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Trimethylarsine oxide in estuary catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) after oral administration of sodium arsenate; and as a natural component of estuary catfish
The Science of the Total Environment, 64 (1987) 317-323 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
317
TRIMETHYLARSINE OXIDE IN ESTUARY CATFISH (CNIDOGLANIS MACROCEPHALUS) AND SCHOOL WHITING (SILLAGO BASSENSIS) AFTER ORAL ADMINISTRATION OF SODIUM ARSENATE; AND AS A NATURAL COMPONENT OF ESTUARY CATFISH
JOHN S. EDMONDS and KEVIN A. FRANCESCONI Western Australian Marine Research Laboratories, P.O. Box 20, North Beach 6020, Western Australia (Australia) (Received September 17th, 1986; accepted October 10th, 1986)
ABSTRACT Oral administration of sodium arsenate to estuary catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) resulted in an accumulation of trimethylarsine oxide in their tissues. The levels of arsenobetaine, which occurs naturally in these fish, did not appear to be affected by the oral dosing with sodium arsenate. Trimethylarsine oxide also occurred as a natural component of estuary catfish and its presence may be related to the mode of feeding of this fish. INTRODUCTION M a r i n e a n i m a l s c o n t r i b u t i n g to the h u m a n diet c o n t a i n arsenic at conc e n t r a t i o n s up to and o c c a s i o n a l l y exceeding 4 0 m g kg -1. The m a j o r or sole a r s e n i c a l c o n t r i b u t i n g to this burden, in fish a n d c r u s t a c e a at least, is ars e n o b e t a i n e ( G E S A M P , 1986). However, in a d d i t i o n to studies t h a t h a v e established the v i r t u a l u b i q u i t y of a r s e n o b e t a i n e as the m a j o r n a t u r a l l y - o c c u r r i n g a r s e n i c c o m p o u n d in seafoods, a limited n u m b e r of studies h a v e e x a m i n e d the m e t a b o l i s m by fish of a d m i n i s t e r e d i n o r g a n i c a r s e n i c (Penrose, 1975; Oladimeji et al., 1979). Oral a d m i n i s t r a t i o n of i n o r g a n i c p e n t a v a l e n t a r s e n i c to fish has resulted in its rapid c o n v e r s i o n to o r g a n i c forms, but in no case h a s the m a j o r o r g a n i c arsenic c o m p o u n d been identified. P e n r o s e (1975) showed t h a t o r a l l y administered radio-labelled sodium a r s e n a t e was c o n v e r t e d into two o r g a n i c arsenic c o m p o u n d s , r e v e a l 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 h i c h a c c u m u l a t e d in the muscle of the b r o w n t r o u t (Salmo trutta). No identification of these c o m p o u n d s was c a r r i e d out, a l t h o u g h it was n o t e d t h a t t h e y were h y d r o p h i l i c and cationic. Oladimeji et al. (1979) d e m o n s t r a t e d the p r o d u c t i o n of m o n o m e t h y l a t e d arsenic and two u n k n o w n a r s e n i c c o m p o u n d s in r a i n b o w t r o u t r e c e i v i n g oral doses of radio-labelled a r s e n i c acid. A f t e r a period of 96h, i n o r g a n i c arsenic, monom e t h y l a t e d a r s e n i c a n d the m i n o r unidentified a r s e n i c m e t a b o l i t e e a c h cons t i t u t e d < 10% of t o t a l arsenic. On the o t h e r hand, the m a j o r o r g a n i c a r s e n i c
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318 metabolite (also unidentified) represented > 84% of total arsenic in all tissues and was the only arsenic compound detected in muscle after 96 h. It is interesting to speculate on the relevance of these studies to the occurrence of arsenobetaine. Both studies (Penrose, 1975; Oladimeji et al., 1979) showed t hat the major organic metabolites of orally-administered inorganic arsenic, alt h ou g h unidentified, were not arsenobetaine. Penrose (1975) considered t h a t the conversion of inorganic arsenic to the absorbed/retained organic forms was mediated by the intestinal flora of the fish. The failure to demonstrate conversion of orally-administered inorganic arsenic to arsenobtaine suggests t hat arsenobetaine is not synthesized de novo by the fish (through the mediation of gut bacteria or otherwise) but is likely, as has been previously suggested, to pass to the fish via the food chain from more complex organo-arsenic compounds found in algae (Edmonds et al., 1982). The study reported here was u n d e r t a k e n to identify metabolites in fish of orally-administered inorganic arsenic, and thereby to clarify the role, if any, of such metabolites in the production of arsenobetaine. E s t u a r y catfish and school whiting were chosen for experimentation because of their availability and the ease with which they could be maintained in aquaria. MATERIALS AND METHODS
Arsenic analysis Arsenic determinations were made and arsenic located in chromatographic fractions by vapour generation or electrothermal atomic absorption spectrophotometry using Varian instrumentation.
Arsenate dosing experiments E s t u a r y catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) were collected from Whitford's Beach 20km nort h of Pert h and transferred to the aquarium in oxygenated seawater. They were maintained in r u nn in g seawater on a diet of mussels for some weeks before the start of experimentation. Four whiting and three catfish received elevated levels of arsenic in their food and four whiting and a single catfish served as controls. Fish were dosed by feeding once each day with earth worms (Eisenia foetida) immediately after the worms had been injected with 100pl or 250pl sodium arsenate solution containing 1 #g gl 1 arsenic. The worms were offered to the fish by suspending them in the water with forceps, whereupon they were rapidly t a k e n or rejected by the fish. In this way known quantities of arsenate were quickly administered to the fish and rejected doses were easily discounted. Fish were supplied with as many worms as they would eat immediately. Consequently the arsenate was injected into the worms and swallowed by the fish within seconds, allowing no time for biotransformation of the arsenate by
319
the worms or leaching of the arsenic into ambient water. No arsenic was detectable in undosed worms. Whiting (four fish) consumed 77 worms containing 7.7mg arsenic over 17 days, and catfish (three fish) consumed 276 worms containing 39.9 mg arsenic over 44 days. Three days were allowed after the final meal before the fish were killed to allow the gut tract to empty. Thus after 20 days the whiting, and after 47 days the catfish, were killed by freezing.
Extraction and chromatographic analyses Whiting The four experimentally-dosed whiting (8.0, 10.9, 14.7 and 18.0 g) were blended with water (200 ml) to produce a grey liquid slurry (58.5 ~g As). Methanol (600ml) was added to the slurry and after standing for 4 h it was filtered to produce a pale filtrate and a grey residue. Methanol washings (200 ml) of the residue were added to the filtrate, which on evaporation yielded a gummy solid (2.2 g, 56/~g As). The aqueous methanol-insoluble residue was dried to produce a grey powder (5.1g, 8.1ttg As). The aqueous methanol-soluble material was partitioned between water and ether to yield, after evaporation, a watersoluble fraction (1.81 g, 44.7 ttg As) and an ether-soluble fraction (570 mg, 2.5 ttg As). Passage of the water-soluble fraction through a Sephadex LH-20 column (840 x 26 mm, elution with water/methanol, 50:50) produced material (800mg, 43.1 ttg As) clean enough for thin layer chromatographic (TLC) analysis. Thus a portion was applied to a single TLC plate (cellulose, E. Merck, Darmstadt; 200 x 200 x 0.1mm) and after development (butan-l-ol, acetic acid, water; 60:15:25) the plate was scribed, divided and analysed as shown in Fig. 1A. The four control whiting (9.2, 9.6, 11.5 and 11.7 g) were treated in identical manner to produce an initial aqueous methanol-soluble fraction (1.9 g, 33.4/~g As) and an insoluble residue (4.7 g dried, 1.75/~g As). Partitioning of the soluble material between water and ether yielded a water-soluble fraction (1.44g, 43.3/~g As) and an ether-soluble fraction (370 mg, 4.75 gg As). The water-soluble fraction was passed through the Sephadex LH-20-water/methanol column to yield material enriched in arsenic (690 mg, 38.1/~g As), which was subjected to TLC under the same conditions as the equivalent fraction from the experimentally dosed whiting (see Fig. 1B).
Catfish The three experimentally dosed catfish (27.9, 39.5 and 49.4 g) were treated as for the whiting to yield a water-soluble fraction (2.45 g, 249.2 ~g As), an ethersoluble fraction (480 mg, 2.3/~g As) and an insoluble residue (17.2 g dried, 55.9 ~g As). A small portion of the water-soluble material (containing ~ 1/~g As) was subjected to TLC as for the whiting (Fig. 1C). The control catfish (39.8g) treated in identical manner yielded a watersoluble fraction (470mg, 15.3ttg As), an ether-soluble fraction (140mg, 2.3t~g As) and an insoluble residue (5.6 g dried, 0.65 ~g As). A portion of the watersoluble material was subjected to TLC analysis (Fig. 1D).
320 80
80
70
70
60
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Fig. 1. Thin-layer chromatographic plates analysed for arsenic. (A) Experimentally-dosed whiting. (B) Control whiting. (C) Experimentally-dosed catfish. (D) Control catfish.
Isolation of arsenic compound from experimentally-dosed catfish The final water-soluble fraction from the experimentally dosed catfish (2.45g, 249ttg As) was subjected to gel permeation chromatography (GPC) (Sephadex LH-20, e l u t i o n with water/methanol, 50:50; 840 x 26 mm column) to yield an arsenic-enriched fraction (1.17 g, 232 pg As), w h i c h was again subjected to GPC under identical conditions. The a r s e n i c - c o n t a i n i n g fractions (300mg, 216pg As) were c h r o m a t o g r a p h e d on cellulose layers (seven of
321 200 × 200 × 0.1mm, developed with butan-l-ol, acetic acid, water; 60:15:25), and the arsenic-containing material (R~ 0.69) again subjected to GPC. Repeat TLC (single 200 × 200 × 0.1mm cellulose plate; development as previously) and GPC afforded further purification and 1H NMR and mass spectra were taken on this material (< I mg, 130 pg As). RESULTS Very little of the administered arsenic was absorbed and/or retained by the fish. If the assumption is made t h a t the experimentally-dosed whiting contained the same quantity of " n a t i v e " arsenic as the control fish, then only some 20pg arsenic was retained out of 7.7mg administered (0.27%). In the case of the catfish some 300 pg arsenic was retained out of 39.9 mg administered (0.75%). Of the retained arsenic a substantial proportion (13.8% in the case of whiting; 15.2% for the catfish) became insoluble/inextractable, presumably because of binding to insoluble proteins. Again this is in contrast to "native" arsenic where 4.8% (whiting) and 2.5% (catfish) remained inextractable. Partitioning of extractable soluble arsenic between water and ether resulted in the bulk of the arsenic, in each case, in the aqueous phase (94.7 and 90.1% for experimentally-dosed and control whiting; 99.0 and 86.9% for experimentally dosed and control catfish). Only the material in the aqueous phase was further examined in this study. Results of thin layer chromatographic analysis of the partially purified arsenic compounds from the water-soluble fractions of whiting and catfish are shown schematically in Fig. 1A-D. It can be seen that control whiting contained a single arsenic compound at Rv 0.50. This was assumed, from its chromatographic position (compared with standard material) and from a previous study (Edmonds and Francesconi, 1981) to be arsenobetaine; but two additional compounds were present in the experimentally dosed animals (R~ 0.25 and 0.69). It is probable that the compound at Rv 0.25 corresponded to a small quantity of residual administered arsenate (again by comparison with standard material), but that at Rv 0.69 was evidently an organic metabolite of arsenate. In the case of the catfish, it can be seen that the control animal contained arsenic compounds chromatographing at RF 0.51 (presumably arsenobetaine) and R~ 0.69, with that at 0.51 predominating in a ratio of ~ 60:40. However, the experimentally dosed animals contained a considerable excess of the organic metabolite at Rv 0.69 but no administered arsenate (Rv 0.25) was apparent. No significant increase in arsenobetaine concentrations in arsenate-dosed over control fish was observed for either catfish or whiting. The organic arsenic metabolite (RF 0.69) was, after isolation and purification, identified as trimethylarsine oxide by NMR spectroscopy (300 MHz 1H, D20) (~ 1.77; and mass spectrometry (electron impact, 70°/35 eV) 136 (100%), 130 (6.7), 121 (89.4), 120 (8.7), 107 (8.7), 106 (5.8), 105 (12.5), 104 (9.6), 103 (19.2), 94 (20.2), 93 (62.5), 91 (64.4), 90 (7.7), 89 (13.5).
322 DISCUSSION
Although only trimethylarsine oxide from experimentally dosed estuary catfish was isolated and rigorously identified in this study, the presence of arsenobetaine in school whiting and estuary catfish (both experimentally dosed with inorganic arsenic and control) was confidently inferred from thin layer chromatographic coordinates. Indeed the presence of arsenobetaine in school whiting has been reported previously (Edmonds and Francesconi, 1981). The presence of trimethylarsine oxide in control estuary catfish was inferred from thin layer chromatographic coordinates. Only a single compound (trimethylarsine oxide) was recovered at RF 0.69 from the TLC plates of experimentally dosed catfish. If a naturally-occurring compound other t han trimethylarsine oxide but having the same R E value was masked by the trimethylarsine oxide, some evidence for multiplicity of compounds would have been expected to become apparent during later isolation and purification stages. This did not occur and was taken as further evidence t hat the arsenic compound having TLC RF value 0.69 in control catfish was trimethylarsine oxide. Norin et al. (1985) reported the presence of trimethylarsine oxide in four species of Baltic Sea fish. In all cases trimethylarsine oxide represented a small proportion of total arsenic, but Norin et al. (1985) considered t hat higher concentrations were found in fish t ha t had been kept frozen t han in fresh fish. Trimethylarsine oxide was identified by a mass fragmentographic technique and in no case was it isolated from the sample. The work reported here shows t hat trimethylarsine oxide can accumulate in the tissues of fish as a result of the oral administration of inorganic arsenic, and it seems likely, as suggested by Penrose (1975), that its formation is dependent upon bacterial action in the gut t r a c t of the fish. The observation t hat estuary catfish contains 40% of its native arsenic as trimethylarsine oxide may well reflect its feeding habits; mouthfuls of sand or silt appear to be ingested when this fish is seeking food, and, inevitably, some finds its way into the gut tract in addition to food organisms. Inorganic arsenic, universally contained in sediments, may therefore be subjected to bacterial action in the fish gut. In contrast, school whiting have a more selective method of feeding and will pick food organisms from sediment without the intake of bottom or detrital material (R.C.J. Lenanton, personal communication, 1986). We have found no evidence t h at trimethylarsine oxide is more likely to occur in stored material, frozen or otherwise, th an in fresh material. Indeed, experiments designed to measure the breakdown of arsenobetaine in stored fish tissue with time failed to produce any trimethylarsine oxide (Edmonds and Francesconi, unpublished observations). The demonstration t ha t the intake of inorganic arsenic results in an accumulation of small quantities of trimethylarsine oxide does not conflict with the hypothesis t h a t arsenobetaine accumulates in marine organisms from a food chain source based on algal-derived arseno-sugars. Support is given to this view by the observation t ha t no increase in arsenobetaine levels occurs with inorganic arsenic ingestion. It appears t h a t the accumulation of ar-
323 s e n o b e t a i n e is n o t r e l a t e d to t h a t of t r i m e t h y l a r s i n e oxide, a n d t h e l a t t e r is n o t formed, in fish t i s s u e s a t l e a s t , by d e g r a d a t i o n of a r s e n o b e t a i n e . C o n v e r s e l y , a r s e n o b e t a i n e is n o t f o r m e d in fish t i s s u e s from i n g e s t e d i n o r g a n i c a r s e n i c , by g u t b a c t e r i a l a c t i o n or by o t h e r m e a n s . T h e p r e s e n c e of t r i m e t h y l a r s i n e o x i d e in e s t u a r y c a t f i s h t i s s u e s r a i s e s toxic o l o g i c a l q u e s t i o n s . A l t h o u g h s t u d i e s of t h e m e t a b o l i s m of a r s e n o b e t a i n e by e x p e r i m e n t a l a n i m a l s a r e r e a s s u r i n g from a t o x i c o l o g i c a l v i e w p o i n t ( V a h t e r et al., 1983), t h e t o x i c i t y of t r i m e t h y l a r s i n e o x i d e is u n k n o w n . T h e e a s e w i t h w h i c h it c a n be r e d u c e d to t h e h i g h l y t o x i c t r i m e t h y l a r s i n e m a y g i v e c a u s e for c o n c e r n . W h i t f i e l d et al. (1983) r e p o r t e d t h a t t h e p r e s e n c e of t r a c e q u a n t i t i e s of t r i m e t h y l a r s i n e in s o m e s p e c i e s of p r a w n s w a s sufficient to p r o d u c e a n "offf l a v o u r " . T h e h i g h e s t r e p o r t e d v a l u e (0.98 pg k g - 1 ) in r e d p r a w n s (Aristeomorpha foliacea) w o u l d be u n l i k e l y to p r e s e n t a t o x i c h a z a r d . It seems p o s s i b l e t h a t t h e p r e s e n c e of t r i m e t h y l a r s i n e in c r u s t a c e a m a y be t h e r e s u l t of t h e r e d u c t i o n of s m a l l q u a n t i t i e s of t r i m e t h y l a r s i n e o x i d e t h a t m a y c o - e x i s t w i t h ars e n o b e t a i n e a n d m a y n o t be m e t a b o l i c a l l y c o n n e c t e d w i t h t h e a r s e n o b e t a i n e .
REFERENCES Edmonds, J.S. and K.A. Francesconi, 1981. The origin and chemical form of arsenic in the school whiting. Mar. Pollut. Bull., 12(3): 92 96. Edmonds, J.S., K.A. Francesconi and J.A. Hansen, 1982. Dimethyloxarsylethanol from anaerobic decomposition of brown kelp Ecklonia radiata: a likely precursor of arsenobetaine in marine fauna. Experientia, 38: 643-644. GESAMP, 1986. Working Group on Review of Potentially Harmful Substances. Hazard Evaluation for Arsenic. World Health Organisation, Geneva. In press. Norin, H., A. Christakopoulos and M. Sandstr6m, 1985. Mass fragmentographic estimation of trimethylarsine oxide in aquatic organisms. Chemosphere, 14(3/4): 313-323. Oladimeji, A.A., S.U. Qadri, G.K.H. Tam and A.S.W. DeFreitas, 1979. Metabolism of organic arsenic to organoarsenicals in rainbow trout (Salmo gairdneri ). Ecotoxicol. Environ. Saf., 3(4): 394~400. Penrose, W.R., 1975. Biosynthesis of organic arsenic compounds in brown trout (Salmo trutta). J. Fish Res. Board Can., 32(12): 238~2390. Vahter, M., E. Marafante and L. Dencker, 1983. Metabolism of arsenobetaine in mice, rats and rabbits. Sci. Total Environ., 30:197 211. Whitfield, F.B., D.J. Freeman and K.J. Shaw, 1983. Trimethylarsine: an important off-flavour component in some prawn species. Chem. Ind., (20): 786-787.
317
TRIMETHYLARSINE OXIDE IN ESTUARY CATFISH (CNIDOGLANIS MACROCEPHALUS) AND SCHOOL WHITING (SILLAGO BASSENSIS) AFTER ORAL ADMINISTRATION OF SODIUM ARSENATE; AND AS A NATURAL COMPONENT OF ESTUARY CATFISH
JOHN S. EDMONDS and KEVIN A. FRANCESCONI Western Australian Marine Research Laboratories, P.O. Box 20, North Beach 6020, Western Australia (Australia) (Received September 17th, 1986; accepted October 10th, 1986)
ABSTRACT Oral administration of sodium arsenate to estuary catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) resulted in an accumulation of trimethylarsine oxide in their tissues. The levels of arsenobetaine, which occurs naturally in these fish, did not appear to be affected by the oral dosing with sodium arsenate. Trimethylarsine oxide also occurred as a natural component of estuary catfish and its presence may be related to the mode of feeding of this fish. INTRODUCTION M a r i n e a n i m a l s c o n t r i b u t i n g to the h u m a n diet c o n t a i n arsenic at conc e n t r a t i o n s up to and o c c a s i o n a l l y exceeding 4 0 m g kg -1. The m a j o r or sole a r s e n i c a l c o n t r i b u t i n g to this burden, in fish a n d c r u s t a c e a at least, is ars e n o b e t a i n e ( G E S A M P , 1986). However, in a d d i t i o n to studies t h a t h a v e established the v i r t u a l u b i q u i t y of a r s e n o b e t a i n e as the m a j o r n a t u r a l l y - o c c u r r i n g a r s e n i c c o m p o u n d in seafoods, a limited n u m b e r of studies h a v e e x a m i n e d the m e t a b o l i s m by fish of a d m i n i s t e r e d i n o r g a n i c a r s e n i c (Penrose, 1975; Oladimeji et al., 1979). Oral a d m i n i s t r a t i o n of i n o r g a n i c p e n t a v a l e n t a r s e n i c to fish has resulted in its rapid c o n v e r s i o n to o r g a n i c forms, but in no case h a s the m a j o r o r g a n i c arsenic c o m p o u n d been identified. P e n r o s e (1975) showed t h a t o r a l l y administered radio-labelled sodium a r s e n a t e was c o n v e r t e d into two o r g a n i c arsenic c o m p o u n d s , r e v e a l 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 h i c h a c c u m u l a t e d in the muscle of the b r o w n t r o u t (Salmo trutta). No identification of these c o m p o u n d s was c a r r i e d out, a l t h o u g h it was n o t e d t h a t t h e y were h y d r o p h i l i c and cationic. Oladimeji et al. (1979) d e m o n s t r a t e d the p r o d u c t i o n of m o n o m e t h y l a t e d arsenic and two u n k n o w n a r s e n i c c o m p o u n d s in r a i n b o w t r o u t r e c e i v i n g oral doses of radio-labelled a r s e n i c acid. A f t e r a period of 96h, i n o r g a n i c arsenic, monom e t h y l a t e d a r s e n i c a n d the m i n o r unidentified a r s e n i c m e t a b o l i t e e a c h cons t i t u t e d < 10% of t o t a l arsenic. On the o t h e r hand, the m a j o r o r g a n i c a r s e n i c
0048-9697/87/$03.50
© 1987 Elsevier Science Publishers B.V.
318 metabolite (also unidentified) represented > 84% of total arsenic in all tissues and was the only arsenic compound detected in muscle after 96 h. It is interesting to speculate on the relevance of these studies to the occurrence of arsenobetaine. Both studies (Penrose, 1975; Oladimeji et al., 1979) showed t hat the major organic metabolites of orally-administered inorganic arsenic, alt h ou g h unidentified, were not arsenobetaine. Penrose (1975) considered t h a t the conversion of inorganic arsenic to the absorbed/retained organic forms was mediated by the intestinal flora of the fish. The failure to demonstrate conversion of orally-administered inorganic arsenic to arsenobtaine suggests t hat arsenobetaine is not synthesized de novo by the fish (through the mediation of gut bacteria or otherwise) but is likely, as has been previously suggested, to pass to the fish via the food chain from more complex organo-arsenic compounds found in algae (Edmonds et al., 1982). The study reported here was u n d e r t a k e n to identify metabolites in fish of orally-administered inorganic arsenic, and thereby to clarify the role, if any, of such metabolites in the production of arsenobetaine. E s t u a r y catfish and school whiting were chosen for experimentation because of their availability and the ease with which they could be maintained in aquaria. MATERIALS AND METHODS
Arsenic analysis Arsenic determinations were made and arsenic located in chromatographic fractions by vapour generation or electrothermal atomic absorption spectrophotometry using Varian instrumentation.
Arsenate dosing experiments E s t u a r y catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) were collected from Whitford's Beach 20km nort h of Pert h and transferred to the aquarium in oxygenated seawater. They were maintained in r u nn in g seawater on a diet of mussels for some weeks before the start of experimentation. Four whiting and three catfish received elevated levels of arsenic in their food and four whiting and a single catfish served as controls. Fish were dosed by feeding once each day with earth worms (Eisenia foetida) immediately after the worms had been injected with 100pl or 250pl sodium arsenate solution containing 1 #g gl 1 arsenic. The worms were offered to the fish by suspending them in the water with forceps, whereupon they were rapidly t a k e n or rejected by the fish. In this way known quantities of arsenate were quickly administered to the fish and rejected doses were easily discounted. Fish were supplied with as many worms as they would eat immediately. Consequently the arsenate was injected into the worms and swallowed by the fish within seconds, allowing no time for biotransformation of the arsenate by
319
the worms or leaching of the arsenic into ambient water. No arsenic was detectable in undosed worms. Whiting (four fish) consumed 77 worms containing 7.7mg arsenic over 17 days, and catfish (three fish) consumed 276 worms containing 39.9 mg arsenic over 44 days. Three days were allowed after the final meal before the fish were killed to allow the gut tract to empty. Thus after 20 days the whiting, and after 47 days the catfish, were killed by freezing.
Extraction and chromatographic analyses Whiting The four experimentally-dosed whiting (8.0, 10.9, 14.7 and 18.0 g) were blended with water (200 ml) to produce a grey liquid slurry (58.5 ~g As). Methanol (600ml) was added to the slurry and after standing for 4 h it was filtered to produce a pale filtrate and a grey residue. Methanol washings (200 ml) of the residue were added to the filtrate, which on evaporation yielded a gummy solid (2.2 g, 56/~g As). The aqueous methanol-insoluble residue was dried to produce a grey powder (5.1g, 8.1ttg As). The aqueous methanol-soluble material was partitioned between water and ether to yield, after evaporation, a watersoluble fraction (1.81 g, 44.7 ttg As) and an ether-soluble fraction (570 mg, 2.5 ttg As). Passage of the water-soluble fraction through a Sephadex LH-20 column (840 x 26 mm, elution with water/methanol, 50:50) produced material (800mg, 43.1 ttg As) clean enough for thin layer chromatographic (TLC) analysis. Thus a portion was applied to a single TLC plate (cellulose, E. Merck, Darmstadt; 200 x 200 x 0.1mm) and after development (butan-l-ol, acetic acid, water; 60:15:25) the plate was scribed, divided and analysed as shown in Fig. 1A. The four control whiting (9.2, 9.6, 11.5 and 11.7 g) were treated in identical manner to produce an initial aqueous methanol-soluble fraction (1.9 g, 33.4/~g As) and an insoluble residue (4.7 g dried, 1.75/~g As). Partitioning of the soluble material between water and ether yielded a water-soluble fraction (1.44g, 43.3/~g As) and an ether-soluble fraction (370 mg, 4.75 gg As). The water-soluble fraction was passed through the Sephadex LH-20-water/methanol column to yield material enriched in arsenic (690 mg, 38.1/~g As), which was subjected to TLC under the same conditions as the equivalent fraction from the experimentally dosed whiting (see Fig. 1B).
Catfish The three experimentally dosed catfish (27.9, 39.5 and 49.4 g) were treated as for the whiting to yield a water-soluble fraction (2.45 g, 249.2 ~g As), an ethersoluble fraction (480 mg, 2.3/~g As) and an insoluble residue (17.2 g dried, 55.9 ~g As). A small portion of the water-soluble material (containing ~ 1/~g As) was subjected to TLC as for the whiting (Fig. 1C). The control catfish (39.8g) treated in identical manner yielded a watersoluble fraction (470mg, 15.3ttg As), an ether-soluble fraction (140mg, 2.3t~g As) and an insoluble residue (5.6 g dried, 0.65 ~g As). A portion of the watersoluble material was subjected to TLC analysis (Fig. 1D).
320 80
80
70
70
60
50
z,O
30 o
20
20
o i,
10
I
1
SF
S,c
0
110
100
90
~ 7o
D
6o
•~
50
'~
30
5
50
8 3O
o
ir
20
~ 20 10
SF
SF
Fig. 1. Thin-layer chromatographic plates analysed for arsenic. (A) Experimentally-dosed whiting. (B) Control whiting. (C) Experimentally-dosed catfish. (D) Control catfish.
Isolation of arsenic compound from experimentally-dosed catfish The final water-soluble fraction from the experimentally dosed catfish (2.45g, 249ttg As) was subjected to gel permeation chromatography (GPC) (Sephadex LH-20, e l u t i o n with water/methanol, 50:50; 840 x 26 mm column) to yield an arsenic-enriched fraction (1.17 g, 232 pg As), w h i c h was again subjected to GPC under identical conditions. The a r s e n i c - c o n t a i n i n g fractions (300mg, 216pg As) were c h r o m a t o g r a p h e d on cellulose layers (seven of
321 200 × 200 × 0.1mm, developed with butan-l-ol, acetic acid, water; 60:15:25), and the arsenic-containing material (R~ 0.69) again subjected to GPC. Repeat TLC (single 200 × 200 × 0.1mm cellulose plate; development as previously) and GPC afforded further purification and 1H NMR and mass spectra were taken on this material (< I mg, 130 pg As). RESULTS Very little of the administered arsenic was absorbed and/or retained by the fish. If the assumption is made t h a t the experimentally-dosed whiting contained the same quantity of " n a t i v e " arsenic as the control fish, then only some 20pg arsenic was retained out of 7.7mg administered (0.27%). In the case of the catfish some 300 pg arsenic was retained out of 39.9 mg administered (0.75%). Of the retained arsenic a substantial proportion (13.8% in the case of whiting; 15.2% for the catfish) became insoluble/inextractable, presumably because of binding to insoluble proteins. Again this is in contrast to "native" arsenic where 4.8% (whiting) and 2.5% (catfish) remained inextractable. Partitioning of extractable soluble arsenic between water and ether resulted in the bulk of the arsenic, in each case, in the aqueous phase (94.7 and 90.1% for experimentally-dosed and control whiting; 99.0 and 86.9% for experimentally dosed and control catfish). Only the material in the aqueous phase was further examined in this study. Results of thin layer chromatographic analysis of the partially purified arsenic compounds from the water-soluble fractions of whiting and catfish are shown schematically in Fig. 1A-D. It can be seen that control whiting contained a single arsenic compound at Rv 0.50. This was assumed, from its chromatographic position (compared with standard material) and from a previous study (Edmonds and Francesconi, 1981) to be arsenobetaine; but two additional compounds were present in the experimentally dosed animals (R~ 0.25 and 0.69). It is probable that the compound at Rv 0.25 corresponded to a small quantity of residual administered arsenate (again by comparison with standard material), but that at Rv 0.69 was evidently an organic metabolite of arsenate. In the case of the catfish, it can be seen that the control animal contained arsenic compounds chromatographing at RF 0.51 (presumably arsenobetaine) and R~ 0.69, with that at 0.51 predominating in a ratio of ~ 60:40. However, the experimentally dosed animals contained a considerable excess of the organic metabolite at Rv 0.69 but no administered arsenate (Rv 0.25) was apparent. No significant increase in arsenobetaine concentrations in arsenate-dosed over control fish was observed for either catfish or whiting. The organic arsenic metabolite (RF 0.69) was, after isolation and purification, identified as trimethylarsine oxide by NMR spectroscopy (300 MHz 1H, D20) (~ 1.77; and mass spectrometry (electron impact, 70°/35 eV) 136 (100%), 130 (6.7), 121 (89.4), 120 (8.7), 107 (8.7), 106 (5.8), 105 (12.5), 104 (9.6), 103 (19.2), 94 (20.2), 93 (62.5), 91 (64.4), 90 (7.7), 89 (13.5).
322 DISCUSSION
Although only trimethylarsine oxide from experimentally dosed estuary catfish was isolated and rigorously identified in this study, the presence of arsenobetaine in school whiting and estuary catfish (both experimentally dosed with inorganic arsenic and control) was confidently inferred from thin layer chromatographic coordinates. Indeed the presence of arsenobetaine in school whiting has been reported previously (Edmonds and Francesconi, 1981). The presence of trimethylarsine oxide in control estuary catfish was inferred from thin layer chromatographic coordinates. Only a single compound (trimethylarsine oxide) was recovered at RF 0.69 from the TLC plates of experimentally dosed catfish. If a naturally-occurring compound other t han trimethylarsine oxide but having the same R E value was masked by the trimethylarsine oxide, some evidence for multiplicity of compounds would have been expected to become apparent during later isolation and purification stages. This did not occur and was taken as further evidence t hat the arsenic compound having TLC RF value 0.69 in control catfish was trimethylarsine oxide. Norin et al. (1985) reported the presence of trimethylarsine oxide in four species of Baltic Sea fish. In all cases trimethylarsine oxide represented a small proportion of total arsenic, but Norin et al. (1985) considered t hat higher concentrations were found in fish t ha t had been kept frozen t han in fresh fish. Trimethylarsine oxide was identified by a mass fragmentographic technique and in no case was it isolated from the sample. The work reported here shows t hat trimethylarsine oxide can accumulate in the tissues of fish as a result of the oral administration of inorganic arsenic, and it seems likely, as suggested by Penrose (1975), that its formation is dependent upon bacterial action in the gut t r a c t of the fish. The observation t hat estuary catfish contains 40% of its native arsenic as trimethylarsine oxide may well reflect its feeding habits; mouthfuls of sand or silt appear to be ingested when this fish is seeking food, and, inevitably, some finds its way into the gut tract in addition to food organisms. Inorganic arsenic, universally contained in sediments, may therefore be subjected to bacterial action in the fish gut. In contrast, school whiting have a more selective method of feeding and will pick food organisms from sediment without the intake of bottom or detrital material (R.C.J. Lenanton, personal communication, 1986). We have found no evidence t h at trimethylarsine oxide is more likely to occur in stored material, frozen or otherwise, th an in fresh material. Indeed, experiments designed to measure the breakdown of arsenobetaine in stored fish tissue with time failed to produce any trimethylarsine oxide (Edmonds and Francesconi, unpublished observations). The demonstration t ha t the intake of inorganic arsenic results in an accumulation of small quantities of trimethylarsine oxide does not conflict with the hypothesis t h a t arsenobetaine accumulates in marine organisms from a food chain source based on algal-derived arseno-sugars. Support is given to this view by the observation t ha t no increase in arsenobetaine levels occurs with inorganic arsenic ingestion. It appears t h a t the accumulation of ar-
323 s e n o b e t a i n e is n o t r e l a t e d to t h a t of t r i m e t h y l a r s i n e oxide, a n d t h e l a t t e r is n o t formed, in fish t i s s u e s a t l e a s t , by d e g r a d a t i o n of a r s e n o b e t a i n e . C o n v e r s e l y , a r s e n o b e t a i n e is n o t f o r m e d in fish t i s s u e s from i n g e s t e d i n o r g a n i c a r s e n i c , by g u t b a c t e r i a l a c t i o n or by o t h e r m e a n s . T h e p r e s e n c e of t r i m e t h y l a r s i n e o x i d e in e s t u a r y c a t f i s h t i s s u e s r a i s e s toxic o l o g i c a l q u e s t i o n s . A l t h o u g h s t u d i e s of t h e m e t a b o l i s m of a r s e n o b e t a i n e by e x p e r i m e n t a l a n i m a l s a r e r e a s s u r i n g from a t o x i c o l o g i c a l v i e w p o i n t ( V a h t e r et al., 1983), t h e t o x i c i t y of t r i m e t h y l a r s i n e o x i d e is u n k n o w n . T h e e a s e w i t h w h i c h it c a n be r e d u c e d to t h e h i g h l y t o x i c t r i m e t h y l a r s i n e m a y g i v e c a u s e for c o n c e r n . W h i t f i e l d et al. (1983) r e p o r t e d t h a t t h e p r e s e n c e of t r a c e q u a n t i t i e s of t r i m e t h y l a r s i n e in s o m e s p e c i e s of p r a w n s w a s sufficient to p r o d u c e a n "offf l a v o u r " . T h e h i g h e s t r e p o r t e d v a l u e (0.98 pg k g - 1 ) in r e d p r a w n s (Aristeomorpha foliacea) w o u l d be u n l i k e l y to p r e s e n t a t o x i c h a z a r d . It seems p o s s i b l e t h a t t h e p r e s e n c e of t r i m e t h y l a r s i n e in c r u s t a c e a m a y be t h e r e s u l t of t h e r e d u c t i o n of s m a l l q u a n t i t i e s of t r i m e t h y l a r s i n e o x i d e t h a t m a y c o - e x i s t w i t h ars e n o b e t a i n e a n d m a y n o t be m e t a b o l i c a l l y c o n n e c t e d w i t h t h e a r s e n o b e t a i n e .
REFERENCES Edmonds, J.S. and K.A. Francesconi, 1981. The origin and chemical form of arsenic in the school whiting. Mar. Pollut. Bull., 12(3): 92 96. Edmonds, J.S., K.A. Francesconi and J.A. Hansen, 1982. Dimethyloxarsylethanol from anaerobic decomposition of brown kelp Ecklonia radiata: a likely precursor of arsenobetaine in marine fauna. Experientia, 38: 643-644. GESAMP, 1986. Working Group on Review of Potentially Harmful Substances. Hazard Evaluation for Arsenic. World Health Organisation, Geneva. In press. Norin, H., A. Christakopoulos and M. Sandstr6m, 1985. Mass fragmentographic estimation of trimethylarsine oxide in aquatic organisms. Chemosphere, 14(3/4): 313-323. Oladimeji, A.A., S.U. Qadri, G.K.H. Tam and A.S.W. DeFreitas, 1979. Metabolism of organic arsenic to organoarsenicals in rainbow trout (Salmo gairdneri ). Ecotoxicol. Environ. Saf., 3(4): 394~400. Penrose, W.R., 1975. Biosynthesis of organic arsenic compounds in brown trout (Salmo trutta). J. Fish Res. Board Can., 32(12): 238~2390. Vahter, M., E. Marafante and L. Dencker, 1983. Metabolism of arsenobetaine in mice, rats and rabbits. Sci. Total Environ., 30:197 211. Whitfield, F.B., D.J. Freeman and K.J. Shaw, 1983. Trimethylarsine: an important off-flavour component in some prawn species. Chem. Ind., (20): 786-787.