Pesticide and Xenobiotic Metabolism in Aquatic Organisms - American

13

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Modeling Aquatic Ecosystems for Metabolic Studies ALLAN R. ISENSEE, PHILIP C. KEARNEY, and GERALD E. JONES U.S. Department of Agriculture, SEA, AR, Agricultural Environmental Quality Institute, Pesticide Degradation Laboratory, Beltsville, MD 20705

M o d e l e c o s y s t e m s have b e e n u s e d f o r a b o u t 8 y e a r s t o meas ure t h e d i s t r i b u t i o n and f a t e o f p e s t i c i d e s i n t h e a q u a t i c e n v i r o n m e n t . Over t h a t p e r i o d o f t i m e numerous d e s i g n c h a n g e s have e v o l v e d t h a t h a v e i n c r e a s e d t h e v e r s a t i l i t y o f t h e e c o s y s tem a n d i m p r o v e d s i m u l a t i o n o f e n v i r o n m e n t a l c o n d i t i o n s . I n o u r l a b o r a t o r y , we have u s e d t h e s t a t i c model e c o s y s t e m p r i m a r i l y t o model t h e pond o r s m a l l l a k e e n v i r o n m e n t , a n d t o s i m u l a t e t h e l i k e l y r a t e s a n d modes o f p e s t i c i d e e n t r y ( 1 ) . More r e c e n t l y , we have d e v e l o p e d l a r g e r s y s t e m s c a p a b l e o f p r o v i d i n g s u f f i c i e n t biomass f o r a c c u m u l a t i o n and d i s s i p a t i o n r a t e d e t e r m i n a t i o n s (2) and f o r m e t a b o l i c s t u d i e s ( 3 ) . The p r i m a r y p u r p o s e o f t h i s p r o j e c t was t o d e m o n s t r a t e t h a t a q u a t i c model e c o s y s t e m s c o u l d b e f u r t h e r s c a l e d up i n s i z e t o p r o v i d e g r e a t e r amounts o f t h e components ( b i o m a s s , s o i l a n d water) t o s a t i s f a c t o r i l y study metabolism k i n e t i c s . We u s e d t r i f l u r a l i n , a d i n i t r o a n i l i n e herbicide, since i t s metabolic p a t h w a y s a r e w e l l known a n d t h e m e t a b o l i t e s were r e a d i l y a v a i l able. Methods and M a t e r i a l s C h e m i c a l s a n d E x p e r i m e n t a l Chambers The c h e m i c a l name a n d s t r u c t u r e o f t r i f l u r a l i n a n d t h e e i g h t m e t a b o l i t e s used i n t h i s study are g i v e n i n T a b l e I . A l l n i n e compounds h a d a c h e m i c a l p u r i t y g r e a t e r t h a n 98.7% a n d t h e [ B e r i n g ] t r i f l u r a l i n ( s p e c i f i c a c t i v i t y 45.25 yCi/mg) h a d a r a d i o p u r i t y g r e a t e r than 97%. F o u r t e e n k g o f M a t a p e a k e s i l t loam (pH 5.3, 1.5% O.M.; s a n d , s i l t , a n d c l a y c o n t e n t s o f 38.4, 49.4, a n d 12.7% r e s p e c t i v e l y ) were t r e a t e d w i t h [ - ^ c ] t r i f l u r a l i n a t t h e r a t e o f 10 ppm a n d i n t r o d u c e d i n t o t h e e c o s y s t e m t a n k s a s d e s c r i b e d b e l o w . The c o n t r o l tank contained f o u r t e e n kg o f u n t r e a t e d s o i l . G l a s s a q u a r i a , m e a s u r i n g 75 χ 29 χ 45 cm, were u s e d a s t h e e c o s y s t e m chambers ( F i g u r e s 1 a n d 2 ) . T w e n t y - f o u r s o i l s a m p l i n g

This chapter not subject to U.S. copyright. Published 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Compound No.

3

C

N0

NH

α,α,α-Trifluoro-2,6-dinitro-N(3h y d r o x y p r o p y l ) -p_- t o l u i d i n e

a,a,a-Trifluoro-N ,N -dipropyltoluene 3,4,5-triamine

4

N0

a,a,a-Trifluoro-2,6-dinitro-N(2h y d r o x y p r o p y 1 ) -p_- t o l u i d i n e

4

N0

2-Ethyl-7-nitro-l-peopyl-5(trifluoromethyl)-benzimidazole

2

2

2

2

2

7

:

2

NH 3

2

^-(C H )

2

2

N0

N0

3

HN-CH CH CH OH

HN-CH CHOHCH

and i t s M e t a b o l i t e s .

N0

Name

F

I. Trifluralin

2-Ethyl-4-nitro-6-(trifluoromethyl benzimidazole

Table

2

13.

isENSEE E T A L .

x

CM

ΓΜ

o

2

Modeling

Χ

2

(N

2

U

to

I

I

χ

CM ο

CM

/—\

/—\

υ

Χ to

Χ to U

2

2

ν—'

2

(Ν

CM

S 2

Ecosystems

2

(Ν

Χ

Aquatic

(Ν

Q 2

ο 2

21

^

21

I

0

Ζ|·Η

ô •Η G I LO I ο

i I

% · to I ω u c ο ο

T3

c

•H

r-H ΜΗ •Η μ Ει

δ*

r—(

Ο

4-> ΓΗ

Χ

ΡΗ

ο

?Η

ο +->

•Η Ρ! •Η Τ3 I (D νΟ •Η (Ν Ό I •Η

Ο u ι-Η ο Ο +->

ι-Η ΜΗ a i •Η I ι-Η ι

ΡΗ

Ο

rH

ΡΗ

δ

ιι ° 2| I c •Η

lit g 2| ι ^3 ι •Η ο Π3 ΓΗ I 4-> ^ · •Η to ι I LQ ω I

Ο u

ο r—(

ι-Η ΜΗ •Η rH Ε ι

δ

Ο

-Ρ ι—ι >% ΡΗ

Ο

ΡΗ

·\ Ό•Η

2| I ι

Ο •Η •Η Τ3 I •Η \Ω Τ3 ·> •Η CM 3 I ι-Η ο Ο

u ο

ι-Η ΜΗ •Η rH Ε Ι

+->

pj ι rH Χ ΡΗ

Ο

rH

a ΡΗ •Η δ Ό ·\ δ

c

8

•8 E-


197

198

PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

Figure 1.

Side view of ecosystem chamber detailing rehtive proportion and distribution of ecosystem components

REFERENCE ,··* ELECTRODE

REDOX

ELECTRODES

END VIEW Figure 2.

End view of ecosystem chamber showing detail of daphnid chamber and positioning of redox electrodes


13.

ISENSEE E T A L .

Modeling

Aquatic

Ecosystems

199

t u b e s (2.5 cm. d i a χ 4 cm h i g h ; F i g u r e 1) w e r e r a n d o m l y l o c a t e d on t h e b o t t o m o f t h e t a n k s . A n aluminum w i r e hoop was cemented ( s i l i c o n s e a l a n t ) t o each t u b e t o a i d sample r e t r i e v a l . A t one end o f t h e c o n t r o l t a n k ( F i g u r e 2) t h r e e h o l e s were d r i l l e d , 1, 2, a n d 3 cm f r o m t h e b o t t o m a n d 2 cm a p a r t ( h o r i z o n t a l l y ) . Two p l a t i n u m d i s c ( t o p and bottom h o l e s ) and one c a l o m e l r e f e r e n c e ( c e n t e r h o l e ) e l e c t r o d e s were cemented i n p l a c e ( s i l i c o n r u b b e r sealant). T h e s e e l e c t r o d e s were u s e d t o measure t h e Eh o f t h e soil. The s o i l ( t r e a t e d o r u n t r e a t e d ) was u n i f o r m l y d i s t r i b u t e d over the bottom o f the tanks and i n the sampling tubes. Only t h e hoop o n t h e s a m p l i n g t u b e s p r o t r u d e d f r o m t h e d i s t r i b u t e d soil. The t h r e e t a n k s were t h e n f i l l e d w i t h 84 l i t e r s o f w a t e r . One day a f t e r f l o o d i n g , 75 b l u e g i l l f i s h (Lepomis m a c r o c h i n u s ) , 60 s n a i l s ( H e l i s o m a s p . ) , 2 grams a l g a e (Oedogonium c a r d i a cum) a n d s e v e r a l h u n d r e d d a p h n i d s ( D a p h n i a magna) w e r e added t o t h e chambers. The d a p h n i d s were p l a c e d i n a s p e c i a l g l a s s t a n k (25 χ 20 χ 15 cm) s u s p e n d e d n e a r t h e w a t e r s u r f a c e i n t h e l a r g e t a n k ( F i g u r e 2 ) . An o p e n i n g i n t h e t a n k b o t t o m was c o v e r e d w i t h a s t a i n l e s s s t e e l s c r e e n w i t h a mesh s u f f i c i e n t l y s m a l l (0.38 mm) t o r e s t r i c t t h e p a s s a g e o f d a p h n i a . A p e r c o l a t o r w a t e r pump c o n t i n u o u s l y pumped w a t e r i n t o t h e d a p h n i d t a n k , e n s u r i n g u n i f o r m m i x i n g o f t h e w a t e r a n d t r a n s p o r t o f f o o d t o d a p h n i d s . The e x p e r i m e n t was c o n d u c t e d i n t h e g r e e n h o u s e u s i n g n a t u r a l l i g h t a t a n a v e r a g e t e m p e r a t u r e o f 27 ί 3 C. S a m p l i n g and A n a l y s i s . W a t e r s a m p l e s ( t r i p l i c a t e 1 ml) w e r e t a k e n a t 2-day i n t e r v a l s a n d a n a l y z e d b y s t a n d a r d l i q u i d s c i n t i l l a t i o n (LS) methods f o r t o t a l C ; 100 m l samples were t a k e n 2, 5, 9, 15, 2 2 , 3 0 , 4 3 , 4 8 , a n d 58 days a f t e r t h e s t a r t o f t h e e x p e r i m e n t , ; t h e s e were e x t r a c t e d t w i c e w i t h 50 m l o f e t h y l a c e t a t e : h e x a n e (7:3 v / v ) a n d t h e e x t r a c t s were r e d u c e d t o 20 m l and a n a l y z e d b y LS a n d T L C . Two s o i l s a m p l i n g t u b e s were removed f r o m e a c h t a n k 2, 5, 9, 15, 27, 3 0 , 4 3 , a n d 58, a n d 72 days a f t e r t h e s t a r t o f t h e e x p e r iment, then f r o z e n and s t o r e d f o r l a t e r a n a l y s e s . For a n a l y s e s , t h e f r o z e n s o i l c o r e s were removed f r o m t h e g l a s s t u b e s (by b r i e f i m m e r s i o n i n h o t w a t e r ) , t h e n s e c t i o n e d i n t o f o u r 1-cm c y l i n d e r s r e p r e s e n t i n g 0-1, 1-2, 2-3, a n d 3-4 cm s o i l d e p t h s . Samples f r o m each d e p t h were s h a k e - e x t r a c t e d w i t h 100 ml e t h y l a c e t a t e : h e x a n e o v e r n i g h t , a n d a g a i n w i t h 100 m l m e t h a n o l o v e r n i g h t . Extracts w e r e f i l t e r e d , c o n c e n t r a t e d t o 20 m l a n d a n a l y z e d b y LS a n d TLC as d e s c r i b e d b e l o w . Samples o f o r g a n i s m s (7 f i s h , 6 s n a i l s , 0.5 g a l g a e , a n d 0.5 t o 1.5 g d a p h n i d s ) were t a k e n a f t e r 2, 5, 9, 1 5 , 2 2 , a n d 30 d a y s . A l l r e m a i n i n g o r g a n i s m s were removed a f t e r 42 d a y s a n d a d d i t i o n a l o r g a n i s m s were a d d e d (27 f i s h , 30 s n a i l s , 2 g a l g a e , a n d s e v e r a l h u n d r e d d a p h n i d s ) t h e same day. Samples o f o r g a n i s m s were a g a i n t a k e n o n Days 44, 4 8 , 58 a n d 72. A l l s a m p l e s w e r e w e i g h e d , t h e n f r o z e n f o r l a t e r p r o c e s s i n g . A l g a e s a m p l e s were o x i d i z e d t o de t e r m i n e t o t a l l ^ C . F i s h , s n a i l s , and daphnids were homogenized i n m e t h a n o l a n d a n a l y z e d b y LS a n d T L C . 1 4


200

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

The i d e n t i t y o f t r i f l u r a l i n and compounds 1-8 were d e t e r m i n e d by c o - c h r o m o t o g r a p h y as f o l l o w s . E x t r a c t s f r o m s o i l , w a t e r , and o r g a n i s m s were r e d u c e d ( u n d e r N ) t o 0.1 m l , c o m b i n e d w i t h 10 t o 20 u l e a c h o f compounds 1-9; t h e n s p o t t e d on s i l i c a g e l TLC p l a t e s (20 χ 20 cm FG-254, E. M e r c k , D a r m s t a d t ) . The p l a t e s were d e v e l oped two d i m e n s i o n a l l y , f i r s t i n b e n z e n e f o r 15 cm, t h e n i n b e n zene: e t h y l a c e t a t e : a c e t i c a c i d (60:40:1). Spots c o r r e s p o n d i n g t o t h e n i n e compounds were l o c a t e d v i s i b l y and by UV l i g h t , t h e n s c r a p e d and a n a l y z e d by LS. I n a d d i t i o n , t h e o r i g i n and a d i f f u s e zone b e t w e e n t h e o r i g i n and compound 1 were s c r a p e d and a r e t e r m e d " p o l a r " and " n o n p o l a r " m e t a b o l i t e s , r e s p e c t i v e l y ( F i g u r e 3 ) . 2

R e s u l t s and

Discussion

A l l o r g a n i s m s t h r i v e d i n t h e s y s t e m s w i t h no l o s s o f f i s h or s n a i l s . Numerous egg c l u s t e r s and s m a l l s n a i l s were e v i d e n t i n a l l t a n k s by Day 42 (when t h e r e m a i n i n g o r g a n i s m s were h a r v e s ted). T h e s e egg c l u s t e r s and s m a l l s n a i l s were n o t removed s i n c e t h e s e c o n d s e t o f s n a i l s was s u f f i c i e n t l y l a r g e t h a t i d e n t i t y a t h a r v e s t was n o t a p r o b l e m . The a l g a e w e i g h t i n c r e a s e d f r o m 2 g ( i n i t i a l ) t o an a v e r a g e o f 9.4 g i n t h e t r e a t m e n t t a n k s and 26.3 g i n the c o n t r o l . However, f o r t h e s e c o n d s e t o f a l g a e , t h e r e was no g r o w t h d i f f e r e n c e d u r i n g t h e e x p o s u r e p e r i o d (Days 42 - 7 2 ) . The c o n c e n t r a t i o n o f t r i f l u r a l i n i n w a t e r d e c r e a s e d r a p i d l y w i t h t i m e ( w h i c h w i l l be d i s c u s s e d i n d e t a i l l a t e r i n t h i s p a p e r ) . Thus any i n i t i a l e f f e c t o f r e d u c i n g a l g a e g r o w t h was l o s t w i t h time. D a p h n i d s r e p r o d u c e d ( i n b o t h t r e a t m e n t and c o n t r o l t a n k s ) q u i c k l y i n c r e a s i n g t h e i r mass a t l e a s t 10 t i m e s i n 15 days and t h e n m a i n t a i n e d t h i s d e n s i t y f o r up t o 42 d a y s . The s e c o n d s e t of daphnids behaved s i m i l a r i l y . D e g r a d a t i o n o f T r i f l u r a l i n i n Submerged S o i l . The r e d o x p o t e n t i a l o f t h e s o i l became n e g a t i v e a f t e r o n l y 3 days and r e a c h e d a low o f -450 mv a f t e r 32 days ( T a b l e I I ) . T h e r e was l i t t l e d i f f e r e n c e i n t h e measurements between t h e 1 and 3 cm s o i l d e p t h s . We t h e r e f o r e assume t h a t a l l s o i l s a m p l e s ( e x c e p t p o s s i b l y a t Day 2) were a n a e r o b i c . O n l y a b o u t 6% o f t h e o r i g i n a l C a p p l i e d t o s o i l as Ct r i f l u r a l i n was l o s t a f t e r 72 d a y s ( F i g u r e 4 ) . A l s o , t h e a c e t a t e : hexane and m e t h a n o l e x t r a c t s ) s t e a d i l y d e c r e a s e d w i t h t i m e t o a low o f a b o u t 58% a f t e r 72 d a y s . ( V a l u e s shown i n F i g u r e 4 a r e t h e a v e r a g e o f t h e f o u r d e p t h s s i n c e t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n t h e C c o n t e n t b e t w e e n t h e m ) . T h e s e r e s u l t s show t h a t t h e d i f f u s i o n o f t r i f l u r a l i n a n d / o r i t s m e t a b o l i t e s f r o m a submer ged s o i l i s a s l o w p r o c e s s , w h i l e a t t h e same t i m e , c o n v e r s i o n t o t h e "bound" o r n o n e x t r a c t a b l e form (35 t o 40% a t 72 d a y s ) o c c u r s . P r o b s t e t a l . (4) f o u n d more r a p i d c o n v e r s i o n t o bound r e s i d u e s : n e a r l y 60% o f t h e t o t a l C f r o m a submerged s o i l was u n e x t r a c t a b l e a f t e r o n l y 14 d a y s . The a n a l y s i s o f t h e s o i l e x t r a c t s by TLC i s shown i n T a b l e III. (The p e r c e n t a g e v a l u e s a r e b a s e d on t h e d i s t r i b u t i o n o f C 1 4

1 4

1 4

1 4


13.

ISENSEE E T A L .

Modeling

Aquatic

201

Ecosystems

15

t II Benzene

60

Et A c

40

HOAc

1

H O

h5 1 Non

Polar

Polar

jjjjjjjjj

Metabolites

iijjSj:

Metabolites

''l''

w

LQ

Benzene

-ι—ι 10

15

Figure 3.

ι 5

I 0

TLC system used to separate trifluralin and metabolites extracted from ecosystem components


PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

202

T a b l e I I . Redox p o t e n t i a l s ( e x p r e s s e d i n m i l l i v o l t s ) a t two d e p t h s i n a f l o o d e d s o i l .

Soil

depth

a

Days

1 cm

3 cm

0

+300

+300

1

+205

+220

2

+ 15

+40

5

-210

-80

6

-260

-140

7

-290

-250

8

-320

-270

9

-340

-300

12

-360

-320

16

-380

-350

19

-405

-370

22

-430

-395

25

-450

-420

30

-460

-440

Measurements t a k e n 1 a n d 3 cm b e l o w s o i l

surface.

Days a f t e r s t a r t o f e x p e r i m e n t .


ISENSEE ET AL.

Modeling

Aquatic

203

Ecosystems

50"

4

0

10

20

30

40

50

60

70

DAYS Figure 4.

Residual and extractable C from C-trifluralin-treated 14

14

anaerobic soil



0

0.3 3.4 0.3 1.1 1.1 0.5 0.1 2.1 0.1 89.4

0.4

1.2

.9 5.0 0.5 0.6 3.6 0.6 0.2 1.8 2.8 78.9

3.0

2.1

i 4

2.1 2.5 0.9 0.9 2.2 2.0 0.2 7.4 13.3 47.9

15.0

5.6

4.2 1.6 1.0 1.0 2.0 4.6 0.3 2.7 13.0 44.8

18.0

6.8

4

Values represent percent o f t o t a l C recovered from s o i l a c e t a t e : h e x a n e a n d m e t h a n o l ) b y TLC m e t h o d s . 1 C r e c o v e r e d from t h e o r g i n . 14c r e c o v e r e d i n a zone b e t w e e n o r g i n a n d compound 1. 14c r e c o v e r e d b e t w e e n compounds 1 a n d 2.

0

P o l a r metabolites Nonpolar metabolites 1 Unknown^ 2 3 4 5 6 7 8 Trifluralin

extracts

16.4 1.6 1.0 1.2 2.2 6.2 0.4 3.0 7.9 40.4

11.7

8.0

(ethyl

14.3 1.2 1.0 1.3 2.5 5.9 0.3 4.5 1.3 39.3

10.6

17.8

Table I I I . Degradation o f [ C ] t r i f l u r a l i n i n a f l o o d e d s o i l (expressed as percent of extracted r a d i o a c t i v i t y ) . Days a f t e r s t a r t o f e x p e r i m e n t 9 22 30 42 71 Compounds 2

ISENSEE E T A L .

13.

Modeling

Aquatic

205

Ecosystems 4

r e c o v e r e d f r o m t h e i n d i v i d u a l TLC p l a t e s , r e p r e s e n t i n g t h e ^ C i n the e x t r a c t s , not thet o t a l s o i l ) . T r i f l u r a l i n appeared t o deg r a d e b o t h b y s e q u e n t i a l r e d u c t i o n o f t h e n i t r o g r o u p s (Compounds 8 a n d 5) a n d b y d e a l k y l a t i o n (Compound 7 ) , T a b l e I I I , F i g u r e 3. These a r e a l l r e c o g n i z e d d e c o m p o s i t i o n p r o d u c t s i n the a n a e r o b i c d e g r a d a t i o n p a t h w a y a s p r o p o s e d b y P r o b s t £t a l _ ( 4 ) . The i n t e r m e d i a t e s i n t h e f o r m a t i o n o f Compound 1 i n o u r s y s t e m a r e unknown, s i n c e i t c o u l d form e i t h e r from t r i f l u r a l i n d i r e c t l y ( v i a s e v e r a l i n t e r m e d i a t o r s ) o r f r o m Compound 7 ( F i g u r e 5 ) . However, o u r r e c o v e r y o f Compound 1 s u p p o r t s a p r e v i o u s o b s e r v a t i o n (5) t h a t i t forms under a n a e r o b i c c o n d i t i o n s , b u t p r o b a b l y i s a minor pathway. The a c c u m u l a t i o n o f p o l a r a n d n o n p o l a r m e t a b o l i t e s a l s o agreed w i t h p r e v i o u s s t u d i e s . Compounds 2, 3, 4, a n d 6 w e r e p r o b a b l y n o t p r e s e n t i n o u r system as i n d i c a t e d b y t h e low r e c o v e r y of C (1 t o 3 . 6 % ) . The l e v e l o f C was t o o l o w t o c o n f i r m t h e p r e s e n c e o f t h e s e compounds b y T L C . In g e n e r a l , t h e d e g r a d a t i o n p r o d u c t s we o b t a i n e d f r o m t h e submerged s o i l o f o u r e c o s y s t e m w e r e v e r y s i m i l a r t o t h o s e r e p o r t ed b y p r e v i o u s i n v e s t i g a t o r s ( 4 , 6 ) . However, t h e r a t e o f t r i f l u r a l i n d e g r a d a t i o n was much s l o w e r . F o r e x a m p l e , a b o u t 8% o f t h e total C i n t h e s o i l was t r i f l u r a l i n a f t e r 58 d a y s ( c o n f i r m e d by e l e c t r o n c a p t u r e g a s c h r o m a t o g r a p h y ) a s compared t o a b o u t 6% a f t e r 14 d a y s b y P r o b s t et_ a l _ . (4) . S o i l o r g a n i c m a t t e r may h a v e been responsible f o r thedegradation rates. P a r r a n d S m i t h (6) m e a s u r ed t h e e x t e n t o f t r i f l u r a l i n d e g r a d a t i o n i n a s i l t loam, amended and unamended w i t h 1% a l f a l f a m e a l , u n d e r a n a e r o b i c c o n d i t i o n s . A f t e r 20 d a y s , t h e amount o f t r i f l u r a l i n r e c o v e r e d was 1% a n d 6 8 % i n t h e amended a n d unamended s o i l s , r e s p e c t i v e l y . The o r g a n i c m a t t e r i n o u r s o i l may n o t h a v e p r o m o t e d r a p i d m i c r o b i a l d e g r a d a t i o n and t h e r e f o r e r e s u l t e d i n a slower r a t e o f d e g r a d a t i o n . 1 4

1 4

%

1 4

1 4

D e g r a d a t i o n o f T r i f l u r a l i n i n W a t e r . The amount o f C r e c o v e r e d from w a t e r w i t h t i m e i s shown i n T a b l e I V . A b o u t h a l f of t h e t o t a l C ( d i r e c t c o u n t a n a l y s i s ) i n w a t e r was r e c o v e r e d by e x t r a c t i n g t w i c e w i t h e t h y l a c e t a t e : h e x a n e ( 7 : 3 ) , i n d i c a t i n g t h a t p o l a r m e t a b o l i t e s r a p i d l y formed. A l s o , t h e c o n c e n t r a t i o n of C i n w a t e r i n c r e a s e d most r a p i d l y d u r i n g t h e f i r s t 22 d a y s , a f t e r which t h e r a t e decreased. A n a l y s i s o f t h e water e x t r a c t s by TLC i s shown i n T a b l e V. T r i f l u r a l i n d i s a p p e a r e d v e r y r a p i d l y , d e c r e a s i n g t o n o n d e t e c t a b l e l e v e l s b e t w e e n 9 a n d 22 d a y s . Even as e a r l y as 2 d a y s o n l y 4 6 % o f t h e r e c o v e r e d C was t r i f l u r a l i n . A s e q u e n c i a l r e d u c t i o n o f t h e n i t r o group (decrease i n t h e concent r a t i o n o f Compound 8 w i t h t i m e f o l l o w e d by a n a c c u m u l a t i o n o f Compound 5) i s i n d i c a t e d b y t h e r e s u l t s ( F i g u r e 6 ) . Compound 1 a l s o i n c r e a s e d r a p i d l y i n c o n c e n t r a t i o n e a r l y i n t h e experiment t h e n m a i n t a i n e d a l o w e r b u t c o n s t a n t c o n c e n t r a t i o n b e t w e e n Days 22 and 5 8 . P o l a r m e t a b o l i t e s r a p i d l y i n c r e a s e d t o 5 2 % o f t h e t o t a l r e c o v e r e d C b y Day 9, t h e n g r a d u a l l y d e c r e a s e d w h i l e t h e c o n c e n t r a t i o n o f nonpolar metabolites continuously increased w i t h time. 1 4

i 4

1 4

1 4


PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

206

N(C,H ) 7

° S N(C H ) 3

7

2 N

CF,

2

l^vjUCompound 8 CF

2

N 0

rîï ' k^Trifluralin \

\^

\

H-N-C3H7

\

LJJCompound 7 \

3

N(C,H ), 7

CF

3

« NO

2

Compound 1

^Compound 5 CF

1

3

I

Polar and Non Polar Metabolites Figure 5.

Postulated pathway of trifluralin degradation in an anaerobic soil from a model ecosystem


isENSEE E T A L .

Table IV.

Days

a

Modeling

Aquatic

207

Ecosystems

Concentration of C i n ecosystem water (expressed on ppb p a r e n t compound).

D i r e c t count ^

Extraction

2

4.7

2.8

5

7.5

3.5

9

11.0

5.4

15

19.1

11.0

22

27.5

9.9

30

29.9

13.6

43

34.8

15.4

48

36.2

14.7

58

38.1

-

72

37.3

-

c

Days a f t e r s t a r t o f e x p e r i m e n t l ^ c r e c o v e r e d f r o m 1 ml w a t e r 1 4

samples.

C r e c o v e r e d from water e x t r a c t e d 2 times w i t h acetate:hexane (7:3).

ethyl


PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

rj- r f

oo

•

LQ

· O

O

O

00

·O

O

O

O

CN

vO •M

00 rf

CD

tO

to vO • · o to m

00 o o · o vo

CM rH

\D LO • · o rH LO

o

CM

00 CTi "3LO • · · o o « o o o o LO Vû

o

o

o

-M υ cd rH +->

CD •Η

U ω

o

CM « o o o o 00

X

CD

rH

CD

+->

& CD

ο| +-> rH cd

PH

B o υ

rH vO 00 O tO 00 CM • · · o · · · o 00 LO rH 00 rH

U

CD

rH

o

cd

O υ

CD

bû CM rH ο τ3

u u CJ>tOOvOCJ*CMrH\OCMrH o CM rH CM LO vO CM rH

+J

MH -H •H >

CM

LO

CD cd

cd +-> · O w ο

00 vO vO to LO to to • • • •o • • • • • rr 00 rf CM O CM CM rH

CM

• 00 rH

• 00

ΜΗ

Λ

o +-> CD

+c-> e CD CJ

O J rH F-

1 o rO cd «P (D SrO 0

1 u

rH cd rH O O.

LO CM LO to rH rH

o

• o rH

CD

+-> -H rH

CM rH O

o

vO

CM to

O

\D CM rf

o CD

u cd •H rH rH O O P O rH CM to rf L0 vO 1 cd c -M r * o ω 1 2 e

G •H rH cd rH 00 rH > > rH O O O υ υ υ CD CD CD t/) rH U U 0 3 rH CD U u u cd o r f r f rf > Cd rH rH rH CD

cd


13.

iSENSEE E T A L .

Modeling

Aquatic

N(CH ) 7

^ ^ ET N(CH ) 7

0

t

N

rîî

N

H

Ecosystems

F

Trifluralin

CF, \

T

2

*

L j J C o m p o u n d 8\ CF,

\

'

\

N(C,H ) 7

2

^/Compound 5

NO,

Compound 1

CF,^>^-

N

CF,

i

ι

Polar a n d Non Polar Metabolites Figure 6.

Postulated pathway of trifluralin degradation in water from a model ecosystem


209

210

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

T r i f l u r a l i n i s known t o u n d e r g o r a p i d p h o t o d e c o m p o s i t i o n on p h y s i c a l s u r f a c e s (7) and d e g r a d e s r a p i d l y i n s h a l l o w w a t e r e x p o s ed t o s u n l i g h t ( 8 ) . The r e c o v e r y o f o n l y 46% o f t h e t o t a l C as t r i f l u r a l i n a f t e r 2 days s u g g e s t s t h a t p h o t o d e g r a d a t i o n s i g n i f i c a n t l y c o n t r i b u t e d t o t r i f l u r a l i n d e g r a d a t i o n i n our ecosystem. The d a t a s u p p o r t s t h i s c o n c l u s i o n s i n c e Compound 1, a known p h o t o p r o d u c t o f t r i f l u r a l i n ( 9 ) , r e p r e s e n t s a s u b s t a n t i a l amount o f t h e t o t a l ^ C d u r i n g the f i r s t 9 days o f the experiment ( c o r r e s p o n d i n g to the h i g h e s t c o n c e n t r a t i o n o f t r i f l u r a l i n ) . D e g r a d a t i o n o f T r i f l u r a l i n i n A q u a t i c O r g a n i s m s . The r a p i d degradation of t r i f l u r a l i n i n water s i g n i f i c a n t l y a f f e c t e d the t y p e and amount o f m e t a b o l i t e s r e c o v e r e d from f i s h , s n a i l s , daphn i d s and a l g a e ( T a b l e s V I - I X ) . I n g e n e r a l , t h e amount o f t r i f l u r a l i n r e c o v e r e d was h i g h e s t f o r t h e i n i t i a l s a m p l i n g p e r i o d s , t h e n d e c r e a s e d t o n o n d e t e c t a b l e l e v e l s between 22 and 42 d a y s . This trend c l o s e l y f o l l o w s the t r i f l u r a l i n c o n c e n t r a t i o n i n water, where t r i f l u r a l i n a c c o u n t e d f o r a b o u t 25% o f t h e t o t a l (1.3 ppb) on Day 2. T h i s may a l s o e x p l a i n why p o l a r m e t a b o l i t e s a c counted f o r the h i g h e s t p r o p o r t i o n of the r e c o v e r e d , even f o r t h e f i r s t s a m p l i n g p e r i o d s . Compound 1 was t h e n e x t most p r e v a l e n t p r o d u c t r e c o v e r e d from organisms. The 14c d i s t r i b u t i o n was more c o m p l e x i n d a p h n i d s ( T a b l e V I I I ) t h a n f o r f i s h , s n a i l s , o r a l g a e , where compounds 8, 2 and n o n p o l a r m e t a b o l i t e s were a l s o detected. (No d a t a was shown i n T a b l e V I I I b e f o r e Day 15 s i n c e f o r TLC a n a l y s i s was i n s u f f i c i e n t ) . I n t e r p r e t a t i o n of the a q u a t i c o r g a n i s m d a t a i s d i f f i c u l t s i n c e we c o u l d n o t d e t e r m i n e w h e t h e r s p e c i f i c compounds ( p r i m a r i l y t h e p o l a r m e t a b o l i t e s ) w e r e f o r m e d i n t h e o r g a n i s m o r were a b s o r b e d f r o m w a t e r . However, w a t e r was p r o b a b l y t h e m a j o r s o u r c e o f t h e r e c o v e r e d compounds s i n c e l i t t l e d i f f e r e n c e was n o t e d b e t w e e n o r g a n i s m s a n a l y z e d on Day 42 and 44, r e p r e s e n t i n g e x p o s u r e t i m e s o f 42 v s . 2 d a y s , r e spectively. ( A l l o r g a n i s m s were removed from t h e t a n k s on Day 42 and r e p l a c e d w i t h a new s e t t h e same d a y ) . F a r more t r i f l u r a l i n was i n i t i a l l y r e c o v e r e d (Days 2 and 5) f r o m a l g a e t h a n any o f t h e o t h e r o r g a n i s m s ( T a b l e I X ) . However, a f t e r 30 days t h e p o l a r and n o n p o l a r m e t a b o l i t e s a c c o u n t e d f o r 75% o r more o f t h e r e c o v e r e d i n d i c a t i n g t h a t t h e a l g a e were a l s o r e s p o n d i n g t o t h e r a p i d l o s s o f t r i f l u r a l i n from w a t e r . The i n i t i a l r e l a t i v e l y h i g h c o n c e n t r a t i o n o f t r i f l u r a l i n may account f o r the lower accumulation o f a l g a e biomass i n the t r e a t e d tanks as compared t o t h e c o n t r o l . O n l y one o t h e r s t u d y has b e e n c o n d u c t e d t o e v a l u a t e t h e f a t e o f t r i f l u r a l i n i n an a q u a t i c model e c o s y s t e m ( 1 0 ) . I n t h e i r s y s tem, t r i f l u r a l i n p e r s i s t e d much l o n g e r i n w a t e r t h a n i n o u r s t u d y ( p r o b a b l y due t o l e s s p h o t o d e g r a d a t i o n t h r o u g h t h e use o f a r t i f i cial light). As a r e s u l t , t h e y r e p o r t e d much h i g h e r c o n c e n t r a t i o n s o f t r i f l u r a l i n i n s n a i l s and f i s h t h a n we f o u n d , b u t no residues i n daphinds. They a l s o r e p o r t e d t h e p r e s e n c e o f Compound 7 p l u s s e v e r a l o t h e r known and unknown m e t a b o l i t e s . 1 4

4



0

3.8 3.9 1.2 1.5 1.7 1.8 0 0 24.2

3.3

58.6

4.9 4.4 0.2 1.2 0.8 1.1 0 0 14.7

5.2

67.5

1 4

7.0 3.3 1.4 0.6 2.7 4.3 0 0 20.5

5.6

54.6

a

13.8 3.4 3.2 2.2 0.8 1.0 0 0 10.6

4.1

60.9

20.6 2.1 1.4 1.8 1.7 1.1 0 0 4.5

3.9

62.9

29.8 4.0 1.3 2.0 1.1 0.4 0 0 2.5

5.6

53.3

c

25.6 6.0 0.5 0.7 0.9 0.4 0 0 0

1.8

63.4

( m e t h a n o l ) by

28.8 4.6 0.9 1.2 0.4 0.6 0 0 1.8

1.5

60.2

Degradation o f [ C ] t r i f l u r a l i n i n b l u e g i l l f i s h (expressed as percent of e x t r a c t e d radioactivity) Days a f t e r s t a r t o f e x p e r i m e n t 2 5 9 22 15 30' 42 44

Values represent percent o f t o t a l C r e c o v e r e d from f i s h e x t r a c t s TLC methods, k r e c o v e r e d from t h e o r g i n . r e c o v e r e d i n a zone b e t w e e n t h e o r g i n a n d compound 1.

a

b

P o l a r metabolites Nonpolar metabolites 1 2 3 4 5 6 7 8 Trifluralin

Compound

Table V I .


a

0

22.2 3.5 6.9 3.5 0 0 0 2.5 4.7

56.9

2

12.6 10.1 3.9 4.4 0 2.6 0 4.3 1.9

60.4

5

a

9

12. 0 7. 8 0 4. 9 0 2. 3 1. 8 3. 6 2. 6

C r e c o v e r e d from t h e o r g i n .

i n snails

1 4

C

15.5 6.6 3.5 6.7 0 0 0 3.3 6.0

57.5 8. 8 5. 6 2. 5 1. 3 10. 2 0 0 0 0

71. 6 10.9 5.3 2.4 3.0 1.1 0.8 0 0 0

76.5

44

21.9 2.1 0.6 0 2.0 0 0 0 0

73.6

48

16.6 2.7 1.4 2.0 1.6 0 0 0 0

75.4

58

10.3 2.1 4.6 2.7 0.9 0 0 0 0

79.4

72

( m e t h a n o l ) b y TLC methods.

18.7 4.6 0 1.4 0 0 0 0 0

75.8

r e c o v e r e d from s n a i l e x t r a c t s

7. 7 1. 0 1. 4 0 6. 2 0 1. 0 2. 8 1. 1

79. 0

experiment 42

( e x p r e s s e d as p e r c e n t o f e x t r a c t e d

Days a f t e r s t a r t o f 22 30 15

C]trifluralin

62. 7

Degradation of [ radioactivity)

Values represent percent o f t o t a l

Polar metabolites 1 2 3 4 5 6 7 8 Trifluralin

Compounds

Table V I I .


0

14

3.9 12.1 0.3 1.5 0.9 0.8 0 7.5 9.7

3.6

1 4

9.8 15.7 0.3 2.8 1.7 0 0 2.3 4.7

8.1

54.6

7.0 6.7 0 1.4 1.0 0 0 0.7 1.5

7.8

73.9

16.0 5.5 0 0 6.0 0 0 0 0

7.9

64.6

9.4 15.8 0.4 1.6 1.2 1.0 0 0 0

8.3

62.3

0

1 4

Values represent percent of t o t a l C r e c o v e r e d from daphnid e x t r a c t s TLC m e t h o d s , b c r e c o v e r e d from t h e o r g i n . C r e c o v e r e d i n a zone b e t w e e n t h e o r g i n a n d Compound 1.

a

7.8 16.6 4.0 0 1.0 1.0 2.3 12.0 36.0

5.2

59.7

8.9 7.0 0.4 2.3 0 0 0 0 0

15.5

(methanol) by

11.2 11.0 0 1.2 0 0.7 0 0 0

10.0

65.9

65.9

1 3

14.1


Compounds

D e g r a d a t i o n o f [ C ] t r i f l u r a l i n i n daphnids (expressed as p e r c e n t o f extracted radioactivity) . Days a f t e r s t a r t o f e x p e r i m e n t 72 58 48 42 44 30 22 15

Table V I I I .


C

5.4 1.8 0 0 0 0 11.0 5.4 74.5

0

0.6 1.7 0 0 1.7 1.2 7.6 1.2 82.0

0.6

3.4

5

14.3 0 8.6 0 5.7 0 0 0 28.6

8.6

34.2

15

i n algae

1 4

11.4 2.3 8.9 0 0 0 6.8 2.3 11.4

9.1

47.7

2.3 0 0 0 0 0 0 0 19.3

22.6

54.8

6.1 0 8.2 0 0 0 0 0 6.1

20.4

54.2

7.1 0 0 0 0 0 0 0 0

28.6

64.3

experiment 42 44

3.0 0 0 0 0 0 0 0 0

21.2

75.8

48

5.8 2.0 0 0 0 0 0 0 0

28.7

61.5

58

( e x p r e s s e d as p e r c e n t o f e x t r a c t e d

Days a f t e r s t a r t o f 22 30

C]trifluralin

13.1 2.0 0 0 0 0 0 0 0

27.7

57.2

72

b

Values represent percent o f t o t a l C r e c o v e r e d f r o m a l g a e e x t r a c t s ( m e t h a n o l ) b y TLC methods. 14c r e c o v e r e d f r o m t h e o r g i n . c 14 r e c o v e r e d i n a zone b e t w e e n t h e o r g i n a n d Compound 1.

a

0

1.8


b

2

a

Degradation of [ radioactivity)

Compounds

Table IX.

13.

isENSEE E T A L .

Modeling

Aquatic

215

Ecosystems

The o b j e c t i v e o f t h i s i n v e s t i g a t i o n , t o d e m o n s t r a t e t h a t a q u a t i c model e c o s y s t e m s c a n b e s c a l e d up i n s i z e t o p e r f o r m t i m e r e l a t e d , m e t a b o l i c s t u d i e s , was o n l y p a r t l y a c h i e v e d . We s u c c e s s f u l l y demonstrated t h a t t h e d e g r a d a t i v e pathways a n d r a t e s o f t r i f l u r a l i n m e t a b o l i s m i n an a n a e r o b i c s o i l c a n b e d e t e r m i n e d i n an e c o s y s t e m j u s t as w e l l a s t h e y c a n i n l a b o r a t o r y s t u d i e s ( 4 ) . A l s o , t h e r e c o v e r y o f Compound 1 f r o m w a t e r s u b s t a n t i a t e d t h e s i g n i f i c a n c e o f p h o t o d e g r a d a t i o n , which had p r e v i o u s l y been measured u n d e r l a b o r a t o r y c o n d i t i o n s ( 9 ) . However, m e t a b o l i c s t u d i e s i n a q u a t i c o r g a n i s m s were n o t v e r y s u c c e s s f u l b e c a u s e ( i ) t h e t r i f l u r a l i n i n water decreased v e r y r a p i d l y , and ( i i ) t h e o r i g i n o f t h e few m e t a b o l i t e s t h a t were r e c o v e r e d was n o t c l e a r . A c c u m u l a t i o n o f t h e C - l a b e l e d compounds i n w a t e r b y t h e o r g a n i s m s was a p p a r e n t l y the predominant source. (The m a j o r p r o b l e m i n s t u d y i n g t h e m e t a b o l i s m o f p e s t i c i d e s i n a q u a t i c o r g a n i s m s r e t r i e v e d f r o m model ecosystems i s i d e n t i f y i n g t h e source o f the m e t a b o l i t e s , i . e . , were t h e y f o r m e d i n t h e o r g a n i s m s t h r o u g h some m e t a b o l i c p r o c e s s o r were t h e y a b s o r b e d f r o m w a t e r ? ) . T h e s e r e s u l t s i n d i c a t e t h a t o u r s c a l e d - u p model e c o s y s t e m s a r e more u s e f u l f o r s t u d y i n g s y s t e m p r o c e s s e s t h a n p r o c e s s e s t h a t f u n c t i o n i n i n d i v i d u a l components o f t h e e n v i r o n m e n t . In this r e g a r d , a p r e l i m i n a r y l a r g e s c a l e ecosystem study c o u l d be v e r y u s e f u l t o i n d i c a t e parameter l i m i t s such as o v e r a l l degradation r a t e s a n d l i k e l y c o n c e n t r a t i o n s o f p a r e n t compounds p l u s metabol i t e s over time. Such i n f o r m a t i o n w o u l d be u s e f u l i n t h e d e s i g n o f m e t a b o l i c s t u d i e s i n v a r i o u s components o f t h e e c o s y s t e m . I n a d d i t i o n , t h e l a r g e s c a l e ecosystem study c o u l d a l s o be used t o d e t e r m i n e i f p r o c e s s e s d e r i v e d under l a b o r a t o r y c o n d i t i o n s cont i n u e t o f u n c t i o n a n d / o r p r e d o m i n a t e when combined i n a c o m p l e x system. 1 4

Abstract This project was designed to demonstrate that the static water model ecosystems can be scaled up in size to provide sufficient amounts of biomass, s o i l , and water to study metabolism kinetics of pesticides. Fourteen kg of s o i l , treated with [ 4C]trifluralin at 10 ppm, was flooded with 84 liters water. Bluegill fish, snails, daphnids, and algae were exposed to this system for 72 days. Samples of s o i l , water, and organisms were periodically analyzed for trifluralin and eight metabolites. The recovered metabolites and their rate of formation closely followed previously reported values for anaerobic soil and water, substantiating the u t i l i t y of the model ecosystem for metabolic studies. However, residues in the biomass were apparently derived primarily through absorption from water rather than metabolism within the organisms themselves. It was concluded that the scaled-up model ecosystem is more useful for studying system process than processes that function in individual components of the environment. 1


216

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

Literature cited 1. Isensee, A. R., Intern. J. Envirnomental Studies (1976) 10, 35. 2. Isensee, A. R., E. R. Holden, E. A. Woolson, G. E. Jones, J. Agric. Food Chem. (1976) 24, 1210. 3. Ambrosi, D., A. R. Isensee, J. A. Macchia, J. Agric. Food Chem. (1978) 26, 50. 4. Probst, G. W., T. Golab, R. J. Herberg, F. J. Holzer, S. J. Parka, C. V. D. Schans, J. B. Tepe, J. Agric. Food Chem. (1967) 15, 592. 5. Probst, G. W., T. Golab, W. L. Wright, "Herbicide: Chemistry, Degradation and Mode of Action" Vol. 1, p. 453 Marcel Dekker, Inc. New York, 1975. 6. Parr, J. F., S. Smith, Soi Sci. (1973) 115, 55. 7. Wright, W. L. G. F. Warren, Weeds (1965) 13, 329. 8. Kearney, P. C., A. R. Isensee, A. Kontson, Pest. Biochem. Physiology (1977) 7, 242. 9. Leitis, E., D. G. Crosby, J. Agric. Food Chem. (1974) 22, 842. 10. Metcalf, R. L., J. R. Sandborn, Illinois Natural History Survey Bull. (1975) 31, 381. RECEIVED

January 2, 1979.


Pesticide and Xenobiotic Metabolism in Aquatic Organisms - American

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