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Respiratory impairment in crustaceans and molluscs due to exposure to heavy metals
Camp. B&hem.
Printedin Great
Physiol. Britain
Vol. lOOC,No. 3, pp. 339-342,
1991 0
0306-4492/91 $3.00 + 0.00 199 1 PergamonPressplc
MINI-REVIEW RESPIRATORY IMPAIRMENT IN CRUSTACEANS AND MOLLUSCS DUE TO EXPOSURE TO HEAVY METALS JOHNI. SPICRRand ROY E. WEBER Department of Zoophysiology, University of Aarhus, Aarhus, Denmark, DK 8000 (Received 2 January 1991) Abstract-l. We have assessed, using current literature, the respiratory consequences of water-borne heavy metal exposure in crustaceans and molluscs. 2. We suggest that in lethal and sub-lethal concentrations the essential metals Cu and Zn act on the respiratory system primarily by disrupting gill function which results in the development of internal hypoxia, although reparation can be accomplished even at “high” sub-lethal concentrations. 3. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) may interfere with the respiratory system at every level of organisation including cellular respiration itself.
INTRODUCTION Heavy metals (HM) are normal constituents of aquatic environments. All HM are toxic at some concentration but this is normally of little biological importance. In coastal and fresh waters, however, the concentrations of HM may be orders of magnitude greater than found in open waters as a result of anthropogenic input (Preston, 1973). Some of the more important HM, due to their toxicity at “low” concentrations, are the essential metals copper (Cu) and zinc (Zn) and the xenobiotes mercury (Hg) and cadmium (Cd). In this paper we will examine the respiratory consequences (pathological and compensatory) of water-borne HM exposure for crustaceans and mollusts, animals which are not only important as food for man and other animals, but also as vectors of disease in fresh water habitats. HM exposure, therefore, may either occur in the form of “pollution” or in the deployment of molluscicides which often rely on the toxic action of HM (particularly Cu and even Zn and Cd; Duncan, 1975) for their effectiveness. We will briefly review the pathological effects of HM exposure on the respiratory system at various levels of organisation before assessing the ecotoxicogical significance of this data. OXYGEN
CONSUMPTION
In general, oxygen consumption (MO,) decreased when molluscs and crustaceans were acutely exposed to HM (Jones, 1942; Corner and Sparrow, 1956; Brown and Newell, 1972; DeCourcey and Vernberg, 1972; Vernberg and Vernberg, 1972; Cheng and Sullivan, 1973a, 1975; McInnes and Thurberg, 1973; Address for correspondence: Dr J. I. Spicer, Department of Animal and Plant Sciences, School of Bi6logical Sciences, University of Sheffield, P.O. Box 601, Sheffield SlO ZUQ, U.K.
Saliba and Vella, 1977; Depledge, 1984; Papathanassiou, 1983) although in some cases either no change (Scott and Major, 1972; Depledge and Phillips, 1987; Spicer and Weber, 1991) or even an increase (Baghdiguian and Riva, 1985) was observed. A priori any compensatory change, immediate or long term, will require 0,. Therefore if some degree of physiological compensation is possible (or at least attempted), there may be an initial enhancement of MO,. Given that a decrease in MOz is indicative of HM-induced pathological damage, the effect could be due to interference with a number of respiratory processes viz. (a) a decrease in ventilation, (b) impeded gas exchange at respiratory surfaces, (c) disrupted perfusion, (d) impaired respiratory gas transport to/from the tissues, or (e) direct inhibition of cellular respiration. Of course, none of these are mutually exclusive and the effect may depend on the identity, as well as the ionic species/complex, of the metal.
TISSUE RESPIRATION
Little information is available on the effects of HM on tissue respiration. Divalent Cu reduces the MO2 of isolated gill tissues from crabs e.g. Carcinus maenas and molluscs e.g. Myths edulis at high concentrations ( > 120 mg 1-l) very quickly (Kerkut and Munday, 1962; Brown and Newell, 1972). At lower, although still considerably elevated, levels (0.1-2.5 mg l-l), however, there was no effect, even in animals that had been acclimated to the medium for many days (Thurberg et al., 1973; Spicer and Taylor, unpublished observation). Brown and Newell (1972) concluded that the decrease in MO2 they observed for Myth edulis was due principally to the action of Cu on gill cilia rather than the inhibition of cellular respiration. Even exposure to high concentrations of Zn ( > 500 mg 1-l) had no effect on 339
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isolated gill tissue from M. edulis (Brown and Newell, 1972). Cd, on the other hand, has been shown to depress MO2 of isolated gill tissue from the crabs Carcinus maenas (Cd > 0.5 mg 1-l) and Cancer irroratus (Cd > 0.12mg 1-l) (Thurberg et al., 1973). Some indirect evidence on Hg toxicity suggests that this ion, like Cd may depress tissue MO, at moderately high concentrations. It appears, at least for crustaceans, that a decrease in organismic MO, is heralded by exposure to Cu and Zn concentrations orders of magnitude lower than those affecting the tissues. This may not necessarily be so for Cd and Hg. VENTILATION
AND PERFUSION
Although exposure to HM has been shown to markedly affect both ventilation and perfusion in fish (Skidmore, 1970; Sellers et al., 1975; Hughes and Adeney, 1977), with early suggestions that ventilation rate could be used as an indicator of pollution (Belding, 1929) this is not so well established for shellfish species such as Mytilus edulis and Cancer pagurus, particularly at low (sublethal) concentrations (Scott and Major, 1972; Manley, 1983; Spicer and Weber, 1991). No attempt has previously been made to differentiate between pathological effects and “normal” compensatory responses in these parameters, making an assessment of literature difficult, although a (sublethal) HM-induced change in cardiac output may occur in the crab Cancer pagurus during hypoxia (Spicer and Weber, 1991). At more lethal concentrations, however, a definite decrease in heart rate occurs in both crustaceans (Kerkut and Munday, 1962: Depledge, 1984) and molluscs (Cheng and Sullivan, 1973b). Depledge (1984) has suggested that the neurotoxicity of Cu and Hg may impair hormonal/neuronal control of ventilation perfusion. Although there is little evidence for this effect in vivo the suggestion merits study, particularly with regard to chronic exposure.
EFFECI- ON STRUCTURE AND FUNCTION RESPIRATORY SURFACES
OF
The respiratory surfaces are most likely the first target of water-borne HM. Early investigators of pollution effects in fish attributed the death of animals exposed to lethal concentrations of HM to the coagulation or precipitation of mucous on the gills which increased the diffusion distances for oxygen and resulted in death by suffocation. In following years, however, cytological damage, rather than mucous accumulation, was cited as the reason for the increased diffusion distance between water and the blood (Evans, 1987; Mallat, 1985). This cytological damage takes the form of separation of intact epithelial cells and cellular necrosis. Acute exposure of crustaceans and molluscs to HM results in profound changes in gill and other respiratory surface ultrastructure and the production of “slime” on the outer surface (Chang and Sullivan, 1975; Bubel, 1976; Couch, 1977; Ghate and Mulberkar, 1979; Roesjadi, 1980; Anderson and Baatrup, 1988; Boitel, 1990; Spicer and Taylor, unpublished observations). Fur-
R. E. WEBER thermore, HM can be accumulated on the gill surface of crustaceans although it is lost at ecdysis. In the crab and shrimp species examined, cytological damage takes the form of a thickening of the branchial epithelium and profound changes in haemolymph flow pattern in the gill concomitant with increased vaculization and reduced haemolymph spaces causing perfusion stagnation. It is interesting, however, that after exposure to sub-lethal concentrations of Cu (and Zn), this condition was reversible (or at least appears to be in the case of the former) after 18 days in Carcinus maenas (Boitel, 1990) and 30days in Cancer pagurus (Spicer and Weber, 1991) even in the continued presence of the contaminant. A similar pattern of recovery has also been demonstrated for the branchial crown of the sabellid polychaete, Eudistylia vancouveri (Young and Roesijadi, 1983) which coincides with metallothionein production in the gill tissue. On exposure to lethal levels, however, the damage to crustacean respiratory tissue is more extensive and irreversible.
0, TRANSPORT
In fish, damage to the gills is accompanied by an internal hypoxia (e.g. Skidmore, 1970). It has been demonstrated that in the crab Carcinus maenas gill damage, even at sub-lethal concentrations, can be correlated with a decrease in arterial O2 partial pressure (Boitel, 1990). More recently, it has been shown in Cancer pagurus that despite the absence of overt physiological dysfunction, characteristic of physiological studies employing supra-lethal concentrations of HM (e.g. Depledge, 1984), there was good evidence for a degree of respiratory impairment which was manifest only during acute hypoxic exposure (Spicer and Weber, 1991). The impairment described by us for Cancer pagurus after 7 days pre-exposure to Zn and Cu salts (0.4 mg. l-l), was due solely to an increase in the diffusion barrier thickness at the respiratory surfaces and was reversible even during continued exposure to HM. Oxygen is transported for the gills to the tissues by the haemolymph. The haemolymph of molluscs and crustaceans can obtain haemoglobin (Hb) in the case of the former and Hb or haemocyanin (Hc) in the case of the latter. The effect of various HM salts on the O2 binding and transporting properties of these respiratory pigments has attracted some attention, albeit often working under non-physiological conditions. To date, only Hg looks likely to disrupt the respiratory function of both Hb (Weber, 1990) and Hc (Kuiper et al., 1981), although more precise ecotoxicological studies are required to test this. Other HM such as Cu, Zn and Cd either have a small, or no effect on Hc 0, affinity or cooperativity (Brouwer et al., 1982; Bellelli et al., 1985; Boitel, 1990; Weber, unpublished observations). Most work would suggest that nearly all of the HM ions are bound in the haemolymph (by the Hc) reducing the potential effector action of these HM on the respiratory function of the pigment. Depledge (personal communication) has suggested that HM may affect more the synthesis of respiratory pigments rather than their function.
Respiratory impairment in crustaceans and molluscs ECOTOXICOLOGY As the objective of pollution studies should be to ensure the well being and reproductive potential of marine organisms, the detection of early departure from the healthy state is essential (Depledge, 1989). Adopting this approach, the cause of death may be unimportant while the mechanism by which normal physiological processes are disrupted is crucial. Many of the studies cited above have employed levels of HM that will elicit clear-cut physiological responses and, as a result are ecologically meaningless: the physiological state of a person with a predilection for almonds and after consuming a bottle of arsenic are fortunately not the same. Furthermore, the susceptibility of respiratory processes to HM is modified both by intrinsic, e.g. life stage (DeCourcey and Vernberg, 1972) sex (Vernberg and Vernberg, 1972) and extrinsic factors, e.g. hypoxia and salinity stress (Depledge, 1987). There appears to be little evidence so far for synergistic effects of metals on MO* (e.g. no synergism observed exposing Artemiu to Cu and Cr (Verriopoulos et al., 1988) but this may just reflect the paucity of the data. In general, there is a dearth of good comparative data on the effects of (environmentally realistic) HM exposure on respiration in crustaceans and molluscs, particularly with regard to synergistic effects. Very little effort has been made to differentiate between compensatory and pathological responses at sublethal concentrations. Consequently, we must he cautious in our interpretation of the HM “effects” noted above as this may be all that they are, effects. For example Waiwood and Beamish (1978) working on fish found that, although 5 days acclimation to 40 pg l- ’ Cu resulted in an increase in MO*, during activity there was a reduction in maximum sustainable MO, noted which implied that there was an inhibition in the O2 transfer system; not a conclusion one would automatically draw form the initial observation. Furthermore, the recent suggestion that the differential response of a large number of individuals of the same species to HM exposure may be important in our understanding of pollution-related disturbance should stimulate an examination of intraspecific variation in physiological responses to HM exposure (Depledge, 1990): up until now such variation has been regarded almost as a mere nuisance in pollution studies. On the basis of the studies presented above it is suggested that the essential metals Cu and Zn (when water-borne) act pathologically on the respiratory system primarily by disruption of gill function resulting in internal hypoxia, although reparation can be accomplished even at “high” sub-lethal concentrations. At these high sublethal concentrations cardiac output may also be affected. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) interfere with the respiratory system at every level of organisation including cellular respiration itself. The differences in the responses to these two groups of metals remains to be examined fully although the answer is likely to lie in the different degrees of lipid solubility of these metal ions/ complexes (Comer and Rigler, 1958). Furthermore,
341
in nature the scope for synergistic interactions is large and should, together with the history of any wntamination and cognizance of the physiological condition of the individuals constituting the affected population/community, be taken into account before attempting to specify the amount of water-borne HM contamination that is required to effect respiratory impairment in situ. Acknowledgements-This
study was supported by the Danish Natural Science Research Council, the Danish Environ-
mental Ministry and the Commission of the European Communities’ 4th Research and Development Programme (EV4V-0121-DK).
REFERENCES Anderson J. T. and Baatrup E. (1988) Ultrastructure localisation of mercury accumulations in the gills, hepatopancress, midgut and antenna1 glands of the brown shrimp, Crangon crangon. Aquat. Toxic. 13, 309-324.
Baghdiguian S. and Riva A. (1985) Metabolic modifications brought by synergistic action of cadmium and experimental starvation of clams Ruditapes decussatus. Mar. environ. Res. 17, 288.
Belding G. (1929) The respiratory movements of fish as an indicator of a toxic enviroment. Trans. Am. Fish. Sot. 59. 238-245.
Bellelli A., Zolla L., Giardina B., Costatini S., Cau A. and Brunori M. (1985) Hemocvanin form Palinurus eleohas: general propkrites’ and effects of heavy metals. Eioihim. biophys. Acta 830, 325-331.
Boitel F. (1990) Action des mbtaux lourds chez les animaux aquatiques: etude des effects du cuivre sur la respiration et les &tats ionique et acide-base de l’hemolymphe chez le crabe vert Carcinlcr maenas. Ph.D. thesis, University of Bordeaux (No. 459), 87 pp. Brouwer M., Bonaventura C. and Bonaventura J. (1982) Heavy metal ion interactions with Callinectes sapidus hemocyanin: structural and functional changes induced by a variety of heavy metal ions. Biochemistry 21, 2529-2538.
Brown B. E. and Newell R. C. (1972) The effect of copper and zinc on the metabolism of the mussel Mytitus edulis. Mar. Biol. 16, 108-118. Bubel A. (1976) Histological and electron microscopical observations on the effects of different salinities and heavy metal ions, on the gills of Jaera nora?nanni (Rathke) (Crustacea: Isopoda). Cell. Tissue Res. 167, 65-95. Cheng T. C. and J. T. Sullivan (1973a) A comparative study of the effects of two copper compounds on the respiration and survival of Biomphalaria glabrata. Gen. camp. Pharmat. 4, 315-320.
Cheng T. C. and J. T. Sullivan (1973b) The effect of copper on the heart rate of Biomphalaria glabrata. Gen. camp. Pharmac. 4, 3741.
Cheng T. C. and J. T. Sullivan (1975) Mode of entry, action and toxicity of copper molluscicides. In, Moiiuscitides in Schistosomiasts Control (Edited by Cheng T. C.) Academic Press, 266 pp. New York. Corner E. D. S. and Rigler F. H. (1958) The modes of action of toxic agents. III Mercuric chloride and N-amylmercurie chloride on crustaceans. J. mar. biol. Ass. U.K. 37, 85-96.
Corner E. D. S. and Sparrow B. W. (1956) The mode of action of toxic agents 1. Observations on the poisoning of certain crustaceans by copper and mercury. J. mar. biol. Ass. U.K. 35, 531-548.
Couch J. A. (1977) Ultrastructure study of lesions in gills of a marine shrimp exposed to cadmium. J. invertebr. Pathol. 29, 267-288.
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DeCourcey P. J. and Vemberg W. B. (1972) Effects of mercury on survival, metabolism and behaviour of larval Uca pugilator (Brachyura) Oikos 23, 241-247. Depledge M. H. (1984) Disruption of circulatory and respiratory activity in shore crab (Carcinus maenas 1.) exposed to heavy metal pollution. Comp. Biochem. Physiol. 78C, 445459.
McInnes J. R. and Thurberg F. P. (1973) Effects of metals on the behaviour and oxygen consumption of the mud snail. Mar. Pollut. Bull. 4,~185-187. _ Panathanassiou E. (1983) Effects of cadmium and mercury ions on respiration and survival of the common prawn Palaemon serratus (Pennant). Rev. Int. Oceanogr. Med. 70, 21-35.
Depledge M. H. (1987) Enhanced copper toxicity resulting from environmental stress factor synergies. Comp. Biothem. Physiol. UC, 15-19. Depledge M. H. (1989) The rational basis for detection of the early effects of marine pollutants using physiological indicators. Ambio 18, 301-302. Depledge M. H. (1990) The use of intra-specific physiological variability to investigate the effects of ecological disturbance on natural populations of marine invertebrates. Ambio (In press): _ Denledae _ _ M. H. and Phillins D. J. H. (1987) \ , Arsenic uncouples cardiac and respiratory responses of Hemifusus tuba (Gmelin) to thermal stress. Asian mar. Bioi. 4, 91-96. Duncan J. (1975) A review of the development and application of mollucicides in schistosomiasis control. In Molluscicides in Schistosomiasis Control (Edited by Cheng T. C.) Academic Press, 266 pp. New York. Evans D. H. (1987) The fish gill: site of action and model for toxic effects of environmental pollutants. Environ. Health Perspect. 71, 47-58.
Ghate H. V. and Mulberkar L. (1979) Histological changes in the gills of two freshwater prawn species exposed to copper sulphate. Ind. J. exp. Bioi. 17, 838-840. Hughes G. M. and Adeney R. J. (1977) The effects of zinc on the cardiac and ventilatory rhythms of rainbow trout (Saimo gairdneri Richardson) and their responses to environmental hypoxia. Water Res. 11, 1069-1077. Jones J. R. E. (1942) The effects of ionic copper on the oxygen consumption of Gammarus pulex and Polycelis _ n&a. J. exp. Biol. 18, 153-161. Kerkut G. A and Mundav K. A. (1962) . , The effect of couner on the tissue respiration of the crab Carcinus maenas. 11
Cah. Bioi. Mar. 3, 27-35.
Kuiper H. A., Zolla L., Calabrese L., Vecchini P., Costantini S. and Brunori M. (1981) Effects of mercuric chloride on the structural and functional properties of Panulirus interruptus hemocyanin. Comp. Biochem. Physiol. 69C, 253-257.
Mallatt J. (1985) Fish gill structural changes induced by toxicants and other irritants: statistical review. Can. J. Fish. Aquat. Sci. 42, 630-648.
Manley A. R. (1983) The effect of copper on the behaviour, respiration, filtration and ventilatory activity of Mytilus edulis. J. mar. biol. Ass U.K. 63, 205-222.
WEBER
Preston A. (1973) Heavy metals in British waters. Nature, Lond. 242, 95-97.
Roesijadi G. (1980) Influence of copper on the clam Protothaca staminea: effects on gills and occurence of copperbinding proteins. Bioi. Bull. 158, 233-247. Saliba L. J. and Vella M. G. (1977) Effects of mercury on the behaviour and oxygen consumption of Monodonata articula. Mar. Biol. 43, 277-282.
Scott D. M. and Major C. W. (1972) The effect of copper (II) on survival, respiration and heart rate in the common blue mussel Myth& edulis. Bioi. Bull. 143, 679-688. Sellers C. M.. Heath A. G. and Bass M. L. (1975) The effect of sublethal concentrations of copper and zinc. on ventilatory activity, blood OXand pH in rainbow trout (Salmo gairdneri).
Water Res. 9, 401408.
Skidmore J. F. (1970) Respiration and osmoregulation in rainbow trout with gills damaged by zinc sulphate. J. exp. Biol. 52, 484494.
Spicer J. I. and Weber R. E. (199 1) Respiratory impairment by water-borne copper and zinc in the edible crab Cancer pagurus during hypoxic exposure. Mar. Biol. (In press). Thurberg F. P., Dawson M. A. and Collier R. S. (1973) Effects of copper and cadmium on osmoregulation and oxygen consumption in two species of estuarine crab. Mar. Biol. 23, 171-175. Vernberg W. B. and Vemberg J. (1971) The synergistic effects of temperature, salinity and mercury on survival and metabolism of the adult fiddler crab. Uca pugilator. Fishery Bull. 70, 415420.
Verriopoulos G., Milliou E. and Moraitou-Apostolopaulou M. (1988) Joint effects of four pollutants (copper, chromium, oil, oil dispersant) on respiration of Artemia. Arch. Hydrobiol. 112, 475-480.
Waiwood K. G. and Beamish F. W. H. (1978) Effects of copper, pH and hardness on the critical swimming performance of rainbow trout (Salmo gairdneri Richardson). Water Res. 12, 611-619. Weber R. E. (1991) Effects of mercury on the functional properties of haemoglobins from the bivalve mollusc Scapharca 39-48.
inaequivalvis. J. exp. mar. Biol. Ecol. 144,
Young J. S and Roesijadi G. (1983) Reparatory adaptation to copper induced injury and occurrence of copper binding protein in the polychaete, Eudistyiia vancouveri. Mar. Pollut. Bull. 14, 30-32.
Printedin Great
Physiol. Britain
Vol. lOOC,No. 3, pp. 339-342,
1991 0
0306-4492/91 $3.00 + 0.00 199 1 PergamonPressplc
MINI-REVIEW RESPIRATORY IMPAIRMENT IN CRUSTACEANS AND MOLLUSCS DUE TO EXPOSURE TO HEAVY METALS JOHNI. SPICRRand ROY E. WEBER Department of Zoophysiology, University of Aarhus, Aarhus, Denmark, DK 8000 (Received 2 January 1991) Abstract-l. We have assessed, using current literature, the respiratory consequences of water-borne heavy metal exposure in crustaceans and molluscs. 2. We suggest that in lethal and sub-lethal concentrations the essential metals Cu and Zn act on the respiratory system primarily by disrupting gill function which results in the development of internal hypoxia, although reparation can be accomplished even at “high” sub-lethal concentrations. 3. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) may interfere with the respiratory system at every level of organisation including cellular respiration itself.
INTRODUCTION Heavy metals (HM) are normal constituents of aquatic environments. All HM are toxic at some concentration but this is normally of little biological importance. In coastal and fresh waters, however, the concentrations of HM may be orders of magnitude greater than found in open waters as a result of anthropogenic input (Preston, 1973). Some of the more important HM, due to their toxicity at “low” concentrations, are the essential metals copper (Cu) and zinc (Zn) and the xenobiotes mercury (Hg) and cadmium (Cd). In this paper we will examine the respiratory consequences (pathological and compensatory) of water-borne HM exposure for crustaceans and mollusts, animals which are not only important as food for man and other animals, but also as vectors of disease in fresh water habitats. HM exposure, therefore, may either occur in the form of “pollution” or in the deployment of molluscicides which often rely on the toxic action of HM (particularly Cu and even Zn and Cd; Duncan, 1975) for their effectiveness. We will briefly review the pathological effects of HM exposure on the respiratory system at various levels of organisation before assessing the ecotoxicogical significance of this data. OXYGEN
CONSUMPTION
In general, oxygen consumption (MO,) decreased when molluscs and crustaceans were acutely exposed to HM (Jones, 1942; Corner and Sparrow, 1956; Brown and Newell, 1972; DeCourcey and Vernberg, 1972; Vernberg and Vernberg, 1972; Cheng and Sullivan, 1973a, 1975; McInnes and Thurberg, 1973; Address for correspondence: Dr J. I. Spicer, Department of Animal and Plant Sciences, School of Bi6logical Sciences, University of Sheffield, P.O. Box 601, Sheffield SlO ZUQ, U.K.
Saliba and Vella, 1977; Depledge, 1984; Papathanassiou, 1983) although in some cases either no change (Scott and Major, 1972; Depledge and Phillips, 1987; Spicer and Weber, 1991) or even an increase (Baghdiguian and Riva, 1985) was observed. A priori any compensatory change, immediate or long term, will require 0,. Therefore if some degree of physiological compensation is possible (or at least attempted), there may be an initial enhancement of MO,. Given that a decrease in MOz is indicative of HM-induced pathological damage, the effect could be due to interference with a number of respiratory processes viz. (a) a decrease in ventilation, (b) impeded gas exchange at respiratory surfaces, (c) disrupted perfusion, (d) impaired respiratory gas transport to/from the tissues, or (e) direct inhibition of cellular respiration. Of course, none of these are mutually exclusive and the effect may depend on the identity, as well as the ionic species/complex, of the metal.
TISSUE RESPIRATION
Little information is available on the effects of HM on tissue respiration. Divalent Cu reduces the MO2 of isolated gill tissues from crabs e.g. Carcinus maenas and molluscs e.g. Myths edulis at high concentrations ( > 120 mg 1-l) very quickly (Kerkut and Munday, 1962; Brown and Newell, 1972). At lower, although still considerably elevated, levels (0.1-2.5 mg l-l), however, there was no effect, even in animals that had been acclimated to the medium for many days (Thurberg et al., 1973; Spicer and Taylor, unpublished observation). Brown and Newell (1972) concluded that the decrease in MO2 they observed for Myth edulis was due principally to the action of Cu on gill cilia rather than the inhibition of cellular respiration. Even exposure to high concentrations of Zn ( > 500 mg 1-l) had no effect on 339
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isolated gill tissue from M. edulis (Brown and Newell, 1972). Cd, on the other hand, has been shown to depress MO2 of isolated gill tissue from the crabs Carcinus maenas (Cd > 0.5 mg 1-l) and Cancer irroratus (Cd > 0.12mg 1-l) (Thurberg et al., 1973). Some indirect evidence on Hg toxicity suggests that this ion, like Cd may depress tissue MO, at moderately high concentrations. It appears, at least for crustaceans, that a decrease in organismic MO, is heralded by exposure to Cu and Zn concentrations orders of magnitude lower than those affecting the tissues. This may not necessarily be so for Cd and Hg. VENTILATION
AND PERFUSION
Although exposure to HM has been shown to markedly affect both ventilation and perfusion in fish (Skidmore, 1970; Sellers et al., 1975; Hughes and Adeney, 1977), with early suggestions that ventilation rate could be used as an indicator of pollution (Belding, 1929) this is not so well established for shellfish species such as Mytilus edulis and Cancer pagurus, particularly at low (sublethal) concentrations (Scott and Major, 1972; Manley, 1983; Spicer and Weber, 1991). No attempt has previously been made to differentiate between pathological effects and “normal” compensatory responses in these parameters, making an assessment of literature difficult, although a (sublethal) HM-induced change in cardiac output may occur in the crab Cancer pagurus during hypoxia (Spicer and Weber, 1991). At more lethal concentrations, however, a definite decrease in heart rate occurs in both crustaceans (Kerkut and Munday, 1962: Depledge, 1984) and molluscs (Cheng and Sullivan, 1973b). Depledge (1984) has suggested that the neurotoxicity of Cu and Hg may impair hormonal/neuronal control of ventilation perfusion. Although there is little evidence for this effect in vivo the suggestion merits study, particularly with regard to chronic exposure.
EFFECI- ON STRUCTURE AND FUNCTION RESPIRATORY SURFACES
OF
The respiratory surfaces are most likely the first target of water-borne HM. Early investigators of pollution effects in fish attributed the death of animals exposed to lethal concentrations of HM to the coagulation or precipitation of mucous on the gills which increased the diffusion distances for oxygen and resulted in death by suffocation. In following years, however, cytological damage, rather than mucous accumulation, was cited as the reason for the increased diffusion distance between water and the blood (Evans, 1987; Mallat, 1985). This cytological damage takes the form of separation of intact epithelial cells and cellular necrosis. Acute exposure of crustaceans and molluscs to HM results in profound changes in gill and other respiratory surface ultrastructure and the production of “slime” on the outer surface (Chang and Sullivan, 1975; Bubel, 1976; Couch, 1977; Ghate and Mulberkar, 1979; Roesjadi, 1980; Anderson and Baatrup, 1988; Boitel, 1990; Spicer and Taylor, unpublished observations). Fur-
R. E. WEBER thermore, HM can be accumulated on the gill surface of crustaceans although it is lost at ecdysis. In the crab and shrimp species examined, cytological damage takes the form of a thickening of the branchial epithelium and profound changes in haemolymph flow pattern in the gill concomitant with increased vaculization and reduced haemolymph spaces causing perfusion stagnation. It is interesting, however, that after exposure to sub-lethal concentrations of Cu (and Zn), this condition was reversible (or at least appears to be in the case of the former) after 18 days in Carcinus maenas (Boitel, 1990) and 30days in Cancer pagurus (Spicer and Weber, 1991) even in the continued presence of the contaminant. A similar pattern of recovery has also been demonstrated for the branchial crown of the sabellid polychaete, Eudistylia vancouveri (Young and Roesijadi, 1983) which coincides with metallothionein production in the gill tissue. On exposure to lethal levels, however, the damage to crustacean respiratory tissue is more extensive and irreversible.
0, TRANSPORT
In fish, damage to the gills is accompanied by an internal hypoxia (e.g. Skidmore, 1970). It has been demonstrated that in the crab Carcinus maenas gill damage, even at sub-lethal concentrations, can be correlated with a decrease in arterial O2 partial pressure (Boitel, 1990). More recently, it has been shown in Cancer pagurus that despite the absence of overt physiological dysfunction, characteristic of physiological studies employing supra-lethal concentrations of HM (e.g. Depledge, 1984), there was good evidence for a degree of respiratory impairment which was manifest only during acute hypoxic exposure (Spicer and Weber, 1991). The impairment described by us for Cancer pagurus after 7 days pre-exposure to Zn and Cu salts (0.4 mg. l-l), was due solely to an increase in the diffusion barrier thickness at the respiratory surfaces and was reversible even during continued exposure to HM. Oxygen is transported for the gills to the tissues by the haemolymph. The haemolymph of molluscs and crustaceans can obtain haemoglobin (Hb) in the case of the former and Hb or haemocyanin (Hc) in the case of the latter. The effect of various HM salts on the O2 binding and transporting properties of these respiratory pigments has attracted some attention, albeit often working under non-physiological conditions. To date, only Hg looks likely to disrupt the respiratory function of both Hb (Weber, 1990) and Hc (Kuiper et al., 1981), although more precise ecotoxicological studies are required to test this. Other HM such as Cu, Zn and Cd either have a small, or no effect on Hc 0, affinity or cooperativity (Brouwer et al., 1982; Bellelli et al., 1985; Boitel, 1990; Weber, unpublished observations). Most work would suggest that nearly all of the HM ions are bound in the haemolymph (by the Hc) reducing the potential effector action of these HM on the respiratory function of the pigment. Depledge (personal communication) has suggested that HM may affect more the synthesis of respiratory pigments rather than their function.
Respiratory impairment in crustaceans and molluscs ECOTOXICOLOGY As the objective of pollution studies should be to ensure the well being and reproductive potential of marine organisms, the detection of early departure from the healthy state is essential (Depledge, 1989). Adopting this approach, the cause of death may be unimportant while the mechanism by which normal physiological processes are disrupted is crucial. Many of the studies cited above have employed levels of HM that will elicit clear-cut physiological responses and, as a result are ecologically meaningless: the physiological state of a person with a predilection for almonds and after consuming a bottle of arsenic are fortunately not the same. Furthermore, the susceptibility of respiratory processes to HM is modified both by intrinsic, e.g. life stage (DeCourcey and Vernberg, 1972) sex (Vernberg and Vernberg, 1972) and extrinsic factors, e.g. hypoxia and salinity stress (Depledge, 1987). There appears to be little evidence so far for synergistic effects of metals on MO* (e.g. no synergism observed exposing Artemiu to Cu and Cr (Verriopoulos et al., 1988) but this may just reflect the paucity of the data. In general, there is a dearth of good comparative data on the effects of (environmentally realistic) HM exposure on respiration in crustaceans and molluscs, particularly with regard to synergistic effects. Very little effort has been made to differentiate between compensatory and pathological responses at sublethal concentrations. Consequently, we must he cautious in our interpretation of the HM “effects” noted above as this may be all that they are, effects. For example Waiwood and Beamish (1978) working on fish found that, although 5 days acclimation to 40 pg l- ’ Cu resulted in an increase in MO*, during activity there was a reduction in maximum sustainable MO, noted which implied that there was an inhibition in the O2 transfer system; not a conclusion one would automatically draw form the initial observation. Furthermore, the recent suggestion that the differential response of a large number of individuals of the same species to HM exposure may be important in our understanding of pollution-related disturbance should stimulate an examination of intraspecific variation in physiological responses to HM exposure (Depledge, 1990): up until now such variation has been regarded almost as a mere nuisance in pollution studies. On the basis of the studies presented above it is suggested that the essential metals Cu and Zn (when water-borne) act pathologically on the respiratory system primarily by disruption of gill function resulting in internal hypoxia, although reparation can be accomplished even at “high” sub-lethal concentrations. At these high sublethal concentrations cardiac output may also be affected. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) interfere with the respiratory system at every level of organisation including cellular respiration itself. The differences in the responses to these two groups of metals remains to be examined fully although the answer is likely to lie in the different degrees of lipid solubility of these metal ions/ complexes (Comer and Rigler, 1958). Furthermore,
341
in nature the scope for synergistic interactions is large and should, together with the history of any wntamination and cognizance of the physiological condition of the individuals constituting the affected population/community, be taken into account before attempting to specify the amount of water-borne HM contamination that is required to effect respiratory impairment in situ. Acknowledgements-This
study was supported by the Danish Natural Science Research Council, the Danish Environ-
mental Ministry and the Commission of the European Communities’ 4th Research and Development Programme (EV4V-0121-DK).
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