Camp, Biochem. Physiol. Vol. 82C, No. 1, pp. 227.-230, 1985 0306~4492/85 $3.00 + 0.00 Printed in Great Britain 0 1985 Pergamon Press Ltd

EFFECTS OF MERCURY ON ION AND OSMOREGULATION IN THE SHORE CRAB CARCINUS iMAENAS (L.)

POUL BJERREGAARD and TONE VISLIE Institute of Biology, Odensc University, Campusvej 55, DK-5230 Odense M, Denmark.

Telephone: (09) 15-8600

(Received 5 March 1985)

Abstract-l. The effects of lethal mercury concentrations on regulation of hemolymph electrolytes in the shore crab Car&us maenas were investigated.

2. In most experiments mercury exposure reduced hemolymph osmolality and Na+, Cl- and K+ levels to 5590% of controls in 48 hr.

3. Mercury exposure augmented calcium levels to 120-300% of controls. 4. The effect of mercury on all of the electrolvtes varied during the vear. especially on maenesium levels

which showed significant increases or decreases, or

At salinities less than 33% the shore crab Curcin~s muenas (L.) normally maintains osmofahty and Na+, Cl-, K+ and Ca2+ concentrations above those of ambient seawater (Webb, 1940; Robertson, 1960; Shaw, 1961; Theede, 1969; Greenaway, 1976; Zan- ders, 1980a), while Mg2+ concentrations are held substantially below ambient seawater concentrations in IS-150~0 seawater (Lockwood and Riegel, 1969; Zanders, 1980b).

The toxicity of mercury towards marine and estu- arinc crustaceans increases with decreasing salinity (Roesijadi et at., 1974; Jones, 1975). Regulation of hemolymph osmolality in the isopod Jaera al~l~o~ is affected by mercury exposure (Jones, 1975). Copper and cadmium affect osmoregulation in C. maenas (Thurberg ef al., 1973; Bjerregaard and Vislie, 1985a,b), but the effect of mercury on regulation of osmolality and individual hemolymph electrolytes has never been investigated in C. maenas.

The present study investigates the effects of dis- solved mercury on regulation of hemolymph os- molality and Na+, Cl-, K+, CaZ+ and Mg2+ concen- trations in C. maenas.

MATERIALS AND METHODS

Adult, male shore crabs (Carcinus maenas) were obtained

-~,. . remained unaffected in different expehments.

from Little Belt. Animals used for experiments in November were caught in October and kept in flowing seawater aquaria at the Marine Biological Station, Bagebjerg, N.E. Funen. In the May, June and August experiments freshly caught crabs were used.

Prior to experiments the crabs were acclimated to the experimental salinity (400 & 10 mOsm) and temperature (15.5 k O.YC) for 7-8 days. Experimental seawater was made by diluting Great Belt seawater with distilled water. The crabs were not fed during acclimation and experimental periods.

In four experimental series groups of four to six crabs were exposed to mercury (added as HgCl,) in 101. poly- styrene aquaria; water was changed every second day. Table 1 summa&es experimental conditions in the different ex- perimental series.

Hemolymph samples of approximately 200 ~1 were drawn through the arthrodial membrane with hypode~ic needles. Crabs exposed to 0.25, OS and 1.0 mg Hgjl were sampled at 0 and 48 hr (and in a single group at 96 hr). Crabs exposed to 10 mg Hg/l were sampled after 0 and 24 hr. To prevent coagulation the hemolymph samples were immediately transferred to 0°C and maintained at this temperature until osmolality measurements and dilutions for cation deter- minations had taken place. The remaining part of the hemolymph samples were stored at - 18°C for subsequent chloride determination.

Osmolality was measured with a Knauer semimicro os- mometer and sodium, potassium, calcium and magnesium were determined by atomic absorption spectroscopy (Beck- mann 1248 and Perkin-Elmer 2380). Mixed standards with

Table I. Car&us nzoenas. Exposure concentrations and numbers of crabs in individual experiments. H~molymph parameters (mean k SD) at day 0 in control crabs of individua1

exoeriments are &en

Experimental series 1-3 June 22-26 Aug. 28-30 Nov. 15-17 May __” . .._.___ ..-_ -..._

N (control) 8 12 10 10 N (0.25 mg Hg/l) * 6 * 5 N (0.50 mg Hg/l) * 6 5 l

N (1 .O Hg/l) mg 5 6 5 5

N (10mg Hg/l) 5 l * *

Osmolality (mOsm) 694 i: 40 735 + 36 659 + 23 765 + 23 Na+ (mM) 361i to 349* 15 3121 II 384 k 14 Cl- (mM) 366 rt 30 321 f 32 342 + 23 383 Itr 22 K+ (mM) 8.2 i: 0.6 8.5 i 0.6 7.5 jI 0.8 8.3 k 0.2 Ca*+ (mM) 7.8 rt 0.6 7.7 + 0.8 7.9 t_ 1.1 8.8 & 1.0 Mg2+ (mM) 11.7i.O.8 10.4 f 1.4 10.9 Sr 1.6 11.3 & 1.0

*Not tested.

227

228 POUL BJERREGAARD and TONE VISLIE

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Fig. 1. Carcinus maenm. Hemolymph parameters in crabs exposed to 0.25 (C)), 0.5 (o), 1 .O (A) and 10 (0) mg Hg/l. Mean values in all of the groups are set to 100% at day 0. Parameters in the exposed groups are expressed relative to values in the control group during experiments. a, l l and I) 0 0 indicate

that differences from the control group are significant at 0.05, 0.01 and O,OOt level, respectively.

ion concentrations similar to crab hemolymph were em- ployed to eliminate interactions between the determination of individual ions. La@, (final concentration: O.l’k La) was added to prevent phosphate interactions. Chloride deter- minations were performed with a Radiometer CMT f0 chloride titrator.

The statistical significance of differences between exposed groups and control groups were calculated by means of two-tailed Student’s 1 tests.

RESULTS

Crabs exposed to 10, 1, 0.5 and 0.25 mg Hg/l showed median survival times of approximately 1, 3, 5 and 7 days, respectively.

Hemolymph parameters of the crabs at day 0 in each experiment are shown in Table 1.

Exposure to 10 mg Hg/l significantly reduced os- modality and Na’, Cl- and K” levels in 24 hr (Fig. 1). I mg Hg/l reduced K+ levels after 48 br in ah of the experiments. Na+ and Cl- levels and osmolalilty were reduced by 1 mg Hg/l in May, June and Novem- ber, while no effect was observed on these three parameters in August (Fig. 1). Exposure to 0.5 mg Hgjl did not affect osmoiality and Na*, Cl- and Ki levels in August, while osmolality and Na+ levels were significantly reduced in November (Fig. 1). 0.25 mg Hg/l caused a transient increase in Na+ levels after 48 hr exposure in August, while K* and

Hg affects Curcinus osmoregulation 229

plays a minor, yet significant role (Shaw, 1961). Siebers e# ul. (1982) showed that Na +-K +-ATPase activity in the three posterior gills increased when the crabs were exposed to dilute seawater and they concluded that this Na+-K+-ATPase plays an im- portant role in ionic regulation.

50' My June July Aug Se@ Ott WV Dn:

Tim of year

Fig. 2. Curcinus maenas. Hemolymph parameters in crabs exposed to 1 mg Hg/l for 48 hr. Expressed as a percentage of hemolymph parameters in control groups. Osmolality (01, Na+ (LL), Cl- (0). K+ (01, Cal+ (Y) and Mg’+ (0).

Mercury has been shown to affect transport of Na+, Cl- and K+ in aquatic organisms in several ways. Bouquegneau (1977) concluded that loss of the NaCl balance in seawater-adapted eels (~~g~~~~u an - guilfa) after exposure to mercury could be attributed to inhibition of gill Na+--K+-ATPase activity. Mer- cury reduces ion gradients in flounder (Pseudo- pleuronrctes americanus) renal tubules (Miller, 1981) by inhibiting Na+-K+-ATPase activity as well as increasing membrane permeability to Na+ and K+. Lock et al. (1981) concluded that reductions in plasma osmolality and Na+ and Cl- concentrations in freshwater-acclimated rainbow trout Saho gai- rdneri exposed to sublethal mercury concentrations can be attributed to increases in gill water perme- ability rather than to inhibition of gill Na+-K+- ATPase activity, although the latter is observed at lethal mercury concentrations.

Cl- levels and osmolality were not affected during four days exposure; in May, however, 0.25mgHgjl reduced Na+ and K+ levels, but not osmolaliIty and Cl- levels (Fig. 1).

Exposure to 0.25-10 mg Hg/l caused significant increases in the Ca ‘+ levels in all of the experimental series (Fig. 1).

In June and November marked increases of mag- nesium concentrations were observed after exposure to 10 and 1 mg Hg,/l (June) and 0.5 and I mg Hg/l (November) (Fig. 1). In August, however, I mg Hg/l significantly reduced magnesium levels, while 0.25 and 1 mg Hg/l caused no effects in May (Fig. 1). In August 0.25 and 0.5 mg Hg/l did not significantly alter magnesium levels, although a diminishing trend after 2 days was observed (Fig. 1).

The changes in osmolality and Na+, Cl- and K+ concentrations caused by mercury exposure in C. maenas may be attributed to inhibition of gill Na+-K+-ATPase activity as well as to increased ion permeabilities of the gills. Time constants for Na+ efflux in C. maenas in 40% seawater vary between 11.1 hr (Zanders, t980a) and 6.4 hr (Shaw, 1961), and the time constant for Cl- efflux is approximately two to three times higher (time constants for Na+ and Cl- efflux in lOOP:, seawater: 10.3 and 25.3 hr, re- spectively; Zanders, 1980a). With these flux rates even the fastest observed changes in hemolymph Na+ and Cl- concentrations induced by mercury could be explained by inhibition of gill Na+-K+-ATPase and subsequent flux of Na+, Cl- and K+ from hemo- lymph to seawater. Further studies are, however. necessary to show if gill permeability or maybe the structural integrity of the gills (Bubel, 1976) could be affected by mercury exposure.

In all of the four experimental series crabs were exposed to 1 mg Hg/l and Fig. 2 shows how the effects of this exposure concentration varied during the year. Calcium concentrations in the hemolymph were affected far more in June than in May, August and November, and fluctuations in the effects on calcium concentrations seemed to be followed by fluctuations in the effects on magnesium concen- trations. Effects on osmolality and Na+, K+ and Cll concentrations showed less variation during the year.

Bouquegneau (1973, 1977) concluded that the lethal effect of mercury towards seawater-adapted eels (A. anguilla) could be attributed to the dis- ruption of the NaCl balance in the animal. Although mercury does affect regulation of osmolality and Na+, Cl- and K+ concentrations in C. maenas, it is not reasonable to assume that the effects on these hemolymph electrolytes could be the direct cause of death after mercury exposure, since crabs exposed to 0.5 and 0.25 mg Hg/l (August) began to die after 3 and 5 days exposure, respectively, without any changes in these parameters.

DISCUSSION

Exposure to lethal mercury concentrations gener- ally reduces hemolymph osmolality and Na+, Cll and K+ concentrations in C. maenas and the effects of these four parameters tend to follow each other.

Regulation of Na+, K+ and Cl- concentrations in the hemolymph of shore crabs in dilute seawater takes place mainly by an increased uptake over the gills (Webb, 1940; Shaw, 1961; Zanders, 1980a), while reabsorption of these ions from the primary urine

Carcinus maenas normally maintains its hemo- lymph calcium concentrations higher than ambient calcium concentrations. Part of the hemoIymph cal- cium is bound to proteins (approximately 32%; Greenaway, 1976), but at 400 mOsm crabs maintain free calcium concentrations in the hemolymph (4.8 rt: 0.7 mM; Bjerregaard and Vislie, 1985a) that are higher than free calcium concentrations in the seawater (approximately 3.7 mM; Greenaway, 1976).

The mechanism for mercury’s augmenting effect on hemolymph calcium levels is unknown. It does not

230 Pout BJERREGAARD and TONE VISLIE

seem very likely that exposure to mercury should stimulate the calcium transporting system of the gills. On the contrary, Shephard and Simkiss (1976) found that 0.4 mg Hg/l inhibited roach (Ruti~~s mfih) gill Ca-ATPase in vitro.

Another possible explanation for the increase in calcium concentrations in the hemolymph may be that mercury exposure induces release of calcium from the major calcium storing tissues-exoskeleton and hepatopancreas----to the hemolymph. Roer (1980) has shown that active transport of calcium over the hypodermis takes place by means of a Ca-ATPase and a Na/Ca exchange mechanism. If mercury inhibits active transport of calcium from hemolymph to exoskeleton, hemolymph calcium lev- els would increase due to passive influx of calcium from the exoskeleton (Roer, 1980). Mercury could reduce active calcium transport directly by inhibiting the Ca-ATPase or indirectly by inhibiting the oua- bain sensitive Na+ transport and thereby the Na/Ca exchange pump.

The effect of exposure to mercury on hemolymph calcium levels resembles the effect of injection of molt-promoting hormone from the Y organ (Carlisle, 1957). In the intermolt stage secretion of the molt- promoting hormone is inhibited by the molt- inhibiting hormone-a poly~ptide-produced in the X organ-sinus gland complex (Carlisle, 1957). If mercury affects the production or function of the molt-inhibiting hormone the observed changes in calcium levels would be expected in association with the partial resorption of the exoskeleton initiating the molt processes (Robertson, 1937; Roer, 1980). Male C. maenns in Danish waters molt in June-July (Ras- mussen, 1973) and the stronger effect of mercury in the premolt-mob period would be expected if mer- cury exerted its effect both on hormonal regulation and directly on calcium transport processes in the hypodermis.

Fluctuations in the effects of mercury on mag- nesium levels during the year partly seem to follow fluctuations in the effects on calcium levels (Fig. 2). However, the mechanism lying behind mercury’s effect on calcium as well as magnesium levels in C. maenas needs further investigations.

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