Fluoride research, 1985: selected papers from the 14th Conference of the International Society for Fluoride Research, Morioka, Japan, 12-15 June 1985

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

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

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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 (ReO. 05

O. 05

/

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 , pstGxylin-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 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

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Ln

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c

0 c

W

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4

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0

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aJ

c

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a

.-c Q r 8

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W v) 3

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m 0

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n 0

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m

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+ W + 0

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r

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c, m

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*0 0 CL

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r' r

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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

-

N N

Lc m

0

0 W N

+I

N

m c

W

c

m

N

W

d

T

0

W

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

h

0

v, +I

a,

m

K

E

E

v

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

m

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E 0 N

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1 0 c

E 1 In

E

-a

K (5,

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d

c

+I

In

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d

0

m +I

a, c

+I

W

0

m +I

rd

W +I

a

c

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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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rd +I N

+I

m

+I W

m W

c

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0

0

m

r-

N 0 N

r+I

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7

d +I

7

0

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c

r-

m 0 c

d

0

.r

N

+I

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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

+I

N c

d

c c

m

a m ,

m

m

W In

+I

a

-J

+I

r

m

c

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m mr m

0 m

m

c

N

c

m

m N

m m

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a

0

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In In

00 c

W

m

+I

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0

m

% d

m

r--

c

d

+I

c

0

m

m

m

+I

c\I

d

+I

0

m

m

a c

r-

0

m

+I In

W

m 0

m

W

c

c

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a a

m

E

In

a

r\l

a E

a

c

Q

m

7

E

r-

m

Q

c

In

c

0

c

a m

E

0

0

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0

c,

c

0

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

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