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Uptake, metabolism, and depuration of PCBs by the polychaete Nereis virens
416 II: N o . 2
Rapid determination of zinc, copper and cadmium organometallics in mussels by gel-permeation high pressure liquid chromatography and in-line detection by inductively coupled plasma atomic emission spectrometry. G . M a z z u c o t e l l i x a n d A . V i a r e n g o 2. Istituto di Chimica Generale, Genova, ItalyX; Istituto di Fisiologia Generale, Genova, Italy 2. The biological importance of the role that Zn and Cu organometallic compounds play in the cells of marine organisms often exposed to variable environmental concentrations of heavy metals and, in particular, the important role that metallothioneins exert in the homeostasis of biological divalent cations (Cu, Zn) and in the detoxification of cytotoxic xenobiotics such as Cd and Hg is now well established. The gel-permeation high pressure liquid chromatography (HPLC) has been successfully used to separate high molecular weight compounds and organometallics; however, the sensitivity necessary for these metallic detections is so high that only electrothermal atomic absorption spectrophotometry (ETAAAS) gives the accuracy and reproducibility needed in these determinations. Unfortunately ETA-AAS is almost impossible to collect in-line with HPLC, so a fraction collection is necessary, with a high increase in manipulation and analytical errors; moreover, a continuous plot of the experiments cannot be obtained. In our labs we have performed an in-line assemblage of a HPLC-ICP (inductively coupled plasma) apparatus. The HPLC eluates are directly aspirated into the plasma torch at a constant flow of less than 1 mL/min, through a nebulization Jobin-Yvon chamber equipped with a Meinhard glass nebulizer. Before the introduction in nebulization chamber eluates pass through the microcell of an UV HPLC-detector: two chromatographic tracks are so obtained relative to protein (UV) and metallorganic compounds (ICP) present in the analyzed sample. The ICP chromatogram is obtained by checking a direct position (the PROFILE option of the program) at the analytical wavelength of the analyzed element and the chromatogram is directly registered by a strip chart recorder; the variations of intensity at the maximum peak, due to the influence of temperature on the optical geometry, are tested by intermediate scanning around the theorical wavelength by a direct aspiration of known (Zn, Cu or Cd) solutions. Repeated injections (20-100 microliters) of sample allow the subsequent detection of the various metals. The present method allows analysis using a minimum amount of sample, also giving for each metal the UV track, in less than half an hour. Data are presented concerning the evaluation of the (Zn, Cu) and (Zn, Cd) thionein content in different tissues of mussels, a lamellibranch mollusc often utilized as a biological indicator in monitoring programmes. II: N o . 3
Uptake, metabolism, and deputation of PCBs by the polychaete Nereis virens. A . E . M c E l r o y ~ a n d J . C . M e a n s 2. Environmental Sciences Program, University oJ Massachusetts, Boston, MA 02125, USAt; Chesapeake Biological Laboratory oJ Maryland, Solomons, MD 20688, U,S.A. 2 Three ~4C-labeled PCB congeners (4-chlorobiphenyl [MCBP], 2,4,2' ,4'-tetrachlorobiphenyl [TCBP], and 2,4,5,2',4',5'-hexachlorobiphenyl [HCBP]) were fed to polychaete worms which had colonized glass tubes. During the 6 wk uptake phase, worms were fed alternately with radiolabeled food followed by unlabeled food on a six day cycleT-Worms were collected the day after receiving unlabeled food. An eight week depuration phase followed, during which the worms were fed only unlabeled food. Total accumulated radioactivity, and radioactivity transformed into polar metabolites and bound residues were measured in individual worms regularly throughout the uptake and depuration period. Fecal pellets were collected, pooled and analyzed w6~kly for total radioactivity over the entire course of the experiment. Total accumulated radioactivity in worms fed TCBP and HCBP increased throughout the uptake
417 phase and remained high during the depuration phase. Total accumulated radioactivity in worms fed MCBP followed a different pattern, increasing only slightly during the uptake phase (relative to the other two congeners) with a rapid depuration to 18% of maximum accumulated dose. Significant evidence of metabolism was observed only in worms fec~ MCBP. After 6 wk of accumulation, most radioactivity recovered from the worms fed MCBP was present as unextractable residues, with less than 10% found as parent compound. Total radioactivity recovered in fecal pellet samples increased for all three congeners during the accumulation phase, following the same general trends observed in worm tissue. After the cessation of the radioactive diet (wk 6), activity in fecal material dropped precipitously to near background levels within 2 wk, even though body burdens of TCBP and HCBP remained elevated. This suggests that depuration via fecal pellet production is not a major pathway for PCB elimination in these animals. Examination of selected fecal pellet samples indicated the presence of metabolic products only in extracts from worms fed MCBP. The major differences observed between accumulation and depuration of the individual PCB congeners appeared to result from differential metabolism. An unexpected finding was that a significant fraction of the isotope fed to the worms was not recovered in either worm tissue or fecal pellets. Investigation of radioactivity lost during ingestion, or from leaching of PCBs from fecal pellets, indicates that excretion of accumulated radioactivity via a 'dissolved' pathway may be responsible for rapid removal of all three PCB congeners from these worms. I: NO. 4
Physiological predictors of reduced production in estuarine mysid populations with chronic pesticide exposure. C . L . M c K e n n e y , J r . U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL 32561 U.S.A. A variety of life functions of an estuarine mysid were examined during chronic exposure to representative pesticides of three distinct classes: the organochlorine endrin, the carbamate thiobencarb, and the organophosphate fenthion. During life-cycle toxicity tests of these three pesticides with Mysidopsis bahia, different life stages were subsampled and measurements made of various aspects of their energy metabolism. Dose-response relationships of individual physiological rate functions (oxygen consumption, ammonia excretion, and growth rates) and their combined bioenergetic and physiological indices were compared with alterations in survival and reproduction during the chronic exposure period. Elevated respiration rates of juvenile mysids after only 1 to 4 days exposure predicted, for all three pesticides, those concentrations which eventually reduced survival in isolated populations with exposure through an entire life cycle (approximately 3 weeks). In accordance with lower net growth efficiency (K2 values), increased metabolic demands on juveniles with sublethal pesticide exposure, as expressed by higher rates of oxygen consumption, reduced the amount of assimilated energy available for production of new tissue. For all three classes of pesticides, retarded growth rates of juveniles were apparent after only 4 days exposure to pesticide concentrations equivalent to those resulting in decreases in total young production of populations exposed through release of their first brood. I(2 values provided a stronger dose-response correlation than growth per se and, as such, should provide better predictive capabilities for those pesticide concentrations with the potential of lowering reproductive capacity of exposed populations. As indicated by the atomic ratio of oxygen consumed to nitrogen excreted (O:N), energy metabolism of juvenile mysids was based primarily on utilization of lipid substrates, but shifted during maturation to greater usage of protein as an energy source. Higher O:N ratios during the maturation of pesticideexposed mysids suggest a greater reliance on the more energy-rich lipid substrates in order to support the elevated rates of oxidative metabolism, resulting in less lipid material being available for gamete production. The results of these studies suggest that short-term measurements of alterations in the energy
Rapid determination of zinc, copper and cadmium organometallics in mussels by gel-permeation high pressure liquid chromatography and in-line detection by inductively coupled plasma atomic emission spectrometry. G . M a z z u c o t e l l i x a n d A . V i a r e n g o 2. Istituto di Chimica Generale, Genova, ItalyX; Istituto di Fisiologia Generale, Genova, Italy 2. The biological importance of the role that Zn and Cu organometallic compounds play in the cells of marine organisms often exposed to variable environmental concentrations of heavy metals and, in particular, the important role that metallothioneins exert in the homeostasis of biological divalent cations (Cu, Zn) and in the detoxification of cytotoxic xenobiotics such as Cd and Hg is now well established. The gel-permeation high pressure liquid chromatography (HPLC) has been successfully used to separate high molecular weight compounds and organometallics; however, the sensitivity necessary for these metallic detections is so high that only electrothermal atomic absorption spectrophotometry (ETAAAS) gives the accuracy and reproducibility needed in these determinations. Unfortunately ETA-AAS is almost impossible to collect in-line with HPLC, so a fraction collection is necessary, with a high increase in manipulation and analytical errors; moreover, a continuous plot of the experiments cannot be obtained. In our labs we have performed an in-line assemblage of a HPLC-ICP (inductively coupled plasma) apparatus. The HPLC eluates are directly aspirated into the plasma torch at a constant flow of less than 1 mL/min, through a nebulization Jobin-Yvon chamber equipped with a Meinhard glass nebulizer. Before the introduction in nebulization chamber eluates pass through the microcell of an UV HPLC-detector: two chromatographic tracks are so obtained relative to protein (UV) and metallorganic compounds (ICP) present in the analyzed sample. The ICP chromatogram is obtained by checking a direct position (the PROFILE option of the program) at the analytical wavelength of the analyzed element and the chromatogram is directly registered by a strip chart recorder; the variations of intensity at the maximum peak, due to the influence of temperature on the optical geometry, are tested by intermediate scanning around the theorical wavelength by a direct aspiration of known (Zn, Cu or Cd) solutions. Repeated injections (20-100 microliters) of sample allow the subsequent detection of the various metals. The present method allows analysis using a minimum amount of sample, also giving for each metal the UV track, in less than half an hour. Data are presented concerning the evaluation of the (Zn, Cu) and (Zn, Cd) thionein content in different tissues of mussels, a lamellibranch mollusc often utilized as a biological indicator in monitoring programmes. II: N o . 3
Uptake, metabolism, and deputation of PCBs by the polychaete Nereis virens. A . E . M c E l r o y ~ a n d J . C . M e a n s 2. Environmental Sciences Program, University oJ Massachusetts, Boston, MA 02125, USAt; Chesapeake Biological Laboratory oJ Maryland, Solomons, MD 20688, U,S.A. 2 Three ~4C-labeled PCB congeners (4-chlorobiphenyl [MCBP], 2,4,2' ,4'-tetrachlorobiphenyl [TCBP], and 2,4,5,2',4',5'-hexachlorobiphenyl [HCBP]) were fed to polychaete worms which had colonized glass tubes. During the 6 wk uptake phase, worms were fed alternately with radiolabeled food followed by unlabeled food on a six day cycleT-Worms were collected the day after receiving unlabeled food. An eight week depuration phase followed, during which the worms were fed only unlabeled food. Total accumulated radioactivity, and radioactivity transformed into polar metabolites and bound residues were measured in individual worms regularly throughout the uptake and depuration period. Fecal pellets were collected, pooled and analyzed w6~kly for total radioactivity over the entire course of the experiment. Total accumulated radioactivity in worms fed TCBP and HCBP increased throughout the uptake
417 phase and remained high during the depuration phase. Total accumulated radioactivity in worms fed MCBP followed a different pattern, increasing only slightly during the uptake phase (relative to the other two congeners) with a rapid depuration to 18% of maximum accumulated dose. Significant evidence of metabolism was observed only in worms fec~ MCBP. After 6 wk of accumulation, most radioactivity recovered from the worms fed MCBP was present as unextractable residues, with less than 10% found as parent compound. Total radioactivity recovered in fecal pellet samples increased for all three congeners during the accumulation phase, following the same general trends observed in worm tissue. After the cessation of the radioactive diet (wk 6), activity in fecal material dropped precipitously to near background levels within 2 wk, even though body burdens of TCBP and HCBP remained elevated. This suggests that depuration via fecal pellet production is not a major pathway for PCB elimination in these animals. Examination of selected fecal pellet samples indicated the presence of metabolic products only in extracts from worms fed MCBP. The major differences observed between accumulation and depuration of the individual PCB congeners appeared to result from differential metabolism. An unexpected finding was that a significant fraction of the isotope fed to the worms was not recovered in either worm tissue or fecal pellets. Investigation of radioactivity lost during ingestion, or from leaching of PCBs from fecal pellets, indicates that excretion of accumulated radioactivity via a 'dissolved' pathway may be responsible for rapid removal of all three PCB congeners from these worms. I: NO. 4
Physiological predictors of reduced production in estuarine mysid populations with chronic pesticide exposure. C . L . M c K e n n e y , J r . U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL 32561 U.S.A. A variety of life functions of an estuarine mysid were examined during chronic exposure to representative pesticides of three distinct classes: the organochlorine endrin, the carbamate thiobencarb, and the organophosphate fenthion. During life-cycle toxicity tests of these three pesticides with Mysidopsis bahia, different life stages were subsampled and measurements made of various aspects of their energy metabolism. Dose-response relationships of individual physiological rate functions (oxygen consumption, ammonia excretion, and growth rates) and their combined bioenergetic and physiological indices were compared with alterations in survival and reproduction during the chronic exposure period. Elevated respiration rates of juvenile mysids after only 1 to 4 days exposure predicted, for all three pesticides, those concentrations which eventually reduced survival in isolated populations with exposure through an entire life cycle (approximately 3 weeks). In accordance with lower net growth efficiency (K2 values), increased metabolic demands on juveniles with sublethal pesticide exposure, as expressed by higher rates of oxygen consumption, reduced the amount of assimilated energy available for production of new tissue. For all three classes of pesticides, retarded growth rates of juveniles were apparent after only 4 days exposure to pesticide concentrations equivalent to those resulting in decreases in total young production of populations exposed through release of their first brood. I(2 values provided a stronger dose-response correlation than growth per se and, as such, should provide better predictive capabilities for those pesticide concentrations with the potential of lowering reproductive capacity of exposed populations. As indicated by the atomic ratio of oxygen consumed to nitrogen excreted (O:N), energy metabolism of juvenile mysids was based primarily on utilization of lipid substrates, but shifted during maturation to greater usage of protein as an energy source. Higher O:N ratios during the maturation of pesticideexposed mysids suggest a greater reliance on the more energy-rich lipid substrates in order to support the elevated rates of oxidative metabolism, resulting in less lipid material being available for gamete production. The results of these studies suggest that short-term measurements of alterations in the energy