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The uptake and metabolism by goldfish Carassius auratus of Pentachloro-2-[Chloromethyl Sulphonamido]Diphenyl ether (6-PCSD), the major component and active ingredient of the mothproofing agent, Eulan WA New
Environmental Pollution (Series A ) 36 (1984) 271-281
The Uptake and Metabolism by Goldfish Carassius auratus of Pentachloro-2-[Chloromethyl
Sulphonamido]Diphenyl Ether (6-PCSD), the Major Component and Active Ingredient of the Mothproofing Agent, Eulan WA New
Andrew Machon, Michael J. North, Nicholas C. Price Department of Biological Science, University of Stirling, Stirling FK9 4LA, Scotland, Great Britain
& David E. Wells Freshwater Fisheries Laboratory, Faskally, Pitlochry, Perthshire PHI6 5LB, Scotland, Great Britain
ABSTRACT Goldfish absorbed pentachloro-2-[chloromethyl sulphonamido] diphenyl ether, 6-PCSD (the major component of the active ingredients oJ the mothproofing agent, Eulan WA New)from the surrounding water by branchial and cutaneous routes. The liver was the tissue in which the corresponding amine, and probable metabolite, pentachloro-2-aminodiphenyl ether, 5-PAD, was first detected. The 5-PAD concentration increased during the first 2 h of the dosing period. The liver tissue is thereJore a primary site oJ 5-PAD Jbrmation in vivo. The uptake and metabolism of 6-PCSD by the goldfish is similar to that observed in the pike after dosing ,[br short periods with 6-PCSD. However, the goldfish provides a more suitable system in which to study this metabolism in vitro. 271 Environ. Pollut. Ser. A. 0143-1471/84/$03.00 ,~ Elsevier Applied Science Publishers Ltd. England, 1984. Printed in Great Britain
272
Andrew Machon, Michael J. North, Nicholas C. Price. David E. Wells
INTRODUCTION Eulan WA New is a widely used textile mothproofing agent. The major components and active ingredients are polychloro-2-[chloromethyl sulphonamido] diphenyl ethers (PCSDs, Fig. 1) which bind avidly to keratin fibres and so prevent their digestion by wool-feedingpests (Anon., 1967). This mothproofing agent has been detected in aquatic environments arising directly or indirectly from the release of textile effluents and both PCSDs and their proposed metabolites, polychloro-2-aminodiphenyl ethers (PADs, Fig. 1), have been identified in the tissues of fish from environments contaminated with Eulan WA New (Wells & Cowan, 1983). The uptake, tissue distribution and metabolism of Eulan WA New by pike E s o x lucius have been studied, both by oral dosing and by dosing of the surrounding water (Machon et al., 1984a,b). An increase in the ratio 5PAD:6-PCSD was observed in most tissues with increasing time of exposure of pike to Eulan WA New. This trend has also been observed in carp on exposure to the mothproofing agent in surrounding water
c, o c, NHR
NH2
a
b
Cl
Cl
Cl
c, o c, el
Cl
I
l
SO,CH=Cl c
Cl
So=CH2Cl d
Fig. 1. Structures of active ingredients and some minor components of Eulan WA New formulation. (a) General formula, where R may be H or SO2CH2CI, x + y may be 4, 5 or 6, denoting the number of chlorine atoms in the component. (b) 5-PAD (2',4',2,3,4-Pentachloro-6-aminodiphenyl ether) main impurity. (c) 6-PCSD (2',4',2,3,4Pentachloro-6-(chloromethyl-sulphonamido)diphenyl ether, the main active ingredient ( 2 0 ~ in formulation). (d) 7-PCSD (2',4',2,3,4,5-Hexachloro-6-(chloromethyl-sulphonamido) diphenyl ether.
Uptake oI" Eulan New by gold/ish
273
(Hamburger et al., 1981). Taken together, these data provide evidence for the in vivo metabolism of 6-PCSD to 5-PAD in tissues of certain fish species. Because of the difficulties of obtaining a regular supply of pike and subsequent husbandry problems, it was necessary to choose an alternative fish species in which to study the metabolism of 6-PCSD to 5PAD. The proposed in vivo metabolism of Eulan WA New by carp (Hamburger et al., 1981) suggested the goldfish Carassius auratus, a carp relative which has the advantage of being easy to obtain and maintain, as a possible choice. The present study is concerned with the uptake, tissue distribution and metabolism of 6-PCSD in the goldfish. High performance liquid chromatography (H PLC) was routinely used for analysis of PCSD and PAD in fish tissues in preference to the alternative gas-liquid chromatography-electron capture detector (GLC) method. In the latter method, methylation is necessary prior to analysis in order to stabilise thermolabile PCSD components (Wells, 1979); by contrast no derivatisation is necessary with HPLC. However, tissue coextractants absorbing at 230nm interfere with the determination of PCSD by H PLC and, in order to remove these coextractants, it was necessary to modify the published tissue extract 'clean up' procedure of Wells & Cowan (1981); these modifications are described.
E X P E R I M E N T A L SECTION Materials
Eulan WA New was supplied by Bayer (Shipley, UK). All chemicals for sample 'clean up' were those described by Wells & Cowan ( 1981). Solvents used were HPLC grade supplied by Rathburn Chemicals Ltd (Walkerburn, UK). Methods
Quantitative analysis Quantitative analysis of 6-PCSD and 5-PAD was performed by H PLC as described by Wells & Johnstone ( 1981) and by GLC as described by Wells & Cowan (1981). For GLC analysis decachlorobiphenyl was added as an internal standard.
274
Andrew Machon, Michael J. North, Nicholas C. Price, David E. Wells
Preparation oJ purified 6-PCSD and its alkaline hydrolysis products A solution of purified 6-PCSD for dosing was prepared from Eulan WA New by the HPLC semi-preparative method (Machon et al., 1984b). Alkaline hydrolysis products of 6-PCSD were prepared on an analytical scale by adapting the method of West66 & Noren (1977). One milligram of purified 6-PCSD in 500 pl dimethylformamide (DMF) was mixed with 500/A 50"/,, (w/v) aqueous potassium hydroxide and the mixture was heated to 100°C in a pyrex glass test tube for 24h. A small aliquot (50#1) of the sample was then diluted with distilled water (1:100) and further
/-II
Fig. 2. HPLC profiles of (a) purified 6-PCSD and (b) alkaline hydrolysis products of 6-PCSD. The conditions for H PLC analysis were: flow rate, 2 m l m i n - l ; solvent, methanol/water (85/15 (v/v)), pH 3.6; wavelength monitored, 230nm; Chart speed, 2 m m m i n - t ; absorbance, 0.1. Five minutes after injection the profiles show a peak absorbing strongly at 230nm. This is the absorbance due to dimethylformamide, the solvent in which the sample for HPLC analysis is injected. In the HPLC profiles, peaks 1 and 11 refer to 6-PCSD and 5-PAD, respectively.
Uptake oJ Eulan New by goldfish
275
extracted with 2 x 5 ml portions of methyitertiary butyl ether (MTBE). The extract was taken to dryness under a stream of air and redissolved in methanol or D M F for H PLC analysis. H P LC profiles of 6-PCSD and its hydrolysis products are shown in Fig. 2. 6-PCSD is seen as an incompletely resolved doublet peak by HPLC analysis (Fig. 2a); two hydrolysis products are formed (Fig. 2b), apparently arising from these two components. The chemical structure of the component forming the second minor peak of the doublet (Fig. 2a) is at present unknown.
Dosing goldfish with purified 6-PCSD Two litres of distilled water were placed in each of two glass tanks (of 3litre capacity). To one tank was added a solution of purified 6-PCSD in DMF, giving an initial concentration of 0.26 mg litre - 1 6-PCSD, and the second (control) tank an equivalent volume of DMF. Eight sexually immature goldfish (weighing between 3.3 and 6.6g and measuring between 6.5 and 8.0cm in length) were obtained from a local supplier. Four goldfish were added to each tank. After dosing periods of 10 min, 30 min, 2 h and 6.5 h, goldfish were removed from each tank and killed by severing the spinal cord. Samples of water were also taken from each tank for analysis. The goldfish were dissected and the liver plus bile, skin plus muscle, digestive tract and gill tissues, removed. These samples were stored at - 2 0 °C before tissue extraction, 'clean up' and analysis.
Extraction, "clean up" and analysis oJ goldfish tissues Tissues were weighed, ground with anhydrous sodium sulphate (4 g) to give a free-flowing powder, and then extracted with 3 x 5 ml portions of MTBE, with a 30-min period of mixing after each addition of extraction solvent. The total MTBE extract from each tissue was evaporated carefully to just dryness under a stream of air and the residue redissoived in 100 pl MTBE prior to application to a mixed alumina column for'clean up'. The necessary modification of the published 'clean up' procedure (Wells & Cowan, 1981) prior to HPLC analysis is described below. RESULTS AND DISCUSSION Modification of the published 'clean up' procedure for HPLC analysis of tissue extracts A preliminary study involving the HPLC analysis of extracts of dosed goldfish tissue, without prior clean up, on mixed alumina columns
276
Andrew Machon, Michael J. North, Nicholas C. Price, David E. Wells"
s h o w e d a n u m b e r o f c o m p o n e n t s which a b s o r b at 230 n m in a d d i t i o n to 6P C S D (Fig. 3b). T h e s e a d d i t i o n a l c o m p o n e n t s are n o t a d e q u a t e l y resolved f r o m 6 - P C S D a n d c o n s e q u e n t l y affect q u a n t i t a t i o n o f the latter. A s e p a r a t e e x p e r i m e n t , in w h i c h a sample o f liver f r o m a fish n o t e x p o s e d to 6 - P C S D was e x t r a c t e d a n d a n a l y s e d by H P L C , showed t h a t these c o m p o n e n t s are c o e x t r a c t a n t s a n d not p r o d u c t s o f m e t a b o l i s m o f 6P C S D (Fig. 3d). F u r t h e r investigation revealed that the c o e x t r a c t a n t s
l l I ÷ polar coextractants
polsa" ooextractants
Fig. 3. The presence of components absorbing at 230nm, with similar retention times to 6-PCSD, revealed by HPLC profiles of fish tissue extracts. HPLC profiles: (a) and (c) 6-PCSD standards. (b) A liver extract from a goldfish dosed with 6-PCSD, without prior 'clean up" on a mixed alumina column. (d) A liver extract from a goldfish not dosed with 6-PCSD and not subjected to "clean up" on a mixed alumina column. Conditions as in Fig. 2.
Uptake of Eulan New by gold/ish
277
polar
Ipolar
~'act~
i c
L
b
Fig. 4. The separation of 6-PCSD, 5-PAD and polar coextractants from a goldfish liver tissue extract. HPLC profiles of: (a) The eluate collected from a mixed alumina column, loaded with a liver extract from a goldfish dosed with 6-PCSD for 2h, obtained by elution with 25 ml MTBE. (b) The subsequent eluate collected from the column described in (a) by elution with 25 ml methanol. (c) The next eluate from the column described in (a) and (b), collected, after acidification of the column, by elution with 10ml MTBE. (d) The eluate collected from a mixed alumina column loaded with a liver extract from a control goldfish (i.e. not dosed with 6-PCSD), obtained by elution with 25ml MTBE. (e) The subsequent eluate collected from the column described in (d) by elution with 25 ml methanol. (f) The next eluate from the column described in (d) and (e), collected, after acidification of the column, by elution with 10ml MTBE. Conditions as Fig. 2.
278
Andrea' Machon, Michael J. North, Nicholas C. Price, David E. Wells
I!
Fig. 5. The identification of 6-PCSD in tissues of goldfish dosed with 6-PCSD. HPLC profiles of: (a) 6-PCSD standard. (b) The eluate collected by elution of a mixed alumina column, loaded with liver extract from a goldfish dosed with 6-PCSD for 2h, with 25ml methanol (see text). (c) As (b) but from a column loaded with digestive tissue extract. Conditions as in Fig. 2.
Fig. 6. The identification of 5-PAD in the liver of goldfish dosed with 6-PCSD. HPLC profiles of: (a) 5-PAD standard. (b) The eluate collected by elution of a mixed alumina column, loaded with liver extract from a goldfish dosed with 6-PCSD for 2h, with 25ml MTBE. (c) A blank, in which anhydrous sodium sulphate was extracted with MTBE and subjected to "clean up' on a mixed alumina column. The profile shown refers to the eluate collected by elution with 25ml MTBE. Conditions as in Fig. 2.
Uptake of Eulan New by goldfish
279
appear, together with 6-PCSD, in the E5 eluate (diethyl ether/hexane; 30/70 (v/v)) from a mixed alumina column (Wells & Cowan, 1981). However, it was possible to separate them from 6-PCSD by selective elution of the latter with methanol prior to acidification of the column. A simplified elution procedure was eventually developed to separate PADs, PCSDs and the coextractants. This procedure consisted of elution with 25 ml MTBE (to remove PADs selectively), followed by elution with 25 ml methanol (to remove PCSDs selectively); the coextractants could then be removed by elution with 10ml MTBE following acidification of the column. This procedure is illustrated in Fig. 4 for a liver sample taken from a goldfish dosed with 6-PCSD for 2h. The identification and quantitation of 6-PCSD and 5-PAD in dosed tissues was based on comparison with standards (Figs 5 and 6).
Analysis of tissues from dosed goldfish The concentration of 6-PCSD in the surrounding water declined only slightly during the dosing period (from 0 . 2 6 m g l i t r e - ' initially to 0.23 mg litre- 1 after 6.5 h). The concentrations of 6-PCSD and 5-PAD in the tissues of goldfish are shown in Table 1. The first tissues to accumulate 6-PCSD are the gills and skin/muscle; these represent the major route of uptake of 6-PCSD from surrounding water. The uptake of 6-PCSD by pike appears to be also by the gills and the skin (Machon et al., 1984b). The liver/bile is the first sample in which 5-PAD was detected (after 10 minutes' dosing). A trend of increasing 5-PAD concentration in this tissue was observed for up to 2 h. The observation of 5-PAD at the earliest dosing times implicates the liver as a primary tissue of 5-PAD formation in the goldfish; a similar conclusion has been reached from studies on pike (Machon et al., 1984b). After the shortest dosing times, the concentration of 6-PCSD in the goldfish liver was below the detection limit by GLC (approximately 0.01 # g g - ' tissue), suggesting a high turnover rate to 5PAD during its in vivo metabolism. 6-PCSD accumulated in all the tissues studied with increasing dosing time (Table 1). In summary, these data provide evidence for the in vivo metabolism of 6-PCSD to 5-PAD in goldfish, with the liver implicated as a primary site of 5-PAD formation. The results are similar to those obtained from the dosing of pike with 6-PCSD (Machon et al., 1984b). Thus, because of its ease of husbandry, the goldfish provides an advantageous system in which to study further the metabolism of 6-PCSD in vivo and to characterise the 5-PAD forming activity in vitro. Such studies are in progress.
280
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Uptake of Eulan New hy goldhsh
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ACKNOWLEDGEMENTS This work was supported by the Natural Environmental Research Council, Grant No. GR3/4129. The dosing studies, under licence No. Ed 4276, and G L C analysis were completed at the Freshwater Fisheries Laboratory. The assistance of Messrs A. E. G. Christie, G. Hanslip and D. M o o r e is gratefully acknowledged.
REFERENCES Anon. (1967). Wool dyeing-wool protection, mixtures of wool and man-made fibres. Bayer Farben Revue, Special Edition, No. 8. Hamburger, B., Kuck, M. & Weis, H. (1981). Eulan WA New hioaccumulation in.hsh. Leverkusen, Bayer A. G. L. E. Umweltschutz/AWALU Fe-Di zentrale Analytik. Machon, A., North, M. J., Price, N. C. & Wells, D. E. (1984a). Dietary accumulation of the mothproofing agent Eulan WA New and its tissue distribution in the northern pike (Esox lucius). Environ. Pollut. Ser. A, 33, 379-85. Machon, A., North, M. J., Price, N. C. & Wells, D. E. (1984b). Accumulation, metabolism and tissue distribution of the mothproofing agent Eulan WA New in the northern pike (Esox lucius). Environ. Pollut. Set. A, 33, 275-89. Wells, D. E. (1979). The isolation and identification of polychloro-2-(chloromethyl sulphonamido) diphenyl ether isomers and their metabolites from Eulan WA New and fish tissue by gas chromatography-mass spectrometry. Analyt. Chirn. Acta, 104, 253-66. Wells, D. E. & Cowan, A. A. (1981). Determination of the mothproofing agent Eulan WA New in fish tissue using gas-liquid chromatography following extractive methylation. Analyst, Lond., 106, 862-8. Wells, D. E. & Cowan, A. A. (1983). Fate and distribution of mothproofing agents Dieldrin and Eulan WA New in Loch Leven, Kinross, 1964-79. Environ. Pollut. Ser. B, 7, 11-33. Wells, D. E. & Johnstone, S. J. (1981). High performance liquid chromatography of polychloro-2-[dichloromethyl sulphonamido] diphenyl ethers and their impurities in the mothproofing agent, Eulan WA New and in water. J. Chromatogr., 19, 137-43. West66, G. & Nor~n, K. (1977). Polychiorinated 2-aminodiphenyl ethers in fish. Ambio, 6, 232-4.
The Uptake and Metabolism by Goldfish Carassius auratus of Pentachloro-2-[Chloromethyl
Sulphonamido]Diphenyl Ether (6-PCSD), the Major Component and Active Ingredient of the Mothproofing Agent, Eulan WA New
Andrew Machon, Michael J. North, Nicholas C. Price Department of Biological Science, University of Stirling, Stirling FK9 4LA, Scotland, Great Britain
& David E. Wells Freshwater Fisheries Laboratory, Faskally, Pitlochry, Perthshire PHI6 5LB, Scotland, Great Britain
ABSTRACT Goldfish absorbed pentachloro-2-[chloromethyl sulphonamido] diphenyl ether, 6-PCSD (the major component of the active ingredients oJ the mothproofing agent, Eulan WA New)from the surrounding water by branchial and cutaneous routes. The liver was the tissue in which the corresponding amine, and probable metabolite, pentachloro-2-aminodiphenyl ether, 5-PAD, was first detected. The 5-PAD concentration increased during the first 2 h of the dosing period. The liver tissue is thereJore a primary site oJ 5-PAD Jbrmation in vivo. The uptake and metabolism of 6-PCSD by the goldfish is similar to that observed in the pike after dosing ,[br short periods with 6-PCSD. However, the goldfish provides a more suitable system in which to study this metabolism in vitro. 271 Environ. Pollut. Ser. A. 0143-1471/84/$03.00 ,~ Elsevier Applied Science Publishers Ltd. England, 1984. Printed in Great Britain
272
Andrew Machon, Michael J. North, Nicholas C. Price. David E. Wells
INTRODUCTION Eulan WA New is a widely used textile mothproofing agent. The major components and active ingredients are polychloro-2-[chloromethyl sulphonamido] diphenyl ethers (PCSDs, Fig. 1) which bind avidly to keratin fibres and so prevent their digestion by wool-feedingpests (Anon., 1967). This mothproofing agent has been detected in aquatic environments arising directly or indirectly from the release of textile effluents and both PCSDs and their proposed metabolites, polychloro-2-aminodiphenyl ethers (PADs, Fig. 1), have been identified in the tissues of fish from environments contaminated with Eulan WA New (Wells & Cowan, 1983). The uptake, tissue distribution and metabolism of Eulan WA New by pike E s o x lucius have been studied, both by oral dosing and by dosing of the surrounding water (Machon et al., 1984a,b). An increase in the ratio 5PAD:6-PCSD was observed in most tissues with increasing time of exposure of pike to Eulan WA New. This trend has also been observed in carp on exposure to the mothproofing agent in surrounding water
c, o c, NHR
NH2
a
b
Cl
Cl
Cl
c, o c, el
Cl
I
l
SO,CH=Cl c
Cl
So=CH2Cl d
Fig. 1. Structures of active ingredients and some minor components of Eulan WA New formulation. (a) General formula, where R may be H or SO2CH2CI, x + y may be 4, 5 or 6, denoting the number of chlorine atoms in the component. (b) 5-PAD (2',4',2,3,4-Pentachloro-6-aminodiphenyl ether) main impurity. (c) 6-PCSD (2',4',2,3,4Pentachloro-6-(chloromethyl-sulphonamido)diphenyl ether, the main active ingredient ( 2 0 ~ in formulation). (d) 7-PCSD (2',4',2,3,4,5-Hexachloro-6-(chloromethyl-sulphonamido) diphenyl ether.
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273
(Hamburger et al., 1981). Taken together, these data provide evidence for the in vivo metabolism of 6-PCSD to 5-PAD in tissues of certain fish species. Because of the difficulties of obtaining a regular supply of pike and subsequent husbandry problems, it was necessary to choose an alternative fish species in which to study the metabolism of 6-PCSD to 5PAD. The proposed in vivo metabolism of Eulan WA New by carp (Hamburger et al., 1981) suggested the goldfish Carassius auratus, a carp relative which has the advantage of being easy to obtain and maintain, as a possible choice. The present study is concerned with the uptake, tissue distribution and metabolism of 6-PCSD in the goldfish. High performance liquid chromatography (H PLC) was routinely used for analysis of PCSD and PAD in fish tissues in preference to the alternative gas-liquid chromatography-electron capture detector (GLC) method. In the latter method, methylation is necessary prior to analysis in order to stabilise thermolabile PCSD components (Wells, 1979); by contrast no derivatisation is necessary with HPLC. However, tissue coextractants absorbing at 230nm interfere with the determination of PCSD by H PLC and, in order to remove these coextractants, it was necessary to modify the published tissue extract 'clean up' procedure of Wells & Cowan (1981); these modifications are described.
E X P E R I M E N T A L SECTION Materials
Eulan WA New was supplied by Bayer (Shipley, UK). All chemicals for sample 'clean up' were those described by Wells & Cowan ( 1981). Solvents used were HPLC grade supplied by Rathburn Chemicals Ltd (Walkerburn, UK). Methods
Quantitative analysis Quantitative analysis of 6-PCSD and 5-PAD was performed by H PLC as described by Wells & Johnstone ( 1981) and by GLC as described by Wells & Cowan (1981). For GLC analysis decachlorobiphenyl was added as an internal standard.
274
Andrew Machon, Michael J. North, Nicholas C. Price, David E. Wells
Preparation oJ purified 6-PCSD and its alkaline hydrolysis products A solution of purified 6-PCSD for dosing was prepared from Eulan WA New by the HPLC semi-preparative method (Machon et al., 1984b). Alkaline hydrolysis products of 6-PCSD were prepared on an analytical scale by adapting the method of West66 & Noren (1977). One milligram of purified 6-PCSD in 500 pl dimethylformamide (DMF) was mixed with 500/A 50"/,, (w/v) aqueous potassium hydroxide and the mixture was heated to 100°C in a pyrex glass test tube for 24h. A small aliquot (50#1) of the sample was then diluted with distilled water (1:100) and further
/-II
Fig. 2. HPLC profiles of (a) purified 6-PCSD and (b) alkaline hydrolysis products of 6-PCSD. The conditions for H PLC analysis were: flow rate, 2 m l m i n - l ; solvent, methanol/water (85/15 (v/v)), pH 3.6; wavelength monitored, 230nm; Chart speed, 2 m m m i n - t ; absorbance, 0.1. Five minutes after injection the profiles show a peak absorbing strongly at 230nm. This is the absorbance due to dimethylformamide, the solvent in which the sample for HPLC analysis is injected. In the HPLC profiles, peaks 1 and 11 refer to 6-PCSD and 5-PAD, respectively.
Uptake oJ Eulan New by goldfish
275
extracted with 2 x 5 ml portions of methyitertiary butyl ether (MTBE). The extract was taken to dryness under a stream of air and redissolved in methanol or D M F for H PLC analysis. H P LC profiles of 6-PCSD and its hydrolysis products are shown in Fig. 2. 6-PCSD is seen as an incompletely resolved doublet peak by HPLC analysis (Fig. 2a); two hydrolysis products are formed (Fig. 2b), apparently arising from these two components. The chemical structure of the component forming the second minor peak of the doublet (Fig. 2a) is at present unknown.
Dosing goldfish with purified 6-PCSD Two litres of distilled water were placed in each of two glass tanks (of 3litre capacity). To one tank was added a solution of purified 6-PCSD in DMF, giving an initial concentration of 0.26 mg litre - 1 6-PCSD, and the second (control) tank an equivalent volume of DMF. Eight sexually immature goldfish (weighing between 3.3 and 6.6g and measuring between 6.5 and 8.0cm in length) were obtained from a local supplier. Four goldfish were added to each tank. After dosing periods of 10 min, 30 min, 2 h and 6.5 h, goldfish were removed from each tank and killed by severing the spinal cord. Samples of water were also taken from each tank for analysis. The goldfish were dissected and the liver plus bile, skin plus muscle, digestive tract and gill tissues, removed. These samples were stored at - 2 0 °C before tissue extraction, 'clean up' and analysis.
Extraction, "clean up" and analysis oJ goldfish tissues Tissues were weighed, ground with anhydrous sodium sulphate (4 g) to give a free-flowing powder, and then extracted with 3 x 5 ml portions of MTBE, with a 30-min period of mixing after each addition of extraction solvent. The total MTBE extract from each tissue was evaporated carefully to just dryness under a stream of air and the residue redissoived in 100 pl MTBE prior to application to a mixed alumina column for'clean up'. The necessary modification of the published 'clean up' procedure (Wells & Cowan, 1981) prior to HPLC analysis is described below. RESULTS AND DISCUSSION Modification of the published 'clean up' procedure for HPLC analysis of tissue extracts A preliminary study involving the HPLC analysis of extracts of dosed goldfish tissue, without prior clean up, on mixed alumina columns
276
Andrew Machon, Michael J. North, Nicholas C. Price, David E. Wells"
s h o w e d a n u m b e r o f c o m p o n e n t s which a b s o r b at 230 n m in a d d i t i o n to 6P C S D (Fig. 3b). T h e s e a d d i t i o n a l c o m p o n e n t s are n o t a d e q u a t e l y resolved f r o m 6 - P C S D a n d c o n s e q u e n t l y affect q u a n t i t a t i o n o f the latter. A s e p a r a t e e x p e r i m e n t , in w h i c h a sample o f liver f r o m a fish n o t e x p o s e d to 6 - P C S D was e x t r a c t e d a n d a n a l y s e d by H P L C , showed t h a t these c o m p o n e n t s are c o e x t r a c t a n t s a n d not p r o d u c t s o f m e t a b o l i s m o f 6P C S D (Fig. 3d). F u r t h e r investigation revealed that the c o e x t r a c t a n t s
l l I ÷ polar coextractants
polsa" ooextractants
Fig. 3. The presence of components absorbing at 230nm, with similar retention times to 6-PCSD, revealed by HPLC profiles of fish tissue extracts. HPLC profiles: (a) and (c) 6-PCSD standards. (b) A liver extract from a goldfish dosed with 6-PCSD, without prior 'clean up" on a mixed alumina column. (d) A liver extract from a goldfish not dosed with 6-PCSD and not subjected to "clean up" on a mixed alumina column. Conditions as in Fig. 2.
Uptake of Eulan New by gold/ish
277
polar
Ipolar
~'act~
i c
L
b
Fig. 4. The separation of 6-PCSD, 5-PAD and polar coextractants from a goldfish liver tissue extract. HPLC profiles of: (a) The eluate collected from a mixed alumina column, loaded with a liver extract from a goldfish dosed with 6-PCSD for 2h, obtained by elution with 25 ml MTBE. (b) The subsequent eluate collected from the column described in (a) by elution with 25 ml methanol. (c) The next eluate from the column described in (a) and (b), collected, after acidification of the column, by elution with 10ml MTBE. (d) The eluate collected from a mixed alumina column loaded with a liver extract from a control goldfish (i.e. not dosed with 6-PCSD), obtained by elution with 25ml MTBE. (e) The subsequent eluate collected from the column described in (d) by elution with 25 ml methanol. (f) The next eluate from the column described in (d) and (e), collected, after acidification of the column, by elution with 10ml MTBE. Conditions as Fig. 2.
278
Andrea' Machon, Michael J. North, Nicholas C. Price, David E. Wells
I!
Fig. 5. The identification of 6-PCSD in tissues of goldfish dosed with 6-PCSD. HPLC profiles of: (a) 6-PCSD standard. (b) The eluate collected by elution of a mixed alumina column, loaded with liver extract from a goldfish dosed with 6-PCSD for 2h, with 25ml methanol (see text). (c) As (b) but from a column loaded with digestive tissue extract. Conditions as in Fig. 2.
Fig. 6. The identification of 5-PAD in the liver of goldfish dosed with 6-PCSD. HPLC profiles of: (a) 5-PAD standard. (b) The eluate collected by elution of a mixed alumina column, loaded with liver extract from a goldfish dosed with 6-PCSD for 2h, with 25ml MTBE. (c) A blank, in which anhydrous sodium sulphate was extracted with MTBE and subjected to "clean up' on a mixed alumina column. The profile shown refers to the eluate collected by elution with 25ml MTBE. Conditions as in Fig. 2.
Uptake of Eulan New by goldfish
279
appear, together with 6-PCSD, in the E5 eluate (diethyl ether/hexane; 30/70 (v/v)) from a mixed alumina column (Wells & Cowan, 1981). However, it was possible to separate them from 6-PCSD by selective elution of the latter with methanol prior to acidification of the column. A simplified elution procedure was eventually developed to separate PADs, PCSDs and the coextractants. This procedure consisted of elution with 25 ml MTBE (to remove PADs selectively), followed by elution with 25 ml methanol (to remove PCSDs selectively); the coextractants could then be removed by elution with 10ml MTBE following acidification of the column. This procedure is illustrated in Fig. 4 for a liver sample taken from a goldfish dosed with 6-PCSD for 2h. The identification and quantitation of 6-PCSD and 5-PAD in dosed tissues was based on comparison with standards (Figs 5 and 6).
Analysis of tissues from dosed goldfish The concentration of 6-PCSD in the surrounding water declined only slightly during the dosing period (from 0 . 2 6 m g l i t r e - ' initially to 0.23 mg litre- 1 after 6.5 h). The concentrations of 6-PCSD and 5-PAD in the tissues of goldfish are shown in Table 1. The first tissues to accumulate 6-PCSD are the gills and skin/muscle; these represent the major route of uptake of 6-PCSD from surrounding water. The uptake of 6-PCSD by pike appears to be also by the gills and the skin (Machon et al., 1984b). The liver/bile is the first sample in which 5-PAD was detected (after 10 minutes' dosing). A trend of increasing 5-PAD concentration in this tissue was observed for up to 2 h. The observation of 5-PAD at the earliest dosing times implicates the liver as a primary tissue of 5-PAD formation in the goldfish; a similar conclusion has been reached from studies on pike (Machon et al., 1984b). After the shortest dosing times, the concentration of 6-PCSD in the goldfish liver was below the detection limit by GLC (approximately 0.01 # g g - ' tissue), suggesting a high turnover rate to 5PAD during its in vivo metabolism. 6-PCSD accumulated in all the tissues studied with increasing dosing time (Table 1). In summary, these data provide evidence for the in vivo metabolism of 6-PCSD to 5-PAD in goldfish, with the liver implicated as a primary site of 5-PAD formation. The results are similar to those obtained from the dosing of pike with 6-PCSD (Machon et al., 1984b). Thus, because of its ease of husbandry, the goldfish provides an advantageous system in which to study further the metabolism of 6-PCSD in vivo and to characterise the 5-PAD forming activity in vitro. Such studies are in progress.
280
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ACKNOWLEDGEMENTS This work was supported by the Natural Environmental Research Council, Grant No. GR3/4129. The dosing studies, under licence No. Ed 4276, and G L C analysis were completed at the Freshwater Fisheries Laboratory. The assistance of Messrs A. E. G. Christie, G. Hanslip and D. M o o r e is gratefully acknowledged.
REFERENCES Anon. (1967). Wool dyeing-wool protection, mixtures of wool and man-made fibres. Bayer Farben Revue, Special Edition, No. 8. Hamburger, B., Kuck, M. & Weis, H. (1981). Eulan WA New hioaccumulation in.hsh. Leverkusen, Bayer A. G. L. E. Umweltschutz/AWALU Fe-Di zentrale Analytik. Machon, A., North, M. J., Price, N. C. & Wells, D. E. (1984a). Dietary accumulation of the mothproofing agent Eulan WA New and its tissue distribution in the northern pike (Esox lucius). Environ. Pollut. Ser. A, 33, 379-85. Machon, A., North, M. J., Price, N. C. & Wells, D. E. (1984b). Accumulation, metabolism and tissue distribution of the mothproofing agent Eulan WA New in the northern pike (Esox lucius). Environ. Pollut. Set. A, 33, 275-89. Wells, D. E. (1979). The isolation and identification of polychloro-2-(chloromethyl sulphonamido) diphenyl ether isomers and their metabolites from Eulan WA New and fish tissue by gas chromatography-mass spectrometry. Analyt. Chirn. Acta, 104, 253-66. Wells, D. E. & Cowan, A. A. (1981). Determination of the mothproofing agent Eulan WA New in fish tissue using gas-liquid chromatography following extractive methylation. Analyst, Lond., 106, 862-8. Wells, D. E. & Cowan, A. A. (1983). Fate and distribution of mothproofing agents Dieldrin and Eulan WA New in Loch Leven, Kinross, 1964-79. Environ. Pollut. Ser. B, 7, 11-33. Wells, D. E. & Johnstone, S. J. (1981). High performance liquid chromatography of polychloro-2-[dichloromethyl sulphonamido] diphenyl ethers and their impurities in the mothproofing agent, Eulan WA New and in water. J. Chromatogr., 19, 137-43. West66, G. & Nor~n, K. (1977). Polychiorinated 2-aminodiphenyl ethers in fish. Ambio, 6, 232-4.