Investigation of Xenobiotic Metabolism in Intact Aquatic Animals

14 Investigation of Xenobiotic Metabolism in Intact Aquatic

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch014

Animals D. G. CROSBY, P. F. LANDRUM, and C. C. FISCHER Department of Environmental Toxicology, University of California, Davis, CA 95616

More than 130 years ago, Keller (1) reported the isolation of hippuric acid (benzoylglycine) from the urine of horses fed pure benzoic acid and so ushered in our modern era of metabolism investigations on xenobiotics (foreign substances in the environment). In addition to the valuable basic knowledge of the biological processes of terrestrial animals provided by such studies, the advent of regulations controlling the use of pesticides stimulated research on the disposition of these chemicals by both mammals and insects (2). Among the requirements for registration of pesticides in the United States, the 1978 guidelines proposed by the U.S. Environmental Protection Agency (3) list general metabolism studies "in at least one mammalian species, preferably the laboratory rat . . . " Although similar tests have been conducted on other terrestrial species with increasing frequency, the small rodents have remained the principal source of metabolism data from intact animals. Standardized techniques and equipment for such investigations are in widespread use. Unfortunately, the same cannot be said for metabolism investigations in aquatic animals. Most of the world's animals exclusive of the insects --over 200,000 known species - - live at least a part of their lives in water; over 100 species have major economic importance; and they form the populations most often at risk of exposure to a growing number of chemical pollutants, but science remains largely ignorant of the disposition of xenobiotics by intact, living specimens of even the most common of the aquatic animals. In t h i s a r t i c l e , we propose to discuss some reasons f o r t h i s l a c k o f i n f o r m a t i o n , compare c h a r a c t e r i s t i c s o f the p r i n c i p a l experimental systems, and d e s c r i b e our current research on a s c i e n t i f i c protocol and technique which would provide the f u n c t i o n a l e q u i v a l e n t o f the standard t e r r e s t r i a l metabolism study but a p p l i e d to aquatic s p e c i e s .

0-8412-0489-6/79/47-099-217$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.

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Laboratory Metabolism Chambers According to the EPA g u i d e l i n e s mentioned above, metabolism studies (in rodents) have the f o l l o w i n g purposes: (1) To i d e n t i f y a n d , to the extent p o s s i b l e , q u a n t i f y significant metabolites; (2) To determine any p o s s i b l e bioaccumulation and/or b i o r e t e n t i o n of the t e s t substance and/or m e t a b o l i t e s ; (3) To determine [ p e s t i c i d e ] absorption as a f u n c t i o n of d o s e ; (4) To c h a r a c t e r i z e routes and r a t e s of [ p e s t i c i d e ] excretion; ( 5 ) To r e l a t e [ p e s t i c i d e ] absorption to the duration of exposure of the animal ; and (6) To o b t a i n an estimate of binding of the t e s t substance and/or i t s metabolites by t a r g e t macromolecules i n p o t e n t i a l t a r g e t organs. The r e q u i r e d data g e n e r a l l y are obtained by administering a measured dose o f the candidate compound - - often i s o t o p i c a l l y labelled to the r a t or mouse e i t h e r by i n j e c t i o n or per ojs_. The animal i s housed i n a glass metabolism "cage" wïïëre i t r e c e i v e s f o o d , w a t e r , and clean a i r , and i t s u r i n e , f e c e s , and r e s p i r e d gases are c o l l e c t e d and examined f o r the parent chemical and i t s m e t a b o l i t e s . Eventual postmortem t i s s u e a n a l y s i s and c a l c u l a t i o n of material balance complete the measurements necessary to s a t i s f y the above purposes of metabolism and pharmacokinetic experiments. While in v i t r o biochemical s t u d i e s are important a d j u n c t s , i t i s a l s o apparent that only experiments with i n t a c t , h e a l t h y , l i v i n g animals w i l l s u f f i c e to meet EPA c r i t e r i a . Why i s i t t h a t so l i t t l e of t h i s information e x i s t s f o r important aquatic species? The f o l l o w i n g represent some of the r e a s o n s : (1) Lack o f experimental animals. Highly standardized r a t s and mice are r e a d i l y a v a i l a b l e i n l a r g e numbers commercially, but few aquatics are s i m i l a r l y a v a i l a b l e a n d , when they a r e , suitable transportation i s d i f f i c u l t . (2) Maintenance and environmental c o n t r o l . While d i e t , b r e a t h i n g , temperature, and waste removal are v i r t u a l l y taken f o r granted i n most rodent work, they form s e r i o u s problems with aquatic a n i m a l s . Knowledge of d i e t a r y requirements and prepared d i e t s g e n e r a l l y are nonexistent f o r most s p e c i e s ; oxygen must be s u p p l i e d and t o x i c gases removed; temperature maintenance and water composition are very important; and the decay of food waste and excreta must be a v o i d e d . (3) Dosing. Rodents commonly are dosed by i n t u b a t i o n , i n t r a p e r i t o n e a l i n j e c t i o n , or i n measured amounts of d i e t . Each of these routes can become extremely d i f f i c u l t to use with most aquatic species - - e s p e c i a l l y very small forms - and absorption from the surrounding water becomes the primary

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


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route of exposure ( l a r g e l y analogous to i n h a l a t i o n exposure i n mammals). (4) Control o f remetabolism. Contamination of bedding and coprophagy undoubtedly were drawbacks i n e a r l i e r rodent work, but modern metabolism chambers remove excretory products r a p i d l y . The excretory products of aquatics are released into the medium and so may be a v a i l a b l e f o r repeated reabsorption and remeta bol i s m . (5) Metabolite c o l l e c t i o n . Whereas the separation and c o l l e c t i o n of u r i n e , f e c e s , and r e s p i r e d gases i s a simple mat t e r with r o d e n t s , the q u a n t i t a t i v e i s o l a t i o n of microgram amounts of complex metabolites from l a r g e volumes of aqueous medium - - e s p e c i a l l y seawater - - has posed a major hurdle to s t u d i e s i n aquatic organisms. Experimental aquatic metabolism systems have taken one of four forms - - s t a t i c water i n ordinary a q u a r i a , j a r s , or beakers; s t a t i c "model ecosystems"; s t a t i c outdoor ponds; and c o n t i n u o u s l y - f l o w i n g systems (Table 1 ) . A very rough comparison of t h e i r advantages i s shown i n Table II. For example, while the s t a t i c aquaria doubtless are by f a r the Table II.

Evaluation of aquatic metabolism chambers.

SA

Τ est s i m p l i c i t y Environment c o n t r o l Species f l e x i b i l i t y Dosing Remetabol ism control Metabolite c o l l e c t i o n a

a

+++ ++ ++ ++ + ++

ME

Ρ

F

+ ++ + + + +

++ + ++ + + +

++ +++ +++ +++ +++ +++

S A = s t a t i c aquarium, ME = model ecosystem Ρ = pond, and F = flow-through system

s i m p l e s t to s e t up and m a i n t a i n , they provide l e s s control of oxygenation, chemical l o s s , and other environmental f a c t o r s than do flow-through systems, as well as l e s s f l e x i b i l i t y i n the species used ( e . g . , some aquatic species are very s e n s i t i v e to accumulated waste p r o d u c t s ) . S o - c a l l e d "model ecosystems" (12) o f f e r environmental c o n t r o l s i m i l a r to that i n s t a t i c aquaria but are much more complex and so f a r have been l i m i t e d to a few c a r e f u l l y s e l e c ted and compatible species - - t y p i c a l l y , m i c r o c r u s t a c e a n s , small s n a i l s , and minnows - - which are hardy and t o l e r a t e each other i n reasonable b a l a n c e . Remetabolism and c o l l e c t i o n of



5 Water Tissues Dealkylated and reduced analogs

Green S u n f i s h

Trifluralin

11 Bile Glucuronide

Sea lamprey Trout

3-Trifluoromethyl4-nitrophenol(TMF)

5 Water

Green S u n f i s h

Methoxychlor

Demethylated and dehydrochlori nated analogs

Bluegill

Methoprene

9

acid

Phthalic

Metabolite pool

phthalate

Four f r e s h water species

10

Dioctyl

Water Muscle

8 Water Tissues

3-Hydroxy analog and other phenols

Frog

4,4'-Di chlorobiphenyl

Tissue

7

Tissues

Chlorosalicyclic acid

Midge

2 ,5-Dichloro-4'nitrosalicylanilide

1

6

Water Tissues

2,4-D

Trout, bluegill , channel c a t f i s h

5

Water Tissues

D i e l d r i n and hydroxy analogs

Reference

Green S u n f i s h

in

4

Identified

Tissues

Metabolites

Dieldrin

Major

Formed by F r e e - L i v i n g Aquatic Animals

Fresh-water C r u s t a c e a n s , mussel , s n a i l

Animal

Examples of Metabolites

2,4-D Butoxyethyl E s t e r

Aldrin

Chemical

Table I.



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i d e n t i f i a b l e q u a n t i t i e s o f metabolites are s t i l l a s e r i o u s problem, and while the i n i t i a l input of chemical to the system may be known, the actual extent of exposure of each species p r e s e n t l y i s unmeasurable. Ponds can be f a i r l y simple and o f f e r the advantage of c o n s i d e r a b l e species f l e x i b i l i t y and a more natural environment - - there seems to be no reason why s i z e o f the t e s t animal should be l i m i t i n g i n such a system — although problems of dosing and metabolite c o l l e c t i o n can be s e v e r e . However, ponds add environmental degradation and evaporation processes which may obscure whether product formation and l o s s i s real or an a r t i f a c t . Closed flow-through l a b o r a t o r y - s c a l e systems appear to have the g r e a t e s t p o t e n t i a l f o r analogy to current t e r r e s t r i a l metabolism chambers. While obviously more complex than s t a t i c a q u a r i a , they allow c l o s e r control of environmental v a r i a b l e s such as oxygenation and v o l a t i l i z a t i o n , a d a p t a b i l i t y to a wide range o f s p e c i e s , maximum freedom from remetabolism, and improved c o l l e c t i o n of waste p r o d u c t s . Dosing can be accomplished through immersion o r , beforehand, by i n j e c t i o n or i n t u b a t i o n where a p p r o p r i a t e . On the other hand, none of the four types of metabolism system i s ideal , and the most complete data probably w i l l be derived from a combination of methods. Aquatic Metabolism Protocol We have developed and tested a metabolism system and regimen which allows c o l l e c t i o n of data comparable to those from t e r r e s t r i a l a n i m a l s . The key to our experiments i s a metabolism chamber, d e s c r i b e d p r e v i o u s l y (13, 14) ( F i g . 1 ) , which can be operated i n e i t h e r the s t a t i c or flow-through mode. B r i e f l y , i n d i v i d u a l s or groups of animals are held at constant temperature i n the jacketed g l a s s chamber ( A ) , on a s t a i n l e s s s t e e l screen ( B ) , while pure water or t e s t s o l u t i o n i s passed over them (or held under s t a t i c c o n d i t i o n s ) . S o l i d wastes are separated i n a jacketed container (C) held near 0°C to minimize m i c r o b i a l a c t i o n , and the e f f l u e n t containing d i s s o l v e d metabolites i s passed onto a column of nonionic m a c r o r e t i c u l a r adsoprtion r e s i n where organic s o l u t e s are adsorbed from s o l u t i o n (D). The general experimental protocol i s summarized i n Table III.

Table III. Step Step Step Step

1. 2. 3. 4.

Proposed t e s t protocol f o r aquatic metabolism T o x i c i t y rangefinding ( s t a t i c ) Metabolism/pharmacodynamics rangefinding ( s t a t i c ) S t e a d y - s t a t e metabolism and d i s p o s i t i o n Metabolite i d e n t i f i c a t i o n and measurement





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A f t e r determining a concentration of t e s t compound which e l i c i t s no v i s u a l l y detectable response or e f f e c t i n the aquatic species over a period o f 48 hours (Step 1 ) , f r e s h animals are placed in the chamber, exposed to known concentrations of t e s t chemical ( u s u a l l y ' ^ C - l a b e l l e d ) , and the uptake r a t e and major metabolites determined (Step 2 ) . Depuration r a t e from the dosed animals a l s o can be estimated at t h i s point by t r a n s f e r to untreated water. Fresh animals a l s o can be exposed to a constant flow of t e s t s o l u t i o n u n t i l an a b s o r p t i o n - e x c r e t i o n e q u i l i b r i u m (steady s t a t e ) has been e s t a b l i s h e d , dosed b r i e f l y with l a b e l l e d compound, and r e l e a s e (turnover) r a t e determined (Step 3 ) . Excreted metabolites are c o l l e c t e d on the r e s i n column as a r e s u l t of both s t a t i c and s t e a d y - s t a t e exposures, and t h e i r separation i s accomplished by t h i n - l a y e r , g a s - l i q u i d , and/or high-pressure l i q u i d chromatography of the eluted residue (Step 4 ) . Separated metabolites (and parent compound) are quantitated by f i r s t scanning developed t h i n - l a y e r chromatographic p l a t e s to l o c a t e r a d i o a c t i v e spots by means of a radiochromatogram scanner and then a c c u r a t e l y measuring r a d i o a c t i v i t y by s c i n t i l l a t i o n c o u n t i n g . Unknowns are i d e n t i f i e d by the usual chemical and spectrometric methods. Although previous a p p l i c a t i o n s of t h i s technique in our l a b o r a t o r y had been concerned with aquatic animal metabolism of p e s t i c i d e s such as DDT, p a r a t h i o n , c a r b a r y l , and t r i f l u r a l i n (14, 1 5 ) , we a l s o became i n t e r e s t e d i n comparing metabolic routesTby means o f a "metabolic p r o b e " . Such a compound i d e a l l y should be s t a b l e to n o n b i o l o g i c a l d e g r a d a t i o n , of low t o x i c i t y to maximize the d o s e , and subject to as many major routes of metabolism as p o s s i b l e without undue a n a l y t i c a l c o m p l e x i t y . p - N i t r o a n i s o l e , whose metabolism i n the mouse r e c e n t l y was i n v e s t i g a t e d by Trautman (16), provided a s a t i s f a c t o r y probe in which O-demethylation, r i n g - o x i d a t i o n , n i t r o r e d u c t i o n , and 0 - and N-conjugation might l o g i c a l l y be observed. Consequentl y , the metabolism of p - n i t r o a n i s o l e i n marine i n v e r t e b r a t e s was chosen f o r comparison with that of the mammal. p - N i t r o a n i s o l e Pharmacodynamics i n the Sea Urchin The aquatic species chosen was Strongylocentrotus p u r p u r a t u s , the purple sea u r c h i n . T h i s animal i s a common r e s i d e n t of the C a l i f o r n i a c o a s t , a frequent pest i n commercial kelp c u l t u r e , and a s p e c i a l t y food item o f growing i n t e r e s t . Phylogenetically, these echinoderms are considered to be in the i n v e r t e b r a t e c l a s s most d i r e c t l y l i n k e d to the v e r t e b r a t e s . -,, p - N i t r o a n i s o l e (PNA), uniformly r i n g - l a b e l l e d with C, was prepared from commercial T^C-p-nitrophenol by the method of Ross (17) and p u r i f i e d by t h i n - l a y e r chromatography i n benzene-methanol (9:1 v / v ) . Sea urchins were c o l l e c t e d at S a l t P o i n t , C a l i f o r n i a , and transported i n natural sea water


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to our Davis laboratory where they were t r a n s f e r r e d to a r t i f i c i a l sea water (Instant Ocean ) at the o r i g i n a l ocean temperature of 1 0 ° C . The acute t o x i c i t y of PNA to the sea urchin was measured by immersion o f the animals at 12°C i n Instant Ocean containing known l e v e l s of t e s t compound (Table I V ) . A f t e r 24 h o u r s , the animals were observed and t r a n s f e r r e d to untreated w a t e r , R

R

Table IV.

Acute t o x i c i t y of p - n i t r o a n i s o l e i n S_. p u r p u r a t u s

(PNA)


a

Concentration (mg/1)

24 h r .

48 h r . b

10

No e f f e c t

No e f f e c t

50

2/3 Not a t t a c h e d ,

No e f f e c t

None a t t a c h e d , Spines i n d i s a r r a y

A l l attached weakly, Spines in rows

None a t t a c h e d , Spines i n d i s a r r a y

A l l dead, Sloughing spines

100

C

500

C

a

Ν = 3

b

R 24 H r s . i n untreated Instant Ocean

c

S o l u b i l i t y of PNA i s 71 .8 mg/1 at 15°C

and observed again a f t e r 24 hours. PNA showed no e f f e c t on sea urchins at concentrations of 10 mg/1 (65 μ Μ ) , and our f u r t h e r experiments were conducted below t h i s l e v e l . For the metabolism rangefinding experiments, sea urchins were dosed by i n j e c t i o n at a l e v e l of 2.2 mg/kg of body weight with 14c-PNA i n t o the c e n t r a l c a v i t y , held i n untreated water at 1 2 ° C , and both the water and coelomic f l u i d sampled p e r i o d i c a l l y over the course o f 8 h o u r s . Most of the PNA was eliminated r a p i d l y i n t o the water ( F i g . 2 ) , with a p s e u d o - f i r s t order h a l f - l i f e of appearance of 12 minutes; h a l f of the t e s t compound was l o s t from the coelomic f l u i d i n about 24 minutes. To determine absorption r a t e , another group of animals was immersed i n a 5 mg/1 s o l u t i o n of PNA in Instant 0cean (closed chamber) and the water sampled at i n t e r v a l s to measure the l o s s o f r a d i o a c t i v i t y compared to c o n t r o l s ( F i g . 3 ) . The data were normalized f o r t i s s u e weight, and e q u i l i b r i u m was reached i n about 8 hours at a body burden of 10 ug/g (10 ppm) e q u i v a l e n t to a bioconcentration f a c t o r o f 2.0 (winter animals). In another experiment, animals were c o l l e c t e d at i n t e r v a l s , s a c r i f i c e d , and t h e i r t i s s u e analyzed f o r 14c R


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HOURS Figure 3.

1

Absorption of PNA by S. purpuratus from a 5-mg/L solution.

1

0

Figure 4.

1

1

1

4 HOURS

1

1

1

Γ

1

Absorption rate of PNA from a 5-mg/L solution


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(Fig. 4). The i n i t i a l uptake r a t e was s i m i l a r f o r both types of samples, but the b i o c o n c e n t r a t i o n f a c t o r in the second instance was 4.0 (summer a n i m a l s ) . When placed i n pure w a t e r , the animals c o n t a i n i n g s t e a d y s t a t e concentrations of PNA l o s t i t at a rate of 9.4 u g / g / h r with a h a l f - l i f e (appearance i n water) of 22 minutes; when they were placed i n a 5 mg/1 s o l u t i o n of unlabel led PNA, the r a t e of l o s s (turnover) of C was s i m i l a r to the depuration rate ( F i g . 5). Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch014

1 4

p - N i t r o a n i s o l e Metabolism The s o l u b l e metabolites excreted from animals dosed by i n j e c t i o n were c o l l e c t e d on A m b e r l i t e XAD-4 r e s i n , the r e s i n eluted s e q u e n t i a l l y with d i e t h y l e t h e r , acetone, and methanol, and the s o l u t e s separated by t h i n - l a y e r chromatography on s i l i c a gel and quantitated by l i q u i d s c i n t i l l a t i o n c o u n t i n g . The products were i d e n t i f i e d by t h e i r chromatographic c h a r a c t e r i s t i c s i n comparison with authentic standards ( F i g . 6) and represented 72% of the o r i g i n a l dose; a c i d d i q e s t i o n of the s e a - u r c h i n t i s s u e released another 21.5% of 1ÏC bound as unknown p r o d u c t s , f o r a t o t a l a c c o u n t a b i l i t y of 93.5%. According to Trautman (]J5), PNA was converted almost e x c l u s i v e l y to p-nitrophenol i n the mouse with 24 h o u r s , nearly a l l of which was excreted i n the urine as g l u c u r o n i d e , s u l f a t e , g l u c o s i d e , and unextractable p r o d u c t s ; the mouse t i s s u e r e t a i n e d only about 1% of the o r i g i n a l dose. By comparison, the sea urchin metabolized PNA s l o w l y ; p-nitrophenol and i t s conjugates accounted f o r o n l y about 6% o f the metabolites and most of the remainder (90%) was p - a n i s i d i n e (p-methoxyaniline) and i t s N-acetyl d e r i v a t i v e . R

Conclusions It i s apparent that the aquatic echinoderm and t e r r e s t r i a l mammal deal with a chemical probe by very d i f f e r e n t metabolic pathways. The e x c l u s i v e formation of o x i d i z e d (Oedèmethylated) product i n the mouse may p a r t l y r e f l e c t the animal 's h i g h l y o x i d i z i n g environment, while the r e l a t i v e l y anoxic marine environment i s represented i n the observed reduced metabolites of the sea u r c h i n . One purpose of metabolic transformations has been assumed to be conversion of a x e n o b i o t i c into more w a t e r - s o l u b l e form (18); as shown by p a r t i t i o n c o e f f i c i e n t s (19), the h y d r o p h i l i c i t y of p-nitrophenol (Kp 81) i s much greater than that o f PNA (Kp 107), and that of the corresponding nitrophenyl s u l f a t e and g l y c o s i d e s must be greater s t i l l . On the other hand, while p - a n i s i d i n e (Kp 9) i s r e l a t i v e l y s o l u b l e compared to PNA, the advantage would seem to be l o s t in conversion to " i n s o l u b l e p-methoxyacetanilide (Kp 1 4 ) . 11




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Figure 6.

PNA metabolites excreted into water by S. purpuratus during 8 hr



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PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

Our research shows t h a t the described system allows the measurement o f b a s i c pharmacodynamic p r o p e r t i e s (not e x p l i c i t l y included i n the EPA g u i d e l i n e s ) and i d e n t i f i c a t i o n of x e n o b i o t i c metabolites from excreta i n much the same way as they are obtained with t e r r e s t r i a l mammals. The o r i g i n a l purposes of general metabolism s t u d i e s , o u t l i n e d at the beginning of t h i s a r t i c l e , can be s a t i s f i e d metabolite i d e n t i f i c a t i o n and q u a n t i t a t i o n , b i o a c c u m u l a t i o n , absorption and e x c r e t i o n , and binding to t i s s u e as shown with a chemical probe such as p - n i t r o a n i s o l e . While i n v i t r o measurements a l s o give important i n s i g h t and have r e c e i v e d the major emphasis in aquatic metabolism s t u d i e s (20, 21_), i t i s obvious that t h e i r r e s u l t s p r e s e n t l y are not so~cTirectly a p p l i c a b l e . The system i s not without major needs and d i f f i c u l t i e s : (1) More uniform animals w i l l be r e q u i r e d . The w i l d animals (such as S_. purpuratus) are quite v a r i a b l e , and the s t r e s s of c o l l e c t i o n and t r a n s p o r t a t i o n remains a problem; (2) The system and protocol r e q u i r e extensive s t a n d a r d i z a t i o n and the s p e c i f i c a t i o n of optimum (or at l e a s t g e n e r a l l y s u i t a b l e ) c o n d i t i o n s of temperature, o x y g e n a t i o n , water c o m p o s i t i o n , flow r a t e s , e t c . ; (3) Comparison of r e s u l t s between these and the other major experimental chambers w i l l be important, both because the others are i n c u r r e n t use and because each can o f f e r unique b e n e f i t s ; (4) The model systems e v e n t u a l l y must be compared against natural h a b i t a t s . Although the r e s u l t s of the c o n t r o l l e d , standardized l a b o r a t o r y t e s t s give important b a s i c and p r a c t i c a l information (as do those from t e r r e s t r i a l metabolism chambers), there i s no reason to b e l i e v e t h a t they represent q u a n t i t a t i v e l y the behavior of the animals in Nature; (5) Model systems e v e n t u a l l y must be extended to accommodate and i n v e s t i g a t e populations of a species and communities composed of a number of s p e c i e s . Even more than with t e r r e s t r i a l a n i m a l s , the aquatic communities provide o p p o r t u n i t i e s f o r a wide range of modified exchange, uptake, e x c r e t i o n , and metabolism which represent more than the sum o f the a c t i v i t i e s o f the component species alone.

Literature Cited 1. Keller, W., Liebig's Ann., (1842), 43, 108. 2. Menzie, C.M., "Metabolism of Pesticides--an Update", Spec. Sci. Report Wildlife 184, USDI, Washington, D.C., 1974. 3. EPA, "Proposed Guidelines for Registering Pesticides in the United States, Part II", Federal Register, (1978), 43, (63), 37395.


14.

4. 5.

CROSBY

ET

AL.

Intact Aquatic Animals

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Khan, M.A.Q., A. Kamal, R.J. Wolin, and J. Runnels, Bull. Environ. Contam. Toxicol., (1972), 8, 219. Reinbold, K.A., and R.L. Metcalf, Pestic. Biochem. Physiol., (1976), 6, 401. Rogers, C.A., and D.L. Stalling, Weed Sci., (1972), 20,


6. 101. 7. Kawatski, J.A., and A.E. Zittel, in "Investigations in Fish Control", Fish and Wildlife Service, USDI, La Crosse, Wisc., No. 79 (1977). 8. Tulp, M.T.M., G. Sundstrom, andO.Hutzinger, Chemosphere, (1976), 6, 425. 9. Metcalf, R.L., G.M. Booth, C.K. Schuth, D.J. Hansen, and P.-Y. Lu, Environ. Health Perspect., (1973), 4, 27. 10. Quistad, G.B., D.A. Schooley, L.E. Staiger, B.J. Bergot, B.H. Sleight, and K.J. Macek, Pestic. Biochem. Physiol., (1976), 6, 523. 11. Lech, J.J., and C.N. Statham, Toxicol. Appl. Pharmacol., (1975), 31, 150. 12. Metcalf, R.L., in "Essays in Toxicology" (W.J. Hayes, Jr., ed.), (1974), 5, 17. 13. Crosby, D.G., Pure Appl. Chem., (1975), 42, 233. 14. Garnas, R.L., Ph.D. Thesis, University of California, Davis, CA., 1975. 15. Fischer, C.C., and D.G. Crosby, Abstr. N W Meeting, Amer. Chem. Soc., Honolulu, Hawaii, 1975. 16. Trautman, T.D., Ph.D. Thesis, University of California, Davis, CA. 1978. 17. Ross, J.H., University of California, Davis, CA., Personal communication, 1977. 18. Baldwin, E., "An Introduction to Comparative Biochemistry", Cambridge University Press, Cambridge, 1964. 19. Leo, Α., C.H. Hansch, and D. Elkins, Chem. Rev., (1971), 71, 555. 20. Chambers, J.E., and J.D. Yarbrough, Comp. Biochem. Physiol., (1976), 55, 77. 21. Adamson, R.H., and S.M. Sieber, in "Survival in Toxic Environments" (M.A.Q. Khan and J.P. Bederka, eds.), Academic Press, New York, 1974, p. 203. RECEIVED

January 2, 1979.


Investigation of Xenobiotic Metabolism in Intact Aquatic Animals

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