In Vivo and In Vitro Studies of Mixed-Function Oxidase in an Aquatic

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21 In Vivo and In Vitro Studies of Mixed-Function Oxidase in an Aquatic Insect, Chironomus riparius J. F. ESTENIK and W. J. COLLINS Departments of Zoology and Entomology, The Ohio State University, Columbus, OH 43210

Introduction Few insects have been studied in detail in regard to the metabolism of insecticides by mixed function oxidases (MFO) (1). Most of those studies dealt with terrestrial insects. Information on the metabolism of insecticides by non-target aquatic insects is fragmentary. High population densities of midge larvae are found in "polluted" waters, i.e. waters having very l i t t l e dissolved oxygen and/or a high biological oxygen demand. MFO enzymes require oxygen. Hence, investigation of an oxygen-requiring enzyme system is especially important in midges, since they reside in habitats that may be oxygen deficient. Moreover, midges are among the most sensitive aquatic insects, responding to insecticides in the ug/L range. Thus, fundamental studies on midge metabolism of insecticides would be elucidating. In addition to the above, Chironomus riparius was selected as the test organism for the following reasons: 1) C. riparius is a common aquatic insect having a wide distribution; 2) A recent review of the taxonomic status of C. riparius Meigen and C. thummi Kieffer concluded that the designations are synonymous, the former being the correct name (2). Larvae of both names have been used extensively in physiological, biochemical and genetical research. 3) Chironomids are considered target or nontarget species. The adults of certain midge species are pests in several areas of the United States, while midge larvae comprise a major portion of the diet of certain fish species. Information about midge-insecticide interactions may relate to more effective control measures or ecological effects on midges. Experiments in this study, done exclusively with midge larvae, include: 1) 24-hr toxicity data for representative insecticides, with and without synergists; 2) in vivo absorptive uptake and metabolic studies of aldrin and dieldrin, with and without piperonyl butoxide (PBO); 3) body depuration rate (loss to water) for dieldrin; 4) determination of optimal in vitro 0-8412-0489-6/79/47-099-349$05.25/0 © 1979 American Chemical Society

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a s s a y c o n d i t i o n s f o r a l d r i n e p o x i d a t i o n by MFO s w i t h w h o l e b o d y homogenates; and 5) measurement o f a l d r i n e p o x i d a t i o n w i t h l a r v a l m i c r o s o m e s , w i t h and w i t h o u t PBO. M a t e r i a l s and Methods Insects. M i d g e l a r v a e were c o l l e c t e d a t t h e J a c k s o n P i k e sewage t r e a t m e n t p l a n t i n Columbus, O h i o and r e a r e d t o t h e a d u l t stage i n the laboratory. From them, a c o l o n y o f C h i r o n o m u s r i p a r i u s (Meigen) was e s t a b l i s h e d and m a i n t a i n e d i n a e r a t e d t a p w a t e r (21 - 2°C) i n a b i n , 48 X 37 X 23 cm, c o v e r e d w i t h a s c r e e n f l i g h t c a g e , 50 X 35 X 75 cm. The l a r v a e were f e d p u l v e r i z e d H a r t z M o u n t a i n Dog Y u m m i e s and r e a r e d a c c o r d i n g t o a d e s c r i b e d method (3) w i t h o u t t h e a d d i t i o n o f s u b s t r a t e . The l a r v a e were m a i n t a i n e d f o r 3 y e a r s as a l a b o r a t o r y c u l t u r e p r i o r t o e x p e r i mentation. R

Chemicals. I n s e c t i c i d e s , a t l e a s t 9 5 % p u r e , were p r e p a r e d as a c e t o n e s o l u t i o n s : p - p DDT, l i n d a n e , p a r a t h i o n , p a r a o x o n , malathion, malaoxon, propoxur, c a r b a r y l , L a n d r i n , aminocarb, m e x a c a r b a t e , a l l e t h r i n ( 9 0 % ) , p i p e r o n y l b u t o x i d e (PBO) and sesamex. A l d r i n was 9 8 . 5 % and d i e l d r i n was 99+% p u r e . A l l o t h e r c h e m i c a l s were a n a l y t i c a l r e a g e n t g r a d e . A l l s o l v e n t s were r e d i s t i l l e d i n g l a s s . f

R

Immersion T o x i c i t y : A s s a y P r o c e d u r e , C r i t e r i a o f Response and D a t a A n a l y s i s . The a s s a y p r o c e d u r e was a m o d i f i c a t i o n o f p u b l i s h e d methods (£, 5 ) . T o x i c i t y a s s a y s were c o n d u c t e d a t 21 - 2°C i n n a r r o w - m o u t h , q u a r t (0.95 L) g l a s s j a r s c o n t a i n i n g c o n d i t i o n e d Columbus t a p w a t e r (pH 7.5 t o 8.5, aged 24 h r ) . No f o o d , s u b s t r a t e , o r a e r a t i o n were u s e d d u r i n g t h e t e s t . Cannib a l i s m d i d n o t o c c u r w i t h w e l l - f e d l a r v a e and s h o r t t e r m (24 h r ) assays. A l l aqueous s o l u t i o n s o r s u s p e n s i o n s were p r e p a r e d b y a d d i n g i n s e c t i c i d e i n 0.5 ml o f a c e t o n e t o 500 ml o f c o n d i t i o n e d t a p w a t e r and v i g o r o u s l y s h a k i n g t h e c a p p e d c o n t a i n e r . New a c e t o n e s o l u t i o n s o f i n s e c t i c i d e were p r e p a r e d f o r e a c h e x p e r i m e n t . E a c h a s s a y c o n t a i n e d u n t r e a t e d c o n t r o l s and s o l v e n t c o n t r o l s . Twenty f o u r t h - i n s t a r midge l a r v a e were p l a c e d i n t e s t c o n t a i n e r s , 10 l a r v a e / c o n t a i n e r , 2 c o n t a i n e r s / i n s e c t i c i d e c o n c e n tration. M o r t a l i t y was r e c o r d e d a f t e r 24 h r , m o r i b u n d l a r v a e b e i n g r e c o r d e d as d e a d . L a r v a e u s e d i n s y n e r g i s m e x p e r i m e n t s were p r e t r e a t e d f o r 1 h r i n 1 mg/L PBO o r sesamex ( s u b - l e t h a l doses). P r e t r e a t e d l a r v a e were t r a n s f e r r e d t o j a r s c o n t a i n i n g i n s e c t i c i d e w i t h s y n e r g i s t , and m o r t a l i t y was r e c o r d e d a f t e r 24 h r . The s y n e r g i s t i c r a t i o (SR) was o b t a i n e d b y d i v i d i n g t h e LC50 o f t h e i n s e c t i c i d e a l o n e b y t h e LC50 o f t h e i n s e c t i c i d e synergist mixture. LC50 v a l u e s a r e f r o m p o o l e d d a t a o f 3 e x p e r i ments p e r f o r m e d on d i f f e r e n t d a y s . O r g a n i s m s t h a t p u p a t e d d u r i n g t h e a s s a y p e r i o d o r t e s t c o n c e n t r a t i o n s where m o r t a l i t y was 0% o r

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100% were e x c l u d e d f r o m a n a l y s i s . T o x i c i t y a s s a y s were c o r r e c t e d f o r c o n t r o l m o r t a l i t y ( 6 ) . The L C v a l u e s and t h e i r 95% c o n f i d e n c e l e v e l were d e t e r m i n e d b y c o m p u t e r ( 7 ) . V a r i o u s c r i t e r i a have b e e n u s e d t o d e f i n e midge m o r t a l i t y i n c l u d i n g l a c k o f movement when t o u c h e d w i t h a p r o b e , i n a b i l i t y t o make u n d u l a t i n g movements, a n d c o l o r changes (£, S_, 8_, 9 ) . We u s e d m o b i l i t y changes as a measure o f midge m o r t a l i t y . N o r m a l m i d g e s e x h i b i t b o d y movements t h a t we d e f i n e a s a swimming c y c l e w h i c h , i n c o m p o s i t e , resembles a f i g u r e e i g h t . Normal l a r v a e g e n e r a t e c o n t i n u o u s f i g u r e e i g h t s a s t h e y swim. A n y l a r v a w h i c h c o u l d n o t r e s p o n d w i t h 3 swimming c y c l e s when p i n c h e d w i t h t w e e z e r s i n t h e r e g i o n o f t h e a n a l p a p i l l a e was c o n s i d e r e d moribund. 5 0

Homogenizing Procedure. L a r v a e i n w h o l e b o d y homogenate o r s u b c e l l u l a r f r a c t i o n a s s a y s were h o m o g e n i z e d i n T r i s - H C l b u f f e r unless otherwise noted. I n uptake o r depuration experiments, m i d g e s were r i n s e d once w i t h w a t e r a n d h o m o g e n i z e d i n 5 m l o f d i s t i l l e d w a t e r . A l l homogenates were p r e p a r e d i n a g l a s s P o t t e r - E l v e h e j m t i s s u e g r i n d e r a t a low speed f o r a p p r o x i m a t e l y 20 s e c , 10 p a s s e s t h r o u g h t h e b r i e . E x t r a c t i o n P r o c e d u r e . We m o d i f i e d t h e e x t r a c t i o n p r o c e d u r e o f N e l s o n ert a l _ (10) . B r i e a c i d i f i e d w i t h 2 ml o f 5% t r i c h l o r a c e t i c a c i d (TCA) was e x t r a c t e d 3 t i m e s w i t h 20 m l o f p e t r o l e u m ether. The c o m b i n e d e x t r a c t s were r e d u c e d t o 5 ml i n a r o t a t i n g e v a p o r a t o r , r e t u r n e d t o t h e s e p a r a t o r y f u n n e l , a n d combined w i t h 60 m l e a c h o f a c e t o n i t r i l e a n d d i s t i l l e d w a t e r . The a c e t o n i t r i l e - w a t e r - i n s e c t i c i d e m i x t u r e was e x t r a c t e d t w i c e w i t h 60 m l o f p e t r o l e u m e t h e r a n d a n h y d r o u s N a S 0 was added t o t h e c o m b i n e d 120 m l e x t r a c t . The e x t r a c t was e v a p o r a t e d j u s t t o d r y n e s s a n d t h e r e s i d u e was d i s s o l v e d i n b e n z e n e f o r a n a l y s i s b y g a s - l i q u i d chromatography (GLC). E x t r a c t i o n e f f i c i e n c i e s i n s p i k e d e x p e r i ments were 7 3 % ( a l d r i n ) a n d 8 3 % ( d i e l d r i n ) . W a t e r s a m p l e s were e x t r a c t e d 3 t i m e s w i t h 50 m l o f p e t r o l e u m e t h e r a n d a n h y d r o u s Na2S04 was added t o t h e c o m b i n e d 150 m l e x t r a c t . The e x t r a c t was e v a p o r a t e d j u s t t o d r y n e s s a n d d i s s o l v e d i n b e n z e n e f o r GLC a n a l y s i s . The e x t r a c t i o n e f f i c i e n c y f o r d i e l d r i n i n w a t e r was 9 5 % . 2

4

G a s - L i q u i d C h r o m a t o g r a p h y ( G L C ) . A V a r i a n A e r o g r a p h GLC M o d e l 1440 e q u i p p e d w i t h a H S c e l e c t r o n c a p t u r e d e t e c t o r a n d a 150 cm X 2 mm ( i . d . ) g l a s s column p a c k e d w i t h 3% SE-30 o n Gas Chrom Q was u s e d . The f o l l o w i n g o p e r a t i n g p a r a m e t e r s were e m p l o y e d : i n j e c t o r 240°C, column 200°C, d e t e c t o r 260°C, a n d a N f l o w o f 37.5 m l / m i n . The chromatogram was r e c o r d e d on a V a r i a n M o d e l A-25 r e c o r d e r w i t h a c h a r t s p e e d o f 0.1 i n / m i n . Insectic i d e s were q u a n t i f i e d b y peak h e i g h t u s i n g d a i l y s t a n d a r d c u r v e s . E a c h a n a l y s i s was d e t e r m i n e d b y a v e r a g i n g t h e p e a k s o f t h r e e injections. 3

2

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I n s e c t i c i d e Absorption, Conversion and Depuration. Aqueous suspensions o f i n s e c t i c i d e s were prepared as f o r t o x i c i t y assays. Fourth i n s t a r l a r v a e , 20 p e r c o n t a i n e r , were exposed f o r 2 h r to 20 ug/L o f a l d r i n o r d i e l d r i n . When a s y n e r g i s t was used, 20 midges were p r e t r e a t e d f o r 1 h r i n 1 mg/L PBO, then exposed f o r 2 h r to 20 ug/L o f a l d r i n o r d i e l d r i n and 1 mg/L PBO. In depuration experiments, 60 midges were exposed f o r 1 h r t o 20 ug/L d i e l d r i n . The water and groups o f 20 midges each were analyzed f o r d i e l d r i n content a f t e r being h e l d f o r 0, 3 h r and 6 h r i n clean water a f t e r exposure. E x t r a c t i o n and a n a l y s i s o f midges and water were as p r e v i o u s l y d e s c r i b e d . Mean values were computed from two separate experiments. Assay o f Homogenate f o r A l d r i n Epoxidation. The f o l l o w i n g experimental sequence was designed t o determine the optimum i n v i t r o c o n d i t i o n s f o r a l d r i n epoxidation i n l a r v a l whole body homogenates: 1) the e f f e c t o f component chemicals g e n e r a l l y i n c l u d e d i n an i n c u b a t i o n mixture, 2) a pH p r o f i l e , 3) a temperature p r o f i l e , 4) a m o l a r i t y p r o f i l e , 5) a r e a c t i o n time p r o f i l e , 6) a l a r v a l c o n c e n t r a t i o n (enzyme concentration) p r o f i l e , 7) a s u b s t r a t e c o n c e n t r a t i o n p r o f i l e , and 8) a restudy o f the e f f e c t s o f component chemicals i n the i n i t i a l i n c u b a t i o n mixture (Step 1) upon a l d r i n epoxidation under optimum c o n d i t i o n s as d e f i n e d by steps 2-7 above. The e f f e c t o f PBO, FMN, and FAD upon enzyme a c t i v i t y was a l s o t e s t e d . In Step 1, an i n c u b a t i o n mixture (11) was t e s t e d using 20 midge l a r v a e homogenized i n 8.3 Χ 10" M T r i s - H C l b u f f e r , pH 7.5. Each 5 ml i n c u b a t i o n mixture contained 20 homogenized midges, 5.0 Χ 1 0 " M T r i s HC1 b u f f e r , pH 7.5, 2.4 Χ Ι Ο " M glucose 6-phosphate (G-6-P), 1.6 u n i t s glucose 6-phosphate dehydrogenase (G-6-P dH), 5.1 Χ 10" M NADP, and 2.7 Χ 10" M KCl. In a d d i t i o n , the f o l l o w i n g chemicals were i n c l u d e d i n the f i n a l concentration i n d i c a t e d : 5.1 Χ 10" M NADH, 1% (W/V) bovine serum albumin (BSA), and 1.0 mg a l d r i n i n 0.1 ml ethanol. Whole body homogenate experiments i n c l u d e d a l l o f the above chemicals unless otherwise noted. Reaction mixtures were incubated with s w i r l i n g i n t e s t tubes at 30 - 1°C. Reactions i n Steps 1-4 o f the experimental sequence were stopped a f t e r 1 h r and Steps 6-8 a f t e r 15 min, by the a d d i t i o n o f 2 ml 5% TCA. 2

2

3

5

3

5

Microsome P r e p a r a t i o n . Fourth i n s t a r midge l a r v a e , 1,000/ experiment, were weighed t o the nearest 0.1 mg and homogenized i n 8.3 Χ 10" M T r i s - H C l b u f f e r , pH 7.5. Preparatory c e n t r i f u g a t i o n s were performed i n a S o r v a l l Model RC2-B c e n t r i f u g e with a SS-34 r o t o r . The homogenate was sedimented a t 2,400g max (4,500 RPM) f o r 15 min at 1 - 1°C t o remove large c e l l fragments and d e b r i s . The supernatant was c e n t r i f u g e d a t 20,000g max (13,000 RPM) f o r 15 min at 1 ± 1°C to i s o l a t e the m i t o c h o n d r i a l p e l l e t . The post-mitochondrial super­ natant was sedimented i n a Beckman Model L c e n t r i f u g e with a 50 1

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r o t o r a t 128,000g max (40,000 RPM) t o i s o l a t e t h e m i c r o s o m e s . A p o r t i o n o f t h e m i c r o s o m e p e l l e t was r e s u s p e n d e d i n 1.5 Χ 1 0 " M K C l , pH 7.5 a n d c e n t r i f u g e d a s b e f o r e (washed m i c r o s o m e s ) . A l l e q u i p m e n t , s o l u t i o n s , a n d g l a s s w a r e were p r e c o o l e d a n d a l l p r o c e s s e d m a t e r i a l was k e p t i n c r u s h e d i c e u n t i l i n c u b a t e d . 1

Assay o f S u b c e l l u l a r F r a c t i o n s f o r A l d r i n Epoxidation. M i t o c h o n d r i a l a n d m i c r o s o m a l p e l l e t s were r e s u s p e n d e d i n T r i s - H C l buffer. Each 5 ml i n c u b a t i o n m i x t u r e c o n t a i n e d the f o l l o w i n g : 2.4 X 10"" M G-6-P, 1.6 u n i t s G-6-P dH, 5.1 X 1 0 ' M NADP, 1.0 mg a l d r i n i n 0.2 m l e t h a n o l when u s e d a l o n e , o r 1.0 mg a l d r i n a n d 1.0 mg PBO, e a c h i n 0.1 m l e t h a n o l , when u s e d i n c o m b i n a t i o n . I n s y n e r g i s m e x p e r i m e n t s , m i x t u r e s were p r e t r e a t e d w i t h PBO f o r 3-5 min p r i o r t o t h e a d d i t i o n o f s u b s t r a t e . The r e a c t i o n m i x t u r e s were i n c u b a t e d w i t h a g i t a t i o n i n t e s t t u b e s a t 30 - 1°C i n a w a t e r b a t h s h a k e r f o r 15 m i n . R e a c t i o n s were s t o p p e d b y a c i d i f y i n g w i t h 2 m l 5% TCA. The a c i d i f i e d m i x t u r e s were e x t r a c t e d a n d a n a l y z e d b y GLC. 3

5

P r o t e i n A n a l y s i s . P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d w i t h a S p e c t r o n i c 20 s p e c t o p h o t o m e t e r e m p l o y i n g BSA a s a s t a n d a r d ( 1 2 ) . E a c h 0.1 m l sample was s p o t t e d o n 3 c m Whatman No. 42 f i l t e r paper and a i r d r i e d . Samples were s t a i n e d w i t h X y l e n e B r i l l i a n t C y a n i n G (Κ a n d Κ L a b o r a t o r i e s , C l e v e l a n d , O h i o ) , a n d t h e a b s o r b a n c e a t 610 nm was r e c o r d e d a g a i n s t a b l a n k c o n t a i n i n g d i s t i l l e d water. Samples were c o r r e c t e d u s i n g c o n t r o l s c o n t a i n ­ i n g a l l components e x c e p t p r o t e i n . 2

Results a n c

T o x i c i t y Assays. The computed L C 5 0 * 95% c o n f i d e n c e i n t e r v a l a n d t h e s y n e r g i s t i c r a t i o (SR) f o r e a c h i n s e c t i c i d e a r e c o n t a i n e d i n T a b l e I . C o n t r o l m o r t a l i t y was 5% o r l e s s i n a l l experiments. In g e n e r a l , o r g a n o c h l o r i n e and organophosphate i n s e c t i c i d e s had L C 5 Q v a l u e s i n t h e same o r d e r o f m a g n i t u d e ( L C 5 Q s f r o m 0.5 ug/L t o 6.2 u g / L ) , w h i l e c a r b a m a t e i n s e c t i c i d e s were g e n e r a l l y l e s s t o x i c ( L C Q S f r o m 12.2 ug/L t o 376.6 u g / L ) . D i e l d r i n , t h e o x i d a t i v e m e t a b o l i t e o f a l d r i n , was t h e most t o x i c o f a l l i n s e c t i c i d e s i n t h i s s t u d y b u t was o n l y s l i g h t l y more t o x i c t h a n i t s p a r e n t compound. The o x i d a t i v e m e t a b o l i t e s o f p a r a t h i o n a n d m a l a t h i o n , p a r a o x o n a n d m a l a o x o n , were s l i g h t l y l e s s t o x i c t h a n t h e i r p a r e n t compounds. The r a n g e f o r t h e r e g r e s s i o n c o e f f i c i e n t s o f a l l i n s e c t i ­ c i d e s was g e n e r a l l y medium t o h i g h (3.0-10.9) e x c e p t f o r p a r a t h i o n a n d c a r b a r y l w h i c h were 2.1 a n d 2.6, r e s p e c t i v e l y . Low s y n e r g i s t i c r a t i o s i n d i c a t i n g a n t a g o n i s m b y PBO ( r a t i o s f r o m 0.15 t o 0.33) were o b t a i n e d w i t h a l d r i n , p a r a t h i o n , m a l a t h i o n , a m i n o c a r b , a n d m e x a c a r b a t e . PBO s t r o n g l y s y n e r g i z e d a l l e t h r i n (SR 1 0 2 ) . A s s a y s where s y n e r g i s m was weak o r a b s e n t f

5

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Table I .

LC5Q values (24 h r ) f o r f o u r t h i n s t a r l a r v a l Chironomus r i p a r i u s

LC Insecticide

9 5 %

5 0

1

( C- *)» ug/L

Synergistic rati

Organochlorine DDT DDT+Sesamex Aldrin Aldrin+PBO Dieldrin Dieldrin+PBO Lindane

4.7 2.9 0.8 2.2 0.5 0.3 3.6

(4.2-5.3) (2.6-3.2) (0.7-0.8) (1.9-2.6) (0.4-0.6) (0.3-0.4) (3.1-4.0)

1.63

-

0.33

1.39

-

Organophosphate Parathion Parathion+PBO Paraoxon Paraoxon+PBO Malathion Malathion+PBO Malaoxon Malaoxon+Sesamex

2.5 17.1 6.2 5.5 1.9 6.8 5.4 1.9

(1.7-4.1) (14.3-21.6) (5.8-6.7) (4.8-6.5) (1.7-2.2) (6.0-7.8) (4.9-5.9) (1.6-2.3)

_

0.15

1.13

-

0.28

-

2.78

Carbamate Carbaryl Carbaryl+PBO Landrin Landrin +PBO Aminocarb Aminocarb+PBO Mexacarbate Mexacarbate+PBO Propoxur Propoxur+PBO R

R

104.5 62.4 51.4 45.7 376.6 1,172.2 12.2 59.3 64.4 29.2

(83.3-151.7) (54.0-72.9) (45.0-59.3) (37.5-54.5) (329.7-428.5) (1,072.0-1,308.1) (11.0-13.5) (52.9-66.2) (59.5-69.4) (25.2-33.8)

_

1.68

1.12

0.32

-

0.20

-

2.21

Synthetic B o t a n i c a l Allethrin Allethrin+PBO

41.9 (38.6-45.3) 0.4 (0.3-0.5)

102.10

21.

ESTENiK A N D C O L L I N S

Mixed-Function

Oxidase

355

( r a t i o s from 0.81 to 2.78) included: malaoxon or DDT with sesamex; propoxur, c a r b a r y l , d i e l d r i n , paraoxon, or L a n d r i n PBO.

R

with

I n s e c t i c i d e Absorption, Conversion and Depuration. Midge l a r v a e immersed i n 20 ug/L a l d r i n f o r 2 hr absorbed 25.4 ng o f i n s e c t i c i d e per l a r v a , converting 58% o f i t to d i e l d r i n (Table I I ) . With 1.0 mg/L PBO present, midges absorbed 18.4 ng o f a l d r i n per l a r v a , but no a l d r i n was converted to d i e l d r i n . With o r without PBO, midge larvae absorbed d i e l d r i n at the same r a t e as a l d r i n (Table I I ) .

Table I I .

The

absorption

o f i n s e c t i c i d e s by C_. r i p a r i u s larvae

Body Content, ng/larva Insecticide

Aldrin

Aldrin

10.7

(0.8)

Dieldrin Aldrin+PBO Dieldrin+PBO a

18.4

(1.5)

Dieldrin

(ug/g) Total

14.7

(1.1)

25.4

(1.9)

23.9

(1.9)

23.9

(1.9)

18.4

(1.5)

24.9

(2.0)

a

N.D.

24.9

(2.0)

N o t Detected

A l l i n s e c t i c i d e s a f f e c t e d the m o b i l i t y o f £. r i p a r i u s larvae i n a s i m i l a r manner. A normal swimming motion was g e n e r a l l y reduced to 1 c y c l e at the onset o f t o x i c symptoms. The e f f e c t o f the t o x i c a n t increased u n t i l the l a r v a l o s t a l l a b i l i t y to move. Death soon followed. S i m i l a r symptoms o f i n s e c t i c i d e poisoning have been reported f o r s t o n e f l y naiads (13, 14). Changes i n l a r v a l c o l o r were u n r e l i a b l e f o r determining t o x i c a f f e c t . Toxic symptoms were observed i n approximately 50-75% o f the midge larvae immersed i n 20 ug/L a l d r i n at the end o f the 2 h r exposure but no e f f e c t s were noted with a l d r i n plus PBO. A l l larvae immersed i n d i e l d r i n , or d i e l d r i n with PBO were moribund. The depuration of d i e l d r i n from midge larvae was r e l a t i v e l y slow (Table I I I ) . Larvae t r a n s f e r r e d to clean water f o r 3 h r r e l e a s e d 0.011 ng o f d i e l d r i n / l a r v a / h r , or 0.2% o f the t o t a l d i e l d r i n absorbed. Another group o f larvae t r a n s f e r r e d to clean water f o r a t o t a l o f 6 hr, released d i e l d r i n to the water at approximately the same r a t e , 0.014 ng/larva/hr, or 0.5% o f the t o t a l d i e l d r i n absorbed.

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

356

Table I I I .

Depuration o f d i e l d r i n by £. r i p a r i u s larvae

Time Hours

Dieldrin, ng/midge

0

19.0

3

19.5

0.65

0.011

6

17.8

1.65

0.014

D i e l d r i n i n water,

28

Loss, ng/midge/hr

E s t a b l i s h i n g Optima f o r A l d r i n Epoxidation Using Whole Body Homogenates. The a d d i t i o n o f a l l component chemicals increased d i e l d r i n p r o d u c t i o n g r e a t e r than 3X compared to the unmodified homogenate (Table IV). As the optimum o f each f a c t o r was establ i s h e d , i t was used i n a l l subsequent experiments.

Table IV. A l d r i n epoxidase requirements o f £. r i p a r i u s whole body homogenates

Incubation Medium

nmoles d i e l d r i n / m i n

% Maximum

0.015

28

+ 1% BSA

0.016

30

+ 1% BSA G-6-P (2.4 X 10-3 M) G-6-P dH (1.6 u n i t s ) NADH (5.1 X 10-5 M) NADP (5.1 X 10-5 M)

0.38

72

+ KCl (2.7 X 10-3 M) and a l l o f above

0.053

Crude Homogenate

a

a

100

2 0 midges/5 ml, 0.05 M T r i s b u f f e r , pH 7.5, 1 mg a l d r i n . A l l incubations at 30°C f o r 1 h r . Not optimum c o n d i t i o n s (Table V ) ; mean o f 2 experiments.

21.

ESTENIK AND COLLINS

Table V.

Mixed-Function

357

Oxidase

A l d r i n epoxidase requirements o f £. r i p a r i u s whole body homogenates under optimum c o n d i t i o n s a

Incubation Medium

nmoles d i e l d r i n / m i n

Crude Homogenate

% Maximum

0.116

36

0.324

100

+ KCl (2.7 Χ 10" M) and e l e c t r o n generator

0.314

97

+ 0.1% BSA, KCl and e l e c t r o n generator

0.279

86

+ NADH (5.1 Χ Ι Ο " M) and a l l o f above

0.275

85

+ ( e l e c t r o n generator) G-6-P (2.4 Χ 10" M) G-6-P dH (1.6 u n i t s ) NADP (5.1 Χ 10" M) 3

5

3

5

1

aLarvae were homogenized i n 8.3 Χ 10" M T r i s - H C l , pH 7.5 b u f f e r . Complete i n c u b a t i o n medium ( f i n a l concentrations): 3 ml o f homogenate (20 l a r v a e ) ; 5.0 Χ 10" M T r i s HC1 b u f f e r , pH 7.5; 1.0 mg a l d r i n i n 0.5 ml ethanol; t o t a l volume, 5 ml. Reaction mixtures were incubated at 30°C f o r 15 min. Mean values from 2 experiments. 1

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

358

The e f f e c t o f pH on in_ v i t r o a l d r i n epoxidase a c t i v i t y was e s t a b l i s h e d over a pH range 6.5-8.5 (Figure 1). A pH o f 7.5 was used as optimum. The e f f e c t o f temperature on i n v i t r o a l d r i n epoxidase a c t i v i t y was determined over a range o f 20°-40°C (Figure 2). An optimum i n c u b a t i o n temperature o f 30°C was used. The maximum epoxidase a c t i v i t y was a t t a i n e d at a T r i s - H C l b u f f e r concentration o f 5.0 Χ Ι Ο " M (Figure 3). A f t e r e s t a b l i s h i n g optimum r e a c t i o n c o n d i t i o n s , the e f f e c t o f chemical supplements upon epoxidase a c t i v i t y was re-examined. Several chemicals used i n p r e l i m i n a r y incubation mixtures and throughout o p t i m i z i n g experiments had no e f f e c t upon d i e l d r i n production under optimum c o n d i t i o n s . These are BSA, KCl and NADH (Table V ) . O v e r a l l , there was a 2 2 - f o l d enhancement o f i n v i t r o epoxidase a c t i v i t y when i n i t i a l c o n d i t i o n s (crude homogenate, Table IVJ are compared t o optimum c o n d i t i o n s (homogenate p l u s e l e c t r o n generator, Table V). FMN decreased, FAD s l i g h t l y increased and PBO completely i n h i b i t e d i n v i t r o epoxidase a c t i v i t y (Table V I ) . 1

Table VI.

E f f e c t o f FMN, FAD, and PBO upon i n v i t r o C. r i p a r i u s a l d r i n epoxidase i n whole body homogenates a

Incubation Medium

nmoles d i e l d r i n / midge/min

Homogenate

Percent

0.081

100

FMN

(10"

3

M)

0.075

92

FAD

(10~

3

M)

0.089

110

PBO

(6.0 X 10~

4

M)

optimum c o n d i t i o n s o f Table V. b

Standard

c

N o t Detected

C

N.D.

b

0

Mean values o f 2 experiments

The cumulative amount o f d i e l d r i n increased during incuba­ t i o n times o f 15 min and 30 min, then approached a p l a t e a u over the f o l l o w i n g 45 min (Figure 4 ) . The p l o t o f d i e l d r i n / i n s e c t / min vs. time o f i n c u b a t i o n had a negative slope (Figure 4 ) . A 15 min i n c u b a t i o n time was used as optimum.

21.

ESTENIK AND COLLINS

Figure

1.

Mixed-Function

Effect of pH on aldrin

Oxidase

epoxidation by midge homogenate experiments)

(mean of

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

360

Figure 2.

ORGANISMS

Effect of temperature on aldrin epoxidation by midge homogenate (mean of 2 experiments)

21.

ESTENiK

Mixed-Function

A N D COLLINS

20 "

I

« // 0.05

9

f

BUFFER Figure 3.

1 0.2 5

Oxidase

1 0.5 0

CONCENTRATION:

1 0.75

i _ 1.0

MOLAR

Effect of buffer concentration on aldrin epoxidation by midge homog note (mean of 2 experiments)

362

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

ORGANISMS

TIME : MINUTES Figure 4. Effect of incubation time on aldrin epoxidation by midge homogenate (mean of 2 experiments). Rate analysis: ( and left ordinate), nmol/larva/min and ( and right ordinate), cumulative aldrin epoxidized, nmol/larva.

21.

ESTENIK AND

COLLINS

Mixed-Function

363

Oxidase

The i n v i t r o a l d r i n epoxidase a c t i v i t y was l i n e a r l y c o r r e l a t e d with homogenate concentration (enzyme concentration) over a range o f 1 t o 5 larvae/ml (Figure 5 ) . D i e l d r i n production increased from 0.39 to 1.91 nmoles/ i n s e c t when the substrate concentration was increased from 0.01 mg t o 1.0 mg i n 5 ml o f incubation mixture (Table V I I ) .

Table V I I .

The e f f e c t o f a l d r i n concentration and BSA upon a l d r i n epoxidase o f £. r i p a r i u s whole body homogenates^

mg a l d r i n / 5 ml

% BSA

nmoles dieldrin/midge

1.0

1.0

1.91

1.0

0.1

2.12

0.1

1.0

1.20

0.01

1.0

0.39

a

0ptimum c o n d i t i o n s o f Table V. experiments.

Mean values from 2

Assay o f S u b c e l l u l a r F r a c t i o n s f o r A l d r i n Epoxidation. There was considerable epoxidase a c t i v i t y i n the mitochondrial f r a c t i o n but the highest a c t i v i t y was i n the washed microsome f r a c t i o n . Microsomes washed i n KCl were more a c t i v e than unwashed microsomes. No epoxidase a c t i v i t y was detected i n the post-microsomal supernatant, o r when PBO was added t o microsomes (Table V I I I ) . Discussion Allowing f o r d i f f e r e n c e s due to s p e c i e s , assay techniques and the s u b j e c t i v e nature o f assessing midge m o r t a l i t y , the s u s c e p t i b i l i t y o f £. r i p a r i u s t o i n s e c t i c i d e s (Table I) i s g e n e r a l l y s i m i l a r t o other chironomid s p e c i e s . £. r i p a r i u s and £. tentans (5) e x h i b i t s i m i l a r s u s c e p t i b i l i t y t o two organoc h l o r i n e s , d i e l d r i n and DDT. Likewise, the L C S Q ' S o f s e v e r a l organophosphates t o £. r i p a r i u s , Tanypus grodhausi (4) and £. tentans (5) are i n the same range, although LC50 values higher than 10 ug/L have been reported f o r other organophosphates and Chironomus sp. 51, Goe1dichironomus holoprassinus and Chironomus

364

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

30000

LU

5

/

25000

ο Ο

/

20000

ο Ο ce Û. 1 5 0 0 0 oc Q

UJ

Q

10000

O

ζ 5000

1

2

3

4

5

6

LARVAL NUMBER/ ML Figure 5.

Effect of enzyme concentration (hrvae/mL) on aldrin epoxidation by midge homogenate (mean of 2 experiments)

21.

ESTENIK AND COLLINS

Table V I I I .

Mixed-Function

365

Oxidase

S u b c e l l u l a r l o c a l i z a t i o n o f a l d r i n epoxidase a c t i v i t y i n £. r i p a r i u s l a r v a eà

pmoles d i e l d r i n / m g p r o t e i n / m i n (pmoles d i e l d r i n / l a r v a l e q u i v . / m i n )

Fraction

Homogenate

236

(86.7)

Mitochondrial

757

(6.6)

Microsomal

798

(19.4)

Microsomal

(washed)

1,303

(23.8)

Microsomal+PBO

N.D.

b

Post-microsomal Supernatant

N.D.

b

a

0 p t i m u m c o n d i t i o n s o f T a b l e V. experiments. Not D e t e c t e d .

Mean v a l u e s o f 2

366

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

sp. (£, 15). In a comparison o f s p e c i f i c i n s e c t i c i d e s , the L C 5 0 values f o r parathion (4), malathion (4_, 5) and a l l e t h r i n (5) among s e v e r a l midge species c l o s e l y correspond to the r e s p e c t i v e LC5Q values f o r £. r i p a r i u s . However, carbamates appear to be approximately 10X more t o x i c to £. tentans (5) than to £. r i p a r i u s . The i n s e c t i c i d e - P B O assays provide evidence that £. r i p a r i u s larvae have an a c t i v e MFO system: 1) PBO, a s e l e c t i v e i n h i b i t o r o f MFO, antagonized a l d r i n , two phosphorothioates (parathion and malathion) and two carbamates (mexacarbate and aminocarb) which are metabolized by MFO,to products that are more t o x i c o r more potent a c e t y l c h o l i n e s t e r a s e i n h i b i t o r s than the parent compound (16, 17, 1J3, 1£, 20) and 2) a l l e t h r i n was s t r o n g l y synergized by PBO, which i n h i b i t s a l l e t h r i n d e t o x i c a t i o n (21). A high a l l e t h r i n - P B O r a t i o with £. r i p a r i u s c o n t r a s t s with £. tentans (5) and other i n s e c t s (21), i n d i c a t i n g a s i g n i f i c a n t metabolic d i f f e r e n c e with respect t o a l l e t h r i n i n two r e l a t e d midge s p e c i e s . None o f the carbamates were synergized very much by PBO i n c l u d i n g c a r b a r y l , a carbamate used to assay f o r MFO i n j o i n t a c t i o n s t u d i e s (22). C. tentans a l s o e x h i b i t e d a low PBOc a r b a r y l r a t i o (5). Paraoxon i s not apparently metabolized very much by MFO, i n c o n t r a s t to malaoxon. I n d i r e c t evidence f o r an a c t i v e MFO system i n midges was provided by f i n d i n g d i e l d r i n i n l a r v a e exposed to a l d r i n (23). Aminocarb and mexacarbate, both antagonized by PBO i n midges, were demethylated at the phenyl-N-dimethyl group by r a t l i v e r MFO, forming more potent a c e t y l c h o l i n e s t e r a s e i n h i b i t o r s than the parent compounds (20). L a n d r i n , with no phenyl-Ndimethyl group but otherwise s i m i l a r to aminocarb and mexacarbate, was u n a f f e c t e d by PBO i n midges. Although no metabolites were i d e n t i f i e d i n the present study, the midge t o x i c i t y data and enzyme i n h i b i t i o n s t u d i e s (20) suggest that demethylation o f phenyl-N-dimethyl groups o f carbamates occurs i n midges as an activation reaction. Midges exposed to a l d r i n o r d i e l d r i n (20 ug/L,2 hr) c o n t a i n ed (body weight b a s i s ) 95X the aqueous c o n c e n t r a t i o n . Furthermore, midges l o s t l e s s than 0.1% o f absorbed d i e l d r i n per hr i n a 6 hr depuration experiment. Therefore, C_. r i p a r i u s r a p i d l y concentrate d i e l d r i n from the water and r e t a i n most o f the absorbed dose. Midges exposed to 0.02 ug/L a l d r i n or 0.05 ug/L DDT accumulated them by f a c t o r s (dry weight b a s i s ) o f > 12,000 ( a l d r i n ) and 7,800 (DDT) i n 24 h r (23). In a longer exposure with a d i f f e r e n t midge, body accumulation o f DDE increased f o r 30 days and had not completely e q u i l i b r a t e d at t e r m i n a t i o n (24). Thus, r a p i d p e n e t r a t i o n p l u s bioaccumulation may c o n t r i b u t e to the high t o x i c i t y o f i n s e c t i c i d e s to midges, at l e a s t f o r the organochlorines. Midges converted 58% o f absorbed a l d r i n to d i e l d r i n but midge epoxidase was completely i n h i b i t e d i n v i t r o and i n vivo by PBO, demonstrating that PBO i s a potent iïïKibitor o f midge MFO R

21.

ESTENIK AND COLLINS

Mixed-Function

Oxidase

367

and supporting the e a r l i e r d i s c u s s i o n o f MFO i n j o i n t a c t i o n s t u d i e s . Under our c o n d i t i o n s , no i n v i v o conversion o f d i e l d r i n was detected and d i e l d r i n was the only detectable metabolite o f a l d r i n in_ vivo and i n v i t r o . In another study, midges converted l e s s than 25% o f a l d r i n to d i e l d r i n and d i e l d r i n a l s o was the only product detected (23). An optimum pH o f 7.5-8.0 f o r midges i s s i m i l a r to the s l i g h t l y a l k a l i n e optima f o r a l d r i n epoxidase o f other i n s e c t s (11, 25, 26, 27, 28) i n c l u d i n g c a d d i s f l i e s , an aquatic species (29). An optimum temperature o f 30°C f o r midge epoxidase i s also near that o f other i n s e c t s (1_, 11^, 2£, 30), although considerable midge epoxidase a c t i v i t y , 80-89% o f maximum, was obtained at 20-25°C. C a d d i s f l y a l d r i n epoxidase was near maximum at 20-25°C (29) and mosquito larvae homogenates o x i d a t i v e l y metabolized more propoxur at 25°C than at 30 C (3T). Wilkinson and B r a t t s t e n (1) speculated that aquatic i n s e c t s , l i v i n g at lower ambient temperatures, may have lower optimum temperatures f o r MFO than t e r r e s t r i a l i n s e c t s and midge data do not s t r o n g l y r e f u t e that n o t i o n . The temperature p r o f i l e o f midge epoxidase i s not much d i f f e r e n t than a l d r i n epoxidase o f c a d d i s f l y f a t body (29). Maximum epoxidase a c t i v i t y was obtained with 5 Χ 10~* M T r i s b u f f e r . KCl enhanced a c t i v i t y only at suboptimal b u f f e r concentrations, probably due to i o n i c strength e f f e c t s (1). NADH, FMN and FAD had l i t t l e or no e f f e c t on midge epoxidase. In v i t r o d i e l d r i n production increased p r o p o r t i o n a l l y to l a r v a l concentration (enzyme concentration) up to 5 larvae/ml and each 10X increase i n substrate ( a l d r i n ) concentration t r i p l e d and doubled, r e s p e c t i v e l y , d i e l d r i n formation. An approximation o f 2 Χ ΙΟ" M a l d r i n as the Km value f o r midge epoxidase was obtained from a double r e c i p r o c a l p l o t o f data i n Table VII, which c l o s e l y corresponds to values f o r a l d r i n epoxidase i n the house f l y (28) and the southern armyworm (11). D i e l d r i n accumulated i n p r o p o r t i o n to incubation time during the f i r s t 30 min and d e c l i n e d t h e r e a f t e r , l i k e the b i p h a s i c curves f o r a l d r i n epoxidation i n other i n s e c t s (11, 26). The r a t e curve d e c l i n e d continuously to 50% o f maximum a f t e r 60 min o f incubation at 30°C. BSA d i d not increase epoxidase a c t i v i t y i n 15 min incubations (Table V) or 60 min incubations (Table IV). Consequently, reduced epoxidase a c t i v i t y i s probably not due to endogenous proteases i n the homogenate (1). The higher a c t i v i t y o f washed microsomes i s probably due to the removal o f i n a c t i v e p r o t e i n and/or endogenous MFO i n h i b i t o r s . Based on r e s u l t s with whole body homogenates, BSA was not used i n s u b c e l l u l a r s t u d i e s . The l i g h t pink c o l o r o f unwashed microsomes i s undoubtedly due to midge hemoglobin. Hemoglobin, porphyrins and heme compounds may bind to mammalian microsomes (32, 33, 34) and such b i n d i n g may i n h i b i t MFO a c t i v i t y (34). Besides the p o s s i b l e i n h i b i t i o n by hemoglobin, we have no evidence o f any endogenous i n h i b i t o r s i n midge homogenates. The s p e c i f i c a c t i v i t y ( p r o t e i n b a s i s ) o f a l d r i n epoxidase Ô

5

368

PESTICIDE

A N D XENOBIOTIC

METABOLISM

IN AQUATIC

ORGANISMS

i n midge m i c r o s o m e s i s h i g h among o t h e r i n s e c t s o r i n s e c t t i s s u e s ( 1 1 , 2 5 , 26, 2 7 , 2 8 , 2 9 , 35-43) b u t optimum c o n d i t i o n s were n o t e s t a b l i s h e d i n a l l o f t h o s e s t u d i e s . The e p o x i d a s e a c t i v i t y o f midge b o d y homogenates i s much g r e a t e r t h a n t i s s u e p r e p a r a t i o n s o f another a q u a t i c i n s e c t ( 2 9 ) , p r o v i d i n g evidence f o r extremes among a q u a t i c i n s e c t s i n t h i s r e g a r d . The r e l a t i v e l y h i g h l e v e l s o f MFO a c t i v i t y i n m i d g e s may be an e v o l u t i o n a r y c o n s e q u e n c e o f the g e n e r a l l y p o l l u t e d c o n d i t i o n s o f t h e i r n a t u r a l h a b i t a t . E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n o f midge m i c r o s o m e s p r e pared by a s l i g h t l y d i f f e r e n t procedure than Table V I I I r e v e a l e d a homogeneous m i x t u r e o f v e s i c l e s d e r i v e d f r o m r o u g h a n d smooth e n d o p l a s m i c r e t i c u l u m , r i b o s o m e s and a few m i t o c h o n d r i a . Midge p r e p a r a t i o n s are s i m i l a r i n composition t o microsomal f r a c t i o n s o f s o u t h e r n armyworm f a t body a n d g u t ( 4 4 ) . Summary 1) 2) 3)

4)

C h i r o n o m u s r i p a r i u s l a r v a e were s u s c e p t i b l e t o i n s e c t i c i d e s i n ug/L c o n c e n t r a t i o n s . R e s u l t s o f j o i n t a c t i o n e x p e r i m e n t s w i t h PBO were t y p i c a l o f an i n s e c t w i t h an a c t i v e MFO s y s t e m . M i d g e l a r v a e r a p i d l y a c c u m u l a t e a l d r i n o r d i e l d r i n and r e a d i l y epoxidize a l d r i n t o d i e l d r i n without f u r t h e r conversion. W i t h i n e x p e c t e d v a r i a t i o n , t h e optimum c o n d i t i o n s f o r i n v i t r o e p o x i d a s e a c t i v i t y o f midges a r e t y p i c a l o f o t h e r i n s e c t s . Maximum a c t i v i t y was o b t a i n e d w i t h 1 mg a l d r i n i n 5 m l homogenate, an e l e c t r o n g e n e r a t o r s y s t e m w i t h NADP, pH 7.5 b u f f e r o f 5 X 1 0 " M and i n c u b a t i o n f o r 15 m i n a t 30°C. M i d g e a l d r i n e p o x i d a s e i s h i g h l y a c t i v e a n d may be c o m p l e t e l y i n h i b i t e d i n _ v i v o o r i n v i t r o b y PBO. The e a s e o f p r e p a r a t i o n , m i n i m a l endogenous i n h i b i t o r p r o b l e m s p l u s p o i n t s 2, 4 and 5 above s u g g e s t t h a t midge p r e p a r a t i o n s may be a c o n v e n i e n t , e f f e c t i v e t o o l f o r i n s e c t MFO s t u d i e s . 1

5) 6)

Acknowledgement Dr. N. W. B r i t t a n d Mr. R. S t o f f e r i d e n t i f i e d t h e midge species i n t h i s research.

21.

ESTENiK A N D C O L L I N S

Mixed-Function

Oxidase

369

Literature Cited 1. Wilkinson, C. F. and Brattsten, L. B. Drug Metab. Rev. (1973) 1:153-227. 2. Credland, P. F. J. Nat. Hist. (1973) 7:209-216. 3. Biever, K. D. Ann. Entomol. Soc. Amer. (1965) 58:135-136. 4. Mulla, M. S. and Khasawinah, A. M. J. Εcon. Entomol. (1969) 62:37-41. 5. Karnak, R. E. and Collins, W. J. Bull. Environ. Contam. Toxicol. (1974) 12:62-69. 6. Abbott, W. S. J. Econ. Entomol. (1925) 18:265-267. 7. Finney, D. J. "Probit Analysis", 333 pp. Cambridge Univ. Press. London (1971). 8. Augenfeld, J. M. Physiol. Zool. (1967) 40:149-158. 9. Sturgess, Β. T. and Goulding, R. L. Ann. Entomol. Soc. Amer. (1968) 61:903-906. 10. Nelson, P. Α., Stewart, R. R., Morelli, M. A. and Nakatsugawa, T. Pesticide Biochem. Physiol. (1976) 6:243-253. 11. Krieger, R. I. and Wilkinson, C. F. Biochem. Pharmacol. (1969) 18:1403-1415. 12. Bramhall, S., Noack, N., Wu, M. and Loewenberg, J. R. Analytical Biochem. (1969) 31:146-148. 13. Jensen, L. D. and Gaufin, A. R. Trans. Amer. Fish. Soc. (1964) 93:357-363. 14. Sanders, H. O. and Cope, O. B. Limnol. Oceanog. (1968) 13:112-117. 15. A l i , A. and Mulla, M. S. J. Econ. Entomol. (1976) 69:509513. 16. Perry, A. S. J. Agric. Food Chem. (1960) 8:266-272. 17. Sun, Y. P. and Johnson, E. R. J. Agric. Food Chem. (1960) 8:261-266. 18. Brooks, G. T. and Harrison, A. Nature (1963) 198:1169-1171. 19. Metcalf, R. L. Ann. Rev. Entomol. (1967) 12:229-256. 20. Oonnithan, E. S. and Casida, J. E. J. Agric. Food Chem. (1968) 16:28-44. 21. Casida, J. E., ed. "Pyrethrum, the Natural Insecticide", 329 pp. Academic Press. New York (1973). 22. Brattsten, L. B. and Metcalf, R. L. J. Econ. Entomol. (1970) 63:101-104. 23. Johnson, B. T., Saunders, C. R. and Sanders, H. O. J. Fish Res. Bd. Can. (1971) 28:705-709. 24. Derr, S. K. and Zabik, M. J. Trans. Amer. Fish Soc. (1972) 101:323-329. 25. Terrier, L. C. and Yu, S. J. Pesticide Biochem. Physiol. (1976) 6:223-228. 26. Benke, G. M. and Wilkinson, C. F. Pesticide Biochem. Physiol. (1971) 1:19-31. 27. Benke, G. M., Wilkinson, C. F. and Telford, J. N. J. Econ. Entomol. (1972) 65:1221-1229.

370

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

Ray, J. W. Biochem. Pharmacol. (1967) 16:99-107. Krieger, R. I. and Lee, P. W. J . Econ. Entomol. (1973) 66:1-6. Tsukamoto, M. and Casida, J . E. J . Econ. Entomol. (1967) 60:617-619. Shrivastava, S. P., Georghiou, G. P. and Fukuto, T. R. Entomol. Exp. and Appl. (1971) 14:333-348. Garfinkel, D. Arch. Biochem. Biophys. (1958) 77:493-509. Petermann, M. L. and Pavlovec, A. J . Biol. Chem. (1961) 236:3235-3239. Maines, D. M. and Kappas, A. J . Biol. Chem. (1975) 250: 2363-2369. Krieger, R. I., Gilbert, M. D. and Wilkinson, C. F. J . Econ. Entomol. (1970) 63:1322-1323. Krieger, R. I. and Wilkinson, C. F. Biochem. J . (1970) 116:781-789. Gilbert, M. D. and Wilkinson, C. F. Pesticide Biochem. Physiol. (1974) 4:56-66. Reed, W. T. J . Econ. Entomol. (1974) 67:150-152. Chan, Τ. Μ., Gillett, J . W. and Terriere, L. C. Comp. Biochem. Physiol. (1967) 20:731-742. Lewis, S. E., Wilkinson, C. F. and Ray, J. W. Biochem. Pharmacol. (1967) 16:1195-1210. Khan, M. A. Q. J . Econ. Entomol. (1969) 62:723-725. Kahn, M. A. Q., Chang, J . L., Sutherland, D. J., Rosen, J. D. and Kamal, A. J . Econ. Entomol. (1970) 63:1807-1813. Williamson, R. L. and Schechter, M. S. Biochem. Pharmacol. (1970) 19:1719-1727. Cassidy, J . D., Smith, E. and Hodgson, E. J . Insect Physiol. (1969) 15:1573-1578.

RECEIVED January 2, 1979.