A Terrestrial-Aquatic Model Ecosystem for Evaluating the

12 A Terrestrial-Aquatic Model Ecosystem for Evaluating the Environmental Fate of Drugs and Related Residues

Downloaded by FUDAN UNIV on January 10, 2017 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch012

in Animal Excreta S. H. CABALLA, M. PATTERSON, and I. P. KAPOOR Agricultural Division, American Cyanamid Company, P. O. Box 400, Princeton, NJ 08540 When farm animals are treated with drugs both as a prophylactic or curative measure, majority of the drug or drug related residues are eliminated i n the excreta. Poultry as well as farm animal excreta is allowed to compost into manure and the manure is used on the farm land. The objective of the present study was to design a terrestrial-aquatic model ecosystem for evaluating the environmental fate of drugs and related residues i n the animal excreta used as manure. Metcalf et al. (1) developed a model ecosystem consisting of a terrestrial/aquatic interface and a seven-element food chain for obtaining valuable information on the biodegradability and ecological fate of numerous pesticides. This study, using a modified system, was initiated to determine the ecological fate of robenidine hydrochloride and related residues present i n turkey excreta. ROBENZ® Robenidine hydrochloride, (Figure 1), has been found to be an effective and safe feed additive product for the prevention of coccidiosis i n broiler chickens (2). For comparison, a parallel experiment using turkey excreta fortified with carbon-14 DDT as a positive control was conducted since the biodegradability and ecological fate of DDT and i t s analogs have already been extensively studied (1, 3). Materials and Methods Robenidine hydrochloride labeled with carbon-14 i n the amino guanidine carbon atom had a specific activity of 18.61 yCi/mg. Carbon-14 DDT, labeled i n the ring, had a specific activity of 83.4 uCi/mg.

®

R e g i s t e r e d Trademark o f A m e r i c a n Cyanamid Company 0-8412-0489-6/79/47-099-183$05.00/0 © 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

184

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

ROBENZ®


Cl-f

^ D E N O T E S

ORGANISMS

ROBENIDINE HYDROCHLORIDE

"\ CC HH == NN HH NN CC **NN HH NN == CC HH --/ \>-NH- HCI

\ - CI

CARBON-14

Figure 1

Figure 2. Schematic of the modified model ecosystem detailing a complete terrestrial/aquatic environment for the study of drug biodegradability and ecological magnification

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


12.

CABALLA ET AL.

Terrestrial-Aquatic

Model

Ecosystem

185

I n t h i s s t u d y , t h r e e e x p e r i m e n t a l t a n k s were e m p l o y e d : Tank A c o n t a i n i n g e x c r e t a from a t u r k e y t r e a t e d w i t h carbon-14 r o b e n i d i n e ; Tank Β c o n t a i n i n g e x c r e t a f o r t i f i e d w i t h c a r b o n - 1 4 DDT w h i c h was u s e d a s a p o s i t i v e c o n t r o l a n d Tank C c o n t a i n i n g u n t r e a t e d e x c r e t a w h i c h was u s e d a s a c o n t r o l . To o b t a i n e x c r e t a f o r t h i s s t u d y , a t u r k e y was f e d 250 grams o f a b a s a l d i e t c o n t a i n i n g 66 ppm r o b e n i d i n e h y d r o c h l o r i d e f o r t e n d a y s . On t h e e l e v e n t h d a y , t h e t u r k e y was d o s e d w i t h 17 mg o r 316.37 u C i o f c a r b o n - 1 4 r o b e n i d i n e i n a c a p s u l e . E x c r e t a was c o l l e c t e d f o r two d a y s a f t e r t r e a t m e n t . Excreta c o l l e c t e d p r i o r t o t h e t e n - d a y c o n d i t i o n i n g p e r i o d was a l s o c o l l e c t e d a n d u s e d f o r t h e c o n t r o l a n d DDT e x p e r i m e n t a l t a n k s . The m o d e l e c o s y s t e m was e s s e n t i a l l y t h e same a s t h a t p r e v i o u s l y d e s c r i b e d b y M e t c a l f et_ a l . (1) e x c e p t t h a t t h e t e r r e s t r i a l p o r t i o n c o n s i s t e d o f u n s t e r i l i z e d sandy loam s o i l i n s t e a d o f w h i t e q u a r t z s a n d . I n p r a c t i c e , 4.5 k g o f a q u a r i u m g r a v e l was washed t h o r o u g h l y t o remove f i n e s and m o l d e d i n t o a s u p p o r t s h e l f m e a s u r i n g 2 χ 6 χ 12 i n c h e s a t t h e b o t t o m o f a 2 0 - g a l l o n g l a s s a q u a r i u m . A s o i l s h e l f was t h e n m o l d e d o n t o p o f t h e g r a v e l c o n s i s t i n g o f 7 k g o f P r i n c e t o n s a n d y loam s o i l . The t o p 2 i n c h e s o f t h e s o i l s h e l f was m o l d e d f r o m 2.5 k g o f s o i l m i x e d t h o r o u g h l y i n a b a l l m i l l w i t h t h e d r i e d t u r k e y e x c r e t a . The amount o f e x c r e t a added c o r r e s p o n d e d t o a n a p p l i c a t i o n r a t e o f 5 tons/acre. The t o t a l a r e a o f t h e p l a t e a u was 78 s q u a r e i n c h e s (6.5 χ 12 i n c h e s ) w i t h a t o t a l h e i g h t o f 6 i n c h e s ( F i g u r e 2 ) . The t o t a l r a d i o a c t i v i t y a p p l i e d t o t h e s o i l a s c a r b o n - 1 4 r o b e n i d i n e r e s i d u e s i n e x c r e t a was 167.54 m i c r o c u r i e s . F o r Tank B, 100 m i c r o c u r i e s o f c a r b o n - 1 4 DDT a n d 9 mg o f u n l a b e l e d DDT w e r e m i x e d i n a b a l l m i l l w i t h 2.5 k g o f s o i l a n d 52g o f c o n t r o l t u r k e y e x c r e t a . The amount o f DDT added t o t h e s o i l c o r r e s p o n d e d to a n a p p l i c a t i o n r a t e o f a p p r o x i m a t e l y 1.5 l b s / a c r e . F o r Tank C, c o n t r o l t u r k e y e x c r e t a was m i x e d w i t h t h e t o p two i n c h e s of s o i l as p r e v i o u s l y described. A f t e r t h e t e r r e s t r i a l p o r t i o n was f o r m e d , 8 l i t e r s o f r e f e r e n c e w a t e r (4) w e r e added t o t h e s y s t e m . The e x c r e t a was a l l o w e d t o age f o r 4 weeks. D u r i n g t h e a g i n g p e r i o d , d i s t i l l e d w a t e r was added whenever needed t o keep t h e l e v e l o f t h e a q u a t i c portion constant. A t t h e e n d o f 4 w e e k s , f i f t y sorghum (sorghum h a l p e n s e ) s e e d s were sowed i n 5 rows a l o n g t h e f l a t t e n e d t e r r e s t r i a l end. A f t e r 3 t o 4 d a y s when t h e s e e d s h a d g e r m i n a t e d , 3 l i t e r s more o f r e f e r e n c e w a t e r were added a n d t h e l e v e l o f w a t e r was k e p t c o n s t a n t t h r o u g h o u t t h e r e m a i n d e r o f t h e s t u d y . At this p o i n t , t h e f o l l o w i n g w e r e added t o t h e a q u a t i c p o r t i o n : 100 D a p h n i a magna, 10 G y r a u l i s s n a i l s , a s t r a n d o f a l g a e ( R h i z o ^ c I o n i u m and L y n g b i a ) a n d 10 m i l l i l i t e r s o f pond w a t e r w h i c h p r o v i d e d t h e p l a n k t o n c u l t u r e . When t h e s e e d l i n g s w e r e 3 weeks o l d , ten e a r l y f i f t h i n s t a r s a l t marsh c a t e r p i l l a r (Estigmene a c r e a ) l a r v a e w e r e p l a c e d o n t h e sorghum p l a n t s . Two t o t h r e e s e e d l i n g s were removed p r i o r t o a d d i t i o n o f t h e l a r v a e i n o r d e r to d e t e r m i n e t h e g r o s s u p t a k e o f r a d i o a c t i v i t y b y t h e p l a n t s .


186

PESTICIDE AND

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

A f i n e mesh w i r e s c r e e n was t h e n f i t t e d o v e r t h e t a n k s t o c o n f i n e t h e l a r v a e on t h e p l a n t s . The c a t e r p i l l a r s consumed t h e p l a n t s w i t h i n 3 t o 4 days and c o n t a m i n a t e d t h e w a t e r w i t h t h e i r e x c r e t a and l e a f f r a s s . I n t h e i r s e a r c h f o r more f o o d , t h e c a t e r p i l l a r s a l s o ended up c o n t a m i n a t i n g t h e w a t e r t h e m s e l v e s . Approximately 300 m o s q u i t o l a r v a e ( A n o p h e l e s q u a d r i m a c u l a t u s ) were added t o t h e e c o s y s t e m a f t e r 26 days and 4 days l a t e r 50 were removed f o r d e t e r m i n a t i o n of gross r a d i o a c t i v i t y . At t h i s p o i n t , t h r e e m o s q u i t o f i s h (Gambusia a f f i n i s ) were i n t r o d u c e d and a l l o w e d t o e a t t h e r e m a i n i n g m o s q u i t o l a r v a e and t h e D a p h n i a . A f t e r t h r e e d a y s (Day 3 3 ) , t h e e x p e r i m e n t was t e r m i n a t e d and t h e d i f f e r e n t components o f t h e s y s t e m were a n a l y z e d f o r c a r b o n - 1 4 r e s i d u e s .


R e s u l t s and

Discussion

A p p r o x i m a t e l y 60% o f t h e c a r b o n - 1 4 r o b e n i d i n e a d m i n i s t e r e d to t h e t u r k e y was r e c o v e r e d i n t h e e x c r e t a w i t h i n 48 h o u r s . About 84% o f t h e r a d i o a c t i v i t y i n t h e e x c r e t a was e x t r a c t e d w i t h m e t h a n o l , a n o t h e r 9% was e x t r a c t e d w i t h a 1% h y d r o c h l o r i c a c i d / m e t h a n o l m i x t u r e l e a v i n g a b o u t 7% u n e x t r a c t e d . I t was f o u n d by TLC t h a t 75% o f t h e m e t h a n o l - s o l u b l e r a d i o a c t i v i t y was due t o t h e p r e s e n c e o f unchanged r o b e n i d i n e ( F i g u r e 3 ) . Metabolites 1, 2, and 3 a c c o u n t e d f o r 7, 2 and 0.7% o f t h e e x t r a c t a b l e radioactivity, respectively. Sorghum s e e d l i n g s r a d i o a s s a y e d f o r c a r b o n - 1 4 r e s i d u e s a t t h e t i m e o f l a r v a l f e e d i n g showed l o w l e v e l s o f 0.004 and 0.013 ppm f o r c a r b o n - 1 4 DDT and c a r b o n - 1 4 r o b e n i d i n e , r e s p e c t i v e l y . R e s i d u e l e v e l s o f c a r b o n - 1 4 i n w a t e r were l o w , e s p e c i a l l y i n t h e c a s e o f c a r b o n - 1 4 DDT, i n d i c a t i n g t h a t D D T - r e l a t e d r e s i d u e s r e m a i n bound t o t h e s o i l ( F i g u r e 4 ) . A f t e r an i n i t i a l c o n c e n t r a t i o n o f 0.009 ppb a t Day 1, t h e c o n c e n t r a t i o n o f c a r b o n - 1 4 DDT r e s i d u e s r e a c h e d an e q u i l i b r i u m o f a b o u t 0.02 ppb by t h e t h i r d day and t h e n d r o p p e d o f f s l i g h t l y t o 0.012 to 0.013 ppb a t t h e t i m e t h e m o s q u i t o l a r v a e and f i s h w e r e i n t r o d u c e d . Carbon-14 r e s i d u e s i n w a t e r d e r i v e d f r o m c a r b o n - 1 4 r o b e n i d i n e showed an i n i t i a l c o n c e n t r a t i o n o f 0.344 ppb and t h e n r e m a i n e d f a i r l y c o n s t a n t a t a b o u t 1 ppb t h r o u g h o u t t h e s t u d y , i n d i c a t i n g t h a t r o b e n i d i n e - r e l a t e d residues are p o l a r i n nature and r e a d i l y m i g r a t e i n t o t h e w a t e r p h a s e and r e a c h e q u i l i b r i u m very r a p i d l y . The n a t u r e o f t h e r a d i o a c t i v i t y i n t h e w a t e r , s o i l and f i s h f r o m t h e c a r b o n - 1 4 DDT e x p e r i m e n t was e x a m i n e d by t h i n - l a y e r c h r o m a t o g r a p h y as shown i n F i g u r e 5. The r a d i o a c t i v i t y i n t h e w a t e r was v e r y p o l a r i n n a t u r e and d i d n o t m i g r a t e a p p r e c i a b l y from the o r i g i n . About 78% o f t h e r a d i o a c t i v i t y i n t h e s o i l was e x t r a c t e d w i t h m e t h a n o l . The m a j o r m e t a b o l i t e i n t h e e x t r a c t a b l e f r a c t i o n was DDD w h i c h r e p r e s e n t e d 33% o f t h e t o t a l r a d i o activity. The r e d u c t i v e d e c h l o r i n a t i o n o f DDT t o DDD i s a known pathway u n d e r a n a e r o b i c c o n d i t i o n s and has been shown t o be due to m i c r o b i a l m e t a b o l i s m ( 5 ) . S i n c e c a r b o n - 1 4 DDT was i n c o r -



CABALLA ET AL.

Figure 3.

Terrestrial-Aquatic

Model

Ecosystem

TLC of the extractable radioactivity from the excreta of turkeys


188

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

CONCENTRATION IN

AT

SORQHUM

OF

CARBON-14

DIFFERENT PLANTS

BY

RESIDUES

DAYS

(CALCULATED

FOLLOWING

SALT

MARSH

LARVAL

A8

PARENT)

FEEDING

OF

CATERPILLAR


THE

WATER

14C-ROBENIDINE

0.01 L _ B _

10

15

20

25

30

DAYS Figure 4


35


CABALLA ET AL.

T erre striai-Aquatic

Model

Ecosystem

189

Figure 5. TLC of the fish and soil extractable radioactivity and water from the C-DDT experiment. Solvent system used was petroleum etheridiethyl ether (9:1).

14



190

PESTICIDE AND

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

porated i n t o the s o i l w i t h the e x c r e t a i t i s l i k e l y t h a t a n a e r o b i c c o n d i t i o n s p r e v a i l e d . P a r e n t DDT a c c o u n t e d f o r 44% of the e x t r a c t a b l e r a d i o a c t i v i t y w i t h t r a c e s (1.1%) of DDE. A b o u t 13% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y was a component more p o l a r t h a n DDT and t h e r e m a i n i n g r a d i o a c t i v i t y s t a y e d a t o r near the o r i g i n . E x t r a c t i o n o f t h e f i s h showed t h a t 8 1 % o f t h e c a r b o n - 1 4 r e s i d u e s were o r g a n o s o l u b l e , 13% w e r e p o l a r w a t e r - s o l u b l e p r o d u c t s and 6% u n e x t r a c t a b l e . Chromatography of the organos o l u b l e r a d i o a c t i v i t y showed t h a t a l a r g e p r o p o r t i o n (87%) was s t i l l v e r y p o l a r i n n a t u r e w i t h DDT a c c o u n t i n g f o r 8%, DDE 3% and DDD 2%. T h i n - l a y e r chromatography of the water from the carbon-14 r o b e n i d i n e s t u d y showed a b o u t 12% (0.138 ppb) p a r e n t compound, 27% (0.319 ppb) o f M e t a b o l i t e No. 2 and 6 1 % (0.734 ppb) p o l a r r a d i o a c t i v i t y which d i d not m i g r a t e f a r from the o r i g i n (Figure 6). The r e s u l t s i n d i c a t e t h a t e v e n t h o u g h r o b e n i d i n e was t h e p r e d o m i n a n t component o f t h e t u r k e y e x c r e t a , i t was readily and e x t e n s i v e l y d e g r a d e d i n t o p o l a r compounds w h i c h end up i n the a q u a t i c phase. A b o u t 20% o f t h e r a d i o a c t i v i t y i n t h e s o i l f r o m t h e c a r b o n 14 r o b e n i d i n e t a n k was e x t r a c t a b l e a t t h e end o f t h e e x p e r i m e n t w i t h 80% r e m a i n i n g u n e x t r a c t a b l e . Chromatography of the e x t r a c t a b l e r a d i o a c t i v i t y showed e x t e n s i v e d e g r a d a t i o n o f t h e compound as shown i n F i g u r e 7. R o b e n i d i n e , w h i c h was t h e m a j o r component i n t h e e x c r e t a , r e p r e s e n t e d a b o u t 10% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y i n t h e soil. I n terms of t o t a l carbon-14 r e s i d u e s i n the s o i l , p a r e n t compound r e p r e s e n t e d 2.0%. M e t a b o l i t e 2, w h i c h was p r e s e n t o n l y i n t r a c e q u a n t i t i e s i n t h e e x c r e t a , a c c o u n t e d f o r 21% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y o r 4.2% o f t h e t o t a l c a r b o n - 1 4 r e s i d u e s i n the s o i l . T h i s m e t a b o l i t e was a l s o t h e o n l y s i g n i f i c a n t compound f o u n d i n t h e w a t e r . T h r e e o t h e r m e t a b o l i t e s a c c o u n t e d f o r a b o u t 18% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y i n t h e s o i l , n a m e l y , M e t a b o l i t e 3, 5.3%, M e t a b o l i t e 6, 7.6% and M e t a b o l i t e 10, 4.9%. P o l a r m a t e r i a l w h i c h was n o t r e s o l v e d f r o m t h e o r i g i n r e p r e s e n t e d 25% o f t h e e x t r a c t a b l e r a d i o a c t i v i t y i n t h e s o i l . The r e m a i n i n g r a d i o a c t i v i t y was d i s t r i b u t e d among t e n m i n o r components. I n t h e f i s h , t h e l a s t e l e m e n t i n t h e f o o d c h a i n web, m e t h a n o l e x t r a c t e d a b o u t 58% o f t h e r a d i o a c t i v i t y r e s u l t i n g f r o m t h e c a r b o n - 1 4 r o b e n i d i n e t r e a t m e n t l e a v i n g 42% u n e x t r a c t e d , i n d i c a t i n g t h a t r o b e n i d i n e was b e i n g e x t e n s i v e l y d e g r a d e d by f i s h into very polar nonextractable products. T h i n - l a y e r chromatography of the e x t r a c t a b l e r a d i o a c t i v i t y d i d n o t show any p a r e n t compound e v e n t h o u g h r o b e n i d i n e was one o f t h e components i n t h e e n v i r o n m e n t ( w a t e r ) ( F i g u r e 8 ) . The c o n c e n t r a t i o n o f c a r b o n - 1 4 r e s i d u e s i n t h e d i f f e r e n t components o f t h e m o d e l e c o s y s t e m i s compared i n T a b l e 3,· The b i o c o n c e n t r a t i o n f a c t o r ( B C F ) , d e f i n e d as t h e r a t i o o f t h e



CABALLA ET AL.

Figure 6.

Figure 7.

T err est rial-Aquatic

Model

Ecosystem

TLC of the water from the C-robenidine model ecosystem 14

TLC of the methanol-soluble radiactivity in the aged soil/excreta mixture from the C-robenidine model ecosystem 14


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


192

Figure 8.

ORGANISMS

TLC of the methanol-soluble radioactivity in the fish from the robenidine model ecosystem


C-

lA

12.

CABALLA ET AL.

Terrestrial-Aquatic

Model

193

Ecosystem

Table I .


COMPARISON

OF

THE

CONCENTRATIONS*'

AND

BIOCONCENTRATION

AND

CARBON-14

FACTOR

ROBENIDINE

(BCF>

MODEL

2 /

OF IN

CARBON-14 THE

PPM

BCF

CARBON-14

EC08Y8TEM8

14C-•DDT

14C-ROBENIDINE COMPONENT

RESIDUES

PPM

BOP

WATER

0.001

ALQAE

0.030

33

0.0022

138

0.0124

10

0.0034

214

M08QUITOLARVAE

0.181

178

0.0038

314

FISH

0.081

88

0.0180

1120

8NAIL8

1/ CALCULATED

2

'

B C F

AS

0.000014

PARENT

. CONCENTRATION CONCENTRATION

OF CARBON-14 RESIDUES IN THE OF CARBON-14 RESIDUES IN THE

ORGANISM WATER


DDT

194

PESTICIDE

A N D XENOBIOTIC

METABOLISM

IN AQUATIC

ORGANISMS

c o n c e n t r a t i o n o f carbon-14 r e s i d u e s i n t h e organism t o t h e c o n c e n t r a t i o n o f c a r b o n - 1 4 r e s i d u e s i n t h e w a t e r , i s a l s o shown. The d a t a c l e a r l y i n d i c a t e s t h a t t h e BCF v a l u e s o b t a i n e d i n a l l t h e e l e m e n t s o f t h e f o o d c h a i n were h i g h e r f o r t h e c a r b o n - 1 4 DDT e x p e r i m e n t compared w i t h t h o s e f o r t h e c a r b o n - 1 4 r o b e n i d i n e e x p e r i m e n t . T h i s d i f f e r e n c e i s more p r o m i n e n t i n t h e c a s e o f s n a i l s a n d f i s h , t h a t i s , 214 v s 10 and 1129 v s 6 8 , r e s p e c t i v e l y . A l l t h e s e r e s u l t s s u g g e s t t h a t t h e u s e o f e x c r e t a a s manure f r o m b i r d s k e p t on a d i e t c o n t a i n i n g r o b e n i d i n e w i l l n o t r e s u l t i n any b i o l o g i c a l m a g n i f i c a t i o n o f r o b e n i d i n e - r e l a t e d r e s i d u e s i n the elements o f t h e environment.


Conclusion The m o d i f i e d t e r r e s t r i a l - a q u a t i c m o d e l e c o s y s t e m d e s c r i b e d h e r e h a s b e e n f o u n d t o be a u s e f u l t o o l i n s t u d y i n g t h e e n v i r o n m e n t a l f a t e o f d r u g s and r e l a t e d r e s i d u e s p r e s e n t i n a n i m a l e x c r e t a u s e d as manure. The o p e r a t i o n o f t h e e c o s y s t e m i s r e l a t i v e l y s i m p l e and y e t i t a l l o w s one t o s t u d y t h e complex metabolic transformations o f a drug o r r e l a t e d r e s i d u e s i n i t s v a r i o u s components. E s p e c i a l l y i n t e r e s t i n g i s t h e s t u d y o f t h e d e g r a d a t i o n o f a compound i n t h e s o i l i n t h e p r e s e n c e o f m i c r o organisms found i n t h e animal e x c r e t a . This i n f o r m a t i o n i s i m p o r t a n t s i n c e i t e v e n t u a l l y d e t e r m i n e s w h e t h e r a compound a n d / or i t s m e t a b o l i t e s w i l l bioaccumulate i n t h e v a r i o u s elements o f the environment.

Literature Cited 1. Metcalf, R. L., Sangha, G. Κ., and Kapoor, I. P., Environ mental Science and Technology, (1971), 5, 709-713. 2. Kantor, S., Kenneth, R. L., Jr., Waletzky, E., Tomcufcik, A. S., Science, (1970), 168, 373. 3. Kapoor, I. P., Metcalf, R. L., Nystrom, R. F., and Sangha, G. K., J. Agr. Food Chem., (1970), 18, 1145. 4. Freeman, L., Sewage Ind. Wastes, (1953), 25, 845, 1331. 5. Burge, W. D., J. Agr. Food Chem., (1971), 19 375-378. RECEIVED

January 10, 1979.


A Terrestrial-Aquatic Model Ecosystem for Evaluating the

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