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Short-term cadmium effects on gill tissue metabolism
Marine Pollution Bulletin 0025-326X/84 $3.00+(}.oo O 1984 PergamonPress Ltd.
Marine Pollution Bulletin, Vol. 15, No. 12, pp. 448-450, 1984
Printed in Great Britain
Short-term Cadmium Effects on Gill Tissue Metabolism L. TORT, P. TORRES and J. HIDALGO Fisiologia Animal, Facuhat de Ciencies, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain
ATP, lactate and respiratory rate in dogfish gill tissue were analysed after 50 ppm cadmium treatment. ATP levels decrease significantly after two days treatment but no differences with respect to the control values were detected after three, four or six days. Lactate increased after one day treatment and values were maintained after two and three days, recovering the control values from the fourth day. Oxygen consumption rate agree with the former patterns. After two days treatment a low rate (49% of controls) was recorded, while the respiratory rate after six days recovered in the 82% of the controls.
Heavy metals have been shown to produce a wide range of effects from molecular to physiological and even to behavioural and ecological levels (Vernberg et al., 1977), the effects depending on the metal itself, the species affected, and the environment. Cadmium present in the environment from the metal manufacture, coal combustion or waste disposal (HuRon, 1983) at concentrations higher than normal has been shown to produce cardiovascular impairment (Majewski & Giles, 1981), disturbances at molecular level (Morselt et al., 1983), bioaccumulation (Dickson et al., 1982) and changes in the metabolism and structure of tissues (Couch, 1977; Engel & Fowler, 1979). However, at certain subacute cadmium concentrations, no significant changes are often observed in physiological or biochemical parameters (Bilinski & Jonas, 1973; Dawson et al., 1977; Majewski & Giles, 1981). Gill disturbances have also been observed after metal exposure and impairment has been found in gill structure (Couch, 1977) as well as in gill function (Engel & Fowler, 1979; Tucker, 1979). As the first barrier the metal reaches when entering the organism, disturbances in gills are important as they produce serious impairment on the oxygen transfer to the blood system and the respiratory physiology. In this way several authors have stated that metals would generate hypoxia at tissue level (Skidmore, 1970; Burton et al., 1972). The metabolic status of dogfish gill tissue is shown in the present work by describing changes in tissue respiration, and ATP and lactate concentration. ATP or adenylate concentration have been shown to be good indicators of energetic balance (Vetter & Hodson, 1982). Lactate concentration is examined as an important endproduct of anaerobic metabolism (Van den Thillart, 1982) and oxygen consumption of gill tissue as a measure of the aerobic pathway. 448
Materials and M e t h o d s
Forty-eight dogfish Scyliorhinus canicula L. males and females of 150-350 g body weight were used. Fish were collected off the coast of Barcelona during January-May 1983 and were kept in an open seawater circulation tank for one month before experiments. After this time fish were transferred to closed seawater circulation tanks for treatment. Seawater and cadmium solutions were replaced every 48 h in order to prevent changes in water characteristics, metal reactions to the container and to remove fish excretions (Eisler & Hennekey, 1977; Henning & Greenwood, 1981; Braun et al., 1983). Cadmium was administered as cadmium chloride added to seawater. Six groups of eight fish in each group were used. One group was used as a control and the remaining groups were treated with 50 ppm of cadmium during 1, 2, 3, 4 or 6 days. Unpublished results from our laboratory show that 24 h LCso for the dogfish is about 200 ppm. Although 50 ppm is not a short-term lethal concentration, it produces relevant physiological and metabolic changes. After treatment, fish were quickly killed by decapitation and gill tissue was removed for metabolite determination. After homogenization in cold (4°C) 7% trichloracetic acid and centrifugation, lactate level was analysed by the method described by Lang & Michal (1974), and ATP by means of the bioluminiscence method with firefly luciferine-luciferase (Cheer et al., 1974). Tissue oxygen consumption measurements were determined by Warburg manometry (Umbreit et al., 1959) following the procedure used in Tort et al. (1982).
% ATP
100-
50-
C
1
2
3
4
6 DAYS
Fig. ! ATP levels after 1, 2, 3, 4 and 6 days referred to the controls.
Mean and standard error of the mean. [] • • A Significant difference (0.05 level).
Volume 15/Number 12/December 1984
have been shown to be a rapid and clear response against depletion of energy caused by lack of oxygen (Van den Thillart, 1982). These responses have also been observed after metal exposure (Burton et aL, 1972; Hodson, 1976). A decrease in respiratory rate and also in ATP level, 200, and a high increase in lactate concentration, are found in the present work, suggesting that cadmium exerts an action in dogfish gills resulting in a depletion of energy and an important increase in anaerobic pathway rates. However, about day six of exposure, lactate levels slowly returned to control values and recovery in ATP values 100. and respiratory rate is also observed. ATP depletion, after cadmium exposure, has also been shown by other authors (Majewski & Giles, 1981; Dickson et al., 1982). These authors found decrease of ATP and respiratory rate at 7 days of cadmium exposure in crayfish, but at 14 days return to control values was observed both in gill C 1 2 3 4 6 DAYS tissue respiration and ATP concentration. Lactate increase seems to be a short-term response of Fig. 2 Lactate levels after 1, 2, 3, 4 and 6 days referred to the controls. the anaerobic metabolism against a depleted aerobic Mean and standard error of the mean. ~r/~ ~7 [] • • Significant difference (0.05 level). energy situation, and by the sixth day, lactate concentration would recover the control levels. As has been proposed (Van den ThiUart, 1982), long-term strategy Student t-tests were used for assessing differences for maintaining energy supply would involve more effibetween treatments. 0.05 was taken as the level in which cient mechanisms, as for example increases of oxygen significance was accepted. extraction in gills or higher respiratory chain turnover (Mela et aL, 1977). Other types of mechanisms are related with adaptations in energy expenditure as reducResults tions of metabolic rate (Mathur, 1967) or motor activity Although 50 ppm is not a lethal concentration for the (Lomholt & Johansen, 1979). dogfish at short term exposure, cadmium produces signiAfter metal exposure some hypotheses have been ficant effects on gill metabolism as shown in Figs 1-3. suggested to explain the metal action: disfunction in Figure 1 shows ATP levels referred to the control after 1, physiological processes as the osmoregulatory, cardio2, 3, 4 and 6 days treatment, the mean control value being vascular or respiratory physiology have been proposed 3.26 ~rnol g-l wet wt. No differences are found after 24 h, (Majewski & Giles, 1981; Skidmore, 1970). Other but significant decrease is observed after 2 days. Results authors emphasize that metals could affect the structure, found after 3, 4 and 6 days show a return to the control and effects observed may be a consequence of damage levels and no significant differences are found between (Bilinski & Jonas, 1973; Couch, 1977). Hypothesis of control and 3, 4 and 6 days treatment. Lactate levels hypoxia caused by metals has also been suggested (mean control value of 1.26 ~tmol g-t wet wt) are quickly (Burton et al., 1972) and there are important similarities and strongly increased at 24 h. From the third day a pro- between effects caused by both hypoxia and metals. So, a gressive decrease takes place and control levels are situation of low oxygen availability for the tissues would recovered about day 6. Values in Fig. 3 show the oxygen be produced after metal exposure. Cardiorespiratory consumption of gill tissue after 2 and 6 days treatment. The results agree with the former patterns for ATP and lactate: at day 2 a lower rate of oxygen consumption is observed (49% of controls) but only a slight difference is _1 observed at day 6 (82% of controls). /UL.MG % LACTATE
!
!
Discussion Adenylate concentration as well as adenylate energy charge or ATP concentration are often used as indicators of energetic status in fish because of its easy measurement, ubiquity in all living cells and its participation in almost all metabolic pathways. Wijsman (1976) and Vetter & Hodson (1982) demonstrated that ATP concentration, total adenylates or adenylate energy charge appear to be useful indicators of environmental stress. Lactate as a measure of anaerobic metabolism has also been widely used and increases of anaerobic metabolism
0,65-
0,52-
//6
C
0,39-
2 0,26.
0,13.
HOURS
Fig. 3 Respiratory rate of gill tissue in control (C), 2 days cadmiumtreated (2) and 6 (6) days cadmium-treated fish.
449
Marine Pollution Bulletin
responses to both situations such as increase in ventilatory frequency and volume, increase in stroke volume and decrease in heart rate have been described (Hughes & Adeney, 1977). Other responses are related with increases of anaerobic metabolism and even alternative pathways have been suggested in fish (Van den Thillart, 1982). Improved gas transfer efficiency and reduction in energetic requirements can be also seen as compensatory mechanisms, and recovery of normal diffusing capacity of gills within 21 days after metal treatment has been observed (Hughes et al., 1979), allowing the fish to recover the normal gill function. Other mechanisms could act as a detoxifiers: metal binding proteins have been found in several fish species (Kito et al., 1982a) playing a protective role against heavy metal toxicity (Kito et al., 1982b). In addition, Cdbinding proteins have been recently detected in dogfish (Flos & Hidalgo, 1983) increasing their level with increasing time treatment and accumulating cadmium, that could partly explain the decrease of metal effects in tissue metabolism. Thanks are given to Dr P. Art6 from the Institut d'Investigacions Pesqueres (Barcelona) for facilities in fish supply and storing. This work was supported by a grant from Comision Asesora de Invcstigacion Cientifica y Tecnica.
Bilinsky, E. & Jonas, R. E. E. (1973). Effects of cadmium and copper on the oxidation of lactate by Rainbow trout Salmo gairdnerigills. J. Fish. Res. Bd Can., 31), 1553-1558. Braun, H. E., Morrow, D. C. & Ripley, B. D. (1983). Efficiency of water rinsing for the decontamination of used pesticide containers. Archs Envir. Contain. Toxic., 12, 257-264. Burton, D. T., Jones, A. H. & Cairns, J. Jr. (1972). Acute zinc toxicity to Rainbow trout Salmo gairdneri: confirmation that the hypothesis that death is related to tissue hypoxia. J. Fish. Res. Bd Can., 29, 14631466. Couch, J. A. (1977). Ultrastructural study of lesions in gills of a marine shrimp exposed to cadmium. J. Invert Path., 29, 267-288. Cheer, S., Gentile, J. H. & Hegre, C. S. (1974). Improved methods for ATP analysis. Analyt. Biochem., 60, 102-114. Dawson, M. A., Could, E., Thurberg, E P. & Calabrese, A. (1977). Physiological response to juvenile stripped bass, Momne saxatilis, to low levels of cadmium and mercury. Chesapeake Sci.; 15, 353-359. Dickson, G. W., Giesy, J. P. & Briese, L. A. (1982). The effect of chronic cadmium exposure on phosphoadenylate concentration and adenylate energy charge of gills and dorsal muscle tissue of crayfish. Envir. Toxic. Chem., i, 147-156. Eisler, R. & Hennekey, R. J. (1977). Acute toxicities of Cd 2+, Cr 6+, Hg 2÷, Ni z+, and Zn 2+ to estuarine macrofauna. Archs Envir. Contain. Toxic., 6, 315-323. Engel, D. W. & Fowler, B. A. (1979). Copper and cadmium induced changes in the metabolism and structure of molluscan gill tissue. In
450
Marine Pollutiorc Functional Research (W. B. Vernberg, E E Thurberg, A. Calabrese & E S. Vernberg, eds.), pp. 239-255. Flos, R. & Hidalgo, J. (1983). Cd metaliothionein in elasmobranchs: its presence in the dogfish. Fifth Conference European Society for Comparative Physiology and Biochemistry. September. Henning, H. E & Greenwood, P. J. (1981). The loss of cadmium and zinc from sea water during accumulation experiments: its implication on toxicity threshold concentrations. Mar. Pollut. Bull., 12, 47-50. Hodson, P. V. (1976). Temperature effects on lactate-glycogen metabolism in zinc-intoxicated Rainbow trout Salmo gairdneri. J. Fish. Res. Bd Can., 33, 1393-1397. Hughes, G. M. & Adeney, R. J. (1977). The effects of zinc on the cardiac and ventillatory rhythms of Rainbow trout, Salmo gairdneri, Richardson; and their responses to environmental hypoxia. Wat. Res., 11, 1069-1077. Hughes, G. M., Perry, S. E & Brown, V. M. (1979). A morphometric study of effects of nickel, chromium and cadmium on the secondary lamellae of Rainbow trout gills. Water Res., 13, 665-679. Hutton, M. (1983). Sources of cadmium in the environment. Ecotoxic. envir. Safety, 7, 9-14. Kito, H., Tazawa, T., Sato, T. & Ishikawa, T. (1982a). Formation of metalIothionein in fish. Comp. Biochem. PhysioL, 73, 129-134. Kito, H., Tazawa, T., Ose, Y., Sato, T. & Ishikawa, T. (1982b). Protection by metallothioneins against cadmium toxicity. Comp. Biochem. Physiol., 73, 135-139. Lang, G. & Michal, G. (1974). Methods of Enzymatic Analysis, 2nd edn. Academic Press, New York. Lomholt, J. P. & Johansen, K. (1979). Hypoxia acclimation in carp. How it affects 02 uptake, ventilation and 02 extraction from water. PhysioL Zool., 52, 38-50. Majewski, H. S. & Giles, M. A. (1981). Cardiovascular-respiratory responses of rainbow trout Salmo gairdneriduring chronic exposure to sublethal concentrations of cadmium. Wat. Res., 15, 1211-1217. Mathur, G. B. (1967). Anaerobic respiration in a cyprinoid fish, Rasbom daniconius. Nature, Lond., 214, 318-319. Mela, L., Goodwin, C. W. & Miller, L. D. (1977). In vivo adaptation of 02 availability: comparison of adult and newborn mitochondria. In Oxygen and Physiological Function (E Jobsis, ed.), pp. 283-291. Morselt, A. E W., Copuis Peereboom-Stegeman, J. H. J., JongstraSpaapen, E. J. & James, J. (1983). Investigation of the mechanism of cadmium toxicity at cellular level. 2. An electron microscopical. Archs Toxic., 52, 99-104. Skidmore, J. E (1970). Respiration and osmoregulation in rainbow trout with gills damaged with zinc sulphate. J. exp. Biol., 52,481-494. Tort, L., Crespo, S. & Balasch, J. (1982). Oxygen consumption of the dogfish gill tissue following zinc treatment. Comp. Biochem. Physiol., 72C, 145-148. Tucker, R. K. (1979). Effects of"in vivo" cadmium exposure on ATPases in gill of the lobster Homarus americanus. Bull. envir. Contam. Toxic., 23, 33-35. Umbreit, W. W., Burris, R. H. & Stauffer, J. E (1959). Manometric Techniques, pp. 1-338. Burgess, Minneapolis. Van den ThiUart, G. (1982). Adaptations of fish energy metabolism to hypoxia and anoxia. Molec. Physiol., 2, 49-61. Vernberg, J., Calabrese, A., Thurberg, E P. & Vernberg, W. B. (1977). Physiological Responses of Marine Biota to Pollutants, pp. 1-462. Academic Press, New York. Vetter, R. D. & Hodson, R. E. (1982). Use of adenylate concentration and adenylate energy change as indicators of hypoxic stress in estuarine fish. Can. J. Fish. aquat. Sci., 39, 535-542. Wijsman, T. C. M. (1976). ATP content and mortality in Mytilus edulis from different habitats in relation to anaerobiosis. Neth. J. Sea Res., 10, 140-148.
Marine Pollution Bulletin, Vol. 15, No. 12, pp. 448-450, 1984
Printed in Great Britain
Short-term Cadmium Effects on Gill Tissue Metabolism L. TORT, P. TORRES and J. HIDALGO Fisiologia Animal, Facuhat de Ciencies, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain
ATP, lactate and respiratory rate in dogfish gill tissue were analysed after 50 ppm cadmium treatment. ATP levels decrease significantly after two days treatment but no differences with respect to the control values were detected after three, four or six days. Lactate increased after one day treatment and values were maintained after two and three days, recovering the control values from the fourth day. Oxygen consumption rate agree with the former patterns. After two days treatment a low rate (49% of controls) was recorded, while the respiratory rate after six days recovered in the 82% of the controls.
Heavy metals have been shown to produce a wide range of effects from molecular to physiological and even to behavioural and ecological levels (Vernberg et al., 1977), the effects depending on the metal itself, the species affected, and the environment. Cadmium present in the environment from the metal manufacture, coal combustion or waste disposal (HuRon, 1983) at concentrations higher than normal has been shown to produce cardiovascular impairment (Majewski & Giles, 1981), disturbances at molecular level (Morselt et al., 1983), bioaccumulation (Dickson et al., 1982) and changes in the metabolism and structure of tissues (Couch, 1977; Engel & Fowler, 1979). However, at certain subacute cadmium concentrations, no significant changes are often observed in physiological or biochemical parameters (Bilinski & Jonas, 1973; Dawson et al., 1977; Majewski & Giles, 1981). Gill disturbances have also been observed after metal exposure and impairment has been found in gill structure (Couch, 1977) as well as in gill function (Engel & Fowler, 1979; Tucker, 1979). As the first barrier the metal reaches when entering the organism, disturbances in gills are important as they produce serious impairment on the oxygen transfer to the blood system and the respiratory physiology. In this way several authors have stated that metals would generate hypoxia at tissue level (Skidmore, 1970; Burton et al., 1972). The metabolic status of dogfish gill tissue is shown in the present work by describing changes in tissue respiration, and ATP and lactate concentration. ATP or adenylate concentration have been shown to be good indicators of energetic balance (Vetter & Hodson, 1982). Lactate concentration is examined as an important endproduct of anaerobic metabolism (Van den Thillart, 1982) and oxygen consumption of gill tissue as a measure of the aerobic pathway. 448
Materials and M e t h o d s
Forty-eight dogfish Scyliorhinus canicula L. males and females of 150-350 g body weight were used. Fish were collected off the coast of Barcelona during January-May 1983 and were kept in an open seawater circulation tank for one month before experiments. After this time fish were transferred to closed seawater circulation tanks for treatment. Seawater and cadmium solutions were replaced every 48 h in order to prevent changes in water characteristics, metal reactions to the container and to remove fish excretions (Eisler & Hennekey, 1977; Henning & Greenwood, 1981; Braun et al., 1983). Cadmium was administered as cadmium chloride added to seawater. Six groups of eight fish in each group were used. One group was used as a control and the remaining groups were treated with 50 ppm of cadmium during 1, 2, 3, 4 or 6 days. Unpublished results from our laboratory show that 24 h LCso for the dogfish is about 200 ppm. Although 50 ppm is not a short-term lethal concentration, it produces relevant physiological and metabolic changes. After treatment, fish were quickly killed by decapitation and gill tissue was removed for metabolite determination. After homogenization in cold (4°C) 7% trichloracetic acid and centrifugation, lactate level was analysed by the method described by Lang & Michal (1974), and ATP by means of the bioluminiscence method with firefly luciferine-luciferase (Cheer et al., 1974). Tissue oxygen consumption measurements were determined by Warburg manometry (Umbreit et al., 1959) following the procedure used in Tort et al. (1982).
% ATP
100-
50-
C
1
2
3
4
6 DAYS
Fig. ! ATP levels after 1, 2, 3, 4 and 6 days referred to the controls.
Mean and standard error of the mean. [] • • A Significant difference (0.05 level).
Volume 15/Number 12/December 1984
have been shown to be a rapid and clear response against depletion of energy caused by lack of oxygen (Van den Thillart, 1982). These responses have also been observed after metal exposure (Burton et aL, 1972; Hodson, 1976). A decrease in respiratory rate and also in ATP level, 200, and a high increase in lactate concentration, are found in the present work, suggesting that cadmium exerts an action in dogfish gills resulting in a depletion of energy and an important increase in anaerobic pathway rates. However, about day six of exposure, lactate levels slowly returned to control values and recovery in ATP values 100. and respiratory rate is also observed. ATP depletion, after cadmium exposure, has also been shown by other authors (Majewski & Giles, 1981; Dickson et al., 1982). These authors found decrease of ATP and respiratory rate at 7 days of cadmium exposure in crayfish, but at 14 days return to control values was observed both in gill C 1 2 3 4 6 DAYS tissue respiration and ATP concentration. Lactate increase seems to be a short-term response of Fig. 2 Lactate levels after 1, 2, 3, 4 and 6 days referred to the controls. the anaerobic metabolism against a depleted aerobic Mean and standard error of the mean. ~r/~ ~7 [] • • Significant difference (0.05 level). energy situation, and by the sixth day, lactate concentration would recover the control levels. As has been proposed (Van den ThiUart, 1982), long-term strategy Student t-tests were used for assessing differences for maintaining energy supply would involve more effibetween treatments. 0.05 was taken as the level in which cient mechanisms, as for example increases of oxygen significance was accepted. extraction in gills or higher respiratory chain turnover (Mela et aL, 1977). Other types of mechanisms are related with adaptations in energy expenditure as reducResults tions of metabolic rate (Mathur, 1967) or motor activity Although 50 ppm is not a lethal concentration for the (Lomholt & Johansen, 1979). dogfish at short term exposure, cadmium produces signiAfter metal exposure some hypotheses have been ficant effects on gill metabolism as shown in Figs 1-3. suggested to explain the metal action: disfunction in Figure 1 shows ATP levels referred to the control after 1, physiological processes as the osmoregulatory, cardio2, 3, 4 and 6 days treatment, the mean control value being vascular or respiratory physiology have been proposed 3.26 ~rnol g-l wet wt. No differences are found after 24 h, (Majewski & Giles, 1981; Skidmore, 1970). Other but significant decrease is observed after 2 days. Results authors emphasize that metals could affect the structure, found after 3, 4 and 6 days show a return to the control and effects observed may be a consequence of damage levels and no significant differences are found between (Bilinski & Jonas, 1973; Couch, 1977). Hypothesis of control and 3, 4 and 6 days treatment. Lactate levels hypoxia caused by metals has also been suggested (mean control value of 1.26 ~tmol g-t wet wt) are quickly (Burton et al., 1972) and there are important similarities and strongly increased at 24 h. From the third day a pro- between effects caused by both hypoxia and metals. So, a gressive decrease takes place and control levels are situation of low oxygen availability for the tissues would recovered about day 6. Values in Fig. 3 show the oxygen be produced after metal exposure. Cardiorespiratory consumption of gill tissue after 2 and 6 days treatment. The results agree with the former patterns for ATP and lactate: at day 2 a lower rate of oxygen consumption is observed (49% of controls) but only a slight difference is _1 observed at day 6 (82% of controls). /UL.MG % LACTATE
!
!
Discussion Adenylate concentration as well as adenylate energy charge or ATP concentration are often used as indicators of energetic status in fish because of its easy measurement, ubiquity in all living cells and its participation in almost all metabolic pathways. Wijsman (1976) and Vetter & Hodson (1982) demonstrated that ATP concentration, total adenylates or adenylate energy charge appear to be useful indicators of environmental stress. Lactate as a measure of anaerobic metabolism has also been widely used and increases of anaerobic metabolism
0,65-
0,52-
//6
C
0,39-
2 0,26.
0,13.
HOURS
Fig. 3 Respiratory rate of gill tissue in control (C), 2 days cadmiumtreated (2) and 6 (6) days cadmium-treated fish.
449
Marine Pollution Bulletin
responses to both situations such as increase in ventilatory frequency and volume, increase in stroke volume and decrease in heart rate have been described (Hughes & Adeney, 1977). Other responses are related with increases of anaerobic metabolism and even alternative pathways have been suggested in fish (Van den Thillart, 1982). Improved gas transfer efficiency and reduction in energetic requirements can be also seen as compensatory mechanisms, and recovery of normal diffusing capacity of gills within 21 days after metal treatment has been observed (Hughes et al., 1979), allowing the fish to recover the normal gill function. Other mechanisms could act as a detoxifiers: metal binding proteins have been found in several fish species (Kito et al., 1982a) playing a protective role against heavy metal toxicity (Kito et al., 1982b). In addition, Cdbinding proteins have been recently detected in dogfish (Flos & Hidalgo, 1983) increasing their level with increasing time treatment and accumulating cadmium, that could partly explain the decrease of metal effects in tissue metabolism. Thanks are given to Dr P. Art6 from the Institut d'Investigacions Pesqueres (Barcelona) for facilities in fish supply and storing. This work was supported by a grant from Comision Asesora de Invcstigacion Cientifica y Tecnica.
Bilinsky, E. & Jonas, R. E. E. (1973). Effects of cadmium and copper on the oxidation of lactate by Rainbow trout Salmo gairdnerigills. J. Fish. Res. Bd Can., 31), 1553-1558. Braun, H. E., Morrow, D. C. & Ripley, B. D. (1983). Efficiency of water rinsing for the decontamination of used pesticide containers. Archs Envir. Contain. Toxic., 12, 257-264. Burton, D. T., Jones, A. H. & Cairns, J. Jr. (1972). Acute zinc toxicity to Rainbow trout Salmo gairdneri: confirmation that the hypothesis that death is related to tissue hypoxia. J. Fish. Res. Bd Can., 29, 14631466. Couch, J. A. (1977). Ultrastructural study of lesions in gills of a marine shrimp exposed to cadmium. J. Invert Path., 29, 267-288. Cheer, S., Gentile, J. H. & Hegre, C. S. (1974). Improved methods for ATP analysis. Analyt. Biochem., 60, 102-114. Dawson, M. A., Could, E., Thurberg, E P. & Calabrese, A. (1977). Physiological response to juvenile stripped bass, Momne saxatilis, to low levels of cadmium and mercury. Chesapeake Sci.; 15, 353-359. Dickson, G. W., Giesy, J. P. & Briese, L. A. (1982). The effect of chronic cadmium exposure on phosphoadenylate concentration and adenylate energy charge of gills and dorsal muscle tissue of crayfish. Envir. Toxic. Chem., i, 147-156. Eisler, R. & Hennekey, R. J. (1977). Acute toxicities of Cd 2+, Cr 6+, Hg 2÷, Ni z+, and Zn 2+ to estuarine macrofauna. Archs Envir. Contain. Toxic., 6, 315-323. Engel, D. W. & Fowler, B. A. (1979). Copper and cadmium induced changes in the metabolism and structure of molluscan gill tissue. In
450
Marine Pollutiorc Functional Research (W. B. Vernberg, E E Thurberg, A. Calabrese & E S. Vernberg, eds.), pp. 239-255. Flos, R. & Hidalgo, J. (1983). Cd metaliothionein in elasmobranchs: its presence in the dogfish. Fifth Conference European Society for Comparative Physiology and Biochemistry. September. Henning, H. E & Greenwood, P. J. (1981). The loss of cadmium and zinc from sea water during accumulation experiments: its implication on toxicity threshold concentrations. Mar. Pollut. Bull., 12, 47-50. Hodson, P. V. (1976). Temperature effects on lactate-glycogen metabolism in zinc-intoxicated Rainbow trout Salmo gairdneri. J. Fish. Res. Bd Can., 33, 1393-1397. Hughes, G. M. & Adeney, R. J. (1977). The effects of zinc on the cardiac and ventillatory rhythms of Rainbow trout, Salmo gairdneri, Richardson; and their responses to environmental hypoxia. Wat. Res., 11, 1069-1077. Hughes, G. M., Perry, S. E & Brown, V. M. (1979). A morphometric study of effects of nickel, chromium and cadmium on the secondary lamellae of Rainbow trout gills. Water Res., 13, 665-679. Hutton, M. (1983). Sources of cadmium in the environment. Ecotoxic. envir. Safety, 7, 9-14. Kito, H., Tazawa, T., Sato, T. & Ishikawa, T. (1982a). Formation of metalIothionein in fish. Comp. Biochem. PhysioL, 73, 129-134. Kito, H., Tazawa, T., Ose, Y., Sato, T. & Ishikawa, T. (1982b). Protection by metallothioneins against cadmium toxicity. Comp. Biochem. Physiol., 73, 135-139. Lang, G. & Michal, G. (1974). Methods of Enzymatic Analysis, 2nd edn. Academic Press, New York. Lomholt, J. P. & Johansen, K. (1979). Hypoxia acclimation in carp. How it affects 02 uptake, ventilation and 02 extraction from water. PhysioL Zool., 52, 38-50. Majewski, H. S. & Giles, M. A. (1981). Cardiovascular-respiratory responses of rainbow trout Salmo gairdneriduring chronic exposure to sublethal concentrations of cadmium. Wat. Res., 15, 1211-1217. Mathur, G. B. (1967). Anaerobic respiration in a cyprinoid fish, Rasbom daniconius. Nature, Lond., 214, 318-319. Mela, L., Goodwin, C. W. & Miller, L. D. (1977). In vivo adaptation of 02 availability: comparison of adult and newborn mitochondria. In Oxygen and Physiological Function (E Jobsis, ed.), pp. 283-291. Morselt, A. E W., Copuis Peereboom-Stegeman, J. H. J., JongstraSpaapen, E. J. & James, J. (1983). Investigation of the mechanism of cadmium toxicity at cellular level. 2. An electron microscopical. Archs Toxic., 52, 99-104. Skidmore, J. E (1970). Respiration and osmoregulation in rainbow trout with gills damaged with zinc sulphate. J. exp. Biol., 52,481-494. Tort, L., Crespo, S. & Balasch, J. (1982). Oxygen consumption of the dogfish gill tissue following zinc treatment. Comp. Biochem. Physiol., 72C, 145-148. Tucker, R. K. (1979). Effects of"in vivo" cadmium exposure on ATPases in gill of the lobster Homarus americanus. Bull. envir. Contam. Toxic., 23, 33-35. Umbreit, W. W., Burris, R. H. & Stauffer, J. E (1959). Manometric Techniques, pp. 1-338. Burgess, Minneapolis. Van den ThiUart, G. (1982). Adaptations of fish energy metabolism to hypoxia and anoxia. Molec. Physiol., 2, 49-61. Vernberg, J., Calabrese, A., Thurberg, E P. & Vernberg, W. B. (1977). Physiological Responses of Marine Biota to Pollutants, pp. 1-462. Academic Press, New York. Vetter, R. D. & Hodson, R. E. (1982). Use of adenylate concentration and adenylate energy change as indicators of hypoxic stress in estuarine fish. Can. J. Fish. aquat. Sci., 39, 535-542. Wijsman, T. C. M. (1976). ATP content and mortality in Mytilus edulis from different habitats in relation to anaerobiosis. Neth. J. Sea Res., 10, 140-148.