4
279.2
197.8 f 14.0
91.4
280.9
198.5 k 58.4
~0.05
94.6
320.9
226.3 f 21.7
-
A
NF F
gb 15
aSD s t a n d a r d d e v i a t i o n b e x c e p t one animal d i e d d u r i n g t h e e x p e r i m e n t a l p e r i o d
As f o r abnormal c o n d i t i o n s ,
t w o o f t h e r a t s i n groups NF showed abdominal
s w e l l i n g w i t h i n one t o t h r e e weeks,
and a n o t h e r i n t h e same group d i e d on
t h e 5 3 r d day o f t h e e x p e r i m e n t a f t e r s u f f e r i n g d i a r r h e a .
One o f t h e r a t s
316 i n g r o u p nF showed d y s p n e a and r h i n o r r h e a . M a c r o s c o p i c o b s e r v a t i o n o f i n c i s o r enamel M o s t mai-ked c h a n g e s o c c u r r e d i n f o u r o u t o f t h e s i x a n i m a l s i n g r o u p NF, i.e., l i n e a r w h i t e c o l o r c h a n g e s w e r e o b s e r v e d i n i n c i s o r enamel.
r e s t o f t h e groups,
I n the
w h i t e d i s c o l o r a t i o n o r i r r e g u l a r w h i t e spots were o f t e n
observed. Occurrence o f d e n t a l c a r r i e s The number and d e g r e e o f c a r r i e s o c c u r r i n g i n t h e m o l a r a r e shown i n F i g u r e 1. number
The f l u o r i d e - t r e a t e d
of
carious
g r o u p s a l l showed a s i g n i f i c a n t l y s m a l l e r
t e e t h compared t o t h e c o n t r o l
groups.
The non-
f l u o r i d a t e d n e p h r i t i s g r o u p ( g r o u p NA) a l s o s h o w e d a s i g n i f i c a n t l y l o w e r number o f c a r i o u s m o l a r s , controls.
carious incidence,
and c a r i o u s e x t e n t t h a n t h e
The number o f c a r i o u s m o l a r s and c a r i e s e x t e n t a r e s i g n i f i c a n t l y
l o w e r i n g r o u p NF o r i n g r o u p nF t h a n i n g r o u p F. F l u o r i d e c o n t e n t s o f h a r d t i s s u e s and some s o f t t i s s u e s The f l u o r i d e c o n t e n t s o f m o l a r s w e r e h i g h e r t h a n i n c i s o r s i n n o n - f l u o r i d a t e d g r o u p s (A
- MA),
fluoride-treated
w h i l e those o f i n c i s o r s were h i g h e r than molars i n
g r o u p s (nF,
NF - F ) ( F i g .
2).
The f l u o r i d e c o n t e n t o f
f e m u r s was h i g h e r t h a n m a n d i b l e s i n g r o u p s A and NA.
The f l u o r i d e c o n t e n t
o f m a n d i b l e s was h i g h e r t h a n f e m u r s i n g r o u p s NF and F t h a n i n o t h e r s . each o f t h e hard t i s s u e s , mandibles),
g r o u p NA was h i g h e r t h a n g r o u p A ( e x c e p t f o r
a n d g r o u p n F w a s h i g h e r t h a n g r o u p F.
This indicates that
3.0
2.0 I
T II 1.0
0
carious molars
carious incidence
0group A I g r o u p nF F i g u r e 1.
In
carious extent
=group
F
0
carious grade
!Ilgroup l
group NF
The number and e x t e n t o f c a r i e s i n m o l a r s .
NA
317
0
200
400
600
800
1000
F-ppm
Femurs Mandibles Molars Incisors Kidneys Liver Heart Lungs Testicles Spleen Pancreas Submandibular salivary glands
F i g u r e 2.
Ogroup =group -group group group
A NA
nF NF F
F l u o r i d e c o n t e n t o f s e v e r a l t i s s u e s and organs.
a n i m a l s w i t h e x p e r i m e n t a l n e p h r i t i s t e n d t o have h i g h e r l e v e l s o f f l u o r i d e i n hard tissues. I n the f l u o r i d e content o f s o f t tissues, observed.
no marked d i f f e r e n c e s were
G e n e r a l l y speaking, f l u o r i d a t e d g r o u p s showed h i g h e r f l u o r i d e
l e v e l s t h a n n o n - f l u o r i d a t e d groups.
F l u o r i d e l e v e l i t s e l f , however, was
n o t r e l a t e d t o t h e presence o f n e p h r i t i s . H i s t o l o g i c a l observation o f s o f t t i s s u e s Kidney.
H i s t o p a t h o l o g i c a l changes o f t h e k i d n e y i n t h e r a t w i t h induced
n e p h r i t i s a r e shown i n F i g u r e s 3-1 t h r o u g h 4. F i g u r e 4.
The d a t a a r e summarized i n
P r e v a l e n t c h a n g e s a r e a t r o p h y and d e g e n e r a t i o n o f g l o m e r u l i ,
e x u d a t i o n and r o u n d c e l l i n f i l t r a t i o n i n i n t e r s t i t i u m o r c a p s u l e , and d e g e n e r a t i o n and n e c r o s i s o f tubules.
These changes a r e marked i n groups
318
F i g u r e 3-1. H i s t o p a t h o l o g i c a l changes o f t h e r a t k i d n e y o f g r o u p NA. I n f i l t r a t i o n o f r o u n d c e l l s i n i n t 2 r s t i t i u n a:>:: ? i e o t \ ? i ? . c r \ s t s i : i t h e t u b u l e s ( l e f t , r 2 - t :ic. 3), an:: f i ' 2 r i n o i : l
F i g u r e 3-2. Kidney o f group NF, r a t no. 4. F i b r o u s l e s i o n o f g l o m e r u l i , h y p e r p l a s t i c changes o f m a l p i g h i a n corpuscles, r e g e n e r a t i o n o f t u b u l a r e p i t h e l i u m and i n f l a m m a t o r y round c e l l i n f i l t r a t i o n on i n t e r s t i t i u m .
319
F i g u r e 3-3. K i d n e y o f g r o u p nF, r a t no. 4. Marked i n f i l t r a t i o n o f r o u n d c e l l s and f i b r o s i s i n t h e i n t e r s t i t i u m , p r o t e i n c a s t s i n t h e tubules, hyperplasia o f rnalpighian corpuscles, atrophy and f i b r o s i s o f PI----
’,.
F i g u r e 3-4. K i d n e y o f g r o u p nF, r a t no. 2. I n f i l t r a t i o n o f r o u n d c e l l s i n i n t e r s t i t i u m , marked d e g e n e r a t i o n and n e c r o s i s o f t u b u l e s , f i b r o p l a s t i c h y p e r p l a s i a o f m a l p i g h i a n c o r p u s c l e s and These changes a r e d i a g n o s e d as a t r o p h i c s c l e r o t i c atrophy o f glomeruli. kidney.
rnultinuclear Fibrous hypertrophy embo 1i sm
inside
capsule
adhesion with glomerulus scar c h a n g e compensatory h y p e r t r o p h y
F i g u r e 4. H i s t o l o g i c a l o b s e r v a t i o n s o f t h e kidney.
l-l
321 NF,
nF and NA,
b u t t h e d e g r e e and s i t e s o f c h a n g e s d i f f e r e d i n e a c h a n i m a l
e v e n w i t h i n t h e same group.
Some o f t h e s e c h a n g e s w e r e a l s o n o t e d i n
and F g r o u p s .
control
Other orgaE.
Microscopic observation o f the tissues from the liver,
heart, lungs, spleen, pancreas, t h y r o i d , s a l i v a r y glands, adrenal glands and t e s t e s ,
r e v e a l e d s e v e r a l h i s t o l o g i c a l changes,
and m e s e n c h y m a l components. l o w , g r o u p s N F > n F > a n d F.
m a i n l y i n blood vessels
The d e g r e e o f t h e s e c h a n g e s w e r e ,
from high t o
However, t h e r e s u l t s w e r e n o t s t a t i s t i c a l l y
significant.
-____ Urine analysis.
P r o t e i n u r i a was o b s e r v e d i n g r o u p NF r a t s a t an e a r l y
p e r i o d o f t h e e x p e r i m e n t , a n d d u r i n g t h e l a s t 4 0 d a y s i n g r o u p s nF a n d N F Some w e r e a l s o n o t e d i n g r o u p A.
as w e l l .
No s i g n i f i c a n t d i f f e r e n c e s were
observed between groups.
-__Blood analysis. range,
Serum t o t a l p r o t e i n l e v e l s w e r e m o s t l y w i t h i n n o r m a l
except f o r t h e f i n a l days o f t h e experiment.
were noted between groups, urea nitrogen,
however (Fig.
and n o n - p r o t e i n
5).
No marked d i f f e r e n c e s
Data on serum c r e a t i n i n e .
n i t r o g e n (NPN)
were f o u n d t o be m o s t l y
w i t h i n a n o r m a l range. I n t h e i n d u c e d n e p h r i t i s g r o u p , b l o o d c h l o r i n e t e n d e d t o i n c r e a s e f r o m 30 d a y s t o 50 d a y s , w h e n t h e c o n c e n t r a t i o n s o f g r o u p NA w e r e 1.5 t i m e s , a n d t h o s e o f g r o u p s nF a n d N F w e r e t w i c e t h o s e o f t h e c o n t r o l s . h a l f o f t h e experimental period,
I n the last
t h e c h l o r i n e l e v e l s were decreased t o
n o r m a l ( F i g . 6).
P
; 5
L Q)
0
10
0
30
u)
W group D--W
F i g u r e 5.
A,
g r o u p NF,
50
60
70
80
90
100
Experimental period, days group NA, h - - A group
nF.
C-. g r o u p F
Serum t o t a l p r o t e i n o f e x p e r i m e n t a l r a t s .
322
*ool W 150
E
0
60
7 0 80 90 1 0 0
Experimental period, days
O---C
qCOUp
)-*group
F i g u r e 6.
50
30
10
A.6-dgpuop NA,t--Agroup
NF,-group
nF,
F
Blood c h l o r i n e c o n t e n t o f experimental r a t s .
DISCUSSION F l u o r i d e a b s o r p t i o n i n a n i m a l s i s c o n s i d e r e d t o be m a i n l y v i a t h e s m a l l i n t e s t i n e and s t o m a c h t h r o u g h p a s s i v e d i f f u s i o n (2).
W h i l e some f l u o r i d e
i s bound t o h a r d t i s s u e s , m o s t o f t h e i n g e s t e d f l u o r i d e i s e x c r e t e d t h r o u g h t h e kidney i n t o urine. I n h e a l t h y persons,
f l u o r i d e i n t a k e a t t h e u s u a l f l u o r i d a t i o n l e v e l may
n o t a f f e c t renal functions. however,
I n patients s u f f e r i n g renal dysfunction,
t h e body f l u o r i d e b u r d e n m i g h t b e i n c r e a s e d , and i t i s supposed
t h a t t h e t h r e s h o l d s a f e t y range m i g h t be narrowed.
It i s a l s o considered
t h a t r e n a l d y s f u n c t i o n has c l o s e and i m p o r t a n t i n t e r a c t i o n s w i t h f l u o r i d e m e t a b o l i s m and t h i s a f f e c t s s o f t t i s s u e s .
The p r e s e n t s t u d y was d e s i g n e d
i n view o f these considerations. D r i n k i n g w a t e r c o n t a i n i n g 10 ppm F i n r a t s i s c o n s i d e r e d t o be e q u i v a l e n t t o 1 p p m F i n h u m a n s (6,7). I n 1970.
Hodge a n d T a v e s (8) r e p o r t e d t h e p r o b a b i l i t y and d a n g e r o f
s a f e t y margin narrowing i n i n d i v i d u a l s s u f f e r i n g renal disturbances. kin
Yud-
g t gl. ( 9 ) o b s e r v e d n o s i g n i f i c a n t d i f f e r e n c e s i n u r i n a r y f l u o r i d e
l e v e l s between t w o groups o f p a t i e n t s w i t h r e n a l dysfunction,
one group
c o n s u m i n g w a t e r f l u o r i d a t e d w i t h 1.0 ppm F and a n o t h e r g r o u p w i t h 0.1 ppm
F.
Smith
g . (10)
out nephritis,
a l s o r e p o r t e d t h a t among o l d e r p e o p l e w i t h and w i t h -
t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e l e v e l o f u r i n -
a r y f l u o r i d e c o n c e n t r a t i o n s b e f o r e and a f t e r f l u o r i d a t i o n .
Dialysis
323 patients with renal failure,
on t h e o t h e r hand,
s e v e r a l t i m e s h i g h e r serum f l u o r i d e
were r e p o r t e d t o have
l e v e l s t h a n h e a l t h y i n d i v i d u a l s (11).
h a v e o t h e r p r o b l e m s s u c h as n o n - d e s i r e d
Dialysis patients w i l l
membranous
exchange o f f l u o r i d e w i t h o t h e r e l e m e n t s i n c l u d i n g c a l c i u m ions. The r e s u l t s o f t h i s s t u d y d e m o n s t r a t e d t h a t l o w c o n c e n t r a t i o n s o f f l u o r i d e s u c h a5 f o u n d i n m u n i c i p a l f l u o r i d a t i o n m i g h t n o t a f f e c t t h e c o n d i t i o n and s e v e r i t y o f g l o m e r u l o n e p h r i t i s . affect
histological
features
of
many s o f t
Also, n e p h r i t i s m i g h t n o t tissues.
There
were
no
d i f f e r e n c e s i n body w e i g h t and m o s t o f t h e b l o o d c h e m i s t r y d a t a b e t w e e n g r o u p s nF,NF and NA.
T h i s s u g g e s t s t h a t c o n s u m p t i o n o f 10 ppm f l u o r i d e b y
n e p h r i t i c r a t s does n o t a d d i t i o n a l l y a f f e c t t h e g r o w t h and p a t h o l o g i c a l c o n d i t i o n s o f t h e kidneys.
I t m u s t be m e n t i o n e d t h a t t h e r a t , a r o d e n t , shows d i f f e r e n t d e n t i t i o n t h a n humans, a s i t s i n c i s o r s c o n t i n u e t o e r u p t .
The f l u o r i d e l e v e l ana-
l y z e d i n t h i s s t u d y may n o t be f u l l y a p p l i c a b l e t o humans.
Since f l u o r i d e
c o n t e n t s i n bones and t e e t h w e r e e l e v a t e d i n n e p h r i t i c r a t s , s u g g e s t i n g f l u o r i d e accumulation i n t h e hard tissue.
Decrease o f d e n t a l c a r i e s i n c i -
d e n c e and o t h e r c a r i e s i n d e x w i l l b e a r e f l e c t i o n o f t h i s phenomenon. CONCLUSION The e f f e c t s o f d r i n k i n g f l u o r i d a t e d w a t e r o n r a t s w i t h i n d u c e d g l o m e r u l o n e p h r i t i s were studied. I n c r e a s e i n b o d y w e i g h t was d e p r e s s e d s 1 i g h t : y compared t o t h e c o n t r o l groups,
i n the experimental r a t s
b u t i t was n o t i n f l u e n c e d b y d r i n k i n g
f l u o r i d a t e d w a t e r . D a t a o n u r i n e and b l o o d a n a l y s i s showed t h a t t h e c h a r a c t e r i s t i c f e a t u r e s o f n e p h r i t i s were n o t enhanced b y f l u o r i d a t i o n .
Both
n e p h r i t i c c o n d i t i o n s and f l u o r i d e supplements i n c r e a s e d t h e f l u o r i d e content o f hard tissues.
T h i s may h a v e r e s u l t e d i n t h e d e c r e a s e o f d e n t a l
c a r i e s observed i n these exoeriments. REFERENCES 1.
WHO:
WHO T e c h Rep, No 582. WHO, Geneva, 1973
2.
S a t o T ( 1 9 7 8 ) B u l l S t o m a t o l o g y K y o t o U n i v 18:16-27
3. S a t 0 T (1971) J D e n t H l t h 21:339-365 4.
B i t t n e r W (1968) D t s c h Z a h n a r z t l Z 23:123-128
5. S m i t h FA, G a r d n e r DE ( 1 9 6 6 ) P h a r m a c o l o g y o f F l u o r i d e s . N.Y. P a r t I , p 1 2 9 6.
M i y a z a k i Y, K y o t o , p 80
7.
S a t 0 T (1963) B u l l S t o m a t o l o g y K y o t o
Springer-Verlag,
I s h i k a w a G, A k i y o s h i M ( 1 9 5 4 ) O r a l P a t h o l o g y I. N a g a s u e , U n i v 3:244-275
324
8. Hodge HC, Taves DR (1970) I n : F l u o r i d e s and Human H e a l t h , WHO Monogr Ser No 59, pp 249-255 9. Y u d k i n EP.
WHO, Geneva,
C z e r n i e j e w s k i J, B l a y n e y JR (1954) J Dent Res 33:691-692
10. S m i t h FA, Gardner DE,
Hodge HC (1955) A r c h I n d u s t r H e a l t h 11:2-10
11. Taves DR. Freeman RB. Kamm DE, Ramos CP, SOC A r t i f I n t Organs 14:412-414
S c r i b n e r BS (1968)
Trans Am
325
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 325-332 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
MECHANISM OF FLUORIDE ABSORPTION FROM THE GASTROINTESTINAL TRACT IN RATS T.I\KUMI
S A T O ~ , KAZUSADA YOSHITAKE~
HI TO MI^
AND GONJIRO
' D e p a r t m e n t o f O r a l and M a x i l l o f a c i a l S u r g e r y , S c i e n c e , O t s u . S h i g a , 520-21 J a p a n ' D e p a r t m e n t o f D e n t i s t r y and O r a l S u r g e r y , 910-11 J a p a n
Shiga U n i v e r s i t y o f Medical
Fukui Medical College,
Fukui.
ABSTRACT The m e c h a n i s m i n v o l v e d i n t h e a b s o r p t i o n o f f l u o r i d e b y t h e g a s t r o i n t e s t i n a l t r a c t has been s t u d i e d b y t h e g a s t r o i n t e s t i n a l r e f l u x method i n s i t u o f r a t . R e f l u x was a p p l i e d t o t h e stomach, mouth o f each animal.
small intestine,
rectum, and
A t certain intervals during the reflux. a portion o f
t h e r e f l u x s o l u t i o n was t a k e n f o r d e t e r m T n a t i o n o f f l u o r i d e u p t a k e . t h e stomach
When
o r t h e s m a l l i n t e s t i n e was r e f l u x e d f o r 1 hour, t h e r a t e o f
f l u o r i d e a b s o r p t i o n was d e c r e a s e d w i t h i n c r e a s e i n f l u o r i d e c o n c e n t r a t i o n s , and i n t h e p r e s e n c e o f d i n i t r o p h e n o l o r s a l i n e .
These r e s u l t s i n d i c a t e
t h a t t h e u p t a k e o f f l u o r i d e f r o m t h e g a s t r o i n t e s t i n a l t r a c t o f t h e r a t may n o t be l i m i t e d t o p a s s i v e t r a n s p o r t alone.
INTRODUCTION F l u o r i d e (F) i s w i d e l y u s e d f o r t h e p r e v e n t i o n o f d e n t a l c a r i e s .
Fluori-
d a t i o n h a s b e e n a d o p t e d i n many c o u n t r i e s a s a p o s i t i v e means f o r t h e p r e v e n t i o n o f d e n t a l c a r i e s i n p u b l i c h e a l t h p r o g r a m s a n d WHO h a s p r o p o s e d t h a t t h i s method be p r a c t i c e d as w i d e l y as p o s s i b l e .
Although t h e dynamics
o f F i n v i v o h a v e b e e n s t u d i e d .by m a n y i n v e s t i g a t o r s ( 1 - 5 ) . r e s u l t s have n o t been s a t i s f a c t o r y .
Consequently,
experimental
it i s difficult to
d e t e r m i n e an a d e q u a t e d o s e o f F f o r f l u o r i d a t i o n o f c o m m u n i t y d r i n k i n g water.
It
i s i m p o r t a n t t o c l a r i f y t h e mechanism o f F a b s o r p t i o n f r o m t h e
g a s t r o in t e s t ina 1 t r a c t . I n t h i s paper, e x p e r i m e n t s were conducted t o s t u d y t h e a b s o r p t i o n o f F f r o m t h e g a s t r o i n t e s t i n a l t r a c t i n s i t u o f r a t s b y means o f a r e f l u x method.
MATERIALS AND METHODS M a l e r a t s w e i g h i n g 120-140
g,
a f t e r 24 h o u r s f a s t i n g ,
were anesthesized
w i t h u r e t h a n a n d p l a c e d i n a r e f l u x a p p a r a t u s a s s h o w n i n F i g u r e 1.
A
326
Figure 1: Front cutaway diagram of apparatus used f o r r e f l u x experiments of r a t stomach and i n t e s t i n e .
r e f l u x i n g f l u i d ( 7 5 ml) containing NaF s o l u t i o n s of k n o w n
concentrations
was refluxed through cne stomach and small i n t e s t i n e . P r i o r t o t h e experiment on F absorption from t h e stomach, d i s t i l l e d water was used a s t h e r e f l u x f l u i d and t h e c h l o r i d e c o n t e n t of t h e r e f l u x was measured o v e r a 90 min p e r i o d .
The amount of c h l o r i d e s e c r e t e d from t h e
stomach w a l l i n t o t h e r e f l u x f l u i d d u r i n g t h i s p e r i o d was a p p r o x i m a t e l y
1,500 ug/dl.
T h i s i s n e g l i g i b l e compared w i t h t h e c h l o r i d e c o n t e n t of
physiological s a l i n e . Consequently, t h e amount o f c h l o r i d e s e c r e t e d i n t o t h e g a s t r i c j u i c e was not taken i n t o account.
Sodium f l u o r i d e dissolved in
d i s t i l l e d w a t e r o r p h y s i o l o g i c a l s a l i n e was used f o r r e f l u x a t v a r y i n g Dinitrophenol (DNP),
concentrations.
an uncoupler of o x i d a t i v e phosphory-
l a t i o n , was used a t Z X I O - ~ M a n d ~ x I O - ~ M . The concentrations of F- and C1ions i n t h e r e f l u x were measured by t h e ion e l e c t r o d e method. Time 0 was defined a s 15 min a f t e r the beginning of infusion.
A t 15,
30
45, a n d 60 min t h e r e a f t e r , two 0.5 ml r e f l u e n t samples were taken, one f o r t h e d e t e r m i n a t i o n of F c o n c e n t r a t i o n and t h e o t h e r f o r t h e c o r r e c t i o n of concentration f o r t h e f l u c t u a t i o n in water content of t h e r e f l u x during t h e course of t h e experiment. To c o r r e c t t h e f l u c t u a t i o n in F concentration
i n
t h e r e f l u x , phenol red,
considered t o be s c a r c e l y absorbed by t h e g a s t r o i n t e s t i n a l t r a c t , was added t o the reflux fluid.
Fluoride concentration was corrected according t o the
following equation:
corrected concentration
=
measured x concentration
absorbance of phenol red a t t h e beginning of r e f l u x measured absorbance of phenol red
327 I n t h e e x p e r i m e n t o n a b s o r p t i o n f r o m t h e r e c t u m , a v i n y l t u b e was conn e c t e d t o t h e u p p e r p a r t o f t h e r e c t u m and anus. a b s o r p t i o n f r o m t h e mouth,
F o r t h e e x p e r i m e n t on
a s p e c i a l l y p r e p a r e d a d a p t e r shown i n F i g u r e 2
was f i x e d t o t h e mouth w i t h c y a n o a c r y l a t e ,
and t h e r e f l u x a p p a r a t u s was
c o n n e c t e d t o it. The e x p e r i m e n t a l m e t h o d s u s e d f o r r e f l u x i n g t e s t s o l u t i o n s ,
and f o r
d e t e r m i n i n g t h e l o s s o f F f r o m r e f l u x f l u i d i n c h r o n o l o g i c a l sequence,
and
F absorption from physiological saline containing varying concentrations o f
F w e r e t h e same a s w i t h t h e s t o m a c h and s m a l l i n t e s t i n e .
Rec i r c u -
nm g;,e s e r v o i r
rnm F i g u r e 2.
D i a g r a m o f a p p a r a t u s u s e d i n r e f l u x e x p e r i m e n t o f r a t mouth.
RESULTS A b s o r p t i o n f r o m t h e stoE& T i m e 0 was d e f i n e d a s 15 m i n a f t e r t h e b e g i n n i n g o f t h e r e f l u x . shows
Figure 3
t h e l o g a r i t h m o f t h e c o r r e c t e d F c o n c e n t r a t i o n s as a b s o r b a n c e o f t h e
r e f l u x a t 15, 30, 45, a n d 60 r n i n t h e r e a f t e r f o r s a m p l e s c o n t a i n i n g 50 a n d
100 u g F / m l .
An a p p r o x i m a t e l y r e c t i l i n e a r r e s p o n s e was seen.
The r a t e o f F
2 0 .-235000 [ LI
$0.150
-n
0
15 3 0 4 5 6 0 Time i n m i n u t e 5 0 yg/ml o----o1 0 0 yg/ml
0-0
F i g u r e 3. R e l a t i o n s h i p b e t w e e n F c o n c e n t r a t i o n s and r e f l u x t i m e o f f l u o r i d e s o l u t i o n f o r f l u o r i d e a b s o r p t i o n f r o m t h e stomach o f t h e rat.
328 a b s o r p t i o n was a f f e c t e d b y t h e F c o n c e n t r a t i o n i n t h e r e f l u x f l u i d . l i q u i d was i n c r e a s e d , c o n s t a n t v a l u e (Fig.
t h e r a t e o f a b s o r p t i o n decreased,
4).
The r a t e o f NaF a b s o r p t i o n f r o m s a l i n e was l o d e r
t h a n t h a t f r o m d i s t i l l e d water,
e s p e c i a l l y a t lower F concentrations.
t h e F c o n c e n t r a t i o n o f t h e r e f l u x was increased. became s m a l l e r ,
c
When
approaching a
however,
As
the difference
and a l m o s t disappeared a t 900 ugF/ml.
I
.:Absorption from d i s t i l l e d water 0 : A b s o r p t i o n from 0 . 9 % N a C l s o l u t i o n F i g u r e 4. Effect of f r o m t h e stomach.
f l u o r i d e c o n c e n t r a t i o n on t h e a b s o r p t i o n o f f l u o r i d e
Absorption from t h e small i n t e s t i n e F l u o r i d e i n t h e r e f l u x f l u i d was d e t e r m i n e d e v e r y 1 5 m i n and t h e l o g a r i t h m o f c o n c e n t r a t i o n s p l o t t e d a g a i n s t t i m e showed a r e c t i l i n e a r r e g r e s s i o n as w i t h t h e stomach.
The r e l a t i o n s h i p between F c o n c e n t r a t i o n i n t h e
r e f l u x and t h e r a t e o f a b s o r p t i o n a t t h e end o f t h e 1 hour r e f l u x p e r i o d i s shown i n F i g u r e 5.
W h i l e t h e a b s o r p t i o n i n t h e s m a l l i n t e s t i n e was gener-
a l l y more e f f i c i e n t t h a n i n t h e stomach, dependent.
i t was a l s o c o n c e n t r a t i o n -
W i t h an i n c r e a s e i n F c o n c e n t r a t i o n s i n t h e r e f l u x ,
F a b s o r p t i o n decreased.
the rate of
The presence o f c h l o r i d e g e n e r a l l y decreased t h e
r a t e o f a b s o r p t i o n ( F i g . 5).
The d e c r e a s e i n t h e r a t e o f a b s o r p t i o n was
more pronounced a t l o w e r F c o n c e n t r a t i o n s ,
i n d i c a t i n g a diminished i n f l u -
ence o f c h l o r i d e w i t h i n c r e a s i n g F c o n c e n t r a t i o n s .
A d d i t i o n o f DNP t o t h e
r e f l u x s o l u t i o n decreased t h e r a t e o f F a b s o r p t i o n (Fig.
5).
329
.
20
c . P i
E
15.o
0
w
c
.-l
;10 . o
a
LI
0 m
5.o
0
4
1
0
:Fluoride :Fluoride A:Fluoride A :Fluoride 0 0
in in in in
200 400 600 800 1000 Fluoride c o n c e n t r a t i o n ( p g / m l ) distilled 0.9% N a C l distilled distilled
water solution water c o n t a i n i n g 2 x 1 0 - 4 DNP w a t e r c o n t a i n i n g 4 x 1 0 - 4 DNP
Figure 5. Effect of fluoride concentration in the reflux on the absorption of fluoride from the small intestine.
Absorption from the rectum The rate of F disappearance from the reflux to aqueous solution with varying concentrations of NaF over a period of 60 m i n was plotted against the initial concentration of the reflux. Unlike those of the stomach and
0
C
nC LI
0 ~1
n
4
4.0
I
L
200 1000 1400 Flucride concentration (
p / m U
do
0 : F l u o r i d e i n d i s t i l l e d water i n 0.9% NaCl s o l u t i o n .:Fluoride
Figure 6. Effect of fluoride concentration on the absorption of fluoride from the rectum.
330
small intestine, the rate remained approximately constant with increases
in
F concentrations in the reflux.
When NaF-containing saline was refluxed, little or no influence of chloride on F absorption was observed (Fig. 6). Absorption from the mouth When the rate o f F absorption was plotted against time, a rectilinear relationship was seen over a 1 hour period, as with the rectum.
As shown
in Figure 7, the rate of absorption remained approximately constant at 2%. The absorption of F from the oral mucosa was unaffected by changes in Fion or C1- ion concentrations (Fig. 7).
0 200 4 0 0 6 0 0 8 0 0 Flurode concentration ( pg/ml) o : F l u o r i d e i n d i s t i l l e d water .:Fluoride i n 0.9% NaCl s o l u t i o n Figure 7. Effect of fluoride concentration on the absorption o f fluoride from the mouth.
DISCUSSION As shown in Figure
3 for the reflux through the stomach, the rectilinear
relationship between the logarithm of F concentrations o f the reflux fluid and time suggests that F was absorbed according to first-order kinetics. And the results shown in Figures 4 and 5 indicate that F absorption from the stomach and small intestine depended on F concentrations, being more efficient at lower concentrations. With increasing F concentrations, however, the rate of absorption approached a constant value. The rate of F absorption from saline was generally lower than that from distilled water, and the influence of C1- ions was especially pronounced at lower F concentrations. The decrease of F absorption in the presence of C1- ions may be explained by their inhibitory effect on F absorption. The data on F absorption were employed in the Lineweaver-Burk equation (6). assuming that active transport operated mainly within the concentration range studied. The results are shown in Figure 8. Rectilinear regressions were obtained with and without chloride. The two lines, moreover, were found to intersect at the ordinate. This indicates a competi-
331
0.0020
1 0.0015V
..
0.0010-
0.010
0.005
0.015 o*020
S:Initial concentration (Absorbance) V:Rate of transport (Absorbance/hr.) 0:Fluoride in distilled water :Fluoride in NaCl solution F i g u r e 8. L i n e w e a v e r - B u r k i n t e s t i n e o f rat.
tive inhibition a c t i o n o f C1-
plot:
Transport o f f l u o r i d e through the small
i n a n enzyme r e a c t i o n and,
ions on F absorption.
i n t h i s case, an i n h i b i t o r y
Even t h o u g h e n z y m a t i c a c t i o n on F
a b s o r p t i o n c a n n o t be d e f i n i t e l y c o n c l u d e d ,
t h e presence o f a rate-1 i m i t i n g
s t e p and c o m p e t i t i v e i n h i b i t i o n b y c o e x i s t i n g c h l o r i d e i s l i k e l y i n F absorption from the gastrointestinal tract.
Decrease i n t h e r a t e o f F
a b s o r p t i o n i n t h e p r e s e n c e o f DNP a l s o s u g g e s t s i n v o l v e m e n t o f an e n e r g y r e q u i r i n g mechanism. t i o n by,
C o n c e n t r a t i o n dependency o f ,
or competitive inhibi-
c h l o r i d e suggests t h a t a c t i v e t r a n s p o r t operates i n F absorption.
On t h e o t h e r hand,
t h e o b s e r v a t i o n t h a t t h e r a t e o f a b s o r p t i o n approached a
constant l e v e l w i t h increasing F concentrations, inhibitors,
o r i n t h e presence o f
suggests absorption by s i m p l e d i f f u s i o n .
It i s p o s s i b l e t h a t
b o t h t h e s e m e c h a n i s m s may be i n v o l v e d i n t h e a b s o r p t i o n s f F f r o m t h e gastrointestinal
t r a c t (Fig.
8).
F a b s o r p t i o n f r o m t h e m o u t h and r e c t u m showed no c o n c e n t r a t i o n dependency under t h e experimental conditions.
The i n f l u e n c e o f c h l o r i d e i o n s was
s l i g h t and F was f o u n d t o be a b s o r b e d f r o m t h e s e s i t e s a l m o s t a l o n g t h e c o n c e n t r a t i o n g r a d i e n t b y a s i m p l e mechanism.
Parkins
&
d.( 7 , 8 )
e v e r t e d s a c t e c h n i q u e t o d e m o n s t r a t e t h e a c t i v e t r a n s p o r t o f F,
and s t a t e d
opinions c o n t r a r y t o t h e conventional concept o f s i m p l e d i f f u s i o n . paper,
u s e d an In this
t h e m e t h o d o f i n t e s t i n a l r e f l u x i n s i t u was used t o e l u c i d a t e t h e
mechanism o f F a b s o r p t i o n f r o m each p a r t o f t h e d i g e s t i v e t r a c t under identical host factors,
and a b s o r p t i o n b y a c t i v e t r a n s p o r t was shown t o
332 occur i n a d d i t i o n t o absorption by s i m p l e d i f f u s i o n i n t h e s m a l l i n t e s t i n e and stomach. CONCLUSION The m e c h a n i s m o f F a b s o r p t i o n f r o m t h e g a s t r o i n t e s t i n a l t r a c t was s t u d i e d by u s i n g t h e r e f l u x method i n s i t u o f r a t s .
The e x p e r i m e n t a l r e s u l t s
showed t h a t t h e a b s o r p t i o n o f F f r o m t h e stomach and s m a l l i n t e s t i n e occurred not only through passive diffusion.
b u t t h r o u g h a mechanism
involving active transport or facilitated diffusion.
The a n t a g o n i s t i c
e f f e c t o f C1- i o n s on F a b s o r p t i o n was a l s o d e m o n s t r a t e d .
REFERENCES 1.
C a r l s o n CH, 104: 235-23'7
2.
S t o o k e y GK, 301
Armstrong
WD,
S i i l g e r L (1960)
P r o c S o c e x p B i o l Med
C r a n e DB, M u h l e r JC ( 1 9 6 4 ) P r o c S o c e x p B i o l Med 1 1 5 : 2 9 1 -
3.
Wagner MJ ( 1 9 6 2 ) J d e n t Res 41:667-671
4.
Wallace-Durbin
5.
Z i p k i n I,
6.
N a t h a n s D,
7.
P a r k i n s FM. H o l l i f i e l d JW, M c C a s l i n AJ, L i u Wu S-M, B i o c h e m B i o p h y s A c t a 126:513-524
P ( 1 9 5 4 ) J d e n t Res 33:789-800
L i k i n s RC ( 1 9 5 7 ) Am J P h y s i o l 191:549-550 T a p l e y DF,
Ross JE ( 1 9 6 0 ) B i o c h i m B i o p h y s A c t a 41:271
8. P a r k i n s F M ( 1 9 7 1 ) B i o c h i m B i o p h y s A c t a 241:507-512
F a u s t RG (1966)
H. Tsunoda a n d M.-H. Yu (Editors)
335
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 335-339 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
OSSIFICATIONS AND CALCIFICATIONS OF MUSCLE AND TENDON INSERTIONS I N HUMAN INDUSTRIAL FLUOROSIS
JURGEN FRANKE D e p a r t m e n t o f O r t h o p e d i c S u r g e r y , M e d i c a l Academy E r f u r t . R e g i e r u n g s s t r 42. DDR-5010, German D e m o c r a t i c R e p u b l i c F l u o r o s i s i s a c h r o n i c f l u o r i d e i n t o x i c a t i o n i n humans and a n i m a l s . There are f o u r types
of
human f l u o r o s i s a c c o r d i n g
to
the
source o f
fluorides:
I.
Endemic f l u o r o s i s . T h i s i s caused b y a d r i n k i n g water.
high fluorine
Examples a r e found i n I n d i a .
content i n
and i n N o r t h and S o u t h
Africa.
2.
Wine f l u o r o s i s .
T h i s was r e p o r t e d b y S o r i a n o o f S p a i n i n 1965(1).
Here h i g h amounts o f f l u o r i d e were added i n t o t h e w i n e t o p r e v e n t abnormal f e r m e n t a t i o n .
3.
Neiqhborhood f l u o r o s i s .
T h i s o c c u r s v e r y r a r e l y i n humans and c a t t l e
near f l u o r i d e e m i t t i n g industries.
4.
Industrial fluorosis. This i s a
rare
o c c u p a t i o n a l d i s e a s e among
w o r k e r s of f l u o r i n e p r o c e s s i n g o r m a n u f a c t u r i n g i n d u s t r i e s . Human f l u o r o s i s i s c h a r a c t e r i z e d b y t h r e e phenomena, s i s w i t h f o r m a t i o n o f exophytes, i n s e r t i o n s and j o i n t c a p s u l e s ;
i.e..
(1) p e r i o s t o -
w i t h o s s i f i c a t i o n s o f ligaments,
muscle
(2) e n d o s t o s i s ; and (3) s c l e r o s i s o f t h e
spongiosa. Radiologically,
i n b e g i n n i n g s t a g e s t h e r e a p p e a r s t o be a c o n d e n s a t i o n o f
t h e bone s t r u c t u r e ,
an e n l a r g e m e n t o f t h e bone t r a b e c u l a e i n t h e l u m b a r
s p i n e and p e r i o s t e a l a p p o s i t i o n s o n t h e bones o f f o r e a r m s and l o w e r l e g s . I n advanced stages,
t h e x-rays
o f t h e s p i n e show an e x t r e m e m a r b l e - l i k e
s c l e r o s i s o f t h e bones w i t h o s s i f i c a t i o n o f t h e l o n g i t u d i n a l ligaments. The f o r m a t i o n o f o u t g r o w t h s and s p u r s a t t h e m u s c u l a r i n s e r t i o n s and o s s i f i c a t i o n s o f the ligaments o f the p e l v i c f l o o r complete t h i s picture. O u t g r o w t h s l i k e a saw b l a d e a l s o d e v e l o p b e t w e e n t h e r a d i u s and u l n a and t h e t i b i a and f i b u l a . Pathologic-anatomical a r e as f o l l o w s :
f i n d i n g s f r o m a severe case o f i n d u s t r i a l f l u o r o s i s
o n t h e s p i n e we s e e a c o m p l e t e o s s i f i c a t i o n o f t h e l o n g i -
t u d i n a l ligaments;
t h e s m a l l v e r t e b r a l j o i n t s and c o s t o v e r t e b r a l j o i n t s a r e
ankylosed by o s s i f i c a t i o n o f t h e capsules, p r o t r u d e as exostoses.
t h e muscular
attachments
These changes a r e seen c l e a r l y o n t h e p e l v i s : a
336 projection
o f t h e m u s c u l a r a t t a c h m e n t s on t h e i l i a c c r e s t and p u b i c bone,
and o s s i f i c a t i o n s o f t h e s a c r o i l i a c j o i n t s , o f the pelvic floor.
Peripherally,
p u b i c symphysis.
saw-blade-like
and l i g a m e n t s
a p p o s i t i o n s between t h e
r a d i u s and u l n a (Fig. 1) o r between t h e t i b i a and f i b u l a a r e found.
The
l o n g bones show an i n c r e a s e o f t h e bone c r o s s - s e c t i o n w i t h d i s t i n c t t h i c k e n i n g o f t h e w h o l e c o r t i c a l i s b y p e r i o s t o s i s and e n d o s t o s i s . H i s t o l o g i c a l f i n d i n g s i n t h i s s t u d y w e r e b a s e d o n t h e a n a l y s i s o f 41 i l i a c c r e s t b i o p s i e s and 3 a u t o p s i e s a t d i f f e r e n t s t a g e s o f f l u o r o s i s (2-4).
These f i n d i n g s i n c l u d e :
1.
c o a r s e n i n g and c o n d e n s a t i o n o f t h e s p o n g y b o n e
2.
p e r i o s t e a l f o r m a t i o n o f f i b r o u s bone w i t h t r a n s f o r m a t i o n i n t o l a m e l -
3.
t h i c k e n i n g and s p o n g i o s a t i o n o f t h e c o r t i c a l i s
l a r bone l e a d i n g t o t h e f o r m a t i o n o f e x o p h y t e s 4.
irregular matrix formation w i t h a high turnover rate
5.
f o c i w i t h a coarse f i b r o u s structure, charides,
6.
a h i g h c o n t e n t o f mucopolysac-
and w i t h an i n c l i n a t i o n t o c a l c i f i c a t i o n
i n some cases,
an i n c r e a s e and t h i c k e n i n g o f t h e o s t e o i d seams
D i f f e r e n t phenomena w e r e f o u n d e s p e c i a l l y on t h e p e r i o s t e a l s u r f a c e : 1.
s u b p e r i o s t e a l f i b r o u s bone f o r m a t i o n ,
sometimes w i t h t r a n s f o r m a t i o n
i n t o l a m e l l a r bone 2.
new bone f o r m a t i o n i n t o t h e c o n n e c t i v e t i s s u e o f t h e p e r i o s t e u m ,
3.
ossification o f inserting periosteal fibers w i t h transformation i n t o
4.
s o m e t i m e s some f i b r o u s s t r u c t u r e s a r e n o t o s s i f i e d b u t e n c l o s e d by
m o s t l y i n t o l i g a m e n t s and m u s c u l a r i n s e r t i o n s ( F i g .
2)
l a m e 1 1 a r bone n e w l y f o r m e d bone and t h e n i n c r u s t e d w i t h m i n e r a l s
5.
t h e s e c a l c i f i e d f o c i ( i n c r u s t a t i o n f o c i ) a r e t o be f o u n d i n t h e n e w l y formed c o r t i c a l i s j u s t below t h e periosteum; coarse,
t h e f o c i a r e PAS p o s i t i v e ( F i g .
the fiber structure i s
3) and e x h i b i t a b l u i s h c o l o r
upon s t a i n i n g w i t h a l i z a r i n b l u e and d a r k b l u e w i t h t o l u i d i n e b l u e I n t h e m i c r o r a d i o g r a m t h e s e f o c i show a h i g h m i n e r a l c o n t e n t ( F i g . 4). The s c a n n i n g - e l e c t r o n - m i c r o s c o p i c
f i n d i n g s (SEM)
(5) a r e b a s e d on exam-
i n a t i o n s o f bones o f t h r e e p e r s o n s i n d i f f e r e n t s t a g e s o f i n d u s t r i a l f l u o rosis.
The s t u d i e s w e r e c a r r i e d o u t o n t h e p e r i o s t e a l and f r a c t u r e s u r -
faces o f ribs, fluorosis,
t i b i a e and v a u l t s o f cranium.
I n t h e case o f beginning
a r e a s w i t h s w e l l i n g o r edema o f t h e c o l l a g e n o u s f i b e r s on t h e
p e r i o s t e a l s u r f a c e o f t h e r i b w e r e found. Sometimes t h e s e f i b e r s a r e i m p r e g n a t e d w i t h g l o b u l a r and c r y s t a l l i n e material,
b u t on t h e s k u l l s u r f a c e
areas w i t h d i s t i n c t m i n e r a l i z a t i o n o f
c o l l a g e n o u s f i b e r s i n s e r t i n g i n t o t h e bone were a l s o found.
A l l these
337
New b o n e f o r m a t i o n a t t h e p e r i o s + ? ; l ( + ) I:.!. x 1 2 5
F i g u r e ?. ,:,:e.in -
p
7
s,>rf-.:c
?nd i n t h e n e r i -
338
Figure 4. M i c r o r a d i o g r a m o f i l i a c crest, f l u o r o s i s s t a g e 1 1 1 ; typical "calcified focus" ( t ) . x 125
Figure 5. SEM: periosteal surface o f tibia in moderately advanced fluorosis: highly-mineralized, broken-off insertion of a tendon. x 2250
339 s t r u c t u r e s r e p r e s e n t a s t a g e b e t w e e n n o r m a l c o n n e c t i v e t i s s u e and i m m a t u r e , s l i g h t l y m i n e r a l i z e d bone t i s s u e . I n m o d e r a t e f l u o r o s i s i t was o b s e r v e d t h a t t h e p e r i o s t e a l s u r f a c e o f t h e t i b i a had a m o r e p r o n o u n c e d edema o f t h e s t i l l d i r e c t e d c o l l a g e n o u s f i b e r s . The h i g h l y - m i n e r a l i z e d , impressive (Fig.
broken-off
i n s e r t i o n s o f t e n d o n s i n t o t h e bone w e r e
5).
I n advanced f l u o r o s i s on t h e r i b s u r f a c e ,
the collagenous f i b e r s are
a b n o r m a l l y t h i n , i r r e g u l a r l y o r i e n t e d and p a r t i a l l y covered b y a t y p i c a l Under h i g h e r m a g n i f i c a t i o n ,
matrix.
s i n g l e f i b e r s as w e l l as t h e w h o l e
bone s u r f a c e h a v e a g r a n u l a r - l i k e c o v e r i n g . I n conclusion,
a t f i r s t a s l i g h t edema and i m p r e g n a t i o n w i t h g l o b u l a r and
c r y s t a l l i n e m a t e r i a l i n t h e p e r i o s t e a l c o l l a g e n o u s f i b e r s ' were observed. The edema and i m p r e g n a t i o n s i n c r e a s e a s t h e d i s e a s e a d v a n c e s and, fluorosis,
i n severe
a c o m p l e t e l y i r r e g u l a r o r i e n t a t i o n o f a b n o r m a l l y t h i n f i b e r s and
t h i c k c r y s t a l l i n e c o v e r i n g s on t h e bone s u r f a c e o c c u r s .
The f i b e r s o f t h e
m u s c u l a r a t t a c h m e n t s and t h e t e n d o n i n s e r t i o n s w e r e o b v i o u s l y t h e f i r s t t o be m i n e r a l ized. Some S E M f i n d i n g s a r e c l o s e l y c o m p a r a b l e w i t h t h e n o r m a l h i s t o l o g y o f fluorosis:
The a p p o s i t i o n a l p e r i o s t e a l o s s i f i c a t i o n o b s e r v e d i n b e g i n n i n g
f l u o r o s i s corresponds t o t h e i m p r e g n a t i o n o f s o f t t i s s u e i n t h e SEM p i c t u r e , w h i c h i s an i n t e r m e d i a t e s t a g e b e t w e e n c o n n e c t i v e t i s s u e and i m m a t u r e bone.
It c o r r e s p o n d s t o t h e f o r m a t i o n o f i m m a t u r e p e r i o s t e a l f i b r o u s bone
i n t h e normal h i s t o l o g i c a l p i c t u r e , i n t o l a m e l l a r bone.
w h i c h a f t e r w a r d s w i l l be t r a n s f o r m e d
I n t h i s p r o c e s s t h e t e n d i n o u s and m u s c u l a r i n s e r t i o n s
a r e a f f e c t e d most severely.
I n t h e normal h i s t o l o g i c a l p i c t u r e , areas w i t h
p u r e i n c r u s t a t i o n s o f c o n n e c t i v e t i s s u e w i t h o u t t r a n s f o r m a t i o n i n t o bone t i s s u e were a l s o observed, However,
t h e so-called
" c a l c i f i e d o r encrustation foci."
t h i s phenomenon c a n b e o b s e r v e d m o r e o f t e n i n t h e SEM p i c t u r e .
The c o m p l e t e l y a t y p i c a l
s t r u c t u r e and a r r a n g e m e n t o f c o l l a g e n o u s f i b e r s
on t h e p e r i o s t e a l bone s u r f a c e i n s e v e r e f l u o r o s i s i n t h e SEM p i c t u r e a r e i n accordance w i t h t h e i r r e g u l a r m a t r i x i n t h e normal h i s t o l o g i c a l p i c t u r e . REFERENCES 1.
S o r i a n o M ( 1 9 6 5 ) Rev C l i n E s p a n o l a 97:375-388
2.
F r a n k e J (1972)
3.
F r a n k e J , R a t h F.
F l u o r i d e 5:182-198 R u n g e H,
F e n g l e r F,
A u e r m a n n E,
Lenart G
(1975)
F l u o r i d e 8:61-83 4.
F r a n k e J,
5.
F r a n k e J. R u n g e H, F e n g l e r F ( 1 9 7 8 ) I n : C o u r v o i s i e r B. D o n a t h A, Baud, CA ( e d s ) Symposium CEMO 11: F l u o r i d e and Bone. Ed M'edecine e t H y g i e n e , Geneva, p p 129-143
H o r n V (1976) F l u o r i d e 9:127-137
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 341-346 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
341
TOPOGRAPHICAL LOCALIZATION OF FLUORIDE I N BONE TISSUE
SEONG BANG AND CHARLES A.
BAUD
I n s t i t u t de M o r p h o l o g i e . C e n t r e M i d i c a l U n i v e r s i t a i r e , Servet, 1211 Genhve 4, S w i t z e r l a n d
1. r u e M i c h e l -
ABSTRACT The e l e c t r o n m i c r o p r o b e was used t o demonstrate t h e t o p o g r a p h i c a l d i s t r i b u t i o n o f f l u o r i d e (F) a t t h e m i c r o s c o p i c a l l e v e l i n a n i m a l and human bone tissues. The f e m o r a l bones o f w e a n l i n g m i c e m a i n t a i n e d on f l u o r i d a t e d d r i n k i n g w a t e r f o r p e r i o d s o f up t o 12 months were analyzed. graph o f F
Kcl
The c o m p o s i t e photo-
X-ray images showed heavy d e p o s i t s o f F i n t h e p e r i o s t e a l and
e n d o s t e a l a r e a s o f bone t i s s u e f o r m e d d u r i n g t h e e x p e r i m e n t .
Moderate
q u a n t i t i e s o f F were a l s o found i n t h e remnants o f woven bone formed b e f o r e t h e b e g i n n i n g o f t h e experiment. I n t h e i l i a c bone b i o p s i e s f r o m o s t e o p o r o t i c p a t i e n t s t r e a t e d w i t h 12 months,
F
for
a h i g h l e v e l o f F was observed i n t h e bone l a y e r s formed d u r i n g
the treatment,
whereas i n t h e l a m e l l a r bone t i s s u e formed b e f o r e t r e a t m e n t ,
t h e F l e v e l was f o u n d t o b e s i m i l a r t o t h a t i n t h e c o n t r o l s e x c e p t i n a t h i n layer l i n i n g the surface i n the v i c i n i t y o f the c a p i l l a r y blood vessels. Thus, i t c o u l d be p o s t u l a t e d t h a t s y s t e m i c a l l y i n g e s t e d F i s d e p o s i t e d p r i m a r i l y i n t h e s i t e s o f new bone formed d u r i n g t h e p e r i o d s o f F i n g e s t i o n i n b o t h cases,
and a l s o s e c o n d a r i l y b y a d i f f u s i o n p r o c e s s i n t h e w h o l e
p r e - e x i s t i n g woven bone i n m i c e , and i n a n a r r o w s u b s u r f a c e b a n d o f t h e l a m e l l a r bone formed b e f o r e t r e a t m e n t i n human s u b j e c t s . INTRODUCTION S y s t e m i c a l l y i n g e s t e d f l u o r i d e (F) i s e i t h e r e x c r e t e d i n t h e u r i n e and feces,
o r d e p o s i t e d p r i n c i p a l l y i n t h e c a l c i f i e d t i s s u e s o f t h e body.
A l t h o u g h e x c e s s i v e i n t a k e may induce f l u o r o s i s ,
F has been employed i n t h e
t r e a t m e n t o f o s t e o p o r o s i s and i n t h e p r o p h y l a x i s a g a i n s t d e n t a l c a r i e s . Thus,
t h e r e i s a c o n s i d e r a b l e i n t e r e s t i n t h e l o c a l i z a t i o n o f absorbed F.
This i n t e r e s t arises from both the possible therapeutic o r prophylactic a c t i o n o f t h i s e l e m e n t and a l s o t h e p o t e n t i a l h a z a r d o f an i n c r e a s e d F accumulation i n t h e c a l c i f i e d tissues. T h i s paper i s a r e p o r t on t h e F d i s t r i b u t i o n p a t t e r n o f t h e bones o f m i c e w h i c h had been g i v e n
F
i n t h e i r drinking
water,
and o f i l i a c c r e s t
342 b i o p s i e s t a k e n f r o m o s t e o p o r o t i c p a t i e n t s who had been t r e a t e d w i t h sodium fluoride. MATERIALS AND METHODS E x p e r i m e n t a l Animals 21-day-old
m i c e were m a i n t a i n e d on f l u o r i d a t e d d r i n k i n g w a t e r (100 ppm F)
f o r up t o 12 months.
A t t h e b e g i n n i n g o f t h e experiment,
i n j e c t e d i n t r a p e r i t o n e a l l y f o r bone l a b e l i n g .
The femoral
t e t r a c y c l i n e was bones o f m i c e
s a c r i f i c e d a t d i f f e r e n t p e r i o d s o f t i m e were p r e p a r e d f o r e l e c t r o n probe Xr a y m i c r o a n a l y s i s (1-4). Human Bone B i o p s i e s I l i a c c r e s t biopsies,
t a k e n f r o m o s t e o p o r o t i c p a t i e n t s who had been
t r e a t e d w i t h NaF ( 3 0 mg F / d a y ) f o r 1 2 months,
were analyzed by e l e c t r o n
m i croprobe.
RESULTS A c o m p o s i t e X-ray
image o f F Ka e m i s s i o n o f t h e f e m o r a l bone o f a mouse
g i v e n f l u o r i d a t e d w a t e r f o r 45 days showed d e p o s i t s o f F i n t h e p e r i o s t e a l and e n d o s t e a l l a y e r s , as i n d i c a t e d b y a r r o w s a and b i n F i g u r e 1. These l a y e r s a r e l a b e l e d w i t h t e t r a c y c l i n e as shown i n F i g u r e 2,
corresponding t o
t h e bone t i s s u e formed d u r i n g t h e e x p e r i m e n t a l period. I n t h e l o n g - t e r m (12 months) F - i n g e s t e d
mice,
t h e F was d i s t r i b u t e d r a t h e r h o m o g e n e o u s l y
t h r o u g h o u t t h e bone s e c t i o n s i n c e t h e woven bone was b e i n g r e p l a c e d by new l a m e l l a r b o n e ( F i g s . 3 a n d 4). A n a l y s i s o f t h e human bone b i o p s i e s showed a h i g h c o n c e n t r a t i o n o f F i n t h e p e r i o s t e a l and e n d o s t e a l l a y e r s , osteons.
i n t h e t r a b e c u l a r bone, and i n c e r t a i n
These were a l l formed d u r i n g t h e p e r i o d o f F-treatment.
However,
i n t h e l a m e l l a r b o n e t i s s u e f o r m e d b e f o r e t h e t r e a t m e n t , t h e F l e v e l was f o u n d t o be s i m i l a r t o t h a t i n t h e c o n t r o l s e x c e p t i n a t h i n l a y e r l i n i n g the surface i n the v i c i n i t y o f t h e c a p i l l a r y blood vessels
( F i g s . 5 and
6). DISCUSSION Chemical analyses o f t h e F c o n t e n t o f whole bone as used by Weidmann and Weatherell (5) t o i n v e s t i g a t e t h e uptake o f F i n t h e various s k e l e t a l t i s s u e s o f r a b b i t d o n o t p r o v i d e d e t a i l s on t h e d i s t r i b u t i o n o f F i n t h e
By u t i l i z i n g t h e r a d i o a c t i v e i s o t o p e o f f l u o r i n e (F18) f o r t h e a u t o r a d i o g r a p h i c s t u d i e s i n b o n e s and t e e t h , V o l k e r d.(6).
bone t i s s u e i t s e l f .
W a l l a c e - D u r b i n (7).
Perkinson
ad.(8)
and E r i c s s o n
gfl.( 9 )
reported
t h a t i n e x p e r i m e n t a l a n i m a l s t h e a r e a o f g r e a t e s t F18 c o n c e n t r a t i o n was i n
7
W
r
m
3
L
Ln
L L
Ln
4
c
0 c
W
V
c, v1
W c 0
n W E VI
aJ
*
r
+ 0 L
4
a
r
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L
0
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.r
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aJ
c
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a)
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0 3 7
LL
343
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r
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0
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8 0
W
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a
.-c Q r 8
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W v) 3
0
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m 0
Lc
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n 0
L
m
7
E
L
0
+ W + 0
C
aJ aJ
n 73
m
0
I
L 3
r
w
K
Q
CL
c, m
u
L
r
*0 0 CL
0
v)
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0
E
m
c
L 4-
m
Y aJ
h
c, v)
Q r
0
n e v) aJ L u u m
r i
I
r' r
v)
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345
346 t h e most r e c e n t l y c a l c i f i e d bone,
and t h e y i n d i c a t e d t h a t t h e a c c u m u l a t i o n
i n b o n e seemed t o d e p e n d u p o n t h e v a s c u l a r i t y o f t h e bone a n d i t s g r o w t h a c t i v i t y . Although t h e autoradiographic technique i s n o t s u i t a b l e f o r a long-term
F-experiment
t h e i r find-
because o f t h e s h o r t h a l f - l i f e o f F18,
ings are q u i t e s i m i l a r t o those o f t h e e l e c t r o n microprobe analysis. I n t h e p r e s e n t study,
a t t e m p t s w e r e made t o show some o f t h e X-ray
e m i s s i o n images i n mosaic f o r m t o p r o v i d e a maximum o f i n f o r m a t i o n f r o m e x t e n s i v e a r e a s o f t h e bone s e c t i o n . The
F
distribution pattern o f the
e x p e r i m e n t a l mouse was s i m i l a r t o t h a t o f t h e human bone t i s s u e .
The
i n g e s t e d F d e p o s i t e d i n t h e n e w l y f o r m e d bone: F a l s o d i f f u s e d f r o m t h e b l o o d v e s s e l s t o t h e bone 1ayers. Thus,
i t may be p o s t u l a t e d t h a t s y s t e m i c a l l y - i n g e s t e d
F
i s deposited
p r i m a r i l y i n t h e s i t e s o f new bone formed d u r i n g t h e p e r i o d s o f
F
ingestion
i n b o t h cases, and, s e c o n d a r i l y , b y a d i f f u s i o n p r o c e s s i n t h e w h o l e p r e e x i s t i n g woven bone i n mice,
and i n a n a r r o w s u b s u r f a c e band o f t h e l a m e l -
l a r bone formed b e f o r e t r e a t m e n t i n human subjects. REFERENCES 1.
Bang S (1976) Revue mens s u i s s e Odonto-Stomatol
2.
Bang S ( 1 9 7 8 ) I n : C o u r v o i s i e r B, D o n a t h A, Baud CA ( e d s ) F l u o r i d e and Bone. Huber, Bern. pp 77-81
3.
Baud CA, Bang S (1970) I n : Huber. Bern, pp 27-36
4.
Baud CA, Bang S (1972) I n : S h i n o d a G. K o h r a K, I c h i n o k a w a T ( e d s ) Proceedings o f t h e S i x t h I n t e r n a t i o n a l Conference on X-ray O p t i c s and Microanalysis. U n i v e r s i t y o f Tokyo Press, pp 841-846.
5.
Weidmann SM, W e a t h e r e l l JA (1959) J P a t h o l B a c t e r i o l 78:243-255
6.
V o l k e r JF,
7.
Wallace-Durbin P (1954) J Dent Res 33:789-800
8.
P e r k i n s o n JD, W h i t n e y I B . M o n r o e RA. L o t z WE, Comar CL (1955) Amer J P h y s i o l 182:383-359
9.
E r i c s s o n Y, U l l b e r g S. A p p e l g r e n LE ( 1 9 6 0 ) A c t a O d o n t o l Scand 18:253261
Sognnaes RF.
86:838-863
V i s c h e r ThL (ed) F l u o r i d e i n M e d i c i n e .
B i b b y GB (1941) Amer J P h y s i o l 132707-712
347
H. Tsunoda and M.-H. Y u (Editors)
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 347-355 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands
BONE STATIC AND D Y N A M I C HISTOMORPHOMETRY IN E N Q E M I C SKELETAL FLUOROSIS SURENDRA P.
S. TEOTIA, M O H I N I TEgTIA, AND DHARAM P. S I N G H
P o s t G r a d u a t e D e p a r t m e n t o f Human M e t a b o l i s m and E n d o c r i n o l o g y , R a i M e m o r i a l M e d i c a l C o l l e g e , M e e r u t - 2 5 0 102, I n d i a
Lala Lajpat
ABSTRACT Quantitative tetracycline-based
a n a l y s i s o f i l i a c bone b i o p s y was p e r -
f o r m e d i n 17 p a t i e n t s w i t h endemic s k e l e t a l f l u o r o s i s . endemic f l u o r o s i s areas s i n c e t h e i r b i r t h . d u r a t i o n o f symptoms 7.6 y e a r s . :!ster
A l l had l i v e d i n
The mean age was 25.6 y e a r s and
The mean f l u o r i d e c o n t e n t o f t h c d r i n k i n g
was 17.8 ppm and t h e mean d a i l y i n t a k e o f f l u o r i d e was 26.2 mg.
o f t h e p a t i e n t s had V i t a m i n D d e f i c i e n c y ,
None
i n t e s t i n a l malabsorption o r renal
dysfunction. P e r t i n e n t b i o c h e m i c a l f i n d i n g s w e r e : r a i s e d p l a s m a le?.ls a l k a l i n e p h o s p h a t a s e and iPTH.
o f fluoride,
and i n c r e a s e d u r i n a r y f l u o r i d e e x c r e t i o n .
S t a t i c and d y n a m i c h i s t o m o r p h o m e t r i c m e a s u r e m e n t s r e v e a l e d t h e p r o f i l e s o f o s t e o m a l a c i a and s e c o n d a r y h y p e r p a r a t h y r o i d i s m i n v a r y i n g c o m b i n a t i o n s i n all
cases.
I n c r e a s e s i n o s t e o i d volume,
osteoid surfaces,
o s t e o i d seams
w i d t h , r e s o r p t i o n s u r f a c e s and m i n e r a l i z a t i o n l a g t i m e and d e c r e a s e s i n corrected apposition rate,
b o n e f o r m a t i o n r a t e s and t e t r a c y c l i n e l a b e l e d
s u r f a c e s w e r e t h e v a r i a b l e f e a t u r e s r e c o r d e d i n each case. D y n a m i c bone h i s t o r n o r p h o m e t r y has f u r t h e r i n c r e a s e d t h e u n d e r s t a n d i n g o f t h e e f f e c t s o f f l u o r i d e on i n t r a s k e l e t a l m e c h a n i s m s a f f e c t i n g t h e s t r u c t u r e and m e t a b o l i s m o f t h e bone b u t i t d o e s n o t a l l o w c l e a r - c u t d i f f e r e n t i a t i o n f r o m m e t a b o l i c bone d i s e a s e .
End>mic s k e l e t a l f l u o r o s i s ,
therefore,
may be
r e g a r d e d as a p a r a m e t a b o l i c bone d i s e a s e .
INTRODUCTION Endemic s k e l e t a l f l u o r o s i s c o n t i n u e s t o r e m a i n a c h a l l e n g i n g n a t i o n a l h e a l t h p r o b l e m i n m o s t p a r t s o f t h e \ w o r l d (1). s t u d i e s on i t s c l i n i c a l .
biochemical,
a s p e c t s h a v e been r e p o r t e d . s k e l e t a l f l u o r o s i s a r e sparse.
metabolic,
I n e a r l i e r w o r k s (2-8).
e n d o c r i n e and r a d i o l o g i c a l
R e p o r t s o n bone h i s t o m o r p h o m e t r y i n e n d e m i c The o n l y r e p o r t s p r o v i d i n g a d e q u a t e d a t a on
human e n d e m i c s k e l e t a l f l u o r o s i s a r e t h o s e o f T e o t i a and T e o t i a (9-13).
348
The hi stopathogenesi s and hi stomorphometry of bone disease produced by chronic fluoride intoxication still remain incompletely understood. There appear to be no published reports o n human e n d e m i c skeletal fluorosis employing a comprehensive histomorphometric analysis of non-decalcified sections of bone that have been labeled at mineralization zones with double tetracycline labels. This extended work on quantitative analysis of labeled iliac crest biopsies will serve t w o major needs: 1. Is bone hlstomorphometry useful for the diagnosis of skeletal fluoro51 5 7
2. To achieve a better understanding of bone disease and toxic effects of fluoride on intraskeletal mechanisms, structure and metabolism of the bone. MATERIALS AND METHODS Seventeen patients of endemic skeletal fluorosis were studied during the period 1972-1984 (Table I).
All had been living in endemic fluorosis areas
since their hi rth.
TABLE I CLASSIFICATION OF SEVERITY OF FLUOROSIS C1 inical Mi
Id:
Moderate:
Generalized bone and joint pains Generalized
bone and joint pains, stiffness and rigidity,
restricted movements at spine and joints. Severe:
Symptoms of moderate
with deformities of spine and limbs,
knock knees, crippl ing/bed-ridden state.
Mi Id:
Radiological Only osteosclerosis
Mod era te :
Osteosclerosis. periosteal bone formation, calcifications o f interosseous membrane, ligaments. capsules, muscular attachments, tendons.
Severe:
Findings as in moderate,
exostoses, osteophytosis and
associated metabol i c bone disease.
349 P e r t i n e n t 1a b o r a t o r y i n v e s t i g a t i o n s i n c l u d e d d e t e r m i n a t i o n s >f p l a s m a fluoride,
calcium,
phosphate,
h y d r o x y c h o l e c a l c i f e r o l (25-OHD), (IPTH).
Twenty-four
alkaline
phosphatase,
hour u r i n e d e t e r m i n a t i o n s o f f l u o r i d e . n e p h r o g e o u s c y c l i c AMP (Nc'AMP)
a t i n i n e c l e a r a n c e (Ccr),
s o r p t i o n o f p h o s p h a t e (TRP) w e r e made. accepted p u b l i s h e d procedures.
25-
a n d i m m u n o r e a c t i v e p a r a t h y r o i d hornlo:? calcium,
cre-
and t u b u l a r r e a b -
A l l t h e e s t i m a t i o n s w e r e made u s i n g
F l u o r i d e c o n c e n t r a t i o n s were measured w i t h
a f l u o r i d e s e l e c t r o d e u s i n g a PHM64 r e s e a r c h r a d i o m e t e r . Bone b i o p s y :
P r i o r t o b i o p s y e a c h p a t i e n t was g i v e n o x y t e t r a c y c l i n e
h y d r o c h l o r i d e 500 mg 8 h o u r l y f o r 3 d a y s , w i t h no m e d i c a t i o n , f o r 3 days.
f o l l o w e d b y an 11 day i n t e r v a l
and t h e n d e m e c l o c y c l i n e h y d r o c h l o r i d e 300 mg 8 h o u r l y
T h r e e d a y s a f t e r t h e s e c o n d l a b e l a f u l l t h i c k n e s s b i o p s y was
o b t a i n e d f r o m t h e i l i a c c r e s t u n d e r l o c a l a n e s t h e s i a u s i n g a 7.5 m m i n t e r nal diameter trephine. end,
T h i s produced a c o r d o f bone w i t h c o r t e x a t each
t r a b e c u l a r bone and m a r r o w i n between.
The w h o l e s p e c i m e n was p r e -
served i m m e d i a t e l y i n 70 p e r c e n t e t h a n o l . S e c t i o n i n g ,
embedding,
staining,
m i c r o s c o p i c e x a m i n a t i o n and q u a n t i t a t i v e h i s t o m o r p h o m e t r y w e r e p e r f o r m e d u s i n g s t a n d a r d p r o c e d u r e s and p r i n c i p l e s (10). s t u d i e d i n c l u d e d s t r u c t u r a l measurements,
The v a r i o u s p a r a m e t e r s
s u r f a c e measurements,
formation
and r e s o r D t i o n i n d i c e s . RESULTS The d i a g n o s i s o f e n d e m i c s k e l e t a l f l u o r o s i s was c o n f i r m e d i n a l l p a t i e n t s b y c h a r a c t e r i s t i c r a d i o l o g i c a l f i n d i n g s and h i g h c o n c e n t r a t i o n s o f f l u o r i d e i n t h e i r p l a s m a and u r i n e and i n d r i n k i n g w a t e r . The c l i n i c a l and r a d i o l o g ical severity o f skeletal
4
0
.
20
40
f l u o r o s i s was c l a s s i f i e d a s m i l d ,
6 0 00
OSTEOID SURFACE
( % TOTAL s uR FAC E )
100
m o d e r a t e and
--
0
0.2
0.4
CORRECTED
0.6
0 8 1.0
APPOSITION
I?
RATE
(ym/d)
F i g u r e 1. Bone h i s t o m o r p h o m e t r i c i n d i c e s i n e n d e m i c s k e l e t a l f l u o r o s i s a s c o m p a r e d t o t h e p a r a m e t e r s seen i n m e t a b o l i c bone d i s e a s e .
w
cn
0
TABLE I 1 CLINICAL DETAILS OF PATIENTS STUDIED Case Age No. (Yrs)
Sex
Fluoride i n water (PPm)
Fluoride intake (rng/d)
Duration o f symptoms (Years)
Severity o f Skeletal Fluorosis Clinical
Radiological
Dental F 1u o r o s i s (Grade)
1
20
M
25
36
5
Severe
Severe
3
2
10
M
25
39
Severe
Severe
3
3
23
M
25
30
Severe
Severe
3
4
21
M
25
28
3 3 4
Severe
Severe
5 6
65 13
M
50 32
20
Severe
Severe
M
25 25
3 4
10
Severe
Severe
0
7
30
M
25
40
Severe
Severe
4
8
32
F
25
36
6 5
Moderate
Moderate
3
9
35
F
32
6
Moderate
Severe
3
10
42
F
25 8.5
12
4
MiI d
Moderate
3
11
15
F
25
27
8
Severe
Severe
2
12
F
18 8
20
3
Severe
Severe
1
13
9 18
16
6
Severe
Severe
2
14
23
M
6.5
14
20
Moderate
Severe
2
15
30 30
F
8.5
Severe
Moderate
Mild
3 1
29
F
5.5 4.5
15 5
Severe
F
21 11
9
6
MiId
MiI d
1
16 17
F
351
TABLE 111 PLASMA COMPOSITION Case Fluoride No.
(wM/1)
Calcium (mg/dl)
Alk Ptase (KAU/dl)
~
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17
8.5 12.0 1.5 5.8 12.7 10.5 8.0 5.6 6.6 3.2 9.5 4.2 6.5 10.2 7.5 3.2 3.4
9.5 10.0 9.6 10.2 11.0 10.0 9.0 9.8 10.5 10.0 9.5 9.8 7.6 8.4 1.7 10.4 8.9
3.5 4.0 3.7 4.8 3.0 4.2 4.0 3.2 4.0 3.4 3.2 5.8 5.0 4.2 3.6 3.8 4.9
35 30 16 31 76 57 46 32 25 28 45 45 53 18 116 27 1"
15.0 16.5 12.0 17.5 23.0 18.0 14.0 25.5 20.0 15.0 12.0 12.0 14.0 20.0 16.0 14.0 l".-
1400 2275 1300 1150 5050 2700 1225 1350 1050 1200 1625 1500 900 1200 2800 ~600 1lQO
18 20 15 16 22 20 15 30 22 18 16 18 19 30 16 22 15
Figure 2. Wide osteoid seams and hook resorption of the trabeculae. Findi n g s are suggestive o f osteomalacia a n d secondary hyperparathyroidism (undecalcified villanueva osteochrome section 100 x ) .
352
TABLE I V URINARY COMPOSITION -
Case
No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Fluoride (mg/d)
11.0 16.8 5.0 13.4 36.0 15.0 13.0 9.4 5.8 5.8 13.0 1.5 11.5 9.2 6.8 6.8 5.4
Calcium
TRP
(mg/d)
(%)
Nc'AMP (nm/mg Cr)
Ccr (ml/min)
78 64 80 70 68 75 82 83 68 85 74 82 78 84 51 82 86
1.32 6.75 3.20 2.23 5.56 4.89 1.29 1.35 4.25 1.39 2.85 2.62 1.65 3.85 7.95 2.25 1.7'
130 120 128 120 125 123 126 118 120 135 116 122 135 126 122 134
41 62 32 40 76 56 35 46 50 44 58 57 53 68 85 74 130
1 q,>
F i g u r e 3. Undecalcified section s a m e a s Figure 2. Photomicrograph taken under fluorescence microscope revealed only a few areas of tetracycline labels indicating lack of mineralization zones.
353 severe (Table I). rigidity,
A l l w e r e s y m p t o m a t i c and had vague p a i n s , s t i f f n e s s ,
and l i m i t a t i o n s i n s p i n a l movements,
movements and i n a b i l i t y t o c l o s e f i s t s .
backache,
restricted joint
The g r o s s l y l i m i t e d movements o f
t h e spine, f l e x i o n d e f o r m i t i e s a t t h e h i p s and knees s u g g e s t t h a t severe f l u o r o s i s was p r e s e n t i n 11 p a t i e n t s . C l i n i c a l and b i o c h e m i c a l o b s e r v a t i o n s a r e summarized i n Tables I
-
I V and
r e s u l t s o f h i s t o m o r p h o m e t r y a r e summarized i n T a b l e V and F i g u r e s 1-4. TABLE V
BONE HISTOMORPHOMETRIC MEASUREMENTS S t a t i c Variables O s t e o i d Volume ( % TT) O s t e o i d S u r f a c e (%TS) O s t e o i d Seam Width (u) R e s o r p t i o n Lacunae w i t h O s t e o c l a s t s ( % TBS)
(MEAN f SD)
E. S. F. 4.64 f 1.97 55.04 18.78 30.29 f 21.53 4.00 f 3.58 _+
Dynamic V a r i a b l e s C o r r e c t e d a p p o s i t i o n r a t e (urn/d) Mineralization lag time (d) Labeled s u r f a c e ( % 0s) Bone f o r m a t i o n r a t e (pm3/um2)
0.30 0.14 276.50 f 165.62 8.89 21.17 5.20 f 3.01 +_
+_
Normal 1.8 13.5 11.25 2.9
0.7 t 9.0 f 5.15 f 0.2 f
*
0.7 0.2 26.25 f 10.15 63.5 f 17.2 21.5 f 16.2
F i g u r e 4. Radiograph showing g e n e r a l i z e d o s t e o s c l e r o s i s and m u l t i p l e c y s t i c r e s o r p t i o n a r e a s . The p r e s e n c e o f h y p e r p a r a t h y r o i d i s m s e c o n d a r y t o t h e f l u o r o s i s i s suggested.
354 DISCUSSION C l i n i c a l d e t a i l s o f t h e p a t i e n t s s t u d i e d a r e g i v e n i n T a b l e 11. t h e p a t i e n t s had d i e t a r y v i t a m i n - D renal dysfunction.
deficiency,
None o f
i n t e s t i n a l malabsorption o r
P l a s m a b i o c h e m i c a l f i n d i n g s showed r a i s e d f l u o r i d e ,
a l k a l i n e p h o s p h a t a s e and iPTH l e v e l s ( T a b l e 111). f l u o r i d e and Nc'AMP w e r e i n c r e a s e d ,
Twenty-four
hour u r i n e
TRP was l o w and c r e a t i n i n e c l e a r a n c e
was n o r m a l ( T a b l e I V ) . Q u a n t i t a t i v e m e a s u r e m e n t s w e r e made o f s t a t i c and d y n a m i c v a r i a b l e s on undecalcified sections o f tetracycline-based
i l i a c c r e s t biopsies obtained
f r o m 1 7 p a t i e n t s o f e n d e m i c s k e l e t a l f l u o r o s i s ( T a b l e V).
The p a r a m e t e r s
s t u d i e d r e v e a l e d t h e p r o f i l e s o f o s t e o m a l a c i a and s e c o n d a r y h y p e r p a r a t h y r o i d i s m i n v a r y i n g c o m b i n a t i o n s i n a l l cases. Bone h i s t o m o r p h o m e t r i c m e a s u r e m e n t s showed i n c r e a s e d v a l u e s o f o s t e o i d volume,
o s t e o i d surfaces,
o s t e o i d seams w i d t h and r e s c r p t i o n l a c u ' n a e con-
t a i n i n g o s t e o c l a s t s . D y n a m i c m e a s u r e m e n t s r e v e a l e d d e c r e a s e d a p p o s i t i o n and bone f o r m a t i o n r a t e s ,
t e t r a c y c l i n e l a b e l i n g and i n c r e a s e d m i n e r a l i z a t i o n
l a g t i m e ( F i g s . 1-3).
I n t h e p r e s e n c e o f d e p r e s s e d a p p o s i t i o n and bone
formation rates. osteosclerosis i n skeletal fluorosis could r e s u l t from the increased volume o f p a r t i a l l y m i n e r a l i z e d osteoid.
Bone h i s t o m o r p h o m e t r i c
changes i n a l l t h e cases s t u d i e d c o r r e l a t e d w i t h d a i l y f l u o r i d e plasma l e v e l s o f f l u o r i d e ,
intakes,
a l k a l i n e p h o s p h a t a s e and iPTH a n d a l s o w i t h t h e
f i n d i n g s i n t h e i r s k e l e t a l radiographs (Fig. I n p r e v i o u s r e p o r t s (9-13).
4).
i t was shown t h a t t h e h i s t o p a t h o l o g i c a l
p i c t u r e o f s k e l e t a l f l u o r o s i s w a s c h a r a c t e r i z e d b y i n c r e a s e d o s t e o i d and supernormal p r o p o r t i o n s o f i n c o m p l e t e l y m i n e r a l i z e d bone ( o s t e o m a l a c i a ) . i n c r e a s e d t r a b e c u l a r r e s o r p t i o n o f bone w i t h o s t e o c l a s t s and m a r r o w f i b r o s i s (secondary hyperparathyroidism). d o m i n a n t l y woven bone,
increased production o f immature pre-
p o o r l y f o r m e d h a v e r s i o n s y s t e m s and d i s o r d e r e d
l a m e l l a r o r i e n t a t i o n o f t h e bone.
I n u n c o m p l i c a t e d c a s e s t r a b e c u l a e may
appear t h i c k w i t h excess calcium.
S i m i l a r h i s t o l o g i c a l f e a t u r e s h a v e been
reported i n patients o f industrial
f l u o r o s i s (14).
Histomorphometric
a n a l y s i s o f i l i a c bone i n s k e l e t a l
fluorosis did not
p r o v i d e any p r e c i s e o r c h a r a c t e r i s t i c i n f o r m a t i o n t o p e r m i t p e n e t r a t i n g s e p a r a t i o n f r o m m e t a b o l i c bone disease. profiles,
however.
H i s t o m o r p h o m e t r i c and dynamic
provided a better viewpoint,
understanding,
and i n s i g h t
i n t o t h e t o x i c e f f e c t s o f c h r o n i c i n g e s t i o n o f n a t u r a l f l u o r i d e on i n t r a s k e l e t a l mechanisms,
s t r u c t u r e and m e t a b o l i s m o f t h e bone.
This informa-
t i o n has a l l o w e d t h e i n s t i t u t i o n o f a p p r o p r i a t e p r e v e n t i v e and t h e r a p e u t i c m e a s u r e s a d a p t e d t o f l u o r i d e i n d u c e d bone d i s e a s e ( o s t e o m a l a c i a and seconda r y h y p e r p a r a t h y r o i d ism).
355 It i s b e l i e v e d t h a t s i m i l a r h i s t o d y n a m i c b e h a v i o r o f bone may o c c u r i n p a t i e n t s o f o s t e o p o r o s i s t r e a t e d w i t h sodium f l u o r i d e ,
and o s t e o m a l a c i a and
s e c o n d a r y h y p e r p a r a t h y r o i d i s m may be t h e p o t e n t i a l r i s k s o f t h i s t h e r a p y . ACKNOWLEDGEMENT We a r e g r a t e f u l t o P r o f e s s o r A. M. P a r f i t t . D i r e c t o r , B o n e a n d M i n e r a l D i v i s i o n , H e n r y F o r d H o s p i t a l . D e t r o i t , USA, a n d t o l a t e P r o f e s s o r C.
E.
D e n t o f U n i v e r s i t y C o l l e g e H o s p i t a l M e d i c a l S c h o o l London f o r t h e i r k i n d h e l p i n t h e s t u d y o f t h e bone h i s t o m o r p h o m e t r y and t h e d e t e r m i n a t i o n s
of
25-OHD and p a r a t h y r o i d hormone. REFERENCES 1.
T e o t i a SPS.
2.
T e o t i a SPS,
3.
T e o t i a M,
4.
T e o t i a SPS,
5.
T e o t i a M ( 1 9 8 4 ) J Assoc Phys I n d i a 32:347-352 K u n w a r KB, T e o t i a SPS,
T e o t i a M (1969) F l u o r i d e 2:142-152 Kunwar K B ( 1 9 7 1 ) A r c h D i s C h i l d 46:686-691
T e o t i a M ( 1 9 7 3 ) B r i t Med J 1:637-640
T e o t i a M, T e o t i a SPS,
K u n w a r K B ( 1 9 7 3 ) I n : F r a m e 6, P a r f i t t A M a n d
Duncan H ( e d s ) C l i n i c a l A s p e c t s o f M e t a b o l i c Bone D i s e a s e . E x c e r 7 t a M e d i c a , Amsterdam, pp 232-238
6. T e o t i a SPS, T e o t i a M, S i n g h RK, T a v e s OR. H e e l s S ( 1 9 7 8 ) J A s s o c P h y s I n d i a 26:995-1000 7.
T e o t i a SPS. T e o t i a M. S i n g h R K , T e o t i a N P S , T a v e s DR, H e e l s S, D ' M e l -
8.
T e o t i a M,
9.
T e o t i a SPS, T e o t i a M, S i n g h DP, A n a n d V.
low,
V P ( 1 9 7 8 ) F l u o r i d e 12:115-119 T e o t i a SPS,
S i n g h RK (1979) F l u o r i d e 12:58-64 S i n g h CV. Tomar NPS (1984)
F1 u o r i d e 17: 14-22 10.
F a c c i n i JM,
T e o t i a SPS (1974) C a l c T i s s Res 16:45-57
11.
T e o t i a SPS,
T e o t i a M,
12.
ASSOC
63:207-211
Teotia
SPS, T e o t i a M.
R o h t a g i VK,
S i n g h RK, T e o t i a NPS ( 1 9 7 4 ) I n : P r o c e e d i n g s o f
t h e s y m p o s i u m on f l u o r o s i s .
Hyderabad,
13.
T e o t i a SPS,
14.
F r a n k e J ( 1 9 7 2 ) F l u o r i d e 5:182-199
T e o t i a M.
T e o t i a N P S ( 1 9 7 4 ) J I n d i a n Med
T e o t i a NPS
I n d i a , p p 425-434
(1976) F l u o r i d e 9:91-98
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 357-367 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
357
I N V E S T I G A T I O N S ON THE RELEVANCE O F DEFLUORIDATED WATER AND NUTRITIONAL SUPPLEMENTS I N FLUOROSIS ENDEMIC AREAS I N ANDHRA PRADESH, I N D I A I(I3LA RAJYALAKSHMI,
N. V.
RAMAMOHAN RAO, AND NEELAM K R I S H N A
I n s t i t u t e o f P r e v e n t i v e M e d i c i n e , P u b l i c H e a l t h L a b o r a t o r i e s and Food ( , i e a l t h ) A u t h o r i t y . A.P., H y d e r a b a d 500 029 I n d i a
ABSTRACT Nalgonda i n Andhra P r a d e s h i s one o f t h e d i s t r i c t s i n I n d i a w h i c h i s severely a f f e c t e d by f l u o r o s i s . disease.
Several hundred pcople a r e c r i p p l e d b y t h e
C o n t r o l l e d s t u d i e s w e r e made t o d e t e r m i n e t h e e f f e c t i v e n e s s o f
p r o v i d i n g d e f l u o r i d a t e d d r i n k i n g w a t e r and n u t r i t i o n a l supplements b o t h s e p a r a t e l y as w e l l as i n combination.
Seventy-two p a t i e n t s i n each o f f o u r
v i l l a g e s who w e r e i n t h e age g r o u p s o f 1-5,
5-10,
10-18.
and above 1 8 y e a r s
o l d w e r e p r o v i d e d d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l s u p p l e m e n t s f o l l o w i n g b a s e l i n e s t u d i e s o n d i e t a r y i n t a k e and c l i n i c a l , ical profiles.
radiological,
a n d biochemi-
The r e s u l t s o f i n v e s t i g a t i o n s c o n d u c t e d o n c e i n t h r e e
m o n t h s e a c h y e a r a f t e r commencement o f t h e i n t e r v e n t i o n p r o g r a m w e r e comp a r e d w i t h i n i t i a l data.
A g r a d u a l r e l i e f i n t h e c l i n i c a l symptonis o f b o d y p a i n s and j o i n t p a i n s was o b s e r v e d .
Radiological p r o f i l e s o f the patients indicated that fluo-
r i l e r e m o b i l i z a t i o n f r o m bones i s r e v e r s i b l e b u t t h e r a t e o f r e m o v a l i s slow.
S t a t i s t i c a l evaluation o f biochemical data revealed s i g n i f i c a n t
r e d u c t i o n i n a l k a l i n e p h o s p h a t a s e and s e r u m f l u o r i d e c o n c e n t r a t i o n s .
The
r e s u l t s o f t h e i n v e s t i g a t i o n h a v e shown t h a t t h e a f f l i c t i o n c a n be s i g w i f i c a n i l y reduced through t h e simultaneous consumption o f d e f l u o r i d a t e d d r i n k i n g w a t e r and n u t r i t i o n a l s u p p l e m e n t s .
INTRODUCTION 5jalgonda d i s t r i c t i n A n d h r a P r a d e s h i s one o f t h e a r e a s i n I n d i a w h i c h i s severely a f f e c t e d by f l u o r o s i s .
A number o f v i l l a g e s i n t h i s d i s t r i c t h a v e
e x c e s s i v e f l u o r i d e i n t h e i r d r i n k i n g w a t e r and s e v e r a l t h o u s a n d i n h a b i t a n t s are suffering from skeletal fluorosis.
Epidemiological surveys revealed
t h a t persons o f l o w income g r o u p s w i t h i n a d e q u a t e n u t r i t i o n a r e more a f f e c t e d t h a n t h o s e i n h i g h e r i n c o m e g r o u p s who h a v e a m o r e b a l a n c e d d i e t . !t i s a l s o o b s e r v e d t h a t a f f e c t e d p e o p l e a s w e l l a s c a t t l e r e c o u p t h e i r
h e a l t h on m i g r a t i o n t o a r e a s w h e r e t h e f l u o r i d e c o n t e n t i n w a t e r i s l o w .
358 As a r e s u l t o f t h e s e o b s e r v a t i o n s and o t h e r r e p o r t s i n t h e l i t e r a t u r e (1-
5). t h e e f f e c t i v e n e s s o f p r o v i d i n g n u t r i t i o n a l s u p p l e m e n t s a n d d e f l u o r i dated water separately,
as w e l l as i n c o m b i n a t i o n ,
t o fluorosis patients
who h a d b e e n c o n s u m i n g d r i n k i n g w a t e r c o n t a i n i n g h i g h l e v e l s o f f l u o r i d e was s t u d i e d . MATERIALS AND METHODS
A t o t a l o f 207 p a t i e n t s w e r e s e l e c t e d f r o m t h e v i l l a g e s o f B a t l a p a l l y , S i v a n n a g u d a and M a r r i g u d a w h e r e t h e d r i n k i n g w a t e r s o u r c e c o n t a i n e d f l u o r i d e i n t h e r a n g e s o f ( a ) 1.1-4.0 and above.
mg/l,
( b ) 4.1-8.0
mg/l,
a n d ( c ) 8.1 m g / l
P e o p l e i n a non-endemic v i l l a g e w e r e used as a c o n t r o l group.
S a m p l e s of d r i n k i n g w a t e r i n t h e f o u r v i l l a g e s w e r e a n a l y z e d f o r v a r i o u s c h e m i c a l p a r a m e t e r s f o l l o w i n g t h e p r o c e d u r e s recommended b y A.P.H.A.
(6).
D i e t a r y surveys were conducted t o evaluate d e f i c i e n c i e s i n calories, protein,
carbohydrates,
calcium,
p h o s p h o r u s and v i t a m i n s .
Dietary intake
o f f o o d m a t e r i a l s was d e t e r m i n e d b y w e i g h t metbfod, and n u t r i t i o n a l paramet e r s w e r e c a l c u l a t e d i n a c c o r d a n c e w i t h t h e I C M R m a n u a l (7). C l i n i c a l examination o f p a t i e n t s I n c i d e n c e o f d e n t a l f l u o r o s i s was s t u d i e d u n d e r t h e f o l l o w i n g c l a s s i f i c a tions: Grade 0
-
n o r m a l , t r a n s l u c e n t , smooth and g l o s s y t e e t h
Grade 1
-
white opacities,
Grade 2
-
c h a n g e s o f Grade 1 and b r o w n s t a i n
Grade 3
-
b r o w n l i n e , p i t t i n g a n d c h i p p e d o f f edges
Grade 4
-
b r o w n b l a c k and l o s s o f t e e t h
Incidence o f
skeletal
f a i n t yellow l i n e
fluorosis
was
categorized by t h e
following
criteria:
0 - normal 1
-
m i l d s y m p t o m a t i c ; r a d i o g r a p h s w i t h i n c r e a s e d bone d e n s i t y
2
-
moderate symptomatic; s t i f f n e s s , r i g i d i t y ,
3
-
severe:
p a i n , aches
symptomatic w i t h o s t e o p h y t o s i s e x o s t o s i s marked l i m i t a t i o n
o f s p i n e and j o i n t s F o r r a d i o l o g i c a l e v a l u a t i o n r a d i o g r a p h s o f f o r e a r m , knee j o i n t s , and s p i n e were taken. Biochemical s t u d i e s B l o o d and u r i n e samples were c o l l e c t e d f r o m b o t h normal persons o f t h e c o n t r o l g r o u p and f r o m f l u o r o s i s o a t i e n t s and a n a l y z e d f o r b i o c h e m i c a l parameters. A f t e r c o l l e c t i n g b a s e l i n e d a t a and e v a l u a t i n g t h e r e s u l t s , t i o n p r o g r a m was i n i t i a t e d b y p r o v i d i n g t h e f o l l o w i n g :
an i n t e r v e n -
359 Group 1 - d e f l u o r i d a t e d w a t e r Group 2
-
n u t r i t i o n a l supplements
Group 3
-
d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l s u p p l e m e n t s
Group 4
-
no d e f l u o r i d a t e d water o r n u t r i t i o n a l supplements ( c o n t r o l )
The d e f l u o r i d a t i o n p r o c e s s d e v e l o p e d a t t h e I n s t i t u t e c o n s i s t e d o f a d d i n g c a l c i u m c a r b o n a t e p o w d e r t o t h e r a w w a t e r f o l l o w e d b y t h e a d d i t i o n o f known q u a n t i t i e s o f s a t u r a t e d f i l t e r e d a l u m s o l u t i o n and m i x i n g f o r 1 0 m i n u t e s . N u t r i t i o n a l supplements
A m i x t u r e o f "Hyderabad Mix",
which i s manufactured l o c a l l y ,
contains the
f o l l o w i n g r a w f o o d s and was s u p p l i e d t o t h e p a t i e n t s i n a p p r o p r i a t e q u a n t i ties: 1.
2. 3. 4.
70 9 35 g 23 9 12 4
wheat b e n g a l gram jaggery groundnut
140 g Following the intervention the biochemical,
c l i n i c a l and r a d i o l o g i c a l
p r o f i l e s o f t h e p a t i e n t s were studied. RESULTS AND DISCUSSION
A t o t a l o f 207 p a t i e n t s r e p r e s e n t i n g b o t h s e x e s i n d i f f e r e n t age g r o u p s ( a g e s 0-5.
5-10,
10-18,
and 18+) w e r e s t u d i e d i n t h e t h r e e v i l l a g e s .
o f t h e p a t i e n t s e x a m i n e d a r e shown i n F i g u r e s 1-4.
Some
C l i n i c a l and r a d i o l o g i -
c a l p r o f i l e s o f t h e p a t i e n t s revealed t h a t t h e y were s u f f e r i n g from dental and s k e l e t a l f l u o r o s i s . degree o f s e v e r i t y , contents o f water,
Several f a c t o r s appear i m p o r t a n t i n a f f e c t i n g t h e
i n c l u d i n g n u t r i t i o n a l status,
TABLE I
INCIDENCE
0-5
IN
OF FLUOROSIS
Age G r o u p (Years)
f l u o r i d e and a l k a l i n i t y
age and o c c u p a t i o n .
I 2
BAT LAP ALLY^ Dental Grade I1 I11 -
-
IV
MiI d
-
2
-
Moderate
Severe
-
-
-
-
5-10
-
2
10-1 8
-
1
3
7
4
18+
-
-
4
11
1
6 6
8
2
3
14
20
5
12
9
Total
7
Sk e l e t a 1
' F l u o r i d e c o n t e n t i n d r i n k i n g w a t e r was ~ 8 . 0m g / l .
1
FIG. I
PATIENTS WITH
KNOCK K N E E AND NOW LEGS
FIG. 2
FIG. 4 FIG. 3 PATIENT HAVING KEPHOSIS AND BOW LEGS
PATIENT WITH RIGID SPINAL COLUMN
FEMALE HAVING BOW LEGS
361 T a b l e I shows t h e i n c i d e n c e o f d i s e a s e among s u b j e c t s o f B a t l a p a l l y v i l l a g e w h e r e t h e maximum f l u o r i d e c o n c e n t r a t i o n i n d r i n k i n g w a t e r r a n g e d f r o m 8.0-8.5
mg/l.
T a b l e s I 1 and I 1 1 show i n c i d e n c e o f d i s e a s e among
s u b j e c t s o f S i v a n n a g u d a and M a r r i g u d a w h e r e t h e f l u o r i d e c o n t e n t s i n t h e w a t e r w e r e 4.1 t o 8.0 m g / l
a n d 1.1 t o 4.0 m g / l ,
respectively.
M o s t o f t h e v i l l a g e r s s u r v e y e d w e r e f o u n d t o h a v e p o o r n u t r i t i o n . The f o o d t h e y consumed was d e f i c i e n t i n s e v e r a l e s s e n t i a l n u t r i e n t s .
The
d i e t a r y s t a t u s o f t h e p e o p l e i n t h e t h r e e v i l l a g e s i s shown i n T a b l e I V .
TABLE I 1
INCIDENCE
OF FLUOROSIS
IN SIVANNAGUDA~
Age Group (Years)
Dental Grade
Skel e t a l
I
I1
0-5
2
-
-
-
-
-
-
5-1 0
1
7
4
4
9
-
-
10-1 8
Total
IV
MiI d
Floderate
Severe
8
8
10
2
10
6
-
-
12
16
-
17
10
3
15
24
30
11
27
16
~
18+
111
' F l u o r i d e c o n t e n t i n d r i n k i n g w a t e r was 4 . 1 t o 8 . 0 m g / l
TABLE I 1 1
INCIDENCE
OF FLUOROSIS
Aqe G r o u p (Years)'
IN MARRIGUDA~
I
Dental Grade I1 I11
0-5
2
-
5-1 0
-
6
-
2
Sk e l e t a l IV
MiId
-
-
-
4
Moderate ~
Severe -
10-18
-
6
10
6
6
-
-
18+
-
4
17
20
10
4
2
2
16
29
26
20
4
2
Total
' F l u o r i d e c o n t e n t i n d r i n k i n g w a t e r was 1 . 1 t o 4.0 m g / l .
362 TABLE IV DIETARY STATUS OF PATIENTS Village
Proteins (9)
(K calorie)
Calcium (mg)
9.9-27.7
584-1464
127-1 63
243-675
4.1-13.9
Sivannaguda
14.7-29.1
691 -1 4 9 0
71-158
268-675
5.7-13.2
Batlapally
22.1-42.2
998-2007
72-222
657-1203
8.8-20.0
19.2-53.7
1350-2500
400-800
--
1.5-28.0
:larriguda
N i n i,num
requirements
Energy
Phosphorus (m9)
Iron (mg)
Biochemical a n a l y s i s The b l o o d p r o f i l e s o f t h e p a t i e n t s showed e l e v a t e d a l k a l i n e p h o s p h a t a s e v a l u e s r a n g i n g f r o m 15-59 i n c r e a s e o f 5-10
units,
f o l d (0.15-0.8)
and a serum f l u o r i d e c o n c e n t r a t i o n
c o m p a r e d t o v a l u e s o f t h e non-endemic
area
c o n t r o l g r o u p (0.01-0.06). The c o n c e n t r a t i o n o f f l u o r i d e i n t h e u r i n e o f t h e s u b j e c t s r a n g e d f r o m 7.5-31
m g / l w h i l e t h a t o f t h e c o n t r o l g r o u p was 0.8-0.9
c a n t c h a n g e s c o u l d be o b s e r v e d i n u r i n a r y p h o s p h a t e ,
mg/l.
No s i g n i f i -
creatin~ne, o r creati-
n i n e c l e a r a n c e values. I n t e r v e n t i o n proqram After intervention,
t h e b l o o d and u r i n e samples o f t h e p a t i e n t s were
a n a l y z e d f o r d i f f e r e n t p a r a m e t e r s o n c e i n t h r e e months. subjected t o s t a t i s t i c a l analysis. t h e b l o o d urea, sium,
phosphate,
creatinine.
The r e s u l t s w e r e
No s i g n i f i c a n t c h a n g e s w e r e o b s e r v e d i n
p h o s p h o r u s and magnesium,
c r e a t i n i n e , and c r e a t i n i n e c l e a r a n c e .
o r i n u r i n a r y magne-
A moderate increase
i n p r o t e i n and c a l c i u m p r o f i l e s o f t h e p a t i e n t s w e r e observed,
but the
v a r i a t i o n s w e r e n o t f o u n d t o be s t a t i s t i c a l l y s i g n i f i c a n t .
A summary o f s e r u m f l u o r i d e ,
a l k a l i n e p h o s p h a t a s e and c a l c i u m v a l u e s i n
p a t i e n t s o f B a t l a p a l l y v i l l a g e i s s h o w n i n T a b l e V. s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n G r o u p s 11,
The d a t a i n d i c a t e a
111 a n d I V a n d t h e
c o n t r o l g r o u p (Group I).
T h e r e d u c t i o n i s s i g n i f i c a n t ( ~ ~ 0 . 0 5f )o r G r o u p
I V ( n u t r i t i o n a l supplement
+
defluoridation).
A l t h o u g h u r i n a r y c a l c i u m and
f l u o r i d e c o n t e n t s s h o w a g e n e r a l r e d u c t i o n i n a l l g r o u p s ( T a b l e V), differences are not significant.
the
The u r i n a r y f l u o r i d e v a l u e s a r e u n i f o r m l y
h i g h compared t o t h o s e o f t h e c o n t r o l group.
It i s e v i d e n t t h a t t h e
f l u o r i d e a l r e a d y absorbed i s r e m o b i l i z e d and e x c r e t e d i n t h e u r i n e even a f t e r c e s s a t i o n o f excessive f l u o r i d e i n g e s t i o n t h r o u g h d r i n k i n g water.
363 TABLE V
SUMMARY OF BIOCHEMICAL PROFILE
OF
BATLAPALLY PATIENTS
Initial Mean f S.D.
A t 12 m o n t h f o l l o w - u p Mean f S.D.
SERUM F l u o r i d e (mg/l) Group I
0.18
f
0.04
0.14 f 0 . 0 3
Group I I
0.19
f
0.04
0.13
Group 1 1 1
0.18
f
0.03
0.12 i 0 . 0 2
G r o u p IV
0.17
f
0.03
0.12
f
Group I
26.07
f
9.71
15.66
f
Group I 1
25.81
?
8.89
G r o u p I11
37.57 f 12.87
23.06
?
10.34
Group I V
32.28 i 10.91
18.96
f
8.69
f
0.03
0.02
A l k Phost (KAU)
8.32
12.88 i 8.72
URINE C a l c i u m (mg % )
9.81
2.68
5.76
f
1.83
10.89 i 3.37
5.23
f
Group I11
9.91 f 3.08
4.10
*
1.57
G r o u p IV
9.95 i 3.24
5.80
f
1.46
10.91
f
2.48
1.17
9.40
f
1.62
1.88
10.95
f
2.55
Group I Group I 1
f
1.03
F l u o r i d e (mg/l) Group I
11.05
Group I 1
11.03
*
Group I 1 1
14.18
?
Group IV
12.04 f 1.57
f
1.39
1 0 . 9 3 f 2.16
The c h a n g e s i n t h e b i o c h e m i c a l p r o f i l e s o f c a l c i u m ,
f l u o r i d e and a l k a l i n e
p h o s p h a t a s e a r e a l s o d i s c e r n i b l e i n i n d i v i d u a l p a t i e n t s o f d i f f e r e n t age g r o u p s c o m p a r e d t o t h o s e o f t h e non-endemic a t t h e 12 month f o l l o w - u p
area.
I n i t i a l v a l u e s and t h o s e
o f serum a l k a l i n e phosphatase,
s e r u m and u r i n a r y
f l u o r i d e and u r i n a r y c a l c i u m i n s u b j e c t s o f B a t l a p a l l y v i l l a g e and t h e N a c h a r a m c o n t r o l g r o u p a r e s h o w n i n T a b l e VI.
It i s seen f r o m t h e t a b l e
t h a t t h e r e d u c t i o n i n t h e r e s p e c t i v e parameters a r e s i g n i f i c a n t i n d i f f e r e n t groups.
However,
s e r u m a l k a l i n e p h o s p h a t a s e and f l u o r i d e l e v e l s a r e
364 much h i g h e r t h a n t h o s e o f t h e c o n t r o l s even 12 months a f t e r t h e i n t e r v e n t i o n was i n j t i a t e d .
The r e s u l t s o f t h e b i o c h e m i c a l s t u d i e s t h u s p o i n t o u t
i m p o r t a n t phenomenon: ( 1 ) p r o v i d i n g d e f l u o r i d a t e d w a t e r o r n u t r i t i o n a l supplements b r i n g a b o u t s i g n i f i c a n t changes i n serum f l u o r i d e and a l k a l i n e phosphatase,
i.e..
r e d u c t i o n o f these parameters,
and (2) s u b j e c t s r e c e i v -
i n g d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l supplements e x c r e t e more f l u o r i d e i n u r i n e t h a n t h o s e r e c e i v i n g n u t r i t i o n a l supplements alone.
TABLE V I INDIVIDUAL BLOOD AND U R I N E PROFILES OF BATLAPALLY SUBJECTS Parameters
Age
Serum
0-5
Group 1
Initial
A t 12 month follow-up
Nacharam c o n t r o l group
N
63.6
34.0
N+D
31.0
20.0
5-1 0
N+D
25.0
16.0
16.0
10-18
N
28.0
14.0
18.5
N+D
33.0
20.0
18+
N+D
48.0
24.0
18+
A1 k a l i n e
1.5
Phosphatase (MU)
20.5
D
0.2
0.1
0.06
F1u o r i de
N
0.2
0.1
0.04
(mgll)
N+D
0.4
0.1
0.06
N+D
9.8
4.0
9.2
Serum
Urinary
10-18
Calcium (mg%)
Urinary
18+
10-18
Fluoride
N+D
11.0
9.4
10.0
D
17.0
11.6
9.8
N
28.6
17.5
1.4
5.0
16.0
1.6
N+D
(mgll)
'D
=
d e f l u o r i d a t e d water
N = n u t r i t i o n a l supplements
365 C l i n i c a l examination
A l l t h e p a t i e n t s e x a m i n e d s h o w e d mod'erate t o good' r e l i e f f r o m t h e c T i n i c a l symptoms.
F o r t h e s u b j e c t s o f B a t l a p a l l y . t h e r e l i e f was g r e a t e s t
among t h o s e r e c e i v i n g n u t r i t i o n a l s u p p l e m e n t s i n a d d i t i o n t o d e f l u o r i d a t e d water.
The r e s u l t s o f c l i n i c a l e x a m i n a t i o n a f t e r i n t e r v e n t i o n a r e shown i n
T a b l e s V I I t h r o u g h X.
TABLE V I I SALIENT CLINICAL FEATURES - BATLAPALLY CONTROL GROUP
S.
Age 5-10 10-18
F
C l i n i c a l Features Initial Final
M
-
-
2
0
vague body p a i n and j o i n t p a i n s
complaints o f pains only
-
3
-
2
1
b o d y aches, back ache, arthralgia, and s t i f f n e s s
c o m p l a i n t s o f back a c h e and s t i f f n e s s
-
-
9
8
1
generalized body p a i n s , b a c k ache, stiffness, swelling o f joints, limitat i o n o f movements, rigidity o f the s p i n e and numbness
complaints o f s t i f f ness. swelling of j o i n t s , n o change i n l i m i t a t i o n o f movements, rigidity of t h e spine, slight i m p r o v e m e n t i n numbness
0-5
1.
-
2
2.
-
3.
-
TABLE
Sex
i8+
No.
joint
vrrr
S A L I E N T CLINICAL FEATURES - BATLAPALLY DEFLUORIDATION GROUP
S. NO.
Age
0-5 5-10 10-18 18+
M
Sex
F
C l i n i c a l Features Initial Final
1.
-
1
-
-
1
0
vague body j o i n t pains
2.
-
-
3
-
2
1
Body aches, back aches, arthralgia, and s t i f f n e s s
c o m p l a i n t s o f back a c h e and s t i f f n e s s
3.
-
-
-
9
4
5
generalized body pains, backache, s t i f f ness. s w e l l in g joints, r i g i d i t y of t h e s p i n e and numbness
complaints o f s t i f f of ness, swelling j o i n t s , no change i n l i m i t a t i o n o f movements, rigidity of spine, slight imp r o v e m e n t o f numbness
and
complaints o f pains only
joint
366 TABLE I X SALIENT CLINICAL FEATURES
S.
NO.
Age
0-5 5-10 10-18 18+
1.
-
1
2.
-
-
3.
-
-
BATLAPALLY NUTRITION GROUP
-
M
Sex
C l i n i c a l Features Initial Final
F
-
0
1
vague pains
7
-
5
2
body aches, back aches, arthralgia, and s t i f f n e s s
no c o m p l a i n t s
-
2
1
1
generalized body pains, backaches, stiffness, swelling of joints, limitat i o n o f movements, rigidity of the s p i n e , and numbness
no c o m p l a i n t s
-
body
joint
no c o m p l a i n t s of body j o i n t p a i n s
I t was o b s e r v e d t h a t p a t i e n t s i n t h e c o n t r o l g r o u p c o n t i n u e d t o s u f f e r f r o m a l l t h e i n i t i a l symptoms.
T h e r e has b e e n s i g n i f i c a n t i m p r o v e m e n t i n
t h e p h y s i c a l d i s a b i l i t i e s experienced by t h e s u b j e c t s r e c e i v i n g n u t r i t i o n a l supplements alone o r i n c o m b i n a t i o n w i t h d e f l u o r i d a t e d w a t e r .
Patients
r e c e i v i n g t h e s e a l o n e were r e l i e v e d o f vague p a i n s b u t j o i n t p a i n s pers i s t e d . R e s u l t s s u g g e s t t h a t some o f t h e s y m p t o m s e x p e r i e n c e d b y t h e p e o p l e c a n be a t t r i b u t a b l e t o m a l n u t r i t i o n .
TABLE X SALIENT CLINICAL FEATURES
S.
NO.
Age
0-5 5-10 10-18 18+
-
M
BATLAPALLY NUTRITION AND DEFLUORIDATION GROUP Sex
F
C l i n i c a l Features Initial Final
1.
3
-
-
-
1
2
malnutrition
moderate improvement
2.
-
3
-
-
1
2
b o d y p a i n s and j o i n t pa1 n s
no c o m p l a i n t s
3.
-
-
2
-
2
0
b o d y ache, b a c k a c h e , artbralgia
no c o m p l a i n t s
4.
-
-
2
2
0
generalized body pains, backache, stiffness. swelling o f j o i n t s . numbness
no c o m p l a i n t s
-
367 Radiological examination F i n d i n g s o f r a d i o l o g i c a l p r o f i l e s r e v e a l no change and i n c l u d e l i t t l e regression i n coarse t r a b e c u l a t i o n pattern, formations,
decreased o s t e o s c l e r o s i s ,
o s s e o u s membrane.
d e c r e a s e d p e r i o s t e a l new bone
and d e c r e a s e d o s s i f i c a t i o n o f i n t e r -
The r e s u l t s i n d i c a t e t h a t t h e p r o c e s s o f m o b i l i z a t i o n o f
skeletal f l u o r i d e i s reversible but it takes a long time f o r substantial r e c t i f i c a t i o n o f skeletal abnormalities. C l i n i c a l d a t a f r o m r a d i o g r a p h s and b i o c h e m i c a l p r o f i l e s o f b l o o d and u r i n e s a m p l e s i n d i c a t e t h a t s i g n i f i c a n t r e l i e f c a n be o b t a i n e d i n p a t i e n t s w i t h s e v e r e f l u o r o s i s s y m p t o m s b y u s i n g d e f l u o r i d a t e d w a t e r and b y e a t i n g a nutritional diet.
REFERENCES 1.
Ramamohan Rao NV. R a j y a l a k s h m i K ( 1 9 7 4 ) I n : P r o c S i g n F l u o r o s i s I n d Acad Geo SOC, p p 273-284
2.
Incidence o f Fluorosis i n R a j y a l a k s h m i K, Ramamohan Rao N V ( 1 9 8 3 ) N a l g o n d a D i s t r i c t , A.P. i n r e l a t i o n t o C h e m i c a l C h a r a c t e r i s t i c s o f P o t a b l e W a t e r and S t a p l e Foods. Paper p r e s e n t e d a t 1 3 t h Conference o f I S F R Nov 14-17, New D e l h i
3.
R a j y a l a k s h m i K, New D e l h i
4.
R a j y a l a k s h m i K, V e n k i a h KR, Ramamohan Rao NV, K r i s h n a N ( 1 9 8 4 ) R e l e v a n c e o f d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l s u p p l e m e n t s t o F l u o r o s i s . R e p o r t s u b m i t t e d t o IDRC. Canada
5.
Ramamohan Rao NV, R a j y a l a k s h m i K, S u r y a p r a k a s h Rao K, N a v n e e t h a Rao T (1983) S t u d i e s on Geo-chemistry o f f l u o r i d e d i s t r i b u t i o n i n w a t e r s o u r c e s o f N a l g o n d a D i s t r i c t , A.P. Paper p r e s e n t e d a t 1 3 t h Conference o f ISFR Nov 14-17, New D e l h i
6.
S t a n d a r d M e t h o d s f o r t h e E x a m i n a t i o n o f W a t e r and Waste Water, APHA, AWWA, WPCF. USA (1981).
7.
N u t r i t i v e V a l u e s o f Food M a t e r i a l s (1975) I n d i a n C o u n c i l o f M e d i c a l Research. New D e l h i
Ramamohan Rao NV ( 1 9 8 3 ) I b i d - r e p o r t s u b m i t t e d t o DOE
1 4 ed
369
H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 369-378 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands
FLUORIDE UPTAKE BY DECIDUOUS ENAMEL FROM FLUORIDE MOUTHRINSE AND NATURAL FLUORIDE I N TtIE WATER YOUICHI
IIJIMA
AND TSUYOSHI KATAYAMA
D e p a r t m e n t of P r e v e n t i v e D e n t i s t r y , D e n t i s t r y , Morioka, Japan
I w a t e Medical U n i v e r s i t y School o f
ABSTRACT The e f f e c t s o f f l u o r i d e m o u t h r i n s e (F:500 ppm. t i o n o f n a t u r a l w a t e r w i t h 0.3-3.2
5 t i m e s / w e e k ) and consump-
ppm F on f l u o r i d e u p t a k e b y d e c i d u o u s
e n a m e l w e r e i n v e s t i g a t e d u s i n g 135 e x f o l i a t e d t e e t h . F l u o r i d e m o u t h r i n s e i n c r e a s e d t h e f l u o r i d e c o n c e n t r a t i o n o f t h e t o p 1 vm l a y e r o f t h e e n a m e l f r o m 4,300 t o 7,300 ppm w h e n t h e r i n s i n g p e r i o d w a s i n c r e a s e d f r o m one t o f o u r years.
No s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e s
i n f l u o r i d e c o n c e n t r a t i o n were found i n t h e enamel 5
urn
f r o m t h e surface.
These r e s u l t s show t h a t f l u o r i d e u p t a k e b y m o u t h r i n s e was m o s t l y r e s t r i c t e d t o t h e o u t e r m o s t l a y e r s o f t h e enamel. F l u o r i d e c o n c e n t r a t i o n s i n t h e d e c i d u o u s enamel s u r f a c e o f i n h a b i t a n t s consuming n a t u r a l f l u o r i d e i n t h e water were s i g n i f i c a n t l y higher than t h o s e i n t h e enamel o f s u b j e c t s consuming w a t e r w i t h v e r y l o w f l u o r i d e o r those i n enamel t r e a t e d w i t h f l u o r i d e mouthrinse.
Fluoride levels o f
a p p r o x i m a t e l y 10,000 pprn w e r e f r e q u e n t l y f o u n d i n t h e t o p 1 Um l a y e r o f t h e e n a m e l f r o m s u b j e c t s l i v i n g i n h i g h f l u o r i d e a r e a s ( o v e r 1.0 ppm). t h e f l u o r i d e c o n c e n t r a t i o n s a t an e n a m e l d e p t h o f 5 Um f r o m
an
However,
area w i t h
0.3 ppm F i n t h e d r i n k i n g w a t e r w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e of
t h e cont.rol.
H i g h f l u o r i d e p e n e t r a t i o n was f o u n d i n b o t h t h e o u t e r m o s t
and d e e p e r l a y e r s o f t h e d e c i d u o u s enamel.
INTRODUCTION F l u o r i d e i s w i d e l y used i n p r e v e n t i v e d e n t a l programs. r i d e i n c o m m u n i t y p r e v e n t i v e measures,
The u s e o f f uo-
i n d i v i d u a l l y based procedures,
i n d i v i d u a l s e l f c a r e h a v e been e x t e n s i v e l y r e v i e w e d r e c e n t l y (1,Z).
and
F uo-
r i d e decreases t h e a c i d s o l u b i l i t y o f enamel by c o n v e r t i n g enamel hydroxya p a t i t e t o f l u o r a p a t i t e ( 3 ) . Because o f t h i s , an i n c r e a s e i n enamel f l u o r i d e c o n c e n t r a t i o n has been s u g g e s t e d t o have c a r i o s t a t i c b e n e f i t s , and t h e r e i s much e v i d e n c e f a v o r i n g a n t i c a r i e s e f f e c t s o f e n a m e l f l u o r i d e
(4,5).
370 It i s general Iv
aLL~2)Lcii
hat t n e a m o u n t o f f l u o r i d e a c q u i r e d i n t h e
enamel v a r i e s c o n s i d e r a b l y w i t h t h e l e n g t h o f t h e p r e - e r u p t i v e stage.
maturation
D e c i d u o u s t e e t h have a s h o r t e r p e r i o d o f enamel m a t u r a t i o n and
t h e r e f o r e a c q u i r e much l e s s f l u o r i d e t h a n t h e p e r m a n e n t t e e t h (6).
However,
l i t t l e e v i d e n c e i s a v a i l a b l e a s t o how n e u t r a l f l u o r i d e m o u t h r i n s e and n a t u r a l f l u o r i d e i n t h e water a f f e c t t h e f l u o r i d e uptake i n deciduous enamel.
I n a p r e v i o u s r e p o r t , i t was d e m o n s t r a t e d t h a t e x t r e m e l y h i g h
f l u o r i d e l e v e l s , a p p r o x i m a t e l y 10,000 ppm, w e r e f r e q u e n t l y f o u n d a t a d e p t h o f 1 p m i n t h e s u r f a c e e n a m e l o f i n d i v i d u a l s f r o m an a r e a w i t h a h i g h l e v e l o f n a t u r a l f l u o r i d e i n t h e w a t e r ( o v e r 1.0 ppm)(7). The p r e s e n t s t u d y was c a r r i e d o u t t o i n v e s t i g a t e f l u o r i d e u p t a k e b y deciduous enamel f r o m m o u t h r i n s e and n a t u r a l f l u o r i d e i n t h e d r i n k i n g water. MATERIALS AND METHODS S p e c i mens Deciduous enamel specimens employed i n t h i s study were d e r i v e d from a t o t a l o f 135 e x f o l i a t e d i n c i s o r s and m o l a r s f r o m s c h o o l c h i l d r e n who had r e s i d e d c o n t i n u o u s l y s i n c e b i r t h f o r 6-11 y e a r s i n one o f t w o a r e a s : one a r e a w i t h 0.3
t o 3.2
ppm n a t u r a l f l u o r i d e i n t h e d r i n k i n g w a t e r ,
and
a n o t h e r a r e a w i t h l e s s t h a n 0.1 ppm i n i t s w a t e r s u p p l y ( T a b l e I). F i f t e e n t e e t h w i t h no h i s t o r y o f t o p i c a l f l u o r i d e a p p l i c a t i o n s f r o m t h e l o w f l u o r i d e community were d e s i g n a t e d as t h e c o n t r o l group,
and a n o t h e r 60 t e e t h
o b t a i n e d f r o m t h e same a r e a b u t t r e a t e d w i t h f l u o r i d e m o u t h r i n s e ppm,
5 times/week)
(F:500
were d i v i d e d e q u a l l y i n t o 4 groups a c c o r d i n g t o r i n s i n g
p e r i o d s o f one t o f o u r y e a r s .
A n o t h e r 60 t e e t h o b t a i n e d f r o m t h e c o m m u n i t y
w i t h n a t u r a l f l u o r i d e i n t h e w a t e r were d i v i d e d i n t o 4 groups according t o
TABLE I
DECIDUOUS ENAMEL SPECIMENS EMPLOYED I N THIS STUDY Enamel specimens
F content o f d r i n k i n g water (PPm)
Type o f t e e t h (surface)
No. o f teeth
Age (Yr)
Control
< 0.1
Molars
15
8
F-mouthrinse ( 5 0 0 ppm F 5 t imes/week)
< 0.1
Incisors (labial)
60
6 - 7
Molars (buccal )
60
8
Natural F
0.3
-
3.2
-
-
10
11
371 The t e e t h w e r e c l e a n e d f o r 60
t h e f l u o r i d e l e v e l s i n t h e d r i n k i n g water.
seconds w i t h a r o t a t i n g b r u s h w i t h o u t pumice, p l a c e d i n a beaker c o n t a i n i n g and s t i r r e d f o r 24 h o u r s w i t h a m a g n e t i c s t i r r e r .
d i s t i l l e d water,
Speci-
mens w i t h o u t c a r i e s l e s i o n s o r o t h e r d e f e c t s o n t h e e n a m e l s u r f a c e w e r e employed i n t h i s study. A n a l y t i c a l procedures s i x successive l a y e r s o f enamel (sampling area: a p p r o x i m a t e l y
20
mm2)
w e r e d i s s o l v e d i n a p l a s t i c t u b e c o n t a i n i n g 1.0 m l o f 0.5M p e r c h l o r i c a c i d w i t h g e n t l e s h a k i n g . T h e f i r s t t h r e e l a y e r s w e r e e t c h e d f o r 5, 10, a n d 1 5 seconds,
respectively,
a n o t h e r 30 seconds.
and t h e l a s t t h r e e l a y e r s were each e t c h e d f o r
E t c h i n g was t e r m i n a t e d b y w a s h i n g t h e e t c h e d s u r f a c e
w i t h d i s t i l l e d w a t e r f o r 1 5 s e c o n d s . 4.0 m l o f a 0.5M t r i s o d i u m c i t r a t e s o l u t i o n was t h e n added t o t h e e t c h e d s o l u t i o n . Concentrations e l e c t r o d e (96-09,
of
fluoride
were determined
O r i o n R e s e a r c h Inc..
by the
Cambridge,
c a l c i u m b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y (508A,
Mass.)
F-specific and t h o s e o f
H i t a c h i , Tokyo).
The
a m o u n t o f e n a m e l r e m o v e d and t h e d e p t h o f e a c h l a y e r w e r e c a l c u l a t e d assumi n g t h e c a l c i u m c o n t e n t o f e n a m e l t o b e 3 7 w e i g h t % a n d t h e d e n s i t y 2.95 gIcm3. T h e r e was a c l o s e a g r e e m e n t b e t w e e n t h e f l u o r i d e d i s t r i b u t i o n c u r v e s e x p e r i m e n t a l l y o b t a i n e d and t h e o r e t i c a l
exponential curves(8)
w h e r e Y i s f l u o r i d e c o n c e n t r a t i o n i n ppm.
[Y
=
and X i s t h e d e p t h i n
2 X-b, um].
Therefore,
t h e e q u a t i o n was o b t a i n e d b y a p p l y i n g t h e l e a s t s q u a r e s method.
Once
t,
and
a r e e x p e r i m e n t a l l y determined,
i t i s p o s s i b l e t o c a l c u l a t e and
compare f l u o r i d e c o n c e n t r a t i o n s a t a g i v e n depth. w e r e c a l c u l a t e d f o r 1,
5,
10,
20. 3 0 ,
Fluoride concentrations
a n d 40 Um l a y e r s o f t h e e n a m e l
surface. Statistical analysis The mean f l u o r i d e c o n c e n t r a t i o n s and t h e s t a t i s t i c a l s i g n i f i c a n c e o f t h e d i f f e r e n c e s among t h e c o n t r o l ,
f l u o r i d e mouthrinse,
g r o u p s w e r e c o m p a r e d b y t h e Newman-Keuls
and n a t u r a l f l u o r i d e
t e s t f o r m u l t i p l e c o m p a r i s o n s (9).
RESULTS F l u o r i d e mouthrinse group The mean f l u o r i d e c o n c e n t r a t i o n s and t h e d i s t r i b u t i o n o f t h e e n a m e l l a y e r s t r e a t e d w i t h f l u o r i d e m o u t h r i n s e a r e shown i n T a b l e I 1 and F i g u r e 1, respectively.
T r e a t m e n t w i t h f l u o r i d e mouthrinse increased t h e f l u c r i d e
c o n c e n t r a t i o n o f t h e 1 um l a y e r o f enamel f r o m 4,300 t o 7,300 r i n s i n g p e r i o d was i n c r e a s e d f r o m one t o f o u r y e a r s . t r a t i o n was p r o p o r t i o n a l t o t h e l e n g t h o f m o u t h r i n s i n g .
ppm when t h e
The f l u o r i d e concenF l u o r i d e uptake by
372
r
L
w
u
n+c, E O w 3 w z
r. +I
W
W
+I
m
r.
+I
c
d
+I
d
c
m
N
c
Lc
7
(u
+I 0 W N
W W
c
W
m +I
m
r.
c
0
c
+I
c
m
r. r-
W c
N
r. d +I N
0
m
+I
+I N 0 N
N
P
m
m 7
N
N 0
N
W 0
+I
c
m r.
F
W N
+I
rc
+I
m
d
m +I 03 0 d W
c
W
+I
m
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0 W N
+I
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m c
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c
m
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W
d
T
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r.
W
+I
m
W
+I
r-
0,
+I 00 N r-
m +I c
d
d
d
c c
W
W W
0
m
7
W
m
+I
W 0
m
c
N
d
+I
c
W
03 m m
c
+I
0 c-. N
d
W W
+I
m W m
0
m
m
0 N r-
(u
+I
W
W 0
c
r.
r-
d W W
+I
W
N
W
d
Ln c
N
m
Lc
c
m d
7
W N
-s
m
c
+I
+I
d
+I
-
+I
-
7
LD
W 0 W c
c
m
m
m
W
m
-
c
m
c
T
0
c
0
c,
u
373 t h e enamel i n t h i s g r o u p was t h r e e t o f o u r t i m e s h i g h e r t h a n t h a t o f t h e c o n t r o l s ( T a b l e 11).
While t h e f l u o r i d e c o n c e n t r a t i o n s o f t h e t w i c e as h i g h as t h o s e o f t h e c o n t r o l , between t h e enamel beyond 5
5 urn l a y e r s o f e n a m e l w e r e o n l y
the difference i n f l u o r i d e contents
and t h a t o f t h e c o n t r o l was o n l y s l i g h t .
A
d i m i n i s h e d f l u o r i d e u p t a k e was o b s e r v e d i n e a c h o f t h e s u c c e e d i n g l a y e r s (Fig.
1).
0
1
1
5
10
20
30
40
F i g u r e 1. Fluoride distribution i n deciduous t e e t h t r e a t e d w i t h f l u o r i d e r n o u t h r i n s e . o : c o n t r o l ; A : m o u t h r i n s e f o r 1 yr; A: r n o u t h r i n s e f o r 2 y r ; A: m o u t h r i n s e f o r 3 yr: A : m o u t h r i n s e f o r 4 yr.
Natural f l u o r i d e group F l u o r i d e c o n c e n t r a t i o n s and d i s t r i b u t i o n o f t h e e n a m e l f r o m a r e a s w i t h d i f f e r e n t l e v e l s o f n a t u r a l f l u o r i d e i n t h e d r i n k i n g w a t e r a r e shown i n T a b l e I11 and F i g u r e 2.
The c o n c e n t r a t i o n s i n t h e e n a m e l f r o m t h i s g r o u p
were s i g n i f i c a n t l y higher than those i n t h e controls.
An i n c r e a s e i n
f l u o r i d e c o n t e n t i n t h e d r i n k i n g w a t e r was p o s i t i v e l y a s s o c i a t e d w i t h i n c r e a s e i n f l u o r i d e c o n c e n t r a t i o n o f t h e o u t e r l a y e r s o f enamel. more,
Further-
h i g h c o n c e n t r a t i o n s o f f l u o r i d e i n t h e o u t e r m o s t l a y e r s o f t h e enamel
were always associated w i t h r e l a t i v e l y h i g h concentrations i n t h e i n t e r i o r r e g i o n , w i t h t h e e x c e p t i o n s o f t h e c o n t r o l s and t h o s e f r o m an a r e a w i t h 0.3 ppm f l u o r i d e i n t h e d r i n k i n g w a t e r .
The mean f l u o r i d e c o n c e n t r a t i o n s o f
t h e 40 um l a y e r s o f t h e e n a m e l s h o w e d t w o
t o f i v e times higher fluoride
u p t a k e t h a n t h o s e o f t h e c o n t r o l s and 0.3 ppm f l u o r i d e g r o u p ( T a b l e 111). S a m p l e s f r o m a r e a s w i t h g r e a t e r t h a n 1.0 p p m n a t u r a l f l u o r i d e i n t h e d r i n k i n g water r e s u l t e d i n t h e h i g h e s t f l u o r i d e uptake:
approximately
314
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a,
m
K
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a a (r
r
0
m
c,
a, V
L c, K K
0 V
a,
u L
.r
0 2
,-LL
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E 1 3 d
E 1 0
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E 0 N
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1 0 c
E 1 In
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-a
K (5,
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d
c
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In
N
W
d
0
m +I
a, c
+I
W
0
m +I
rd
W +I
a
c
N
a, W
c
m W
c
W +I
m d
c
c
N
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0 m r-
0 c
0
N
W
a +I
L D
W
W
d
r-
c
m
m c
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c
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r-
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d +I
7
0
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r-
c
r-
m 0 c
d
0
.r
N
+I
W
m c
In
N
m
c
c
c
r-
N
LD
W
r-
N
m +I
N
N
+I
r-
W 0 In
In
0
W N
m m +I 7
+I
m
m
m m
u3
d
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N c
d
c c
m
a m ,
m
m
W In
+I
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r
m
c
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m
c
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c
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c
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c
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a
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a
c
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r-
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375 1 0 , 0 0 0 ppm f l u o r i d e i n t h e t o p 1 urn l a y e r o f t h e e n a m e l w a s f r e q u e n t l y observed.
I n contrast,
e n a m e l f l u o r i d e u p t a k e s o f t h e c o n t r o l s and t l i e 0.3
ppm f l u o r i d e g r o u p w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r ( F i g .
1
5
10
20
30
40
Deplhbm)
.:
F i g u r e 2. Fluoride distributions i n deciduous t e e t h f r o m areas w i t h natur a l f l u o r i d e i n t h e w a t e r . N a t u r a l f l u o r i d e i n t h e w a t e r : o : <0.1 p p m 1.0 ppm; m: 1.7 ppm; 3.2 ppm. ( c o n t r o l ) ; 0 :0.3 ppm;
a:
Statistical evaluation The mean f l u o r i d e c o n c e n t r a t i o n s and t h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s between t h e c o n t r o l s , f l u o r i d e mouthrinse, c o m p a r e d ( T a b l e s I V a n d V).
and n a t u r a l f l u o r i d e g r o u p s w e r e
The d a t a on f l u o r i d e c o n t e n t s i n t h e 1 p m
l a y e r o f t h e samples i n d i c a t e d t h a t a s i g n i f i c a n t l y h i g h e r f l u o r i d e uptake o c c u r r e d i n b o t h f l u o r i d e m o u t h r i n s e and n a t u r a l w a t e r g r o u p s t h a n i n t h e controls.
Although f l u o r i d e uptake b y samples f r o m areas w i t h more than
1.0 ppm f l u o r i d e w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m one a n o t h e r , w e r e s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e o f t h e 0.3
they
ppm f l u o r i d e and f l u o -
r i d e mouthrinse groups (Table IV). However,
s i g n i f i c a n t d i f f e r e n c e s between t h e c o n t r o l s ,
f l u o r i d e mouth-
r i n s e , a n d t h e 0.3 p p m f l u o r i d e g r o u p s w e r e n o t s h o w n f o r t h e 5 t o 40 um l a y e r s ( T a b l e V).
A somewhat s m a l l e r u p t a k e o f f l u o r i d e i n t o deeper l a y e r s
o f e n a m e l was f o u n d w i t h t h e 0.3 ppm f l u o r i d e group. A p a r t f r o m t h i s g r o u p , a c o n s i d e r a b l e u p t a k e o f f l u o r i d e i n t o t h e d e e p e r l a y e r s o f enamel was f o u n d ( T a b l e V).
376
TABLE I V NEWMAN-KEULS TEST ON DIFFERENCES BETWEEN ALL PAIRS TRATIONS I N THE 1 Urn LAYER OF THE ENAMEL
1 Urn l a y e r
F mouthrinse (year)
Control
1
~~~~~~~
2
3
OF MEAN FLUORIDE CONCEN-
N a t u r a l f l u o r i d e v a l u e (ppm) 4
~~
0.3
~
aMR-l: M o u t h r i n s e f o r 1 y e a r bFW-0.3: 0.3 ppm F i n d r i n k i n g w a t e r
1.0
itP<0.O5
1.7
3.2
i"cp
TABLE V NEWMAN-KEULS TEST ON DIFFERENCES BETWEEN ALL PAIRS TRATIONS I N THE 5-40 urn LAYER OF THE ENAMEL
5-40 urn l a y e r
F mouthrinse (year) Control
1
2
3
OF
MEAN FLUORIDE CONCEN-
N a t u r a l f l u o r i d e v a l u e (ppm) 4
0.3
1.0
Control MR-1 a MR-2 MR-3 MR-4 F W-0.3b
FW-1.0 FW-1.7 FW-3.2
aMR-l: M o u t h r i n s e f o r 1 y e a r bFW-0.3: 0.3 ppm F i n d r i n k i n g w a t e r
"p
+"i'p
1.7
3.2
377 OISCUSSION T h e r e i s s u f f i c i e n t e v i d e n c e t o i n d i c a t e t h a t o l d e r (10).
more h i g h l y
m i n e r a l i z e d enamel (11) o r enamels c o n t a i n i n g h i g h f l u o r i d e c o n c e n t r a t i o n s (12) can a c q u i r e l e s s f l u o r i d e w h i l e porous enamel (13) o r n e w l y erupted
It i s conceivable,
t e e t h ( 1 0 ) a c q u i r e more.
therefore,
t h a t f l u o r i d e uptake
b y m o u t h r i n s i n g may be r e s t r i c t e d t o t h e o u t e r m o s t l a y e r s o f t h e enamel. The e n a m e l s p e c i m e n s e m p l o y e d i n t h i s s t u d y w e r e n o t t r e a t e d w i t h f l u o r i d e mouthrinse immediately a f t e r eruption.
By t h e t i m e s c h o o l - b a s e d m o u t h r i n s e
was f i r s t a p p l i e d , t h e d e c i d u o u s t e e t h h a d b e e n e x p o s e d t o an o r a l e n v i r o n ment f o r about 5 years.
Despite this,
f l u o r i d e concentrations were
increased i n t h e 1 pm l a y e r w i t h increase i n t h e length o f mouthrinsing. T h i s o b s e r v a t i o n i s i n c o n t r a s t w i t h t h e f i n d i n g o f M e l l b e r g g t ~ l ( .1 4 ) . They d i d n o t remove t h e o u t e r m o s t enamel l a y e r (1 p m ) and c o n c l u d e d t h a t f l u o r i d e p e n e t r a t i o n d i d n o t e x t e n d b e y o n d a p p r o x i m a t e l y 5 pm. F l u o r i d e u p t a k e b y t h e e n a m e l i n a r e a s w i t h m o r e t h a n 1.0 ppm f l u o r i d e i n t h e d r i n k i n g w a t e r was h i g h e r t h a n t h a t o f t h e c o n t r o l s as w e l l as enamel t r e a t e d w i t h f l u o r i d e mouthrinse.
the
From b i r t h t o e r u p t i o n o f t h e
d e c i d u o u s t e e t h t h e s u r f a c e o f t h e e n a m e l a p p e a r s t o be i n c o m p l e t e l y m i n eralized.
Consequently,
t h e o u t e r m o s t and d e e p e r l a y e r s o f e n a m e l w i l l be
able t o acquire additional f l u o r i d e through contact w i t h t i s s u e f l u i d s (pre-eruptive
acquisition).
As t h e m a t u r a t i o n p r o c e s s p r o c e e d s ,
z a t i o n becomes m o r e c o m p l e t e ,
o u t e r m o s t enamel l a y e r s w i l l d i m i n i s h (pre- and/or tion)(l5).
minerali-
and t h e a m o u n t o f f l u o r i d e a c q u i r e d i n t o t h e post-eruptive
acquisi-
T h e r e f o r e t h e h i g h f l u o r i d e u p t a k e f o u n d i n t h e o u t e r m o s t and
d e e p e r l a y e r s o f e n a m e l i n t h i s s t u d y may b e d u e t o b o t h p r e - e r u p t i v e post-eruptive
and
acquisition o f fluoride.
V a r i o u s f a c t o r s s u c h a s t o p i c a l l y a p p l i e d f l u o r i d e (16). p l a q u e (18),
f o o d s t u f f (17),
and i n c i p i e n t c a r i e s ( 1 9 ) may p o s s i b l y a f f e c t f l u o r i d e u p t a k e
b y d e c i d u o u s enamel as w e l l as b y permanent t e e t h .
This suggests t h a t
e n a m e l may a c q u i r e f l u o r i d e t h r o u g h o u t t h e l i f e o f t h e t e e t h . REFERENCES 1. B u r t BA, H a u g e j o r d e n 0, H e i n W , H o r o w i t z HS, H u n t e r PB. M o r r i s o n EC, O ' B r e i n - M o r a n E, Stamm J (1984) I n t e r D e n t J 34:141-158
2.
E r i c s s o n Y (ed) (1978) C a r i e s Res 1 2 ( s u p p 1):7-64
3.
M i r NA,
4.
R e t i e f DH, Bishoff
H i g u c h i W I (1969) A r c h s o r a l B i o l 14:904-920 B r a d l e y EL, B a r b a k o w FH, F r i e d m a n M, V a n d e r M e r w e EHM.
J I (1979) J O r a l P a t h o l 8:224-236
5. Speake JD,
C u t r e s s TW,
B a l l M E (1979) N Z d e n t J 75:94-106
6. M e l l b e r g JR ( 1 9 7 7 ) J P r e v e n t D e n t 4:8-20 7.
I i j i m a Y,
Katayama
T
(1985) C a r i e s Res 19:262-265
378 8. A t h a n a s s o u l i TM, 7: 368-373 9.
P a p a s t a t h o p o u l o s DS.
W i n e r BJ ( 1 9 7 1 ) S t a t i s t i c a l P r i n c i p l e s i n E x p e r i m e n t a l D e s i g n . 2 n d Ed. McGraw-Hill, New York, p p 201
10. Mellberg JR, 11. Brudevold
F.
12. Nicholson C R , 13. Aasenden R. 14. Mellberg JR.
Nicholson C R (1968) J D e n t Res 4/:17b Aasenden R.
Bakhos Y (1982)
C a n e s Res 16:243-248
Mellberg JR (1969) J D e n t Res 48:302-306 Brudevold
F.
MaCann H G (1968) Archs o r a l B i o l 13:543-552
Nicholson C R (1974) Caries Res 8:148-154
15. W e a t h e r e l l JA, D e u t s c h D, R o b i n s o n C, 11 ( s ~ p p1 ):85-115 16.
H a d j i i o a n n o u TP (1973) C a r i e s Res
H a l l s w o r t h A S ( 1 9 7 7 ) C a r i e s Res
K i r k e g a a r d E (1977) C a n e s Res 11:16-23
17. B a u m e LJ, V u l l i e m o z JP ( 1 9 7 0 ) A r c h s o r a l B i o l 1 5 : 4 3 1 - 4 4 3 18. T u r t o l a L O (1977) Scand J D e n t Res 85:373-379 19. T a k a e s u Y , I i j i m a Y ( 1 9 7 6 ) I n : S u g a S. I s h i i T ( e d s ) C a r i e s S u s c e p t i b i l i t y . S t r u c t u r e and Composition o f t h e S u r f a c e Enamel. K o k u h Hoken Kyokai. Tokyo, p p 117-132
H. Tsunoda and M.-H. Yu (Editors)
379
Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 379-388 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
A S T U D Y ON THE APPEARANCE
OF
DENTAL FLUOROSIS I N RELATION
TO
ENAMEL
DEVELOPMENT AND FLUORIDE INTAKE TAKUO I S H I I AND HARUO NAKAGAKI D e p a r t m e n t o f P r e v e n t i v e D e n t i s t r y and D e n t a l P u b l i c H e a l t h , A i c h i - G a k u i n U n i v e r s i t y S c h o o l o f D e n t i s t r y , 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464. Japan
ABSTRACT F l u o r i d e - r i c h (7.8
ppm) w a t e r was i n a d v e r t e n t l y s u p p l i e d t o t h e I k e n o
a r e a o f A i c h i P r e f e c t u r e i n J a p a n f r o m 1 9 6 0 t o 1973. examination,
D u r i n g t h e 1973
n o r m a l e n a m e l o n t h e u p p e r i n c i s o r was f o u n d i n c h i l d r e n who
w e r e m o r e t h a n 38 m o n t h s o l d when t h e w a t e r s u p p l y was s t a r t e d .
Limited
o p a c i t y o n t h e c e r v i c a l p o r t i o n o f t h e u p p e r i n c i s o r was o b s e r v e d among c h i l d r e n who w e r e b o r n d u r i n g a p e r i o d 29 m o n t h s p r i o r t o , a f t e r t h e w a t e r s u p p l y was s t a r t e d .
and 8 months
I n t h e 1984 e x a m i n a t i o n ,
children
e x p o s e d t o t h e w a t e r u n t i l a b o u t 35 m o n t h s o f age showed s e v e r e o r m o d e r a t e d e n t a l f l u o r o s i s on t h e u p p e r i n c i s o r s ; m o n t h s showed m i l d o r v e r y m i l d f l u o r o s i s :
c h i l d r e n exposed f o r 24 t o 33 and c h i l d r e n who consumed t h e
w a t e r f o r 1 1 t o 22 m o n t h s showed q u e s t i o n a b l e f l u o r o s i s o r a n o r m a l condition.
T h e s e f i n d i n g s seemed t o p r e s e n t some i n f o r m a t i o n o n t h e
c o r r e l a t i o n b e t w e e n f l u o r i d e a c t i o n and t h e s t a g e o f e n a m e l f o r m a t i o n .
INTRODUCTION T h e r e have been more t h a n 300 r e p o r t s on t h e e p i d e m i o l o g i c a l s t u d y o f d e n t a l f l u o r o s i s i n J a p a n s i n c e 1925.
D e n t a l f l u o r o s i s h a s been f o u n d i n
a l m o s t a l l o f t h e p r e f e c t u r e s o f Japan,
b u t o n l y f o u r areas have been
s t u d i e d d u r i n g t h e p e r i o d o f t i m e i n which cases o f d e n t a l f l u o r o s i s d e c r e a s e d t o t h e i r p r e s e n t l o w l e v e l (1).
Moreover,
t h e r e are few r e p o r t s
d e s c r i b i n g t h e changes i n appearance o f d e n t a l f l u o r o s i s caused b y exposure t o w a t e r w i t h a h i g h f l u o r i d e c o n t e n t f o r l i m i t e d b u t known p e r i o d s d u r i n g t o o t h formation. From December 1960 t o F e b r u a r y 1973, d r i n k i n g w a t e r c o n t a i n i n g a h i g h l e v e l o f f l u o r i d e (7.8 p p m ) w a s i n a d v e r t e n t l y s u p p l i e d t o t h e I k e n o a r e a (Pop.
423) o f A i c h i P r e f e c t u r e i n Japan.
The h i g h l e v e l o f f l u o r i d e was
n o t r e c o g n i z e d u n t i l 1 0 y e a r s l a t e r when d e n t a l f l u o r o s i s was shown among
380 I n February 1973,
school c h i l d r e n .
t h e w a t e r s u p p l y was r e p l a c e d w i t h one
c o n t a i n i n g l e s s t h a n 0.2 ppm f l u o r i d e .
The t e e t h o f t h e a f f e c t e d c h i l d r e n
h a v e been e x a m i n e d a n n u a l l y s i n c e 1973 (2,3).
1 2 t o 15 years,
I n 1984. 1 6 c h i l d r e n , aged
were re-examined as a f o l l o w - u p s t u d y (4).
The p u r p o s e o f t h e p r e s e n t s t u d y was t o a s s e s s t h e c o r r e l a t i o n b e t w e e n t h e degree o f d e n t a l f l u o r o s i s and t h e d u r a t i o n o f f l u o r i d e exposure. MATERIALS AN0 METHODS The s t u d y i n c l u d e d e x a m i n a t i o n o f 101 c h i l d r e n f r o m t h e I k e n o area. w e r e b o r n i n t h e a r e a b e t w e e n 1959 and 1972, f l u o r i d e - r i c h w a t e r u n t i l 1973.
They
and h a d been c o n s u m i n g
They w e r e e x a m i n e d a n n u a l l y w h i l e t h e y
w e r e a t t e n d i n g t h e l o c a l e l e m e n t a r y s c h o o l . V i s u a l and t a c t i l e d e n t a l e x a m i n a t i o n s o f t h e d e n t a l f l u o r o s i s were p e r f o r m e d i n accordance w i t h t h e c r i t e r i a d e s c r i b e d b y Dean (5). The t e e t h w e r e n o t d r i e d b u t w e r e c l e a n e d w i t h gauze as r e q u i r e d .
Intra-oral
photographs were t a k e n a t each examina-
tion. RESULTS The r e s u l t s f r o m t h e surveys o f 1973 and 1980. dental f l u o r o s i s ,
i n d i c a t i n g t h e changes i n
a r e p r e s e n t e d i n Tables I and 11,
o r severe dental f l u o r o s i s
respectively.
Moderate
was seen i n c h i l d r e n exposed t o f l u o r i d e f r o m
l e s s t h a n t h r e e y e a r s o f age.
No c h i l d r e n who were younger t h a n t w o y e a r s
TABLE I
PERCENTAGE O I S T R I B U T I O N OF DENTAL FLUOROSIS I N SUBJECTS I N 1973
Age'
n
Very M i l d
(%)
C l a s s i f i c a t i o n (Dean) MiId Moderate
(%)
Severe
(%)
(%I
50.0
0-7
100.0
100.0
100.0
0-8
100.0
100.0
100.0
57.1
0-9
100.0
100.0
66.7
33.3
0-1 0 0-1 1
100.0
100.0
100.0
75.0
100.0
100.0
100.0
0-1 2
100.0
100.0
100.0
50.0 28.6
1-1 3 2-1 4
100.0
100.0
66.7
33.3
100.0
100.0
33.3
3-1 5
100.0
100.0 33.3
33.3
0.0
' B e g i n n i n g and e n d i n g ages o f c h i l d r e n exposed t o F - r i c h water
381 TABLE I 1 PERCENTAGE DISTRIBUTION OF DENTAL FLUOROSIS I N SUBJECTS IN 1980
Age’
n
-
10
Very M i l d
(2)
C l a s s i f i c a t i o n (Dean) MiId Moderate
Severe
(2)
(2)
(70)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0 0.0
0
3
0.0
0-1
5
80.0
0-2
8
100.0
0-3
7
100.0
0.0 20.0 100.0 100.0
0-4
7
100.0
100.0
100.0 100.0
71.4
100.0
85.7
l B e g i n n i n g and e n d i n g ages o f c h i l d r e n exposed t o F - r i c h w a t e r
o f age when t h e w a t e r was changed showed moderate o r s e v e r e d e n t a l f l u o r o -
sis. The c h i l d r e n w e r e d i v i d e d i n t o t h r e e g r o u p s a c c o r d i n g t o t h e s t a g e o f t o o t h development d u r i n g t h e p e r i o d of exposure t o f l u o r i d e - r i c h water. G r o u p A.
The e n a m e l o f t h e p e r m a n e n t t e e t h had been p a r t i a l l y f o r m e d
b e f o r e 1961. Group B.
The enamel of t h e permanent t e e t h developed d u r i n g t h e p e r i o d
o f exposure t o h i g h f l u o r i d e . Group C.
A g r e a t e r p a r t o f t h e enamel o f t h e permanent t e e t h was formed
a f t e r t h e w a t e r s u p p l y was changed (Table 111).
A t y p i c a l example o f d e n t a l f l u o r o s i s observed among s u b j e c t s i n group A i s shown i n F i g u r e 1.
The c h i l d was b o r n i n S e p t e m b e r 1958 a n d was 2 7
months o l d when t h e f l u o r i d e - r i c h w a t e r s u p p l y was s t a r t e d .
The p r e m o l a r s
showed m o d e r a t e f l u o r o s i s , b u t t h e d i f f e r e n c e b e t w e e n i n c i s a l l y p l a c e d normal enamel and g i n g i v a l l y p l a c e d f l u o r o s e d enamel was c l e a r l y seen on t h e c a n i n e s and m a x i l l a r y i n c i s o r s .
S i m i l a r o b s e r v a t i o n s w e r e made i n
c h i l d r e n b o r n d u r i n g a p e r i o d 29 m o n t h s b e f o r e , and 8 m o n t h s a f t e r s t a r t o f t h e w a t e r supply. fluoride-rich
the
C h i l d r e n o l d e r t h a n 38 months who r e c e i v e d t h e
w a t e r showed d e n t a l f l u o r o s i s i n some p r e m o l a r s b u t n o t i n
incisors.
A l l t h e c h i l d r e n i n g r o u p B, b o r n a f t e r t h e w a t e r was s u p p l i e d , showed severe o r moderate f l u o r o s i s on a l m o s t a l l o f t h e i r teeth.
F i g u r e 2 shows
t h e t e e t h of a boy b o r n i n March 1963 who had been exposed t o t h e f l u o r i d e r i c h w a t e r p r e n a t a l l y u n t i l e i g h t y e a r s o f age.
T h e r e was s e v e r e d e n t a l
f l u o r o s i s on t h e i n c i s o r s and even on t h e deciduous molars.
382
TABLE 111 RELATION BETWEEN THE PERIOD OF FLUORIDE-RICH WATER SUPPLY AND THE SUBJECTS
OF THIS STUDY '59'601
1 2 3 4 5 6 7 8 9'701 2
13
4
5
6
calender year 7 8 9 '80 84
r
-
.year of birth
11
- 6 aye
pa,
F i g u r e 1. Teeth o f a 14-year-old g i r l was 27 months old.
girl.
F l u o r i d e exposure s t a r t e d when t h e
A l m o s t a l l o f t h e i n c i s o r s o f c h i l d r e n i n group C showed m i l d o r moderate fluorosis;
t h e p r e m o l a r s showed o n l y q u e s t i o n a b l e changes.
However,
the
f i r s t m o l a r s and c e n t r a l i n c i s o r s showed severe f l u o r o s i s i n c h i l d r e n who were more t h a n t h r e e y e a r s o f age when t h e w a t e r s u p p l y was changed. F i g u r e 3 i l l u s t r a t e s t y p i c a l f l u o r o s i s p a t t e r n s f r o m each group.
Inci-
s o r s and m o l a r s i n group A showed f l u o r o s e d enamel on t h e c e r v i c a l p o r t i o n o f t h e t o o t h crown, t o o t h surface.
and changed e n a m e l on p r e m o l a r s r e a c h e d a l l o f t h e
On t h e o t h e r hand,
i n c i s o r s i n g r o u p C showed w h i t e and
s t a i n e d enamel c l o s e t o t h e i n c i s a l t i p , o f t h e g i n g i v a l margin,
and normal enamel i n t h e p o r t i o n
b u t t h e d i f f e r e n c e b e t w e e n c h a n g e d e n a m e l and
normal enamel was n o t as c l e a r as i n group A.
383
F i g u r e 2. Teeth o f an 8-year-old u n t i l t h e boy was 8 y e a r s old.
Tooth Group A
Group B
Group C
14
16
boy.
11
F l u o r i d e exposure s t a r t e d p r e n a t a l l y
n~or~
a00a a (3 0 ........
.:.>:.......
..... .... ...... ... .....
........... ..:.:.>>
....... ........ ....... ............. ...
.. ........ ..
. . . .::. . ...... ..:
filj.
or
MO
r.10
0r
s
........... ........... .....
,:
F i g u r e 3. Appearance o f s e l e c t e d t e e t h r e l a t i n g t h e p e r i o d o f exposure t o f l u o r i d e - r i c h w a t e r s u p p l y u s i n g Dean's c l a s s i f i c a t i o n .
The s u b j e c t s i n group C were d i v i d e d i n t o t h r e e sub-groups i n a f o l l o w - u p s t u d y c o n d u c t e d i n 1984.
C h i l d r e n e x p o s e d t o f l u o r i d e f o r m o r e t h a n 35
months showed t h e most severe e f f e c t s on t h e upper i n c i s o r s . The enamel o f t h e i n c i s o r s was d u l l , e x a m i n a t i o n o f 1980, g r e a t (Fig.
4).
chalky-white
and compared w i t h t h e r e s u l t s o f t h e
l o s s e s o f o u t e r m o s t e n a m e l and s t a i n e d a r e a s w e r e
Loss o f enamel and m a g n i f i c a t i o n o f s t a i n e d areas were n o t
f o u n d i n y o u n g e r c h i l d r e n e x p o s e d t o t h e w a t e r f o r l e s s t h a n 3 3 months. F o l l o w i n g these findings,
t h e c h i l d r e n exposed f o r more t h a n 35 months were
separated from o t h e r children.
The i n c i s o r s o f c h i l d r e n exposed f o r 24 t o
33 m o n t h s w e r e c l a s s i f i e d as m o d e r a t e , m i l d , o r v e r y m i l d . t h e second sub-group
o f Group C.
They w e r e i n
384
Fig. 4-1
Fig. 4-2
Fig. 4-3
Figure 4. Teeth o f a girl exposed to fluoride-rich water prenatally until 40 months old. Figure 4-1. X-ray photograph of teeth when the subject was 51 months old ( 1 973.11). Figure 4-2. (1980.5) Figure 4-3. (1984.6)
385
F i g . 5-1
F i g . 5-2
F i g . 5-3
F i g u r e 5. T e e t h o f a b o y exposed t o f l u o r i d e - r i c h w a t e r p r e n a t a l l y u n t i l 33 m o n t h s old. F i g u r e 5-1. X-ray p h o t o g r a p h of t e e t h when t h e s u b j e c t was 44 m o n t h s o l d (1973.11). (1980.5) F i g u r e 5-2. F i g u r e 5-3. (1984.6)
386 F i g u r e 5 shows t h e t e e t h o f a boy who r e c e i v e d f l u o r i d e - r i c h
water u n t i l
33 months o f age. H i s upper c e n t r a l i n c l s o r s were c l a s s i f i e d as moderate, b u t t h e c o n d i t i o n s were n o t a g g r a v a t e d f o r t h e l a s t f o u r years.
X-ray
photographs of t h e t e e t h showed t h a t t h e permanent i n c i s o r s had developed m o r e t h a n h a l f o f t h e i r t o o t h c r o w n s when t h e w a t e r s u p p l y was c h a n g e d . , B u t t h e d i f f e r e n c e i n t h e c o n d i t i o n b e t w e e n i n c i s a l l y p l a c e d e n a m e l and g i n g i v a l l y p l a c e d e n a m e l was n o t c l e a r l y seen.
The t h i r d s u b - g r o u p o f
Group C c o n s i s t e d o f c h i l d r e n who r e c e i v e d t h e f l u o r i d e - r i c h t o 22 months.
These c h i l d r e n showed v e r y m i l d ,
enamel changes (Fig.
w a t e r f o r 11
q u e s t i o n a b l e o r normal
6).
F i g . 6-1
F i g . 6-2
F i g u r e 6. Teeth o f a g i r l exposed t o f l u o r i d e - r i c h 22 months old. F i g u r e 6-1. (1980.5) F i g u r e 6-2. (1984.6)
water p r e n a t a l l y u n t i l
387
DISCUSSION Schour and S m i t h ( 6 ) f o u n d t h r o u g h a n i m a l e x p e r i m e n t s t h a t f l u o r i d e e x e r t e d a d i r e c t l o c a l a c t i o n on e n a m e l - f o r m i n g h a v e been v o i c e d r e c e n t l y a b o u t t h e s t a g e o r
cells.
Different opinions
s t a g e s o f enamel
formation
t h a t a r e more s e n s i t i v e t o f l u o r i d e . M a n y e x p e r i m e n t s w i t h r a t s p r o d u c e d m o r p h o l o g i c a l changes 1n i n c i s o r s when f l u o r i d e was a d m i n i s t e r e d a t t h e s e c r e t o r y s t a g e o f enamel f o r m a t i o n
(7-9).
Suga and K a t a g i r i
( 1 0 ) r e p o r t e d t h a t a h y p o m i n e r a l i z e d l e s i o n was
i n d u c e d b y a s i n g l e i n j e c t i o n o f NaF i n t h e e a r l y m a t u r a t i o n z o n e o f t h e i n c i s o r enamel of a r a t .
S h i n o d a (11) r e p o r t e d t h a t t h e l a t e f o r m a t i v e
a m e l o b l a s t s were p a r t i c u l a r l y s e n s i t i v e t o f l u o r i d e .
F e j e r s k o v and h i s co-
w o r k e r s ( 1 2 ) s u g g e s t e d t h a t t h e a m e l o b l a s t s a r e a b l e t o l a y down m a t r i x , b u t i t i s d u r i n g t h e f o l l o w i n g m i n e r a l i z a t i o n p r o c e s s t h a t d i s t u r b a n c e s may occur. The p r e s e n t s t u d y h a s d e m o n s t r a t e d t h a t e f f e c t s o f w a t e r c o n t a i n i n g 7.8 ppm f l u o r i d e d i d n o t a p p e a r on t h e u p p e r i n c i s o r s o f c h i l d r e n c o n s u m i n g t h e w a t e r when t h e y w e r e more t h a n 3 8 m o n t h s o l d . ages when t h e w a t e r s u p p l y was changed,
Regarding the childrens'
t h o s e who w e r e m o r e t h a n 33 m o n t h s
o l d had s e v e r e d e n t a l f l u o r o s i s on t h e i r u p p e r i n c i s o r s . It i s i n t e r e s t i n g t h a t , on c o m p a r i n g t h e X-ray photographs o f c h i l d r e n
exposed t o f l u o r i d e - r i c h
w a t e r u n t i l 33 and 4 0 m o n t h s o f age,
the differ-
e n c e i n t o o t h g r o w t h was n o t g r e a t b u t t h e d i f f e r e n c e i n t h e g r a d e o f d e n t a l f l u o r o s i s w a s v e r y c l e a r ( F i g s . 3 a n d 4). p o r t e a r l i e r r e p o r t s b y Dean
Gal.
(13).
These o b s e r v a t i o n s sup-
Approximately ten years a f t e r
t h e change i n t h e communal w a t e r s u p p l y , b a u x i t e was r e s u r v e y e d . t e n 13-year
o l d c h i l d r e n who had r e c e i v e d t h e f l u o r i d e - r i c h
f o r t h r e e years,
e i g h t were affected.
Among t h e s i x 1 2 - y e a r
who had been e x p o s e d t o t h e w a t e r f o r t w o y e a r s , m o t t l e d enamel b u t o f t h e seven 11-year-old water f o r o n l y a year, fluorosis.
O f the
water supply old children
t h r e e showed e v i d e n c e o f
c h i l d r e n who had consumed t h e
o n l y one showed a d e g r e e o f " v e r y
mild" dental
These f i n d i n g s i n d i c a t e t h a t t h e l a t t e r p h a s e o f e n a m e l f o r m a -
t i o n h a s an i m p o r t a n t i n f l u e n c e on t h e a p p e a r a n c e o f d e n t a l f l u o r o s i s . During a period o f high f l u o r i d e intake, (14).
f l u o r i d e i s s t o r e d i n t h e bones
F l u o r i d e f r o m such sources m i g h t produce t h e i n d i s t i n c t d i f f e r e n c e
b e t w e e n f l u o r o s e d e n a m e l p l a c e d on t h e i n c i s o r p o r t i o n and n o r m a l enamel p l a c e d on t h e g i n g i v a l p o r t i o n i n group C o f t h i s study. o f f l u o r i d e ' s i n f l u e n c e occurred,
I f t h i s extension
t h e l a t t e r s t a g e o f enamel f o r m a t i o n
seems t o be m o r e i m p o r t a n t . The o u t e r l a y e r o f enamel b e g i n s t o m i n e r a l i z e v e r y r a p i d l y d u r i n g t h e l a t t e r s t a g e o f m a t u r a t i o n and f i n a l l y shows t h e h i g h e s t d e g r e e o f m i n e r a l i z a t i o n (15).
The f l u o r i d e c o n c e n t r a t i o n i n c r e a s e d t o a peak a t t h e e a r l y
388 s t a g e o f m a t u r a t i o n o f t h e enamel, m a t u r a t i o n p r o c e e d e d (16,17).
and t h e n t e n d e d t o f a l l as enamel
D u r a t i o n o f t h e s e c r e t o r y and maturation
phases o f enamel d e v e l o p m e n t i n human p e r m a n e n t t e e t h i s n o t known.
The
f i n d i n g s i n t h e p r e s e n t s t u d y suggest a c o r r e l a t i o n between f l u o r i d e a c t i o n and p e r m a n e n t enamel d e v e l o p m e n t i n humans. I n t h i s o b s e r v a t i o n t h e g r a d e s o f d e n t a l f l u o r o s i s w e r e changed by t h e d u r a t i o n of f l u o r i d e intake,
g i v e n t h e same c o n c e n t r a t i o n o f f l u o r i d e .
When p a r t o f t h e f l u o r i d e i n d u c e d change o c c u r s d u r i n g t o o t h f o r m a t i o n ,
it
a p p e a r s d i f f i c u l t t o c a t e g o r i z e enamel change a c c o r d i n g t o Dean's c l a s s i f i cation.
A s y s t e m f o r c l a s s i f y i n g enamel d e f e c t s based on t h e i r appearance
(DDE I n d e x F D I ) ( 1 8 )
as u s e d b y I s h i i and S u c k l i n g (4).
c l a s s i f i c a t i o n s f o r d e n t a l f l u o r o s i s (19,20)
or alternative
w o u l d be u s e f u l t o assess
enamel changes s i m i l a r t o t h o s e o b s e r v e d i n t h i s s t u d y . REFERENCES S a k a k i b a r a Y (1982) J D e n t a l H e a l t h 32:78-102
1.
I s h i i T,
K a t o K,
2.
I s h i i T,
S a k a k i b a r a Y (1983) IADR,
3.
I s h i i T, N a k a g a k i H ( 1 9 8 4 ) I n : F e a r n h e a d RW, S u g a S ( e d s ) T o o t h Enamel I V . E l s e v i e r S c i e n c e Pub1 i s h e r s , Amsterdam, p p 338-341
P r o g - A b s t 61:651
Suckling G (submitted)
4.
I s h i i T,
5.
Dean HT (1934) JADA 21:1421-1426
6.
Schour I ,
7.
Weber D,
8.
Ishida T (1970) J a p J O r a l Biol 12:88-107
9.
S m i t h MC (1935) JADA 22:796-813 Yaeger JA (1964) JDR 43:50-56
W a l t o n RE,
Eisenmann DR (1974) A r c h s o r a l B i o l 19:171-182
M
10.
Suga S.
11.
S h i n o d a H (1975) C a l c i f T i s s Res 18:91-100
12.
F e j e r s k o v 0, S i l v e r s t o n e LM, M e l s e r B. M b ' l l e r I J ( 1 9 7 5 ) C a r i e s Res 9~190-219
13.
Dean HT,
14.
Angmar-mansson
15.
Suga S,
Katagiri
McKay FS.
(1967) JDR 46:134-135
E l v o v e E (1938) Pub H e a l t h r e p 53:1736-1748
B, L i n d h V.
Kamio N,
Y o s h i d a S,
W h i t f o r d GM (1985) I A D R A b s t 64:675 Doh A (1977) Jap J O r a l B i o l 19:219-238
G e d a l i a I (1982) C a r i e s Res 16:428-432
16.
D e u t s c h D,
17.
D e u t s c h D, S h a p i r a L. A l a y o f f A, L e v i e l D, Y o e l i Z, A r a d A ( 1 9 8 4 ) I n : Fearnhead RW, Suga S (eds) T o o t h Enamel I V . E l s e v i e r Science Publishers, Amsterdam, p p 234-239
18.
T h y l u s t r u p A,
19.
FDI T e c h n i c a l R e p o r t No 1 5 (1982) I n t D e n t J 32:159-167
20.
H o r r o w i t z HS. H e i f e t z SB, D r i s c o l l WS, JADA 109: 37-41
F e j e r s k o v 0 (1978) Comm D e n t O r a l E p d e m i o l 6:315-328 K i n g m a n A,
M e y e n s RJ ( 1 9 8 4 )
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 389-394 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
389
GASTROINTESTINAL ABSORPTION OF FLUORIDE I N HUMANS - A COMPARATIVE STUDY OF NaF AND CaF2
NORIKO TSUNODA, S H I R O SAKURAI AND HUM10 TSUNODA D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , U n i v e r s i t y , M o r i o k a 020, J a p a n
School o f Medicine,
I w a t e Medical
ABSTRACT Twenty-three
male s u b j e c t s p a r t i c i p a t e d i n a study i n which d i f f e r e n c e s
i n t h e g a s t r o i n t e s t i n a l a b s o r p t i o n o f f l u o r i d e were examined based on changes
i n serum
and u r i n a r y
fluoride
concentrations
subsequent t o
i n g e s t i o n o f NaF o r CaF2. R a p i d f l u o r i d e a b s o r p t i o n was o b s e r v e d w i t h NaF.
and t h e s e r u m f l u o r i d e
c o n c e n t r a t i o n r e a c h e d a p e a k 30 m i n u t e s a f t e r f l u o r i d e a d m i n i s t r a t i o n .
With CaF2,
however,
f l u o r i d e a b s o r p t i o n was much l o w e r t h a n t h a t o f NaF,
accounting f o r o n l y e i g h t t o t e n percent o f the l a t t e r ,
and t h e serum
f l u o r i d e l e v e l d i d n o t peak u n t i l t w o hours f o l l o w i n g i t s a d m i n i s t r a t i o n . A m a r k e d d i f f e r e n c e i n t h e e x t e n t o f f l u o r i d e a b s o r p t i o n was a l s o o b s e r v e d w i t h CaF2,
suggesting t h e presence o f i n d i v i d u a l v a r i a t i o n .
INTRODUCTION The t o x i c i t y o f o r a l l y a d m i n i s t e r e d f l u o r i d e i s d e p e n d e n t on i t s c h e m i c a l form.
Presumably, t h i s i s r e l a t e d t o t h e v a r y i n g r a t e s o f f l u o r i d e absorp-
t i o n due t o d i f f e r e n c e s i n c h e m i c a l various fluorine-containing
properties,
including solubility,
of
compounds (1).
W i t h t h e advent o f r a p i d advances i n t h e m i c r o a n a l y s i s o f f l u o r i d e s i n r e c e n t years,
i t i s now p o s s i b l e t o d e t e r m i n e w i t h h i g h a c c u r a c y t r a c e
amounts o f f l u o r i d e ,
such as t h o s e found i n t h e serum
(2-4).
This paper i s concerned w i t h a study i n which t h e r a t e o f f l u o r i d e a b s o r p t i o n t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t i n humans was i n v e s t i g a t e d f o l l o w i n g t h e i n g e s t i o n o f NaF o r CaF2.
t w o f l u o r i d e compounds w i t h con-
t r a s t i n g s o l u b i 1 it i e s .
MATERIALS AND METHODS Twenty-three t h i s study.
h e a l t h y male adults,
a l l i n t h e i r twenties,
participated i n
They w e r e d i v i d e d i n t o t w o g r o u p s : NaF and CaF2.
Subjxts i n
390 t h e NaF g r o u p (n=12)
w e r e o r a l l y a d m i n i s t e r e d a 200 m l s o l u t i o n c o n t a i n i n g
F
1 0 mg F a s NaF. w h i l e t h o s e i n t h e CaF2 ( n = l l ) g r o u p r e c e i v e d 20 mg p o w d e r e d CaF2 and 200 m l w a t e r . t h i s group,
as
A h i g h e r a m o u n t o f F was a d m i n i s t e r e d i n
as a p r e l i m i n a r y s t u d y r e v e a l e d no a p p r e c i a b l e changes i n
f l u o r i d e e x c r e t i o n s u b s e q u e n t t o a d m i n i s t r a t i o n o f 1 0 mg F a s CaF2.
Fluo-
r i d e s were a d m i n i s t e r e d i n t h e morning w h i l e t h e s u b j e c t s were i n a f a s t i n g state.
Experiments were c a r r i e d o u t d u r i n g t h e w i n t e r t o avoid excessive
p e r s p i r a t i o n by t h e subjects,
t h u s i n f l u e n c i n g t h e e x p e r i m e n t a l d a t a (5.6).
B l o o d s a m p l e s w e r e t a k e n p r i o r t o f l u o r i d e a d m i n i s t r a t i o n , and a t 15 m i n u t e , 3 0 m i n u t e , 1,
2.
6. 9. 12, a n d 2 4 h o u r i n t e r v a l s , r e s p e c t i v e l y ,
4,
f o r f l u o r i d e determination.
The v o l u m e and c o n c e n t r a t i o n o f e a c h o f t h e
u r i n e samples were a l s o determined.
F l u o r i d e c o n c e n t r a t i o n was d e t e r m i n e d
by use o f t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e method (2,3,7). During t h e experimental period, t h e 12-hour
r e g u l a r meals were p r o v i d e d o n l y a f t e r
b l o o d and u r i n e s a m p l e s had been t a k e n ,
while drinking water
was p r o v i d e d ad l i b i t u m .
RESULTS Changes i n serum f l u o r i d e c o n c e n t r a t i o n s a t d i f f e r e n t t i m e i n t e r v a l s f o l l o w i n g t h e o r a l a d m i n i s t r a t i o n o f f l u o r i d e s a r e shown i n F i g u r e 1.
It
i s c l e a r t h a t s e r u m f l u o r i d e l e v e l s o f t h e NaF g r o u p s h o w e d a r a p i d r i s e immediately following f l u o r i d e administration, a t 30 m i n u t e s .
p e a k i n g a t 0.341fO.076
s t i l l h i g h e r t h a n t h o s e shown p r i o r t o f l u o r i d e i n g e s t i o n , restored t o
ppm
A l t h o u g h f l u o r i d e l e v e l s d i m i n i s h e d subsequently, t h e y were and d i d n o t
n o r m a l v a l u e s u n t i l a f t e r 24 h o u r s .
1
2
4
9
12
24
Time, hr F i g u r e 1: Changes i n serum f l u o r i d e a d m i n i s t r a t i o n o f NaF and CaF2.
concentrations following oral
391 On t h e o t h e r h a n d , t h e s e r u m f l u o r i d e l e v e l s o f t h e CaF2 g r o u p d i d n o t show a p r o n o u n c e d r i s e f o l l o w i n g i n d i v i d u a l subjects.
f l u o r l d e i n t a k e and
p e a k e d a t t w o h o u r s s h o w i n g a l o w e r v a l u e o f 0.073+0.065 F i g u r e 1,
varied widely w i t h
An e n h a n c e d l e v e l w a s s e e n a f t e r o n e h o u r , ppm.
and i t
As shown i n
t h e p a t t e r n and peak l e v e l s o f s e r u m f l u o r i d e f o l l o w i n g a d m i n i s -
t r a t i o n o f NaF and CaF2 v a r i e d m a r k e d l y .
A t t h e peak l e v e l ,
o f CaF2 a t a n a m o u n t e q u i v a l e n t o f 1 0 mg F.
administration
a s w a s t h e c a s e w i t h NaF.
r e s u l t e d i n a s e r u m f l u o r i d e l e v e l w h i c h was o n l y 10.7% o f t h a t e x h i b i t e d b y t h e NaF g r o u p . much s l o w e r ,
I t a p p e a r s t h a t a b s o r p t i o n o f f l u o r i d e f r o m CaF2 w a s
and i t s r a t e much l o w e r t h a n t h a t o f NaF.
Two s u b j e c t s i n t h e CaF2 g r o u p showed a g r e a t e r i n c r e a s e i n s e r u m f l u o r i d e l e v e l s t h a n t h a t d e m o n s t r a t e d b y t h e r e s t o f t h e group, c o n c e n t r a t i o n s a v e r a g e d 0.045+0.021
ppm ( F i g .
2).
whose peak
Whereas t h e l a t t e r v a l u e
w a s 6.6% o f t h e c o r r e s p o n d i n g v a l u e o f t h e NaF g r o u p , t h e v a l u e s s h o w n b y t h e t w o s u b j e c t s w e r e as h i g h a s 21% and 31%. r e s p e c t i v e l y . i n a l l cases,
Nevertheless,
p e a k v a l u e s w e r e n o t a t t a i n e d u n t i l t w o h o u r s a f t e r CaF2
administration.
m u c h l a t e r t h a n t h a t o b s e r v e d w i t h t h e NaF g r o u p .
s h o u l d b e m e n t i o n e d t h a t no o t h e r e f f e c t s w e r e o b s e r v e d ,
It
s u g g e s t i n g an
i n d i v i d u a l v a r i a t i o n i n t h e a b s o r p t i o n o f f l u o r i d e when CaF2 was used. T a b l e I shows t h e c u m u l a t i v e u r i n a r y f l u o r i d e e x c r e t i o n a s t h e p e r c e n t a g e o f f l u o r i d e o r a l l y ingested.
F o r t h e NaF g r o u p ,
f l u o r i d e a m o u n t e d t o a b o u t 20% a t t w o h o u r s , o t h e r hand,
f o r t h e CaF2 g r o u p ,
urinary excretion o f
and 48.1% a t 24 h o u r s .
On t h e
o n l y 8.5% o f t h e i n g e s t e d f l u o r i d e w a s
e x c r e t e d w i t h i n 24 h o u r s o f a d m i n i s t r a t i o n . I n a d d i t i o n , as shown i n F i g u r e 3, e x c e p t f o r t h e t w o s u b j e c t s s h o w i n g a n enhanced s e r u m f l u o r i d e
level,
t h e c u m u l a t i v e e x c r e t i o n r a t e was 4.7k1.82.
F o r t h e t w o s u b j e c t s whose s e r u m f l u o r i d e p e a k e d a t 0.17 t h e c o r r e s p o n d i n g v a l u e s w e r e 19.9% and 31.5%.
5 0.301 Q
-
_.
--*--
ppm and 0.22 ppm,
respectively.
Subject A
Subiect 6
Time, hr F i g u r e 2: Changes i n s e r u m f l u o r i d e c o n c e n t r a t i o n s f o l l o w i n g o r a l a d m i n i s t r a t i o n o f CaF2.
392 TABLE I CUMULATIVE RATE OF URINARY FLUORINE EXCRETION FOLLOWING ORAL ADMINISTRATION OF NaF AND CaF2 ( M e a n f S.D.) Time a f t e r a d m i n i s t r a t i o n Hr
NaF ( n = 1 2 )
%
9.9 18.5 _+ 29.4 f 35.3 40.1 f 42 .8 48.1 f
1
_+
2 4 6 9 12 24
*
_+
CaF2 ( n = l l )
%
2.9
0.4
f
0.3
4.0 4.0 4.1 4.1 4.5
4.2 5.6 6.6 7.3 8.5
f
5.4 6.6 7.7 8.3 9.0
3.5
2.1 f 2.6 f
f f f
8 a
401
--o---
Subject A
--0--
Subject B
L
Time, hr F i g u r e 3: Cumulative r a t e o f urinary f l u o r i d e excretion f o l l o w i n g oral a d m i n i s t r a t i o n o f CaF2.
DISCUSSION F l u o r i d e a b s o r p t i o n t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t i n humans depends on t h e s o l u b i l i t y o f f l u o r i d e s . According t o t h e experimental data from metabolic fluoride and McClure
b a l a n c e s t u d i e s o n human a d u l t s r e p o r t e d b y L a r g e n t (8)
gtt. ( 9 ) ,
the rate o f fluoride absorption for soluble fluo-
r i d e s s u c h a s NaF i s 89-97% w h e r e a s f o r i n s o l u b l e f l u o r i d e s s u c h a s CaF2 i t i s 62-69%.
These d a t a w e r e based on t h e t o t a l f l u o r i d e i n t a k e and t h e
f l u o r i d e e x c r e t i o n i n u r i n e and feces. quantitative analysis o f fluoride, mined accurately.
I n view o f this,
As a r e s u l t o f r e c e n t a d v a n c e s i n
serum f l u o r i d e l e v e l s can a l s o be d e t e r t h e p r e s e n t s t u d y i n c l u d e d serum f l u o -
393 r i d e c o n c e n t r a t i o n s a s w e l l a s t h e c o m m o n l y u s e d u r i n a r y f l u o r i d e l e v e l s as an i n d i c a t o r o f
f l u o r i d e absorption,
whereby changes i n t h e r a t e o f f l u o -
r i d e a b s o r p t i o n caused by d i f f e r e n c e s i n t h e s o l u b i l i t y o f f l u o r i d e s have been examined. The r e s u l t s o b t a i n e d f r o m t h i s s t u d y h a v e c o n f i r m e d e a r l i e r f i n d i n g s t h a t i n a d u l t s t h e a b s o r p t i o n o f f l u o r i d e s u c h a s NaF was r a p i d a n d c o n s i d e r a b l e (8-12).
On t h e o t h e r hand,
t h e f l u o r i d e a b s o r p t i o n f r o m compounds s u c h as
CaF2 w a s l o w e r t h a n t h a t r e p o r t e d b y L a r g e n t ( 8 ) a n d M c C l u r e
gal.
(9). A
m a j o r i t y o f t h e s u b j e c t s i n t h e CaF2 g r o u p e x h i b i t e d a n a b s o r p t i o n r a t e t h a t w a s o n l y 6.6% o f t h e v a l u e s h o w n b y NaF w h e n v i e w e d a t p e a k f l u o r i d e c o n c e n t r a t i o n i n t h e serum. the urine,
When v i e w e d a t o f 2 4 - h o u r f l u o r i d e e x c r e t i o n i n
t h e f l u o r i d e a b s o r p t i o n r a t e w a s e s t i m a t e d t o b e o n l y 10%.
a l t h o u g h i n some i n s t a n c e s t h e r a t e was h s h i g h as 20-30% o f t h e c o r r e s p o n d i n g v a l u e o f NaF. i s possible,
t h e g a s t r i c mucosa, (5,13).
The r e a s o n f o r t h e s e d i f f e r e n c e s i s n o t known.
It
t h a t t h e pH o f t h e g a s t r i c j u i c e and t h e t r o u b l e s o f
however,
e.g.,
inflammation o r ulcers,
may h a v e a n e f f e c t
The i n f l u e n c e o f h o s t f a c t o r s s u c h a s t h e s e a w a i t s f u r t h e r s t u d y .
Recently, a c r i t i c a l problem a s s o c i a t e d w i t h a i r b o r n e f l u o r i d e i n d u c e d p o l l u t i o n i n Japan h a s been t h e a c c u m u l a t i o n o f f l u o r i d e i n a g r i c u l t u r a l p r o d u c t s (14,15).
The f i n d i n g s o f t h i s s t u d y s t r e s s e d t h e i m p o r t a n c e o f
i d e n t i f y i n g n o t o n l y the f l u o r i d e content o f a g r i c u l t u r a l products, but also the chemical properties,
including solubility,
i n evaluating the
i n f l u e n c e o f p o l l u t e d f o o d p r o d u c t s on humans. ACKNOWLEDGEMENT T h i s w o r k was s u p p o r t e d b y a G r a n t - i n - A i d 60480193) f o r 1985-1987
f o r Co-operative
R e s e a r c h (No.
f r o m t h e M i n i s t r y o f Education, Science and C u l t u r e
o f t h e Government o f Japan. REFERENCES 1. E a g e r s R Y ( 1 9 6 9 ) T o x i c P r o p e r t i e s o f I n o r g a n i c F l u o r i n e C o m p o u n d s . E l s e v i e r . Essex, p 33 2. T a v e s DR (1968) T a l a n t a 15:1015-1023 3.
F u j i m o r i S,
I t a i K,
Tsunoda H (1984) F l u o r i d e 17:27-35
4. V e n k a t e s w a r l u P (1983) I n : Shupe JL. P e t e r s o n HB, Leone NC ( e d s ) F l u o r i d e s : E f f e c t s o n V e g e t a t i o n , A n i m a l s a n d Humans. Paragon Press I n c . S a l t L a k e City, Utah, pp 21-52 Simons JH (ed) F l u o r i n e C h e m i s t r y , 5. Hodge HC, S m i t h FA (1965) I n : 4. A c a d e m i c P r e s s , New York. p p 137-176 6.
C r e m e r HD, B i t t n e r W ( 1 9 7 0 ) I n : E r i c s s o n Y ( e d ) F l u o r i d e H e a l t h . WHO, Geneva, p p 75-91
7.
F r a n t MS,
Ross JW (1966) S c i e n c e 154:1553-1554
Vol.
a n d Human
394 The H e a l t h A s p e c t s o f F l u o r i n e Compounds. 8. L a r g e n t EJ (1961) F l u o r o s i s : O h i o S t a t e U n i v Press, Columbus, p p 34-39 9. M c C l u r e FJ, M i t c h e l l HH, 27: 159-1 70
H a m i l t o n TS, K i n s e r C A ( 1 9 4 5 ) J i n d u s t r Hyg
10. H e n s c h l e r 0. B i t t n e r W. P a t z J (1975) I n : K u h l e n c o r d t F. K r u s e HP (eds) C a l c i u m M e t a b o l i s m , Bone and M e t a b o l i c Bone Diseases. S p r i n g e r - V e r l a g . B e r l i n , New York, p p 111-121 A l v a n G,
11. E k s t r a n d J. 12: 31 1-317
Borbs,
N o r l i n A (1977)
Eur J C l i n Pharrnacol
12. Spak CJ,
E k s t r a n d J, Z y l b e r s t e i n (1982) C a r i e s Res 16:249-256
13. W h i t f o r d
GM,
14. Tsunoda
H
15. S a k u r a i S.
P a s h l e y DH,
D i r k s e n TR (1982) J D e n t Res 61:291
(1978) N i p p o n D e n t Rev 427:137-150 I t a i K, Tsunoda H (1983) F l u o r i d e 16:175-180
H. Tsunoda and M.-H. Y u (Editors)
395
Fluoride Research 1 9 8 5 , Studies in Environmental Science, Volume 27, pp. 395-400 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
POWER SPECTRUM ANALYSIS OF U R I N A R Y FLUORIDE EXCRETION I N HUMAN ADULTS TOSHITAKA H O R I U C H I ,
I K U O NASU, AND MOT01 MORIMOTO
Nihon U n i v e r s i t y School o f D e n t i s t r y a t Matsudo, D e p a r t m e n t o f D e n t a l P u b l i c H e a l t h , 2-870-1 S a k a e c h o - N i s h i , M a t s u d o - s h i , C h i b a 271, J a p a n
ABSTRACT The p o w e r s p e c t r u m a n a l y s i s o f t h e r h y t h m o b t a i n e d f r o m a s t u d y
n which
6 h e a l t h y a d u l t s were f e d a space f o o d 3 t i m e s a day f o r 6 days i s
eported
here.
Urine samples were c o l l e c t e d every 4 hours f o r determin t i o n o f
fluoride.
The r e s u l t s w e r e u s e d f o r t h e c a l c u l a t i o n o f t h e a u t o c o r r e l a t i o n
c o e f f i c i e n t and p o w e r s p e c t r u m .
The e x p e r i m e n t a l d a t a i n d i c a t e t h a t t h e
r h y t h m e x h i b i t e d by u r i n a r y f l u o r i d e e x c r e t i o n c o n s i s t e d o f a 24-hour
8.
c y c l e w i t h m i n o r p e a k s a t 6,
and 1 2 h o u r s .
main
The d a t a s u g g e s t p e r i o d i c i -
t i e s i n u r i n e v o l u m e and 4 and 6 h o u r f e e d i n g c y c l e s o f a d u l t s u b j e c t s .
INTRODUCTION U r i n e a n a l y s i s i s f r e q u e n t l y used i n s t u d y i n g t h e m e c h a n i s m o f exogenous fluorine excretion. (1,2).
Twenty-four
hour u r i n e samples a r e g e n e r a l l y analyzed
To e v a l u a t e c h a n g e s i n u r i n a r y f l u o r i n e e x c r e t i o n ,
analysis by time
s e r i e s w o u l d seem an e f f e c t i v e m e t h o d b e c a u s e u r i n e i s c o n t i n u o u s l y p r o duced.
A detailed,
s e r i a l a n a l y s i s u s i n g s p o t u r i n e s a m p l e s h a s many
a d v a n t a g e s i n t e r m s o f r e d u c i n g t h e b u r d e n on t h e p a r t o f t h e s u b j e c t s and i n t h e t i m e and c o s t i n v o l v e d , as w e l l a s w i t h t h e p o s s i b i l i t y t h a t m o r e a c c u r a t e i n f o r m a t i o n may be o b t a i n e d .
However,
l i t t l e information i s
a v a i l a b l e c o n c e r n i n g d e t a i l e d a n a l y s i s o f s p o t u r i n e samples. I n p r e v i o u s work,
s p o t u r i n e s a m p l e s w e r e e x a m i n e d and t h e e x i s t e n c e o f
p e r i o d i c i t y i n u r i n a r y f l u o r i n e e x c r e t i o n was r e p o r t e d (3). a power spectrum, d i u m (4,5),
I n t h i s study,
w h i c h i s a method used i n t h e a n a l y s i s o f e l e c t r o m y o c a r -
was e m p l o y e d i n t h e a n a l y s i s o f t h e c o m p o s i t i o n a n d p e r i o d i c i t y
o f f l u o r i n e excreted i n spot u r i n e i n a d u l t subjects.
MATERIALS AND METHODS Six healthy male adults,
1 9 t o 4 0 y e a r s o f age, w e r e g i v e n s p a c e f o o d
m a n u f a c t u r e d b y O r e g o n F r e e z e Dry F o o d s , Inc., o f vegetables, shuttle.
chicken stew,
and c r a c k e r s ,
U.S.A.
The food, composed
i s b e i n g used i n t h e space
I t w a s p r o v i d e d 3 t i m e s d a i l y a t 8:30 A.M.,
1 2 : 3 0 P.M.,
and 6 : 3 0
396
P.M.
Water was given 3 times d a i l y . T o t a l d a i l y w a t e r i n t a k e was 1400 m l ,
i n c l u d i n g t h e h o t w a t e r used f o r r e s t o r i n g t h e f r e e z e - d r i e d food samples.
T h e s u b j e c t s were a l l o w e d t o l e a d r e g u l a r l i v e s e x c e p t t h a t t h e y w e r e housed a t n i g h t i n a l o c a t i o n f a c i l i t a t i n g t h e study. Urine s a m p l e s were c o l l e c t e d e v e r y 4 hours f o l l o w i n g i n g e s t i o n of t h e e x p e r i m e n t a l food f o r t h e e n t i r e e x p e r i m e n t a l p e r i o d of 6 days.
Urinary
f l u o r i d e c o n t e n t was d e t e r m i n e d by t h e f l u o r i d e ion m e t e r method (6,7). To a n a l y z e t h e p e r i o d i c i t y o f u r i n a r y f l u o r i d e e x c r e t i o n ,
t h e power
s p e c t r u m ( 8 . 9 ) was o b t a i n e d by u s e o f t h e a u t o c o r r e l a t i o n c o e f f i c i e n t s described previously (3). RESULTS AND
DISCUSSION
F i g u r e 1 shows t h e amount of f l u o r i d e e x c r e t e d i n t h e u r i n e e v e r y 4 hours by t h e 6
participants during t h e G
day e x p e r i m e n t a l period.
W i t h some
e x c e p t i o n s , u r i n a r y f l u o r i d e l e v e l s p l o t t e d a g a i n s t s a m p l i n g time form an
23
Figure 1.
23
23
23
23
Clock time
Dally u r i n a r y e x c r e t i o n of f l u o r i d e i n s i x a d u l t s
397 M - s h a p e d c u r v e . T h e p e a k s a p p e a r a t t h e 11:OO a n d 19:OO s a m p l i n g t i m e s . The d a t a c o n c e r n i n g t h e a m o u n t o f f l u o r i n e e x c r e t e d i n t h e u r i n e a t e a c h s a m p l i n g t i m e a r e s u m m a r i z e d i n T a b l e I.
I n a previous experiment.
s a m p l e s w e r e c o l l e c t e d e v e r y 2 h o u r s (3).
The amount o f f l u o r i n e e x c r e t e d
urine
i n u r i n e showed a d e f i n i t e t e n d e n c y t o i n c r e a s e i m m e d i a t e l y a f t e r each meal.
I n t h e p r e s e n t study, u r i n e samples were c o l l e c t e d every 4 hours,
and t h e a m o u n t f o r e a c h t i m e p e r i o d was averaged. T h e r e f o r e , a c l e a r - c u t d i f f e r e n c e was n o t observed.
When a d i e t c o n t a i n i n g a n e q u a l a m o u n t o f
f l u o r i n e was i n g e s t e d b y a l l t h e SubJects,
however, t h e d a i l y f l u o r i n e
e x c r e t i o n was a p p r o x i m a t e l y 350 u g i n t h e 1 9 - y e a r - o l d s u b J e c t ( T a b l e 11).
ug i n t h e 40-year-old
Thus,
and a p p r o x i m a t e l y 470
a s s t a t e d b y Tsunoda (10).
t h e amount o f f l u o r i n e e x c r e t e d i n u r i n e t e n d e d t o i n c r e a s e w i t h i n c r e a s i n g age o f t h e SubJect. o l d and 4 0 - y e a r - o l d
B o t h young and m i d d l e - a g e d males,
subjects,
U R I N A R Y FLUORIDE LEVEL AT EACH SAMPLING TIME Mean
Max.
Min.
S. D.
23: 00-07: 00
10.0
14.0
7.4
2.6
07:00-11:00
16.9
21.3
12.2
3.5
11 :00-1 5: 00
20.1
24.7
17.8
3.2
15: 00-1 9: 00
14.4
18.5
11.7
2.3
19: 00-23: 00
22.5
25.1
20.4
3.9
Data i s f o r 6 s u b j e c t s f o r 6 days (ug/hr)
TABLE 11 24-HOUR URINARY EXCRETION OF FLUORIDE I N ADULTS ( p g / d a y ) Subject
Mean
a 19-year-
showed a l m o s t t h e same c i r c a d i a n v a r i a t i o n s .
TABLE I
Time
1.e..
Max.
M i n.
S. D.
TH
349
418
31 2
38
KO
348
417
292
45 34
IG
335
372
292
KH
395
452
280
57
HK
343
369
310
20
HT
472
520
382
45
It
398 i s d i f f i c u l t t o determine the d e f i n i t e p e r i o d i c i t y o f circadian variations as d i f f e r e n t f a c t o r s a r e i n v o l v e d .
The d a t a f r o m t h e q u a n t i t a t i v e a n a l y s i s
o f f l u o r i n e excreted i n u r i n e were then analyzed by t i m e s e r i e s t o evaluate periodicity. The d a t a o n u r i n a r y f l u o r i n e e x c r e t i o n w e r e e x a m i n e d f o r t h e a u t o c o r r e l a t i o n coefficient.
As shown i n F i g u r e 2,
all 6
s u b j e c t s showed p e r i o d i c i t y
w h e r e t h e a u t o c o r r e l a t i o n c o e f f i c i e n t a p p r o a c h e d 1 e v e r y 24 h o u r s . p e r i o d i c i t i e s showed no s p e c i f i c p a t t e r n s as shown p r e v i o u s l y w i t h Na.
The
K.
and C1 (3.11).
Changes i n t h e c o r r e l a t i o n c o e f f i c i e n t s i n t h e (-) a r e a w e r e
o f a W-shape,
i n s t e a d o f a V-shape.
t h e (-)
The c o e f f i c i e n t f o r e v e r y 24 h o u r s i n
a r e a a p p r o a c h e d zero.
To a c c u r a t e l y a s s e s s v a r i a t i o n i n t h e (-) a r e a , f a s t F o u r i e r t r a n s f o r m was u s e d t o d e t e r m i n e t h e p o w e r s p e c t r u m o f t h e a u t o c o r r e l a t i o n c o e f f i c i e n t . A t w o - p e a k p o w e r s p e c t r u m a p p e a r e d w i t h s u b j e c t s T.H. s p e c t r u m w i t h K.O.,
three-peak w i t h H.K.
(Fig.
3).
a n d H.T.,
K.O.,
..
I.G..
a n d H.T..
H.K..
..
. . . . . . .. ... . . . ..-. - . .......... . . . .. -.. ..
. . --. .............. --.
0
F i g u r e 2.
24
~
and a four-peak
a n d I.G..
a
spectrum
The f i r s t peak o f t h e p o w e r s p e c t r a a p p e a r e d i n a 24
h o u r c y c l e i n s u b j e c t s T.H.,
..
K.H.,
I
.* *~
~~
48
I
72
96
I
Time
1M
Autocorrelation coefficient o f urlnary fluoride
The s e c o n d peak
399
I
iii
'
Period(hr) F i g u r e 3.
Power s p e c t r u m o f u r i n a r y f l u o r i d e
a p p e a r e d s o m e t i m e d u r i n g t h e t i m e p e r i o d s o f 12,
8. a n d 6 h o u r s .
p e r i o d i c i t i e s a l l c o r r e s p o n d e d t o d i v i s o r s o f 24 h o u r s . w i t h subject
K.H.
appeared a t 12 hours,
Changes i n t h e p o w e r s p e c t r a o f Na,
K
These
The f i r s t peak
u n l i k e t h a t o f t h e other subjects.
and C1 e x c r e t e d i n t h e u r i n e b y t h i s
s u b j e c t a l s o showed p e r i o d i c i t i e s ( 1 1 ) w h i c h w e r e d i f f e r e n t f r o m t h o s e o f other subjects,
suggesting a different
u r i n e e x c r e t i o n p a t t e r n compared t o
t h a t o f t h e other 5 subjects. Circadian periodicity, urine,
i.e..
the main rhythm o f f l u o r i n e excretion i n
seems t o b e i n a g r e e m e n t w i t h c h r o n o t r o p i s m ( p e r i o d i c v a r i a t i o n s ) i n
t h e n a t u r a l environment,
e.g..
tide.
and w i t h t h e c i r c a d i a n p e r i o d i c i t y o f
u r i n e volume reported by M i l l s e t al.
(12) and Sulzman (13).
g e n e r a t e d b y endogenous o s c i 1l a t o r y systems. t h e p e r i o d i c i t i e s o f 6, 8, i n t a k e (131,
which are
It i s . h i g h l y probable t h a t
and 12 h o u r s a r e due t o t h e rhythm o f food
from the f a c t that the urinary fluorine excretion i s influ-
e n c e d b y t h e f l u o r i n e i n g e s t e d a t meal t i m e (3).
The r h y t h m o f s l e e p i n g
400 and w a k i n g ,
light-dark
cycle,
etc..
c a n be c o n s i d e r e d o t h e r f a c t o r s ,
b u t no
d e f i n i t e c o n c l u s i o n c a n be d r a w n i n t h e p r e s e n t s t u d y .
I t w o u l d seem r e a s o n a b l e t o assume t h a t m e t a b o l i s m a n d e x c r e t i o n o f v a r i o u s m a t e r i a l s a r e repeated i n a temporal p a t t e r n i n t h e l i f e process i n a s h o r t c y c l e r a t h e r than c i r c a d i a n p e r i o d i c i t y o f constant rhythm. u l t r a d i a n rhythm.
F l u o r i n e shows an u l t r a d i a n r h y t h m ,
C1 show a c i r c a d i a n r h y t h m ( 3 , l l ) .
K,
and
These f a c t s s u g g e s t a u n i q u e r e a c t i v i t y
or material property o f fluorine (electronically, ment, and t h r e e - d i m e n s i o n a l l y ,
w h e r e a s Na,
i.e..
t h e most negative ele-
t h e s m a l l e s t atom n e x t t o hydrogen).
CONCLUSION T h i s e x p e r i m e n t a l d a t a i n d i c a t e s t h a t t h e u l t r a d i a n r h y t h m e x h i b i t e d by u r i n a r y f l u o r i n e e x c r e t i o n c o n s i s t s o f a 24,
1 2 , 8,
and 6 h o u r c y c l e
a c c o r d i n g t o t h e p o w e r s p e c t r u m method. T h e s e p e r i o d i c i t i e s s u g g e s t p e r i o d i c i t i e s o f u r i n e v o l u m e and 4 and 6 h o u r f e e d i n g c y c l e s o f a d u l t S u b j e c t s .
REFERENCES
1.
D e r r y b e r r y OM,
6: 503-514
2.
Hodge HC,
3.
H o r i u c h i T,
B a r t h o l o m e w MD,
F l e m i n g RBL (1963)
Arch Environ Health
S m i t h FA (1970) J A i r P o l l A s s o c 20:226-232 Nasu I.
M o r i m o t o M (1984) F l u o r i d e 17:173-177
4. C h a f f i n DB (1969) J Occup Med 11:109-115 5. W a l t o n JH (1952) J N e u r o l N e u r o s u r g P s y c h i a t 15:219-226 6. H o r i u c h i T, K a s a i G, T e r a m o t o K. M o r i m o t o M (1977) J D e n t H e a l t h 27:102
7. O r i o n R e s e a r c h I n c (1971) S p e c i f i c I o n E l e c t r o d e T e c h n o l o g y 3:8 8. A k a i k e K ( 1 9 8 5 )
Power Spectrum A n a l y s i s o f I r r e g u l a r O s c i l l a t i o n . I n s t i t u t e o f S t a t i s t i c a l M a t h e m a t i c s , p p 37-40
9. H i n o M (1980) P o w e r S p e c t r u m A n a l y s i s .
A s a k u r a Shuppan.
p p 40-51
10. Tsunoda H, S a k u r a i S, I t a i K, S a t 0 T, Nakaya S, M i t a M, T a t u m i M (1983) F 1u o r i d e 17: 15971 67 11. H o r i u c h i T.
S t u d y f o r t h e movement o f u r i n a r y f l u o r i d e i n human a d u l t s
(unpublished)
12. M i l l s JN, S t a n b u r y SW (1952) P h y s i o l 117:22 13. S u l z m a n FM, F u l l e r CA, Moore-Ede MC (1977) P h y s i o l Behav 18:775-779
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 401-406 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
401
NORMAL FLUORIDE CONCENTRATIONS I N SPOT SAMPLES OF U R I N E
KAORU KASAHARA, TAKESHI KONDO. AND TAKASHI NAKANE D e p a r t m e n t o f Communi t y D e n t i s t r y , Gobara S h i o j i r i C i t y 399-07, J a p a n
Matsumoto Dental College,
1780 Hirooka
ABSTRACT I n an a t t e m p t t o e v a l u a t e n o r m a l f l u o r i d e c o n c e n t r a t i o n s i n u r i n e , s a m p l e s o f u r i n e w e r e c o l l e c t e d f r o m 1,213
spot
h e a l t h y Japanese m a l e a d u l t s ,
c o n s i d e r e d t o be f r e e f r o m u n u s u a l f l u o r i d e exposure i n t h e i r l i v i n g o r working environment, Mid-morning
and t h e i r f l u o r i d e c o n t e n t s
were determined.
u r i n e s a m p l e s w e r e c o l l e c t e d f r o m 1,047 G r o u n d S e l f - D e f e n s e
F o r c e o f f i c i a l s aged 1 8 t o 58, and t h e f i r s t - m o r n i n g u r i n e s a m p l e s f r o m 172 f a r m e r s aged 50 t o 69.
F l u o r i d e c o n c e n t r a t i o n s w e r e d e t e r m i n e d u s i n g a n F-
e l e c t r o d e a f t e r t h e f l u o r i d e was s e p a r a t e d b y a m i c r o d i f f u s i o n method. S t a t i s t i c a l a n a l y s i s o f t h e d a t a was c a r r i e d o u t f o l l o w i n g l o g a r i t h m i c t r a n s f o r m a t i o n o f t h e values,
since t h e d i s t r i b u t i o n o f f l u o r i d e concentra-
t i o n s was n e a r l y l o g a r i t h m i c a l l y n o r m a l . G e o m e t r i c mean v a l u e s o f t h e u r i n a r y f l u o r i d e c o n c 2 n t r a t i o n s o f t h e S e l f D e f e n s e F o r c e o f f i c i a l s (N=1040) and t h e f a r m e r s (N=164) w e r e e s t i m a t e d t o b e 0.32 m g / l
a n d 0.50 m g / l ,
respectively.
INTRODUCTION U r i n a r y e x c r e t i o n o f f l u o r i d e i s w i d e l y a c c e p t e d as one o f t h e b e s t i n d i c e s o f f l u o r i d e a b s o r p t i o n (1).
F l u o r i d e l e v e l s i n t h e u r i n e samples
o f i n d u s t r i a l w o r k e r s e x p o s e d t o f l u o r i d e have, i n d e x o f h e a l t h c a r e (2.3). tion,
24-hour
therefore,
b e e n u s e d a s an
F o r an a c c u r a t e e v a l u a t i o n o f f l u o r i d e e x c r e -
u r i n e s a m p l e s s h o u l d be used.
However,
s p o t samples o f u r i n e
a r e employed r o u t i n e l y . Normal f l u o r i d e e x c r e t i o n i n t h e 24-hour j e c t s has been r e p o r t e d b y Tsunoda
u r i n e s a m p l e s o f J a p a n e s e sub-
g d.( 3 ) ,
b u t r e p o r t s on t h e normal
f l u o r i d e c o n c e n t r a t i o n o f s p o t samples o f u r i n e have been c o n t r a d i c t o r y . I n an a t t e m p t t o e v a l u a t e normal f l u o r i d e c o n c e n t r a t i o n s i n urine.' s a m p l e s w e r e c o l l e c t e d f o r m 1,219
spot
h e a l t h y Japanese male a d u l t s c o n s i d e r e d
t o be f r e e f r o m f l u o r i d e exposure i n t h e i r l i v i n g o r w o r k i n g environment, and t h e f l u o r i d e c o n c e n t r a t i o n s o f t h e s e u r i n a r y samples were determined.
402 MATERIALS AND METHODS I n t h i s s t u d y , t w o d i f f e r e n t e x p e r i m e n t a l g r o u p s w e r e examined. m o r n i n g u r i n e s a m p l e s (8:30-11:OO Self-Defense Force
a.m.)
(GSDF) o f f i c i a l s
Mid-
w e r e c o l l e c t e d f r o m 1,047 G r o u n d
aged 18 t o 58, and t h e f i r s t - m o r n i n g
u r i n e samples were c o l l e c t e d f r o m 172 farmers,
aged 50 t o 69.
residing i n
d i f f e r e n t areas. F l u o r i d e c o n c e n t r a t i o n s i n t h e d r i n k i n g w a t e r i n b o t h areas were l e s s t h a n 0.1 m g / l and f l u o r i d e i n t h e a i r was n o t d e t e c t a b l e .
To s e p a r a t e t h e f l u o r i n e i n u r i n e ,
(4).
a m i c r o d i f f u s i o n method was employed
A 5 m l u r i n e s a m p l e was d i f f u s e d i n 2.7 M HC104 ( H M D S - s a t u r a t e d ) .
F l u o r i n e was t r a p p e d i n f i l t e r paper t r e a t e d w i t h 0.5
M
NaOH.
The f i l t e r
paper was t h e n p l a c e d i n a t e s t t u b e c o n t a i n i n g 1 m l o f d i s t i l l e d w a t e r and t h e f l u o r i d e was e l u t e d by shaking.
F l u o r i d e concentrations i n the eluates
w e r e m e a s u r e d b y t h e f l u o r i d e i o n - s c l ? c C i v a 31.?c-:r>:.'? . i n l i i ~ o d . F i g u r e 1
shc'~.i; .i:is ~ . : i ; x ~ r o t :? IiZsY ! f a r ,Tic;po?i f-F:!-io?.
F i g u r e 1.
Apparatus used f o r m i c r o d i f f u s i o n .
RESULTS The minimum and maximum f l u o r i d e c o n c e n t r a t i o n s i n t h e mid-morning u r i n e s a m p l e s w e r e f o u n d t o b e 0.046 m g / l and 4.6 m g / l . m o r n i n g u r i n e were 0.070 mg/l and 10 mg/l.
and t h o s e i n t h e f i r s t -
respectively.
m o r n i n g u r i n e s a m p l e s showed m o r e t h a n 1.2 m g F / l .
Seven o f t h e mid-
and n i n e o f t h e f i r s t -
m o r n i n g u r i n e s a m p l e s g a v e v a l u e s h i g h e r t h a n 1.4 m g F / l .
They w e r e ex-
c l u d e d f r o m t h e s t a t i s t i c a l a n a l y s i s , as t h e y were c o n s i d e r e d abnormal. F i g u r e s 2 a n d 3 show t h e h i s t o g r a m s o f f l u o r i d e c o n c e n t r a t i o n s i n t h e mid-morning
and t h e f i r s t - m o r n i n g
urine.
These r e s u l t s suggest t h a t t h e
d i s t r i b u t i o n o f u r i n a r y f l u o r i d e c o n c e n t r a t i o n s i n s p o t samples i s logar i t h m i c a l l y normal.
ik
400
300
200
403
20
1
100
0
0 .24
0
.48
.72
1.2
.96
0
.4
.8
1.2
1.6
F conc. i n u r i n e (mgll)
F conc. i n u r i n e ( m g / l )
F i g u r e 2. (N=l040).
Hi,-togram
o f fluoride concentrations i n t h e mid-morning
urine
F i g u r e 3. (N=l64).
Histogram o f f l u o r i d e concentrations i n the f i r s t - m o r n i n g
urine
(21 99 r
0.05
0.1
0.5
1
5
F concentrotlons In urlne Img/l)
F i g u r e 4. C u m u l a t i v e f r e q u e n c y c u r v e o f f l u o r i d e concentrations i n spot sampling urine. 0 . mid-morning u r i n e ; O , f i r s t - m o r n i n g u r i n e .
404 F i g u r e 4 shows t h e c u m u l a t i v e f r e q u e n c y c u r v e o f u r i n a r y f l u o r i d e concent r a t i o n s on l o g a r i t h m i c p r o b a b i l i t y paper.
The d i s t r i b u t i o n of u r i n a r y
f l u o r i d e c o n c e n t r a t i o n s was c o n f i r m e d t o b e l o g a r i t h m i c a l l y n o r m a l , t h e c u m u l a t i v e f r e q u e n c y gave e s s e n t i a l l y s t r a i g h t l i n e s .
since
Thus s t a t i s t i c a l
a n a l y s i s o f t h e d a t a was p e r f o r m e d a f t e r t h e l o g a r i t h m i c t r a n s f o r m a t i o n of the values. T a b l e s I and I 1 show t h e g e o m e t r i c mean v a l u e s and s t a n d a r d d e v i a t i o n of u r i n a r y f l u o r i d e c o n c e n t r a t i o n s o f s p o t samples.
The g e o m e t r i c mean v a l u e s
o f t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e mid-morning and t h e f i r s t - m o r n i n g u r i n e s a m p l e s w e r e c a l c u l a t e d t o be 0.32 m g / l and 0.50 m g / l , F i g u r e 5 i l l u s t r a t e s t h e n o r m a l r a n g e (Meang
* 2S.D. 4 ) o f
r i d e c o n c e n t r a t i o n s i n d i f f e r e n t age groups.
TABLE I FLUORIDE CONCENTRATIONS ( m g / l ) I N THE M I D - M O R N I N G U R I N E OF MALE ADULTS Age
n
Geometric S. D. Mean
18-1 9
97
0.25
1.57
20-29
447
0.29
1.72
30-39
182
0.36
1.73
40-49
209
0.35
1.60
50-58
105
0.37
1.66
Total
1040
0.32
1.71
TABLE I 1 FLUORIDE CONCENTRATIONS (mg/l) I N THE FIRST-MORNING U R I N E OF MALE ADULTS
n
Geometric Mean S. D.
50-59
a3
0.49
1.65
60-69
81
0.50
1.57
Total
164
0.50
1.61
respectively. urinary fluo-
405
ll
T
T
10’
30’ 40’
20’
50’
60’s
Age group
F i g u r e 5. N o r m a l r a n g e (Mean + 2S.D. ) o f u r i n a r y f l u o r i d e c o n c e n t r a t i o n s i n d i f f e r e n t age groups. , a i d - m o r n g n g ; 0 ,f i r s t - m o r n i n g u r i n e .
DISCUSSION F l u o r i d e c o n c e n t r a t l o n s i n u r i n e samples a r e o f t e n d i r e c t l y determined using a fluoride ion-selective i s e a s y t o use,
e l e c t r o d e p o t e n t i a l i s long,
5).
electrode.
The d i r e c t d e t e r n i n a t i o n m e t h o d
b u t t h e t i m e r e q u i r e d t o r e a c h an e q u i l i b r i u m o f t h e e s p e c i a l l y a t l o w f l u o r i d e c o n c e n t r a t i o n s (2,
I n t h i s s t u d y . f l u o r i n e was s e p a r a t e d f r o m u r i n e s a m p l e s b y m i c r o -
d i f f u s i o n method,
and i t s c o n c e n t r a t i o n d e t e r m i n e d as a s a m p l e f i v e t i m e s
i t s o r i g i n a l concentration.
With t h i s method,
t h e e l e c t r o d e p o t e n t i a l can
be s t a b i l i z e d i n a few minutes.
I n s t a t i s t i c a l analysis o f u r i n a r y f l u o r i d e concentrations,
t h e concent-
r a t i o n s a r e o f t e n r e g a r d e d a s n o r m a l d i s t r i b u t i o n (2.5-7,9).
We h a v e
confirmed that the distribution o f urinary fluoride concentrations i s l o g a r i t h m i c a l l y normal,
as was s u g g e s t e d b y T o y o t a (8).
Thus,
t h e geomet-
r i c mean s h o u l d b e u s e d i n s t e a d o f t h e a r i t h m e t r i c mean f o r t h e s t a t i s t i c a l analysis. T h e r e h a v e b e e n many r e p o r t s c o n c e r n i n g t h e n o r m a l f l u o r i d e c o n c e n t r a t i o n s o f s p o t samples o f u r i n e u r i n e , 0.32 m g / l ,
(2,5-9).
The mean v a l u e o f m i d - m o r n i n g
o b t a i n e d i n t h i s study i s l o w e r than those r e p o r t e d by
o t h e r w o r k e r s (5-7).
T h i s d i s c r e p a n c y may b e d u e p a r t l y t o t h e u s e o f
g e o m e t r i c means i n s t a t i s t i c a l a n a l y s i s and t o t h e u n i q u e l i f e - s t y l e o f t h e p a r t i c i p a t i n g GSDF o f f i c i a l s . A k i n i v i a r e p o r t e d t h e mean f l u o r i d e c o n c e n t r a t i o n o f t h e f i r s t - m o r n i n g u r i n e t o be 0.457 m g / l ( 9 ) . O u r d a t a w e r e s i m i l a r t o t h i s value, s t a n d a r d d e v i a t i o n was v e r y l a r g e .
but the
A possible explanation i s t h a t eating
h a b i t s and l i f e - s t y l e s may h a v e i n f l u e n c e d f l u o r i d e e x c r e t i o n . .
406
CONCLUSION S t u d i e s were made on t h e normal f l u o r i d e c o n c e n t r a t i o n s o f s p o t samples o f u r i n e among Japanese male s u b j e c t s . The d i s t r i b u t i o n o f f l u o r i d e concent r a t i o n s i n s p o t samples was n e a r l y l o g a r i t h m i c a l l y normal.
The g e o m e t r i c
mean f l u o r i d e c o n c e n t r a t i o n o f t h e mid-morning u r i n e samples o b t a i n e d from 1,040 Ground Self-Defense F o r c e o f f i c i a l s was 0.32 mg/l,
while the f i r s t -
m o r n i n g u r i n e samples c o l l e c t e d f r o m 164 f a r m e r s gave a v a l u e o f 0.50 mg/l.
REFERENCES
1.
I s h i k a w a S (1976) J I w a t e Med Ass 28:207-222
2. Y o s h i d a Y, T o y o t a S , Kono K, Watanabe M, I w a s a k i K ( 1 9 7 8 ) B u l l Osaka Med School 24:56-67
3.
Tsunoda H, S a k u r a i S , I t a i K, (1984) F l u o r i d e 17: 159-167 Kondo T (1978)
S a t o T,
Nakaya S,
M i t a M,
Tatsumi
4.
K a m i j o h K,
5.
Yasaki T, Ohhashi K, Dent H l t h 24:191-206
6.
Seki Y.
7.
K o n i s h i M, I w a t a M. Minagawa Y (1975) Jap J I n d H e a l t h 17:238-239
a.
Toyota S (1979) Jap J I n d H e a l t h 21:335-348
9.
A k i n i w a K (1981)
M
Matsumoto Shigaku 4:150-153
Kubota M,
Okudera H.
M i y a s i t a N,
I i z u k a Y (1974) J
I c h i k a w a Y (1975) Jap J I n d H e a l t h 17:236-237
F l u o r i d e R 1:75-77
407
H. Tsunoda a n d M.-H. Y u (Editors)
F l u o r i d e Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 407-414 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
FLUORIDE CLEARANCE I N THE AGING KIDNEY
KOI C H I K O N O ~, Y ASUH ISA Y O S H ID A ~ , HI ROSHI Y O S H I H I R O TAKEDA',
Y A M A G A T A ~ , YOSHIHISA
TANI M U R A ~ .
AKIRA HARADA3, AND KAZUHIDE DO14
'Department o f Environmental Health,
Fukui M e d i c a l School, Japan
' D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , Osaka M e d i c a l C o l l e g e , Osaka 569, J a p a n ( a d d r e s s c o r r e s p o n d e n c e t o O r . Y. Y o s h i d a ) 3Kansai T e c h n i c a l C e n t r e f o r O c c u p a t i o n a l Health, 4Matsushita E l e c t r o n i c s Corporation.
Takatsuki,
Japan
Japan
ABSTRACT Several c h a r a c t e r i s t i c s o f t h e aging process related t o renal function and f l u o r i d e m e t a b o l i s m w e r e i n v e s t i g a t e d . 1088
h e a l t h y s u b j e c t s by 10-year
A cross-sectional
analysis o f
age g r o u p s showed a p r o g r e s s i v e l i n e a r
d e c l i n e i n c r e a t i n i n e c l e a r a n c e ( C C r ) and f l u o r i d e c l e a r a n c e (CF). w a s a s i g n i f i c a n t r e d u c t i o n i n C C r a n d CF a f t e r a g e 80. f l u o r i d e f i l t e r e d t h r o u g h t h e g l o m e r u l i p e r m i n u t e (FF).
There
The a m o u n t o f
t u b u l a r reabsorp-
t i o n o f f l u o r i d e (TRF) a n d i t s r a t e (%TRF), s e e m e d t o r e m a i n s t a b l e u n t i l a g e 79.
The 2 4 - h o u r
w i t h e a c h o t h e r (r=0.83)
e x c r e t i o n o f f l u o r i d e a n d CF w e r e w e l l c o r r e l a t e d i n 7 p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e (CRF) and
11 h e a l t h y s u b j e c t s a f t e r f l u o r i d e a d m i n i s t r a t i o n .
The r e s u l t s s u g g e s t
t h a t r e n a l f l u o r i d e clearance decreased n o t o n l y w i t h the evidence o f k i d n e y d i s e a s e b u t a l s o w i t h a d v a n c i n g age.
For t h e h e a l t h care o f workers
e x p o s e d t o f l u o r i d e and o f p o p u l a t i o n s l i v i n g i n f l u o r i d e p o l l u t e d a r e a s , age may b e an i m p o r t a n t f a c t o r t o be c o n s i d e r e d .
INTRODUCTION The m a j o r p a t h w a y o f f l u o r i d e e x c r e t i o n f r o m t h e human b o d y i s v i a t h e k i d n e y (1,2,3).
Since f l u o r i d e has a s h o r t b i o l o g i c a l
h e a l t h y person,
40 t o 70% o f t h e i n g e s t e d f l u o r i d e a p p e a r s i n t h e u r i n e
d u r i n g a 24-hour
p e r i o d (4.5,6).
l i f e i n a
When r e n a l f u n c t i o n d e t e r i o r a t e s ,
a b i l i t y t o e x c r e t e f l u o r i d e markedly decreases, greater retention o f fluoride.
half
the
possibly resulting i n
Some i n v e s t i g a t o r s d e m o n s t r a t e d t h a t f l u o -
r i d e c o n c e n t r a t i o n s i n t h e plasma and bone s i g n i f i c a n t l y i n c r e a s e d i n u r e m i c p a t i e n t s (7,8.9).
On t h e o t h e r h a n d , i t h a s b e e n s h o w n t h a t m a n y
a s p e c t s o f k i d n e y f u n c t i o n d e c r e a s e w i t h age ( l D , l l , l Z ) .
F l u o r i d e metabo-
408
lism in elderly persons, however, remains incompletely understood.
This
paper deals with the characteristics of the aging process related to renal function and fluoride metabolism. ( S F ) and urine ( U F ) ,
Fluoride concentrations in the serum
the renal clearance of fluoride (CF).
fluoride filtered through the glomeruli per minute (FF). tion of fluoride (TRF) and its rate (%TRF),
the amount o f
tubular reabsorp-
and phenolsulfonphthalein
excretion (PSP, 15 min) were investigated i n healthy subjects living in a rural area of Japan. In addition, the relationship between 24-hour urinary excretion and renal clearance of fluoride was studied i n patients with chronic renal failure (CRF) and healthy controls following fluoride administration. MATERIALS AND METHODS In the first part of the study, 2-hour renal clearance determinations were performed on 1088 healthy individuals (aged 35 to 88) living i n a rural area of Japan. Fasting blood samples and urine specimens were taken from the subjects between 6:DO and 8 : O O a.m. Before the sampling, the subjects were given 300 ml water in order to increase the amount of urine. None of the subjects were known to be on drugs and none had been exposed to any unusually high level of fluoride before the investigation. Water fluoridation is not practiced in Japan, and in the community under study the fluoride level in the drinking water was approximately 0.1 ppm. In the second part of the study, seven patients with chronic renal failure (aged 40 to 78) and 1 1 healthy controls (aged 20 to 57) were selected. After an overnight fasting, the subjects were given 4 mg of fluoride by a single oral administration with water.
Blood samples and 24-hour urine
specimens were collected after the fluoride administration. Renal clearance of fluoride (CF) was calculated according to the following formula (4):
CF (ml/min/1.48 m2)
=
U-F
(ug/ml) x V (ml/min)/S-F (vg/ml) x 1.48 (m2)/A in2
where U-F is the concentration of fluoride in urine at the specific time (ug/ml): S-F is the concentration of fluoride in serum at the same time (vg/ml); V is the urinary flow during 1 minute at the same time (ml/min);
1.48 m2 is the mean body surface area of Japanese: and A m 2 is the body surface area of the subject. The amount of fluoride filtered through the glomeruli per minute (FF) was calculated by the following equation: FF(ug/min/1.48 m2) = S-F (vg/ml) x GFR (ml/min/1.48 m2) where GFR i s the glomerular filtration rate, which is the creatinine clearance (CCr) in this study.
409
The t u b u l a r r e a b s o r p t i o n o f f l u o r i d e (TRF) a n d t h e r a t e o f t u b u l a r r e a b s o r p t i o n o f f l u o r i d e (%TRF) w e r e d e t e r m i n e d f r o m t h e f o l l o w i n g e q u a t i o n : TRF ( u g / m i n / 1 . 4 8
m2)
=
FF (ug/min/1.48
%TRF ( % ) = [l-CF ( m l / m i n / 1 . 4 8
m2)
m2)/GFR
[U-F(ug/ml)
-
(ml/min/1.48
m2)]
x V (ml/min)]
x 100
The e x c r e t i o n r a t i o o f f l u o r i d e i n t h e u r i n e d u r i n g t h e 24-hour was c a l c u l a t e d u s i n g t h e f o r m u l a : ( 2 )
period
( 1 ) u g / d o s e ( 4 0 0 0 u g ) x 100 w h e r e
-
(1) i s t h e base l i n e v a l u e c a l c u l a t e d f r o m t h e u r i n a r y c o n c e n t r a t i o n o f f l u o r i d e b e f o r e t h e e x p e r i m e n t and 24-hour t o t a l e x c r e t i o n v a l u e i n a 24-hour
u r i n e volume,
w h i l e (2)
i s the
experiment.
F l u o r i d e c o n c e n t r a t i o n s i n t h e serum and u r i n e w e r e measured b y t h e f l u o r i d e e l e c t r o d e m e t h o d u s i n g an O r i o n M o d e l 901 i o n a n a l y z e r (13). C r e a t i n i n e c o n c e n t r a t i o n s i n t h e serum and u r i n e were measured b y t h e F o l i n - W u m e t h o d u s i n g a H i t a c h i M o d e l 706D a u t o a n a l y z e r . D i f f e r e n c e s b e t w e e n means w e r e e v a l u a t e d b y S t u d e n t ' s t - t e s t . RESULTS The r e s u l t s o f t h e c r o s s - s e c t i o n a l
a n a l y s e s i n 1088 h e a l t h y s u b j e c t s by
1 0 - y e a r a g e g r o u p s o n s e r u m a n d u r i n a r y f l u o r i d e ( S F a n d UF). and f l u o r i d e c l e a r a n c e ( C C r
creatinine
and CF),
t h e amount o f f l u o r i d e f i l t e r e d
t h r o u g h t h e g l o m e r u l i p e r m i n u t e (FF),
tubular reabsorption o f fluoride
(TRF) and i t s r a t e (%TRF) a r e shown i n T a b l e I.
The mean c o n c e n t r a t i o n s o f
s e r u m and u r i n a r y f l u o r i d e i n t h e s e s u b j e c t s w e r e 14.42 u g / l ( p p b ) and 0.78 u g / m l (ppm), 84.57
r e s p e c t i v e l y . The v a l u e s f o r C C r ,
ml/min/1.48
ug/min/1.48
m2,
a d v a n c i n g age.
in2,
43.30
a n d 38.74%,
ml/min/1.48
respectively.
SF
CF, FF. TRF a n d %TRF w e r e 1.18 u g / m i n / 1 . 4 8
m2,
m2,
0.71
and UF s l i g h t l y i n c r e a s e d w i t h
A p r o g r e s s i v e l i n e a r d e c l i n e w a s o b s e r v e d i n C C r a n d CF.
When e x p r e s s e d a s m e a n s o f d e c a d e a g e g r o u p s ( F i g .
1).
CF was shown t o
r e m a i n s t a b l e u n t i l age 50 and t h e r e a f t e r decreased p r o g r e s s i v e l y .
The
l o w e s t v a l u e s i n C C r and CF w e r e o b s e r v e d a t t h e age i n t e r v a l o f 80 t o 89. T h e r e was a s i g n i f i c a n t r e d u c t i o n i n FF,
TRF,
and %TRF a f t e r age 80.
T a b l e s I 1 and I 1 1 show c r e a t i n i n e and f l u o r i d e c l e a r a n c e s t u d i e s ( C C r , CF, F F . TRF, a n d %TRF) a n d p h e n o l s u l f o n p h t h a l e i n ( P S P ) e x c r e t i o n t e s t i n t h r e e d i f f e r e n t age g r o u p s .
The v a l u e s f o r C C r ,
CF and PSP e x c r e t i o n t e s t
f o r t h e 60-79 y e a r age g r o u p w e r e m a r k e d l y l o w e r t h a n t h o s e f o r t h e 35-59 y e a r age g r o u p (pi0.01). i n t h e age 60-79
A b o u t 1 5 % d e c r e a s e i n t h e s e f u n c t i o n s was shown
y e a r age group, c o m p a r e d w i t h t h e 35-39
T h e r e w a s a s i g n i f i c a n t r e d u c t i o n a f t e r a g e 80. r e m a i n s t a b l e u n t i l a g e 79.
%TRF i n t h e 6 0 - 7 9
y e a r age group.
F F a n d TRF s e e m e d t o
y e a r age g r o u p was h i g h e r
t h a n t h o s e i n o t h e r age g r o u p s . F i g u r e 2 shows t h e r e l a t i o n s h i p b e t w e e n f l u o r i d e c l e a r a n c e (CF) a n d t h e
24-
h o u r u r i n a r y f l u o r i d e e x c r e t i o n (%) f o r 7 p a t i e n t s w i t h c h r o n i c r e n a l
TABLE I STUDIES BY 10-YEAR AGE GROUP I N HEALTHY I N H A B I T A N T S L I V I N G I N
CLEARANCE
N
Age
S- F ugll
U-F vglml
CCr
.
m! / m i n / l 48m2
CF
A RURAL AREA OF JAPAN (MEAN k SD) FF ug/mi n / l .48m
TY
%TRF (%)
~~
~
35-39
78
12.0527.61
0.71k0.48
99.54k24.59
47.69t38.68
1.01k0.89
0.62k0.87
43.02k40.95
40-49
248
13.09k8.18
0.69k0.43
96.30k32.27
46.63t33.28
1.13k0.90
0.66k0.91
41.15k47.98
50-59
403
15.0821 1.92
0.84kO. 56
84.74254.50
39.70k42.56
1.24t1.22
0.70t1.19
31.57156.09
60-69
249
15.09t9.26
0.78tO. 57
81.34t53.07
37.30t31.51
1.22k1.35
0.81k1.31
45.05t49.61
70-79
97
15.1529.63
0.79k0.58
65.00k31.09
31.71k22.02
1.04k1.10
0.65k0.89
44.92k40.81
80-89
13
15.00+7.08
0.79t0.28
53.68t25.00
30.33k20.78
0.96k0.87
0.53t0.64
34.97t30.77
14.42t10.10
0.78k0.53
84.57k46.74
43.30k35.92
1.18k1.15
0.71k1.12
38.74k51.33
T o t a l 1088 or a v e .
411
TABLE I 1
SERUM AND URINARY FLUORIDE, AND FLUORIDE CLEARANCE STUDIES IN THREE GROUPS
35-59 60-79 80-89
729 346 13
14.07k10.45 15.10k9.38 15.00k7.08
0.78k0.52 0.78k0.57 0.79k0.28
91.90k45.51]] 77.01k48.76 53.68+25.00
“
42.45k39.301 35.53k29.41 30.33QO. 78
AGE
_L
”
“P<0.01
failure (CRF) and for 1 1 healthy controls following administration o f 4 mg of fluoride. The 24-hour urinary fluoride excretion and fluoride clearance were well correlated with each other (r=0.83) i n these subjects. I n the healthy SubJeCtS, about 50% of the ingested fluoride was excreted into the urine in a 24-hour period. The excretion rate decreased with the diminution of the fluoride clearance and when fluoride clearance decreased 20%. the 24-hour excretion of fluoride also diminished 20%.
Figure I. Cross-sectional differences in fluoride clearance with age. The number o f subjects in each group is indicated above the abscissa. Values plotted are means ?r SEM.
412
TABLE I 1 1 GLOMERULAR FLUORIDE F I L T R A T I O N , TUBULAR FLUORIDE REABSORPTION AND I T S RATE, AND PHENOLSULFONPHTHALEIN I N THREE AGE GROUPS Age
N
FF
35-59 60-79 80-89
729 346 13
1.19k1.09 1.17t1.29 0.96k0.87
2X
JRF
%TRF
0.68t1.07 0.77t1.22 0.53k0.64
35.96t52.28 45.02k48.871 34.97k30.77
PSP
(%I
pg/mi n/l .48m
% (15 min)
"
32.04k9.60 29.44t10.6I 21.37t7.51
]I7$
50
a,
Y = 0.94X-0.07 r = +0.83
Fluoride clearance ,ml/min/l
.48m2
Figure 2. Correlation between fluoride clearance and rate of urinary fluoride excretion in 24 hours.
DISCUSSION
t
Once renal function is impaired the excretion of fluoride i n the urine decreases (3). A previous report showed that the 24-hour excretion of fluoride in patients with chronic renal failure ( C R F ) was significantly lower (p
413 involution.
Among t h e f a c t o r s c o n t r i b u t i n g t o t h i s d e c r e a s e a r e r e d u c t i o n s
i n t h e r e n a l v a s c u l a r bed w h i c h can be a c c e l e r a t e d b y a r t e r i o s c l e r o s i s (10,14,15).
Creatinine clearance
i s simple.
r e q u i r e a d m i n i s t r a t i o n o f an exogenous s u b s t a n c e .
i n e x p e n s i v e and does n o t Thus,
the determination
o f g l o m e r u l a r f i l t r a t i o n r a t e (GFR) h a s b e e n f o u n d t o b e c l i n i c a l l y u s e f u l . D u r a t i o n o f u r i n e c o l l e c t i o n f o r t h e m e a s u r e m e n t o f C C r v a r i e s f r o m 1 t o 24 hours.
A c c o r d i n g t o Wesson ( 1 6 ) a 2 4 - h o u r c o l l e c t i o n seemed m o s t u s e f u l as
t h e e f f e c t o f d i u r n a l v a r i a t i o n i n GRF c o u l d be a v o i d e d .
However,
i t may
n o t b e p r a c t i c a b l e i n a s t u d y t h a t i n v o l v e s a l a r g e number o f i n d i v i d u a l s o r workers i n a plant.
A two-hour
clearance examination performed i n the
e a r l y morning. as done i n t h i s study, appears t o be s u i t a b l e f o r a r a p i d evaluation o f the renal function. The r e s u l t s o b t a i n e d i n t h i s s t u d y d e m o n s t r a t e a h i g h l y s i g n i f i c a n t ager e l a t e d d e c l i n e i n c r e a t i n i n e c l e a r a n c e and f l u o r i d e c l e a r a n c e (CF) h e a l t h y persons.
T h e f i n d i n g s o n CF,
a b o u t 50% o f C C r ,
agree w i t h t h e r e p o r t b y Waterhause
i n
which i n these subjects averaged
et
gil.. (6).
A
d e c l i n e i n C C r a n d C F among t h e d i f f e r e n t d e c a d e age g r o u p s was w e l l demonstrated
i n this
study.
By age 9 0 e a c h o f t h e s e f u n c t i o n s
has
d e c r e a s e d t o a v a l u e a p p r o x i m a t e l y 50 t o 6 0 % o f w h a t i t w a s a t t h e a g e o f
3 5 t o 39. FF,
TRF and %TRF w e r e a l s o m a r k e d l y d e c r e a s e d a f t e r age 80.
m e n t a l d a t a o f W h i t f o r d (17)
The e x p e r i -
s u g g e s t t h a t 35 t o 45% o f t h e f i l t e r e d
f l u o r i d e was r e a b s o r b e d i n t h e p r o x i m a l t u b u l e ,
and t h e r e a b s o r p t i o n was
i n v e r s e l y r e l a t e d t o t h e pH v a l u e o f t h e t u b u l a r f l u i d .
I t seems t h a t
a b o u t 38% o f t h e f i l t e r e d f l u o r i d e i s r e a b s o r b e d i n h e a l t h y s u b J e c t s . As shown i n t h i s s t u d y ,
w i t h CF.
t h e 24-hour
fluoride
When C F f e l l b e l o w 10 m l / m i n / 1 . 4 8
f l u o r i d e markedly decreased (Fig.
2).
m’,
excretion correlated well t h e 24-hour e x c r e t i o n o f
T h i s i n d l c a t e s t h a t t h e 24-hour
e x c r e t i o n o f f l u o r i d e i s d e p e n d e n t o n CF. The r e s u l t s o b t a i n e d i n t h i s s t u d y s u g g e s t t h a t r e n a l f l u o r i d e c l e a r a n c e decreases n o t o n l y w i t h t h e evidence o f kidney disease b u t a l s o w i t h a d v a n c i n g age.
Therefore,
f o r t h e h e a l t h c a r e o f workers exposed t o f l u o -
r i d e and o f p o p u l a t i o n s l i v i n g i n f l u o r i d e p o l l u t e d a r e a s , age may be an i m p o r t a n t f a c t o r t o be considered. REFERENCES
1.
E k s t r a n d J ( 1 9 7 8 ) C a r i e s Res 1 2 1 2 3 - 1 2 7
2.
S p e n c e r H, L e w i n I , W i a t r o s k i E. S a m o c h s o n J ( 1 9 7 0 ) Am J M e d 4 9 : 8 0 7 813
3.
W h i t f o r d G M , P a s h l e y DH ( 1 9 7 9 ) I n : J o h a n s e n E, T a v e s DR, O l s e n TO ( e d s ) C o n t i n u i n g E v a l u a t i o n o f t h e Use o f F l u o r i d e s . W e s t v i e w P r e s s I n c , B o u l d e r , p p 187-221
414
4.
Kono K, Yoshida Y, 22: 33-40
5.
Macheshwar VS, B r u n e t t i AJ, L e i b i n L, Newbrun E, Hodge HC (1981) A m J C1 i N u t r 34:2679-2684
Watanabe M, Tanimura Y, H i r o t a T (1984) Ind Health
6.
Waterhause C, Taves D. Munzer A (1980) C l i n S c i 58:145-152
7.
S i d d i q u i JY, S i m p s o n SW, E l l i s H E , K e r r DMS, A p p l e t o n O R , R o b i n s o n BH, Hawkins JB, Robertson PW, Taves OR (1971) Proc E u r D i a l y s i s Transp l a n t Ass 8:149-159
8.
Sreepada Rao JK, Friedman EA (1975) Kidney I n t 7:125-129
9.
T a v e s D R , F r e e m a n RB, Kamm DB, Ramos SP, S c r i b n e r BH ( 1 9 6 8 ) T r a n s A m Soc A r t i f Intern Organs 14:412-414
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F r i e d m a n SA, R a i z n e r A E , Rosen H, Solomon N A , Sy W ( 1 9 7 2 ) A n n I n t e r n Med 76:41-45
11.
Lewis WH, Alving AS (1938) A m J P h y s i o l 123:500-515
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Rowe JW, A n d r e s R , T o b i n JD. N o r r i s A H , Shock N W ( 1 9 7 6 ) 31 155-163
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Y o s h i d a Y , T o y o t a S , Kono K, W a t a n a b e M , I w a s a k i K ( 1 9 7 8 ) B u l l Osaka Med School 24:56-67 Sworn MJ, Fox M (1972) Brit 3 Uro 44:377-383
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Wardener HE (1985) In: Wardener H E ( e d ) T h e Kidney. C h u r c h i l l L i v i n g s t o n e , Edinburgh, pp 128-131
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P h y s i o l o g y o f t h e Human
415
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 415-422 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
KINETICS OF FLUORIDE EXCRETION IN HUMAN SALIVA
YASUHISA Y O S H I D A ~ , K O I C H I K O N O ~ . M I S U Z U W A T A N A B E ~ . S U M I E I N O U E ~ . T O S H I Y U K U HIROTAl. MICHIZO MURAOl, AND
HIDEHIRO NAGAIE3
'Department o f Hygiene and P u b l i c Health, Osaka Medical College, T a k a t s u k i . O s a k a , 569. J a p a n 'Department of Environmental Health, F u k u i Medical School, Japan 3Department of P h y s i c a l Education. Osaka Dental U n i v e r s i t y , Japan
ABSTRACT The f l u o r i d e found i n s a l i v a may depend l a r g e l y on serum f l u o r i d e c o n t e n t w h i c h i s a f f e c t e d m a i n l y by t h e d i e t and by t h e k i d n e y f u n c t i o n o f t h e individual.
The p r e s e n t i n v e s t i g a t i o n was concerned w i t h f l u o r i d e c o n t e n t s
of t h e whole and p a r o t i d s a l i v a from r e s i d e n t s of a n o n - f l u o r i d a t e d
com-
munity and from p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e (CRF), whose a b i l i t y t o e x c r e t e f l u o r i d e was markedly decreased. Whole and p a r o t i d s a l i v a and serum s a m p l e s were o b t a i n e d f r o m e i g h t h y d r o f l u o r i c a c i d (HF) p l a n t w o r k e r s ,
f i v e p a t i e n t s w i t h CRF,
h e a l t h y c o n t r o l s . The s a m p l e s were t a k e n a t 6:OD a.m. and t h i s was f o l l o w e d by t h e r e s p e c t i v e f l u o r i d e dose.
and 40
in t h e f a s t i n g s t a t e , S a l i v a r y and serum
s a m p l e s were c o l l e c t e d a t f i x e d i n t e r v a l s o v e r a 48-hour period. I n t h e h e a l t h y s u b j e c t s , a h i g h d e g r e e o f c o r r e l a t i o n ( r = + 0 . 9 9 ) was o b t a i n e d between serum and s a l i v a r y f l u o r i d e l e v e l s .
Changes i n f l u o r i d e
c o n t e n t of t h e whole and p a r o t i d s a l i v a a f t e r f l u o r i d e i n t a k e were s i m i l a r t o t h o s e of t h e s e r a .
The s a l i v a r y f l u o r i d e c o n t e n t of p a t i e n t s w i t h C R F
was t h r e e t o s i x t i m e s h i g h e r t h a n t h a t of h e a l t h y s u b j e c t s a t any t i m e of t h e day. In HF p l a n t workers, p o s t - s h i f t s a l i v a r y f l u o r i d e c o n t e n t was f i v e t o 50 t i m e s h i g h e r t h a n t h a t i n s e r a , p o s s i b l y d u e t o o r a l c o n t a m i n a t i o n from t h e work environment. T h e s e r e s u l t s s u g g e s t t h a t f l u o r i d e c o n t e n t s o f t h e w h o l e and p a r o t i d s a l i v a r e f l e c t e d t h e body b u r d e n o f f l u o r i d e
i n
non-fluoridated healthy
subjects.
INTRODUCTION S e v e r a l i n v e s t i g a t o r s (1-4) have d e t e r m i n e d human s a l i v a r y f l u o r i d e cont e n t s and observed t h a t t h e v a l u e s w e l l r e f l e c t t h e serum f l u o r i d e concen-
416
trations. On the other hand, a correlation exists between serum fluoride levels and the body burden o f fluoride and its excretion. T h e s e characteristics are utilized in biological monitoring of fluoride among indusDetermination of fluoride level in the serum makes it trial workers (5). possible to diagnose certain diseases, such as chronic renal failure (6). In the present investigation, the kinetics o f fluoride excretion in human saliva has been studied.
MATERIALS AND METHODS Forty healthy male and female subjects, eight workers from a hydrofluoric acid (HF) manufacturing plant, and five patients with chronic renal failure, were employed in this study. The patients with chronic renal failure s h o w e d over 5 mg/dl creatinine and over 50 mg/dl BUN. S o m e o f these patients w e r e using artificial dialysis. T h e H F plant workers' routine work involved washing TV picture tubes at in electronics department. The
HF concentration in the working environment was about 3 ppm. Salivary specimens were obtained by stimulation of the top of the subject's tongue w i t h l e m o n drops. T h e fluoride concentrations in parotid saliva, obtained by use o f Curby's plastics cap ( 3 ) with negative pressure, and in mixed saliva, naturally obtained in the oral cavity. w e r e determined. O n e ml o f the saliva s a m p l e w a s centrifuged at 1,000 xg f o r 15 min.
A
0.5 ml aliquot of the supernatant was then used for fluoride determination. The determination was carried out with a microprocessor ion analyzer, Model
901, and total ion strength a d j u s t m e n t buffer T y p e 1 1 1 , o f the U.S. Orion Research Company (7). For the determination of fluoride concentrations i n saliva and serum, s p e c i m e n s w e r e taken at 6:OO a.m. f r o m subjects in the fasting state. After a certain amount of fluoride dosing, specimens were collected at fixed intervals over a period of 48 hours. Portions o f serum and saliva s p e c i m e n s w e r e also used f o r the determination o f calcium, sodium and chloride ions.
RESULTS The correlation between salivary fluoride and serum fluoride levels of adult subjects is shown in Figure 1. Although the salivary specimens were whole saliva, the correlation coefficient between serum fluoride and salivary fluoride was as high as 0.99. Figure 2 s h o w s a survey o f salivary and s e r u m fluoride levels f o r 24 hours following the intake of 5 mg of fluoride by healthy adult subjects.
417
300 3
r
E
c ci
h
m i
2
-?-Ze 0
100 200 S a l i v a r y fluoride, n g / m l
300
n
0
2
4
6
6 1 0 1 2
Time, hr
Figure 1. Correlation between salivary fluoride and serum fluoride levels in human subjects. Figure 2. 5 mg F.
Salivary fluoride and serum fluoride levels following intake of
The serum fluoride level rapidly increased 30 min after fluoride intake and then gradually decreased. It is seen that changes in salivary fluoride level paralleled those of serum fluoride. The correlation between mixed and parotid salivary fluoride levels following the intake of 5 mg fluoride by a healthy subject is shown in Figure 3. The mixed salivary fluoride level changed i n parallel with the parotid saliva fluoride level. It should be pointed out that the parotid saliva fluoride level was always slightly lower than the mixed salivary fluoride level. Figure 4 shows the response of mixed salivary fluoride levels when the intake of fluoride was increased form 3 to 10 mg. A dose-response is clearly shown. Similar results were obtained when the number of subjects was increased to five. Thus, the salivary fluoride level coincided well with that of the serum. Similar results to those of parotid saliva were obtained with the mixed saliva. Calcium, sodium, and chloride ions i n the serum and saliva showed a nearly constant diurnal variation with time. As shown i n Table I . no correlation was observed between fluoride ions and calcium ions. Table I 1 shows the correlation between the salivary fluoride and serum fluoride levels in two patients with chronic renal failure. Changes in the fluoride levels up to 48 hours after the intake of 5 mg fluoride are shown.
418
0
1
2
4
6
Time, h r
Mixed and p a r o t i d s a l i v a r y f l u o r i d e l e v e l s i n a s u b j e c t f o l l o w F i g u r e 3. i n g i n t a k e o f 5 mg F.
T i m e , hr
Response o f mixed s a l i v a r y f l u o r i d e l e v e l s f o l l o w i n g i n t a k e o f F i g u r e 4. v a r i o u s amounts o f f l u o r i d e .
The m i x e d s a l i v a r y f l u o r i d e l e v e l s i n t h e s e p a t i e n t s were t w o t o f o u r t i m e s h i g h e r t h a n t h o s e o f serum f l u o r i d e . T a b l e 111 shows a d i u r n a l v a r i a t i o n o f s a l i v a r y f l u o r i d e l e v e l s i n t h e patients w i t h chronic renal failure.
T h e i r f l u o r i d e l e v e l s w e r e much
h i g h e r t h a n t h o s e o f t h e c o n t r o l group.
Therefore,
i t can be s a i d t h a t t h e
mixed s a l i v a r y f l u o r i d e l e v e l r e f l e c t s t h e s t a t u s o f t h e k i d n e y f u n c t i o n and t h a t i t i s p o s s i b l e t h a t t h e v a l u e s may be u t i l i z e d f o r t h e d i a g n o s i s o f chronic renal failure.
419 TABLE I CHANGES I N SERUM AND SALIVARY F LEVELS I N NORMAL SUBJECTS (N=5) FOLLOWING INTAKE OF 5 'MG F
Time a f t e r F intake (hr)
Serum
Mixed s a l i v a
F ng/ml
T-Ca m E q / l
F ng/ml
T-Ca mEq/l
0
16.0t3.6
4.8420.22
17.7k2. 52
2.3821.07
0.5
236.625.8
4.78t0.05
223.3k4.77
2.85t0.58
1
184.3k4.0
4.68tO. 15
171.7k18.9
2.64t1.20
2
123.3k10
4.27tO. 37
106.0k16.4
2.3321.17
4
72.7k11
3.87k0.66
66.3k7.51
2.31k0.82
12
41.6k5.6
4.77t0.27
40.723.06
2.19k1.57
TABLE I 1 CHANGES I N SERUM AND SALIVARY F LEVELS I N PATIENTS WITH CHRONIC RENAL FAILURE (CRF) FOLLOWING INTAKE OF 5 MG F Time a f t e r F intake (hr)
Fluoride level (vg/l) Patient B Serum Saliva
Patient A Serum Saliva 54
70
40
120
0.5
0
210
244
230
440
4
120
180
120
260
12
90
110
70
145
24
60
80
44
160
48
50
60
50
160
TABLE I 1 1 DIURNAL VARIATION O F SALIVARY F LEVELS I N PATIENTS WITH C H R O N I C RENAL FAILURE ( C R F ) F l u o r i d e l e v e l , ng/ml SubJ e c t
p.m.
a.m.
6
8
10
12
2
4
6
8
10
12
925
17k10
16k9
18k10
17k9
13k6
15k10
2Ot9
1326
12+6
A (dialysis)
86
150
-
105
250
86
80
50
70
-
B (dialysis)
-
180
-
82
C
153 42
72 -
-
-
120 38
132
-
-
C o n t r o l (n=9) Patient
82
-
-
-
420 Among w o r k e r s o f t h e HF m a n u f a c t u r i n g p l a n t , p o s t - s h i f t
salivary fluoride
c o n c e n t r a t i o n s w e r e a l w a y s 5 t o 50 t i m e s h i g h e r t h a n t h e v a l u e s i n serum.
i t w o u l d b e r e a s o n a b l e t o assume t h a t s u c h h i g h v a l u e s w e r e due t o
However,
o r a l contamination o f f l u o r i d e f r o m t h e i r working environment.
DISCUSSION A p r o p e r e v a l u a t i o n o f t h e body burden o f f l u o r i d e , s o u r c e o f burden,
regardless o f the
as a means o f p r e v e n t i n g f l u o r i d e p o i s o n i n g has been
r e c o g n i z e d t o be i n d i s p e n s a b l e .
The m a j o r i t y o f t h e f l u o r i d e t a k e n i n t o
t h e body o r a l l y o r t h r o u g h t h e a i r passage i s e x c r e t e d i n t h e u r i n e w i t h i n hours.
The l e v e l o f u r i n a r y f l u o r i d e has, t h e r e f o r e , been c o n s i d e r e d a
good i n d i c a t o r o f t h e b o d y b u r d e n o f f l u o r i d e . As a s p e c i a l case,
m e a s u r e m e n t o f f l u o r i d e c o n c e n t r a t i o n i n t h e b l o o d has
been c o n s i d e r e d i m p o r t a n t , r i d e clearance
as c o m p a r e d t o t h a t i n u r i n e .
o l d o r patients w i t h chronic renal failure. excretion,
First,
low fluo-
i s o f t e n o b s e r v e d b e c a u s e o f r e n a l h y p o f u n c t i o n among t h e
fluoride concentrations
Due t o d i s t u r b a n c e o f f l u o r i d e
i n t h e b l o o d a r e r e l a t i v e l y h i g h when
compared w i t h t h e amount o f f l u o r i d e d i s c h a r g e d i n t o t h e u r i n e .
Accumula-
t i o n o f f l u o r i d e i n t h e b l o o d can cause f l u o r i d e poisoning. Second, s t u d i e s c o n d u c t e d i n t h i s l a b o r a t o r y (8) s h o w e d t h a t a l a r g e amount o f f l u o r i d e moved i n t o t h e b l o o d f r o m t h e i n j u r e d s k i n o f victims.
I t was p o i n t e d o u t t h a t ,
i n t h i s case,
HF
burn
through reaction with
f l u o r i d e , b l o o d c a l c i u m l e v e l was m a r k e d l y l o w e r e d , p o s s i b l y l e a d i n g t o death.
Clinically.
i t i s n e c e s s a r y t o m o n i t o r t h e b l o o d f l u o r i d e and
c a l c i u m c o n c e n t r a t i o n s and t o a d m i n i s t e r c a l c i u m b y i n t r a v e n o u s i n j e c t i o n i m m e d i a t e l y and r e p e a t e d l y ,
t o m a i n t a i n t h e necessary l e v e l o f calcium t o
save t h e l i f e o f t h e p a t i e n t . I n r o u t i n e m e d i c a l e x a m i n a t i o n o f i n d u s t r i a l HF w o r k e r s and i n t h e t r e a t m e n t o f HF b u r n s , a n a l y s i s o f b l o o d f l u o r i d e i s n e c e s s a r y a n d e f f e c t i v e . However, r e p e a t e d s a m p l i n g o f t h e b l o o d i s o f t e n p a i n f u l t o t h e s u b j e c t .
I f s a l i v a does r e f l e c t t h e f l u o r i d e c o n c e n t r a t i o n i n t h e blood,
i t may be
possible t o s u b s t i t u t e t h i s f o r blood sampling i n b i o l o g i c a l monitoring. S a l i v a i s n o t rnerejy f i l t e r e d blood, s a l i v a r y gland.
Consequently,
b u t i s an e x t e r n a l s e c r e t i o n b y t h e
J e n k i n s (3) n o t e d t h a t g e n e r a l l y t h e r e was
no c o r r e l a t i o n b e t w e e n s e r u m and s a l i v a i n t h e c o n c e n t r a t i o n s o f c a l c i u m , sodium, al.
chloride,
and o t h e r i o n s .
a l l y s i m i l a r t o t h a t i n t h e serum. that,
However,
E r i c s s o n ( 1 ) and H e n s c h l e r
(2) r e p o r t e d t h a t f l u o r i d e c o n c e n t r a t i o n i n t h e s a l i v a was e x c e p t i o n u n l i k e o t h e r ions,
Presumably,
t h i s i s due t o t h e f a c t
f l u o r i d e i o n s a r e n o t re-absorbed b y t h e e x c r e t o r y
d u c t o f t h e s a l i v a r y gland.
421 At first,
i n healthy a d u l t subjects,
t h e c o r r e l a t i o n between mixed s a l i -
v a r y a n d s e r u m f l u o r i d e w a s c a l c u l a t e d a s y = 1 . 0 4 ~+ 1.06,
and r = t0.99.
T h i s c o e f f i c i e n t i s a p p r o x i m a t e l y t h e same as t h a t r e p o r t e d by E k s t r a n d ( 9 ) w i t h p a r o t i d s a l i v a (r =
+0.99) a n d t h a t o f P a t z e t a l . ( r
The t e s t was f o l l o w e d b y a 5 mg f l u o r i d e b u r d e n t e s t .
= +0.98)(10).
I n t h i s t e s t i t was
observed t h a t t h e f l u o r i d e c o n c e n t r a t i o n i n t h e mixed s a l i v a c l o s e l y f o l l o w e d t h a t i n t h e serum w i t h o u t a t i m e lag. I n t h e i r f l u o r i d e b u r d e n t e s t s , E k s t r a n d g J g l . ( l l ) a n d H e n s c h l e r gJ g l . (2)
h a v e shown a d o s e - r e s p o n s e
i n t h e serum.
relationship i n the fluoride concentration
S i m i l a r r e s u l t s were obtained i n t h i s study o f f l u o r i d e
c o n c e n t r a t i o n s i n t h e m i x e d s a l i v a i n r e s p o n s e t o t h e a d m i n i s t r a t i o n o f 3,
5. a n d 1 0 mg o f f l u o r i d e ( F i g . 4). E k s t r a n d ( 9 ) and P a t z ng/ml
aJ.(lO)
r e p o r t e d f l u o r i d e c o n c e n t r a t i o n s o f 9-15
i n t h e p a r o t i d s a l i v a and 20-30
ng/ml i n t h e m i x e d s a l i v a . They a l s o
s t a t e d t h a t t h e p a r o t i d s a l i v a s e r v e d a s an i n d i c a t o r o f t h e f l u o r i d e c o n c e n t r a t i o n i n t h e serum.
Yao and G r d h ( 1 2 ) o b s e r v e d t h a t t h e f l u o r i d e
concentration i n the centrifugally-separated
m i x e d s a l i v a was s i m i l a r t o
t h a t i n t h e p a r o t i d s a l i v a . The c o r r e l a t i o n b e t w e e n f l u o r i d e c o n c e n t r a t i o n s i n t h e m i x e d s a l i v a and t h e p a r o t i d s a l i v a shown i n t h i s s t u d y s u p p o r t t h e
above-mentioned observations. The r e s u l t s o f t h i s s t u d y show t h a t t h e m i x e d s a l i v a , obtainable,
which i s e a s i l y
s e r v e s a s a good d i r e c t i n d i c a t o r o f t h e f l u o r i d e c o n c e n t r a t i o n
i n t h e s e r u m w i t h o u t a t i m e lag.
On t h e o t h e r hand,
with patients suffering
c h r o n i c r e n a l f a i l u r e a n d w o r k e r s i n HF m a n u f a c t u r i n g p l a n t s , d i f f e r e n t r e s u l t s were o b t a i n e d compared w i t h t h o s e f r o m h e a l t h y s u b j e c t s .
Fluoride
concentrations i n t h e mixed s a l i v a o f these p a t i e n t s were always two t o four
t i m e s h i g h e r t h a n t h e c o r r e s p o n d i n g v a l u e s i n t h e serum.
The
i n c r e a s e d f l u o r i d e c o n c e n t r a t i o n i n t h e m i x e d s a l i v a may b e d u e t o an enhanced s e l e c t i v e s e c r e t i o n o f t h e s a l i v a r y g l a n d i n r e s p o n s e t o t h e h i g h serum f l u o r i d e l e v e l , o r due t o an e x t r e m e l y l o w w a t e r i n t a k e because o f r e n a l insufficiency. The d i u r n a l v a r i a t i o n o f t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e s a l i v a o f t h e h e a l t h y s u b j e c t s was 9 t o 20 n g / m l on t h e average.
T h i s was i n f l u e n c e d t o
some e x t e n t b y t h e i n t a k e o f f o o d s a n d t e a c o n t a i n i n g f l u o r i d e . p a t i e n t s s u f f e r i n g c h r o n i c r e n a l fa1 lure, diets,
In
i n spite o f s t r i c t l y controlled
f l u o r i d e c o n c e n t r a t i o n i n t h e s a l i v a o c c a s i o n a l l y reached as h i g h as
ten times that o f the controls.
This suggests a high concentration o f
f l u o r i d e i n t h e blood o f these patients. W i t h i n d u s t r i a l HF w o r k e r s ,
because o f d i r e c t c o n t a m i n a t i o n i n t h e o r a l
c a v i t y by f l u o r i d e i n t h e working environment. c o n c e n t r a t i o n u s i n g s a l i v a may be e r r o n e o u s .
e s t i m a t i o n o f serum f l u o r i d e However,
w i t h acute f l u o r i d e
422 p o i s o n i n g s u c h a s i n HF b u r n s ,
s a l i v a r y f l u o r i d e l e v e l s e r v e s a s a good
i n d i c a t o r o f serum f l u o r i d e c o n c e n t r a t i o n . CONCLUSION S a l i v a r y f l u o r i d e c o n c e n t r a t i o n s w e r e c o m p a r e d w i t h s e r u m f l u o r i d e conc e n t r a t i o n s among h e a l t h y s u b j e c t s , w i t h chronic renal failure.
i n d u s t r i a l HF w o r k e r s , a n d p a t i e n t s
F o r t h e h e a l t h y s u b j e c t s ( N = 40). t h e r e l a -
t i o n s h i p between t h e f l u o r i d e c o n c e n t r a t i o n i n t h e mixed s a l i v a and t h a t i n t h e s e r u m c a n b e e x p r e s s e d w i t h y = 1 . 0 4 ~ + 1.06 r e g r e s s i o n e q u a t i o n and c o r r e l a t i o n c o e f f i c i e n t , A l s o i n t h e f l u o r i d e burden t e s t ,
=
+0.99,
as t h e
s a l i v a r y f l u o r i d e c o n c e n t r a t i o n s were
v e r y s i m i l a r t o t h o s e i n t h e serum. e a s i l y sampled,
and r
respectively.
Therefore,
t h e mixed saliva,
which i s
c a n be u s e d t o e s t i m a t e t h e f l u o r i d e c o n c e n t r a t i o n i n t h e
s e r u m and t o e v a l u a t e t h e f l u o r i d e b u r d e n o f an i n d i v i d u a l . S p e c i a l a t t e n t i o n s h o u l d be p a i d t o t h e f a c t t h a t w i t h p a t i e n t s s u f f e r i n g c h r o n i c r e n a l f a i l u r e , due t o t h e i r c o n c e n t r a t e d s a l i v a , and w i t h indust r i a l HF w o r k e r s i m m e d i a t e l y a f t e r t h e s h i f t , tamination i n the oral cavity.
d u e t o d i r e c t f l u o r i d e con-
a higher salivary fluoride concentration
t h a n t h a t i n t h e serum i s o f t e n obtained.
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I w a s a k i K, H i r o t a T.
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Shibuya
E k s t r a n d J ( 1 9 7 7 ) C a l c i f T i s s Res 23:225-228
10. P a t z VS.
H e n s c h l e r D,
11. E k s t r a n d J. A l v ’ s l G, 12: 31 1-317 12. Yao K,
W u r z b u r g HF (1977) D t s c h Z a h n a r z t l Z 32:482-486 B o r b s LO,
N o r l i n A (1977)
Grdh P (1970) C a r i e s Res 4:321-331
Europ J C l i n Pharmacol
423
H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 423-429 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
AN INTERNATIONAL COMPARISON OF TRACE AMOUNTS OF FLUORIDE CONTAINED I N HUMAN
HAIR YASUHIRO TAKAGI,
YOSHITERU OHKUBO, TSUNEO ASADA, AND SUSUMU MATSUDA
Fukui P r e f e c t u r a l I n s t i t u t e o f P u b l i c Health,
39-4,
Harame, F u k u i . J a p a n
ABSTRACT Fluoride concentrations countries,
i n t h e h a i r s a m p l e s o f 306 r e s i d e n t s f r o m f i v e
i n c l u d i n g Japan,
India,
U.S.A.,
Canada,
and P o l a n d w e r e
d e t e r m i n e d b y means o f t h e o x y g e n b o m b - f l u o r l d e i o n s e l e c t i v e e l e c t r o d e method. Samples f r o m I n d i a n r e s i d e n t s gave h i g h e r f l u o r i d e l e v e l s t h a n t h o s e f r o m other residents.
O n l y s a m p l e s f r o m I n d i a showed d i f f e r e n c e s w i t h sex,
m a l e h a i r samples gave h i g h e r v a l u e s t h a n f e m a l e samples.
and
The I n d i a n m a l e
s a m p l e s a l s o showed an i n c r e a s i n g l e v e l o f f l u o r i d e w i t h i n c r e a s e i n each age group. S m o k i n g and f i s h e a t i n g h a b i t s w e r e shown t o h a v e an e f f e c t on f l u o r i d e a c c u m u l a t i o n i n t h e h a i r , b u t t h e h a i r f l u o r i d e l e v e l was u n a f f e c t e d b y t h e p r a c t i c e o f h a i r d y i n g a n d p e r m a n e n t wave. Some c o r r e l a t i o n b e t w e e n f l u o r i d e and o t h e r m i n e r a l e l e m e n t s i n t h e h a i r samples were a l s o observed.
INTRODUCTION T r a c e e l e m e n t s i n t h e human b o d y h a v e b e e n r e c o g n i z e d as an i m p o r t a n t i n d e x o f e n v i r o n m e n t a l p o l l u t i o n i n r e c e n t y e a r s (1).
Use o f human h a i r has
a d v a n t a g e s i n t h a t t h e c o l l e c t i o n and t r a n s p o r t o f s a m p l e s a r e easy, and t h e c o n c e n t r a t i o n s o f v a r i o u s e l e m e n t s a r e h i g h compared t o t h o s e i n b l o o d and u r i n e .
For t h i s reason,
h a i r s a m p l e s have been used t o s t u d y t h e
i n f l u e n c e o f l o n g t e r m e x p o s u r e t o l o w c o n c e n t r a t i o n s o f p o l l u t a n t s (6.8). However,
l i t t l e i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g f l u o r i d e i n human h a i r .
I n v i e w o f t h e i n t e r e s t i n t h e d i s t r i b u t i o n o f t r a c e e l e m e n t s i n human hair,
an i n t e r n a t i o n a l s u r v e y was c o n d u c t e d t o s t u d y t h e i r d i s t r i b u t i o n i n
populations o f different origin.
H a i r s a m p l e s t a k e n f r o m U.S.A..
Canada,
P o l a n d , Japan, and I n d i a w e r e a n a l y z e d f o r f l u o r i d e c o n t e n t b y means o f t h e oxygen b o m b - f l u o r i d e i o n e l e c t r o d e method. study the correlation,
i f any,
An a t t e m p t w a s a l s o m a d e t o
between e l e m e n t a l c o n c e n t r a t i o n ,
age,
sex
424
and l i v i n g s t y l e o f t h e i n d i v i d u a l s i n a p a r t i c u l a r c o u n t r y .
Some o f t h e
f i n d i n g s a r e r e p o r t e d here. MATERIALS AND METHODS Sampling procedure The n u m b e r o f d o n o r s f r o m s e l e c t e d l o c a t i o n s i n N o r t h A m e r i c a ,
Europe,
a n d A s i a a n d t h e d i s t r i b u t i o n o f t h e s a m p l e s a r e s h o w n i n T a b l e I. -
the participants from India.
a n d U.S.A..
years.
Poland,
Canada,
f r o m 1 9 t o 80
The h a i r s a m p l e f r o m e a c h i n d i v i d u a l was p r o c u r e d b y n o r m a l c u t t i n g
and c o l l e c t e d w i t h c a r e t o a v o i d a n y e x t e r n a l c o n t a m i n a t i o n . a t e l y s e a l e d i n a c l e a n p l a s t i c bag. health,
and i m m e d i -
Each d o n o r was a s k e d t o c o m p l e t e a
p e r s o n a l q u e s t i o n n a i r e d e s c r i b i n g h , s / h e r age,
TABLE
The
70 y e a r s , w h i l e t h o s e o f
a g e s o f t h e J a p a n e s e p a r t i c i p a n t s r a n g e d f r o m 20
sex,
domicile.
general
o c c u p a t i o n and l i v i n g s t y l e .
I
OF
DISTRIBUTION
HAIR SAMPLES TAKEN FROM DIFFERENT POPULATIONS
Country
Location
Male
Fema 1e
Japan
Total
Fukui
29
44
73
India
New D e l h i Bombay
40
62
102
Canada
Mon c t on
33
20
53
U. S. A.
Scranton
21
22
43
P o l and
Poznan
22
13
35 ~
Total
145
161
306
A n a l y t i c a l method H a i r samples f r o m each i n d i v i d u a l were c u t i n t h e l a b o r a t o r y t o about 3 mm l e n g t h s .
again,
They
were washed w i t h acetone and w a t e r , and t h e n acetone
and d r i e d a t room t e m p e r a t u r e .
A p p r o x i m a t e l y 1.0 g o f t h e d r i e d
h a i r s a m p l e was p l a c e d i n t h e p l a t i n u m b o a t o f an o x y g e n bomb c o n t a i n i n g 10 m l 0.5N KOH a s a n a b s o r b e r .
T h e s a m p l e w a s b u r n e d i n t h e b o m b b y t h e m e t h o d o f T a k a g i gJ F o l l o w i n g t h e combustion, water.
a1.(7).
t h e bomb was s h a k e n v i g o r o u s l y and c o o l e d i n i c e
When t h e i n t e r n a l p r e s s u r e o f t h e bomb was r e s t o r e d t o t h a t o f t h e
atmosphere,
t h e c o n t e n t s o f t h e bomb w e r e washed t w i c e w i t h t h e a b s o r b e r
425 and t h e w a s h i n g s w e r e combined. a t 135-145°C
The c o m b i n e d w a s h i n g s w e r e s t e a m d i s t i l l e d
i n t h e presence o f p e r c h l o r i c a c i d - s i l v e r p e r c h l o r a t e u n t i l
t h e d i s t i l l a t e w a s 100 m l .
The i o n i c s t r e n g t h o f t h e d i s t i l l a t e was
a d j u s t e d w i t h t h e same v o l u m e o f TISAB ( s o d i u m c i t r a t e b u f f e r )
solution.
The f l u o r i d e c o n t e n t o f t h e m i x t u r e was t h e n d e t e r m i n e d b y t h e f l u o r i d e i o n s e l e c t i v e e l e c t r o d e method. The c o m p o s i t i o n o f d i f f e r e n t m i n e r a l e l e m e n t s o t h e r t h a n f l u o r i d e was determined by emission spectrum a n a l y s i s and a t o m i c a b s o r p t i o n s p e c t r o metry. RESULTS AND DISCUSSION R e s u l t s o f t h e a n a l y s i s showed t h a t t h e h i s t o g r a m o f f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r was c l o s e t o a n o r m a l d i s t r i b u t i o n .
A
c o m p a r i s o n o f t h e mean f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r s a m p l e s f r o m
e a c h o f t h e f i v e c o u n t r i e s a r e s h o w n i n F i g u r e 1. T h e h a i r s a m p l e s f r o m I n d i a n m a l e s w e r e f o u n d t o c o n t a i n a much h i g h e r f l u o r i d e l e v e l t h a n t h a t i n I n d i a n f e m a l e samples.
I n addition,
t h e f l u o r i d e c o n t e n t was s i g n i f i -
c a n t l y h i g h e r t h a n t h o s e o f samples f r o m o t h e r n a t i o n a l s r e g a r d l e s s o f sex ( ~ ~ 0 . 0 1 ) On . t h e o t h e r hand, t h e r e w e r e no d i f f e r e n c e s i n f l u o r i d e l e v e l s b e t w e e n m a l e and f e m a l e s a m p l e s f r o m o t h e r f o u r
countries.
The f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r s a m p l e s w e r e c o m p a r e d a c c o r d i n g t o d i f f e r e n t age g r o u p s .
The r e s u l t s showed t h a t I n d i a n m a l e s a m p l e s f o r
a l l age g r o u p s h a d h i g h e r f l u o r i d e l e v e l s t h a n t h o s e f r o m o t h e r c o u n t r i e s . a n d t h a t t h e d i f f e r e n c e s b e c a m e h i g h e r w i t h i n c r e a s e i n a g e ( F i g . 2). t h e o t h e r hand,
f e m a l e h a i r samples f r o m a l l f i v e c o u n t r i e s
3).
a n y age e f f e c t o n f l u o r i d e l e v e l s ( F i g .
-
I male
15! C
0
u
LL
0
T
I
female
Y
.,
.
W
-
.
Y
On
d i d n o t show
426
o INDIA rn JAPAN A U.S.A..CANADA POLAND
o, \ 0
1
-
I
/
F i g u r e 2. C o m p a r i s o n o f F c o n c e n t r a t i o n i n human ( m a l e ) h a i r a c c o r d i n g t o age groups.
female
Cn \
0
INDIA
rn J A P A N
07
2
U.S.A.,CANADA POLAND
I
C 0
''
21-30
31;LO
L1-SO Age
51-60
F i g u r e 3. C o m p a r i s o n o f F c o n c e n t r a t i o n i n human ( f e m a l e ) h a i r a c c o r d i n g t o age groups.
The e f f e c t o f s m o k i n g on f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r was s t u d i e d based on t h e r e s u l t s o f t h e q u e s t i o n n a i r e . a n d U.S.A. countries. females),
A s s h o w n i n F i g u r e 4,
smoking.
i n Canada,
U.S.A.,
and I n d i a (except
samples f r o m smokers had h i g h e r f l u o r i d e l e v e l s t h a n t h o s e o f
n o n - s m o k e r s (pi0.01). P 9 I 9 (5).
The d a t a o n s a m p l e s f r o m Canada
were pooled as no d i f f e r e n c e s were found between these two
But no d i f f e r e n c e s were observed w i t h t h e e x t e n t o f
I t h a s b e e n r e p o r t e d t h a t t h e f l u o r i d e c o n t e n t i n t o b a c c o i s 100
-
-
01
\,in.n
Smoking Hobit L_I1 YES
1 1
C
0 ._ c 13
L
I*
c
C
8
C
427
50
1
0
-
NO
T I
u
U
n L INDIA F i g u r e 4.
CANADA
E f f e c t o f smoking on F c o n c e n t r a t i o n s i n t h e h a i r .
E x a m i n a t i o n o f t h e e f f e c t o f f i s h e a t i n g on f l u o r i d e c o n t e n t s o f h a i r samples r e v e a l e d t h a t t h o s e who e a t l i t t l e f i s h had a h i g h e r f l u o r i d e l e v e l t h a n f i s h e a t e r s ( ~ ~ 0 . 0 5 ) . B u t among t h e f i s h e a t e r s ,
the frequency o f
f i s h e a t i n g was n o t f o u n d t o a f f e c t t h e f l u o r l d e l e v e l i n t h e h a i r ( F i g .
5).
I t i s c o n s i d e r e d t h a t t h e c o n t r i b u t i o n o f f i s h e a t i n g t o f l u o r i d e was
insignificant. The p r a c t i c e o f h a i r d y i n g and permanent wave was n o t found t o i n f l u e n c e f l u o r i d e l e v e l s i n t h e hair.
I t i s c o n s i d e r e d t h a t f l u o r i d e i n human h a i r i s r e l a t e d t o i n g e s t i o n o f f o o d and w a t e r c o n t a i n i n g i t .
I n areas where l a r g e q u a n t i t i e s o f
f l u o r i d e c o n t a i n i n g t e a (2) and g r a i n s (3) a r e consumed, zone,
t h e d a i l y f l u o r i d e i n t a k e may b e h i g h ,
Ib NO
F i g u r e 5.
YES
L -4
1-2
high-
and i n t h e t o r r i d
leading t o accumulation o f
5-6
E f f e c t o f f i s h e a t i n g on F c o n c e n t r a t i o n s i n t h e h a i r .
428
TABLE I 1 CORRELATION BETWEEN FLUORIDE AND OTHER MINERAL ELEMENTS I N HUMAN H A I R (MALE) Country
Element
Contents (vglg)
r
Japan
A1 Cr
16.8 0.13
0.448 0.277
India
A1
64.7 0.14 0.67 863.5
0.557 0.451 0.408 0.324
V
Cr Ca
U.S.A.
As Cd Pb
0.048 1.25 10.01
0.418 0.629 0.422
Canada
Sn Cd
co Na
Pb
0.294 0.494 8.34 0.041 15.9
0.474 0.393 0.499 0.383 0.379
Cr
0.45
0.479
P o l and -:+: >,
,,
P
p
. p
TABLE I 1 1 CORRELATION BETWEEN FLUORIDE AND OTHER MINERAL ELEMENTS I N HUMAN HAIR (FEMALE) Country
Element
Contents
r
P
0.519 0.320 0.353 0.308
c
Mg
0.976 10.8 425.3 318.6
India
A1 Cd cu Fe
32.4 0.228 12.1 36.0
0.497 0.372 0.338 0.415
U. S.A.
v
0.184
0.41 6
P o l and
Cd Pb Ni Zn
0.318 2.38 0.333 176.9
0.647 0.580 0.548 0.489
(11919)
Japan
Sb K Na
c ;${c
-:c
429 fluoride i n the hair. h a i r i s very limited,
The l i t e r a t u r e o n t h e e f f e c t o f t o b a c c o o n human b u t t o b a c c o c o n t a i n s a h i g h l e v e l o f f l u o r i d e (5.6).
So i t i s s u g g e s t e d t h a t s m o k i n g h a b i t s may i n f l u e n c e f l u o r i d e c o n c e n t r a -
t i o n s i n human h a i r . T a b l e s I 1 a n d 111 show t h e c o r r e l a t i o n b e t w e e n f l u o r i d e c o n t e n t s and t h o s e o f o t h e r e l e m e n t s i n t h e h a i r samples. common c o r r e l a t i o n was o b s e r v e d ( T a b l e 111), c o r r e l a t i o n s w e r e shown ( T a b l e 11).
W h i l e i n f e m a l e s a m p l e s no i n m a l e s a m p l e s a f e w common
E x a m p l e s a r e s a m p l e s f r o m I n d i a and
J a p a n w h e r e b o t h A1 a n d C r w e r e f o u n d t o b e c o r r e l a t e d . s a m p l e s f r o m U.S.A.
I n contrast, i n
a n d C a n a d a , c o r r e l a t i o n b e t w e e n F a n d Cd a n d Pb w a s
o b s e r v e d ( p < 0.01 ). This
p r e l i m i n a r y survey,
though n o t conclusive,
does s u g g e s t t h a i
f l u o r i d e c o n t e n t s i n h a i r c a n be u s e f u l i n s t u d y i n g t h e i m p a c t o f e n v i r o n m e n t a l a n d d i e t a r y f a c t o r s o n humans i n g e n e r a l . ACKNOWLEDGEMENT We d e e p l y t h a n k Prof.
P r o f . J. A.
Vinson ( U n i v e r s i t y o f Scranton,
M. C. M e h r a ( M o n c t o n U n i v e r s i t y ,
o f S o c i a l M e d i c i n e M e d i c a l Academy, I n s t i t u t e o f Technology, mented on t h e manuscript.
Canada),
Dr. A.
Poland) and Dr.
U.S.A.),
Kaniewski ( I n s t i t u t e B. K. P u r i ( I n d i a n
I n d i a ) who h e l p e d c o l l e c t h a i r s a m p l e s and comT h i s e x p e r i m e n t was,
i n part, supported by a
g r a n t f r o m Nissan Science Foundation.
REFERENCES 1.
F l y n n A ( 1 9 7 7 ) J A p p l N u t r 29:51-57
2.
H a r r i s o n M F (1949) B r i t J N u t r 3:162-167
3.
M c C l u r e F J (1949) Pub H e a l t h Rep 64:1061
4.
Okamoto T,
M a t s u h i s a T (1965) J Food Hyg SOC J a p a n 6(4):382-387
5.
Okamoto T,
M a t s u h i s a T (1967) J p n J Pub H e a l t h 14(3):123-126
6.
R e a v e s RD, 14: 579-584
J o l l y KW,
7.
T a k a g i Y. T a n a k a H, M a s u n a g a S ( 1 9 7 5 ) J Food H y g S O C J a p a n 1 6 ( 2 ) : 1 1 9 123
8.
T a k a g i Y ( 1 9 8 1 ) J p n J Hyg 36:824-829
B u c k l e y PD ( 1 9 7 5 ) B u l l E n v i r o n C o n t a r n T o x i c o l
431
AUTHOR
INDEX Hitomi, G . , 325 Honma, Y . , 1 6 5 H o r i u c h i , T., 395 Hwang, H . L . S . , 203
Abe, T . , 299 H s a d a , PI., 3 0 7 A s a d a , T., 4 2 3
Bagley, C . V . , B a n g , S.,
113
187, 341
Baud, C . A . , 187, 3 4 1 B e s s h o , Y., 73 B h a v s a r , B.S., 1 9 3 B i e r s t e k e r , K . , 135 B o l e i j , J.S.M., 135 Butcher, J.E., 113 C a l l , J.W., 1 1 3 C h a n d r a , S . , 181 C h i k u m a , M., 4 3 Cordonn i e r , J. A . ,
I i d a , M., 2 6 7 , 3 1 3 I i j i m a , Y., 3 6 9 Imai, T., 263 Inoue, Inoue, Ishii, Itai,
43 415 T., 379 K . , 25, 99 A.,
S.,
J a i n , S.K.,
155
231
Kanamoto, Y., 211 Kaneko, Y., 73 K a n t h a r i a , S.L.,
Uate, C.,
307
Demeurisse, C . , 187 Desai, V.K., 193 Uoi, K . ,
407
3, 1 5 ,
K a w a s a k i , T., 2 6 7 , Kohyama, Y . , 3 0 7
59 L e i , W.,
Hattori, T., Hayashi, M.,
277 307 415
Y.,
59 253
Ma, H . , 2 5 3 M a e h a s h i , H.,
15, 31
Heyndrickx, A.M., H i r o t a , T.,
Li,
407
H a r a g u c h i , H.,
313
Krishna, N., 357 K u r i h a r a , H., 307
G i a n n i n i , J . , 241 Gojo, K , , 263 Goto, S., 47 H a r a d a , A.,
401 369
Kondo, T . , 4 0 1 Kono, K . , 4 0 7 , 4 1 5
F r a n k e , J . , 335 Fujiwara, K., 59 F u r u t a n i , IY., 2 6 7 Fuwa, K . ,
Kasahara, K., Katayama, T.,
193
155
Masuoka, M . , M a t s u d a , S.,
277 299 423
Mehta, M.R., 193 MGndez, M . C . , 127 M i l l e r , G.W., 113, 241 Miyajima,
H.,
299
432
Morimoto,
M.,
K.,
Mui,
Murao,
395
307
347
Songpaisan,
415
M.,
D.P.,
Singh, Suga,
Murray,
F.,
91
Nagaie,
H.,
415
173
Y.,
285
S.,
Suketa,
Y.,
Susheela,
211 231
A.K.,
Nakagaki,
H.,
379
Takagi,
Y.,
423
Nakagawa,
T.,
43
Takeda,
Y.,
407
Nakane, Nakao, Nasu,
T.,
143
M.,
Y.,
M.,
K.,
263 257,
M.,
Nomura, Oh,
257
225,
Nishikawa,
263
299
Y.,
Ohkubo, Okada,
S.,
Okayasu, Oliveira,
Tanaka,
H.,
307
ranaka,
H.,
J.A.,
127
P.,
M.,
Teotia,
S.P.S.,
25,
Tsunoda,
K.,
15
Tsunoda,
N.,
107,
Van d e n H e e d e ,
N.V.,
Very,
J.M.,
357
Wada,
K.,
99,
389
Sato,
T.,
267,
313,
Sato,
T.,
257,
263
Schild, Sharma, Shupe,
R.P.,
J.L.,
325
211
A.L.,
113 113
107,
389 155
M.A.,
187
M.,
415
Yamagata,
H.,
407
Yamauchi,
T.,
165
Yanagisawa,
F.,
Yanaka,
81
T.,
Yoshida,
193 127
263
99,
285
Sakurai,
R.G.,
257,
H.,
Watanabe,
D.K.,
307
225,
Tsunoda,
127
Schamschula,
187
H.J.,
M.,
T.,
F.,
Y.,
155
!I., 73
Riet-Correa. S.,
347
J.M.,
357
K.,
Ramamohan Rao,
Saxena,
347
173
241
J.C.,
407
Teotia,
Tsuji,
113
Rajyalakshmi,
Satoh,
43 Y.,
Tanimura,
Tsuchida,
211 I., 307
Phantumvanit, Pushnik,
143
Tomita,
43
Y.,
A.E.,
Olson,
F.,
Tochon-Oanguy,
423
Okabayashi,
225
J.,
Tanaka,
Timperman,
43
K.,
285
Y.,
Takimoto,
395
I.,
Nishida, Niwa,
Taki,
401
173
Yu,
M.H.,
Zaima,
K., 47
O.,
415
313,
203
Y.,
Zenebon,
407,
Y.,
Yoshitake,
307
127
325
1 6 5 , 359
433
KEYWORD I N D E X (Page number r e f e r s t o t h e f i r s t
page o f t h e
A b s o r p t i o n mechanism,
Deciduous enamel,
Accumulation,
325
241
Defluoridation,
Acetylcholine sensitivity, 277 Active transport, A d r e n a l ec tomy, Aging,
attrition,
127
Dental
fluorosis,
165
synthesis,
Drinking water, 91,
107
99,
Airborne fluoride,
73,
15,
165
187 acid,
25
Echinocyte,
91
Electron-probe,
341
277
Electrothermal,
25
agents,
299
Electrothermal graphite furnace,
203
growth,
15
Emission factor,
Blood,
267
Enamel,
3
Biogeochemistry,
187,
CaF2,
389
47,
Enameloid,
415 73
211,
E r y t h r o c y t e membrane,
,
231
395
211
257 127
113
F i e l d work,
379
Fish teeth,
285
Fluoridation,
313
91,
99,
Fluoride, renal failure,
combustion, 3,
Cord serum,
225
415
127
Coated d i f f u s i o n tube, Colorimetry,
401
F-electrode,
113
C a t t l e and sheep,
Coal
127
3
285
Excretory rhythm,
Chronic f l u o r id e toxicosis, Chronic
379
Environmental problem,
341
C a l c iu m - f 1 u x Cattle,
99
285
Evolution,
CAMP,
143
Environmental pollution,
B l o o d serum, Bone,
325
43
Emission sources, Bacterial
intake,
231
Effect o f chloride,
Anti-cholinesterase,
Ascorbic
165
379
Ecosystem,
Anion-exchange resin,
Apatite,
59,
107
155
Anti-neoplastic
81
253
Duration o f fluoride
155
Aluminum m o n o f l u o r i d e , Aluminum p l a n t ,
357
Dental
DNA/RNA
products,
Air pollution,
369
0 i f f us i o n a1 t r a n s p o r t ,
325
211
407
Agricultural
Analysis,
a r t i c l e concerned.)
59
225,
241,
253,
155, 263,
F1 u o r i d e a b s o r p t i o n , 81
-,
analysis,
47,
415
-, c l e a r a n c e , 4 0 7
-,
concentration,
401
187, 285,
325,
341 389
434
-, c o n t e n t , 4 2 3
Human b o n e s ,
-, d e t e r m i n a t i o n , 1 5 , -, e x c r e t i o n , 1 0 7 -, i o n , 4 3 , 3 0 7 -, m e t a b o l i s m , 3 1 3 -, m o u t h r i n s e , 3 6 9 -, p o i s o n i n g , 1 2 7 -, t o x i c i t y , 2 3 1 -, u p t a k e , 3 6 9 -, w o r k e r , 4 0 7 , 4 1 5 F1 u o r i d e s , Fluorine,
155
Human f l u o r o s i s ,
59
Human h a i r ,
335
423
Human s u b j e c t s ,
389
107,
Hydrogen f l u o r i d e sampl i n g ,
81 H y p e r p a r a t h y r o i d ism, Hyperplasia,
307
Indicator plant, Industry,
267
347
99
193
Influencing factors,
73
Fluorine determination, Fluorosis,
181,
347,
Functional
resin,
I n t e r n a t i o n a l comparison,
31
357,
113 423
379 L a m i n a r and t u r b u l e n t f l o w s ,
43
81 Gas c h r o m a t o g r a p h i c d e t e c tion,
Lanthanum complex o f s u l f o n a t e d a1 i z a r i n - c o i n p l e x o n e ,
31
Gas c h r o m a t o g r a p h y , Gaseous f l u o r i d e ,
43
47
Litterfall,
143
Gastrointestinal
absorption,
Localization, Logarithmic
107 Glass f i b r e plant,
tion,
135
G 1 o m e r u 1 o n e p h r it is ,
313
Glucosyl transferase, G l y c o l y t i c enzyme,
267
91 341
normal d is t r i b u -
401
Long c a p i l l a r y c e l l , 47, Low-protein diet,
Low t e m p e r a t u r e a s h i n g ,
257
Lung f u n c t i o n , Lymphocyte,
Hard tissue,
313
Health care,
407,
plasma, HF,
135,
73
135
257
415
H e 1 i um m i c r o w a v e - i n d u c e d
Masugi's nephritis, M a t e r n a l serum,
31
HeLa c e l l s ,
59
203
313
225
Mesophyll protoplast,
263
MIC,
241
High r i s k department,
193
253
267
Microdiffusion,
73,
401
H i stodynamic,
347
Mice,
Histological,
335
Molecular absorption spectro-
H i stomorphometry,
Histopathologic radiographic Hokutolite,
3
347
203
metry,
15,
25
and rnicro-
, 299
NaF,
389
Natural
fluoride,
369
435
kutritional
357
supplements,
Smoking a n d f i s h e a t i n g ,
423
Sodium c a r b o n a t e , 8 1
Oral
administration, 389
Oral microorganisms,
267
Osteomalacia, 347
S o d i u m f l u o r i d e , 277 Sources, 113 395
Space food,
Species tolerance,
113
Parafoll icular c e l l s , 307
S p e c i f i c symptoms,
193
P a r t i c u l a t e f l u o r i d e , 143
Spectroscopy, 3
Pathologic-anatomical,
335
Spot samples of u r i n e , 401
Phosphatase, 257
Subcellular distribution, 241
P l a n t , 193 P 1 asnia e m i s s i o n s p e c t r o m e t r y ,
Symptoms,
31
193
THC-278,
299
29Y
Power s p e c t r u m , 3 9 5
Tegafur,
P r o s p e c t i v e s u r v e y , 99
Tnroughfal 1 , 9 1
77KD P r o t e i n , 2 6 3
T h y r o i d-par at h y r o i d e c t o m y ,
PTH,
211
211
Pyrohydrolysis f l u o r i n e det e r m i n a t i o n , 25
Thyroid g l a n d , 307 Tibia, 203 T i l e works,
143
Rat, 47
Total
Rat incisor, 299
Transmitter release, 277
Rats,
307
Tunnel
reflection, 59 kiln,
143
Recycling, 187 Heflux method, 3 2 5 Removal,
Unpolished r i c e , 9 9
187
Urinary f l u o r i d e excretion,
Renal f u n c t i o n , 4 0 7
181
Urinary f l u o r i d e , Salivary fluoride,
173,
415
173,
395
Urine, 135
School c h i l d r e n , 3 7 9 S e l e c t i v e c o l l e c t i o n , 43 SEM-f indings,
Serum g l u c o s e ,
Vegetables,
135
335 211
Serum a1 k a l i n e p h o s p h a t a s e , 203
S k e l e t a l muscle, 277
Water f l u o r i d e , 1 7 3 Water-Dorne f l u o r i d e , 1 6 5 Water s a m p l e s , 3 1