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Interaction between selenium and cadmium in the hemolymph of the shore crab Carcinus maenas (L.)
Aquatic Toxicology, 13 (1988) 1-12 Elsevier
1
AQT 00282
Interaction between selenium and cadmium in the hemolymph of the shore crab C a r c i n u s m a e n a s (L.) Poul Bjerregaard Institute o f Biology, Odense University, Campusvej 55, DK-5320 Odense M, Denmark (Received 15 June 1987; revision received 8 December 1987; accepted 23 January 1988)
The effects of selenium on survival and binding of cadmium in the hemolymph of cadmium-exposed shore crabs Carcinus maenas (L.) were studied in a series of laboratory experiments. Selenite in the seawater neither prolongs survival time nor counteracts disturbance of calcium regulation in crabs exposed to lethal cadmium concentrations. Crabs in the moulting stage appear more sensitive to cadmium than intermoult crabs. Exposure to selenite concentrations above approximately 300 #g Se-SeO]-/l leads to elevated selenium concentrations in the hemolymph, whereas lower selenite concentrations do not. Selenite concentrations above approximately 200 #g Se-SeO2-/l result in augmented levels of cadmium in the hemolymph of crabs exposed to 200 #g Cd/1. Cadmium is eliminated from the hemolymph of selenite- and non-selenite exposed crabs according to first order kinetics with half-lives of 51 and 7 h, respectively. In the selenite-exposed crabs, higher protein concentrations in the hemolymph were associated with greater retention of cadmium. Initial uptake rates for cadmium in the hemolymph of individual crabs are positively correlated with hemolymph protein concentration. Initial uptake rates for cadmium are not affected by selenite exposure. The binding affinity of the hemolymph for cadmium is positively correlated with the protein concentration of the hemolymph, and is unaffected by selenite exposure. Selenite exposure (from 35 #g Se-SeO]-/1) augments cadmium uptake in the gills, whereas no consistent effect is seen in hepatopancreas, hypodermis and carapace. Key words: Selenium; Cadmium; Hemolymph; Protein; Binding; Carcinus maenas
INTRODUCTION
Since the discovery by Kar et al. (1960) that selenium protects rat testes against the toxic effects of cadmium, the interactions between selenium and cadmium and mercury in mammals have been intensively studied (reviewed by Magos and Webb, 1980). However, the exact mechanism by which selenium both augments uptake and reduces the toxicity of cadmium has not been elucidated (Chmielnicka et al., 1983).
Correspondence to: P. Bjerregaard, Inst. of Biology, Odense University, Campusvej 55, DK-5320 Odense M, Denmark. 0166-445X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
In aquatic invertebrates the interaction between selenium and cadmium is poorly understood. Exposure to selenium augments cadmium uptake in gills and hemolymph of the shore crab C a r c i n u s m a e n a s (L.), while cadmium uptake in other tissues are not affected (Bjerregaard, 1982, 1985). Cadmium taken up in the hemolymph is mainly bound in the high molecular weight proteins (hemocyanin) and selenium exposure augments binding of cadmium in this fraction (Bjerregaard, 1985). Cadmium bound in the hemolymph of C. m a e n a s is normally transferred to hepatopancreas (Wright and Brewer, 1979), but how selenium exposure affects the internal transport of cadmium in C. m a e n a s is unknown. Exposure to 10 mg Cd/1 is lethal for C. m a e n a s within 7 to 16 days (Jennings and Rainbow, 1979; Bjerregaard and Vislie, 1985), and the normal regulation of calcium concentrations in the hemolymph is severely affected (Bjerregaard and Vislie, 1985). Whether or not exposure to selenite can protect C. m a e n a s from the toxic effects of cadmium is unknown. This study investigates the effect of selenium on binding and transport of cadmium in the hemolymph of C. m a e n a s . Furthermore, the protective effect of selenium with regard to the toxic effects of cadmium is investigated. MATERIALS AND M E T H O D S
Adult male shore crabs C. m a e n a s were obtained from Lillebaelt, Denmark. In experiments carried out from May to October freshly caught crabs were used. Crabs used in experiments from October to April were caught in October and kept in flowing sea water aquaria at the Marine Biological Station, Bogeberg, N.E. Funen. Small crabs (15-26 g w.w.) were acclimated to laboratory conditions for one week prior to use in experiments. Experiments 1, 2, 4, and 5 were carried out at a salinity of 400 mOsm (14%0) and Exp. 3 at 670 mOsm (20%o). The experimental temperature was 15.5+0.5°C. Crabs were exposed to cadmium as CdC12 and selenite as Na2SeO3. Water was changed every second or third day. The crabs were not fed. Hemolymph samples were drawn with hypodermic syringes through the arthrodial membrane of the posterior periopod. Cadmium, calcium, and protein concentrations in the hemolymph were determined as described by Bjerregaard (1982, 1985) and Bjerregaard and Vislie (1985) . For selenium analysis, 200 td hemolymph were digested in 2 ml of concentrated nitric acid at approximately 100 °C, evaporated to dryness, and dissolved in 0.2% nitric acid containing 1000 mg nickel/1 (added as Ni (NO3)2). Selenium was determined on a Perkin-Elmer 2380 atomic absorption spectrophotometer fitted with a graphite furnace (Perkin-Elmer HGA 300). For determination of concentrations of free and protein-bound cadmium in the hemolymph, 200 tzl hemolymph were dialyzed against 100/zl crab ringer solution (360 mM NaCI, 8 mM KCI, 22 mM MgSO4, 4 mM NaHCO3, 10 mM CaC12, pH 8.0) in dialysis chambers. Ten microliters a°9Cd (dissolved in the ringer solution) were
added to the hemolymph side and 1°9Cd was allowed to equilibrate at 4 °C. After 4 days the radioactivity on both sides of the dialysis membrane was determined. When sufficient amounts of hemolymph were available from individual crabs, 2 determinations were made. 1°9Cd was obtained from New England Nuclear and radioactivity was determined with a Searle Mark III Liquid Scintillation Counter. Two-tailed Student's t-tests were used in the statistical evaluation of the data. Probit plots of survival times were evaluated according to Litchfield and Wilcoxon's method (1949).
Experiment 1 Four groups of 25 crabs were exposed to 10 mg Cd/l and the following concentrations of selenite: 0, 0.25, 1, and 4 mg Se-SeO]-/1. Twenty-five crabs served as a control group. Mortality rate was recorded and on the 9th day of exposure approximately 0.1 ml of hemolymph was drawn from the surviving crabs (n = 23, 19, 18, 15, and 13 in the control group and groups exposed to 0, 0.25, 1, and 4 mg Se-SeO]-/1) for calcium determination. Exposure was initiated on August 13th.
Experiment 2 Twelve groups of 10 crabs were exposed to 10 mg Cd/1 and the following concentrations of selenite: 0, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 mg SeSeO2-/l. Ten crabs in uncontaminated water served as a control group. Mortality was noted daily and on day 3 and 9 approximately 0.1 ml hemolymph samples were obtained for cadmium analysis. Exposure was initiated on November 4th.
Experiment 3 Eight groups of 6 or 7 crabs were exposed to 200/~g Cd/l and the following concentrations of selenite: 0, 35, 70, 140, 281,562, 1124, and 2248 #g Se-SeO]-/1, corresponding to molar ratios Se:Cd of 0.25, 0.5, 1, 2, 4, 8, and 16 in the Se-exposed groups. After 28 days hemolymph samples were drawn from the surviving crabs which were then killed by freezing. Selenium and cadmium concentrations in the hemolymph and cadmium concentrations in gills, hypodermis, hepatopancreas, and carapace were measured. The exposure was initiated on August 20th.
Experiment 4 Four groups of 5 crabs were exposed to the following combinations of cadmium and selenite (~g Cd/l:/zg Se-SeO2-/1): 0:0, 200:0, 200:2248, and 0:2248. Exposure was initiated on January 10th. After 24 days 5 crabs from each group were placed in separate 4-1 aquaria. Fifty
microliters of crab ringer solution containing approximately 200 000 dpm of l°9Cd were injected into the hemolymph of each crab through the arthrodial membrane of the anterior periopod. After the injection each crab was briefly rinsed in clean seawater and then returned to the aquarium. Thirty minutes after the injection, a sample of 0.15 ml hemolymph was drawn from each crab and elimination of l°9Cd from the hemolymph was followed during the next 28 h by sampling at 3, 10, 21 and 28 h. At 28 h the maximal obtainable amount of hemolymph was drawn and the experiment was stopped. The amount of l°9Cd in hemolymph samples and seawater after 28 h was determined. Protein concentrations and partitioning between protein-bound and free cadmium in the hemolymph were determined.
Experiment 5 On January 10th a group of 5 crabs was exposed to 2248 #g Se-SeO2-/l. Five crabs were used as control. After 38 days the 2 groups were placed in 4 1 seawater containing approximately 4000 dpm l°9Cd/ml. Hemolymph samples of approximately 0.25 ml were drawn after 20 h, 3 days, and 7 days (max. obtainable) and
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Fig. 1. Carcinus maenas. Results from toxicity experiments. (a) Probit plot of mortality in crabs exposed to 10 m g C d / l . Pooled results from groups in which selenite had no effect on survival. El: Exp. 1 ( n = 100), O : Exp. 2 (n= 100), I : mortality in control group from Exp. 1. (b) Median survival time for crabs exposed to 10 mg Cd/1 a n d varying selenite concentrations. 95070 confidence limits are shown. Symbols as in (a). (c) Calcium concentrations in the hemolymph o f control crabs and crabs exposed to 10 m g C d / l and varying selenite concentrations (Exp. 1). Significance levels ( * : 0.05, * * : 0.01, ~ - * * : 0.001) for the difference from the control group are indicated. Symbols as in (a). (d) C a d m i u m concentrations (mean ± SEM) in the hemolymph of crabs exposed to 10 m g Cd/1 and varying selenite concentrations for 3 (lower curve, n = 10) and 9 (upper curve, n as indicated) d a y s . . k indicates that the concentration is significantly ( P < 0.05) different from the group not exposed to selenite.
the amount of 1°9Cd taken up was measured. Protein concentrations in the hemolymph were measured. RESULTS Toxicity experiments In Exp. 2 none of the control crabs died during the experimental period (28 days), while 6 out of 25 control crabs died after moulting in Exp. 1 (Fig. la). For each of the 12 groups exposed to 10 mg Cd/l in Exp. 2, survival times could be fitted to straight lines (P< 0.05) in a probit plot. Median survival times are shown in Fig. lb. Survival times were not affected by selenite concentrations between 0.032 and 8 mg Se-SeO2-/l, while 16 and 32 mg Se-SeO2-/l seemed to reduce the survival times, indicating that these selenite concentrations may be toxic to C. maenas. Probit analyses of cadmium-induced mortality in crabs from the two experiments are shown in Fig. la (data from the two groups exposed to 16 and 32 mg Se-SeO2-/1 in Exp. 2 are omitted). In Exp. 2 mortality began at day 6 and survival times fitted a straight line. In Exp. 1 mortality began at day 1, and 13 crabs died between day 2 and 3. Between day 3 and 8 and day 8 and 28, respectively, survival times could be fitted to straight lines (P < 0.05) with different slopes. Examination of the crabs that died early in the experiment revealed that they had initiated the formation of a new cuticule, indicating an advanced stage in the moulting process (Drach, 1939). Exposure to cadmium augmented the calcium concentrations in the hemolymph, and selenite did not protect the crabs from this effect of cadmium (Fig. lc). There was a trend that the highest concentrations of selenite augmented uptake of cadmium in the hemolymph, but the effect was not seen consistently (Fig. ld). Experiment 3 Three of the 53 crabs died during the experimental period - all after moulting. The selenium concentration in the hemolymph of crabs not exposed to selenite in the sea water was 0.226_+0.046 #g/ml, and exposure to 35, 70, 141, and 281 #g SeSeO2-/l (with 200 #g Cd/l) did not lead to increased selenium concentrations in the hemolymph (Fig. 2a), while selenium concentrations in the hemolymph of crabs exposed to 562, 1124, and 2248 #g Se-SeO2-/1 increased with increasing ambient selenite concentration (Fig. 2a). Cadmium concentration in the hemolymph of crabs exposed to 200 #g Cd/1 was 55 _+22 #g Cd/1 (Fig. 2b). Ambient selenite concentrations at and above 281 #g SeSeO2-/1 augmented cadmium uptake in the hemolymph, while the augmenting effect of lower selenite concentrations was not statistically significant (Fig. 2b). Increases in cadmium and selenium concentrations were parallel, and the molar ratio Se:Cd in the hemolymph was in the range 3 to 8 for all of the groups (Fig. 2c).
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Fig. 2. C a r c i n u s m a e n a s . Selenium (a) and cadmium (b) concentrations and Se:Cd molar ratios (c) in hemolymph of crabs exposed to 0.2 mg C d / l and different selenite concentrations for 28 days. Mean +_ SEM for 5-7 crabs are given. * , * * and * * * indicate statistically significant difference at the 0.05, 0.01, and 0.001 levels from the group not exposed to selenite.
Cadmium uptake in the gills was augmented by all selenite concentrations tested (Fig. 3a), except for 70/~g Se-SeO]-/1 (P= 0.07). Cadmium uptake in the gills increased with increasing ambient selenite concentrations. Cadmium uptake in the carapace was not affected by selenite (Fig. 3b), and cadmium uptake in hepatopancreas and hypodermis was not affected by selenite concentrations up to 1124/zg SeSea]-/1. In hypodermis, cadmium uptake diminished after exposure to 2248 #g SeSea]-/1 (Fig. 3c), and the same trend was seen in hepatopancreas (Fig. 3d) although the difference from the control group was not statistically significant (P= 0.07).
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Fig. 3. C a r c i n u s m a e n a s . Cadmium concentrations (mean _+ SEM for 5-7 crabs) in gills (a), carapace (b), hypodermis (c), and hepatopancreas (d) of crabs exposed to 0.2 mg Cd/l and different selenite concentrations for 28 days. * and * * * indicate statistically significant difference at the 0.05 and 0.001 levels from the group not exposed to selenite.
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Fig. 4. C a r c i n u s m a e n a s . (a) Retention of :°gCd injected into the hemolymph of crabs on day 29 of exposure to 0.2 mg Cd/1 (©), 0.2 m g Cd + 2.248 m g Se-SeO2-/l (4), 2.248 m g Se-SeO2-/l (A) and control crabs (e). Mean _+ SEM for 5 crabs. (b) % ]°gCd retained after 28 h elimination shown against protein concentrations in the hemolymph for individual crabs. Symbols as in (a). (c) % free cadmium
(determined in vitro) against protein concentration in the hemolymph of individual crabs. Symbols as in (a).
Experiment 4 Elimination of cadmium from the hemolymph of unexposed crabs and crabs exposed to 200 #g Cd/1 proceeded identically (Fig. 4a). Between 3 and 28 h cadmium was eliminated according to first order kinetics with a half-life of 6.8 h (data from both groups pooled: In y = 9 1 . 3 - 0.102 x). In the two selenite exposed groups elimination of cadmium from the hemolymph proceeded with identical rates between 3 and 28 h (Fig. 4a). Half-life for cadmium in the hemolymph was 51.5 h (data from both groups pooled: In y = 81.7 - 0.014 x). After 28 h the seawater in the 4 aquaria contained 23 000, 27 000, 48 000, and 17 000 dpm l°9Cd, respectively. This corresponds to 1.7% - 4.8% of the total amount of 1°9Cd injected in the crabs of each aquaria. In the selenite-exposed groups the amount of cadmium remaining in the hemolymph of individual crabs after 28 h (Fig. 4b) was positively correlated with the protein concentration in the hemolymph of the crabs (r= 0.7332, P = 0.016). In the two groups not exposed to selenite no such correlation was found (r = - 0.0724, P = 0.84). TABLE I Carcinus maenas.
Percentage of free cadmium (determined in vitro) in the hemolymph of crabs from
Exp. 4. Mean +_ SD for 5 crabs. Individual values are seen in Fig. 4.
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Dialysis showed that generally 99 to 99.9% of the cadmium in the hemolymph was non-dialyzable. The percentage of free cadmium did not differ in the four groups of crabs (Table I). The percentage of free cadmium in the individual crabs was negatively correlated (r= -0.5844, P = 0.0068) with the protein concentration in the hemolymph (Fig. 4c).
Experiment 5 In the group not exposed to selenite a concentration factor (l°9Cd in hemolymph -- l°9Cd in sea water) of approximately 0.1 for cadmium was reached within 20 h and in 7 days no increase was observed (Fig. 5a). In the selenite-exposed group, the concentration factor increased significantly from 20 h to 3 (P=0.016) and 7 (P=0.013) days (Fig. 5a). After 20 h, uptake of cadmium in the hemolymph in individual crabs of the two groups was positively correlated (r= 0.8566, P = 0.0016) with the protein concentration of the hemolymph (Fig. 5b). During one week's exposure this correlation (day 3: r=0.6173, P=0.057; day 7: r=0.4387, P=0.20) gradually disappeared (Fig. 5c,d). DISCUSSION
While the protective effect of selenium against the toxic effects of cadmium and mercury in mammals has been well documented (reviewed by Magos and Webb, 1980), comparable studies on aquatic organisms are scarce. Selenite tended to protect oyster embryos and crab larvae (Glickstein 1978) and shrimps (Lucu and
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Skreblin 1981) from the acute, lethal effects of mercury, but in neither of these investigations statistically significant results were obtained. Selenite more than doubled the median survival time in the freshwater mollusc L y m n e a stagnalis exposed to a lethal cadmium concentration (Puymbroek et al., 1982). The present results show that selenite neither prolongs survival nor protects the calcium regulation in Carcinus maenas exposed to a lethal cadmium concentration. The results do not exclude the possibility that selenium administered differently (e.g. via the food) may have some protective effect. Moulting is associated with major adjustments of physiological processes especially ionic and osmotic regulation - in crabs (Robertson, 1960). As exposure to toxic concentrations of cadmium interferes with the normal ion and osmoregulatory pattern in Carcinus maenas (Bjerregaard and Vislie, 1985), it is not surprising that crabs in the stages immediately preceding the moult respond more rapidly to lethal cadmium concentrations than intermoult crabs. Rasmussen (1973) also found that the fraction of small, adult male crabs being in the premoult stage should decrease from late July to late August. Cadmium concentrations in the hemolymph of shore crabs exposed to cadmium concentrations lower than 2.4 mg Cd/l rapidly reach a steady state level at or below the ambient cadmium concentration (Wright, 1977a,b; Wright and Brewer, 1979; Bjerregaard and Vislie, 1985), while crabs exposed to cadmium concentrations above 4 mg Cd/1 concentrate cadmium in the hemolymph to levels above those in the water (Bjerregaard and Vislie, 1985). The present data support these observations. Wright and Brewer (1979) and Lyons et al. (1984) suggest that cadmium in the hemolymph of crustaceans is rapidly transferred to other organs (primarily hepatopancreas), giving low turnover times for cadmium in the hemolymph. The present results show that C. maenas exposed to background cadmium concentrations of approximately 25 ng Cd/l (Magnusson and Rasmussen, 1982) and 200 #g Cd/1 eliminate cadmium from the hemolymph at equal rates. The half-life for cadmium in the hemolymph of C. maenas is in the same range as in the freshwater crayfish Austropamobiuspallipes (Lyons et al., 1984). Exposure to selenite markedly lengthens elimination time for cadmium in the hemolymph, while the initial uptake rate for cadmium from the ambient seawater is not increased in the selenite exposed crabs. Thus, selenium leads to increased cadmium accumulation in the hemolymph by diminishing elimination rates, rather than enhancing uptake rates. This agrees with the fact that in vitro the affinity of the hemolymph proteins for added cadmium is identical in selenite- and non-selenite exposed crabs. Concurrent administration of selenite to cadmium-treated rats greatly increases binding of cadmium in the blood plasma (Nishiyama et al., 1987; Gasiewicz and Smith, 1976, 1978). Gasiewicz and Smith (1976, 1978) suggest that the effect of selenite on cadmium binding depends on the metabolic conversion of Se(IV) to Se( - II), which is supposed to form a protein stabilized CdSe complex with a molar
10 ratio Cd:Se close to one. Such a m e c h a n i s m m a y also be responsible for the interaction b e t w e e n selenium a n d c a d m i u m in the h e m o l y m p h o f the crab, a l t h o u g h 1:1 m o l a r ratios b e t w e e n the two elements are n o t necessarily o b t a i n e d (Bjerregaard, 1985). The processes, which t r a n s p o r t c a d m i u m f r o m the h e m o l y m p h to the tissues are largely u n k n o w n . U p t a k e o f c a d m i u m f r o m the h e m o l y m p h into the h e p a t o p a n creas a n d h y p o d e r m i s is n o t affected b y exposure to selenite c o n c e n t r a t i o n s which lead to s u b s t a n t i a l increases in the total c a d m i u m c o n c e n t r a t i o n o f the h e m o l y m p h . This might indicate that t r a n s p o r t into these tissues derives f r o m the free pool o f c a d m i u m (not affected by exposure to selenite) in the h e m o l y m p h . H o w e v e r , this hypothesis w o u l d n o t a c c o u n t for the a p p a r e n t l y lower u p t a k e o f c a d m i u m in the h e p a t o p a n c r e a s a n d the h y p o d e r m i s for the highest selenite c o n c e n t r a t i o n tested. Internal t r a n s p o r t a n d b i n d i n g o f heavy metals require f u r t h e r investigation, ACKNOWLEDGEMENTS I t h a n k Mrs. Vibeke E r i k s e n a n d Mr. Rene Stoving for technical assistance a n d Dr. Michael H. Depledge for critically r e a d i n g the m a n u s c r i p t . The project was supported by grants f r o m the D a n i s h N a t u r a l Science Research C o u n c i l a n d the T h o m a s B. Thriges F o u n d a t i o n . REFERENCES Bjerregaard, P., 1982. Accumulation of cadmium and selenium and their mutual interaction in the shore crab C a r c i n u s m a e n a s (L.). Aquat. Toxicol. 2, 113-125. Bjerregaard, P., 1985. Effect of selenium on cadmium uptake in the shore crab C a r c i n u s rnaenas (L.). Aquat. Toxicol. 7, 177-189. Bjerregaard, P. and T. Vislie, 1985. Effects of cadmium on hemolymph composition in the shore crab C a r c i n u s m a e n a s . Mar. Ecol. Prog. Ser. 27, 135-142. Chmielnicka, J., E.M. Bem and P. Kaszubski, 1983. Organ and subcellular distribution of cadmium in rats exposed to cadmium, mercury, and selenium. Environ. Res. 31,273-278. Drach, P. 1939. Mue et cycle d'intermue chez les Crustaces Decapodes. Ann. Inst. Oceanogr. 19, 103-391. Gasiewics, T.A. and J.C. Smith, 1976. Interactions of cadmium and selenium in rat plasma in vivo and in vitro. Biochim. Biophys. Acta 428, 113-122. Gasiewics, T.A. and J.C. Smith, 1978. Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro. Chem. Biol. Interact. 23, 171-183. Glickstein, N., 1978. Acute toxicity of mercury and selenium to C r a s s o s t r e a gigas embryos and C a n c e r m a g i s t e r larvae. Mar. Biol. 49, 113-117. Jennings, J.R. and P.S. Rainbow, 1979. Studies on the uptake of cadmium by the crab C a r c i n u s m a e n a s in the laboratory. I. Accumulation form seawater and a food source. Mar. Biol. 50, 131-139. Kar, A.B. and R.P. Das and F.N.I. Mukerji, 1960. Prevention of cadmium induced changes in the gonads of rat by zinc and selenium. A study in antagonism between metals in the biological system. Proc. Natl. Inst. Sci. India. Pt. B. 26, (Suppl.) 40-50. Litchfield, J.T. and F. Wilcoxon, 1949. A simplified method of evaluating dose-effect experiments. J. Pharmac. Exp. Ther. 96, 99-113.
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Lucu, C. and M. Skreblin, 1981. Evidence of the interaction of mercury and selenium in the shrimp Palaemon elegans. Mar. Environ. Res. 5, 265-274. Lyon, R., M. Taylor and K. Simkiss, 1984. Ligand activity in the clearance of metals form the blood of the crayfish (Austropotamobiuspallipes). J. Exp. Biol. 113, 19-27. Magnusson, B. and L. Rasmussen, 1982. Trace metal levels in coastal sea water. Investigation of Danish waters. Mar. Pollut. Bull. 13, 81-84. Magos, L. and M. Webb, 1980. The interactions of selenium with cadmium and mercury. CRC Crit. Rev. Toxicol. 8, 1-42. Nishiyama, S., K. Nakamura, and Y. Konishi, 1987. Effect of selenium on blood pressure, urinary sodium excretion and plasma aldosterone in cadmium-treated male rats. Arch. Toxicol. 59, 365-370. Pymbroeck, S.L.C. van, W.J.J. Stips and O.L.J. Vanderborght, 1982. The antagonism between selenium and cadmium in a freshwater mollusc. Arch. Environ. Contam. Toxicol. 11, 103-106. Rasmussen, E., 1973. Systematics and ecology of the Isefjord fauna (Denmark). Ophelia 11, 1-507. Robertson, J.D., 1960. Ionic regulation in the crab Carcinus maenas (L.) in relation to the moulting cycle. Comp. Biochem. Physiol. 1, 183-212. Wright, D.A., 1977a. The effect of salinity on cadmium uptake by the tissues of the shore crab Carcinus maenas. J. Exp. Biol. 67, 137-146. Wright, D.A., 1977b. The uptake of cadmium into the haemolymph of the shore crab Carcinus maenas: the relationship with copper and other divalent cations. J. Exp. Biol. 67, 147-161. Wright, D.A. and C.C. Brewer, 1979. Cadmium turnover in the shore crab Carcinus rnaenas. Mar. Biol. 50, 151-156.
1
AQT 00282
Interaction between selenium and cadmium in the hemolymph of the shore crab C a r c i n u s m a e n a s (L.) Poul Bjerregaard Institute o f Biology, Odense University, Campusvej 55, DK-5320 Odense M, Denmark (Received 15 June 1987; revision received 8 December 1987; accepted 23 January 1988)
The effects of selenium on survival and binding of cadmium in the hemolymph of cadmium-exposed shore crabs Carcinus maenas (L.) were studied in a series of laboratory experiments. Selenite in the seawater neither prolongs survival time nor counteracts disturbance of calcium regulation in crabs exposed to lethal cadmium concentrations. Crabs in the moulting stage appear more sensitive to cadmium than intermoult crabs. Exposure to selenite concentrations above approximately 300 #g Se-SeO]-/l leads to elevated selenium concentrations in the hemolymph, whereas lower selenite concentrations do not. Selenite concentrations above approximately 200 #g Se-SeO2-/l result in augmented levels of cadmium in the hemolymph of crabs exposed to 200 #g Cd/1. Cadmium is eliminated from the hemolymph of selenite- and non-selenite exposed crabs according to first order kinetics with half-lives of 51 and 7 h, respectively. In the selenite-exposed crabs, higher protein concentrations in the hemolymph were associated with greater retention of cadmium. Initial uptake rates for cadmium in the hemolymph of individual crabs are positively correlated with hemolymph protein concentration. Initial uptake rates for cadmium are not affected by selenite exposure. The binding affinity of the hemolymph for cadmium is positively correlated with the protein concentration of the hemolymph, and is unaffected by selenite exposure. Selenite exposure (from 35 #g Se-SeO]-/1) augments cadmium uptake in the gills, whereas no consistent effect is seen in hepatopancreas, hypodermis and carapace. Key words: Selenium; Cadmium; Hemolymph; Protein; Binding; Carcinus maenas
INTRODUCTION
Since the discovery by Kar et al. (1960) that selenium protects rat testes against the toxic effects of cadmium, the interactions between selenium and cadmium and mercury in mammals have been intensively studied (reviewed by Magos and Webb, 1980). However, the exact mechanism by which selenium both augments uptake and reduces the toxicity of cadmium has not been elucidated (Chmielnicka et al., 1983).
Correspondence to: P. Bjerregaard, Inst. of Biology, Odense University, Campusvej 55, DK-5320 Odense M, Denmark. 0166-445X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
In aquatic invertebrates the interaction between selenium and cadmium is poorly understood. Exposure to selenium augments cadmium uptake in gills and hemolymph of the shore crab C a r c i n u s m a e n a s (L.), while cadmium uptake in other tissues are not affected (Bjerregaard, 1982, 1985). Cadmium taken up in the hemolymph is mainly bound in the high molecular weight proteins (hemocyanin) and selenium exposure augments binding of cadmium in this fraction (Bjerregaard, 1985). Cadmium bound in the hemolymph of C. m a e n a s is normally transferred to hepatopancreas (Wright and Brewer, 1979), but how selenium exposure affects the internal transport of cadmium in C. m a e n a s is unknown. Exposure to 10 mg Cd/1 is lethal for C. m a e n a s within 7 to 16 days (Jennings and Rainbow, 1979; Bjerregaard and Vislie, 1985), and the normal regulation of calcium concentrations in the hemolymph is severely affected (Bjerregaard and Vislie, 1985). Whether or not exposure to selenite can protect C. m a e n a s from the toxic effects of cadmium is unknown. This study investigates the effect of selenium on binding and transport of cadmium in the hemolymph of C. m a e n a s . Furthermore, the protective effect of selenium with regard to the toxic effects of cadmium is investigated. MATERIALS AND M E T H O D S
Adult male shore crabs C. m a e n a s were obtained from Lillebaelt, Denmark. In experiments carried out from May to October freshly caught crabs were used. Crabs used in experiments from October to April were caught in October and kept in flowing sea water aquaria at the Marine Biological Station, Bogeberg, N.E. Funen. Small crabs (15-26 g w.w.) were acclimated to laboratory conditions for one week prior to use in experiments. Experiments 1, 2, 4, and 5 were carried out at a salinity of 400 mOsm (14%0) and Exp. 3 at 670 mOsm (20%o). The experimental temperature was 15.5+0.5°C. Crabs were exposed to cadmium as CdC12 and selenite as Na2SeO3. Water was changed every second or third day. The crabs were not fed. Hemolymph samples were drawn with hypodermic syringes through the arthrodial membrane of the posterior periopod. Cadmium, calcium, and protein concentrations in the hemolymph were determined as described by Bjerregaard (1982, 1985) and Bjerregaard and Vislie (1985) . For selenium analysis, 200 td hemolymph were digested in 2 ml of concentrated nitric acid at approximately 100 °C, evaporated to dryness, and dissolved in 0.2% nitric acid containing 1000 mg nickel/1 (added as Ni (NO3)2). Selenium was determined on a Perkin-Elmer 2380 atomic absorption spectrophotometer fitted with a graphite furnace (Perkin-Elmer HGA 300). For determination of concentrations of free and protein-bound cadmium in the hemolymph, 200 tzl hemolymph were dialyzed against 100/zl crab ringer solution (360 mM NaCI, 8 mM KCI, 22 mM MgSO4, 4 mM NaHCO3, 10 mM CaC12, pH 8.0) in dialysis chambers. Ten microliters a°9Cd (dissolved in the ringer solution) were
added to the hemolymph side and 1°9Cd was allowed to equilibrate at 4 °C. After 4 days the radioactivity on both sides of the dialysis membrane was determined. When sufficient amounts of hemolymph were available from individual crabs, 2 determinations were made. 1°9Cd was obtained from New England Nuclear and radioactivity was determined with a Searle Mark III Liquid Scintillation Counter. Two-tailed Student's t-tests were used in the statistical evaluation of the data. Probit plots of survival times were evaluated according to Litchfield and Wilcoxon's method (1949).
Experiment 1 Four groups of 25 crabs were exposed to 10 mg Cd/l and the following concentrations of selenite: 0, 0.25, 1, and 4 mg Se-SeO]-/1. Twenty-five crabs served as a control group. Mortality rate was recorded and on the 9th day of exposure approximately 0.1 ml of hemolymph was drawn from the surviving crabs (n = 23, 19, 18, 15, and 13 in the control group and groups exposed to 0, 0.25, 1, and 4 mg Se-SeO]-/1) for calcium determination. Exposure was initiated on August 13th.
Experiment 2 Twelve groups of 10 crabs were exposed to 10 mg Cd/1 and the following concentrations of selenite: 0, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 mg SeSeO2-/l. Ten crabs in uncontaminated water served as a control group. Mortality was noted daily and on day 3 and 9 approximately 0.1 ml hemolymph samples were obtained for cadmium analysis. Exposure was initiated on November 4th.
Experiment 3 Eight groups of 6 or 7 crabs were exposed to 200/~g Cd/l and the following concentrations of selenite: 0, 35, 70, 140, 281,562, 1124, and 2248 #g Se-SeO]-/1, corresponding to molar ratios Se:Cd of 0.25, 0.5, 1, 2, 4, 8, and 16 in the Se-exposed groups. After 28 days hemolymph samples were drawn from the surviving crabs which were then killed by freezing. Selenium and cadmium concentrations in the hemolymph and cadmium concentrations in gills, hypodermis, hepatopancreas, and carapace were measured. The exposure was initiated on August 20th.
Experiment 4 Four groups of 5 crabs were exposed to the following combinations of cadmium and selenite (~g Cd/l:/zg Se-SeO2-/1): 0:0, 200:0, 200:2248, and 0:2248. Exposure was initiated on January 10th. After 24 days 5 crabs from each group were placed in separate 4-1 aquaria. Fifty
microliters of crab ringer solution containing approximately 200 000 dpm of l°9Cd were injected into the hemolymph of each crab through the arthrodial membrane of the anterior periopod. After the injection each crab was briefly rinsed in clean seawater and then returned to the aquarium. Thirty minutes after the injection, a sample of 0.15 ml hemolymph was drawn from each crab and elimination of l°9Cd from the hemolymph was followed during the next 28 h by sampling at 3, 10, 21 and 28 h. At 28 h the maximal obtainable amount of hemolymph was drawn and the experiment was stopped. The amount of l°9Cd in hemolymph samples and seawater after 28 h was determined. Protein concentrations and partitioning between protein-bound and free cadmium in the hemolymph were determined.
Experiment 5 On January 10th a group of 5 crabs was exposed to 2248 #g Se-SeO2-/l. Five crabs were used as control. After 38 days the 2 groups were placed in 4 1 seawater containing approximately 4000 dpm l°9Cd/ml. Hemolymph samples of approximately 0.25 ml were drawn after 20 h, 3 days, and 7 days (max. obtainable) and
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Fig. 1. Carcinus maenas. Results from toxicity experiments. (a) Probit plot of mortality in crabs exposed to 10 m g C d / l . Pooled results from groups in which selenite had no effect on survival. El: Exp. 1 ( n = 100), O : Exp. 2 (n= 100), I : mortality in control group from Exp. 1. (b) Median survival time for crabs exposed to 10 mg Cd/1 a n d varying selenite concentrations. 95070 confidence limits are shown. Symbols as in (a). (c) Calcium concentrations in the hemolymph o f control crabs and crabs exposed to 10 m g C d / l and varying selenite concentrations (Exp. 1). Significance levels ( * : 0.05, * * : 0.01, ~ - * * : 0.001) for the difference from the control group are indicated. Symbols as in (a). (d) C a d m i u m concentrations (mean ± SEM) in the hemolymph of crabs exposed to 10 m g Cd/1 and varying selenite concentrations for 3 (lower curve, n = 10) and 9 (upper curve, n as indicated) d a y s . . k indicates that the concentration is significantly ( P < 0.05) different from the group not exposed to selenite.
the amount of 1°9Cd taken up was measured. Protein concentrations in the hemolymph were measured. RESULTS Toxicity experiments In Exp. 2 none of the control crabs died during the experimental period (28 days), while 6 out of 25 control crabs died after moulting in Exp. 1 (Fig. la). For each of the 12 groups exposed to 10 mg Cd/l in Exp. 2, survival times could be fitted to straight lines (P< 0.05) in a probit plot. Median survival times are shown in Fig. lb. Survival times were not affected by selenite concentrations between 0.032 and 8 mg Se-SeO2-/l, while 16 and 32 mg Se-SeO2-/l seemed to reduce the survival times, indicating that these selenite concentrations may be toxic to C. maenas. Probit analyses of cadmium-induced mortality in crabs from the two experiments are shown in Fig. la (data from the two groups exposed to 16 and 32 mg Se-SeO2-/1 in Exp. 2 are omitted). In Exp. 2 mortality began at day 6 and survival times fitted a straight line. In Exp. 1 mortality began at day 1, and 13 crabs died between day 2 and 3. Between day 3 and 8 and day 8 and 28, respectively, survival times could be fitted to straight lines (P < 0.05) with different slopes. Examination of the crabs that died early in the experiment revealed that they had initiated the formation of a new cuticule, indicating an advanced stage in the moulting process (Drach, 1939). Exposure to cadmium augmented the calcium concentrations in the hemolymph, and selenite did not protect the crabs from this effect of cadmium (Fig. lc). There was a trend that the highest concentrations of selenite augmented uptake of cadmium in the hemolymph, but the effect was not seen consistently (Fig. ld). Experiment 3 Three of the 53 crabs died during the experimental period - all after moulting. The selenium concentration in the hemolymph of crabs not exposed to selenite in the sea water was 0.226_+0.046 #g/ml, and exposure to 35, 70, 141, and 281 #g SeSeO2-/l (with 200 #g Cd/l) did not lead to increased selenium concentrations in the hemolymph (Fig. 2a), while selenium concentrations in the hemolymph of crabs exposed to 562, 1124, and 2248 #g Se-SeO2-/1 increased with increasing ambient selenite concentration (Fig. 2a). Cadmium concentration in the hemolymph of crabs exposed to 200 #g Cd/1 was 55 _+22 #g Cd/1 (Fig. 2b). Ambient selenite concentrations at and above 281 #g SeSeO2-/1 augmented cadmium uptake in the hemolymph, while the augmenting effect of lower selenite concentrations was not statistically significant (Fig. 2b). Increases in cadmium and selenium concentrations were parallel, and the molar ratio Se:Cd in the hemolymph was in the range 3 to 8 for all of the groups (Fig. 2c).
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Fig. 2. C a r c i n u s m a e n a s . Selenium (a) and cadmium (b) concentrations and Se:Cd molar ratios (c) in hemolymph of crabs exposed to 0.2 mg C d / l and different selenite concentrations for 28 days. Mean +_ SEM for 5-7 crabs are given. * , * * and * * * indicate statistically significant difference at the 0.05, 0.01, and 0.001 levels from the group not exposed to selenite.
Cadmium uptake in the gills was augmented by all selenite concentrations tested (Fig. 3a), except for 70/~g Se-SeO]-/1 (P= 0.07). Cadmium uptake in the gills increased with increasing ambient selenite concentrations. Cadmium uptake in the carapace was not affected by selenite (Fig. 3b), and cadmium uptake in hepatopancreas and hypodermis was not affected by selenite concentrations up to 1124/zg SeSea]-/1. In hypodermis, cadmium uptake diminished after exposure to 2248 #g SeSea]-/1 (Fig. 3c), and the same trend was seen in hepatopancreas (Fig. 3d) although the difference from the control group was not statistically significant (P= 0.07).
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Fig. 3. C a r c i n u s m a e n a s . Cadmium concentrations (mean _+ SEM for 5-7 crabs) in gills (a), carapace (b), hypodermis (c), and hepatopancreas (d) of crabs exposed to 0.2 mg Cd/l and different selenite concentrations for 28 days. * and * * * indicate statistically significant difference at the 0.05 and 0.001 levels from the group not exposed to selenite.
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Fig. 4. C a r c i n u s m a e n a s . (a) Retention of :°gCd injected into the hemolymph of crabs on day 29 of exposure to 0.2 mg Cd/1 (©), 0.2 m g Cd + 2.248 m g Se-SeO2-/l (4), 2.248 m g Se-SeO2-/l (A) and control crabs (e). Mean _+ SEM for 5 crabs. (b) % ]°gCd retained after 28 h elimination shown against protein concentrations in the hemolymph for individual crabs. Symbols as in (a). (c) % free cadmium
(determined in vitro) against protein concentration in the hemolymph of individual crabs. Symbols as in (a).
Experiment 4 Elimination of cadmium from the hemolymph of unexposed crabs and crabs exposed to 200 #g Cd/1 proceeded identically (Fig. 4a). Between 3 and 28 h cadmium was eliminated according to first order kinetics with a half-life of 6.8 h (data from both groups pooled: In y = 9 1 . 3 - 0.102 x). In the two selenite exposed groups elimination of cadmium from the hemolymph proceeded with identical rates between 3 and 28 h (Fig. 4a). Half-life for cadmium in the hemolymph was 51.5 h (data from both groups pooled: In y = 81.7 - 0.014 x). After 28 h the seawater in the 4 aquaria contained 23 000, 27 000, 48 000, and 17 000 dpm l°9Cd, respectively. This corresponds to 1.7% - 4.8% of the total amount of 1°9Cd injected in the crabs of each aquaria. In the selenite-exposed groups the amount of cadmium remaining in the hemolymph of individual crabs after 28 h (Fig. 4b) was positively correlated with the protein concentration in the hemolymph of the crabs (r= 0.7332, P = 0.016). In the two groups not exposed to selenite no such correlation was found (r = - 0.0724, P = 0.84). TABLE I Carcinus maenas.
Percentage of free cadmium (determined in vitro) in the hemolymph of crabs from
Exp. 4. Mean +_ SD for 5 crabs. Individual values are seen in Fig. 4.
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Dialysis showed that generally 99 to 99.9% of the cadmium in the hemolymph was non-dialyzable. The percentage of free cadmium did not differ in the four groups of crabs (Table I). The percentage of free cadmium in the individual crabs was negatively correlated (r= -0.5844, P = 0.0068) with the protein concentration in the hemolymph (Fig. 4c).
Experiment 5 In the group not exposed to selenite a concentration factor (l°9Cd in hemolymph -- l°9Cd in sea water) of approximately 0.1 for cadmium was reached within 20 h and in 7 days no increase was observed (Fig. 5a). In the selenite-exposed group, the concentration factor increased significantly from 20 h to 3 (P=0.016) and 7 (P=0.013) days (Fig. 5a). After 20 h, uptake of cadmium in the hemolymph in individual crabs of the two groups was positively correlated (r= 0.8566, P = 0.0016) with the protein concentration of the hemolymph (Fig. 5b). During one week's exposure this correlation (day 3: r=0.6173, P=0.057; day 7: r=0.4387, P=0.20) gradually disappeared (Fig. 5c,d). DISCUSSION
While the protective effect of selenium against the toxic effects of cadmium and mercury in mammals has been well documented (reviewed by Magos and Webb, 1980), comparable studies on aquatic organisms are scarce. Selenite tended to protect oyster embryos and crab larvae (Glickstein 1978) and shrimps (Lucu and
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Skreblin 1981) from the acute, lethal effects of mercury, but in neither of these investigations statistically significant results were obtained. Selenite more than doubled the median survival time in the freshwater mollusc L y m n e a stagnalis exposed to a lethal cadmium concentration (Puymbroek et al., 1982). The present results show that selenite neither prolongs survival nor protects the calcium regulation in Carcinus maenas exposed to a lethal cadmium concentration. The results do not exclude the possibility that selenium administered differently (e.g. via the food) may have some protective effect. Moulting is associated with major adjustments of physiological processes especially ionic and osmotic regulation - in crabs (Robertson, 1960). As exposure to toxic concentrations of cadmium interferes with the normal ion and osmoregulatory pattern in Carcinus maenas (Bjerregaard and Vislie, 1985), it is not surprising that crabs in the stages immediately preceding the moult respond more rapidly to lethal cadmium concentrations than intermoult crabs. Rasmussen (1973) also found that the fraction of small, adult male crabs being in the premoult stage should decrease from late July to late August. Cadmium concentrations in the hemolymph of shore crabs exposed to cadmium concentrations lower than 2.4 mg Cd/l rapidly reach a steady state level at or below the ambient cadmium concentration (Wright, 1977a,b; Wright and Brewer, 1979; Bjerregaard and Vislie, 1985), while crabs exposed to cadmium concentrations above 4 mg Cd/1 concentrate cadmium in the hemolymph to levels above those in the water (Bjerregaard and Vislie, 1985). The present data support these observations. Wright and Brewer (1979) and Lyons et al. (1984) suggest that cadmium in the hemolymph of crustaceans is rapidly transferred to other organs (primarily hepatopancreas), giving low turnover times for cadmium in the hemolymph. The present results show that C. maenas exposed to background cadmium concentrations of approximately 25 ng Cd/l (Magnusson and Rasmussen, 1982) and 200 #g Cd/1 eliminate cadmium from the hemolymph at equal rates. The half-life for cadmium in the hemolymph of C. maenas is in the same range as in the freshwater crayfish Austropamobiuspallipes (Lyons et al., 1984). Exposure to selenite markedly lengthens elimination time for cadmium in the hemolymph, while the initial uptake rate for cadmium from the ambient seawater is not increased in the selenite exposed crabs. Thus, selenium leads to increased cadmium accumulation in the hemolymph by diminishing elimination rates, rather than enhancing uptake rates. This agrees with the fact that in vitro the affinity of the hemolymph proteins for added cadmium is identical in selenite- and non-selenite exposed crabs. Concurrent administration of selenite to cadmium-treated rats greatly increases binding of cadmium in the blood plasma (Nishiyama et al., 1987; Gasiewicz and Smith, 1976, 1978). Gasiewicz and Smith (1976, 1978) suggest that the effect of selenite on cadmium binding depends on the metabolic conversion of Se(IV) to Se( - II), which is supposed to form a protein stabilized CdSe complex with a molar
10 ratio Cd:Se close to one. Such a m e c h a n i s m m a y also be responsible for the interaction b e t w e e n selenium a n d c a d m i u m in the h e m o l y m p h o f the crab, a l t h o u g h 1:1 m o l a r ratios b e t w e e n the two elements are n o t necessarily o b t a i n e d (Bjerregaard, 1985). The processes, which t r a n s p o r t c a d m i u m f r o m the h e m o l y m p h to the tissues are largely u n k n o w n . U p t a k e o f c a d m i u m f r o m the h e m o l y m p h into the h e p a t o p a n creas a n d h y p o d e r m i s is n o t affected b y exposure to selenite c o n c e n t r a t i o n s which lead to s u b s t a n t i a l increases in the total c a d m i u m c o n c e n t r a t i o n o f the h e m o l y m p h . This might indicate that t r a n s p o r t into these tissues derives f r o m the free pool o f c a d m i u m (not affected by exposure to selenite) in the h e m o l y m p h . H o w e v e r , this hypothesis w o u l d n o t a c c o u n t for the a p p a r e n t l y lower u p t a k e o f c a d m i u m in the h e p a t o p a n c r e a s a n d the h y p o d e r m i s for the highest selenite c o n c e n t r a t i o n tested. Internal t r a n s p o r t a n d b i n d i n g o f heavy metals require f u r t h e r investigation, ACKNOWLEDGEMENTS I t h a n k Mrs. Vibeke E r i k s e n a n d Mr. Rene Stoving for technical assistance a n d Dr. Michael H. Depledge for critically r e a d i n g the m a n u s c r i p t . The project was supported by grants f r o m the D a n i s h N a t u r a l Science Research C o u n c i l a n d the T h o m a s B. Thriges F o u n d a t i o n . REFERENCES Bjerregaard, P., 1982. Accumulation of cadmium and selenium and their mutual interaction in the shore crab C a r c i n u s m a e n a s (L.). Aquat. Toxicol. 2, 113-125. Bjerregaard, P., 1985. Effect of selenium on cadmium uptake in the shore crab C a r c i n u s rnaenas (L.). Aquat. Toxicol. 7, 177-189. Bjerregaard, P. and T. Vislie, 1985. Effects of cadmium on hemolymph composition in the shore crab C a r c i n u s m a e n a s . Mar. Ecol. Prog. Ser. 27, 135-142. Chmielnicka, J., E.M. Bem and P. Kaszubski, 1983. Organ and subcellular distribution of cadmium in rats exposed to cadmium, mercury, and selenium. Environ. Res. 31,273-278. Drach, P. 1939. Mue et cycle d'intermue chez les Crustaces Decapodes. Ann. Inst. Oceanogr. 19, 103-391. Gasiewics, T.A. and J.C. Smith, 1976. Interactions of cadmium and selenium in rat plasma in vivo and in vitro. Biochim. Biophys. Acta 428, 113-122. Gasiewics, T.A. and J.C. Smith, 1978. Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro. Chem. Biol. Interact. 23, 171-183. Glickstein, N., 1978. Acute toxicity of mercury and selenium to C r a s s o s t r e a gigas embryos and C a n c e r m a g i s t e r larvae. Mar. Biol. 49, 113-117. Jennings, J.R. and P.S. Rainbow, 1979. Studies on the uptake of cadmium by the crab C a r c i n u s m a e n a s in the laboratory. I. Accumulation form seawater and a food source. Mar. Biol. 50, 131-139. Kar, A.B. and R.P. Das and F.N.I. Mukerji, 1960. Prevention of cadmium induced changes in the gonads of rat by zinc and selenium. A study in antagonism between metals in the biological system. Proc. Natl. Inst. Sci. India. Pt. B. 26, (Suppl.) 40-50. Litchfield, J.T. and F. Wilcoxon, 1949. A simplified method of evaluating dose-effect experiments. J. Pharmac. Exp. Ther. 96, 99-113.
11
Lucu, C. and M. Skreblin, 1981. Evidence of the interaction of mercury and selenium in the shrimp Palaemon elegans. Mar. Environ. Res. 5, 265-274. Lyon, R., M. Taylor and K. Simkiss, 1984. Ligand activity in the clearance of metals form the blood of the crayfish (Austropotamobiuspallipes). J. Exp. Biol. 113, 19-27. Magnusson, B. and L. Rasmussen, 1982. Trace metal levels in coastal sea water. Investigation of Danish waters. Mar. Pollut. Bull. 13, 81-84. Magos, L. and M. Webb, 1980. The interactions of selenium with cadmium and mercury. CRC Crit. Rev. Toxicol. 8, 1-42. Nishiyama, S., K. Nakamura, and Y. Konishi, 1987. Effect of selenium on blood pressure, urinary sodium excretion and plasma aldosterone in cadmium-treated male rats. Arch. Toxicol. 59, 365-370. Pymbroeck, S.L.C. van, W.J.J. Stips and O.L.J. Vanderborght, 1982. The antagonism between selenium and cadmium in a freshwater mollusc. Arch. Environ. Contam. Toxicol. 11, 103-106. Rasmussen, E., 1973. Systematics and ecology of the Isefjord fauna (Denmark). Ophelia 11, 1-507. Robertson, J.D., 1960. Ionic regulation in the crab Carcinus maenas (L.) in relation to the moulting cycle. Comp. Biochem. Physiol. 1, 183-212. Wright, D.A., 1977a. The effect of salinity on cadmium uptake by the tissues of the shore crab Carcinus maenas. J. Exp. Biol. 67, 137-146. Wright, D.A., 1977b. The uptake of cadmium into the haemolymph of the shore crab Carcinus maenas: the relationship with copper and other divalent cations. J. Exp. Biol. 67, 147-161. Wright, D.A. and C.C. Brewer, 1979. Cadmium turnover in the shore crab Carcinus rnaenas. Mar. Biol. 50, 151-156.