Chlorodioxins: Origin and Fate (Advances in Chemistry Series : No 120)

Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.fw001 Chlorodioxins—Origin and Fate Pub...

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Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.fw001

Chlorodioxins—Origin and Fate


Chlorodioxins—Origin and Fate


Etcyl H. Blair, Editor

A symposium sponsored by the Division of Pesticide Chemistry at the 162nd Meeting of the American Chemical Society, Washington,D.C., Sept. 16-17, 1971.

ADVANCES IN CHEMISTRY SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1973

120


ADCSAJ 120 1-140 (1973)

Copyright © 1973 American Chemical Society All Rights Reserved

Library of Congress Catalog Card 73-84139 ISBN 8412-0181-1 PRINTED IN THE UNITED STATES OF AMERICA

Advances in Chemistry Series


Robert F. Gould, Editor

Advisory Board Bernard D. Blaustein Paul N. Craig Ellis K. Fields Louis Lykken Egon

Matijević

Thomas J. Murphy Robert W. Parry Aaron A. Rosen Charles N. Satterfield


FOREWORD ADVANCES

IN

CHEMISTRY

SERIES

w a s f o u n d e d i n 1949

b y the

A m e r i c a n C h e m i c a l S o c i e t y as a n outlet for s y m p o s i a a n d c o l lections of d a t a i n s p e c i a l areas of t o p i c a l interest that c o u l d not be a c c o m m o d a t e d i n the Society's journals. m e d i u m f o r s y m p o s i a that w o u l d o t h e r w i s e be

It p r o v i d e s a fragmented,

their papers d i s t r i b u t e d a m o n g several journals or not p u b l i s h e d at a l l . P a p e r s are r e f e r r e d c r i t i c a l l y a c c o r d i n g to A C S e d i t o r i a l standards a n d receive the c a r e f u l a t t e n t i o n a n d p r o c essing characteristic of A C S p u b l i c a t i o n s . in

ADVANCES IN

CHEMISTRY

SERIES

Papers p u b l i s h e d

are o r i g i n a l c o n t r i b u t i o n s

not p u b l i s h e d elsewhere i n w h o l e or major p a r t a n d i n c l u d e reports of research as w e l l as r e v i e w s since s y m p o s i a m a y e m b r a c e b o t h types of presentation.

PREFACE ' h l o r i n a t e d d i b e n z o - p - d i o x i n s l o n g h a v e b e e n r e c o g n i z e d as p o s s i b l e ^

b y - p r o d u c t s i n m a n u f a c t u r i n g c e r t a i n c h l o r i n a t e d p h e n o l s ( 1 ).

Cur

Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.pr001

r e n t interest i n c h l o r i n a t e d d i b e n z o - p - d i o x i n s has o r i g i n a t e d b e c a u s e t h e h i g h l y toxic 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n

appeared i n trace

ments i n some samples of the h e r b i c i d e 2 , 4 , 5 - t r i c h l o r o p h e n o x y a c e t i c

ele acid

(2,4,5-T). T h e i n i t i a l c o n c e r n for the p o s s i b l e h a z a r d to h u m a n s exposed to 2,4,5-T w a s p r e c i p i t a t e d b y t e r a t o l o g i c studies c o n d u c t e d b y

Bionetics

R e s e a r c h Institute u n d e r contract f r o m T h e N a t i o n a l C a n c e r

Institute

(2).

I n these studies, large doses of 2,4,5-T w e r e a d m i n i s t e r e d to p r e g

n a n t rats a n d m i c e f o r n i n e of the 21 days of p r e g n a n c y .

T h e incidence

of f e t a l a b n o r m a l i t i e s w a s s l i g h t l y h i g h e r i n the treated a n i m a l s t h a n i n c o n t r o l animals.

L a t e r tests i n d i c a t e d that these a b n o r m a l i t i e s

p a l a t e ) m a y h a v e b e e n c a u s e d b y 27 ±

(cleft

8 p p m of 2,3,7,8-tetrachloro-

d i b e n z o - p - d i o x i n present as a c o n t a m i n a n t i n the 2,4,5-T s a m p l e u s e d i n the Bionetic study (3).

A f t e r the results of the s t u d y w e r e

k n o w n , the P a n e l o n H e r b i c i d e s of the President's

Science

made

Advisory

C o m m i t t e e s t u d i e d the t o t a l 2,4,5-T s i t u a t i o n . T h e r e p o r t of this c o m mittee w a s p u b l i s h e d i n M a r c h , 1971

(4).

I n a d d i t i o n to its e x t r e m e l y h i g h o r a l t o x i c i t y , s k i n contact w i t h substances c o n t a i n i n g 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n m a y a l l o w tox i c i t y i n the f o r m of c h l o r a c n e , a c o n d i t i o n c h a r a c t e r i z e d b y e r u p t i o n s of the s k i n o n the face, neck, a n d b a c k . A l s o , c h l o r i n a t e d d i b e n z o - p - d i o x i n s h a v e b e e n associated w i t h t h e " c h i c k e d e m a f a c t o r , " a disease of c h i c k s associated w i t h c o n t a m i n a t e d fats or oils u s e d i n t h e m a n u f a c t u r e

of

their f e e d . S i n c e 1950 m a n y i n the c h e m i c a l i n d u s t r y h a v e b e e n k e e n l y a w a r e of

the

possibilities of

h i g h l y toxic

2,3,7,8-tetrachlorodibenzo-p-dioxin

f o r m i n g i n the m a n u f a c t u r e of 2 , 4 , 5 - t r i c h l o r o p h e n o l . T h i s p h e n o l is m a d e b y t r e a t i n g 1,2,4,5-tetrachlorobenzene w i t h strong caustic at h i g h t e m peratures f o r several hours.

M a n u f a c t u r i n g p r o c e d u r e s w h i c h d o not

c a r e f u l l y c o n t r o l t e m p e r a t u r e a n d a l k a l i n i t y increase the p r o b a b i l i t y of dioxin formation. H i g h e r c h l o r i n a t e d d i b e n z o - p - d i o x i n s , s u c h as the o c t a c h l o r o d e r i v a tive, c a n b e f o r m e d i n m a n u f a c t u r i n g p e n t a c h l o r o p h e n o l f r o m h e x a c h l o r o ix

benzene.

H o w e v e r , this h i g h l y c h l o r i n a t e d d i o x i n is r e m a r k a b l y different

i n c h e m i c a l , p h y s i c a l , a n d b i o l o g i c a l properties f r o m that of the 2,3,7,8tetrachlorodibenzo-p-dioxin

a n d is m u c h less toxic.

Theoretically,

75

isomers of c h l o r i n a t e d d i b e n z o - p - d i o x i n s are possible, b u t f e w h a v e b e e n synthesized and studied i n detail. T h i s v o l u m e s h o u l d d o m u c h to a d v a n c e the k n o w l e d g e of these types of m o l e c u l e s , a n d a d d i t i o n a l research s h o u l d b e s t i m u l a t e d to ex p l a i n f u r t h e r the c h e m i c a l , p h y s i c a l , a n d b i o l o g i c a l properties

of

the

Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.pr001

chlorinated dibenzo-p-dioxins. Literature

Cited

1. Bauer, H., Schultz, K. H., Schultz, V., Spiegelberg, Χ. X., Arch. Gewerbe pathol. Gewerbehyg. (1961) 18, 538. 2. Higginbotham, G. R., Huang, Α., Firestone, D., Verett, J., Ress, J., Camp bell, A. D., Nature (1968) 220, 702. 3. "Effects of 2,4,5-T on M a n and T h e Environment," Hearings Before T h e Subcommittee on Energy, Natural Resources, and T h e Environment, A p r i l 7, 15, 1970; Serial 91-60, pp. 375-376. 4. "Report on 2,4,5-T," A Report of T h e Panel on Herbicides of T h e President's Science Advisory Committee, Executive Office of T h e President, Office of Science and Technology. ETCYL

Midland, Mich. February

1973

χ

H.

BLAIR

1

The Preparation of Uniformly Labeled 14

C-2,7-Dichlorodibenzo-p-dioxin and

14

Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.ch001

C-2,3,7,8-Tetrachlorodibenzo-p-dioxin

W E S L E Y W. M U E L D E R and LEWIS A. S H A D O F F Ag-Organics Department and Analytical Laboratories, The Dow Chemical Co., Midland, Mich. 48640

2,7-Dichlorodibenzo-p-dioxin

was prepared

potassium 2,4-dichlorophenate

from

isotopic

uniformly labeled with

Ullman conditions gave a 20.5% yield.

14

C.

Small amounts of

dichlorophenoxy chlorophenol were removed from the product by extraction with sodium hydroxide before purification by fractional sublimation and recrystallization from anisole. Chlorination

of 2,7-dichlorodibenzo-p-dioxin

solution containing trace amounts of FeCl

3

in chloroform and I yielded a 2

mixture of tri-, tetra-, and pentachloro substitution Purification by digestion

products.

in boiling chloroform, fractional

sublimation, and recrystallization from anisole was effective in refining this product to 92% 2,3,7,8-tetrachloro isomer, which also contained 7% of the tri- and 1% of the penta-substituted dibenzo-p-dioxin.

Mass spectroscopy was used

exclusively to monitor the quality of the products during the synthesis.

>T - D B p D ) to mass peak 322 ( f r o m 2 , 3 , 7 , 8 - C l D B p D ) w e r e d e t e r m i n e d a n d c o r r e c t e d for the sensitivity factors of the respective p a r e n t ions of these c o m p o u n d s . T h e a p p r o p r i a t e sensitivity factors w e r e p r o v i d e d b y t h e D o w C h e m i c a l C o . ( M i d l a n d , M i c h . ) ( 5 ) . T h e c r y s t a l structure studies, i n v o l v i n g x-ray d i f f r a c t i o n ( 6 ) , w e r e per f o r m e d b y the D o w C h e m i c a l C o . T h e c h e m i c a l p u r i t y a n d i d e n t i t y of the u n l a b e l e d a n d l a b e l e d c h e m i c a l s w e r e d e t e r m i n e d b y i n f r a r e d spectroscopy, mass spectrometry, G L C , and T L C . Unlabeled Chemicals. Samples of u n l a b e l e d D B p D d e r i v a t i v e s w e r e o b t a i n e d f r o m the f o l l o w i n g sources: 2 , 3 , 7 , 8 - C l - D B p D ( a n a l y t i c a l stand8

4

5

8

4

r

4

4

2.

viNOPAL

E T

Τritium-Labeled Dioxins

AL.

9

a r d , > 9 9 % p u r e ) f r o m t h e D o w C h e m i c a l C o . ; 1 - C l - D B p D a n d 2,3,7,8C l - D B p D f r o m A . E . P o h l a n d ( D i v i s i o n o f C h e m i s t r y a n d P h y s i c s , U . S. F o o d a n d D r u g A d m i n i s t r a t i o n , W a s h i n g t o n , D . C . ). D B p D was p r e p a r e d b y s l o w l y h e a t i n g a m i x t u r e o f o - c h l o r o p h e n o l (480 m m o l e s ) , p o t a s s i u m c a r b o n a t e ( 2 4 0 m m o l e s ) , a n d p u r i f i e d c o p p e r p o w d e r ( 5 0 m m o l e s ) i n a 5 0 0 - m l E r l e n m e y e r flask to 1 6 0 ° - 1 8 0 ° C a n d m a i n t a i n i n g this t e m p e r a t u r e f o r 6 hours. D B p D , w h i c h s u b l i m e d t o t h e w a l l s o f t h e flask as i t was f o r m e d , was r e c o v e r e d b y s c r a p i n g a n d w a s r e c r y s t a l l i z e d f r o m absolute e t h a n o l ( 1 4 % y i e l d ; w h i t e needles, m p 1 1 9 ° - 1 2 0 ° C ; reported 1 2 0 ° - 1 2 2 ° C ( 7 ) ; elemental analyses—calcd. C = 78.26, H = 4.34, f o u n d C = 77.98, H = 4 . 4 8 ) . D B p D w a s also p r e p a r e d o n a s u b m i l l i m o l e - s c a l e b y r e d u c t i v e d e c h l o r i n a t i o n of l , 6 - C l - D B p D . I n this m e t h o d l , 6 - C l - D B p D (0.1 m m o l e ) in 2-propanol (5 m l ) containing 1 0 % palladium on powdered charcoal (25 m g ) w a s h y d r o g e n a t e d at a pressure o f 10 lbs/square i n c h w i t h s h a k i n g for 1 h o u r at 25 ° C i n the 2 0 0 - m l pressure bottle o f a c o n v e n t i o n a l hydrogénation a p p a r a t u s ( P a r r I n s t r u m e n t C o . , M o l i n e , 111.). T h e cata lyst was r e m o v e d b y f i l t r a t i o n a n d was w a s h e d w i t h 25 m l o f 2 - p r o p a n o l . T h e c o m b i n e d 2 - p r o p a n o l fractions w e r e e v a p o r a t e d onto F l o r i s i l ( 1 0 g r a m , 60/100 m e s h , F l o r i d i n C o . , B e r k e l e y S p r i n g s , W . V a . ) , a n d t h e m i x t u r e w a s a d d e d to a glass c o l u m n ( 4 . 5 c m d i a m e t e r ) c o n t a i n i n g 30 grams F l o r i s i l . T h e n 25 grams F l o r i s i l w e r e a d d e d to t h e t o p o f t h e c o l u m n , a n d D B p D was e l u t e d w i t h hexane w h i c h left the v e r y p o l a r ( p r o b ably phenolic) breakdown products o n the column. T h e appropriate fractions w e r e c o m b i n e d ( b a s e d o n T L C - a n d G L C - m o n i t o r i n g ) a n d e v a p o r a t e d u n d e r n i t r o g e n to g i v e p u r e D B p D ( 5 5 - 6 5 % y i e l d ; single compound b y G L C and T L C ; m p 119°C, without recrystallization). l , 6 - C l - D B p D w a s p r e p a r e d either b y h e a t i n g 2 , 6 - d i c h l o r o p h e n o l ( 150 m m o l e s ), p o t a s s i u m carbonate, a n d c o p p e r p o w d e r u n d e r t h e c o n d i t i o n s above for D B p D or b y h e a t i n g the p o t a s s i u m phenate ( 35 m m o l e s )


4

2

2

2

Table I.

G L C Characteristics of Dibenzo-/>-dioxin and its Chlorinated Derivatives Retention Time, min

Compound

1%

DBpD 1-Cl-DBpD 1.6- C 1 - D B D 2.7- C l o - D B p D 2,3,7-Cl -DBpD* 2,3,7,8-CL-DBpD 2

P

3

SE-30

a

1.5 3.0 5.9 5.9

C

C C c

0.7 0.7 1.3 2.5

d d

10%

OV-17

b

3.0 6.0 11.0 10.0

e e e e

0.8' 1.4' 2.4' 2.2'

d d

33.5 , 6 . 8 ' e

On Chromosorb Ρ (100/120 mesh), 1.5 m Χ 3 mm stainless steel column On Chromosorb W (80/100 mesh), 1.5 m X 3 mm stainless steel column Injection temperature = 245°C, column temperature = 160°C, N = 25 ml/min Injection temperature = 280°C, column temperature = 195°C, N = 66 ml/min Injection temperature = 280°C, column temperature = 250°C. N = 25 ml/min ' Injection temperature = 280°C, column temperature = 270°C, N = 66 ml/min Obtained by irradiating an ethanol solution of 2,3,7,8-Cl4-DBpD with short-wave length ultraviolet light. a

b c

d

e

2

2

2

2

0

10

CHLORODIOXINS

ORIGIN

AND

FATE

d i r e c t l y w i t h c o p p e r p o w d e r . T h e d e s i r e d p r o d u c t s u b l i m e d i n the f o r m e r m e t h o d at 2 4 0 ° - 2 6 0 ° C a n d i n the latter one at 3 0 0 ° - 3 2 0 ° C . A f t e r r e c r y s t a l l i z a t i o n f r o m m e t h a n o l , the p r o d u c t s o b t a i n e d h a d the f o l l o w i n g characteristics: b y the p h e n o l m e t h o d — 4 . 1 % y i e l d ; w h i t e plates, m p 1 9 7 ° - 1 9 9 ° C ; e l e m e n t a l a n a l y s e s — c a l c d . C — 56.91, H = 2.37, C l = 28.06, f o u n d C = 57.04, H = 2.52, C l = 28.18, a n d b y the p o t a s s i u m phenate p r o c e d u r e — 7 . 8 % y i e l d ; w h i t e needles, m p 1 9 8 ° - 2 0 0 ° C ; e l e m e n t a l a n a l y s e s — f o u n d C = 56.92, H — 2.30, C l = 27.88. 2 , 7 - C l - D B p D was p r e p a r e d b y the same t w o p r o c e d u r e s u s e d for 1 , 6 - C L - D B p D i n v o l v i n g 2 , 4 - d i c h l o r o p h e n o l (75 m m o l e s ) a n d s u b l i m a t i o n at 2 0 0 ° - 2 2 0 ° C or p o t a s s i u m 2 , 4 - d i c h l o r o p h e n a t e (63 m m o l e s ) a n d sub l i m a t i o n at 2 2 0 ° - 2 4 0 ° C . A f t e r r e c r y s t a l l i z a t i o n f r o m p e t r o l e u m ether, the p r o d u c t s o b t a i n e d h a d the f o l l o w i n g characteristics: b y the p h e n o l p r o c e d u r e — 4 . 5 % y i e l d ; w h i t e needles, m p 2 0 6 ° - 2 0 8 ° C ; e l e m e n t a l a n a l y s e s — c a l c d . C = 56.91, H = 2.37, C l = 28.06, f o u n d C = 56.64, H = 2.40, C l = 28.10, a n d b y the p o t a s s i u m p h e n a t e p r o c e d u r e — 5 . 7 % y i e l d ; w h i t e needles, m p 2 0 7 ° - 2 0 8 ° C ; e l e m e n t a l a n a l y s e s — f o u n d C = 56.92, H = 2.52, C l = 28.12 ( 2 , 7 - C L - D B p D p r e p a r e d p r e v i o u s l y f r o m s o d i u m 2 , 4 - d i c h l o r o p h e n a t e gave m p 2 0 1 ° - 2 0 2 ° C ( 8 ) a n d b y c h l o r i n a t i o n of D B p D gave m p 2 0 3 ° C (7) and m p 1 9 5 ° - 1 9 7 ° C (9)). 2 , 3 , 7 , 8 - C l - D B p D was p r e p a r e d o n a s u b m i l l i m o l e - s c a l e b y c h l o r i n a t i o n of D B p D u n d e r c a r e f u l l y s t a n d a r d i z e d c o n d i t i o n s . T r a c e amounts of i o d i n e a n d f e r r i c c h l o r i d e w e r e a d d e d to a s o l u t i o n of p u r e D B p D (0.22-0.24 m m o l e ) i n c h l o r o f o r m (0.5 m l ) i n a 2 5 - m l c o n i c a l test t u b e . A f t e r c o o l i n g to 0 ° - 5 ° C , c h l o r i n e gas w a s b u b b l e d s l o w l y t h r o u g h the c h l o r o f o r m m i x t u r e for 5 - 6 m i n , f o r m i n g a copious p r e c i p i t a t e . T h e p r e c i p i t a t e i n the r e a c t i o n m i x t u r e was w a s h e d t w i c e w i t h 1.5-ml portions of c h l o r o f o r m at 2 5 ° C , u s i n g c e n t r i f u g a t i o n for m a x i m u m separation, to r e m o v e the u n w a n t e d c h l o r o f o r m - s o l u b l e materials. T h e p r e c i p i t a t e w a s t h e n treated b y s h a k i n g it w i t h c h l o r o f o r m ( 5 m l ) a n d w a t e r (1 m l ) , a n d the w a t e r - s o l u b l e p r o d u c t s ( s u c h as f e r r i c c h l o r i d e ) w e r e r e m o v e d , f o l l o w i n g c e n t r i f u g a t i o n . T h e c h l o r o f o r m phase, c o n t a i n i n g some u n d i s s o l v e d m a t e r i a l , w a s e v a p o r a t e d to dryness u n d e r a n i t r o g e n stream, a n d the r e s i d u e was transferred to a 2 5 - m l E r l e n m e y e r flask to w h i c h w a s a d d e d anisole ( 1 0 m l ) . T h e anisole i n the flask w a s h e a t e d to reflux to dissolve most of the r e s i d u e , a n d the u n d i s s o l v e d p o r t i o n was r e m o v e d b y filtering the hot anisole s o l u t i o n . C r y s t a l l i z a t i o n w a s a c h i e v e d b y a l l o w i n g the anisole s o l u t i o n to c o o l to 25 ° C , p u t t i n g i t i n the r e f r i g e r a t o r o v e r n i g h t , a n d filtering to recover 0.0093-0.0185 m m o l e of m a t e r i a l w h i c h c o n t a i n e d ( G L C a n d M S ) 9 0 - 9 4 % 2 , 3 , 7 , 8 - C l - D B p D ( r a n g e f r o m sev eral different p r e p a r a t i o n s ) , the r e m a i n d e r b e i n g m o s t l y C l - a n d C l D B p D . I n one s t u d y three batches of m a t e r i a l (0.050 m m o l e ) f r o m the anisole r e c r y s t a l l i z a t i o n w e r e c o m b i n e d a n d w a s h e d w i t h hot c h l o r o f o r m (8 m l ) . T h e c h l o r o f o r m - i n s o l u b l e p o r t i o n (0.022 m m o l e ) consisted of a single c r y s t a l l i n e m a t e r i a l w h i c h gave a x-ray d i f f r a c t i o n p a t t e r n i d e n t i c a l to that of k n o w n 2 , 3 , 7 , 8 - C l - D B p D , i n d i c a t i n g that the p r o d u c t consisted o n l y of 2 , 3 , 7 , 8 - C l - D B p D a n d d i d not c o n t a i n a n y other i s o m e r i c C l D B p D . ( T h e p r o d u c t also c o n t a i n e d some glass, p o s s i b l y f r o m the coarse f r i t t e d d i s k i n the B u c h n e r f u n n e l u s e d i n filtrations. ) I n another s t u d y the p r o d u c t f r o m the anisole r e c r y s t a l l i z a t i o n (0.013 m m o l e ) w a s c o m b i n e d w i t h a u t h e n t i c 2 , 3 , 7 , 8 - C l - D B p D (0.037 m m o l e , > 9 9 % p u r e ) to


2

4

4

3

r >

4

4

4

4

2.

VINOPAL

E T

11

Tritium-Labeled Dioxins

AL.

o b t a i n sufficient m a t e r i a l to w a s h t w i c e w i t h hot c h l o r o f o r m (8 m l a n d 3 m l ) . T h e final p r o d u c t w a s > 9 9 % 2 , 3 , 7 , 8 - C l - D B p D , b a s e d o n M S analysis. Labeled Chemicals. l , 6 - T - D B p D was s y n t h e s i z e d essentially b y the same p r o c e d u r e u s e d to p r e p a r e u n l a b e l e d D B p D o n a s u b m i l l i m o l e scale b y r e d u c t i v e d e c h l o r i n a t i o n ( a b o v e ) w i t h the f o l l o w i n g exceptions. A f t e r r e d u c t i o n of 1 , 6 - C L - D B p D (0.1 m m o l e ) i n 2 - p r o p a n o l ( 5 m l ) for 1 h o u r b y s t i r r i n g w i t h t r i t i u m gas ( 5 C i ) a n d h y d r o g e n gas (0.5 m l ) at atmos p h e r i c pressure, the r e a c t i o n m i x t u r e was w a s h e d w i t h 2 - p r o p a n o l (2 X 10 m l ) to r e m o v e l a b i l e t r i t i u m ; the catalyst w a s r e m o v e d b y filtration; the solvent was r e m o v e d b y v a c u u m d i s t i l l a t i o n , a n d the p r o d u c t s w e r e r e d i s s o l v e d i n 2 - p r o p a n o l (10 m l ) . ( T h i s p o r t i o n of the p r o c e d u r e w a s c a r r i e d out b y N e w E n g l a n d N u c l e a r C o r p . ( B o s t o n , M a s s . ) . ) T h e t r i t i a t e d m a t e r i a l w a s d i l u t e d ( to o b t a i n an a p p r o p r i a t e scale of r e a c t i o n i n s u b s e q u e n t steps) b y a d d i n g p u r e u n l a b e l e d D B p D (0.082 m m o l e ) i n 2 - p r o p a n o l (25 m l ) , a n d the r e s u l t i n g m i x t u r e w a s c h r o m a t o g r a p h e d (as a b o v e ) to isolate c r u d e l , 6 - T - D B p D (0.17 m m o l e , 1.1 C i / m m o l e ) . A n a l yses b y M S a n d G L C i n d i c a t e d the presence of trace amounts of a c o n t a m i n a n t , l - C l - 6 - T - D B p D , i n the c r u d e l , 6 - T - D B p D . T h e presence of this k i n d of c o n t a m i n a n t i n t h e l a b e l e d p r e p a r a t i o n b u t not i n the u n l a b e l e d p r e p a r a t i o n r e s u l t e d p r o b a b l y f r o m the l o w e r pressure u s e d d u r i n g the r e d u c t i o n step—i.e. 10 lbs/square i n c h f o r the u n l a b e l e d p r e p a r a t i o n a n d a t m o s p h e r i c pressure for the t r i t i u m - p r e p a r a t i o n . A s m a l l p o r t i o n (0.015 m m o l e ) of the c r u d e l , 6 - T - D B p D was r e d u c e d to r e m o v e the trace l e v e l of m o n o c h l o r o i m p u r i t y b y d i s s o l v i n g the sample i n 2 - p r o p a n o l (3.5 m l ) , a d d i n g 1 0 % p a l l a d i u m o n p o w d e r e d c h a r c o a l (3.8 m g ) , a n d s h a k i n g w i t h h y d r o g e n gas at 8 lbs/square i n c h for 15 m i n at 2 5 ° C . T h e catalyst w a s r e m o v e d b y filtration, a n d an a d d i t i o n a l p o r t i o n of p u r e u n l a b e l e d D B p D (0.054 m m o l e ) i n 2 - p r o p a n o l (10 m l ) w a s a d d e d b e f o r e c h r o m a t o g r a p h i c isolation of l , 6 - T - D B p D (138 m C i / m m o l e ; single r a d i o a c t i v e c o m p o u n d b y T L C a n d r a d i o - G L C ; no 1-C1D B p D d e t e c t e d b y M S ) . T h e c a l c u l a t e d specific a c t i v i t y , a s s u m i n g that there is no loss of c o m p o u n d o n r e d u c t i o n , is 239 m C i / m m o l e , i n d i c a t i n g that a p o r t i o n of the l a b e l e d m a t e r i a l is lost d u r i n g the process of the second r e d u c t i o n . 4


2

2

2

2

2

l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D was p r e p a r e d b y c h l o r i n a t i o n of 1,6-T D B p D (0.23 m m o l e , a m i x t u r e of 0.15 m m o l e of the c r u d e l , 6 - T - D B p D d e s c r i b e d above a n d 0.08 m m o l e of u n l a b e l e d D B p D ) i n the same m a n n e r d e s c r i b e d p r e v i o u s l y for p r e p a r i n g u n l a b e l e d 2 , 3 , 7 , 8 - C l - D B p D . T h i s re s u l t e d i n the i s o l a t i o n of c r u d e l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D (0.0115 m m o l e , c a l c u l a t e d as the tetrachloro d e r i v a t i v e a l t h o u g h some C l D B p D a n d a smaller a m o u n t of C l - D B p D w e r e p r e s e n t ) f o l l o w i n g r e c r y s t a l l i z a t i o n f r o m anisole. U n l a b e l e d 2 , 3 , 7 , 8 - C l - D B p D (0.043 m m o l e ) was t h e n a d d e d to the c r u d e l a b e l e d p r o d u c t , a n d the m i x t u r e was w a s h e d w i t h hot c h l o r o f o r m (8 m l ) , as d e s c r i b e d a b o v e for the u n l a b e l e d 2 , 3 , 7 , 8 - C l - D B p D , filtered, a n d w a s h e d a g a i n w i t h hot c h l o r o f o r m ( 3 m l ) to o b t a i n the final l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D p r e p a r a t i o n (0.011 m m o l e , 107 m C i / m m o l e ) . T h e specific a c t i v i t y is b e l o w the t h e o r e t i c a l v a l u e of 152 m C i / m m o l e , c a l c u l a t e d f r o m the v a r i o u s d i l u t i o n factors, p r o b a b l y as a result of i n a c curacies i n w e i g h i n g s or of a s m a l l a m o u n t of glass i n the s a m p l e f r o m the f r i t t e d glass filter. T h e p r o d u c t c o n t a i n e d no r a d i o l a b e l e d i m p u r i t i e s 2

4

2

2

4

2

4

; r

5

4

4

2

4

12

CHLORODIOXINS

ORIGIN

A N D

FATE

d e t e c t a b l e b y T L C a n d r a d i o a u t o g r a p h y or r a d i o - G L C ; there w e r e n o significant u n l a b e l e d c o n t a m i n a n t s e v i d e n t o n G L C , a n d M S i n d i c a t e d a c h e m i c a l p u r i t y of > 9 9 % . Discussion Three

routes

investigated.

for preparing tritium-labeled 2 , 3 , 7 , 8 - C l - D B p D 4

were

T h e first i n v o l v e d a n exchange r e a c t i o n b e t w e e n t r i t i u m

water a n d 2,4,5-trichlorophenol

to o b t a i n t r i t i u m - l a b e l e d

2,4,5-trichloro-

p h e n o l , u s e f u l i n s y n t h e s i z i n g 2 , 3 , 7 , 8 - C l - D B p D b y the salt f u s i o n reac Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.ch002

4

tion.

P r e l i m i n a r y studies w i t h d e u t e r i u m oxide, i n v o l v i n g p r o t o n m a g

netic resonance m o n i t o r i n g , i n d i c a t e d that extensive exchange of a r o m a t i c protons c a n be a c h i e v e d u n d e r basic b u t n o t a c i d i c c o n d i t i o n s .

However,

the degree of exchange does n o t seem to be sufficiently great to p r e p a r e h i g h specific a c t i v i t y m a t e r i a l u p o n r e p l a c i n g the d e u t e r i u m o x i d e w i t h t r i t i u m water.

T h e second route started w i t h l , 6 - C l - D B p D w h i c h w a s 2

c o n v e r t e d to the 1 , 6 - d i l i t h i u m d e r i v a t i v e , a n d this w a s h y d r o l y z e d w i t h t r i t i u m w a t e r to p r o d u c e l , 6 - T - D B p D .

T h e D B p D p r e p a r e d b y this

2

method was always contaminated w i t h about 3 0 % 1 - C l - D B p D , indicating that the o v e r a l l r e a c t i o n w a s n o t sufficiently c o m p l e t e .

A l s o , the separa

t i o n of D B p D a n d 1 - C l - D B p D w a s v e r y difficult. T h e route selected uses t r i t i u m gas instead of t r i t i u m w a t e r f o r l a b e l i n g , a n d this c h a n g e a l l o w s c o m p o u n d s of h i g h e r specific a c t i v i t y t o b e p r e p a r e d . Reductive

d e c h l o r i n a t i o n of l , 6 - C l - D B p D , u s i n g t r i t i u m gas a n d 2

s u b s e q u e n t c h l o r i n a t i o n of t h e l , 6 - T - D B p D , is a c o n v e n i e n t m e t h o d f o r 2

p r e p a r i n g l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D , b u t care must b e exercised i n several 2

4

steps i n v o l v e d i n this s u b m i l l i m o l e - s c a l e radiosynthesis.

Insufficient re

d u c t i o n of l , 6 - C l - D B p D results i n c o n t a m i n a t i o n b y 1 - C l - D B p D w h i l e 2

the a m o u n t of p o l a r p r o d u c t s increases i f the r e d u c t i o n is too drastic. U s i n g exactly 0.5 m l of p r e c o o l e d c h l o r o f o r m f o r the c h l o r i n a t i o n step results i n a m i x t u r e of t h e f o l l o w i n g components, as d e t e r m i n e d b y G L C chromatography 2-Cl-DBpD);

and M S : D B p D ;

a monochloro

2 , 7 - C l - D B p D ; another 2

isomeric

derivative

Cl -DBpD; 2

(probably a

trichloro

d e r i v a t i v e , a s s u m e d to b e 2 , 3 , 7 - C l D B p D ; 2 , 3 , 7 , 8 - C l - D B p D , a n d C l , ; r

DBpD.

4

r

N o h i g h e r c h l o r i n a t e d d e r i v a t i v e s of D B p D w e r e detected.

The

contaminants i n the m i x t u r e are r e m o v e d l a r g e l y i n various p u r i f i c a t i o n stages. W a s h i n g t h e c r u d e r e a c t i o n m i x t u r e w i t h c h l o r o f o r m removes a l l u n r e a c t e d D B p D , most of the 1 - C l - D B p D , a n d some of t h e C l - a n d CI32

D B p D d e r i v a t i v e s . T h e next step of w a s h i n g the c h l o r o f o r m w i t h w a t e r removes the f e r r i c c h l o r i d e catalyst. the

chloroform-soluble

products,

F o l l o w i n g anisole r e c r y s t a l l i z a t i o n of the material

2 , 3 , 7 , 8 - C L i - D B p D a n d 6 - 1 0 % of C l

: r

consists

and C l - D B p D . 5

of 9 0 - 9 4 %

of

T h e final c h l o r o

f o r m washes r e m o v e almost a l l r e s i d u a l C l - a n d C l - D B p D , r e s u l t i n g i n 8

very p u r e 2 , 3 , 7 , 8 - C l - D B p D . 4

5

2.

viNOPAL

E T

13

Τritium-Labeled Dioxins

AL.

T h e samples of l , 6 - T - D B p D a n d l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D are u s e f u l 2

2

4

i n m e t a b o l i s m a n d m o d e of a c t i o n studies. F o r e x a m p l e , w h e n i n c u b a t e d w i t h r a b b i t l i v e r microsomes, l , 6 - T - D B p D is extensively m e t a b o l i z e d to 2

polar product(s)

b u t o n l y w h e n these p r e p a r a t i o n s

reduced nicotinamide-adenine dinucleotide phosphate. conditions l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D 2

4

are f o r t i f i e d w i t h U n d e r the same

is c o m p l e t e l y resistant

to m e t a b o l i c

attack. I n some types of studies, a h i g h e r specific a c t i v i t y p o s s i b l y is d e sirable (i.e., > 1 C i / m m o l e ) , a n d this c a n b e a c h i e v e d , w i t h the m e t h o d


ology a l r e a d y

d e v e l o p e d , b y u s i n g larger

amounts

of t r i t i u m gas or

w o r k i n g o n a larger synthetic scale so that it is not necessary to a d d u n l a b e l e d materials

to assist i n c r y s t a l l i z a t i o n steps w h e r e

a

certain

m i n i m u m a m o u n t of c o m p o u n d is necessary. Acknowledgment T h i s w o r k w a s a i d e d b y P H S grant E S 00049 a n d A E C C o n t r a c t N o . A T ( 0 4 - 3 ) - 3 4 , project agreement N o . 113. T h e authors are i n d e b t e d to J u n e T u r l e y a n d L e w i s Shadoff ( C h e m i c a l P h y s i c s R e s e a r c h L a b o r a t o r y ) and Warren Crummett (Analytical Laboratory, T h e D o w Chemical C o . , M i d l a n d , M i c h . ) a n d to L o r e n D u n h a m ( Z o e c o n C o r p . , P a l o A l t o , C a l i f . ) for a d v i c e a n d assistance i n p r o d u c t analysis. Literature

Cited

1. Wilson, J. G . , et al., "Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency" (May 7, 1971). 2. Schwetz, Β. Α., Norris, J. M . , Sparschu, G. L., Rowe, V. K., Gehring, P. J., Emerson, J. L., Gerbig, C . G . , A D V A N . C H E M . SER. (1973) 120, 55. 3. Muelder, W. W., Shadoff, L . Α., A D V A N . C H E M . SER. (1973) 120, 1. 4. Kovacs, M . F . , Jr., J. Ass. Offic. Agr. Chem. (1963) 46, 884. 5. Dow Chemical Co., private communication. 6. Boer, F. P., Neuman, Μ. Α., van Remoortere, F. P., North, P. P., Rinn, H . W., ADVAN.

C H E M . SER. (1973) 120,

14.

7. Gilman, H . , Dietrich, J. J., J. Amer. Chem. Soc. (1957) 79, 1439. 8. Julia, M . , Baillarge, M., Bull. Soc. Chim. France (1953) 644. 9. Tomita, M . , Ueda, S., Narisada, M . , Yakugaku Zasshi (1959) 79, 186. R E C E I V E D February 18,

1972.

3

X-ray Diffraction Studies of Chlorinated


Dibenzo-p-dioxins

F. P. BOER, M . A. N E U M A N , F . P. V A N R E M O O R T E R E , P. P. N O R T H , and H . W . RINN Analytical Laboratories, The Dow Chemical Co., Midland, Mich. 48640

The

crystal structures of four chlorinated derivatives of di

benzo-p-dioxin have been determined

by x-ray diffraction

from diffractometer data (MoKα radiation). The compounds, their formulae, cell dimensions, space groups, the number of molecules per unit cell, the crystallographic R-factors, and the number of observed reflections are given.

The dioxin

crystal structures were performed to provide absolute stand ards for assignment of isomeric structures and have been of considerable practical use in combination with x-ray powder diffraction analysis.

"Decause

o f t h e present interest i n t h e c h e m i c a l , b i o l o g i c a l , a n d eco-

l o g i c a l properties

of t h e c h l o r i n a t e d

a p p l i e d X - r a y d i f f r a c t i o n methods compounds.

dibenzo-p-dioxins, w e have

to i d e n t i f y a n d characterize

S u c h studies are u s e f u l f o r three

these

reasons:

(1) A b s o l u t e i d e n t i f i c a t i o n o f t h e i s o m e r i c s t a n d a r d materials b y single c r y s t a l t e c h n i q u e s ;

structure

of p r i m a r y

( 2 ) C o r r e l a t i o n of the single-crystal structure results w i t h the p o w d e r d i f f r a c t i o n p a t t e r n , e s t a b l i s h i n g x-ray p o w d e r d i f f r a c t i o n as a c o n v e n i e n t , p o w e r f u l means o f i s o m e r i d e n t i f i c a t i o n ; a n d ( 3 ) G e n e r a t i o n o f a ( u s u a l l y ) accurate, d e t a i l e d p i c t u r e o f t h e m o l e c u l a r geometry to p e r m i t f u t u r e correlations b e t w e e n s t r u c t u r a l features a n d b i o l o g i c a l a c t i v i t y . W e r e p o r t t h e c r y s t a l structures

of f o u r c h l o r i n a t e d

dioxins—the

2 , 7 - d i c h l o r o - , 2 , 8 - d i c h l o r o - , 2,3,7,8-tetrachloro-, a n d o c t a c h l o r o d i b e n z o - p dioxins.

T h u s , five c r y s t a l structures of c h l o r o d i o x i n s are n o w k n o w n . 14

3.

The

BOER

E T

AL.

unexpected

X-ray Diffraction Studies identification

(I)

of

15

1,2,3,7,8,9-hexachlorodibenzo-p-

d i o x i n i n toxic f e e d fats b y C a n t r e l l , W e b b , a n d M a b i s , u s i n g x-ray c r y s t a l l o g r a p h y , first p o i n t e d to the c h l o r o d i o x i n s as a p o t e n t i a l toxicol o g i c a l h a z a r d . T h e f o u r c r y s t a l structures r e p o r t e d here w e r e d e t e r m i n e d b y v e r y s i m i l a r t e c h n i q u e s , a n d t h e i r essential parameters are s u m m a r i z e d i n T a b l e I.

A P i c k e r f o u r - c i r c l e diffractometer was u s e d to

determine

the lattice constants a n d to gather i n t e n s i t y d a t a i n Θ-2Θ scan m o d e , u s i n g m o n o c h r o m a t i c MoKa r a d i a t i o n . T h e structures w e r e s o l v e d f r o m Pat


terson f u n c t i o n s a n d r e f i n e d b y f u l l - m a t r i x least-squares, a s s u m i n g aniso t r o p i c t e m p e r a t u r e factors f o r C I , O , a n d C atoms a n d i s o t r o p i c t e m p e r a ture factors

for H .

H y d r o g e n parameters

were

n o t r e f i n e d for

the

2 , 8 - d i c h l o r o c o m p o u n d for w h i c h o n l y a s m a l l f r a c t i o n of the p o s s i b l e reflections was o b t a i n e d o w i n g to the weakness of s c a t t e r i n g a n d p r o gressive d e t e r i o r a t i o n of the c r y s t a l .

C o r r e c t i o n s w e r e a p p l i e d f o r ab

s o r p t i o n , for a n o m a l o u s scattering b y C I , a n d , w h e r e a p p r o p r i a t e , for secondary extinction.

T a b l e II lists the a t o m i c positions a n d t h e r m a l

parameters for the f o u r structures. T h e m o l e c u l e s of the 2 , 7 - d i c h l o r o , 2,3,7,8-tetrachloro, a n d o c t a c h l o r o structures are a l l l o c a t e d o n c r y s t a l l o g r a p h i c centers of s y m m e t r y a n d are v e r y n e a r l y p l a n a r i n e a c h case.

( I n the 2,3,7,8-tetrachloro structure

there are t w o i n d e p e n d e n t m o l e c u l e s s i t u a t e d o n the 1 elements at t h e o r i g i n a n d at the center of the u n i t c e l l , r e s p e c t i v e l y . )

I n e a c h of these

three structures the m o l e c u l e s f o r m stacks a l o n g t h e short u n i t c e l l transla tions w i t h i n t e r p l a n a r distances b e t w e e n 3 . 4 5 - 3 . 4 9 A .

T h e structure of

the 2 , 8 - d i c h l o r o c o m p o u n d is a different t y p e w i t h the m o l e c u l e d i v i d e d transversely b y a c r y s t a l l o g r a p h i c m i r r o r p l a n e a n d e x h i b i t i n g a slight b e n d i n g at the oxygens, s i m i l a r to that d e s c r i b e d f o r t h e 1,2,3,7,8,9-hexa compound (I).

F i g u r e s 1, 2, 3, a n d 4 i n d i c a t e that the m o l e c u l a r g e o m

etries are q u i t e r e g u l a r w i t h b o n d distances a n d angles a g r e e i n g a c c e p t e d literature values.

with

T h e C — C I distances, h o w e v e r , s h o w a n i n

teresting t r e n d t o w a r d s h o r t e n i n g w i t h i n c r e a s i n g c h l o r i n e s u b s t i t u t i o n o n the rings ( F i g u r e 5 ) .

T h e e s t i m a t e d errors a n d i n t e r n a l agreement of

the b o n d distances g i v e confidence that this effect is r e a l . T h e differences also seem to h o l d w h e n b o n d distance corrections a s s u m i n g r i g i d b o d y m o t i o n are a p p l i e d ( 2 ) .

T h i s result c o u l d arise f r o m a r e d u c t i o n i n the

effective e l e c t r o n e g a t i v i t y difference b e t w e e n C a n d C I as m o r e e l e c t r o n d e n s i t y is d r a w n f r o m the a r o m a t i c r i n g , w h i c h s h o u l d i n t u r n result i n i n c r e a s e d c o v a l e n c y of the C — C I b o n d s a n d g i v e shorter

distances.

A l t e r n a t i v e l y , i n terms of m o l e c u l a r orbitals, the a d d i t i o n of m o r e electro negative c h l o r i n e substituents

c o u l d d r a w electrons

from antibonding

M O ' s associated w i t h the C — C I b o n d s a n d thus increase the net b o n d order of the C — C I b o n d s .

16

CHLORODIOXINS

ORIGIN

A N D

FATE

Table I. 2,7-DC Β D


Space Group

PI

Cell Constants a (A) b (A) c (A) α (deg) β (deg) γ (deg) Ζ Density (g c m ) L i n e a r a b s o r p t i o n coeff. ( c m " ) N o . Reflections M e a s u r e d N o . Reflections A b o v e B a c k g r o u n d Λ Χ = Σ I I F l - l I F I |/Σ | F | R = \Lw{F -F y/JiwF *\ /*

3.878(3) 6.755(9) 10.265(15) 99.46(1) 100.63(3) 99.73(3) 1 1.647 5.26 1152 1030 0.057 0.062

- 3

1

0

2

0

c

e

0

0

1

Table II.

Structure

2,7-Dichlorodibenzo-p-dioxin Atom

Χ

y

ζ

CI 0 C(l) C(2) C(3) C(4) C(5) C(6)

-0.4292(3) 0.0466(7) -0.0668(8) -0.1125(8) -0.1340(9) -0.2219(9) -0.2452(9) -0.2894(8)

-0.3654(1) 0.2155(3) 0.0749(5) -0.1359(5) 0.1467(5) -0.2706(5) 0.0123(5) -0.1955(5)

0.3777(1) 0.0479(2) 0.1228(3) 0.0757(3) 0.2468(3) 0.1534(3) 0.3255(3) 0.2782(3)

H(3) H(4) H(5)

-0.094(4) -0.240(9) -0.283(9)

0.293(5) -0.411(6) 0.058(5)

0.278(3) 0.124(4) 0.406(4)

2,8-Dichlorodibenzo-p-dioxin Atom CI 0(1) 0(2) C(l) C(2) C(3) C(4) C(5) C(6)

X

0.3003(6) 0.2586(20) -0.1758(22) 0.1492(25) -0.0638(22) 0.2607(25) -0.1596(20) 0.1541(31) -0.0581(27)

y

ζ

0.0705(7) 0.0973(21) -0.0780(22) 0.0443(18) -0.0372(18) 0.0804(21) -0.0843(18) 0.0298(19) -0.0489(23)

0.4541(1) 0.25 0.25 0.2969(6) 0.2971(5) 0.3449(6) 0.3460(6) 0.3937(5) 0.3944(6)

3.

BOER

E T

17

X-ray Diffraction Studies

A L .

Crystal D a t a


2,8-DCBD

OCBD

2,3,7,8-TCBD

Pnam

Pi

P2i/c

5.983(6) 7.114(10) 24.637(36) 90. 90. 90. 4 1.602 5.12 1206 293 0.064 0.065

3.783(3) 9.975(9) 15.639(15) 94.14(2) 95.20(4) 92.77(4) 2 1.827 9.79 2666 2381 0.036 0.038

12.009(9) 3.828(3) 16.297(9) 90. 101.14(3) 90. 2 2.077 15.05 1640 1460 0.058 0.053

Parameters "

10«pii 945(9) 1110(25) 587(26) 569(26) 694(29) 599(28) 640(29) 453(24)

10 β 4

2 2

240(2) 134(5) 159(7) 165(2) 166(8) 160(9) 213(9) 208(8)

10^33

106(1) 72(2) 66(3) 66(3) 78(4) 84(4) 77(4) 89(4)

10 βι 4

2

85(3) 82(8) 74(11) 77(11) 74(12) 63(11) 110(12) 89(11)

10*βΐ3

10 β

142(2) 114(6) 36(7) 35(7) 64(8) 41(8) 84(8) 55(7)

63(1) 15(3) 30(4) 21(4) 3(4) 32(4) 25(5) 58(4)

10» β„

10 β

4

2 3

Β 3.5(8) 4.4(9) 4.0(9)

10 βιι

10 &22

29(1) 17(6) 15(5) 20(6) 9(6) 26(6) 10(5) 30(7) 8(5)

37(1) 32(5) 28(5) 10(3) 17(4) 13(3) 9(3) 16(4) 26(4)

3

10 & 3

3

15(1) 10(2) 18(3) 13(4) 17(4) 14(3) 20(3) 10(2) 15(3)

12

-4(1) -10(4) -7 -3(3) -4(4) 0(4) -3(4) 10(5) 4(5)

3

2 3

-1(1)

0(1)

-1(1) 2(2)

0(1) -1(1)

-1(1)

0(1) -1(1) -1(1)

KD KD KD

KD

18

CHLORODIOXINS

ORIGIN

A N D FATE

Table II. X

y

ζ

0.414 -0.311 -0.136

0.141 -0.148 -0.080

0.345 0.346 0.430

Atom H(3) H(4) H(6)

2,3,7,8-Tetrachlorodibenzo-p-dioxin Published on March 1, 1973 on http://pubs.acs.org | doi: 10.1021/ba-1973-0120.ch003

Atom

ζ

X

y

C I (2 A ) C1(3A) C1(2B) C1(3B) 0(A) ' 0(B) C(1A) C(2A) C(3A) C(4A) C(5A) C(6A) C(1B) C(2B) C(3B) C(4B) C(5B) C(6B)

0.24332(15) -0.17456(16) 0.10613(16) 0.17045(17) 0.19947(40) 0.46780(42) 0.20316(53) 0.11584(51) -0.06777(52) -0.17010(53) -0.08608(50) 0.10264(50) 0.29579(55) 0.22673(51) 0.25627(52) 0.35698(56) 0.42831(50) 0.39708(52)

-0.28365(5) -0.47674(5) 0.77903(6) 0.97910(6) 0.08571(14) 0.40986(13) -0.09398(20) -0.22616(20) —0.31053(19) -0.26211(20) -0.13010(19) -0.04631(19) 0.58979(21) 0.72370(21) 0.81109(20) 0.76516(21) 0.63162(20) 0.54373(19)

H(1A) H (4 A ) H(1B) H(4B)

0.3493(57) -0.3102(52) 0.2730(53) 0.3803(53)

-0.0369(23) -0.3208(20) 0.5287(21) 0.8316(21)

0.26900(3) 0.11985(4) 0.77142(3) 0.62508(4) 0.06607(9) 0.56599(9) 0.15576(13) 0.16989(12) 0.10454(13) 0.02588(13) 0.01266(12) 0.07732(12) 0.65628(13) 0.67145(13) 0.60742(13) 0.52812(13) 0.51347(12) 0.57761(12) 0.1996(14) -0.0219(13) 0.7043(13) 0.4815(13)

Octachlorodibenzo-p-dioxin Atom

X

y

ζ

Cl(l) Cl(2) Cl(3) Cl(4) 0 C(l) C(2) C(3) C(4) C(5) C(6)

0.36887(8) 0.44176(8) 0.11407(8) 0.25867(8) -0.0329(2) 0.0773(3) 0.1098(3) 0.1574(3) 0.2222(3) 0.2699(2) 0.3031(3)

0.1125(3) -0.2322(3) 0.3190(3) -0.3520(3) 0.1580(10) 0.0630(12) -0.0887(12) 0.1261(11) -0.1749(11) 0.0359(11) -0.1153(11)

0.23425(6) 0.07846(7) 0.22562(6) -0.08448(6) 0.0705(2) 0.0704(2) 0.0017(2) 0.1419(2) 0.0033(2) 0.1449(2) 0.0758(2)

α

The anisotropic thermal parameters are in the form exp-(/i p + Α; β + Ζ β + 2ΛΑ;β„ + 2hl$ + 2Μβ ) 2

u

2

22

2

33

u

28

3.

BOER

E T

19


A L .

Continued 10 βπ 3

10 β 3

2 2

10 β 4

3

1O 0

12

1 2

3

1O 0 3

13

1O 0 3

1 3

1O 0

3

2

Β 4.0 4.0 4.0

10 β 2

10 &33

1O 0

865(6) 617(5) 924(6) 635(5) 629(15) 620(15) 678(21) 723(21) 545(19) 635(20) 656(20) 587(19) 731(22) 762(22) 594(20) 648(21) 665(20) 604(20)

301(2) 407(2) 310(2) 469(3) 248(6) 244(6) 256(8) 255(8) 325(9) 276(9) 225(8) 253(8) 256(8) 261(8) 346(9) 305(9) 236(8) 267(8)

325(13) -179(13) 455(14) 645(13) 386(35) 682(36) 220(43) 435(43) 108(42) 3(42) 116(41) 119(41) 168(46) 161(44) 265(42) 226(45) 272(41) 242(41)

ΙΟ^π

10 β 2

10*033

49(1) 40(1) 58(1) 59(1) 40(2) 43(3) 45(2) 50(3) 46(3) 42(3) 36(2)

82(11) 82(11) 56(9) 67(10) 103(35) 50(33) 44(32) 36(30) 37(30) 43(31) 43(32)

25(1) 33(1) 22(1) 25(1) 27(1) 23(1) 23(1) 19(1) 23(1) 17(1) 28(2)

10 β π


4

758(5) 841(5) 803(5) 864(5) 800(13) 920(14) 465(14) 464(14) 472(14) 463(14) 437(14) 453(14) 529(16) 446(14) 452(15) 556(16) 446(14) 473(14)

5

2

5

5

1O 0 5

5

2 3

130(8) 152(9) 463(8) 340(10) -217(22) 359(23) 9(27) 98(27) 231(28) 79(27) 87(25) 126(26) 148(28) 122(27) 5(29) 113(29) 68(26) 35(27)

132(3) 78(3) 95(3) 48(3) 16(7) 56(7) -25(10) 76(10) 36(10) -43(10) 12(9) -6(10) 8(11) -74(10) 44(11) 51(11) 5(10) 5(10)

1O*0

10*023

Β 3.6(0.5) 2.3(0.4) 2.9(0.4) 2.7(0.4)

3

2

Isotropic temperature factors (Β) parentheses. b

1O*0

12

-16(2) 34(2) -7(2) 38(2) 47(7) 7(8) 12(8) -1(8) 19(8) -5(8) 2(8)

13

-4(1) 6(1) 7(1) 11(1) -2(1) 4(1) 2(1) 8(2) 8(2)

-KD

9(2)

-12(2) 13(2) -22(1) -18(2) -74(5) -11(6) -7(6) 7(5) 1(6) 14(5) 21(6)

in A ; standard deviations are given in 2


20

CHLORODIOXINS

ORIGIN

A N D FATE

DCBD BONDS

C-C ±0.005 Â

ANGLES C - C - C

±0.3°

C-CI ± 0 . 0 0 4

C-C-CI

±0.3°

C - H ±0.04

C-C-H

±0.004

C-O-C

Figure 1.

±0.3°

C-C-0

C-0

*?· ±03°

Bond distances and angles for 2,7-dichlorodibenzo-p-dioxin. The molecule is situated on an inversion center.

2.8-DCBD BONDS

C-C ± 0 . 0 I - 0 . 0 2 Â C-0 ±0.01 C-CL ±0.01

ANGLES

C-C-C ±1.3° C-C-0 ± l . 3 C-C-CL±l.3 C-O-C ±1.5° e

e

Site Symmetry m

Figure 2.

Bond distances and angles for 2,8-dichlorodibenzo-p-dioxin. The molecule is located on a crystallographic mirror plane.


3.

BOER

E T

A L .

21


TCBD BONDS

C-C C-0 C-CL C-H

*0.003Â *0.003 ±0.003 ±0.02

ANGLES C - C - C C-C-0 C-C-CI C-C-H C-O-C

±0.2' ±0.2* ±0.2° *l.3 ±0.2* e

Figure 3. Bond distances and angles for the two independent molecules in 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both molecules are located on crystallographic inversion centers. X - r a y p o w d e r d i f f r a c t i o n , u s e d w h e r e v e r possible w i t h x-ray singlec r y s t a l d a t a , is a v e r y c o n v e n i e n t m e t h o d f o r i d e n t i f y i n g samples of c h l o r i n a t e d d i o x i n s a n d i n p a r t i c u l a r f o r g i v i n g i n f o r m a t i o n o n t h e isomers present.

Some of the t r a d i t i o n a l m e t h o d s f o r i d e n t i f y i n g o r g a n i c m o l e

cules, s u c h as i n f r a r e d a n d mass spectrometry, are of l i m i t e d u t i l i t y i n i d e n t i f y i n g isomers o f t h e c h l o r o d i o x i n s .

Nuclear magnetic

resonance

m e t h o d s h a v e b e e n h i n d e r e d b y s o l u b i l i t y p r o b l e m s ; they also are less s u i t a b l e f o r w o r k i n g w i t h the v e r y m i n u t e samples that c a n b e a n a l y z e d


22

CHLORODIOXINS

BONDS

Figure 4.

C-C C-0 C-CL

±0.005 Â ±0.004 ±0.004

ANGLES

C-C-C C-C-0 C-C-CL C-O-C

ORIGIN

A N D FATE

±0.4° ±0.3° ±0.3° ±0.3°

Bond distances and angles for octachlorodibenzo-p-dioxin. The molecule is located on an inversion center.

BOND LENGTH

(A)

-2,8-DI

1.750-2,7-DI 1.7402,3,7,8-TETRA .730-Γ

1,2,3,7,8,9- HEXA RANGE 1.69-1.79

Ι.720-

OCTA

1.710 NO. OF C l ' S Figure 5.

Carbon-chlorine bond lengths (A) of chlorinated dibenzo-p-dioxins as a function of the number of chlorine substituents.

3.

BOER

E T

A L .

23


Table III. X - r a y Powder Diffraction Data for 2,3,7,8-Tetrachlorodibenzo-^-dioxin" and 2,7-Dichlorodibenzo-f>-dioxin &

TCBD


C

d(hkl)

I/Ko)

8.728 8.130 6.369 5.901 4.984 4.851 4.739 4.639 4.448 4.343 4.046 3.759 3.736 3.578 3.537 3.456 3.330 3.296 3.266 3.188 3.180 3.035 3.003 2.953 2.916 2.892 2.866 2.850

3 4 23 24 2 3 6 3 6 15 10 10 7 50 2 60 100 38 23 25 22 50 3 17 13 5 4 4

hkl 011 011 012 012 020 021 013 021 013 022 022 100 101, 101, 111, 110, 112 111, 102 103, 024 113, 121 024 103 015, 113 122

023 110 014 111 031 031 015, 112

122, 033

2,7-DCBD d(hkl)

I/I(o)

9.9 6.55 5.00 4.39 3.56 3.46 3.26 3.01 2.92 2.76 2.57 2.48 2.38

13 25 63 5 63 25 100 10 20 5 8 8 5

hkl 001 010 011 012 110 111 101, 022 021, 112, 113 014, 122

020, 111 112 120 004

24

CHLORODIOXINS

ORIGIN

A N D FATE

Table III. Continued 2,7-DCBD d(hkl)


2.30 2.21 2.12 2.07 2.03

I/I(o)

hkl

5 5 18 5 5

104, 031, 114, 130, 031

112 114 123 121

"Obtained on a 115-mm diameter A E G (Allgemeine Elektrizitats-Gesellschaft) Guinier camera using Seeman-Bohlin focusing and Cu Κα, radiation (λ 1.5405 A). Data taken with a 143.2-mm diameter Debye-Scherrer camera using Cu Κα radiation (λ 1.5418 A). Forty additional lines to 1.603 A were recorded 6

c

b y x - r a y d i f f r a c t i o n . T h e o b s e r v e d p o w d e r patterns f o r t h e 2 , 7 - d i c h l o r o a n d t h e 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n s are g i v e n i n T a b l e I I I . S e v e r a l cases h a v e b e e n e n c o u n t e r e d that s h o w t h e u t i l i t y o f x-ray p o w d e r d i f f r a c t i o n as a n a n a l y t i c a l m e t h o d f o r i d e n t i f y i n g d i o x i n s . A n a t t e m p t w a s m a d e to synthesize 2 , 8 - d i c h l o r o d i b e n z o - p - d i o x i n b y h e a t i n g the p o t a s s i u m salt of a t r i c h l o r i n a t e d 2 - h y d r o x y d i p h e n y l ether ( 3 ) . Sur prisingly, the major crystalline product under the initial reaction condi

tions w a s 2 , 7 - d i c h l o r o d i b e n z o - p - d i o x i n suggesting that cleavage of t h e ether a n d its subsequent r e f o r m a t i o n h a d o c c u r r e d u n e x p e c t e d l y .

Some

2 , 8 - d i o x i n c o u l d also b e d e t e c t e d i n the r e a c t i o n p r o d u c t . A second sam ple of 2,8-dioxin contained an u n k n o w n crystalline i m p u r i t y w h i c h c o u l d be p h y s i c a l l y separated o n t h e basis of c r y s t a l m o r p h o l o g y . I n a d d i t i o n , a n interesting, a l t h o u g h negative, result has c o m e f r o m p o w d e r d i f f r a c t i o n studies o f the h e x a c h l o r o c o m p o u n d s . W e h a v e exam i n e d D e b y e - S c h e r r e r p h o t o g r a p h s of several samples k n o w n t o c o n t a i n p r e d o m i n a n t l y h e x a c h l o r o d i b e n z o - p - d i o x i n s a n d h a v e i d e n t i f i e d t h e pat terns o f at least three c r y s t a l l i n e phases t h e r e i n . isomers

of hexachlorodibenzo-p-dioxin. )

These

( T h e r e are 10 possible patterns

have

been

c h e c k e d c a r e f u l l y against the c a l c u l a t e d d-spacings a n d intensities of t h e 1,2,3,7,8,9-hexa isomer d e s c r i b e d b y C a n t r e l l , W e b b , a n d M a b i s ( I ) a n d also against a n o b s e r v e d p a t t e r n s u p p l i e d b y C a n t r e l l a n d b e l i e v e d t o b e f r o m t h e l o w t e m p e r a t u r e phase of t h e same m a t e r i a l .

Y e t t o date w e

3.

BOER

E T

A L .


25

h a v e n o t b e e n a b l e to detect the s i g n a t u r e of either of these 1,2,3,7,8,9h e x a c h l o r o d i o x i n phases i n a n y s a m p l e e x a m i n e d . Acknowledgment T h e authors are p l e a s e d to a c k n o w l e d g e the c o n t r i b u t i o n s of W . W . M u e l d e r a n d O . A n i l i n e , w h o p r o v i d e d the samples a n d m u c h s t i m u l a t i n g


discussion. Literature

Cited

1. Cantrell, J. S., Webb, N. C., Mabis, A. J., Acta Crystallogr. (1969) B25, 150. 2. Schomaker, V., Trueblood, Κ. N., Acta Crystallogr. (1968) B24, 63. 3. Aniline, O., private communication (1971). R E C E I V E D February 8,

1972.

4

Thermal Chemistry of Chlorinated Phenols

H . G . L A N G E R , T. P. BRADY, L . A. D A L T O N , T . W . S H A N N O N , and P. R. BRIGGS


The Dow Chemical Co., Eastern Research Laboratory, Wayland, Mass. 01778

Thermal properties of several chlorinated phenols and derivatives were studied by differential thermal analysis and mass spectrometry and in bulk reactions. Conditions which might facilitate the formation of stable dioxins were emphasized. No two chlorinated phenols behaved alike. given compound the decomposition

For a

temperature and rate

as well as the product distribution varied considerably with reaction conditions.

The phenols themselves seem to pyro-

lyze under equilibrium conditions slowly above 250°C. their alkali salts the onset of decomposition around 350°C.

For

is sharp and

The reaction itself is exothermic.

Prelimi-

nary results indicate that heavy ions such as cupric ion may decrease the decomposition

temperature.

' T p h e literature on formation of halodibenzo-p-dioxins is vague and A

sometimes c o n f u s i n g .

M o s t of t h e i n f o r m a t i o n a v a i l a b l e is s u m m a

r i z e d i n a n a r t i c l e b y M . K u l k a ( 1 ) a n d deals w i t h t h e f o r m a t i o n of octachlorodibenzo dioxin from pentachlorophenol.

W e have investigated the

t h e r m a l properties of several c h l o r i n a t e d p h e n o l s a n d derivatives b y d i f f e r e n t i a l t h e r m a l analysis a n d mass s p e c t r o m e t r y a n d i n b u l k reactions. T h e r e a c t i o n c o n d i t i o n s w e r e chosen to f a v o r the d i o x i n f o r m a t i o n . O f t h e c h l o r i n a t e d p h e n o l s p e n t a c h l o r o p h e n o l is t h e most l i k e l y to produce a dioxin.

Its d i f f e r e n t i a l t h e r m o g r a m ,

reveals n o d e c o m p o s i t i o n .

however,

b y m e l t i n g b e l o w 200 ° C a n d v a p o r i z a t i o n above prolonged heating

(Figure

1)

A s o l i d state t r a n s i t i o n at 7 5 ° C is f o l l o w e d

i n b u l k a n d at temperatures

300 ° C . above

o c t a c h l o r o d i b e n z o d i o x i n b e i d e n t i f i e d i n t h e t a r r y residue.

O n l y after 200°C

could

If the com

p o u n d is h e a t e d i n a sealed c a p i l l a r y , conversions of ~ 5 0 % are o b t a i n e d c l e a r l y i n d i c a t i n g a n e q u i l i b r i u m ( F i g u r e 2 ) . H e r e t h e area of the melt i n g e n d o t h e r m is u s e d to m e a s u r e t h e progress of t h e r e a c t i o n . 26

Only a

LANGER

E T A L .

ATM. Areon/ISCFH

SAMPLE :


27

Thermal Chemistry

CeClsOH

REF. glass beads

Pentachlorophenol

PROGRAM MODE

ORIGIN :

RATE

100

150

Τ SCALE I 50 SETTING

15 T£ .START

200

RUN NO. Δ

*

τ

DATE

1.0

* OPERATOR

°C

250

300

350

400

T. °C (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

Figure 1.

Differential thermogram of pentachlorophenol

80

60

40

+ HC1

CI 8

10

CI

12

TIME (Hours o f r x . a t 250°C)

Figure 2.

Thermal decomposition of

C Cl OH G

5

28

CHLORODIOXINS

ORIGIN

A N D FATE

s m a l l a m o u n t o f d i o x i n w a s d e t e c t e d i n t h e s a m p l e at t h e e n d o f t h e experiment—the

main product

being

the chlorinated

phenoxyphenol,

i n d i c a t i n g a n e q u i l i b r i u m f o r t h e first step of t h e c o n d e n s a t i o n

reaction.

A d r a s t i c a l l y different r e a c t i o n is i n d i c a t e d o n t h e t h e r m o g r a m o f sodium pentachlorophenate at ^ 3 6 0 ° C .

( F i g u r e 3 ) as a strongly e x o t h e r m i c

reaction

U p o n c o o l i n g , essentially p u r e o c t a c h l o r o - p - d i b e n z o d i o x i n


crystallized. SAMPLE:

SIZE_

CeClsONa

REF._

ATM.

micro ( v i a )

Τ

[Sodium Pentachlorophenate PROGRAM MODE ORIGIN:

0

RATE

50

100

Figure 3.

200 ° C ;

SCALE SETTING

15 ^ ,START_

50

RUN NO Δ

τ

1.0

*

DATE *

OPERATOR.

150 200 250 300 350 T, ° C (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

400

450

500

Differential thermogram of sodium pentachlorophenate

F o r 2,3,4,6-tetrachlorophenol above

Areon 1SCFH

hardly any discoloration was detected

instead the sample

vaporized rapidly.

r e a c t e d s i m i l a r l y to s o d i u m p e n t a c h l o r o p h e n a t e

Its s o d i u m

salt

except that i t s exo

t h e r m i c d e c o m p o s i t i o n ( F i g u r e 4 ) is less c l e a r l y d e f i n e d ; n o c r y s t a l l i z a tion occurred o n cooling, a n d the yield of hexachlorodioxins was m u c h l o w e r , t h e r e m a i n i n g p r o d u c t s b e i n g h i g h e r m o l e c u l a r w e i g h t materials. T h e h e x a c h l o r o d i o x i n s w e r e i d e n t i f i e d b y g a s - l i q u i d c h r o m a t o g r a p h y as t w o isomers i n a r a t i o o f 35:65. F o r l o w e r c h l o r i n a t e d phenates t h e reactions complicated.

T h e phenols

vaporize

s o d i u m salts react i n t h e m e l t dichlorophenate.

without

become even

decomposition,

more

a n d the

( F i g u r e 5 ) , as s h o w n f o r s o d i u m 2,4-

T h e r e l a t i v e a m o u n t o f d i o x i n s p r o d u c e d is l o w e r i n

favor of more highly condensed material.


4.

LANGER

100

Figure 4.

150 200 250 300 350 Τ , ° C (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

SIZE

Sodium 2,4-dichloropheni e

3

400

Differential thermogram of sodium 2,3,4,6-tetrachlorophenate

SAMPLE : NaOC H Cl

29

Thermal Chemistry

E T A L .

2

micro

ATM

PROGRAM MODE

ORIGIN:

RATE

1 5 ^ ώ .SIfART

RUN NO.

Arson

I τ

glass beads

°C

SCALE 1 50 SETTING

DAT!

Î -dioxin. Uniformly labeled T C D D - C was synthesized at the Radiochemistry Research Laboratory of The D o w Chemical C o . T h e specific activity was 2.8 /xCi/mg. The T C D D - C sample was analyzed by mass spectrometry and gas—liquid chromatography and indicated a purity of 93.3 and 95.0%, respectively. Dosage. T C D D was dissolved i n acetone; subsequently, one part of the acetone solution was added to and mixed with nine parts of U S P corn oil. The acetone-corn oil solution of T C D D was given to rats at a rate of 5 ml/kg by intubation. This volume of solution provided a dose of 50 fxg/kg and 0.14 C i / k g T C D D - C . Sample Collection. After administering the solution containing T C D D - C , the rats were placed in all glass metabolism chambers which were equipped for separate collection of urine, feces, and expired air. The C 0 i n the exiting air stream was trapped by bubbling it through 3:7 ethanolamine : 2-methoxyethanol. Radioactivity Analysis. Samples of urine, feces, and tissues were combusted to C 0 and analyzed for radioactivity ( 5 ) . B y using this method the recovery of radioactivity from samples spiked with C was 95 ± 5 % . T o determine the radioactivity expired as C 0 , 5-ml aliquots of the solution used to trap the C 0 were added to 15 m l of a scintillation counting solution containing 4 grams 2,5-diphenyloxazole ( P P O ) and 0.1 grams l,4-bis-2(5-phenyloxazolyl)-benzene ( P O P O P ) per liter of 1:1 toluene:2-methoxyethanol. Samples were counted for radioactivity i n a Nuclear Chicago Mark II liquid scintillation counter. Counting efficiency was corrected by the internal standard technique. 1 4

14

1 4

1 4

M

1 4

2

1 4

2

1 4

2

2

Results The percentage of the total dose of radioactivity excreted daily i n the feces, urine, or expired air over a 21-day period following a single oral dose of T C D D - C is shown in Figure 2. Approximately 30% of the 1 4

9.

PIPER E T AL.

87

Excretion and Tissue Distribution

C activity was excreted in the feces during the first 48 hours. Most of this probably represents unabsorbed T C D D - C . O n each of the remain ing 19 days, 1-2% per day of the C activity was excreted i n the feces. A total of 53.2 ± 3.8% of the administered dose was excreted via the feces over the 21-day period. The total cumulative amount excreted i n the urine and expired air was 13.2 db 1.3% and 3.2 d= 0.1%, respectively. 1 4

1 4


1 4

20

r

Q UJ HLU

g

X LU

'5-

Id CO Ο

DAYS Figure 2. Excretion of C activity by rats following a single oral dose of 50 μg/kg (0.14 μα/kg) 2,3,7,8-tetrachlorodibenzo-p-dioxin. Each point represents the mean ± SE for three rats. 14

To determine the overall rate of clearance of C administered as T C D D from the body, the total cumulative amount of C excreted i n feces, urine, and expired air at the end of each day was subtracted from the total dose administered to the animal. These values, representing the percentage of the total dose remaining i n the animal at the end of each day, were then plotted semilogarithmically as a function of time 1 4

1 4

88

CHLORODIOXINS

ORIGIN A N D F A T E

ζ 100* 80. QC 60. Id Ο

3CD

40.

>

Ο Ο


m

20

Ιϋ!

0

1

1

1

1

5

Î0 DAYS

15

20

Figure 3. Clearance of C activity from the body of rats given a single oral dose of 50 ^g/hg (0.14 μα/kg) 2,3,7,8-tetrachlorodibenzo-p-dioxin. Each value represents the mean ± SE for three rats. 14

(Figure 3 ) . Except for the first two days following administration, the clearance of C activity from the body followed apparent first order rate kinetics. The half-life for clearance, t , was 17.4 ± 5.6 days. As previ ously indicated, it was assumed that the relatively large amount excreted during the first 2 days had not been absorbed; therefore, these values were not used i n calculating the clearance rate. Analyses of tissues indicated that the C activity derived from T C D D and/or its breakdown products was located mainly i n the liver and fat (Table I ) . The percentages of the dose per gram of liver at 3, 7, and 21 days following administration were 3.18, 4.49, and 1.33, respec tively. Comparable values for fat were 2.60, 3.22, and 0.43% /gram. Smaller concentrations of C activity were found i n other tissues: muscle, testes, lungs, heart, skin, spleen, stomach, pancreas, brain, bone, kidneys, and adrenals (Table I I ) . Standard errors as large as the mean 1 4

1/2

1 4

1 4

Table I. C Activity Expressed as Percent of Dose per Gram (%/gram) in the Liver and Fat of Rats 3, 7, and 21 Days Following a Single Oral Dose of T C D D - C 1 4

1 4

Time Tissue Liver Fat

3 days 3.18±0.21 (47%) « 2.60=1=0.48

6

Post-Administration 7 days

21 days

4.49d=0.62 (45%) 3.22=fc0.63

1.33±0.70 (11%) 0.43

° Dose—50 μg/kg (0.14 y.Ci/kg); 3 rats/observation. Mean ± standard error. The percent of the total dose found in the entire liver. Mean for 2 rats. 6 c

d

a

d

9.

PIPER E T A L .

89


Table II. C Activity Expressed as Percent of Dose per Gram (%/gram) in Various Tissues of Rats 3, 7, and 21 Days Following a Single Oral Dose of TCDD- C° 1 4

14

Time


Tissue Muscle Testes Lungs Heart Skin Spleen Stomach Pancreas Brain Bone Kidneys Adrenals

21 days

7 days

3 days 0.38=1=0.01 0.38=1=0.03 0.27=1=0.02 0.20=1=0.03 0.19=1=0.10 0.15=1=0.02 0.16=1=0.05 0.11=1=0.06 0.06=1=0.00 0.09=1=0.03 0.00 0.79=L0.79

6

d

Post-Administration 0.20=1=0.12 0.11=1=0.09 0.06=1=0.05 0.09=b0.05 0.09=b0.04 0.22=1=0.22 0.02=b0.02 0.16±0.16 0.01=1=0.01 0.08=1=0.08 0.00 3.69=1=1.77

0.21=1=0.05 0.36=1=0.10 0.39=1=0.14 0.40=1=0.16 0.19=1=0.10 0.95=1=0.53 O.lOiO.OO 0.16 0.13=1=0.09 0.42db0.42 0.34=1=0.17 0.02=1=0.02 c

d

Dose—50 μg/kg (0.14 μΟί/1*£); 3 rats/observation. Mean =b standard error. Mean of 2 rats. No activity above background in all 3 rats. This large value may be experimental error. The DPM'S above background for the adrenals of the three rats were 17, 29 and 90. Since the total amount of tissue was 1 ess than 20 mg, the multiplication factor may have magnified the error many fold. a

h c

d

e

suggest that some of the values presented i n this table may result from experimental error: adrenals—3 days; bone—7 days; spleen—21 days; pancreas—21 days. Radioactivity exceeding background i n these tissues at the indicated time was detected i n only one of three rats. The value given i n Table II for adrenals 21 days following adminis tration suggests that this tissue may concentrate T C D D - C and/or a metabolite. This observation results probably from experimental error. The disintegrations per minute ( D P M ) above background for this tissue were only 17, 29, and 90. Since the total amount of tissue available for analysis was less than 20 mg, the multiplication factor may have magnified the error many fold. Total recovery of the administered C activity was determined for those rats used i n the 21-day experiment. T h e C activity remaining i n the unused carcass was determined b y analyzing an aliquot of a homoge nate of the remaining carcass. The recovery was 96.8 ± 3.0%. 1 4

1 4

1 4

Discussion In this study the tissue distribution and excretion of C activity has been evaluated i n rats following a single oral dose of T C D D - C . Almost 30% of the dose administered was eliminated via the feces during the first 48 hours following treatment. The excretion of C activity via the 1 4

1 4

1 4

90

CHLORODIOXINS


feces after the first 48 hours ranged from 1 - 2 % per day. It appears that T C D D is incompletely absorbed from the gastrointestinal tract. The C activity derived from the absorbed T C D D - C also is excreted mainly via the feces. Once absorbed i n the body, most of the C activity derived from T C D D - C is localized i n the liver and fat. The data suggest that the level i n these tissues is approximately 10-fold greater than that found i n other tissues. T h e C level i n liver and fat seemed to increase between 3 and 7 days following administration; the C activity i n liver and fat decreased more between 7 and 21 days than what would have been predicted by assuming that the rate of clearance from these tissues would be equal to the rate of clearance from the body. Between days 7 and 21 the C level i n muscle remained essentially unchanged. Therefore, re distribution of T C D D or metabolites of T C D D may have been occurring. The apparently high level i n the adrenals 21 days after administration results probably from experimental error. The dose of T C D D given to the male rats used i n this study, 50 /xg/kg, was approximately twice the L D , 23 μ-g/kg. This large dose was necessary because of the low specific activity of the T C D D - C used. In this study rats lost weight, and their physical condition was poor, which typifies the insidious response to T C D D ( 1 ). Survival of the rats for 21 days was not totally unexpected because i n previous studies on the lethality of T C D D deaths frequently occurred 20 or more days fol lowing a single oral dose of similar magnitude ( 1 ). W i t h doses that do not induce untoward effects, the compound may be excreted at a different rate. The results do not differentiate between C activity derived from T C D D and that of possible metabolites. However, small amounts of C activity were detected i n the expired air and urine within the first 10 days following administration. This is evidence that some metabolic alteration or breakdown of T C D D occurs. 1 4

1 4

1 4

1 4

1 4


1 4

1 4

5 0

1 4

1 4

1 4

Acknowledgment The authors thank W . W . Muelder for preparation of tetrachlorodibenzo-p-dioxin. Literature

14

C-2,3,7,8-

Cited

1. Rowe, V. K., Norris, J. M., Sparschu, G. L., Schwetz, Β. Α., Gehring, P. J., ADVAN. C H E M . SER. ( 1 9 7 3 ) 120, 55.

2. Sparschu, G. L., Dunn, F. L., Rowe, V. K., "Study of the Teratogenicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat," Food Cosmet. Toxicol. ( 1 9 7 1 ) 9, 4 0 5 .

3. Kimmig, J., Schulz, Κ. H., Dermatologia ( 1 9 5 7 ) 115, 540.

9.

PIPER E T A L .

91


4. Jones, E. L., Krizek, H., "A Technique for Testing Acnegenic Potency in Rabbits, Applied to the Potent Acnegen, 2,3,7,8-Tetrachlorodibenzo-p-dioxin," J. Invest. Dermatol. (1962) 39, 511. 5. Smith, G. N., Ludwig, P. D., Wright, K. C., Bauriedel, W. R., "Simple Apparatus for Combustion of Samples Containing C -Labeled Pesticides for Residue Analyses," Agr. Food Chem. (1964) 12, 172. 14


RECEIVED

February

8, 1972.

10

An Improved Analysis for Tetrachlorodibenzo-p-dioxins


ROBERT

BAUGHMAN

and M A T T H E W

MESELSON

Department of Chemistry and Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Mass. 02138

A meaningful assessment of the environmental 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD),

levels of

an extraordi-

narily toxic compound present as an impurity in the herbicide 2,4,5-T and in some commercial chlorophenols, can be made only by evaluating representative samples with a sufficiently sensitive analytical method. The sensitivity required is well beyond that available with current methods. We report a procedure using time averaged high resolution mass -12

spectroscopy with a sensitivity (10 an investigation.

gram) suitable for such

Interference from pentachlorobiphenyl

in

certain materials from the environment presently limits attaining

full sensitivity of the

method

although

we

are

working toward a resolution of this problem.

^ V u r interest i n the chlorodioxin problem stems from our work with the Herbicide Assessment Commission of the American Association for the Advancement of Science which was organized i n 1970 to initiate a study of the effects of herbicide use i n Vietnam. As one part of that investigation we are analyzing various samples from Vietnam for T C D D , a known impurity i n 2,4,5-T (1, 2, 3, 4). This herbicide i n a one-to-one mixture with 2,4-D is a component of agent Orange, the herbicide that was used most widely i n Vietnam. O u r aim has been to determine whether T C D D has accumulated i n food chains to any significant extent. W e were surprised to find that no method existed that was sensitive enough to detect T C D D i n animal tissues even after administration i n some species of lethal doses. A n example is the guinea pig, the most susceptible species of the few that have been tested, and therefore a good choice for establishing desirable limits of detection. The lethal single oral dose ( L D ) i n males of this species is 0.6 pg/kg body weight 5 0

92


10.

B A U G H M A N AND MESELSON

93

An Improved Analysis

(5). This means that if all of the T C D D were retained, the level of T C D D would be less than 1 part per billion (ppb) in the whole animal. The lowest reported limit of detection for T C D D in whole tissue is 50 ppb (6). Thus, a guinea pig could be killed with T C D D , and it would be impossible to establish this fact with the analytical procedures in current use. Such analytical procedures are clearly of little value i n monitoring food chains for the buildup of T C D D . This is even more apparent if one considers the possibility of sub-lethal toxic effects and allows a margin for a safety factor. If we provide a factor of about 100 for non-lethal toxicity (6) and a further factor of 10 for a safety margin and to allow for the possible existence of species even more sensitive than the guinea pig, we would require a level of detection of (10" )(10" )(10 ) = 10~ or 1 ppt (1 part i n 10 ) for environmental monitoring. For a 1 gram sample this would require a limit of detection of 1 pg ( 1 0 gram). The limit of detection of T C D D for the electron capture detector, the keystone of current analytical procedures, is not much less than 1 ng ( 1 0 gram ). In addition to high sensitivity, a requirement for any acceptable analytical method is high specificity because at very low levels few confirmatory procedures can be used to establish the identity of a particular compound. A method which uniquely combines high sensitivity with high specificity is high resolution mass spectrometry. W e have used this method as the basis for an approach which we believe w i l l make possible a meaningful assessment of T C D D levels in the environment. Figure 1 shows that the mass spectrum of T C D D is relatively simple. ( A l l the work reported here was done with an Associated Electrical In9

2

_1

12

12

1 2

9

Relative +

(M) -

Intensity (M-COCl) (M)

(M-2COCl)

+

+

(M-2C1)

(M n i (M-Cl)

+

250

+

Trichlorodibenzo-pdioxin*

— I — 300

m/e

Figure 1. Mass spectrum of 2,3,7,8-tetrachhrodibenzo-p-dioxin (TCDD). The molecular ion (M ) is at m/e 320. Ionizing voltage 70 eV, source 150°C. Asterisk denotes impuriiy. +

94

CHLORODIOXINS

dustries MS-9 double focusing mass spectrometer. ) The base peak is the molecular ion at m/e 320. As a result of the various possible combinations of the naturally occurring C 1 and C 1 isotopes in a tetrachloro compound, the signal for the molecular ion is a pentuplet with peaks at m/e 320, 322, 324, 326, and 328 with intensities in the ratio 77:100:49:10:1. In addition, the four chlorine atoms and the limited number of hydrogen atoms make the compound significantly mass deficient ( the m/e 320 peak is actually 319.8956) and, therefore, relatively easily resolved from most other organic residues (for which m/e 320 would be 320.1-320.2). First, we tried scanning the region m/e 310-330 (Figure 2). As the sample was introduced into the mass spectrometer, signals appeared at m/e 320, 322, and 324 and then, as the sample became exhausted, disappeared. Under these conditions the limit of sensitivity was on the order of 100 pg. W e next reduced the scanning interval to about one third of a mass unit. This allows the detector to spend more time in the region of interest, considerably increasing the signal. A t a resolution of 10,000 a series of scans was made, alternately two at 322, two at 314 perfluorotributylamine ( P F A ) reference peak, two at 322, etc. (Figure 3). The P F A was bled in from an external reservoir at a constant rate, providing reference peaks that remain at the same height throughout the analysis while the sample peaks rise and then fall as the sample volatilizes. This procedure with a sensitivity of about 20 pg was still not adequate. It is possible to obtain greater sensitivity from the repeated narrow scans shown in Figure 3 by combining them to produce a single time averaged scan. Procedures accomplishing this under low resolution conditions have been reported previously (7, 8). Under the present conditions a system was devised for doing this using a Varian 1024 averaging computer ( C A T ) in conjunction with the M S - 9 . The result is shown in Figure 4. The signal for a pair of peaks at the limit of detection for a single scan is shown in Figure 4A, and the averaged signal from sixty scans is shown in Figure 4B. The signal-to-noise ratio is expected to improve approximately as the square root of the number of scans (9). W i t h 1 min of scanning at a rate of one scan per second, the observed improvement is approximately that expected. A t very fast scan rates data is inefficiently transferred to the memory of the C A T , and resolution is decreased by damping caused by the time constant of the M S - 9 circuitry. In the present system this limits the maximum scan rate to four scans per second. W i t h very short volatilization times ( < 10 sec) sensitivity is decreased, perhaps i n part because of decreased ionization efficiency. W i t h volatilization times longer than about 60 sec the drift in peak position from scan to scan is large enough to decrease significantly the resolution observed in the time averaged spectrum. The optimum volatilization time is from 30 to 60 sec. 35



37

10.


95



The interfacing of the C A T with the M S - 9 is illustrated i n Figure 5. The ions i n the m/e region of interest, after being focussed, pass b y a small magnet coil which deflects the beam back and forth over the detector slit. After passing through the slit, the ions strike an electron

AXA; 314

314

326

320

322

324

326

328

Figure 2. Repetitive scanning of m/e 310-330 (5 sec/scan). Standard conditions for this and all following figures: ionizing voltage 70 eV, ac celerating voltage 8 W, trap current 300 μ A, multiplier 600, source 150° C.

96

CHLORODIOXINS

u

U


314(PFA)

il

kJ

U


322(TCDD)

1U*

I II

II It 11 I

314 322314 322 314

Figure 3. Top:

Alternating narrow scans

regions scanned on each narrow scan. Bottom: alternating narrow scans (two half-second scans at 314, two at 322, etc.) (200 pg TCDD).

multiplier, producing a signal which is continuously displayed on an oscilloscope on the M S - 9 . This provides a means of monitoring each scan. Simultaneously, the signal is added to the 1024-channel memory of the C A T . A n oscilloscope on the C A T continuously displays the total memory content which makes it possible to monitor the overall course of the analysis. A potential problem of phasing the beam deflection coil with the memory sweep circuit of the C A T is avoided b y using the sweep voltage ramp of the C A T , via an amplifier and appropriate circuits of the M S - 9 , to drive the beam deflection coil. The coil is thus necessarily i n synchrony with the C A T . The procedure we have adopted for introducing samples into the M S - 9 is shown i n Figure 6. It provides reproducible analyses at a high level of sensitivity. The sample tubes are made from 1 mm i d melting point capillaries. A Hamilton 10-μΙ syringe is used to introduce a 3-4 μ\ portion of the residue into the sample tube. W i t h a small flame the sample tube is drawn out just above the level of the liquid to pro duce a capillary constriction about 20 m m long. The solvent is then


10.


97


removed at reduced pressure. Bumping is prevented b y the capillary constriction. The sample tube is then sealed with a flame. A t the time of analysis the capillary is broken off 2-3 m m above the constriction to give the tube configuration shown i n Figure 6. The tubes are introduced into the M S - 9 with a wire holder on the tip of a standard M S - 9 direct insertion probe. T o aid reproducibility a l l analyses are started at the same time after insertion of the sample tube into the M S - 9 source. The temperature of the source heating block is adjusted to give a sample volatilization time of 30 to 60 seconds. The result of combining these various components i n the analysis of a 2-pg sample of T C D D is illustrated i n Figure 7. A n internal standard is given by a P F A fragmentation peak, which is a known distance, 85 mmu (1 millimass unit or m m u = 10~ atomic mass unit), from the T C D D peak. In its present form the M S - 9 - C A T system has a limit of detection for T C D D of about 1 pg. The procedure we have described retains the generality of normal mass spectral analysis. It is particularly suited, however, to compounds 3

314.960

315.040

314.960

m/e

_l_

_L_

315.040

m/e

Figure 4. Improvement in sensitivity with the CAT. PFA and a reference peak at m/e 315. The observed improvement in signal-to-noise ratio results from the longer total scanning time and also the fact that many sweeps are made during this time. The overall improvement in signal-to-noise ratio depends on the detailed power spectrum of the noise (9). Resolution 12,000 here and for all following time averaged spectra Left: one scan. Right: 60 scans (one scan/sec).


98

CHLORODIOXINS


containing atoms with significant mass defects, such as heavy metal or organochlorine compounds, which are easily resolved from other residues. Before the procedure is applied to tissue or other samples from the environment, some potential complications must be taken into account. One is the possibility that other chlorinated organic compounds present in the environment might interfere with or obscure the T C D D peaks. To test this, we obtained mass spectra on the M S - 9 for most of the common organochlorine pesticides including lindane, aldrin, dieldrin, mirex, heptachlor, D D D , D D E , and D D T , as well as various polychlorinated biphenyl ( P C B ) mixtures. In the T C D D mass range D D E from its molecular ion has isotopic isomer peaks at m/e 320, 322, and weakly 324. The molecular ion of pentachlorobiphenyl, a component of some P C B mixtures, has a peak at m/e 324, and this compound has a weak fragmentation peak at m/e 322. D D T has weak fragmentation peaks at m/e 320, 322, and 324. As shown for m/e 322 in Figure 8, all of these compounds can be resolved from T C D D at our normal resolu tion of 12,000 (27 mmu at m/e 322). The relative input amounts of each compound producing the peaks shown are: D D T , 250; D D E , 25; T C D D , 1; P C B (Arochlor 1254), 250. Even though moderately large excesses of these interferences can be tolerated, it is necessary to use highly

Α Ε Ι MS-9

D o u b l e F o c u s s i n g Mass S p e c t r o m e t e r

Electron Oscilloscope

Multiplier

Beam Deflection Coil

Source

Memory XY

Circuits

Oscilloscope

V a r i a n 1024

Figure 5.

CAT

CAT-MS-9 interfacing

Recorder

10.

B A U G H M A N A N D MESELSON

An Improved

99

Analysis

Ionizing beam

Probe shaft


1 cm I 1

Sample tube Figure 6. Internal

Sample

introduction

standard

TCDD

Internal

standard

I

320.000

319.900 m/e Figure 7.

Top:

Limit of detection

2 pg(2 X 10~ gram) TCDD. 12

Bottom: background.

100

CHLORODIOXINS



efficient cleanup procedures to carry out analyses for T C D D at very low levels. Another complicating characteristic of materials from the environ ment is that the size and nature of the residue to be analyzed in the mass spectrometer w i l l change from sample to sample. To determine if this might have an effect on the observed T C D D signal, we analyzed identical samples of T C D D with differing amounts of squalane, a saturated hydro carbon selected as a model for residues obtained from standard extraction and cleanup procedures. As is indicated in Table I (Part A ) , there was

DDT DDE R e l a t i v e amts:

250

t-~—

h 322.000

—ι

TCDD PCB

25

1

•

-~

Stondord

250 -

1

1

1

321.900

321.800

m/e

Figure 8. A:

DDT

Resolution of TCDD from interferences

+ TCDD. B: DDE + TCDD. C: PCB (Arochlor 1254) DDT + DDE + PCB. E: DDT + DDE + PCB +

+ TCDD. TCDD.

D:

10.


Table I. A. Response for


Effect of Size of Total Residue

TCDD

Squalane added (micrograms)

Relative response for 20 pg


Ratio of two components

a

Squalane added (micrograms) 1 3 9

TCDD

100 75 50 15

0 1 5 25 B.

101

TCDD

IΤBB 11 5 1

° Ratio of response for 20 pg T C D D to response for 200 pg of 2,3,5,6-tetrachloro-4bromoethylbenzene ( T B B ) .

a significant effect on the response to T C D D . This eliminates any hope of determining the amount of T C D D present directly from the area of the T C D D peak. In an attempt to resolve this difficulty, we added an internal standard which does not coincide with T C D D or with peaks from any of the potential chlorinated hydrocarbon impurities and mea sured the ratio between T C D D and the standard in the presence of vari ous amounts of squalane. A compound with a suitable mass, 2,3,5,6tetrachloro-4-bromoethylbenzene ( T B B ) , was synthesized for this pur pose. However, as is apparent from Table I (Part B ) , the ratio of its signal to that of T C D D was far from constant, and this procedure was ruled out. Table I also suggests that in order not to cause a significant loss of sensitivity, the total sample size should be kept under 5 /xg. Some alternative method had to be devised to quantify the T C D D measurements. The problem was solved with the observation, illus trated in Figure 9, that the response to T C D D is linear over a wide con centration range as long as the size and nature of the sample matrix remain the same. Thus, it is possible to divide a sample into two equal portions, run one, then add an appropriate known amount of T C D D to the other, run it, and by simply noting the increase i n area caused by the added T C D D to calculate the amount of T C D D present in the first portion. Figure 9 illustrates the reproducibility of the system. E a c h point was obtained from four or five independent analyses with an error (root mean square) of 5-10%, as indicated by the error flags, which is acceptable for the present purposes. To satisfy the requirement of having a total residue of only a few micrograms, the sample cleanup must be very thorough (10). The pro cedure which we have used to accomplish this is the following. A 10-


102

CHLORODIOXINS


TCDD (pg)

Figure 9. Linearity of response and reproducibility. The error flags indicate the root mean square error for five measurements at each value. The average relative error is about 10%. gram sample of human milk was combined with 10 m l of ethanol (all solvents were of pesticide grade) and 20 m l of aqueous 4 0 % K O H and refluxed for 6 hours. This solution was then extracted with four 6-ml portions of 5 % benzene in hexane. The organic phase was extracted with four 8-ml portions of 8 5 % H S 0 , filtered through a 10 m m i d column of 10 grams of powdered N a C 0 , concentrated carefully to about 10 μ\, and preparatively chromatographed by gas-liquid chromatography on a 1 m X 1/4" column of 3 % O V - 1 0 1 methyl silicone polymer liquid phase on 50/60 mesh Anakrom A B solid support (200°C column, 30 ml/min H e ) . The G L C trap consisted of a 150 mm X 1.5 m m i d borosilicate glass tube packed with 30 mm of 100/120 mesh glass beads retained with glass wool plugs ( 11,12). The trap was wetted with hexane and cooled in dry ice. The G L C cleanup is carried out through a thermal conductivity detector. A small amount of an internal standard, m-terphenyl, with a known retention time relative to T C D D was added to make certain that the T C D D collection was carried out at the right retention time. The total residue from the G L C cleanup when divided into twelve fractions provided a suitable sample size. 2

2

4

3

Figure 10 illustrates the results of a typical analysis of a 10-gram sample of human milk to which 0.1 ppb T C D D has been added. Blank


10.


I—ι 322.000

1

103


i 321.900

1

1 321.800

1

m/e Figure 10. TCDD recovery in 10 gram human milk to which 10~ gram (0.1 ppb) TCDD was added. Each trace represents 8% of the total residue. Top: residue (25% recovery). Bottom: residue -f- 20 pg TCDD. 9

samples showed no indication of T C D D at this level. E a c h trace repre sents 8 % of the total sample and since the area under the unknown T C D D peak, calculated from the run with T C D D added, corresponds to approximately 20 pg, the recovery is about 2 5 % . The other signals observed i n the T C D D mass region are probably from D D E and pentachlorobiphenyl. A t the present time the major impediment to extending the procedure to the desired level of 1 ppt is interference from pentachlorobiphenyl. W e are investigating changes i n the cleanup procedure which show excellent promise of reducing the level of this and other interferences i n the final residue. W e are also developing a procedure for directly measuring the recovery of T C D D i n each cleanup by adding chlorine-[ C1] labelled T C D D to the sample before cleanup. 37

Acknowledgment W e would like to thank D a v i d Firestone of the Division of Chemistry and Physics, F D A for providing us with samples of T C D D , Klaus Biemann and Charles Hignite of the Department of Chemistry, Massachu setts Institute of Technology for assistance i n the early stages of this work, D a v i d Parrish of the Department of Chemistry, Harvard U n i versity for assistance i n developing the M S - 9 - C A T system, and W i l l i a m Doering of the Department of Chemistry, Harvard University for the use of laboratory facilities. This work was supported by the Herbicide Assess-

104

CHLORODIOXINS


ment Commission of the American Association for the Advancement of Science and by the F o r d Foundation.


Literature

Cited

1. Brenner, K. S., Müller, K., Sattel, P.,J.Chromatogr. (1972) 64, 39. 2. Elvidge, D. Α., Analyst (1971) 96, 721. 3. Johnson, J. E., Hearings Before the Subcommittee on Energy, Natural Re sources, and the Environment of the Committee on Commerce, United States Senate on "Effects or 2,4,5-T on Man and the Environment," April 7 and 15, 1970. 4. Woolson, Ε. Α., Thomas, R. F., Ensor, P. D. J.,J.Agr. Food Chem. (1972) 20, 351. 5. "Report on 2,4,5-T," A Report of the Panel on Herbicides of the President's Science Advisory Committee, Executive Office of the President, Office of Science and Technology (March 1971). 6. Woolson, Ε. Α., Reichel, W. L., Young, A. L., ADVAN. C H E M . SER. (1973) 120, 112. 7. Biros, F. J., Anal. Chem. (1970) 42, 537. 8. Plattner, J. R., Markey, S. P., Org. Mass Spec. (1971) 5, 463. 9. Ernst, R. R., Rev. Sci. Inst. (1965) 36, 1689. 10. Ress, J., Higginbotham, G. R., Firestone, D.,J.Ass. Offic. Anal. Chem. (1970) 53, 628. 11. Bierl, Β. Α., Beroza, and Ruth, J. M.,J.Gas Chromatogr. (1968) 6, 286. 12. Murray, Κ. E., Shipton, J., Robertson, Α. V., Smyth, M. P., Chem. Ind. (1971) 401. R E C E I V E D February 8, 1972.

11

Environmental Significance of Chlorodioxins

PHILIP C. K E A R N E Y , A L L A N R. I S E N S E E , C H A R L E S S. H E L L I N G ,


E D W I N A . W O O L S O N , and JACK R. P L I M M E R Agricultural Environmental Quality Institute, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Md. 20705

An environmental protocol has been developed to assess the significance of newly discovered hazardous substances that might enter soil, water, and the food chain. lished laboratory procedures and chlorodibenzo-p-dioxin

(TCDD),

14

C-labeled

Using estab2,3,7,8-tetra-

gas chromatography, and

mass spectrometry, we determined mobility of TCDD

by soil

TLC

in five soils, rate and amount of plant uptake in oats

and

soybeans, photodecomposition rate and nature of the

products, persistence in two soils at 1, 10, and 100 ppm, and metabolism rate in soils. We found that TCDD

is immobile

in soils, not readily taken up by plants, subject to photodecomposition, persistent in soils, and slowly degraded in soils to polar metabolites.

Subsequent studies revealed that the

environmental contamination by TCDD

is extremely small

and not detectable in biological samples.

"TVetection of the highly potent impurity, 2,3,7,8-tetrachlorodibenzo-pdioxin ( T C D D ) i n the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), necessitated an environmental assessment of the impact of this contaminate. Information was rapidly needed on movement, persistence, and plant uptake to determine whether low concentrations reaching plants, soils, and water posed any threat to man and his environment. Because of the extreme toxicity of T C D D , utmost precautions were taken to reduce or minimize the risk of exposure to laboratory personnel. Synthesis of uniformly labeled C - T C D D by Muelder and Shadoff ( I ) greatly facilitated T C D D detection i n soil and plant experiments. F o r unlabeled experiments it seemed wise to use only small quantities of diluted solutions i n situations where decontamination was feasible and to rely on the sensitivity afforded by electron capture gas chromatography 1 4

105

106

CHLORODIOXINS


as reported by Woolson et al. (2). In addition to movement, persistence, and plant uptake studies, it was essential to determine whether chlorinated phenols could be condensed to their corresponding chlorodioxins by photochemical or biochemical processes under natural conditions.


Table I.

Properties of Soils Used in Mobility Studies with the Chlorodioxins

Soil Type

Organic Matter,

Norfolk sandy loam Lakeland sandy loam Hagerstown silty clay loam Barnes clay loam Celeryville muck

0.14 0.90 2.50 6.90 90.4

%

Clay,

%

11.3 12.0 39.5 34.4

Field Moisture Capacity,

%

pH

6.5 8.5 25.8 28.5 113

5.1 6.4 6.8 7.4 5.0

Mobility The mobilities of two chlorinated dioxins, 2,7-dichlorodibenzo-pdioxin ( D C D D ) and T C D D , and 2,4,5-T were evaluated by the soil thin layer chromatography ( T L C ) technique developed by Helling and Turner ( 3 ) . This is a simple, safe technique (i.e., it is easy to decontaminate), that gives comparative data for other chemical compounds analyzed i n the same system. The procedure uses the conventional techniques used i n preparing silica gel or alumina thin layer plates. Instead of silica gel, or alumina, however, sieved soils are used as the solid support. Radioisotopically-labeled compounds are spotted at the origin, and the plate is developed with water, dried, and covered with no screen x-ray film to locate the compound. Five soils varying in texture and other properties were tested: Norfolk and Lakeland sandy loams, Hagerstown silty clay loam, Barnes clay loam, and Celeryville muck (Table I ) . These soils gave a cross section of important soil parameters affecting mobility. Both dioxins were immobile i n a l l soils—i.e., they would not be leached into the soil by rainfall or irrigation (4). The relative immobility of the chlorodioxins is expected, based on the very low solubility of these compounds i n water (0.6 /^g/liter). In contrast, the herbicide, 2,4,5-T, is relatively mobile i n sandy soils, but movement decreases as soil organic matter increases. What does this information tell us, and how does it compare with other organic compounds? A mobility scale has been devised for a large number of pesticides ( 3 ). Higher mobility numbers reflect increased compound mobility in soils. The dioxins would be i n Class 1—i.e., they are immobile i n soils and would compare with several chlorinated hydrocarbon insecticides.

11.

KEARNEY E T A L .

107

Environmental Significance


Thus, the dioxins would not threaten groundwater supplies and would be subject to the surface processes affecting pesticides.

20 °

20

40

80

160

350

DAYS

Figure 1.

Persistence of TCDD in Lakeland sandy loam applied at 1, 10, 100 ppm and sampled after 20, 40, 80, 160, and 350 days

ο

tr 40 Ul Q_

20 20

40

80

160

350

DAYS

Figure 2.

Persistence of TCDD in Hagerstown silty clay loam at 1, 10, 100 ppm and sampled after 20, 40, 80, 160, and 350 days

Persistence The objectives of the soil persistence experiments were ( 1 ) to learn the effect of soil type and concentration on the T C D D degradation rate, (2) to isolate and characterize degradation products from D C D D and T C D D , and (3) to determine whether chlorodioxins could be formed from chlorophenol condensation i n the soil environment. This last study was essential since quality control at the manufacturing level could reduce or eliminate the formed dioxin impurity. But the biosynthesis of chlorodioxins by chlorophenol condensation i n the soil environment could not be controlled and would have connotations for all chlorophenol-derived pesticides if formation d i d occur. The same question needed to be answered for photochemical condensation reactions leading to chloro-


108

CHLORODIOXINS


dioxin formation. No evidence of D C D D or T C D D could be found in soils receiving 10, 100, and 1000 ppm of the 2,4-dichlorophenol or the 2,4,5-trichlorophenol, as determined by electron capture gas chromatography. Plimmer and Klingebiel (5) found no evidence for photocondensation of lower chlorophenols to chlorodioxins. Therefore, the possibility of chlorodioxins arising as biochemical or photochemical processes in the environment seems remote. These findings were critical for assessing the continued use of chlorophenolic pesticides or any chlorophenol used on a large scale basis. To determine the persistence of chlorodioxins, concentration rates of 1, 10, and 100 ppm of unlabeled T C D D were established in 300-gram soil samples and then assayed periodically for dioxin residues. Several factors were considered in selecting these rates—they are many times greater in magnitude than would be encountered in the field. First, since the stability of these dioxins in soils was unknown, three concentrations were used to determine the degradation pattern, particularly if the process were rapid. Second, the highest concentration was to be used for product identification studies. Finally, preliminary problems in detection necessitated a range of concentrations exceeding 1 ppm since the limit of detection was ca. 0.2 ppm under the conditions used. Measurements on dioxin residues after 20, 40, 80, 160, and 350 days of incubation of 28 ± 3 ° C in foil sealed beakers indicated a relatively slow degradation process in both soils (Figures 1 and 2). A t 350 days, 56% of the initially applied T C D D was recovered from the sandy soil and 6 3 % from the silty clay loam for all concentrations. A second experiment was conducted using C - T C D D at 1.78, 3.56, and 17.8 ppm in the same two soils. After 350 days the soils were combusted, and C 0 trapped and determined by liquid scintillation. The amount recovered from Lakeland was 67% at 1.78 ppm, 70% at 3.56, and 73% at 17.8 ppm. For Hagerstown the recoveries were 52%, 52%, and 89% over the same concentrations. The C and gas—liquid chromatography ( G L C ) data are not directly comparable although they seem to be in the same range of recovery. The C data might reflect the parent compound plus any labeled metabolites; therefore, the recovery values may be higher than the G L C values. A major metabolite was detected in the ethanol extract of 2,7-dichlorodioxin-treated soils. The metabolite was less mobile than D C D D in benzene-acetonitrile on T L C . The metabolite was eluted from the silica gel and methylated with diazomethane. The methylated metabolite was rechromatographed in benzene and migrated to the solvent front, suggesting a polar group on the non-methylated metabolite. The soil persistence data suggested that T C D D is a relatively persistent compound. This is relative to various pesticides in the same con1 4

1 4

1 4

1 4

2

11.

KEARNEY E T A L .

109


centration range. As pointed out by Kearney et al. (6), a concentration of 1 p p m of T C D D in soil is 10 greater than the amount that would be found in soil receiving 2.24 kg/ha of 2,4,5-T with a contamination level of 1 ppm T C D D . 6


—

PPM (DRY TISSUE %0F TCDD IN SOIL TRANSLOCATED TO TOPS

DAYS AFTER PLANTING

Figure 3. Uptake of C-TCDD from Lakeland sandy loam by soybeans 14

PPM(DRY TISSUE) %0F TCDD IN SOIL TRANSLOCATED TO TOPS

10 15 20 25 30 DAYS AFTER PLANTING

40

Figure 4. Uptake of C-TCDD from Lakeland sandy loam by oats 14

Plant Uptake Plant uptake is one of several routes by which an organic contaminant can enter man's food chain. The amount of uptake depends on plant species, concentration, depth of placement, soil type, temperature, moisture, and many other parameters. Translocation of the absorbed material into various plant parts w i l l determine the degree of man's exposure—i.e., whether the material moves to an edible portion of the plant. Past experience with nonpolar chlorinated pesticides suggested optimal uptake conditions are achieved when the chemical is placed i n a soil with low adsorptive capacity (e.g., a sand), evenly distributed throughout the soil profile, and with oil producing plants. Plant experiments were conducted with one set of parameters that would be optimal for uptake and translocation. The uptake of two dioxins and one phenol ( 2,4-dichlorophenol ( D C P ) ) from one soil was measured i n soybean and oats ( 7 ) . The application rates were D C P = 0.07 ppm, D C D D = 0.10 ppm, and T C D D = 0.06 ppm. The specific activity of the com-

110

CHLORODIOXINS


pounds were: 5.8 /xC/mg, 3.6 /xC/mg, and 2.8 juC/mg, respectively. Oats and soybeans (at all growth stages) accumulated very small quantities of chlorodioxins or the chlorophenol ( Figures 3 and 4 ). A maximum of 0.21% of the phenol and 0.15% of the dioxin present in soils was translocated to the aerial portion of oats and soybeans. N o detectable amounts of C were found i n the grain or soybeans harvested at maturity. The amount of T C D D applied to these soils would be many thousands times greater than that which would occur i n soils from herbicide applications containing a few parts per million of T C D D as an impurity. Even with these excessive rates in soil, significant amounts could not be measured in plants. Based on these studies, we must conclude that soil uptake of T C D D residues by plants is highly unlikely. Next, we attempted to deal with translocation of foliar-applied T C D D . Labeled dioxins were applied to the center leaflet of the first trifoliate leaf of 3-week-old soybean plants and the first leaf blade of 12-day-old oat plants. A l l compounds were applied in an aqueous surfactant solution (Tween 80) to enhance leaf adsorption and to keep the water insoluble dioxins in solution. Plants were harvested 2, 7, 14, and 21 days after treatment, dissected into treated and untreated parts, and analyzed separately. Neither dioxin nor chlorophenol was translocated from the treated leaf. A rapid loss of the dichlorodioxin and dichlorophenol occurred from the leaf surface. This loss may have resulted from volatilization. Very little T C D D was lost from soybean leaves while a gradual loss (38% in 21 days) d i d occur from oat leaves.


1 4

Summary The detection of a potent dioxin impurity in a major herbicide has focused attention on the nature of chlorinated impurities i n pesticides, and i n a larger sense, impurities in all chlorinated industrial compounds used extensively i n man's environment. The present 2,4,5-T controversy is overshadowed by the dioxin problem. Major disagreement still exists on their relative contributions to the teratogenic effects observed in chicks and the validity of interpretation of high dosage rates used to achieve these effects. W e have avoided any assessment of the healthrelated aspects of dioxins but have dealt almost exclusively with dioxins as an environmental entity. Information on dioxins i n the environment was acquired rapidly by using some simple, but safe and reliable techniques developed for chlorinated pesticdes. Based on results of these tests, one should be able to predict whether routes of entry into aquatic and terrestrial food chains are significant, the rate and products of decomposition mechanism, and their general longevity i n the environment.


11.

KEARNEY E T A L .


111

Several facts have emerged from our studies with 2 , 7 - D C D D and 2,3,7,8-TCDD. They are not biosynthesized by condensation of chlorophenols i n soils, and they are not photoproducts of 2,4-dichlorophenol. They do not leach into the soil profile and consequently pose no threat to groundwater, and they are not taken up by plants from minute resi dues likely to occur i n soils. Photodecomposition is insignificant on dry soil surfaces but is probably important i n water. Dichlorodibenzo-pdioxin is lost by volatilization, but T C D D is probably involatile. These compounds are not translocated within the plant from foliar application, and they are degraded i n the soil. Literature

Cited

1. Muelder, W. W., Shadoff, L . Α., ADVAN. C H E M . SER. (1973) 120, 1.

2. Woolson, Ε. Α., Thomas, R. F., Ensor, P. D. J.,J.Agr. Food Chem. (1972) 20, 351. 3. Helling, C . S., Turner, B.C.,Science (1968) 162, 562. 4. Helling, C . S., Soil Sci. Amer. Proc. (1971) 35, 737. 5. Plimmer, J. R., Klingebiel, U. I., Science (1971) 174, 407. 6. Kearney, P.C.,Woolson, Ε. Α., Ellington, C. P. Jr., Environ. Sci. Tech. (1972) 6, 1017. 7. Isensee, A. R., Jones, G. E.,J.Agr. Food Chem. (1971) 19, 1210. R E C E I V E D February 8, 1972.

12

Dioxin

Residues

in

Lakeland

Sand

and

Bald

Eagle Samples

E D W I N A. W O O L S O N and P E T E R D . J. ENSOR


Plant Science Research Division, Agricultural Research Service, U . S. Department of Agriculture, Beltsville, M d . 20705 WILLIAM L . REICHEL U . S. Department of Interior, Patuxent Wildlife Center, Laurel, Md. 20810 ALVIN L.

1

YOUNG

U . S. Air Force, Eglin Air Force Base, Fla. 32542

2,3,7,8-Tetrachlorodibenzo-p-dioxin

(TCDD)

is a

contaminant of 2,4,5-trichlorophenoxyacetic Higher

chlorinated

pentachlorophenol.

dioxins are possible

acid

possible (2,4,5-T).

contaminants of

To assess the ecological importance of

chlorinated dioxins, eagle tissue was examined by electron-capture gas chromatography for the presence of dioxins. No dioxins were detected at a minimum detection limit of 50 ppb.

Eagle samples from various regions in the

States were included. Florida

were analyzed for TCDD

lbs 2,4,5-T/acre

United

Soil samples from a Lakeland sand in after applying up to 947

(between 1962-1969). No TCDD

was de-

tected at a minimum limit of -dioxin. Purified 2,4,5-trichlorophenol (50 grams, 0.26 mole) was converted to its potassium salt and dissolved in 100 m l of b E E E . After addition of the copper catalyst and ethylene diacetate, the mixture was transferred to the bottom of a 300-ml sublimer with chloroform. Sublimation (200°C/2 mm) yielded 14 grams (39% yield) of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mass spectral analysis revealed trace quantities of pentachlorodibenzo-p-dioxin, tetrachlorodibenzofuran, and several unidentified substances of similar molecular weight. The combined impurity peaks were estimated to be < 1 % of the total integrated G L C area. The product was further purified by recrystallizations from o-dichlorobenzene and anisole. The final product had an estimated 260 p p m of trichlorodibenzo-p-dioxin as the only detected impurity. Hexachlorodibenzo-£-dioxin. 2,3,4,6-Tetrachlorophenol was purified by distillation and recrystallization to yield a product containing < 0 . 1 % trichlorophenol impurity. The phenol was dissolved i n toluene and mixed with an equimolar amount of aqueous caustic. Water was azeotropically Table III.

G L C and Mass Spectrometry Analysis

GLC Peak Area, %

Observed Parent Ion m/e

80

252

253

15

288

289.5

Structure

Molecular Weight

H

5

162

OH

163


134

CHLORODIOXINS


distilled. After trituration of the sodium 2,3,4,6-tetrachlorophenate with toluene, the salt was ground to a powder. As i n a typical pyrolytic preparation, powdered sodium tetrachlorophenate ( 30 grams ) was placed evenly on the bottom of a 300-cc Nester-Faust sublimer. The salt was covered with 20 grams of C a O and a glass wool pad. The vessel was immersed i n a sand bath and heated at 350°C under reduced pressure. Temperatures over 380°C induced localized hot spots which caused smoking or bumping. Product crystals began to form on the sublimer walls after 2 hours of heating. Several product samples were isolated from their reaction mixtures by extraction with o-dichlorobenzene. The o-dichlorobenzene extracts were combined and analyzed by G L C . Four peaks were observed under standard G L C conditions in the 10 to 15 min retention time range which is characteristic of hexachlorodibenzo-p-dioxins (sample 1 in Table I V ) . The mixture was fractionally sublimed (120° to 175°C/1 m m ) . The major crop was harvested at 175 °C and recrystallized from anisole. Analysis of this material by G L C indicated that two isomeric hexachlorodibenzo-p-dioxins were present (sample 2). Overall yield (1.5 grams) of the product was 1 - 3 % at 9 9 + % purity, as determined by G L C and mass spectrometry. Table IV.

G L C Analysis of o-Dichlorobenzene Extracts

Sample

Description

Peak

GLC Retention Time, min

% of Total

1

o-dichlorobenzene extract

A Β C D

10.8 12.4 13.4 14.0

16 65 10 9

2

sublimed and recrystallized

Β C

12.0 13.5

55 45

Octachlorodibenzo-^-dioxin. Pentachlorophenol was purified by sublimation and recrystallization to yield a product with the following composition: trichlorophenol, 0.04%; tetrachlorophenol, 0.07%; and pen tachlorophenol, 100.4 ± 1 % . Pentachlorophenol (300 grams, 1.13 mole) was dissolved in 900 m l of trichlorobenzene and chlorinated anhydrously for 18 hours at reflux. Chlorine addition was stopped and the mixture was heated for 28 more hours at reflux. The crystalline product was washed with 2-liter portions of chloroform, I N N a O H , methanol, and water. Analysis by G L C suggested the presence of 5-15% heptachlorodibenzo-p-dioxin. The mixture was carefully added to a cleaning solution of 200 m l water, 3.5 liters sulfuric acid, and 125 grams sodium dichromate. The mixture was heated at 150 °C for six hours. The product was recrystallized from hot o-dichlorobenzene and then from anisole. The purified product (160 grams, mp 329.8° ± 0.5°C) contained

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