1 Metabolism of Organophosphorus Insecticides in Aquatic Organisms, with Special Emphasis on Fenitrothion JUNSHI MIYAMOTO, YOSHIYUKI ΤΑΚΙΜΟΤΟ, and KAZUMASA MIHARA Research Department, Pesticides Division, Institute for Biological Science, Sumitomo Chemical Co., Ltd., 4-2-1 Takatsukasa, Takarazuka, Hyogo 665, Japan
The organophosphorus compounds constitute one major group of insecticides, and a certain portion thereof may be transported to the aquatic environment resulting either from the actual use on paddy fields or from unavoidable transmittance to waterways. However, possibly because of its relatively shorter persistence, the translocation and transformation of organophosphorus compounds in the aquatic environment has not been extensively investigated as compared with more persistent organοchlorine compounds. Fenitrothion, O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate, is widely used for the control of paddy field insects and forest protection in several countries, and since it is rather highly toxic to some aquatic organisms (LC 50 after 48hr exposure, 1.28 ppm for rainbow trout, 2.72 ppm for bluegill, 4.4 ppm for carp, LC 50 after 3 hr exposure, 0.0092 ppm for daphnia and no-effect dosage after 4 week exposure, 0.02 ppm for carp) (1, 2), the knowledge on degradation and metabolism of the compound in the aquatic environment is important for assessing short-term and long-term impacts on the non-target aquatic organisms. In this article metabolism and bioaccumulation of fenitro thion in several aquatic species are dealt with under laboratory conditions. Metabolism in vitro To acquire information on the intrinsic metabolic activity of aquatic organisms, liver of carp (Cyprinus carpio Linnaeus), rainbow trout (Salmo gairdneri) and freshwater snail (Cipangopaludina japonica Martens) was dissected out, homogenized in 0.1M phosphate buffer, pH 7.5, and centrifuged at 105,000 g for 60 min to obtain the microsome-equivalent (described as the microsomal fraction hereafter) fraction. The protein content of microsomal and sub microsomal (supernatant fractions by Lowry's method, micro somal P-450 content (3), activity of aniline hydroxylase (4) and aminopyrine N-demethylase (_5) were determined. Table I shows the results which reveal that the drug0-8412-0489-6/79/47-099-003$05.00/0 © 1979 American Chemical Society
Rat Rainbow trout Carp Snail
37.1 16.5 3.7 2.3 1.8
5.5 99%) was e x a m i n e d b y u s i n g t h e m i c r o s o m a l f r a c t i o n a n d determining o x i d a t i v e d e s u l f u r a t i o n t of e n i t r o o x o n , 0,0-dimethyl 0-(3-methyl-4-nitrophenyl) p h o s p h a t e , o x i d a t i o n o f t h e m-mehtyl g r o u p o f f e n i t r o o x o n i n t h e p r e s e n c e o f NADPH p l u s EDTA, a n d h y d r o l y t i c cleavage o f f e n i t r o o x o n t o 3-methyl-4-nitrophenol, with c a l c i u m i o n a s a c o f a c t o r (6_). A l s o i n t h e p r e s e n c e o f r e d u c e d g l u t a t h i o n e , O - d e m e t h y l a t i o n o f f e n i t r o t h i o n a n d f e n i t r o o x o n was t e s t e d i n the s u p e r n a t a n t f r a c t i o n . D u r i n g i n c u b a t i o n f o r 5-60 min a t 24°C ( f o r a q u a t i c a n i m a l s ) ( 7 ) o r 37°C ( f o r mammals), an a l i q u o t o f t h e i n c u b a t i o n m i x t u r e was s a m p l e d p e r i o d i c a l l y , a n d a n a l y z e d f o r s u b s t r a t e d i s a p p e a r a n c e and t h e r e s p e c t i v e p r o d u c t formed by two-dimensional t h i n l a y e r chromatography, f o l l o w e d b y d e t e r m i n a t i o n o f r a d i o a c t i v i t y o f the s e p a r a t e d s p o t s . T a b l e I I summarizes the r e s u l t s t o g e t h e r w i t h the d e t a i l e d e x p e r i m e n t a l c o n d i t i o n s . As i s e v i d e n t , m e t a b o l i c a c t i v i t i e s w e r e d e t e c t a b l e i n t h e s e 3 a q u a t i c s p e c i e s , b u t t h e r a t e was f a r l o w e r as compared w i t h mammalian h e p a t i c enzume p r e p a r a t i o n s , a n d t h e o x i d a t i v e a c t i v i t i e s i n s n a i l were p a r t i c u l a r l y low a l t h o u g h t h e p o s s i b i l i t y was n o t r u l e d o u t o f t h e p r e s e n c e o f i n h i b i t o r s o f mixed-function oxidases i n the f r a c t i o n s . The O - d e m e t h y l a t i o n r e a c t i o n p r o c e e d s e x t r e m e l y s l o w l y i n t h e enzyme p r e p a r a t i o n o f a q u a t i c a n i m a l s , a t l e s s t h a n one h u n d r e d t h t h a t o f mammals. T h u s , a l t h o u g h o r g a n o p h o s p h o r u s compounds l i k e f e n i t r o t h i o n may b e m e t a b o l i z e d i n a q u a t i c o r g a n i s m s t h r o u g h o x i d a t i v e d e s u l f u r a t i o n , s i d e chain o x i d a t i o n , h y d r o l y t i c cleavage of P - O - a r r y l l i n k a g e , as w e l l a s O - d e m e t h y l a t i o n , t h e t u r n - o v e r r a t e a p p a r e n t l y i s much l o w e r t h a n i n mammals. M e t a b o l i s m and b i o a c c u m u l a t i o n
i n vivo
In order t o obtain metabolic p r o f i l e s o f f e n i t r o t h i o n i n f i s h i n v i v o , 2 y e a r l i n g r a i n b o w t r o u t w e i g h i n g on an a v e r a g e 26.6 g w e r e m a i n t a i n e d i n 10 l i t e r s o f a e r a t e d w a t e r a t 18±0.5°C c o n t a i n i n g 0.1 ppm o f r a d i o a c t i v e f e n i t r o t h i o n l a b e l e d a t t h e m-methyl p o s i t i o n (3.16 mCi/mmole, >99%) ( 8) . A t 6 and 2 4 h r , a n d a l s o a t 24 h r a f t e r t r a n s f e r t o f r e s h w a t e r o f f i s h exposed f o r t h e p r e c e d i n g 24 h r t o t h e fen i t r o t h i o n - c o n t a i n i n g w a t e r (24+24 i n F i g u r e 1 ) , t h e f i s h were s a m p l e d and s u b j e c t e d t o a u t o r a d i o g r a p h y . The p a t t e r n o f d i s t r i b u t i o n o f t h e a b s o r b e d r a d i o a c t i v i t y i s shown i n F i g u r e 1. A f t e r 6 h r s o f e x p o s u r e t h e c o n c e n t r a t i o n o f r a d i o a c t i v i t y was h i g h e s t i n g a l l b l a d d e r and i n t e s t i n e , and t h e 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 i n most t i s s u e s e x c e p t b r a i n a n d h e a r t a f t e r 24 h r . Twenty-four hrs a f t e r t r a n s f e r t o f r e s h water ( 2 4 + 2 4 ) , most o f t h e r a d i o a c t i v i t y i n t i s s u e s h a d d i s a p p e a r e d
13.8 20.5 0.6 0.6 99%) a t 1.0 ppm h a d b e e n i n c o r p o r a t e d i n t o t h e s o i l , and w a t e r p o u r e d o n t o t h e s o i l s u r f a c e , t h e r a d i o c a r b o n was e l u t e d i n t o w a t e r g r a d u a l l y u n t i l the e q u i l i b r i u m (28 ppb) was a t t a i n e d a f t e r 2 8 days a t 25°C. The c o n c e n t r a t i o n o f f e n i t r o t h i o n i n w a t e r r e a c h e d maximum, 4 ppb o r c a . 3% o f the t o t a l i n c o r p o r a t e d r a d i o a c t i v i t y , on t h e 1 s t day, d e c r e a s i n g r a p i d l y t h e r e a f t e r and c o n c u r r e n t l y t h e r a d i o a c t i v e d e g r a d a t i o n p r o d u c t s s u c h as demethylfenitrothion, 3-methyl-4-nitrophenol and N - a c e t y l a m i n o feni troth ion increased. The f i n d i n g s r e f l e c t r a p i d t r a n s f o r m a t i o n o f f e n i t r o t h i o n i n t h e submerged s o i l as has b e e n r e p o r t e d e l s e where ( 1 8 ) , i m p l y i n g t h a t l i t t l e f e n i t r o t h i o n w o u l d be t a k e n up i n t o t h e component o r g a n i s m s u n d e r the g i v e n c o n d i t i o n s . I n a n o t h e r t r i a l , a t 7 days a f t e r b u i l d i n g - u p t h e e c o s y s t e m , 10 ppb e q u i v a l e n t o f t h e r a d i o a c t i v e f e n i t r o t h i o n was added t o w a t e r . Ten ppb o f c a r b o n - 1 4 f e n i t r o t h i o n was added 3 t i m e s a t t h e i n t e r v a l o f one week. The s y s t e m was k e p t a t 25°C i n a g r e e n h o u s e . An a l i q u o t o f w a t e r was s a m p l e d p e r i o d i c a l l y , and t h e c o n t e n t o f t o t a l r a d i o a c t i v i t y as w e l l as f e n i t r o t h i o n was m o n i tored. As i n d i c a t e d i n F i g u r e 4 i r r e s p e c t i v e o f s i n g l e o r m u l t i p l e a p p l i c a t i o n s , f e n i t r o t h i o n i n water disappeared with s i m i l a r
1.
MIYAMOTO E T AL.
Organophosphorus
0.011
Insecticides
15
10
20
days Figure 4. Concentration of C and fenitrothion in water of aquatic model ecosystem: (a) fenitrothion added once to the model ecosystem; (b) fenitrothion added to the model ecosystem 2 more times at 7th and 14th day. 14
13
14
PESTICIDE A N D
XENOBIOTIC M E T A B O L I S M IN AQUATIC
ORGANISMS
r a p i d i t y w i t h a h a l f - l i f e o f ca. 2 days, w h i l e t o t a l r a d i o a c t i v i t y d e c r e a s e d r e l a t i v e l y s l o w l y . A t 3, 7 and 21 days a f t e r s i n g l e a p p l i c a t i o n o r 7 days a f t e r 3 a p p l i c a t i o n s o f f e n i t r o t h i o n , 4 component o r g a n i s m s as w e l l as s o i l s a m p l e s w e r e e x t r a c t e d w i t h methanol. W a t e r was p a s s e d t h r o u g h an XAD-2 column and t h e r a d i o c a r b o n t r a p p e d was e l u t e d w i t h a c e t o n e . The e x t r a c t e d r a d i o c a r b o n was s e p a r a t 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 w i t h t h e a v a i l a b l e a u t h e n t i c r e f e r e n c e compounds. The r a d i o a c t i v i t y i n t h e u n e x t r a c t a b l e r e s i d u e was d e t e r m i n e d a f t e r c o m b u s t i o n . T a b l e V, VI and V I I s u m m a r i z e t h e r e s u l t s o f i d e n t i f i c a t i o n o f the d e g r a d a t i o n p r o d u c t s . The t o t a l r a d i o a c t i v i t y as w e l l as i n t a c t f e n i t r o t h i o n d e c r e a s e d i n s n a i l and f i s h , and 3 c o n s e c u t i v e a p p l i c a t i o n s of f e n i t r o t h i o n d i d not a f f e c t these concentrations v e r y much. I n c o n t r a s t , i n d a p h n i d s and i n a l g a e t o t a l r a d i o a c t i v i t y t e n d e d t o i n c r e a s e w i t h t h e l a p s e o f t i m e , m o s t l y due t o an i n c r e a s e o f u n e x t r a c t a b l e r a d i o a c t i v i t y and u n i d e n t i f i e d p r o d u c t s , a l t h o u g h t h e f e n i t r o t h i o n c o n t e n t was c o n s t a n t l y d e c r e a s i n g B i o a c c u m u l a t i o n r a t i o s o f t o t a l r a d i o c a r b o n and f e n i t r o t h i o n r e l a t i v e t o t h e w a t e r c o n c e n t r a t i o n s a r e shown i n T a b l e V I I I . They r e v e a l t h a t t h e r a t i o i s n o t s o h i g h , a t t h e maximum 180, and i n f i s h i t tends t o decrease w i t h l o n g e r i n c u b a t i o n o f the system, w h e r e a s i n d a p h n i d s and a l g a e t h e b i o a c c u m u l a t i o n r a t i o o f f e n i t r o t h i o n was i n c r e a s i n g u n d e r t h e p r e s e n t s t a t i c c o n d i t i o n s , due to q u i t e r a p i d disappearance of f e n i t r o t h i o n i n water. I n any e v e n t , t h e b i o a c c u m u l a t i o n r a t i o was o b v i o u s l y f a r l e s s t h a n t h a t o f DDT and i t s d e g r a d a t i o n p r o d u c t s . The number o f t h e d e g r a d a t i o n p r o d u c t s i d e n t i f i e d was l a r g e s t i n w a t e r ; t h e y i n c l u d e f e n i t r o o x o n , a m i n o f e n i t r o t h i o n , and i t s N - f o r m y l and N - a c e t y l d e r i v a t i v e s as w e l l as 3 d e m e t h y l a t e d p r o ducts. S e v e r a l p h e n o l i c products are found i n water, t o g e t h e r w i t h a s u l f a t e conjugate of 3-methyl-4-nitrophenol. I n s o i l , no o x y g e n a n a l o g s w e r e d e m o n s t r a t e d , b u t s e v e r a l amino d e r i v a t i v e s were p r e s e n t , c o n s i s t e n t w i t h p r e v i o u s f i n d i n g s (18) . F i s h cont a i n e d amino compounds t h a t h a v e n o t b e e n i d e n t i f i e d i n m e t a b o l i s m s t u d i e s d e s c r i b e d above. They p r o b a b l y r e s u l t f r o m a b s o r p t i o n from the s u r r o u n d i n g water. S n a i l s c o n t a i n e d a f e w e r number o f d e g r a d a t i o n p r o d u c t s , among w h i c h amino d e r i v a t i v e s o f f e n i t r o t h i o n , d e m e t h y l f e n i t r o t h i o n , d e m e t h y l f e n i t r o o x o n as w e l l as 3 - m e t h y l - 4 - n i t r o p h e n o l and i t s s u l f a t e c o n j u g a t e a r e i n c l u d e d . Most o f t h e r a d i o c a r b o n i n d a p h n i d s and s n a i l s i s y e t t o be identified. T h u s , i n t h e s t a t i c e c o s y s t e m t h e t r a n s l o c a t i o n and metabol i s m o f f e n i t r o t h i o n i s v e r y c o m p l i c a t e d , a p p a r e n t l y due t o decomp o s i t i o n by s o i l o r g a n i s m s . N e v e r t h e l e s s , the data presented here may i m p l y t h a t r a p i d b i o a c c u m u l a t i o n o f t h e r a d i o a c t i v e compounds d e r i v e d from f e n i t r o t h i o n i n s n a i l or i n f i s h i s u n l i k e l y t o o c c u r i n the n a t u r a l environment.
1
7.19 5.24 0.97 0.01 0.23 0.02 0.11 0.06 0.03 0.05 0.01 0.48 2.37 0.01 0.03 0.17 0.71 1.95
7.00 5.45 3.02
ο w
ο
Ο W
Μ
ο χ
>
1
à
1
4
75.6 199
46.3 30.9
a )
82.0 251
—
0.85
— — — — — —
315 644
—
15.6
— — — — — —
42.1 30.0
480
6.14 55.3
—
Carp
29.3 28.0
5.83 52.5 1.94 675
—
7 d
1260 1000 972 1230 547 154 82.3 30.1 13.5 25.3 1.23 — 2.46 —
3 d
28.6 21.0
1.07 20.2 7.31 129
— —
236 215 3.01 0.65 18.1 7.09
21 d
a) C - f e n i t r o t h i o n a d d e d t o t h e m o d e l e c o s y s t e m 2 more t i m e s a t 7 and 1 4 t h day. b) N o t d e t e c t e d .
—
8.72
— — — — —
—
3.45
— — — — —
21 d
a )
81.7 56.0
4.69 12.2 44.1 13.1 679
—
994 938 14.1 36.6 51.6 0.94
21 d
i n individual
(ppb o f f e n i t r o t h i o n e q u i v a l e n t )
336 984 85.0 340 2.13 9.19
21 d
Daphnid 7 d
124 333 93.1 134 46.1 49.7 — b) —
3 d
Concentration
D i s t r i b u t i o n of C, f e n i t r o t h i o n a n d i t s d e g r a d a t i o n p r o d u c t s components o f a q u a t i c m o d e l e c o s y s t e m : D a p h n i d a n d c a r p .
14 Total C Extractable C Fenitrothion Deme t h y 1 f e n i t r o t h i o n Deme t h y l a m i n o f e n i t r o t h i o n il/-Ace t y l a m i n o f e n i t r o t h i o n Fenitrooxon N-Acetylaminofeni trooxon Demethylfenitrooxon 3-Me t h y 1 - 4 - n i t r o p h e n o 1 3-Me t h y l - 4 - a c e t y l a m i n o p h e n o l 3-Methyl-4-nitrophenyl-gglucuronide Unidentified ^C Unextractable C
C-compound
Table V I I .
14
15.8
181
48.4
23.7
thion
Fenitro14
12.8
19.2 58.1 5.7
14th
97.7
41.6
159 139
thion 181
c
day.
Fenitro-
180
14
Total
Carp
32.8
9.3
21.8 16.3
8.2
thion
Fenitro-
24.9
c
Total
Snail
ratio
C and f e n i t r o t h i o n i n a q u a t i c
a) " ^ C - f e n i t r o t h i o n a d d e d t o t h e m o d e l e c o s y s t e m 2 more t i m e s a t 7 and
37.8
12.5
57.5
a )
92.6
69.2
59.3
21
21
61.5
51.2
46.3
7
c 42.7
thion
14
Total
15.3
c
Fenitro-
Alga
17.7
14
Total
Daphnid
total
Bioaccumulation
Bioaccumulation r a t i o of model ecosystem.
3
(days)
Period
Table V I I I .
>
§
d
H-I
r
w
η
I
Ο W
χ
α η
>
Θ M
ί
w
00
1.
MIYAMOTO ET AL.
Organophosphorus Insecticides
19
Abstract Although metabolism of fenitrothion, O,O-dimethyl O-(3-methyl4-nitrophenyl) phosphorothioate in subcellular fractions of rain bow trout and carp is i n t r i n s i c a l l y not high, as compared with mammalian hepatic enzyme preparations, rainbow trout can metabo l i z e , in vivo, this organophosphorus compound through oxidative desulfuration, cleavage of the P-O-aryl linkage and O-demethyla tion. The degradation products as well as the parent compound are excreted into the surrounding water. As a result, bioaccumu lation of fenitrothion in the fish species proved not to be high. Southern top-mouthed minnows found not to contain much fenitro thion in tissues. In a s t a t i c model ecosystem, several amino derivatives of fenitrothion, probably derived from the s o i l metabolism, were demonstrated in carp tissues, together with the nitro-containing compounds. The concentration of fenitrothion in carp, snails, daphnids and algae decreased with time, although i t s bioaccumula tion ratio relative to the concentration in water tended to increase gradually in snails, daphnids and algae, presumably due to lower metabolic activity and/or slow excretion. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15.
Miyamoto, J., Botyu-Kagaku (Scientific Pest Control) (1971) 36, 189. Takimoto, Y., Kagoshima, M. and Miyamoto, J., (1978) Unpublished observation. Ohmura, T. and Sato, R., J . Biol. Chem. (1964) 239, 2370. Hilton, J. and Sartonelli, A. C., J. B i o l . Chem. (1970) 245, 4187. Shenkman, J. B., Remmer, H. and Esterbrook, R. W., Mol. Phar macol. (1967) 3, 113. Ohkawa, Η., Mikami, N. and Miyamoto, J., Agr. Biol. Chem. (1977) 41, 369. Adamson, R. Η., Fed. Proc. (1967) 26, 1047. Takimoto, Y. and Miyamoto, J., J. Pesticide S c i . (1976) 1, 261. Miyamoto, J . , Mihara, K. and Hosokawa, S., J. Pesticide S c i . (1976) 1, 9. Benke, G. M., Cheever, K. L., Mirer, F. E. and Murphy, S. D. Toxicol. Appl. Pharmacol. (1974) 28, 97. Lech, J. J., Toxicol. Appl. Pharmacol. (1973) 28, 97. Kanazawa, J., Bull. Environ. Contami. Toxicol. (l975) 14, 346. Lockhart, W. L., Metner, D. A. and G r i f t , Ν., The Manitoba Entomologist (1973) 7, 26. Haque, R., Kearney, P. C. and Freed, V. H., "Pesticides in Aquatic Environment" (Khan, M. A. Q., Ed.), P. 39, Plenum Press, New York, 1977. Kearney, P. C., J. Pesticide S c i . (1975) Inaugural Issue, 43.
PESTICIDE
20
16. 17. 18.
A N D XENOBIOTIC
METABOLISM
I N AQUATIC
ORGANISMS
Ohkawa, H., Kikuchi, R. and Miyamoto, J., Abstract of Papers presented at 3rd Annual Meeting of Pesticide Science Society (Tokyo) No. 317 (1978). Freeman, L., Sewage Ind. Wastes (1953) 25, 845. Takimoto, Y., Hirota, Μ., Inui, H. and Miyamoto, J., J. Pesticide Sci. (1976) 1, 131.
RECEIVED
January 2, 1979.
