Trace metal uptake by the Chinese mitten crab Eriocheir sinensis: the role of osmoregulation

Trace metal uptake by the Chinese mitten crab Eriocheir sinensis: the role of osmoregulation

Marine Environmental Research 53 (2002) 453–464 www.elsevier.com/locate/marenvrev Trace metal uptake by the Chinese mitten crab Eriocheir sinensis: t...

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Marine Environmental Research 53 (2002) 453–464 www.elsevier.com/locate/marenvrev

Trace metal uptake by the Chinese mitten crab Eriocheir sinensis: the role of osmoregulation S.D. Roasta,*, P.S. Rainbowb, B.D. Smithb, M. Nimmoc, M.B. Jonesa a

Plymouth Environmental Research Centre (Department of Biological Sciences), University of Plymouth, Drake Circus, Plymouth, Devon PL8 4AA, UK b Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK c Plymouth Environmental Research Centre (Department of Environmental Sciences), University of Plymouth, Drake Circus, Plymouth, Devon PL8 4AA, UK Received 31 July 2001; received in revised form 2 December 2001; accepted 17 December 2001

Abstract Changes in salinity affect the bioavailability and consequent uptake of trace metals by euryhaline invertebrates. In many cases, salinity-related effects on metal uptake can be explained by changes in chemical speciation but salinity may also influence uptake indirectly through its action on osmoregulatory mechanisms. Specifically, it can be hypothesised that trace metal uptake may be reduced at salinities approaching the isosmotic point of a species because, at this point, there is reduced activity of ionic exchange pumps. The present study tested this hypothesis using the Chinese mitten crab, Eriocheir sinensis, a hyper–hypoosmoregulator with an isosmotic point around 33%. Crabs were exposed to radio-labelled cadmium and zinc at 23, 33 and 43% for 4 days. To eradicate speciation effects, crabs were exposed to the same concentration of the radio-labelled free metal ion (estimated using MineQL computer software) at each salinity. Haemolymph samples were taken daily and radio-labelled metal concentrations were estimated from radioactivity counts and used to provide relative measures of metal uptake. Neither cadmium nor zinc uptake was lowest at the isosmotic point. The uptake of cadmium increased significantly with increase in salinity, while the uptake of zinc showed no significant change with increased salinity. Thus changes in trace metal uptake rates in E. sinensis do not appear to be controlled only by changes in free metal ion concentrations. The different effects of salinity change on the uptake of cadmium

* Corresponding author. Tel.: +44-1752-633469; fax: +44-1752-232970. E-mail address: [email protected] (S.D. Roast). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00090-9

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and zinc (in the absence of free metal ion change) also indicate that physiological responses to osmotic change alone do not control metal uptake rates for this species. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Osmoregulation; Isosmotic point; Eriocheir sinensis; Metal uptake; Bioavailability; Cadmium; Zinc

1. Introduction The effects of salinity on the rates of uptake of trace metals from solution and their toxicity to aquatic invertebrates have been well documented (for reviews see Hall & Anderson, 1995; Wright, 1995). In general, uptake and/or toxicity of many trace metals are increased at low salinity indicating that the free (or aqueous) metal ion is the bioavailable form (Campbell, 1995). For example, the inorganic complexation of cadmium by chloride is necessarily decreased at low salinity due to decreased concentration of chloride ions, leaving a greater concentration of unbound (or free) cadmium (Rainbow, Malik, & O’Brien, 1993; Turner, Whitfield, & Dickson, 1981). Increased availability of the free metal ion correlates well with the apparent increase in toxicity of cadmium at low salinity (Hall & Anderson, 1995). In addition to affecting the speciation and availability of trace metals, however, salinity also affects the physiology of invertebrates through the demands of ionic regulation. Some aquatic invertebrates (e.g. common estuarine invertebrates) respond mechanistically to salinity changes in such a way that their physiological responses interact with changes in free metal ion availabilities to control trace metal uptake rates (Chan, Bjerregaard, Rainbow, & Depledge, 1992; Rainbow, 1995, 1997). Depending on the osmoregulatory physiology of the animal, water and ions (including metals) are exchanged between the organism and the external medium at various rates in relation to the salinity of the external environment. Clearly, this adds another variable to our understanding of the mechanisms and controls of trace metal uptake in aquatic invertebrates. If metal speciation is controlled, however, rates of uptake of trace metals at various salinities can help determine the physiological processes used in metal uptake, and crustaceans have proved to be convenient models to this end (Rainbow, 1995, 1997). An example of the possible control of trace metal uptake during osmoregulation has been demonstrated in the hyperbenthic mysid Neomysis integer (Wildgust & Jones, 1998). N. integer inhabits the upper reaches of estuaries and may be exposed to wide fluctuations in salinity over a single tidal cycle (e.g. Roast, Widdows, & Jones, 1998). N. integer is an extremely efficient hyper–hypo-osmoregulator, i.e. it actively regulates the ionic concentration of its body fluids irrespective of the osmotic concentration of the external environment (Moffat, 1996). Using the median lethal concentration (LC50) as a measure of toxicity, cadmium toxicity was found to be reduced at the salinity corresponding to the isosmotic point of N. integer. The authors concluded that reduced osmoregulatory ionic exchange led to decreased uptake of metal from solution, suggesting that metal uptake may be mediated by osmoregulatory mechanisms (Wildgust & Jones, 1998). A similar reduction

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in toxicity of cadmium at salinities close to the isosmotic point has been reported for the American mysid Americamysis bahia (DeLisle & Roberts, 1988). To test further this hypothesis the present study investigates whether the rate of uptake of cadmium or zinc is reduced at the isosmotic point of the Chinese mitten crab (Eriocheir sinensis), another hyper–hypo-osmoregulator (Scholles, 1933).

2. Materials and methods During September 1998 and 1999, male E. sinensis were collected from the intake filter screens of Lots Road Power Station (Thames Estuary), London, UK (OS grid reference SU 266 772). Only male crabs were used in the experiments to eliminate intergender variation and the potential presence of yolk proteins in the haemolymph. Immediately after collection, the crabs were transferred to holding aquaria maintained at 33 1% and 10 1  C. 2.1. Estimation of the isosmotic point To estimate the isosmotic point, 15 crabs of similar size (45  5 mm carapace width; 40  5 g wet weight) were exposed to five different salinities (three crabs being exposed to each salinity). E. sinensis is reported to be isosmotic in full-strength seawater (ca. 33%; Scholles, 1933), so the salinities used were 25, 30, 35, 40 and 45% made by combining synthetic seawater salts (Instant Ocean, Aquarium Systems, USA) with double-distilled water. Individual crabs were kept in plastic containers (23128 cm) containing 1 l of exposure water. Plastic lids, with holes to allow air to circulate, were used to prevent the crabs from escaping (the use of plastic air-lines was not possible since the crabs attack them, nor did they prove necessary). Haemolymph samples were extracted through the arthrodial membrane at the base of the most posterior walking leg using a disposable 1-ml syringe with a 240.5 mm sterile needle. Haemolymph osmolality was measured using a Westcor 5500C osmometer. Samples of the synthetic seawater medium were also taken to compare haemolymph osmolality with external medium osmolality. Haemolymph osmolality was measured every 24 h until readings were constant, when it was assumed that the haemolymph had attained a steady ionic state (this took ca. 24–48 h). The isosmotic point of E. sinensis was calculated by plotting haemolymph osmolality against medium osmolality, the isosmotic point being where the line of haemolymph osmolality crosses the isosmotic line (Campbell, 1988). The isosmotic point of E. sinensis collected from Chelsea in the Thames Estuary in September was estimated to be 32.7% (Fig. 1). 2.2. Metal uptake Having identified the isosmotic point of E. sinensis, three test salinities were chosen to include the isosmotic point and one on either side of the isosmotic point. The three test salinities used (22.7, 32.7 and 42.7%) were also made by combining

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Fig. 1. Osmoregulatory curve for Eriocheir sinensis. Haemolymph osmolality reaches a steady state after 72 h acclimation to constant salinity. The point where the osmoregulatory curve crosses the isosmotic line corresponds to the isosmotic point of E. sinensis, and is 945 mOsm/kg (equivalent to a salinity of 32.7%).

the Instant Ocean synthetic seawater salts with double-distilled water. The free (hydrated) metal ion is often assumed to be the bioavailable form of trace metals, and the amount of cadmium or zinc available as the free ion is dependent upon the salinity, due mainly to the formation of complexes with chloride ions. The degree of speciation (i.e. the amount of complexation) of cadmium and zinc at the three test salinities was calculated using MineQL software (Environmental Research Software, Maine, USA) programmed with the exact constituents and concentrations supplied by Instant Ocean. Computer-generated data compared well with the data of Turner et al. (1981). From the speciation data, the amount of dissolved metal needed at each salinity to result in the same concentration of free ion could be calculated. 2.3. Cadmium and zinc uptake by E. sinensis Crabs collected from the power station were placed in a recirculating holding aquarium at 33% for 24 h to acclimate to laboratory conditions. Crabs were then separated randomly into three groups of approximately equal numbers and placed in holding aquaria containing synthetic seawater of each test salinity. Crabs were maintained at these salinities over a weekend to ensure that they were fully acclimated to the exposure salinity and their haemolymph was in a steady state. Crabs were then exposed for 4 days to cadmium or zinc (in the form of Analar CdC12 or

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ZnCl2 respectively; BDH Ltd., UK) at the three different salinities. 50 mg l 1 of total metal is a typical concentration for these types of experiments, which are usually run at 33% (Martin & Rainbow, 1998). The free ion concentrations used were 1.65 mg Cd 1 and 25.5 mg Zn l 1, the calculated free ion concentrations of cadmium and zinc at a total concentration of 50 mg l 1 at 33%. Total cadmium concentrations used were 26.83, 49.40 and 82.50 mg l 1; and total zinc concentrations used were 45.41, 49.85 and 55.5 m l 1 at 22.7, 32.7 and 42.7%, respectively. In addition to the stable cadmium or zinc added to the exposure water, 5 mCi of radio-labelled cadmium (109Cd; NEN Life Science Products, USA) or zinc (65Zn; NEN Life Science Products, USA) were also added to each exposure vessel, so that all the added cadmium or zinc in the exposure medium is ‘radio-labelled’ metal at a known specific activity. The amount of metal added as radiotracer is negligible in comparison with the total radio-labelled concentration. When this radio-labelled metal (consisting of radioactive and non-radioactive metal) is taken up into the blood of the crab, the radioactivity can be counted, and this value used to calculate (via the specific activity) the concentration of radio-labelled (i.e. newly taken up) metal in the blood. Haemolymph samples were taken every 24 h, and concentrations of radio-labelled cadmium and zinc were determined by counting each 10 ml sample for 10 min on an LKB Wallac Compugamma counter. Measures of the uptake rates of cadmium and zinc from solution by crabs are provided by features of the accumulation kinetics of either metal in the haemolymph of the crab, as shown by Carcinus maenas by Martin and Rainbow (1998), E. sinensis by Black and Rainbow (unpublished), and used by Rainbow et al. (1999) and Rainbow, Amiard-Triquet, Amiard, Smith, and Langston (2000) for other crustaceans. The radio-labelled cadmium concentration in the haemolymph rapidly reaches a steady state as its rate of removal from the haemolymph (to the hepatopancreas) matches its rate of uptake into the haemolymph (via the gills) under constant exposure (Fig. 2). The steady state concentration of cadmium in the haemolymph (ng ml 1) does, however, increase with increased concentration of cadmium available in solution and provides a surrogate measure of the rate of uptake of cadmium from the medium by the whole crab (Martin & Rainbow, 1998; Rainbow, Amiard-Triquet, Amiard, Smith, Best et al., 1999). The highest value of the four haemolymph counts was taken as the plateau value. The radio-labelled zinc concentration in the haemolymph, on the other hand, continues to increase over the exposure period (Fig. 3). The rate of this increase (the regression coefficient of the best-fit line, expressed in ng ml 1 day 1) is directly proportional to the concentration of available zinc in the exposure solution (and hence rate of zinc uptake into the crab—see Chan & Rainbow, 1993a, 1993b) and can, therefore, be considered to be a surrogate (relative) measure of the crab’s uptake rate of dissolved metal (Black & Rainbow, unpublished; Martin & Rainbow, 1998; Rainbow et al., 1999, 2000). 2.4. Statistical analysis of results Comparisons of metal uptake rates were made by analysis of variance (of the highest of the four daily values used for each cadmium datum; and the regression coefficient for each zinc datum). Where significant differences between the means

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Fig. 2. Kinetics of accumulation of cadmium by Eriocheir sinensis. Radio-labelled cadmium concentrations in the haemolymph reach a steady state as Cd removal (into the hepatopancreas) equals Cd uptake (at the gills). A relative measure of uptake rate is the plateau concentration, which in these experiments was taken as the highest concentration measured in the haemolymph during the 4 days. Examples shown are two crabs held at 32.7% in 1999.

were found, Tukey’s honestly significant difference (HSD) test was used to identify which means were significantly different. All analyses were made using StatGraphics for WindowsTM computer software (Stat Graphics Corp., USA).

3. Results 3.1. Cadmium In 1998, six, seven and four crabs from a starting number of eight survived exposure to cadmium at 22.7, 32.7 and 42.7%, respectively; and in 1999, nine, ten and ten crabs (from ten) survived, respectively. The 1998 data for cadmium are shown in Table 1. There is a highly significant effect of salinity on cadmium uptake (ANOVA; f=15.9; d.f.=2, 14; P=0.0003). Regression analysis showed that cadmium uptake increased significantly with salinity (coefficient=0.170; t=5.77; P < 0.001). However, in the experiment repeated in 1999, there was no significant effect of salinity on cadmium uptake (ANOVA; f=3.01; d.f.=2, 23; P=0.069), a

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Fig. 3. Kinetics of accumulation of zinc by Eriocheir sinensis. Radio-labelled zinc concentrations in the haemolymph increase with length of exposure, and the rate of uptake is proportional to the slope of the best-fit line. A relative measure of uptake rate is taken as the regression coefficient expressed as ng ml 1 day 1. Examples shown are two crabs held at 22.7% in 1998.

contrast perhaps to be expected given the large standard deviation in the 1999 data (Table 1). The data were then checked to see whether uptake rates at each salinity differed significantly between 1998 and 1999. Since there was no significant difference at any salinity (ANOVA; P > 0.05), data for the two years were combined at each salinity and ANOVA repeated. ANOVA of the combined cadmium data revealed a significant difference (ANOVA; d.f.=2, 43; f=6.14; P < 0.005). Regression analysis confirmed that cadmium uptake increased with salinity (coefficient=0.169; r=3.22; P < 0.005). 3.2. Zinc In 1998, five and four crabs (from eight) survived exposure to zinc at 22.7 and 32.7%, respectively, and in 1999, nine, nine and seven (from ten) survived at 22.7, 32.7 and 42.7%, respectively. Data for zinc uptake rates are given in Table 2. In 1998, there was no significant difference in mean zinc uptake rates between crabs at 22.7 and 32.7% (ANOVA; f=4.51, d.f.=1, 7; P=0.07). Although the difference was not significant, zinc uptake rate appeared to be lower at high than at low salinity. In 1999, the same pattern emerged. There was no significant difference (ANOVA; f=1.12, d.f.=2, 22; P=0.35) between zinc uptake rates at the different

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Table 1 Eriocheir sinensis: relative cadmium uptake rates (measured as plateau concentrations of radio-labelled Cd in haemolymph, ng ml 1) from 1.65 mg l 1 free Cd ion at each of three salinities Salinity

n

Mean uptake (ng m1 1 day 1)

Standard deviation

1998

22.7 32.7 42.7

6 7 4

2.79 4.68 6.15

0.68 1.22 0.64

1999

22.7 32.7 42.7

9 10 10

2.13 5.45 6.25

1.73 3.59 4.76

1998+1999

22.7 32.7 42.7

15 17 14

2.33 5.13 6.21

1.36 2.82 3.81

Table 2 Eriocheir sinensis: relative zinc uptake rates [measured as regression coefficients (ng m1 1 day 1) of change of haemolymph radio-labelled Zn concentrations with time] from 25.5 mg l 1 free Zn ion at each of three salinities Salinity

n

Mean uptake rate (ng ml 1 day 1)

Standard deviation

1998

22.7 32.7

5 4

8.37 3.65

3.35 3.26

1999

22.7 32.7 42.7

9 9 7

7.77 6.96 4.04

6.27 4.96 3.29

1998+1999

22.7 32.7 42.7

14 13 7

7.98 5.94 4.04

5.26 4.64 3.29

salinities, although zinc uptake did appear to decrease with increased salinity. Combination of the 1998 and 1999 data sets produced the same conclusion (Table 2). There was a trend of decreasing rate of zinc uptake with increasing salinity but regression analysis showed that this decrease was not significant (coefficient= 0.198; t= 1.892; d.f.=32; P > 0.05).

4. Discussion For many trace metals, uptake and toxicity both increase at low salinity (e.g. Hall & Anderson, 1995; Wright, 1995). Furthermore, it has been shown that increased toxicity at low salinity can be explained by physicochemistry, particularly

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speciation of the metal ion (Rainbow, 1995; Wright, 1995), usually due to decreased complexation of the free metal ion with chloride at low salinity (Turner et al., 1981). In the present study, we have addressed how physiological responses of a crustacean to different salinities may modify trace metal uptake. In order to isolate physiological effects, physico-chemical changes in free ion availabilities have been predicted using computer modelling, and then removed by using different concentrations of total dissolved metal at each salinity such that free ion concentrations remained the same. For the remainder of this discussion, therefore, it is assumed that differences in salinity between experimental treatments had no effect on free metal ion concentrations of cadmium or zinc and that any salinity-related differences in metal uptake rates reported are the result of different physiological responses of the crabs at those salinities. The isosmotic point of E. sinensis collected from the Thames Estuary in September was 32.7%, in good agreement with the published estimate of Scholles (Scholles, 1933). The large male crabs used in the present study are considered to be migrating from fresh water into the more saline parts of the estuary to breed (Clark, Rainbow, Robbins, Smith, Yeomans, & Thomas et al., 1998), and could be expected to be carrying out considerable osmoregulation in this passage through the upper parts of the estuary. Although the isosmotic point of E. sinensis correlates well with previously published values, it is surprisingly high for an animal that spends so much time in a low salinity environment. In general, a depression of the isosmotic point is an indication of adaptation to life in fresh water (Campbell & Jones, 1989), so it is surprising that E. sinensis is isosmotic in normal seawater. There are several routes by which metals may enter marine organisms, and these routes may operate alone or simultaneously, and to greater or lesser extents at different times (Rainbow, 1995, 1997). The uptake route of significance for the present study is that by which metals may enter the cell via an active (i.e. energy dependent) pump, for example, a calcium pump. This type of pump is responsible for ionic exchange during osmoregulatory processes (Mantel & Farmer, 1983). Such pumps are selective in that they will only allow ions of a certain size and charge through the channel, but they are not selective to specific metal ions. For example, the free cadmium ion has a similar ionic radius and charge density to the free calcium ion, hence cadmium ions will pass through calcium pumps (Rainbow, 1995; Wright, 1995). If trace metals are taken up via such pumps, osmoregulatory mechanisms of a crustacean may increase or mediate trace metal uptake depending on the activity of such pumps. The basic principles involved (described in detail by Mantel & Farmer, 1983) are reviewed here briefly to allow comparison of predicted pump activity with trace metal uptake measured in the present study. When a decapod crustacean is placed in a medium of lower osmotic concentration than its body fluids, water enters by osmosis. The extra water is then excreted by increased urine production. The urine, however, is isosmotic with the haemolymph so salts are lost through this route. To counteract this loss of ions, there is a requirement for the active uptake of essential ions (for example calcium and sodium), which are taken up through their respective pumps. Increased activity of ionic pumps leads to increased uptake of these metal ions, but the pumps will also

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allow entry of any ion of the correct size and charge density. Thus, if the activity of calcium pumps is increased, more calcium is obviously taken up but so too is more cadmium since the free cadmium ion will also pass through the pump (Rainbow, 1995; Wright, 1995). Essentially, the reverse of this exchange could happen if the animal is placed in a medium of higher osmotic concentration than that of the body fluids, but trace metals are tightly bound intracellularly and practically no free metal ion is available for any reverse process. Furthermore, hypo-regulation by crustaceans is relatively rare and little is known of the mechanism of how ionic concentrations are maintained. Clearly, water will leave the animal by osmosis and it is presumed that increased drinking, or perhaps active uptake of water, replaces the water lost. To maintain haemolymph osmolality, ions taken up through this extra drinking (for example sodium and chloride) are actively pumped out (Spaargaren, 1971). Increased drinking will again lead to increased uptake of other metals (e.g. cadmium), which may then pass into the cell via passive or facilitated diffusion in the gut. However, at certain salinities (depending on the crustacean species in question), the external medium will be of the same osmotic concentration as the haemolymph (i.e. the isosmotic point). Thus, there is no osmotic movement of water between the haemolymph and the external medium, and pump activity is reduced. This argument predicts that metal uptake is reduced at the isosmotic point. It should be remembered though that ionic exchange would still occur because, although the haemolymph is isosmotic with the external medium, it is not isionic, and diffusion of ions will occur still (Mantel & Farmer, 1983). Results from the present study contradict the hypothesis that rates of trace metal uptake are reduced at the isosmotic point of a crustacean; for both cadmium and zinc uptake rates, there was no minimum at the isosmotic point. This is surprising, as previous work has shown that the toxicity of trace metals is reduced at, or near, the isosmotic points of two mysid species, Americamysis bahia (DeLisle & Roberts, 1988) and Neomysis integer (Wildgust & Jones, 1998). Although statistically significant trends were identified in the data, results from the present study show considerable variation, although this is typical of such metal uptake measurements (e.g. Chan & Rainbow, 1993b). Such variation might be expected to increase under extreme conditions. Regarding trace metal uptake at salinities at 23 and 43%, results from the present study are again surprising. Zinc and cadmium have similar chemistries, and there is evidence that their uptake rates are correlated in individual crustaceans, for example in the caridean Palaemon elegans (Nugegoda & Rainbow, 1995), and in the amphipod Orchestia gammarellus and the crabs Carcinus maenas and Pachygrapsus marmoraus (Rainbow et al., 2000). Thus, it might be concluded that the metals share similar uptake routes to some degree (Nugegoda & Rainbow, 1995; Rainbow et al., 2000). It is unusual, therefore, that the uptake rates of the two metals showed differing responses to salinity change in the present study. The rate of uptake of cadmium increased with increasing salinity while that of zinc did not change significantly, with some evidence of a tendency to decrease. Thus, any physiological changes made by the crab, as salinity changes on either side of the isosmotic point, are affecting the uptake rates of the two metals differentially.

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If trace metal ions are entering the crustacean via ionic pumps (Rainbow, 1995, 1997), then the highest rate of trace metal uptake could be expected at the lowest salinity (in this case 23%) when such pumps are most active replacing essential ions lost through excretion. There is some suggestion of this for zinc, but uptake of cadmium is actually reduced at low salinity. Some crustaceans are able to change their (apparent) water permeability (AWP) in response to changes in salinity with possible consequences for trace metal uptake (Rainbow, 1995, 1997). Decreased permeability to water at low salinity provides a physical barrier preventing the increased uptake of water that one would expect. Clearly, a reduction in AWP would reduce the need for ionic pump activity and consequently reduce metal uptake via such pumps. Although this might account for reduced uptake of cadmium at low salinity it is unlikely, since E. sinensis shows little change of AWP with salinity, the AWP being already very low in correlation with its preceding occupation of fresh water prior to migration (Rainbow & Black, 2001). Since the exact mechanisms of hyperregulation are less well documented for crustaceans, predictions of metal uptake rates compared with ionic pump activity can be made with less certainty. In summary, Eriocheir sinensis appears to be making physiological changes on either side of the isosmotic point affecting the rates of uptake of cadmium and zinc differentially. Thus, changes in trace metal uptake rates in E. sinensis are not controlled only by changes in free metal ion concentrations. The different effects of salinity change on the uptake of cadmium and zinc (in the absence of free metal ion change) also indicate that changes in ionic pump activity alone similarly do not control metal uptake rates.

References Campbell, P. J. (1988) Osmoregulation in the prawn Palaemon longirostris (Caridea, Palaemonidae). PhD thesis, University of Plymouth, Plymouth, UK. Campbell, P. G. C. (1995). Interactions between trace metals and aquatic organisms: a critique of the freeion activity model. In A. Tessier, & D. R. Turner (Eds.), Metal speciation and bioavailabilily in aquatic systems (pp. 45–102). Chichester, UK: John Wiley & Sons. Campbell, P. J., & Jones, M. B. (1989). Osmoregulation of the estuarine prawn Palaemon longirostris (Caridea: Palaemonidae). Journal of the Marine Biological Association of the United Kingdom, 69, 261–272. Chan, H. M., & Rainbow, P. S. (1993a). The accumulation of dissolved zinc by the shore crab Carcinus maenas (L.). Ophelia, 38, 13–30. Chan, H. M., & Rainbow, P. S. (1993b). On the excretion of zinc by the shore crab Carcinus maenas (L.). Ophelia, 38, 31–45. Chan, H. M., Bjerregaard, P., Rainbow, P. S., & Depledge, M. H. (1992). Uptake of zinc and cadmium by two populations of shore crabs Carcinus maenas at different salinities. Marine Ecology Progress Series, 86, 91–97. Clark, P. F., Rainbow, P. S., Robbins, R. S., Smith, B. D., Yeomans, W. E., Thomas, M., & Dobson, G. (1998). The alien Chinese mitten crab, Eriocheir sinensis (Crustacea: Decapoda: Brachyura) in the Thames catchment. Journal of the Marine Biological Association of the United Kingdom, 78, 1215–1221. DeLisle, P. F., & Roberts, M. H. Jr. (1988). The effect of salinity on cadmium toxicity to the estuarine mysid Mysidopsis bahia: role of chemical speciation. Aquatic Toxicology, 12, 357–370.

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