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Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs
Comparative Biochemistry and Physiology Part C 123 (1999) 193 – 199 www.elsevier.com/locate/cbpc
Review
Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs David Sheehan *, Aisling Power 1 Department of Biochemistry, Uni6ersity College Cork, Lee Maltings Complex, Mardyke, Cork, Ireland Received 27 December 1998; received in revised form 15 April 1999; accepted 25 April 1999
Abstract Levels of chemical pollutants in the environment often display wide seasonal variation in response to climatic and other factors. Use of bioindicators such as enzyme activities in biomonitoring studies is complicated by this variation. Many such enzyme activities themselves show considerable seasonal fluctuation and there is known to be seasonality also in natural exposure to oxidative stress. This review attempts to explore some consequences of seasonal variation for biomonitoring studies with bivalve molluscs. It is suggested that independence of seasonal variation should be seen as a desirable feature of a bioindicator molecule. Where such molecules show seasonal variation, however, this should be incorporated into interpretation of biomonitoring studies by the use of appropriate controls. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Xenobiotic; Antioxidant; Defence mechanisms; Detoxification; Seasonality; Biomonitor; Bivalve molluscs
1. Introduction Bivalve molluscs such as Mytilus edulis are commonly used as bioindicators in the assessment of environmental quality. They are filter-feeding organisms which, consequently, may be exposed to large amounts of chemical pollutants even if these are present in fairly dilute concentrations. They are also capable of bioconcentrating xenobiotics to many thousands times background which can facilitate chemical analysis. Mussels have a number of properties which make them useful sentinels for chemical pollution; they have a wide geographical distribution, are stationary, euryhaline and are normally the dominant species in their habitat. In addition they are capable of withstanding baseline levels of pollution and are abundant in estuaries where much human contact with the aquatic environment occurs [53]. * Corresponding author. Tel.: +353-21-904207; fax: + 353-21274034. E-mail address: [email protected] (D. Sheehan) 1 Present address; Neurogenetics Unit, Imperial College School of Medicine at St. Mary’s, Norfolk Place, London W21PG, England.
In recent years, there has been considerable interest in the use of biochemical indices within bivalve molluscs which might provide greater sensitivity and/or more information on how these animals interact with environmental pollutants [39]. Of particular interest in this regard are the levels and activities of enzymes involved in detoxification of xenobiotics such as the Cytochrome P-450 system and glutathione S-transferases (GSTs), binding proteins such as metallothioneins and GSTs and stress-related proteins such as the heat shock proteins. Laboratory and field studies have suggested that measurements of the levels of these proteins may be indicative of exposure to chemical pollution. Another important set of investigations have focused on bioindicators of oxidative stress within the organisms. Such studies suggest that oxidative stress may render mussels more sensitive to toxicity from xenobiotics. An important aspect of mussel physiology which has also come in for attention is seasonality. Depending on availability of nutrients, reproductive status, growth-dilution with season and other factors, levels of important bioindicators of pollution and of oxidative stress
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Fig. 1. The Ecotoxicology Triangle. There is a complex relationship between chemical pollutants, their environment and living organisms in that environment. Several aspects of this relationship are likely to vary in a seasonal manner (adapted from Ref. [53]).
may fluctuate significantly throughout the year. This poses an important limitation on the interpretation of bioindicator data per se as increased levels of a particular bioindicator might merely be a normal part of the molluscan species annual physiological cycle and be quite unrelated to exposure to chemical pollution. The relevance of seasonal variation to environmental monitoring studies of vertebrates such as fish [29] and birds [14,55] has been extensively studied. The present review attempts to explore the relationship between seasonality, pollution bioindicators and oxidative stress in bivalves and suggests that independence from seasonal variation is a desirable feature for biochemical bioindicators of xenobiotic pollution.
2. Seasonality of environmental pollution The ‘Ecotoxicology Triangle’ (Fig. 1) summarises a complex interrelationship between levels and type of chemical pollutant, living organisms within the environment and the environment itself [53]. One of the important interactions in this triangle is that between the pollutant and the environment. In studies of respiratory and cardiovascular diseases in man it has frequently been observed that temporal variations in air pollution correlate closely with the incidence of such conditions [7,19]. Levels of specific pollutants found in environ-
mental samples and in tissues of bioindicator species, such as M. edulis, may be expected to vary in a seasonal manner. This could result from factors such as temperature changes, cycles in industrial activity and annual alterations in climate. In addition, since marine organisms themselves are highly seasonal in terms of their basic physiology and detoxification biochemistry (see below), the levels of xenobiotics detected in tissue samples may be found to follow a variable seasonal pattern [21]. Some recent reports of seasonal variations in environmental pollutant levels are summarised in Table 1. Wide temporal variation has been observed in profiles of atmospheric polychlorinated biphenyls (PCBs) in Southern Chesapeake Bay [13]. Somewhat smaller variations in levels in precipitation samples were observed although these also followed a pattern of seasonal variation which was suggested as being due to the effect of temperature on washout efficiency. In a seasonal study of the German Bight, PCB congeners were measured in M. edulis. This study found generally lower levels of coplanar (i.e. ‘dioxin-like’) and atropisomeric congeners in autumn than during spring [24]. It was tentatively concluded that these results were due to seasonal variation of lipid content, although such variations are thought to be modest. Interestingly, the congener PCB 126 was unusual in that it was elevated or only slightly decreased in autumn. Since this possesses such a large toxic equivalency factor relative to other congeners [50], this resulted in only a comparatively small difference in toxic equivalents between data sets in spring and autumn despite the clear trend towards a large decrease in autumn with most other congeners. These results suggest that seasonal variations of total PCBs may mask important inter-pollutant differences which may be very significant for estimation of variation in toxic threat. Polyaromatic hydrocarbons (PAHs) such as fluoranthene and pyrene have been reported to vary seasonally in the Humber coastal zone with highest concentrations in September and lowest in June [66]. Generally similar results were obtained in a study of the Chesapeake Bay where atmospheric levels of PAHs (in particular, less volatile PAHs) were determined [13].
Table 1 Some examples of seasonal variation in pollutant patterns Pollutant
Level
Factor responsible
Reference
PAH, PCB PCB PAH and dissolved pesticides Metals TBT, DBT Cd, Cu, Pb, Zn
Increased deposition Decreased lipid variation Decreased Increased Decreased Pb increased; others decreased
Atmospheric Seasonal Agricultural runoff Runoff and salinity; change during Monsoon Variation in water flow and sediment deposition Spawning, variation in body weight
[13] [24] [66] [42] [38] [56]
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Seasonal variation in metal pollutants has also attracted considerable investigation. Analysis of tissue samples from marine organisms has revealed significant seasonal variation [11,37,41]. Factors such as high river run-off in winter or during Monsoon conditions (depending on Geography), tidal variations and cycles of industrial activities featuring metals (e.g. mining) may be expected to underlie such variation in estuaries. Seasonally-varied metal levels have been described in the mollusc Sunetta scripta off the coast of India [42]. This was attributed to large increases in river run-off as a result of Monsoon conditions. While this may not be a concern in more economically-affluent regions of the World, it is clearly likely to be important in some of the most densely populated regions in Asia. It should also be noted that comparatively little is known about seasonal variation of metallothioneins [3]. Since these important metal binding proteins (see below) may vary in amount and specificity during a seasonal cycle, this could result in a seasonal variation in the toxicological threat posed by metals. It has been observed that tributyltin (TBT) levels of the water column varies seasonally [15]. TBT and dibutyltin (DBT) have been found to be higher in late spring/early summer and lower in winter in the Tamar Estuary in England [38]. By contrast, these compounds were found to be highest in winter and lowest in spring in both M. edulis (a ten-fold difference) and Nucella lapillus (five-fold difference) sampled near Rekjavik Harbour, Iceland [54] suggesting that different factors might underlie the TBT/DBT levels in these two sampling sites. In the former study, variation is suggested to be due to large (up to eight-fold) differences in the amounts of water flowing through the Tamar in summer and winter. This is thought to cause greater flushing and decreased pollutant transport from downstream via tidal movement of suspended sediment. A secondary explanation of these effects is variation in M. edulis dry weight due to natural fluctuations in carbohydrate and protein content [38]. The pattern observed near Rekjavik Harbour is thought to be due to decreased feeding in winter in response to temperature-dependent, low phytoplankton levels. In summer, the mussels are thought to depurate themselves, leading to lower levels of TBT/DBT [54]. Some seasonal variation has also been observed in levels of TBT/DBT in Mytilus gallopro6incialis in Genoa, Italy, with DBT being three-fold higher in July than in March [48]. Comparison of these studies shows how interrelated biological, climatic and geographical factors can be in interpreting seasonal effects in molluscs. While studies on metal levels in molluscs have concentrated on inshore organisms such as M. edulis [41], it has been found that some offshore organisms collected from the central North Sea had higher concentrations of some metals than those collected near the shore [4].
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In a study of Arctica islandica from Kiel Bay, copper, zinc and cadmium were found to be generally lower in winter compared to summer, while lead, conversely, was highest in winter [56]. These variations were attributed to seasonal changes in reproductive cycle and food availability leading to alterations in body weight and composition. Studies of major pollutants suggest, in summary, that geographical and climatic factors may have an effect on natural biological cycles and result in considerable variation in levels of major chemical pollutants such as PCBs, PAHs and metals. Interpretation of data from biomonitoring studies may be seriously complicated by these factors. It is important, therefore, to take note of such seasonal variation in the interpretation of biomonitoring data.
3. Biological seasonality in molluscs The biochemistry of bivalve molluscs is known to vary in response to food supply, reproductive and pollution status. Early work demonstrated an important seasonal variation in enzymes involved in glycolysis and anaerobic metabolism [1,30,31]. There is known to be a close functional relationship between the digestive system and gonad development and periods of food abundance and gonad development are often coincident [35]. Although there may be some variation with geographical location, in Western Europe the gonads remain resting up to October–November before undergoing ripening culminating in early spring. Partial spawning may occur then but this is followed by further gametogenesis until the gonads are again fully ripe by early summer [52]. Temperature variation with season is thought strongly to influence the relationship between the digestive system and gonad development [6] especially through effects on lysosomes [36,58]. The appearance of enlarged lysosomes has been considered a general stress response to temperature increase or pollutant exposure [8]. Important variables affecting lysosomes include the age of the mollusc [22] and identity/concentration of pollutant [8,27,57]. Reproductive activity and temperature-associated changes in patterns of food storage and utilisation are also likely to cause changes in hormonal and nutritional status which might also be expected to affect the levels of bioindicator molecules. Temperature changes associated with seasonality may have profound effects on bivalve biology including winter-survival of juveniles, recruitment into estuaries and availability/quality of food. In a study mimicking winter conditions in British waters, juvenile Crassostrea gigas showed limited survival at 3°C compared to 6 and 9°C, at which temperatures all animals survived [9]. A statistical study of the Wash on the East coast of
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Table 2 Some bioindicator molecules used in molluscan biomonitoring studies Bioindicator
Organism
Reference
Glutathione S-transferase
Sphaerium corneum M. edulis
[5] [18]
Glutathione Cytochrome P-450 Catalase Superoxide Dismutase Metallothioneins
M. gallopro6incialis M. edulis M. gallopro6incialis M. gallopro6incialis C. 6irginica
[59,60] [26] [60] [60] [49]
England has demonstrated that seasonal variations in temperature over the last century may partly explain differential recruitment of M. edulis and Cerastoderma edule [65]. This effect is probably exerted through currents which, in turn, affect plankton availability. A similar study covering the period 1973 – 1996 in the Dutch Wadden See on Macoma balthica demonstrated a temperature-dependent effect on recruitment which was not related to fecundity (i.e. number of adults and number of eggs) [23]. Temperature tolerance of bivalves is known to be strongly influenced by seasonality as well as by other factors such as geographical latitude [63] and position on the shore [62]. Some environments such as the deep-sea and polar regions are exposed to particularly wide variations of temperature, food-supply and light. This provides a challenge to bivalves which has been met by genetic adaptation [10,25]. In particular, the high levels of dissolved oxygen in polar environments has attracted attention to the potential of antioxidant defence systems in bivalves as bioindicators of pollutant exposure [45 – 47]. While seasonal variation is not as extreme in tropical regions, bivalves are nonetheless affected by variation in temperature and food supply [51]. The particular environmental sensitivity of systems such as coral reefs and mangrove swamps [40] have led to inclusion of bivalves in tropical biomonitoring programmes [2,20].
4. Bioindicator proteins and seasonality Some suggested bioindicator molecules are shown in Table 2. Considerable attention has focused on enzymes involved in the metabolism and detoxification of xenobiotics. By analogy with other species, these enzymes would be expected to be inducible in response to chemical pollutants. Of particular interest in this regard are the Phase I activities associated with the mixedfunction oxygenase (cytochrome P-450) system and the Phase II conjugation activity of the GSTs. The cytochrome P-450 system is mainly localised in digestive gland microsomes in molluscs and consists of
a number of related proteins and enzymatic activities. The levels of cytochrome P-450 activity found in M. edulis are generally lower than those of mammalian species. There is a seasonal variation in benzo(a)pyrene hydroxylase and NADPH-independent 7-ethoxycoumarin O-deethylase activities with these being high in autumn and declining during gametogenesis and spawning. Interestingly, although the level of cytochrome P-450 protein also decreases just prior to gametogenesis, the seasonal variation of this is somewhat different to the enzyme activities suggesting that there is a complex pattern of variation of individual components of the system [26]. It has been reported that cytochrome P-450 activities are not inducible in bivalve digestive gland, unlike the liver of verterbrate fish where they are readily induced by xenobiotics [33]. Phase II of detoxification involves a range of enzyme activities which conjugate xenobiotics to endogenous substrates. The conjugate thus formed is usually more water-soluble than the xenobiotic and this facilitates its exclusion from the cell. Although some Phase II activities are associated with the microsomes, the glutathione S-transferases (GSTs), the most abundant Phase II activity in mammalian species, are principally located in the cytosol. In M. edulis, a number of GSTs are present [16,17]. In common with mammalian GSTs, these proteins also are capable of non-catalytic binding of a wide range of xenobiotics [44]. GSTs are most abundant in the gill and digestive gland tissues of M. edulis [16]. Field studies suggest that cytochrome P-450 activity is a good bioindicator of environmental status [34]. Levels of GST activity also appear to be altered by exposure to xenobiotics [5] and to be elevated in some field studies [18], although not in others [34]. The discrepancies between these findings may be due to the different pollution load in each case. Interestingly, GST activity has not been found to vary in a seasonal manner [43]. Thus, it would seem that molluscan GSTs might, in this regard, be superior to cytochrome P-450 as an index of xenobiotic exposure. Metallothioneins are a class of cysteine-rich proteins which may bind and detoxify heavy metals. They have been identified in a wide range of aquatic species and are inducible by cadmium, copper, mercury, silver and zinc. There are some important differences between the characteristics of mammalian and marine invertebrate metallothioneins such as a higher level of cysteine in the former [61]. A model has recently been proposed suggesting that endogenous zinc plays an important role in regulation of metallothionein induction in Crassostrea 6irginica by interaction with a Zn-sensitive inhibitor [49]. A four-fold annual biological variation in metallothionein level of the Asiatic clam (Corbicula fluminea) was observed in an unpolluted site in France [3]. This variation, which peaked in May, was unrelated to metal
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exposure and seemed to be strongly correlated with the reproductive cycle of this bivalve.
processes leading to oxidative stress in molluscs. The best-known example of this is the Fenton reaction which can result in the production of ·OH.
5. Oxidative stress and seasonality
FeII + H2O2 FeIII + HO − + ·OH
Bivalves are able to survive in a wide range of oxygen concentrations ranging from anoxic to high levels of dissolved oxygen. Variations in this ability has been proposed as an index of environmental stress [12]. Oxyradicals (O2− , H2O2, ·OH) can be highly toxic to aquatic organisms often resulting in lipid peroxidation in membranes, altered pyridine nucleotide redox status and DNA damage [28]. Moreover, many xenobiotics are capable of modulating oxidative stress either by acting directly as redox cycling compounds (e.g. menadione) or as a consequence of biotransformation to quinones which are redox cycling (e.g. benzo(a)pyrene) [32]. The microsomes appear to be an important subcellular location for such activity since this is the site of cytochrome P-450 induction and catalysis. An attractive in vitro system for studying the production of oxyradicals in M. edulis lysosomes using the laser dye dihydrorhodamine 123 is available [64]. A range of antioxidant defence mechanisms are present in bivalve molluscs. These enzyme activities (catalase, superoxide dismutase, GSH peroxidase) and antioxidant compounds (e.g. vitamin E and GSH) are known to be under extensive seasonal control. In digestive gland, levels of these antioxidant activities and molecules appear to be at their lowest in winter (November–March), corresponding to increases in levels of lipid peroxidation products, a useful index of oxidative stress [60]. In gill, the major feeding organ of M. edulis, a generally similar pattern for these activities and molecules exists. However, in this tissue, GST levels (normally some three-fold higher in gill than digestive gland) are at their highest in November–December and decrease to a minimum in March –April [43]. By contrast, gill catalase levels are four-fold lower than those of digestive gland and show little seasonal variation. M. edulis GST1 is known to display a distinct GSH peroxidase activity [17] and it is possible that this may be especially important in gill tissue during the winter months when other antioxidant defences are at their lowest. Oxidative stress is a highly seasonal phenomenon in bivalve molluscs. The modulation of oxidative stress by environmental pollutants is a factor which could seriously complicate interpretation of biomonitoring data.
Many of the toxic effects observed in marine animals exposed to high levels of heavy metals involve membrane-related processes. A possible mechanism for such toxicity is by metal-mediated peroxidation of lipid components of membranes as a result of oxyradicals. It has been demonstrated that Cu may cause a significant increase in malondialdehyde (an indicator of the peroxidative process) and a decrease in GSH while Cd and Zn do not [59]. This increase is associated with an elevation of the levels of lysosomal lipofuscin granules. It is clear from this that a complex relationship may exist between metals, their homeostasis [61], oxidative stress and seasonality. Varying levels of metals could be expected to lead to an altered oxyradical threat to cells. Possible effects of this on bioindicator molecules would obviously need to be taken into account in biomonitoring studies.
7. Conclusions Both pollution inputs to the environment and some bioindicator molecules thought to be sensitive to such inputs may be subject to seasonal variation due to a range of factors such as natural climatic and biological variation. These factors may be independent of direct interaction between xenobiotic pollutants and biomarker organisms such as molluscs. Seasonal variation in both pollutant input and in biochemical processes involving specific bioindicator molecules therefore needs to be factored into biomonitoring studies. An ideal bioindicator would be expected to show no seasonal variation in response to factors such as food supply or reproductive status and would vary only in response to pollutant exposure. Of course, this will often not be the case in practice. In such situations, it is essential that adequate control samples be taken from unpolluted sites contemporaneously and that care be taken in the choice of such sites. Tissue samples from control and test animals should be treated identically throughout the processes of dissection, storage, extraction and analysis.
Acknowledgements 6. Heavy metals and oxidative stress Because of their ability to exist in a variety of oxidation states, metals are capable of becoming involved in
We would like to thank Professor Mike Depledge, University of Plymouth, for critically reading the manuscript.
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[53] Sheehan D, McIntosh J, Power A, Fitzpatrick PJ. Environmental biochemistry. Biochem Soc Trans 1995;23:419 – 22. [54] Skarphedinsdottir H, Olafsdottir K, Svavarsson J, Johannesson T. Seasonal fluctuations of tributyltin (TBT) and dibutyltin (DBT) in the dogwhelk, Nucella lapillus (L.), and the blue mussel, Mytilus edulis L., in Icelandic waters. Mar Pollut Bull 1996;32:358 – 61. [55] Stewart FM, Thompson DR, Furness RW, Harrison N. Seasonal variation in heavy metal levels in tissues of common guillemots, Uria aalge, from Northwest Scotland. Arch Contam Toxicol 1994;27:168 – 75. [56] Swaileh KM. Seasonal variations in the concentrations of Cu, Cd, Pb and Zn in Arctica islandica L. (Mollusca:Bivalvia) from Kiel Bay, Western Baltic Sea. Mar Pollut Bull 1996;32:631–5. [57] Tremblay R, Pellerin-Massicote J. Effect of the tidal cycle on lysosomal membrane stability in the digestive gland of Mya arenaria and Mytilus edulis L. Comp Biochem Physiol 1997;117A:99 – 104. [58] Tremblay R, Myrand B, Guderley H. Temporal variation of lysosomal capacities in relation to susceptibility of mussels, Mytilus edulis, to summer mortality. Mar Biol 1998;132:641–9. [59] Viarengo A, Canesi L, Pertica M, Poli G, Moore MN, Orunesu M. Heavy metal effects on lipid peroxidation in the tissues of Mytilus gallopro6incialis Lam. Comp Biochem Physiol 1990;97C:37 – 42. [60] Viarengo A, Canesi L, Pertica M, Livingstone DR. Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels. Comp Biochem Physiol 1991;100C:187– 90. [61] Viarengo A, Nott JA. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp Biochem Physiol 1993;104C:355– 72. [62] Wilson JG, Elkaim B. Tolerances to high temperature of infaunal bivalves and the effect of geographical distribution, position on the shore and season. J Mar Biol Assoc UK 1991;71:169–78. [63] Wilson JG, Elkaim B. Seasonal and geographical differences in oxygen consumption with temperature of Cerastoderma glaucum (Poiret) and a comparison with C. edule. Estuarine Coastal Shelf Sci 1997;45:571 – 7. [64] Winston GW, Moore MN, Kirchin MA, Soverchia C. Production of reactive oxygen species by hemocytes from the marine mussel, Mytilus edulis: lysosomal localization and effect of xenobiotics. Comp Biochem Physiol 1996;113C:221– 9. [65] Young EF, Bigg GR, Grant A. A statistical study of environmental influences on bivalve recruitment in the Wash, England. Mar Ecol Prog Series 1996;143:121 – 9. [66] Zhou JL, Fileman TW, Evans S, Donkin P, Mantoura RFC, Rowland SJ. Seasonal distribution of dissolved pesticides and polynuclear aromatic hydrocarbons in the Humber estuary and Humber Coastal Zone. Mar Pollut Bull 1996;32:599 – 608.
Review
Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs David Sheehan *, Aisling Power 1 Department of Biochemistry, Uni6ersity College Cork, Lee Maltings Complex, Mardyke, Cork, Ireland Received 27 December 1998; received in revised form 15 April 1999; accepted 25 April 1999
Abstract Levels of chemical pollutants in the environment often display wide seasonal variation in response to climatic and other factors. Use of bioindicators such as enzyme activities in biomonitoring studies is complicated by this variation. Many such enzyme activities themselves show considerable seasonal fluctuation and there is known to be seasonality also in natural exposure to oxidative stress. This review attempts to explore some consequences of seasonal variation for biomonitoring studies with bivalve molluscs. It is suggested that independence of seasonal variation should be seen as a desirable feature of a bioindicator molecule. Where such molecules show seasonal variation, however, this should be incorporated into interpretation of biomonitoring studies by the use of appropriate controls. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Xenobiotic; Antioxidant; Defence mechanisms; Detoxification; Seasonality; Biomonitor; Bivalve molluscs
1. Introduction Bivalve molluscs such as Mytilus edulis are commonly used as bioindicators in the assessment of environmental quality. They are filter-feeding organisms which, consequently, may be exposed to large amounts of chemical pollutants even if these are present in fairly dilute concentrations. They are also capable of bioconcentrating xenobiotics to many thousands times background which can facilitate chemical analysis. Mussels have a number of properties which make them useful sentinels for chemical pollution; they have a wide geographical distribution, are stationary, euryhaline and are normally the dominant species in their habitat. In addition they are capable of withstanding baseline levels of pollution and are abundant in estuaries where much human contact with the aquatic environment occurs [53]. * Corresponding author. Tel.: +353-21-904207; fax: + 353-21274034. E-mail address: [email protected] (D. Sheehan) 1 Present address; Neurogenetics Unit, Imperial College School of Medicine at St. Mary’s, Norfolk Place, London W21PG, England.
In recent years, there has been considerable interest in the use of biochemical indices within bivalve molluscs which might provide greater sensitivity and/or more information on how these animals interact with environmental pollutants [39]. Of particular interest in this regard are the levels and activities of enzymes involved in detoxification of xenobiotics such as the Cytochrome P-450 system and glutathione S-transferases (GSTs), binding proteins such as metallothioneins and GSTs and stress-related proteins such as the heat shock proteins. Laboratory and field studies have suggested that measurements of the levels of these proteins may be indicative of exposure to chemical pollution. Another important set of investigations have focused on bioindicators of oxidative stress within the organisms. Such studies suggest that oxidative stress may render mussels more sensitive to toxicity from xenobiotics. An important aspect of mussel physiology which has also come in for attention is seasonality. Depending on availability of nutrients, reproductive status, growth-dilution with season and other factors, levels of important bioindicators of pollution and of oxidative stress
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Fig. 1. The Ecotoxicology Triangle. There is a complex relationship between chemical pollutants, their environment and living organisms in that environment. Several aspects of this relationship are likely to vary in a seasonal manner (adapted from Ref. [53]).
may fluctuate significantly throughout the year. This poses an important limitation on the interpretation of bioindicator data per se as increased levels of a particular bioindicator might merely be a normal part of the molluscan species annual physiological cycle and be quite unrelated to exposure to chemical pollution. The relevance of seasonal variation to environmental monitoring studies of vertebrates such as fish [29] and birds [14,55] has been extensively studied. The present review attempts to explore the relationship between seasonality, pollution bioindicators and oxidative stress in bivalves and suggests that independence from seasonal variation is a desirable feature for biochemical bioindicators of xenobiotic pollution.
2. Seasonality of environmental pollution The ‘Ecotoxicology Triangle’ (Fig. 1) summarises a complex interrelationship between levels and type of chemical pollutant, living organisms within the environment and the environment itself [53]. One of the important interactions in this triangle is that between the pollutant and the environment. In studies of respiratory and cardiovascular diseases in man it has frequently been observed that temporal variations in air pollution correlate closely with the incidence of such conditions [7,19]. Levels of specific pollutants found in environ-
mental samples and in tissues of bioindicator species, such as M. edulis, may be expected to vary in a seasonal manner. This could result from factors such as temperature changes, cycles in industrial activity and annual alterations in climate. In addition, since marine organisms themselves are highly seasonal in terms of their basic physiology and detoxification biochemistry (see below), the levels of xenobiotics detected in tissue samples may be found to follow a variable seasonal pattern [21]. Some recent reports of seasonal variations in environmental pollutant levels are summarised in Table 1. Wide temporal variation has been observed in profiles of atmospheric polychlorinated biphenyls (PCBs) in Southern Chesapeake Bay [13]. Somewhat smaller variations in levels in precipitation samples were observed although these also followed a pattern of seasonal variation which was suggested as being due to the effect of temperature on washout efficiency. In a seasonal study of the German Bight, PCB congeners were measured in M. edulis. This study found generally lower levels of coplanar (i.e. ‘dioxin-like’) and atropisomeric congeners in autumn than during spring [24]. It was tentatively concluded that these results were due to seasonal variation of lipid content, although such variations are thought to be modest. Interestingly, the congener PCB 126 was unusual in that it was elevated or only slightly decreased in autumn. Since this possesses such a large toxic equivalency factor relative to other congeners [50], this resulted in only a comparatively small difference in toxic equivalents between data sets in spring and autumn despite the clear trend towards a large decrease in autumn with most other congeners. These results suggest that seasonal variations of total PCBs may mask important inter-pollutant differences which may be very significant for estimation of variation in toxic threat. Polyaromatic hydrocarbons (PAHs) such as fluoranthene and pyrene have been reported to vary seasonally in the Humber coastal zone with highest concentrations in September and lowest in June [66]. Generally similar results were obtained in a study of the Chesapeake Bay where atmospheric levels of PAHs (in particular, less volatile PAHs) were determined [13].
Table 1 Some examples of seasonal variation in pollutant patterns Pollutant
Level
Factor responsible
Reference
PAH, PCB PCB PAH and dissolved pesticides Metals TBT, DBT Cd, Cu, Pb, Zn
Increased deposition Decreased lipid variation Decreased Increased Decreased Pb increased; others decreased
Atmospheric Seasonal Agricultural runoff Runoff and salinity; change during Monsoon Variation in water flow and sediment deposition Spawning, variation in body weight
[13] [24] [66] [42] [38] [56]
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Seasonal variation in metal pollutants has also attracted considerable investigation. Analysis of tissue samples from marine organisms has revealed significant seasonal variation [11,37,41]. Factors such as high river run-off in winter or during Monsoon conditions (depending on Geography), tidal variations and cycles of industrial activities featuring metals (e.g. mining) may be expected to underlie such variation in estuaries. Seasonally-varied metal levels have been described in the mollusc Sunetta scripta off the coast of India [42]. This was attributed to large increases in river run-off as a result of Monsoon conditions. While this may not be a concern in more economically-affluent regions of the World, it is clearly likely to be important in some of the most densely populated regions in Asia. It should also be noted that comparatively little is known about seasonal variation of metallothioneins [3]. Since these important metal binding proteins (see below) may vary in amount and specificity during a seasonal cycle, this could result in a seasonal variation in the toxicological threat posed by metals. It has been observed that tributyltin (TBT) levels of the water column varies seasonally [15]. TBT and dibutyltin (DBT) have been found to be higher in late spring/early summer and lower in winter in the Tamar Estuary in England [38]. By contrast, these compounds were found to be highest in winter and lowest in spring in both M. edulis (a ten-fold difference) and Nucella lapillus (five-fold difference) sampled near Rekjavik Harbour, Iceland [54] suggesting that different factors might underlie the TBT/DBT levels in these two sampling sites. In the former study, variation is suggested to be due to large (up to eight-fold) differences in the amounts of water flowing through the Tamar in summer and winter. This is thought to cause greater flushing and decreased pollutant transport from downstream via tidal movement of suspended sediment. A secondary explanation of these effects is variation in M. edulis dry weight due to natural fluctuations in carbohydrate and protein content [38]. The pattern observed near Rekjavik Harbour is thought to be due to decreased feeding in winter in response to temperature-dependent, low phytoplankton levels. In summer, the mussels are thought to depurate themselves, leading to lower levels of TBT/DBT [54]. Some seasonal variation has also been observed in levels of TBT/DBT in Mytilus gallopro6incialis in Genoa, Italy, with DBT being three-fold higher in July than in March [48]. Comparison of these studies shows how interrelated biological, climatic and geographical factors can be in interpreting seasonal effects in molluscs. While studies on metal levels in molluscs have concentrated on inshore organisms such as M. edulis [41], it has been found that some offshore organisms collected from the central North Sea had higher concentrations of some metals than those collected near the shore [4].
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In a study of Arctica islandica from Kiel Bay, copper, zinc and cadmium were found to be generally lower in winter compared to summer, while lead, conversely, was highest in winter [56]. These variations were attributed to seasonal changes in reproductive cycle and food availability leading to alterations in body weight and composition. Studies of major pollutants suggest, in summary, that geographical and climatic factors may have an effect on natural biological cycles and result in considerable variation in levels of major chemical pollutants such as PCBs, PAHs and metals. Interpretation of data from biomonitoring studies may be seriously complicated by these factors. It is important, therefore, to take note of such seasonal variation in the interpretation of biomonitoring data.
3. Biological seasonality in molluscs The biochemistry of bivalve molluscs is known to vary in response to food supply, reproductive and pollution status. Early work demonstrated an important seasonal variation in enzymes involved in glycolysis and anaerobic metabolism [1,30,31]. There is known to be a close functional relationship between the digestive system and gonad development and periods of food abundance and gonad development are often coincident [35]. Although there may be some variation with geographical location, in Western Europe the gonads remain resting up to October–November before undergoing ripening culminating in early spring. Partial spawning may occur then but this is followed by further gametogenesis until the gonads are again fully ripe by early summer [52]. Temperature variation with season is thought strongly to influence the relationship between the digestive system and gonad development [6] especially through effects on lysosomes [36,58]. The appearance of enlarged lysosomes has been considered a general stress response to temperature increase or pollutant exposure [8]. Important variables affecting lysosomes include the age of the mollusc [22] and identity/concentration of pollutant [8,27,57]. Reproductive activity and temperature-associated changes in patterns of food storage and utilisation are also likely to cause changes in hormonal and nutritional status which might also be expected to affect the levels of bioindicator molecules. Temperature changes associated with seasonality may have profound effects on bivalve biology including winter-survival of juveniles, recruitment into estuaries and availability/quality of food. In a study mimicking winter conditions in British waters, juvenile Crassostrea gigas showed limited survival at 3°C compared to 6 and 9°C, at which temperatures all animals survived [9]. A statistical study of the Wash on the East coast of
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Table 2 Some bioindicator molecules used in molluscan biomonitoring studies Bioindicator
Organism
Reference
Glutathione S-transferase
Sphaerium corneum M. edulis
[5] [18]
Glutathione Cytochrome P-450 Catalase Superoxide Dismutase Metallothioneins
M. gallopro6incialis M. edulis M. gallopro6incialis M. gallopro6incialis C. 6irginica
[59,60] [26] [60] [60] [49]
England has demonstrated that seasonal variations in temperature over the last century may partly explain differential recruitment of M. edulis and Cerastoderma edule [65]. This effect is probably exerted through currents which, in turn, affect plankton availability. A similar study covering the period 1973 – 1996 in the Dutch Wadden See on Macoma balthica demonstrated a temperature-dependent effect on recruitment which was not related to fecundity (i.e. number of adults and number of eggs) [23]. Temperature tolerance of bivalves is known to be strongly influenced by seasonality as well as by other factors such as geographical latitude [63] and position on the shore [62]. Some environments such as the deep-sea and polar regions are exposed to particularly wide variations of temperature, food-supply and light. This provides a challenge to bivalves which has been met by genetic adaptation [10,25]. In particular, the high levels of dissolved oxygen in polar environments has attracted attention to the potential of antioxidant defence systems in bivalves as bioindicators of pollutant exposure [45 – 47]. While seasonal variation is not as extreme in tropical regions, bivalves are nonetheless affected by variation in temperature and food supply [51]. The particular environmental sensitivity of systems such as coral reefs and mangrove swamps [40] have led to inclusion of bivalves in tropical biomonitoring programmes [2,20].
4. Bioindicator proteins and seasonality Some suggested bioindicator molecules are shown in Table 2. Considerable attention has focused on enzymes involved in the metabolism and detoxification of xenobiotics. By analogy with other species, these enzymes would be expected to be inducible in response to chemical pollutants. Of particular interest in this regard are the Phase I activities associated with the mixedfunction oxygenase (cytochrome P-450) system and the Phase II conjugation activity of the GSTs. The cytochrome P-450 system is mainly localised in digestive gland microsomes in molluscs and consists of
a number of related proteins and enzymatic activities. The levels of cytochrome P-450 activity found in M. edulis are generally lower than those of mammalian species. There is a seasonal variation in benzo(a)pyrene hydroxylase and NADPH-independent 7-ethoxycoumarin O-deethylase activities with these being high in autumn and declining during gametogenesis and spawning. Interestingly, although the level of cytochrome P-450 protein also decreases just prior to gametogenesis, the seasonal variation of this is somewhat different to the enzyme activities suggesting that there is a complex pattern of variation of individual components of the system [26]. It has been reported that cytochrome P-450 activities are not inducible in bivalve digestive gland, unlike the liver of verterbrate fish where they are readily induced by xenobiotics [33]. Phase II of detoxification involves a range of enzyme activities which conjugate xenobiotics to endogenous substrates. The conjugate thus formed is usually more water-soluble than the xenobiotic and this facilitates its exclusion from the cell. Although some Phase II activities are associated with the microsomes, the glutathione S-transferases (GSTs), the most abundant Phase II activity in mammalian species, are principally located in the cytosol. In M. edulis, a number of GSTs are present [16,17]. In common with mammalian GSTs, these proteins also are capable of non-catalytic binding of a wide range of xenobiotics [44]. GSTs are most abundant in the gill and digestive gland tissues of M. edulis [16]. Field studies suggest that cytochrome P-450 activity is a good bioindicator of environmental status [34]. Levels of GST activity also appear to be altered by exposure to xenobiotics [5] and to be elevated in some field studies [18], although not in others [34]. The discrepancies between these findings may be due to the different pollution load in each case. Interestingly, GST activity has not been found to vary in a seasonal manner [43]. Thus, it would seem that molluscan GSTs might, in this regard, be superior to cytochrome P-450 as an index of xenobiotic exposure. Metallothioneins are a class of cysteine-rich proteins which may bind and detoxify heavy metals. They have been identified in a wide range of aquatic species and are inducible by cadmium, copper, mercury, silver and zinc. There are some important differences between the characteristics of mammalian and marine invertebrate metallothioneins such as a higher level of cysteine in the former [61]. A model has recently been proposed suggesting that endogenous zinc plays an important role in regulation of metallothionein induction in Crassostrea 6irginica by interaction with a Zn-sensitive inhibitor [49]. A four-fold annual biological variation in metallothionein level of the Asiatic clam (Corbicula fluminea) was observed in an unpolluted site in France [3]. This variation, which peaked in May, was unrelated to metal
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exposure and seemed to be strongly correlated with the reproductive cycle of this bivalve.
processes leading to oxidative stress in molluscs. The best-known example of this is the Fenton reaction which can result in the production of ·OH.
5. Oxidative stress and seasonality
FeII + H2O2 FeIII + HO − + ·OH
Bivalves are able to survive in a wide range of oxygen concentrations ranging from anoxic to high levels of dissolved oxygen. Variations in this ability has been proposed as an index of environmental stress [12]. Oxyradicals (O2− , H2O2, ·OH) can be highly toxic to aquatic organisms often resulting in lipid peroxidation in membranes, altered pyridine nucleotide redox status and DNA damage [28]. Moreover, many xenobiotics are capable of modulating oxidative stress either by acting directly as redox cycling compounds (e.g. menadione) or as a consequence of biotransformation to quinones which are redox cycling (e.g. benzo(a)pyrene) [32]. The microsomes appear to be an important subcellular location for such activity since this is the site of cytochrome P-450 induction and catalysis. An attractive in vitro system for studying the production of oxyradicals in M. edulis lysosomes using the laser dye dihydrorhodamine 123 is available [64]. A range of antioxidant defence mechanisms are present in bivalve molluscs. These enzyme activities (catalase, superoxide dismutase, GSH peroxidase) and antioxidant compounds (e.g. vitamin E and GSH) are known to be under extensive seasonal control. In digestive gland, levels of these antioxidant activities and molecules appear to be at their lowest in winter (November–March), corresponding to increases in levels of lipid peroxidation products, a useful index of oxidative stress [60]. In gill, the major feeding organ of M. edulis, a generally similar pattern for these activities and molecules exists. However, in this tissue, GST levels (normally some three-fold higher in gill than digestive gland) are at their highest in November–December and decrease to a minimum in March –April [43]. By contrast, gill catalase levels are four-fold lower than those of digestive gland and show little seasonal variation. M. edulis GST1 is known to display a distinct GSH peroxidase activity [17] and it is possible that this may be especially important in gill tissue during the winter months when other antioxidant defences are at their lowest. Oxidative stress is a highly seasonal phenomenon in bivalve molluscs. The modulation of oxidative stress by environmental pollutants is a factor which could seriously complicate interpretation of biomonitoring data.
Many of the toxic effects observed in marine animals exposed to high levels of heavy metals involve membrane-related processes. A possible mechanism for such toxicity is by metal-mediated peroxidation of lipid components of membranes as a result of oxyradicals. It has been demonstrated that Cu may cause a significant increase in malondialdehyde (an indicator of the peroxidative process) and a decrease in GSH while Cd and Zn do not [59]. This increase is associated with an elevation of the levels of lysosomal lipofuscin granules. It is clear from this that a complex relationship may exist between metals, their homeostasis [61], oxidative stress and seasonality. Varying levels of metals could be expected to lead to an altered oxyradical threat to cells. Possible effects of this on bioindicator molecules would obviously need to be taken into account in biomonitoring studies.
7. Conclusions Both pollution inputs to the environment and some bioindicator molecules thought to be sensitive to such inputs may be subject to seasonal variation due to a range of factors such as natural climatic and biological variation. These factors may be independent of direct interaction between xenobiotic pollutants and biomarker organisms such as molluscs. Seasonal variation in both pollutant input and in biochemical processes involving specific bioindicator molecules therefore needs to be factored into biomonitoring studies. An ideal bioindicator would be expected to show no seasonal variation in response to factors such as food supply or reproductive status and would vary only in response to pollutant exposure. Of course, this will often not be the case in practice. In such situations, it is essential that adequate control samples be taken from unpolluted sites contemporaneously and that care be taken in the choice of such sites. Tissue samples from control and test animals should be treated identically throughout the processes of dissection, storage, extraction and analysis.
Acknowledgements 6. Heavy metals and oxidative stress Because of their ability to exist in a variety of oxidation states, metals are capable of becoming involved in
We would like to thank Professor Mike Depledge, University of Plymouth, for critically reading the manuscript.
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