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Mercury and osmoregulation in the euryhaline crab, Eriocheir sinensis
Camp. Biochem. Physiol. Vol. 113C, No. 2, pp. 149-155, Copyright 0 1996 Elsevier Science Inc.
1996
ISSN 0742-8413/96/$15.00 SSDI 0742s8413(95)02081,0
ELSEVIER
Mercury and Osmoregulation in the Euryhaline Crab, Eriocheir sinensis A. E’iqueux,A. Bianchini
l
and R. Gil/es
LABORATORY OF ANIMAL PHYSIOLOGY, UNIVERSITYOF L&GE, INSTITUTEE. VAN BENEDEN,22, QUAI VAN BENEDEN,B4020 LIBGE, BELGIUM
ABSTRACT. This review will focus on cases where it might be possible that the toxicity of the heavy metal mercury results from an interaction with osmoregulatory mechanisms. It is shown that mercury-induced impairment of osmoregulatory capability in the sense of severe modifications of the blood osmotic concentration is more pronounced in brachyuran decapod species adapted to dilute waters. The rationale for considering these effects is based on a comparison between mercury effects on three species of decapod crustaceans exhibiting various degrees of osmoregulation capability: the strong regulator crab Eriocheir sinensis, the weak regulator Carcinus maenas, both of them being euryhaline, and the stenohaline osmoconformer Cancer pagums. It is established that a synergistic effect exists between salinity and HgC& toxicity in euryhaline species which are hyperregulators in dilute media, that is, E. sinensis and C. mae~s only. Depth study of E. sinensis as a model demonstrates that Na+ and Cl- permeabilities of the gill epithelium is affected by mercury, as well as the Na+ and Cl- active transport processes located at the same level. Evidences are brought showing that mercury drastically disturbs the Na+/K+ pump and the Cl- channels located in the serosal baso-lateral membranes of the posterior gills. COMPBIOCHEMPHYSIOL113c, 149-155, 1996. KEY WORDS. Crustaceans, gills, Eriocheir sinensis, Cur&us difference, active transport, sodium flux, chloride flux
INTRODUCTION
When considering the various sources and kinds of pollutants, it is clear that pollution by heavy metals, either natural or related to human activity, is the type most extensively documented and studied (see for example, 3,13,12,5,7,4). Most of the works devoted to these effects are concerned with the definition of mortality graphs and of tolerance limits, with reports on bioaccumulation, on effects on reproduction, on teratology, so that information on crab-heavy metal interactions at the molecular level remains fragmentary, superficial, essentially scanty. In these studies, it is often impossible to distinguish between real synergistic effects and interactions between pollutants and basic mechanisms concerned with blood or cell osmolarity regulation. It has been reported that estuarine species of crustaceans
maenas, Cancer pagurus, crab, mercury, potential
are particularly susceptible to heavy metals. Moreover, several studies have emphasized the importance of a salinity stress in enhancing heavy metal toxicity (see for example 2,6). Intuitively, these findings support the idea that osmoregulatory processes in crustacean species confronted with dilute media might be among the primary sites to be affected by these toxics. Surprisingly, that question has previously received little attention up to now. This paper will focus on the observation that the toxicity of mercury results from an interaction with osmoregulatory mechanisms. The results originate from experiments conducted in our laboratory on the strong osmoregulator Eriocheir sinmsis (see also 1).
MATERIALS
AND
METHODS
Material Address reprint requests to: A. PCqueux, Lab. of Animal Physiology, Univ. of Liege, Institut E. Van Beneden, 22, quai Van Beneden, B4020 Likge, Belgium *Present address: Universidad do Rio Grande Laboratorio de Zoofisiologia, Rua E.A. HUCH, 475, CATXA Postal 474, 96.201-900 Rio Grande-RSBrasil. Based on a presentation given at the 15th Annual Conference of the European Society for Comparative Physiology and Biochemistry held in Genoa, Italy, in September 1994. The Conference title was: Biochemical and physiologicaleffects of poUution and toxicological assessment of environmentaI wk Received
17 October
1995; accepted
2 November
1995.
Experiments reported in this paper were carried out on three species of crab, that is, the strong hyperregulator Eriocheir sine&, the weak regulator Curcinw maenas and the stenohaline osmoconformer Cancer pagums. However, the largest part of the experiments were performed on isolated and perfused anterior and posterior gills of the euryhaline Chinese crab Erioc&r sinensis. Chinese crabs were caught in autumn in freshwater (FW) lakes near Emden in the North of Germany. They were transferred to the aquarium of the University of Liege (Belgium) and kept in tanks of circulating FW.
150
Methods Chronic poisoning by mercury was achieved by adding HgCl, 0.1 ppm or 1 ppm to the external medium (50 1. FW glass tanks). Medium was renewed each day and regularly controlled. Gills were perfused and the transepithelial potential difference (PD) was measured following procedures already described in detail (8,9,10). Gills were handled in, incubated in and perfused by a “freshwater (FW) saline”, the composition of which corresponded to the ionic composition of the blood of FW crabs. It contained (in mM): NaCl, 240; KCl, 5; MgCl,,5; CaCl,, 12.5; H,BOX, 8.8. The pH was adjusted at 7.6 with Tris-base. When announced, HgClz (10 ppm) was added to the incubation (OUT) or to the perfusion (IN) media. The transepithelial movements of Na+ and Cl-- were estimated using the radioactive tracers 22Naf and ‘Q(0.20 and 0.25 /.&ml-‘, respectively). Tracers were added either to the incubation medium (influx) or to the perfusion saline (efflux). “Na+ was using counted in a y scintillator, 36Cl- in a /3 scintillator, Pica Fluor TM30 (Packard) as scintillation cocktail. Hemolymph Na+ and K+ ions were measured in blood aliquots adequately diluted, by means of a flame photometer (Eppendorf); blood Clwas measured by coulometryamperometry with a Buchler-Cotlove chloridometer. RESULTS AND DISCUSSION Toxicity of Mercury to Whole Crustacean Species In a previous review, the toxicity and biological effects of different groups of chemical pollutants, among which heavy metals and mercury have been considered, included scanty evidence of synergistic actions where toxicity could be related to interactions with osmoregulatory mechanisms (5). More recent studies have been conducted in this laboratory on three crab species, i.e. the strong hyperregulator Eriocbir sinensis, the weak regulator Carcinus maenu-.s, both of them being euryhaline, and the stenohaline osmoconformer Cancer pagurus, chronically poisoned with HgCl, 1 ppm. applied in the external medium. Mercury exposure has always proved to be lethal for the three species under consideration. However, in the Chinese crab Eriocheir sinensis, H&l, has appeared to be much more toxic for FW animals (LTSO: 40 hr) than for SW/2 (18%) (LTSO = 77 hr) or SW (35%ti) animals (LT50 = 75 hr). In the shore crab C. maenas, HgCl, was more toxic too for SW/3 (11%) animals (LT50 = 37 hr) than for SW animals (LT50 = 70 hr). Surprisingly, the SW osmoconformer C. pagurus was more sensitive to HgCl, (LT50 = 43 hr) than other species but it didn’t exhibit any salinity linked sensitivity. These results corroborate the fact that osmoregulating euryhaline species in dilute media are much more susceptible to mercury salts than in full strength seawater. In gills and shell of the euryhaline species, the rate of up take was faster in dilute SW crabs than in SW acclimated
A. Pkqueux et al.
ones, while there was no significant difference in rate of uptake as a function of salinity in the osmoconformer C. pagurus. Moreover, when the digestive tract of E. sine&s was made unfunctional by occlusion of the mouth with dental cement and whatever the salinity of acclimation, Hg+ + accumulation did not change in all organs and tissues studied but the digestive tract itself where mercury content dropped down by mote than 50%. In conclusion, it is thus clear that osmoregulating crab species, when a hemolymph-ambient water gradient exists, are much more sensitive to mercury than conforming species in salinity and that organs involved in ion balance such as the gills accumulate much more pollutant. Intuitively, such a huge accumulation of toxicants at that level must lead to faster impairment of the mechanisms there at work. These findings support the idea that osmoregulatory processes in these species might therefore be among the primary sites to be affected by mercury. This finding prompts us to suggest that the toxicity of HgCl, for hyperregulating crabs could be due to impairment of the maintenance of the blood ionic balance.
Effect of Mercury
on Crustacean
Hemolymph
Composition
Surprisingly, the effects of mercury salts on crustacean hemolymph composition has received little attention and data remain essentially scanty. Among the few most recent studies, Bjerregaard and Vislie (2) reported a decrease of blood osmolarity and inorganic ions content in green crabs Carcinus maeTUISacclimated to reduced salinity (400 mosm.l- ‘) and exposed to 1 and 10 ppm HgC12. In Gammarus, Johnson and Jones (6) established that the depression of hemolymph osmolality was the result of reduced levels of blood sodium. In E. sinensis too, a severe decrease of the Na+ and Cl- blood content occurs during poisoning with 0.1, 1 and 10 ppm HgCl,. Figure 1 illustrates the effects of 1 ppm HgCl, on SW (35%os), SW/2 (18%) and FW acclimated Chinese crabs. It is clear that the decrease in Na+ and Cl- concentration occurs in FW crabs only within hr. There is no significant effect of a several days exposure in SW and SW/2 animals, as there is no effect too in SW and SW12 acclimated stone crabs C. pagurus (Pequeux and Gilles, unpublished results). Undoubtedly, these results corroborate the above hypothesis of a mercury-induced impairment of the maintenance of the blood ionic balance in regulating crabs at ambient concentrations where the hemolymph-ambient seawater difference is highest.
Tissue Physi&gy
4 Regulating
Crabs
In an attempt to understand and explain the mechanism of action of heavy metals that leads to a disturbance of the osmoregulatory function in crustacea, studies have been conducted in this laboratory on the effects of mercury on the gill tissue
Mercury and Osmoregulation in Crabs
151
E. siaensis HgCI’ (1wW
FW
H&f (1ppm)
0
If:::
20
Days (SW andSW/2crabs)
w t
E. sinensis
Ant. gills
10
Na+
0
SW/l l
-
-
__L___.
-10
0
5
/
-20
Hours (F-W crabs) 24
12
T dI
-0-o
T yi-_PyT
T
Post. gills
1
1
-30 -40
I
I
I
I
1
0
1
2
3
4
Time of exposure (days) 0
5
0 ,
1’
Hours (Fw crabs) 24
12
;
;’
; Days(SWrndSW/2crrbs)
‘\t/i-f”-
clF\\
loo
I?
m
0
s
I2
Hours(FN crabs) 24
Time of exposure to HgCI,
1. Effect of HgCl, (1 ppm in external medium) on hemoe lymph Na+, K+ and Cl- contents (mequiv.1 -‘) of Chinese crabs Eriocheir sinensis acclimated to FW, SW/2 and full SW. Time of exposure to HgCl, is hours or days. FW = freshwater; SW = sea water.
FIG. 2. Chronic poisoning of FW Chinese crabs E. sinensis with 1 ppm HgC&: transepithelial electrical potential difference (mV) across epithelium from isolated perfused anterior (Ant.) and posterior (Post.) gills as a function of the exposure time to the pollutant. Gills are bathed on both sides with the same FW saline (Na+ 240 mequiv.l-‘; Cl- 280 mequiv.l-I). Mean red suits of 6 experiments + SD. CHRONIC POISONING OFWHOLECRABS. The transepithelial electrical potential difference measured in vitro across both kinds of gills is progressively abolished as a function of the time of exposure of the crabs to mercury (Fig. 2). In first analysis, this suggests a severe impairment of the permeability and/or of the transport properties of the tissues.
FIG.
2500
r 2000 ‘k
1
T
‘:
of Chinese crabs, E. sinensis. Experiments have been carried out on the perfused preparation of isolated gill that is used in this laboratory to investigate the part played by the gills in the overall osmoregulation function in crabs (11). The anteriorly-located pairs of gills are respiratory and the posteriorly by well-developed saltlocated gills are characterized transporting epithelium and have been demonstrated to be involved primarily in salt transport and hydromineral regulation (11). Two series of experiments have been conducted: (1) experiments on gills isolated from crabs which were poisoned chronically with 1 ppm HgCl,, and (2) experiments where gills from non-poisoned crabs were acutely and directly poisoned with 10 ppm HgCl,.
W
f
I
_-
1500-
__--
+m z IOOO-
l Na+ INFLUX 0
I
I
0
1
I
1
3 2 HgCI, exposure (days)
Na* EFFLUX
I
1
4
5
.g-
FIG. 3. Chronic poisoning of FW Chinese crabs E. sinensis with 0.1ppm HgCl,: Na+ in and effluxes (wquiv Na+ ’ ww.h- ‘) across anterior gills as a function of the exposure time to the pollutant. Gills are bathed on both sides with the same FW saline (see legend of Fig. 2). Mean results of at least 3 experis ments -+ SD.
152
A. Piqueux et al.
l Na’ INFLUX
OJ 900
1
T
,’
* 600s 7UJ
,’
,’
*’
_’
___- -P
+s 69 m 300-
0 Na* EFFLUX
__--- a __o__--
1
P
0’ I
,
0
1
2
,
1
3
4
HgCI, exposure (days)
FIG. 4. Chronic poisoning of FW Chinesecrabs E. sinensiswith 0.1 ppm H&l,: Na+ in and effluxes (wquiv Na+.g-’ ww.h-‘) across anterior gills as a function of the exposure time to the pollutant. Incubation medium: artifkial FW (Na+ 1 mequiv.l- ‘; Cl- 1.1 mequiv.l-9. Perfusion medium: FW saline (Na+ 240 mequiv.l-I; Cl- 280 mequiv.l-I). Mean results of at least 3 experiments f SD.
In agreement with and supporting that hypothesis, transepithelial in and effluxes of Na + , measured in the absence of a transepithelial gradient of concentration, are concomitantly greatly increased in the anterior gills (Fig. 3). In the same gills, there is no further Cl- flux (neither influx, neither efflux) than in control conditions without mercury treatment. In these incubation and perfusion conditions, increased Na+ movements never result in any net transepithelial flux. On the other hand, when the anterior gills are bathed with an external dilute medium such as artificial fresh water (FW), a net Na+ efflux appears, that is considerably increased following mercury exposure (Fig. 4). The transepithelial Na + influx is always reduced in the posterior salt-transporting gills, without or with a transepithelial concentration gradient (Fig. 5). At variance, Cl- efflux is always increased (Fig. 6). The Cl- influx remains unaffected when there is no transepithelial gradient (Fig. 6)) while
,J ~_._____._9._.__.___~_________ r
I
0
1
I
1
2 3 HgCI, expoaun (days)
I
4
FIG. 5. Chronic poisoning of FW Chinesecrabs E. sine&s with 0.1 ppm H&I,: Na+ in and effluxes (wquiv Na+.g-’ ww.h-*) across posterior gills as a function of the time of exposure to the pollutant. A: no transepithelialconcentration gradient: see also legend of Fig. 3. B: with transepithelialconcentration gradient: see also legend of Fig. 4.
it is progressively decreased in the presence of a concentration gradient (Fig. 7). In these last conditions, the net Cl- flux being nearly zero initially, turns progressively into an efflux, hence into a progressive loss of Cl-, when the mercury poisoning is sustained. From these experiments, it appears that most of the ionic movements considered as passive are considerably enhanced following poisoning with HgCl,. This suggests that mercury directly affects the ionic permeability properties of the epithelium membranes. However, further investigation will substantiate an action of the pollutant on the plasma membranes of
Mercury and Osmoregulation in Crabs
153
FW
E. sineasis H&I’ (IO
60-l
ppm)
Ant. gill
.L
0 Cl’ INFLUX 0 Cl- EFFLUX
I
I
I
I
I
I
0
1
2
3
4
5
HgCI, exposure (days)
FIG. 6. Chronic poisoning of FW Chinesecrabs E. sinensis with 0.1 ppm HgCI,: Cl- in and effluxes (ccequiv Cl-. g-’ ww.h-t) across posterior gills as a function of the time of exposure to the polhttant. Incubation conditions: no transepithelial concentration gradient; see also legend of Fig. 3. -60 ’ 80. T c
40-
i
20-
40
80
120
160
200
Time of exposure (min)
6o
r &
‘, .
0
Cl- INFLUX
FIG. 8. Effect of HgCI, ( 10 ppm) on the transepithelialpotential difference (PD, mV) or perfused gigs isolated from FW nonpoisoned Chinese crabs E. &en&s. Mercury is added to the incubation (OUT) or to the perfusion (IN) media. Gii are bathed on both sides with the same FW saline (see legend of Fig. 2). Ant.: anterior; Post: posterior.
O-
400
Ts 300 i k 200 ‘0 B * 100
,’
_4...--:
.- .-
the cells and/or on the cuticle lining the apical membranes. These findings account, at least partly, for the severe decrease of blood NaCl content that follows chronic poisoning with HgCl,. However, our findings do not exclude the possibility of an effect on active processes at work in branchial cells. The results reported above (Figs. 5 and 7) show that active influxes of both Na+ and Cl- ions in posterior gills are significantly decreased and we obtained experimental evidence that this inhibition might be related to a direct effect of mercury on This enzyme has been observed the (Na+ + K+)ATPase. indeed to undergo a 50% inhibition after 24 hr exposure of the animals to the mercury salt.
______---p ___= 4 .-4__--
.-
0
Cl- EFFLUX
tY= 0
;r
;
1
HgCI, exposure (days)
J
i
FIG. 7 Chronic poisoning of FW Chinese crabs E. sinensis with 0.1 ppm HgCl,: Cl- in and effluxes (ccequiv Cl-.g-’ ww.h-‘) across posterior gigs as a function of the time of exposure to the pohutant. Incubation conditions: with transepithelialconcentration gradient; see also legend of Fig. 4.
ACUTE POISONING OF ISOLATED GILLS. In an attempt to better grasp the way mercury salts affect the gill tissue itself, experiments have been carried out on gills isolated from nonpoisoned crabs. In these conditions, an acute exposure to 10 ppm HgCll induces an immediate and severe depolarization of both the anterior and the posterior gills epithelia (Fig. 8). Depolarization occurs only when the mercury is applied at the
A. Pequeux et al.
154
TABLE 1. Acute intoxication with 10 ppm HgClz of Anterior and Posterior perfused gills isolated from nonintoxicated FW Chinese crabs E. sine&s Na+ Influx
Anterior
gills
Control H&l, In HgCl, Out Control HgClz In HgClz Out
Posterior gills
1857 902 873 420 128 391
-t 2 ? 5 + 2
209 301 292 107 72 132
ClEiIIWL (pgHg.gww930 ? 183 897 2 261 1028 ‘-’ 245 Und. Und. Und.
Influx ‘.h- ‘) Und. Und. Und. 1028 ? 252 508 * 188 1214 2 331
H&I2 added to the incubation (Out) or to the perfusion (In) saline. Same W saline on both sides (Na+ 240 mequiv.1 -‘. , Cl- 280 mequiv.l-‘). of at least 3 experiments 2 standard deviation. Und: undetectable.
FW E.shensis HgCl* (10 ppm) ‘;
ii” op
300
+a
3.
z
,,,,
I/
’
0'0 .'e .4
0
l go
t
i
l a z
, ,
A
100
200 0
HI&
,
,
/’
I’
Efflux
Und. Und. Und. 413 2 89 476 4 77 489 * 123 Mean results
serosal side but the mercury remains
ineffective when applied to the apical side. The data reported in Table 1 summarize the results of transepithelial Na+ and Cl- fluxes measured during acute exposures to mercury of crab isolated perfused gills. Amazing is the fact that in and effluxes of Na” are, in no way, affected in the anterior gills, whatever the epithelial side exposed to the 10 ppm HgCl,. In posterior gills, Na+ and Cl- influxes are inhibited only when mercury is applied in the perfusion medium and Cl- efflux remains unaffected (Fig. 9). The results reported above substantiate the idea that the only mechanisms to be disturbed when gills from nonpoisoned crabs are acutely exposed to the mercury salt are the active processes located at the serosal side of the epithelium. Permeability properties do not seem to be affected in these experiments. This suggests that the heavy metal does not only act directly on the mechanisms that govern ionic movements at the branchial level but likely affects also other processes much more indirectly, such as neurohormonal ones that are not located in the gills themselves. Further investigations are needed to substantiate that idea.
This work has been aided by a grnnt EV4V-0123-B from the Commission of the European Communities to R. G.
0
20
40
60
60
100
120
Time of perfusion (min) FIG. 9. Effect of HgCl, ( 10 ppm) on unidirectional Na+ (A) and Cl- (B) influxes (wquiv.g-’ ww.) across perfused postee rior gills of FW nonpoisoned crabs E. sine&s. Gills are bathed on both sides with the same FW saline (see legend of Fig. 2).
References 1. Bianchini, A. Effets du mercure sur l’osmoregulation chez les crustac& decapodes. These de doctorat en Oceanologie, Universite de Liege, Liege, Belgique, 1990: 146 pp. 2. Bjerregard, P.; Vislie, T. Effects of mercury on ion and osmoregulation in the shore crab Carcinus mumm (L. ). Comp. Biothem.
Physiol. 82C:227-230;1985.
3. Cairns, J. Jr.; Lanza, G.R.; Parker, B.C. Pollution related structural and functional changes in aquatic communities with emphasis on freshwater algae and protozoa. Proceed. Acad. Nat. Sci. Philadelphia 124(5):79-127;1972. 4. FGrstner, U.; Schoer, J. Metal pollution in the tidal Elbe River. The Science of the Total Environment. 97/98: 347-368;1990. 5. Gilles, R.; Pbqueux, A. Interactions of chemical and osmotic regulation with the environment. In: Bliss, E.D., Editor-in-
Mercury and Osmoregulation
6.
7.
8.
9.
in Crabs
chief. The Biology of Crustacea, Vol. 8: Environmental adaptations. New York: Academic Press; 1983: 109-177. Johnson, LT.; Jones, M.B. Effect of zinc on osmoregulation of Gumnurrus duebeni (Crustacea: amphipoda) from the estuary and the sewage treatment works at Looe, Cornwall. Ophelia 313: 187-196;1990. Moustafa, E.K.; Moharram, Y.G.; El Sokkary, I.; Telb, A.I. Total mercury and its distribution in blue crab and deep water pink shrimp from Alexandria Coast. Egypt. Nahrung 31:773776;1987. Pequeux, A.; Gilles, R. Osmoregulation of the euryhaline chinese crab Eriocheir sinensis. Ionic transport across isolated perfused gills as related to the salinity of the environment. Proceedings of the 12th EMBS, Stirling, Scotland. Physiology and Behaviour of marine organisms. Oxford: Pergamon Press; 1978: 105-111. Pequeux, A.; Gilles, R. Na+ fluxes across isolated perfused gills of the Chinese crab Eriocheir sinmsis. J. Exp. Biol. 92:173-186; 1981.
155
10. Pequeux, A.; Gilles, R. The transepithelial potential difference of isolated perfused gills of the Chinese crab Eriocheir sinmsis acclimated to fresh water. Comp. Biochem. Physiol. 89A: 163172;1988. 11. Pequeux, A.; Gilles, R. NaCl transport in gills and related structures. Part I: Invertebrates. In: Greger, R., ed. Advances in comparative and environmental physiology, vol. 1. Berlin, Heidelberg: Springer Verlag; 1988: 2-47. 12. Takizawa, Y. Epidemiology of mercury poisoning. In: Nriagu, J.O., ed. The biogeochemistry of mercury in environment. Topics in environmental health, vol. 3. Amsterdam: Elsevier; 1979: 303-323. 13. Waldichuk, M. Some biological concerns in heavy metals pollution. In: Vemberg, F. J.; Verberg, W.B., eds. Pollution and physiology of marine organism. New York: Academic Press; 1974: l-57.
1996
ISSN 0742-8413/96/$15.00 SSDI 0742s8413(95)02081,0
ELSEVIER
Mercury and Osmoregulation in the Euryhaline Crab, Eriocheir sinensis A. E’iqueux,A. Bianchini
l
and R. Gil/es
LABORATORY OF ANIMAL PHYSIOLOGY, UNIVERSITYOF L&GE, INSTITUTEE. VAN BENEDEN,22, QUAI VAN BENEDEN,B4020 LIBGE, BELGIUM
ABSTRACT. This review will focus on cases where it might be possible that the toxicity of the heavy metal mercury results from an interaction with osmoregulatory mechanisms. It is shown that mercury-induced impairment of osmoregulatory capability in the sense of severe modifications of the blood osmotic concentration is more pronounced in brachyuran decapod species adapted to dilute waters. The rationale for considering these effects is based on a comparison between mercury effects on three species of decapod crustaceans exhibiting various degrees of osmoregulation capability: the strong regulator crab Eriocheir sinensis, the weak regulator Carcinus maenas, both of them being euryhaline, and the stenohaline osmoconformer Cancer pagums. It is established that a synergistic effect exists between salinity and HgC& toxicity in euryhaline species which are hyperregulators in dilute media, that is, E. sinensis and C. mae~s only. Depth study of E. sinensis as a model demonstrates that Na+ and Cl- permeabilities of the gill epithelium is affected by mercury, as well as the Na+ and Cl- active transport processes located at the same level. Evidences are brought showing that mercury drastically disturbs the Na+/K+ pump and the Cl- channels located in the serosal baso-lateral membranes of the posterior gills. COMPBIOCHEMPHYSIOL113c, 149-155, 1996. KEY WORDS. Crustaceans, gills, Eriocheir sinensis, Cur&us difference, active transport, sodium flux, chloride flux
INTRODUCTION
When considering the various sources and kinds of pollutants, it is clear that pollution by heavy metals, either natural or related to human activity, is the type most extensively documented and studied (see for example, 3,13,12,5,7,4). Most of the works devoted to these effects are concerned with the definition of mortality graphs and of tolerance limits, with reports on bioaccumulation, on effects on reproduction, on teratology, so that information on crab-heavy metal interactions at the molecular level remains fragmentary, superficial, essentially scanty. In these studies, it is often impossible to distinguish between real synergistic effects and interactions between pollutants and basic mechanisms concerned with blood or cell osmolarity regulation. It has been reported that estuarine species of crustaceans
maenas, Cancer pagurus, crab, mercury, potential
are particularly susceptible to heavy metals. Moreover, several studies have emphasized the importance of a salinity stress in enhancing heavy metal toxicity (see for example 2,6). Intuitively, these findings support the idea that osmoregulatory processes in crustacean species confronted with dilute media might be among the primary sites to be affected by these toxics. Surprisingly, that question has previously received little attention up to now. This paper will focus on the observation that the toxicity of mercury results from an interaction with osmoregulatory mechanisms. The results originate from experiments conducted in our laboratory on the strong osmoregulator Eriocheir sinmsis (see also 1).
MATERIALS
AND
METHODS
Material Address reprint requests to: A. PCqueux, Lab. of Animal Physiology, Univ. of Liege, Institut E. Van Beneden, 22, quai Van Beneden, B4020 Likge, Belgium *Present address: Universidad do Rio Grande Laboratorio de Zoofisiologia, Rua E.A. HUCH, 475, CATXA Postal 474, 96.201-900 Rio Grande-RSBrasil. Based on a presentation given at the 15th Annual Conference of the European Society for Comparative Physiology and Biochemistry held in Genoa, Italy, in September 1994. The Conference title was: Biochemical and physiologicaleffects of poUution and toxicological assessment of environmentaI wk Received
17 October
1995; accepted
2 November
1995.
Experiments reported in this paper were carried out on three species of crab, that is, the strong hyperregulator Eriocheir sine&, the weak regulator Curcinw maenas and the stenohaline osmoconformer Cancer pagums. However, the largest part of the experiments were performed on isolated and perfused anterior and posterior gills of the euryhaline Chinese crab Erioc&r sinensis. Chinese crabs were caught in autumn in freshwater (FW) lakes near Emden in the North of Germany. They were transferred to the aquarium of the University of Liege (Belgium) and kept in tanks of circulating FW.
150
Methods Chronic poisoning by mercury was achieved by adding HgCl, 0.1 ppm or 1 ppm to the external medium (50 1. FW glass tanks). Medium was renewed each day and regularly controlled. Gills were perfused and the transepithelial potential difference (PD) was measured following procedures already described in detail (8,9,10). Gills were handled in, incubated in and perfused by a “freshwater (FW) saline”, the composition of which corresponded to the ionic composition of the blood of FW crabs. It contained (in mM): NaCl, 240; KCl, 5; MgCl,,5; CaCl,, 12.5; H,BOX, 8.8. The pH was adjusted at 7.6 with Tris-base. When announced, HgClz (10 ppm) was added to the incubation (OUT) or to the perfusion (IN) media. The transepithelial movements of Na+ and Cl-- were estimated using the radioactive tracers 22Naf and ‘Q(0.20 and 0.25 /.&ml-‘, respectively). Tracers were added either to the incubation medium (influx) or to the perfusion saline (efflux). “Na+ was using counted in a y scintillator, 36Cl- in a /3 scintillator, Pica Fluor TM30 (Packard) as scintillation cocktail. Hemolymph Na+ and K+ ions were measured in blood aliquots adequately diluted, by means of a flame photometer (Eppendorf); blood Clwas measured by coulometryamperometry with a Buchler-Cotlove chloridometer. RESULTS AND DISCUSSION Toxicity of Mercury to Whole Crustacean Species In a previous review, the toxicity and biological effects of different groups of chemical pollutants, among which heavy metals and mercury have been considered, included scanty evidence of synergistic actions where toxicity could be related to interactions with osmoregulatory mechanisms (5). More recent studies have been conducted in this laboratory on three crab species, i.e. the strong hyperregulator Eriocbir sinensis, the weak regulator Carcinus maenu-.s, both of them being euryhaline, and the stenohaline osmoconformer Cancer pagurus, chronically poisoned with HgCl, 1 ppm. applied in the external medium. Mercury exposure has always proved to be lethal for the three species under consideration. However, in the Chinese crab Eriocheir sinensis, H&l, has appeared to be much more toxic for FW animals (LTSO: 40 hr) than for SW/2 (18%) (LTSO = 77 hr) or SW (35%ti) animals (LT50 = 75 hr). In the shore crab C. maenas, HgCl, was more toxic too for SW/3 (11%) animals (LT50 = 37 hr) than for SW animals (LT50 = 70 hr). Surprisingly, the SW osmoconformer C. pagurus was more sensitive to HgCl, (LT50 = 43 hr) than other species but it didn’t exhibit any salinity linked sensitivity. These results corroborate the fact that osmoregulating euryhaline species in dilute media are much more susceptible to mercury salts than in full strength seawater. In gills and shell of the euryhaline species, the rate of up take was faster in dilute SW crabs than in SW acclimated
A. Pkqueux et al.
ones, while there was no significant difference in rate of uptake as a function of salinity in the osmoconformer C. pagurus. Moreover, when the digestive tract of E. sine&s was made unfunctional by occlusion of the mouth with dental cement and whatever the salinity of acclimation, Hg+ + accumulation did not change in all organs and tissues studied but the digestive tract itself where mercury content dropped down by mote than 50%. In conclusion, it is thus clear that osmoregulating crab species, when a hemolymph-ambient water gradient exists, are much more sensitive to mercury than conforming species in salinity and that organs involved in ion balance such as the gills accumulate much more pollutant. Intuitively, such a huge accumulation of toxicants at that level must lead to faster impairment of the mechanisms there at work. These findings support the idea that osmoregulatory processes in these species might therefore be among the primary sites to be affected by mercury. This finding prompts us to suggest that the toxicity of HgCl, for hyperregulating crabs could be due to impairment of the maintenance of the blood ionic balance.
Effect of Mercury
on Crustacean
Hemolymph
Composition
Surprisingly, the effects of mercury salts on crustacean hemolymph composition has received little attention and data remain essentially scanty. Among the few most recent studies, Bjerregaard and Vislie (2) reported a decrease of blood osmolarity and inorganic ions content in green crabs Carcinus maeTUISacclimated to reduced salinity (400 mosm.l- ‘) and exposed to 1 and 10 ppm HgC12. In Gammarus, Johnson and Jones (6) established that the depression of hemolymph osmolality was the result of reduced levels of blood sodium. In E. sinensis too, a severe decrease of the Na+ and Cl- blood content occurs during poisoning with 0.1, 1 and 10 ppm HgCl,. Figure 1 illustrates the effects of 1 ppm HgCl, on SW (35%os), SW/2 (18%) and FW acclimated Chinese crabs. It is clear that the decrease in Na+ and Cl- concentration occurs in FW crabs only within hr. There is no significant effect of a several days exposure in SW and SW/2 animals, as there is no effect too in SW and SW12 acclimated stone crabs C. pagurus (Pequeux and Gilles, unpublished results). Undoubtedly, these results corroborate the above hypothesis of a mercury-induced impairment of the maintenance of the blood ionic balance in regulating crabs at ambient concentrations where the hemolymph-ambient seawater difference is highest.
Tissue Physi&gy
4 Regulating
Crabs
In an attempt to understand and explain the mechanism of action of heavy metals that leads to a disturbance of the osmoregulatory function in crustacea, studies have been conducted in this laboratory on the effects of mercury on the gill tissue
Mercury and Osmoregulation in Crabs
151
E. siaensis HgCI’ (1wW
FW
H&f (1ppm)
0
If:::
20
Days (SW andSW/2crabs)
w t
E. sinensis
Ant. gills
10
Na+
0
SW/l l
-
-
__L___.
-10
0
5
/
-20
Hours (F-W crabs) 24
12
T dI
-0-o
T yi-_PyT
T
Post. gills
1
1
-30 -40
I
I
I
I
1
0
1
2
3
4
Time of exposure (days) 0
5
0 ,
1’
Hours (Fw crabs) 24
12
;
;’
; Days(SWrndSW/2crrbs)
‘\t/i-f”-
clF\\
loo
I?
m
0
s
I2
Hours(FN crabs) 24
Time of exposure to HgCI,
1. Effect of HgCl, (1 ppm in external medium) on hemoe lymph Na+, K+ and Cl- contents (mequiv.1 -‘) of Chinese crabs Eriocheir sinensis acclimated to FW, SW/2 and full SW. Time of exposure to HgCl, is hours or days. FW = freshwater; SW = sea water.
FIG. 2. Chronic poisoning of FW Chinese crabs E. sinensis with 1 ppm HgC&: transepithelial electrical potential difference (mV) across epithelium from isolated perfused anterior (Ant.) and posterior (Post.) gills as a function of the exposure time to the pollutant. Gills are bathed on both sides with the same FW saline (Na+ 240 mequiv.l-‘; Cl- 280 mequiv.l-I). Mean red suits of 6 experiments + SD. CHRONIC POISONING OFWHOLECRABS. The transepithelial electrical potential difference measured in vitro across both kinds of gills is progressively abolished as a function of the time of exposure of the crabs to mercury (Fig. 2). In first analysis, this suggests a severe impairment of the permeability and/or of the transport properties of the tissues.
FIG.
2500
r 2000 ‘k
1
T
‘:
of Chinese crabs, E. sinensis. Experiments have been carried out on the perfused preparation of isolated gill that is used in this laboratory to investigate the part played by the gills in the overall osmoregulation function in crabs (11). The anteriorly-located pairs of gills are respiratory and the posteriorly by well-developed saltlocated gills are characterized transporting epithelium and have been demonstrated to be involved primarily in salt transport and hydromineral regulation (11). Two series of experiments have been conducted: (1) experiments on gills isolated from crabs which were poisoned chronically with 1 ppm HgCl,, and (2) experiments where gills from non-poisoned crabs were acutely and directly poisoned with 10 ppm HgCl,.
W
f
I
_-
1500-
__--
+m z IOOO-
l Na+ INFLUX 0
I
I
0
1
I
1
3 2 HgCI, exposure (days)
Na* EFFLUX
I
1
4
5
.g-
FIG. 3. Chronic poisoning of FW Chinese crabs E. sinensis with 0.1ppm HgCl,: Na+ in and effluxes (wquiv Na+ ’ ww.h- ‘) across anterior gills as a function of the exposure time to the pollutant. Gills are bathed on both sides with the same FW saline (see legend of Fig. 2). Mean results of at least 3 experis ments -+ SD.
152
A. Piqueux et al.
l Na’ INFLUX
OJ 900
1
T
,’
* 600s 7UJ
,’
,’
*’
_’
___- -P
+s 69 m 300-
0 Na* EFFLUX
__--- a __o__--
1
P
0’ I
,
0
1
2
,
1
3
4
HgCI, exposure (days)
FIG. 4. Chronic poisoning of FW Chinesecrabs E. sinensiswith 0.1 ppm H&l,: Na+ in and effluxes (wquiv Na+.g-’ ww.h-‘) across anterior gills as a function of the exposure time to the pollutant. Incubation medium: artifkial FW (Na+ 1 mequiv.l- ‘; Cl- 1.1 mequiv.l-9. Perfusion medium: FW saline (Na+ 240 mequiv.l-I; Cl- 280 mequiv.l-I). Mean results of at least 3 experiments f SD.
In agreement with and supporting that hypothesis, transepithelial in and effluxes of Na + , measured in the absence of a transepithelial gradient of concentration, are concomitantly greatly increased in the anterior gills (Fig. 3). In the same gills, there is no further Cl- flux (neither influx, neither efflux) than in control conditions without mercury treatment. In these incubation and perfusion conditions, increased Na+ movements never result in any net transepithelial flux. On the other hand, when the anterior gills are bathed with an external dilute medium such as artificial fresh water (FW), a net Na+ efflux appears, that is considerably increased following mercury exposure (Fig. 4). The transepithelial Na + influx is always reduced in the posterior salt-transporting gills, without or with a transepithelial concentration gradient (Fig. 5). At variance, Cl- efflux is always increased (Fig. 6). The Cl- influx remains unaffected when there is no transepithelial gradient (Fig. 6)) while
,J ~_._____._9._.__.___~_________ r
I
0
1
I
1
2 3 HgCI, expoaun (days)
I
4
FIG. 5. Chronic poisoning of FW Chinesecrabs E. sine&s with 0.1 ppm H&I,: Na+ in and effluxes (wquiv Na+.g-’ ww.h-*) across posterior gills as a function of the time of exposure to the pollutant. A: no transepithelialconcentration gradient: see also legend of Fig. 3. B: with transepithelialconcentration gradient: see also legend of Fig. 4.
it is progressively decreased in the presence of a concentration gradient (Fig. 7). In these last conditions, the net Cl- flux being nearly zero initially, turns progressively into an efflux, hence into a progressive loss of Cl-, when the mercury poisoning is sustained. From these experiments, it appears that most of the ionic movements considered as passive are considerably enhanced following poisoning with HgCl,. This suggests that mercury directly affects the ionic permeability properties of the epithelium membranes. However, further investigation will substantiate an action of the pollutant on the plasma membranes of
Mercury and Osmoregulation in Crabs
153
FW
E. sineasis H&I’ (IO
60-l
ppm)
Ant. gill
.L
0 Cl’ INFLUX 0 Cl- EFFLUX
I
I
I
I
I
I
0
1
2
3
4
5
HgCI, exposure (days)
FIG. 6. Chronic poisoning of FW Chinesecrabs E. sinensis with 0.1 ppm HgCI,: Cl- in and effluxes (ccequiv Cl-. g-’ ww.h-t) across posterior gills as a function of the time of exposure to the polhttant. Incubation conditions: no transepithelial concentration gradient; see also legend of Fig. 3. -60 ’ 80. T c
40-
i
20-
40
80
120
160
200
Time of exposure (min)
6o
r &
‘, .
0
Cl- INFLUX
FIG. 8. Effect of HgCI, ( 10 ppm) on the transepithelialpotential difference (PD, mV) or perfused gigs isolated from FW nonpoisoned Chinese crabs E. &en&s. Mercury is added to the incubation (OUT) or to the perfusion (IN) media. Gii are bathed on both sides with the same FW saline (see legend of Fig. 2). Ant.: anterior; Post: posterior.
O-
400
Ts 300 i k 200 ‘0 B * 100
,’
_4...--:
.- .-
the cells and/or on the cuticle lining the apical membranes. These findings account, at least partly, for the severe decrease of blood NaCl content that follows chronic poisoning with HgCl,. However, our findings do not exclude the possibility of an effect on active processes at work in branchial cells. The results reported above (Figs. 5 and 7) show that active influxes of both Na+ and Cl- ions in posterior gills are significantly decreased and we obtained experimental evidence that this inhibition might be related to a direct effect of mercury on This enzyme has been observed the (Na+ + K+)ATPase. indeed to undergo a 50% inhibition after 24 hr exposure of the animals to the mercury salt.
______---p ___= 4 .-4__--
.-
0
Cl- EFFLUX
tY= 0
;r
;
1
HgCI, exposure (days)
J
i
FIG. 7 Chronic poisoning of FW Chinese crabs E. sinensis with 0.1 ppm HgCl,: Cl- in and effluxes (ccequiv Cl-.g-’ ww.h-‘) across posterior gigs as a function of the time of exposure to the pohutant. Incubation conditions: with transepithelialconcentration gradient; see also legend of Fig. 4.
ACUTE POISONING OF ISOLATED GILLS. In an attempt to better grasp the way mercury salts affect the gill tissue itself, experiments have been carried out on gills isolated from nonpoisoned crabs. In these conditions, an acute exposure to 10 ppm HgCll induces an immediate and severe depolarization of both the anterior and the posterior gills epithelia (Fig. 8). Depolarization occurs only when the mercury is applied at the
A. Pequeux et al.
154
TABLE 1. Acute intoxication with 10 ppm HgClz of Anterior and Posterior perfused gills isolated from nonintoxicated FW Chinese crabs E. sine&s Na+ Influx
Anterior
gills
Control H&l, In HgCl, Out Control HgClz In HgClz Out
Posterior gills
1857 902 873 420 128 391
-t 2 ? 5 + 2
209 301 292 107 72 132
ClEiIIWL (pgHg.gww930 ? 183 897 2 261 1028 ‘-’ 245 Und. Und. Und.
Influx ‘.h- ‘) Und. Und. Und. 1028 ? 252 508 * 188 1214 2 331
H&I2 added to the incubation (Out) or to the perfusion (In) saline. Same W saline on both sides (Na+ 240 mequiv.1 -‘. , Cl- 280 mequiv.l-‘). of at least 3 experiments 2 standard deviation. Und: undetectable.
FW E.shensis HgCl* (10 ppm) ‘;
ii” op
300
+a
3.
z
,,,,
I/
’
0'0 .'e .4
0
l go
t
i
l a z
, ,
A
100
200 0
HI&
,
,
/’
I’
Efflux
Und. Und. Und. 413 2 89 476 4 77 489 * 123 Mean results
serosal side but the mercury remains
ineffective when applied to the apical side. The data reported in Table 1 summarize the results of transepithelial Na+ and Cl- fluxes measured during acute exposures to mercury of crab isolated perfused gills. Amazing is the fact that in and effluxes of Na” are, in no way, affected in the anterior gills, whatever the epithelial side exposed to the 10 ppm HgCl,. In posterior gills, Na+ and Cl- influxes are inhibited only when mercury is applied in the perfusion medium and Cl- efflux remains unaffected (Fig. 9). The results reported above substantiate the idea that the only mechanisms to be disturbed when gills from nonpoisoned crabs are acutely exposed to the mercury salt are the active processes located at the serosal side of the epithelium. Permeability properties do not seem to be affected in these experiments. This suggests that the heavy metal does not only act directly on the mechanisms that govern ionic movements at the branchial level but likely affects also other processes much more indirectly, such as neurohormonal ones that are not located in the gills themselves. Further investigations are needed to substantiate that idea.
This work has been aided by a grnnt EV4V-0123-B from the Commission of the European Communities to R. G.
0
20
40
60
60
100
120
Time of perfusion (min) FIG. 9. Effect of HgCl, ( 10 ppm) on unidirectional Na+ (A) and Cl- (B) influxes (wquiv.g-’ ww.) across perfused postee rior gills of FW nonpoisoned crabs E. sine&s. Gills are bathed on both sides with the same FW saline (see legend of Fig. 2).
References 1. Bianchini, A. Effets du mercure sur l’osmoregulation chez les crustac& decapodes. These de doctorat en Oceanologie, Universite de Liege, Liege, Belgique, 1990: 146 pp. 2. Bjerregard, P.; Vislie, T. Effects of mercury on ion and osmoregulation in the shore crab Carcinus mumm (L. ). Comp. Biothem.
Physiol. 82C:227-230;1985.
3. Cairns, J. Jr.; Lanza, G.R.; Parker, B.C. Pollution related structural and functional changes in aquatic communities with emphasis on freshwater algae and protozoa. Proceed. Acad. Nat. Sci. Philadelphia 124(5):79-127;1972. 4. FGrstner, U.; Schoer, J. Metal pollution in the tidal Elbe River. The Science of the Total Environment. 97/98: 347-368;1990. 5. Gilles, R.; Pbqueux, A. Interactions of chemical and osmotic regulation with the environment. In: Bliss, E.D., Editor-in-
Mercury and Osmoregulation
6.
7.
8.
9.
in Crabs
chief. The Biology of Crustacea, Vol. 8: Environmental adaptations. New York: Academic Press; 1983: 109-177. Johnson, LT.; Jones, M.B. Effect of zinc on osmoregulation of Gumnurrus duebeni (Crustacea: amphipoda) from the estuary and the sewage treatment works at Looe, Cornwall. Ophelia 313: 187-196;1990. Moustafa, E.K.; Moharram, Y.G.; El Sokkary, I.; Telb, A.I. Total mercury and its distribution in blue crab and deep water pink shrimp from Alexandria Coast. Egypt. Nahrung 31:773776;1987. Pequeux, A.; Gilles, R. Osmoregulation of the euryhaline chinese crab Eriocheir sinensis. Ionic transport across isolated perfused gills as related to the salinity of the environment. Proceedings of the 12th EMBS, Stirling, Scotland. Physiology and Behaviour of marine organisms. Oxford: Pergamon Press; 1978: 105-111. Pequeux, A.; Gilles, R. Na+ fluxes across isolated perfused gills of the Chinese crab Eriocheir sinmsis. J. Exp. Biol. 92:173-186; 1981.
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10. Pequeux, A.; Gilles, R. The transepithelial potential difference of isolated perfused gills of the Chinese crab Eriocheir sinmsis acclimated to fresh water. Comp. Biochem. Physiol. 89A: 163172;1988. 11. Pequeux, A.; Gilles, R. NaCl transport in gills and related structures. Part I: Invertebrates. In: Greger, R., ed. Advances in comparative and environmental physiology, vol. 1. Berlin, Heidelberg: Springer Verlag; 1988: 2-47. 12. Takizawa, Y. Epidemiology of mercury poisoning. In: Nriagu, J.O., ed. The biogeochemistry of mercury in environment. Topics in environmental health, vol. 3. Amsterdam: Elsevier; 1979: 303-323. 13. Waldichuk, M. Some biological concerns in heavy metals pollution. In: Vemberg, F. J.; Verberg, W.B., eds. Pollution and physiology of marine organism. New York: Academic Press; 1974: l-57.