influence of anoxia and hydrogen sulphide on the energy metabolism of scrobicularia plana (da costa)...

ELSEVIER Journal of Experimental Marine Biology and Ecology

184 (1994) 255-268

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

Influence of anoxia and hydrogen sulphide on the energy metabolism of Scrobicularia plana (da Costa) (Bivalvia)

Rolf Oeschger a,*, Torben Feldsgaard Pedersen b,**

a Universiliit Bremen, Meereszoologie, FB 2. Biirgermeister-Smidt-StraJe 20, D-27568 Bremerhaven, Germany bBiologisk Institut, Aarhus Universitet, Ny Munkegade. DK-8000 AOrhus C, Denmark

Received 24 September 1993; revision received 23 August 1994; accepted 3 1 August 1994

Abstract

Responses to anoxia and hydrogen sulphide exposure are studied in the intertidal clam Scro- bicularia plana. In their habitat the clams encounter varying sulphide concentration in sediment pore water (up to 1.19 mM). After long-term exposure to anoxic conditions (5 days) clams keep an enhanced oxygen uptake for about one day (accumulated.oxygen debt 225 pmol O,.g-‘dry mass), before resuming normoxic consumption rates (MO, = 6.3 k 2.4 pmol O,.g-‘dry mass.h’). Specimens incubated under anoxia and sulphide (200 ,uM) for 5 days exhibit increased recovery oxygen uptake compared to anoxic clams (oxygen debt 385 pmol O,.g-‘dry mass) and accumulate significant elevated levels of succinate in muscular tissues. Even though

this indicates an influence of sulphide on the overall metabolism of Scrobicularia plana, no de- teriorating effect is observed, as clams regain normoxic consumption rates in the same time frame as individuals incubated without sulphide. During anoxic sulphide incubation (200 PM), sulphide

concentration in extrapallial fluid is low throughout 2 days, before reaching an equilibrium with the external concentrations after 8 days. Oxidation of accumulated sulphide accounts for less than 10% of the elevated oxygen uptake. Mantle edge tissue turns black upon sulphide exposure and accumulates high amounts of sulphide, suggesting a temporary function as a periph- eral “sulphide-trap”. Our study suggests, that the disappearance of Scrobiculariaplana from most Danish coastal areas during the last decades is unlikely to be related primarily to increased periods of oxygen depletion and occurrence of hydrogen sulphide in coastal waters.

Keywords: Anoxia; Hydrogen sulphide; Normoxia; Scrobicularia plana

* Corresponding author.

**Present address: Milja- og spildevandsafdelingen, Jomfrustien 2, DK-6270 Tender, Denmark.

0022-0981/94/$7.00 0 1994 Elsevier Science B.V. All rights reserved

SSDI 0022-0981(94)00129-4

256 R. Oeschger, T.F. Pedersen 1 J. Exp. Mar. Biol. Ecol. 184 (1994) 255-268

1. Introduction

Intertidal animals live in an environment which exhibits extreme fluctuations in

physical and chemical conditions. Oxygen availability shows high alternations in the intertidal zone, either due to periodic (imposed by the tidal cycle) or seasonal variations, and anoxic conditions might be experienced by the infauna (Bayne & Newell, 1983). Moreover, oxygen is only present in the upper millimetres of the sediment (Revsbech et al., 1980) and hydrogen sulphide (H2S) may build up in considerable

concentrations (Fenchel & Riedl, 1970). This noxious substance is primarily produced by sulphate reducing bacteria situated in the anoxic layers of the sediment, and is highly toxic to aerobic organisms in nanomolar concentrations (National Research Council, 1979). Thus intertidal animals are living in an environment where they naturally face

the risk of restricted oxygen availability and concomitant sulphide exposure. Marine invertebrates have acquired a variety of adaptations to cope with these conditions. When ambient oxygen availability decreases, some intertidal animals are capable of maintaining an unchanged oxygen uptake over a certain range of oxygen tensions. This has been thoroughly studied in molluscs (e. g. Bayne, 1971; Mangum & van Winkle, 1973; Taylor & Brand, 1975; Newell et al., 1978; Famme & Kofoed, 1980; Pedersen,

1992). Most intertidal bivalves are also capable of keeping a sustained energy production by use of anaerobic pathways (for detailed reviews see de Zwaan, 1977; Storey

& Storey, 1990), enabling them to withstand periods of anoxia (Theede et al., 1969). A long overlooked factor affecting the distribution and survival of marine inverte-

brates is hydrogen sulphide. With the recent discovery of animals constantly exposed to high sulphide levels in the vicinity of hydrothermal vents, research has been stimu-

lated to elucidate those adaptations which enable the macrofauna to cope with hydrogen sulphide exposure. Most of the hydrothermal vent fauna is associated with endo- symbiotic sulphide-oxidising bacteria. This is the basis for survival in such an environment (Somero et al.; 1989; Fisher, 1990). But little is known about how inter-

tidal bivalves without bacterial symbionts survive toxic hydrogen sulphide (Levitt & Arp, 1991; Vismann, 1991, 1993; Oeschger & Storey, 1993).

The deposit feeding tellinid clam, Scrobiculuria plana, is found in intertidal and shallow subtidal areas from Norway to Senegal (Rasmussen, 1973; Tebble, 1976). Scrobicularia plana may encounter high sulphide levels, since the animals live deep in

the substratum at lo-15 cm depth (Zwarts, 1986; Zwarts & Wanink, 1989). The population dynamics of this species have been well studied (for review, see Essink et al., 1991), but only a few studies have been done on the physiology of Scrobiculariaplana

(e. g. Brinkhoff et al., 1983; Worrall & Widdows, 1983; Worrall et al., 1983; Ahmad & Chaplin, 1985). This clam was commonly found in Danish coastal waters in the early century, especially in the Danish Wadden Sea (Thamdrup, 1935) but has not been reported in this area in recent years (K. T. Jensen, pers. comm.). Scrobicufariu pluna

is also disappearing from the German Wadden Sea (Reise, 1982), but there seems to be no general trend or explanation for declining populations in Europe (Essink et al., 1991).

In this paper we report on the first findings of Scrobicularia plana in Arhus Bight, Denmark. We focus on respiration measurements as a parameter to evaluate the in-

R. Oeschger, T.F. Pedersen / .l. Exp. Mar. Bid Ecol. 184 (1994j 255-268 257

fluence of anoxia and hydrogen sulphide on the energy metabolism of the clams. Ad- ditionally we studied the impact of sulphide on selected tissues of this species.

2. Materials and methods

2.1. Sampling

Scrobiculavia pZrrna were collected at L0gten Beach, Arhus Bight, Denmark, during March 1992 (shell length 4.2-4.5 cm, dry mass about 200 mg). This is the first find- ing of Scrobic~lar~a plana in that area of the Danish part of the Baltic Sea. During collection, temperatures at the collection site varied between 4-6 “C and salinity was between 22-26 ppt. Clams were transferred to Ronbjerg Marine Laboratory immedi- ateIy after collection, and kept at 10 it: I “C in running seawater with a of salinity 24 ppt 2-5 days before use, Sediment from the sampling location was collected with a core (internal diameter 4.6 cm) and brought to the laboratory to measure sulphide contents of the pore water. Pore water was obtained immediately by pressure filtration (N2) and analysed for sulphide concentration by the methylene blue method (Cline, 1969). In this paper the term sulphide refers to all forms of hydrogen sulphide (H,S, HS -, S _ - ).

2.2. Anoxic incubation with and without &hide

For anoxic exposure expe~ments animals were incubated in airtight stoppered flasks containing 1000 ml of nitrogen bubbled oxygen-free seawater (10 2 1 “C, salinity 24 ppt, pH adjusted to 7.5) For anoxic strIphide exposure experiments, clams were incubated in airtight stoppered flasks as described above. Sulphide levels were adjusted by adding Na2S*9H,0 crystals to nitrogen bubbled oxygen-free seawater to a given final concentration of 200 PM, pH 7.5. Before sealing the chamber, an argon atmosphere was introduced to seal the surface. During exposure experiments triplicate water samples were regularly taken, and sulphide concentration was measured spectrophotometrically after Cline (1969), which gives immediate results. Due to chemical oxidation processes during incubation (e.g. sulphide precipitation on the shells, some oxygen left initially in the body fluids of the clams), sulphide concentration had to be readjusted several times to the intended level by adding a deoxygenated 5 mM H-S stock solution. Prior to adding aliquots of this solution into the incubation flasks, a stream of argon was used to prevent oxygen penetrating into the anoxic seawater. To avoid accumulation of toxic metabolites, the seawater was changed every second day. Under these conditions sulphide levels were in the range of 200 _t 30 PM. For measurements of sulphide accumulation in the extrapallial fluid of animals exposed to an external concentration of 200 PM sulphide (10 + 1 “C, salinity 24 ppt, pH 7.5), 50 ~1 of extrapallial fluid of individual clams in triplicates were immediately collected. Samples were spun at 12 000 x g for 15 s prior to analysing sulphide contents (Cline, 1969).

258 R. Oeschger, T.F. Pedersen 1 .T. Exp. Mar. Eioi. Ecol. 184 [1994) 255-268

2.3. Oxygen uptake measurements

Oxygen uptake (MO,) was measured in flow respirometers as described previously (Pedersen, 1991; Oeschger et al., 1992). The signals from the oxygen meters were monitored on a computer equipped with a data acquisition card (PCL-7 1 lS, Advantec Co. Ltd., USA) at frequency of 0.2 Hz. MO, was calculated using the integrated re- corded mV signals over intervals of 10 min. After the various treatments, clams were placed in ~ndividu~ chambers of 10 ml volume and perfused with filtered seawater (0.45 pm, salinity 24 ppt) at a flow rate of 50-60 ml-h-’ at 10 J- 0.1 “C. To exclude influ- ences of the light regime, all measurements were made in the dark. The first 2-3 h of each measurement after insertion into the chamber were omitted in calculating MO, to eliminate the effects of handling stress. Oxygen consumption of cleaned empty shells was additionally measured after anoxic and sulphide incubations (200 PM). These values were subtracted from measurements on whole clams. The recovery MO2 after anoxic and sulphidic incubation of various exposure times was calculated as the mean MO, during 24-h measurements. The time course of the recovery MO, after 5 days of anoxic and sulphide exposure was calculated by integrating MO, for 4-h intervals for a total of 32 h. Tissue dry mass of whole clams was determined after 2 days at 108 “C.

2.4. Succinate

Adductor and foot muscle were extracted with 0.6 N HC104 according to Beis & Newsholm (1975). The anaerobic indicator metabolite succinate was measured using the standard spectrophotometric enzymatic determination (Beutler, 1985).

2.5. Determination ofsuiphide in tissues

Quantification of sulphide was done by reversed phase high performance liquid chromatography (HPLC) (Fahey & Newton, 1987; Vetter et al., 1989) with monobro- mobimane (mBBr). This synthetic compound covalently binds reduced sulphur compounds yielding fluorescent derivatives. Samples were run on a computer-controlled Kontron HPLC system, excitation wavelength of the detector was set at 350 nm, emission wavelength on 480 nm. Quantification was achieved by comparing fluores- cence of the samples with a standard. At the end of the experiments tissues were immediately homogenised in an ice cold HEPES buffer (N-Zhydroxyethyl piperazine- N’-Zethane sdphonic acid, 200 ,uM, pH 8.0) containing the mBBr and prepared for further HPLC analysis (Vet&r et al., 1989). Samples were stored at 4 “C prior to analysis.

2.6. Statistical analyses

The MO, of the clams after normoxic, anoxic, and sulphidic incubations was analysed by one way ANOVA. Preceding the analyses of variance, the data were tested for variance homogeneity using Bartlett’s Box-F test (Sokal & Rohlf,1981), at the p ~0.05 level. The mean MO, of the three experimental groups were compared using the

R. Oeschger, T.F. Pedersen /J. Exp. Mar. Bioi. .&xl. 184 cl9941 255-268 259

Student-beak-Keuls test. Succinate data were tested with Student’s t-test for significant differences between anoxic exposure with and without sulphide. A significant difference was inferred when p ~0.05.

3. Results

3.1. &.&hide content of sediment pore water and observations during incubation

Sulphide contents in sediment pore water collected in March 1992 were low, with a maximum con~eniration of 26 PM at 4 cm depth. In June 1992, considerable amounts of sulphide were found in a patchy dist~bution in five cores sampled within an area of 1 m*. Highest amounts reached a concentration of 1.189 mM sulphide at IO-12 cm sediment depth (Table 1).

During anoxic exposure experiments with and without sulphide clams extruded their siphons a few hours after transfer to the incubation flasks. This extrusion was observed throughout the whole experiments. The outer shell layers and the mantle edge of sulphide exposed Scrobicularia plana turned black in the course of one day of incubation. After transferring clams to normoxic conditions again, the blackish colour disappeared within 24 h. The same was observed when the clams were collected: most clams had blackish coloured shells at the time of collection, which disappeared within one day after transfer to aerated seawater.

3.2. Oxygen consumption at normoxia and after anoxic incubations

6f0, of Scrobicularia piana kept at normoxia showed an average uptake of 6.3 + 2.4 pmol O,*g-’ dry mass. h-’ (n = 7) during 24 h of measurements. After one day of anoxic incubation, the clams had a slightly increased h0, after returning to oxic conditions. Average oxygen uptake during the first 24 h of recovery was 7.3 rl: 4.3 pmol 02vg-” dry mass-h-’ (n = 3) and did not differ significantly from oxic clams. After 5 days of anoxic incubation, the mean recovery MO, (16.8 k 3.2 pmol 02.g-’ dry mass*h-‘, n = 6, during 24 h of measurement) was significantly higher than MO, after one day of anoxic exposure.

3.3. Oxygen uptake after anoxic incubations with sulphide

The mean &IO, (11.1 k 3.6 pmol 02*g-’ dry mass.h-‘, n= 5) during the first 24 h of recovery after one day of sulphide incubation was significantly higher compared to normoxic control clams, with increased MOz during the first hour after return to oxic conditions. The mean MO, after 5 days of sulphidic exposure was significantly higher (24.3 1: 3.7 pmol 02.g-’ dry mass*h-‘, n = 6, during 24 h of measurement) compared to one day of exposure.

260 R. Oeschger, T.F. Pedersen /J. Exp. Mar. Biol. Ecol. 184 (1994) 255-268

3.4. Recovery oxygen uptake after 5 days of anoxic exposure with and without sulphide

After 5 days of exposure, anoxic and sulphide exposed clams had a strongly increased

recovery oxygen uptake (calculations are on the basis of averaged 4-h measurements) during the first 20 h of measurements compared to normoxic clams (Fig. 1). During the first 4 h, no significant difference in MO, of the differently treated clams was found, but from 8 to 16 h after transfer to normoxic conditions, anoxic incubated individuals without sulphide had a significantly lower h0, compared to sulphidic clams. After about 24 h of recovery, oxygen uptake of anoxic incubated clams with and without

sulphide was not significantly different from that of normoxic control clams.

3.5. St&hide accumulation in extrapallialfluid

The sulphide content of the extrapallial fluid of Scrobicularia plana during anoxic incubations in 200 PM sulphide is shown in Fig. 2. Sulphide levels were low during the first 2 days (16 and 14 ,uM, respectively). Then sulphide concentration started to increase significantly. After 8 days of anoxic sulphide exposure, sulphide in the extrapallial fluid reached an equilibrium with the external sulphide concentration (186 t 24 PM). Number of determinations depicted in Fig. 2 were n = 5-13.

011 I I , , , I I

0 8 16 24 32

hours

Fig. 1. Scrobiculariaplana: averaged oxygen consumption at air saturation (10 “C, salinity 24 ppt) during 32-h measurements after different treatments. (O), 5 days of sulphidic exposure; (V), 5 days of anoxic exposure; (m), normoxic control clams. Each plot represents the mean oxygen uptake of six individual clams during progressing recovery, calculated from integrated 4-h intervals. For visual clarity, error bars are not shown. Instead, * indicates significant differences (Student-Newman-Keuls test, p < 0.05) between the average rates of the different treatments.

R. Oeschger, T.F. Pedersen 1 J. Exp. Mar. Biol. Ecoi. 184 (1994) 255-268 261

3.6. Accumulation of sulphide in siphon and mantle edge tissue

Sulphide content of the tissue of the inhalant siphon increased from 22 + 8 nmol.g-’

fresh mass (n = 4) in controls to 80.4 k 17.7 nmol.g-’ fresh mass (n = 5) after 2 days of incubation, reaching a maximum of 95.6 + 30.5 nmol.g-r fresh mass (n = 5) after 5 days (Fig. 3). Sulphide content of the mantle edge tissue increased from 41 k 7 (n = 3) in controls markedly during the first 2 days of exposure, reaching a content of 745 k 254 nmol.g-’ fresh mass at day 2 (n = 5) and 517 k 162 nmol.g-’ fresh mass at day 5 (n = 5, Fig. 4).

3.7. Succinate accumulation in adductor and foot muscle

Succinate levels in controls were 0.12 k 0.09 ~rno1.g~’ fresh mass (n = 6) in foot and

0.15 + 0.13 pmol.g-’ fresh mass (n = 6) in adductor. Anoxically incubated clams showed a marked increase in the succinate content of both foot and adductor muscle after one day of exposure, after which the accumulation was almost constant during the

remaining exposure experiments. Succinate contents under anoxia at day 5 were 3.25 k 0.93 prnol.g-’ fresh mass (n = 7) in adductor (Fig. 5) and 3.25 ? 0.92 ~rnol~g~’ fresh mass (n = 7) in foot tissue. After 5 days, sulphide-incubated clams accumulated significantly higher amounts of the anaerobic metabolite succinate during the incubation. Succinate in adductor muscle accumulated to 7.42 + 2.29 pmol*g-’ fresh mass, n = 5 (Fig. 5) and foot muscle contents were 6.10 + 2.42 prnol.g-I fresh mass

(n = 5).

0 2 4 6 8

days

Fig. 2. Scrobicularia plana: sulphide concentration of extrapallial fluid of clams incubated in March 1992 for 8 days in 200 PM sulphide (10 “C, salinity 24 ppt). Values are in PM sulphide k SD, n = 5-13.

262 R. Oeschger, T.F. Pedersen /J. Exp. Mar. Biol. Ecol. 184 (1994) 2X-268

15-a

0 1 2 3 4 5

days

Fig. 3. Scrobiculuriu pluna: sulphide contents in the inhalant siphon tissue of animals incubated in March 1992 anoxically in 200 PM sulphide (10 “C, salinity 24 ppt). Values are in nmol sulphide-g --’ fresh mass + SD,

n = 4 for controls, n = 5 for other measurements.

t 400

0 1 2 3 4 5

days

Fig. 4. Scrobiculario plana: contents of sulphide in mantle edge tissue of clams incubated in March 1992 for up to 5 days in 200 pM sulphide (10 “C, salinity 24 ppt). Values are in nmolg’ fresh mass + SD, n = 3 for controls, n = 5 for other meas~ements.

R. Oeschger. T.F. Pedersen 1 J. Exp. Mar. Biol. Ecol. 184 (1994) 255-268 263

Fig. 5. Scrobicularia plana: succinate contents of adductor tissue of clams incubated in March 1992 for 5 days of anoxia (v) or anoxia with 200 PM sulphide (0) (10 “C, salinity 24 ppt). Values are in prnol succinate.g-’ fresh mass k SD, n = 4 for controls, n = 5-7 for samples of incubated clams. *, Significantly different from anoxic values without sulphide, p < 0.05.

4. Discussion

Marine invertebrates from intertidal sediments are subjected to extreme fluctuations of abiotic factors such as restricted oxygen availability and concomitant development of sulphide. Sulphide contents in pore water from Logten Beach are highly variable. A pore water concentration of 26 PM sulphide was measured in March 1992, while at the same area sulphide concentrations measured were up to 1.19 mM in June 1992

(Table 1). Freshly collected specimens of Scrobiculuriu plana from Logten Beach ex- hibited a blackish coloration of the periostracum. Moreover, white shells of clams

previously kept normoxic turn black upon experimental sulphide exposure. This indicates that, although sulphide concentrations vary, Scrobiculariu plana regularly experi- ences sulphide in its environment. Previous studies showed high survival rates of Scrobicularia plana in the presence of sulphide (up to 18 days during anoxic sulphide exposure at 10 “C, Theede et al., 1969).

To evaluate the impact of sulphide on the overall metabolism of the clams, we firstly measured the normoxic oxygen uptake rates. Scrobiculariaplana from Arhus Bight had an oxygen consumption of 6.3 + 2.4 pmol O,*g-’ dry mass, which corresponds to populations from Great Britain (Worrall et al., 1983). Then we measured the time course and additional oxygen amounts needed to regain pre-exposed consumption rates. Anoxic exposure of Scrobicularia plana with and without sulphide is followed by a subsequent phase of increased oxygen uptake rates. The phenomenon of elevated rates of oxygen uptake after anoxic exposure is well-documented and referred to as the oxygen debt (Herreid, 1980). Increased oxygen uptake after anoxic exposure is due to

264

Table 1

R. Oeschger. T.F. Pedersen /J. Exp. Mar. Bid. Ecol. 184 (1994) 255-268

Sulphide concentration (FM) of pore water obtained from sediment cores (4.6 cm I.D.) collected at Lergten

Beach, June 1992, nm = no measurement

Depth

(cm)

Sulfide (PM)

Sample number:

1 2 3 4 5

o-2 0 1 <5 0 10

2-4 159 17 20 10 10

4-6 1003 <5 289 51 1

6-8 812 6 200 166 7

8-10 310 20 163 230 <5

10-12 1189 35 40 60 20

12-14 23 nm nm nm 40

replenishment of the ATP-pool, oxidation of anaerobic metabolites or re-oxygenation of body fluids (Ellington, 1983).

Total normoxic oxygen uptake of Scrobiculuriu pluna amounted for about 150 pmol

O,.g-’ dry mass during 24 h. During our recovery experiments the calculated oxygen debt was 225 pmol dry mass for anoxic incubated clams and 385 pmol O,.g-’ dry mass after sulphide exposure (integrated measurement during 24 h of recovery respiration,

c.f. Fig. 1). Even though sulphide-incubated Scrobiculuriu plunu have a supplementary oxygen debt of 160 pmol O,*g-’ dry mass, they recovered upon transfer into normoxic seawater within the same time course as anoxic exposed specimens.

To roughly estimate the amount of oxygen needed to oxidise accumulated sulphide during a 5-day exposure, the following assumptions are made: Measured sulphide accumulation in mantle edge and inhalant siphon was about 3.1 pmol.g-’ dry mass (ratio: dry mass/fresh mass w 0.2) and about 140 nmol in the body fluid (c.f. Fig. 2 and

assuming 1 ml of total body fluid). Assuming an oxidation of sulphide to thiosulphate upon transfer to normoxia, about 2.3 pmol oxygen are needed to oxidise this amount of sulphide. Taking into account the whole soft tissue of clams (e.g. a factor of 6 to estimate the entire sulphide contents of the tissues) less than 10% of the elevated oxygen uptake of sulphide vs. mere anoxia (of an additional oxygen debt of 160 pmol O,.g-’ dry mass) is needed. Even an oxidation of some of the accumulated sulphide to sulphate, a process which needs more oxygen per molecule of sulphide, will not increase the oxygen demand substantially. From this consideration it seems, that the clams need most of the additional oxygen demand to adjust their metabolism after a sulphide exposure, although details are yet awaiting a closer study.

The toxicity of hydrogen sulphide is well-known. It is mainly attributed to the block- age of cytochrome c oxidase of the respiration chain and the interference with other crucial enzymes (National Research Council, 1979). A first protection against the detrimental effects of sulphide seems to be a passive, but temporarily effective, exclu- sion by shell closure in bivalves. We observed Scrobiculuriu plunu extending its siphons into the seawater during sulphide exposure. Deposit feeding bivalves, like Scrobiculuriu

R. Oeschger, T.F. Federsen /J. Exp. Mar. Bid. Ecol. 184 (1994) 25-F-268 265

plana, use their siphons to take up organic particles by taking in a stream of water. Such a permanent siphon extrusion as seen during our experiments seems to be related to monitor the su~oundings for better ambient conditions. In nature, sulphide develops in the deeper sediment layers and comes from below the clams, while oxygen will be available from above the clams’ position. Although this behaviour results in a constant exposure of the siphon to high concentrations of ambient sulphide, its sulphide content is comparatively low. Whether the siphons of Scrobicularia plana have a special kind of diffusive barrier against sulphide or even exhibit a different capacity for sulphide detoxification, remains presently unanswered. This assumption derives from our obser- vation during the experiments, because probably the siphons are most frequently exposed to sulphide according to the clams’ mode of living.

Moreover, Scrobicularia plana managed to keep a low sulphide concentration in its extrapallial fluid during the first 2 days of incubation. This seems to be partly due to a reduced ventilation activity, a typical behaviour of bivalves during anoxia. Besides such a reduction of exchange of sulphide loaded seawater, another mechanism appears to be effective. The mantle edge tissue of the clams seems to function as a temporary “sulphide-trap”, involved in a first passive defence reaction due to sulphide precipitation. The mantle edge tissues turns black during sulphide exposure, which is probably due to the formation of a black metallic-sulphide precipitate, e.g. iron-sulphide. Mea- surements revealed that mantle edge tissue of Scrobicularia plana had a high sulphide content. A slight decrease of the sulphide content of the mantle edge tissue after 5 days might be due to an increased solubility of precipitated acid-labile sulphide, connected with an acidification of the tissue due to an elevated accumulation of anaerobic end products. Recently published results on Macoma bafthica support our hypothesis. The mantle edge of this species turns also black upon sulphide exposure and the clams are also able to maintain a sulphide concentration of their body fluid below the ambient experimental conditions (Levitt & Arp, 1991). Ultrastructural studies have shown, that the blackening derives from intra- and extracellular electron dense inclusions, identi- fied as sulphur containing molecules. The proportion of these compounds seems to be higher in animals exposed for a longer time to sulphide (Windoffer & Jahn, 1994).

During progressive or prolonged anoxia, many marine invertebrates rely on the so- called glucose-succinate pathway to maintain a production of metabolic energy (for a review see e. g. Livingstone, 1991). Out of a variety of metabolites formed during anoxia, succinate is a sensitive indicator. Anaerobic working mitochondria reduce fuma- rate to succinate to keep the basic generation of ATP for cellular processes (Schottler et al., 1984). But on the other hand sulphide interacts on mitochondrial processes involved in energy release, i.e. blocking cytochrome c oxidase (National Research Coun- cil, 1979). To evaluate a possible effect of the presence of hydrogen sulphide exposure on that kind of mitochondrial energy formation, we analysed the accumulation of succinate under both conditions in adductor and foot muscle. Muscular tissues of marine bivalves essentially contribute to anaerobic energy production when oxygen is lacking in the environment. Our results reveal, that the clams are able to maintain a working anaerobic mito~hondri~ metabolism under both conditions. This is deduced by the levels of succinate, although its contents are higher in the presence of sulphide compared to mere anoxia. Such an increased steady state concentration might indicate

266 R. Oeschger, T.F. Pedersen / .I. Exp. Mar. Biol. Ecol. 184 (1994) 255-268

an influence of sulphide in such a way, that the clams do not decrease their metabo-

lism as strong as during mere anoxia. This might also explain a reduced survival rate

in the presence of sulphide (Theede et al., 1969). Studies on the polychaete Arenicolu

marina also reveal an elevated accumulation of succinate in the presence of sulphide (Volkel & Grieshaber, 1992) and Hagermann & Vismann (1993) found a decreased survival in the hypoxia tolerant crustacean Saduria entomon during anoxic sulphide exposure. But this seems not to be a general feature of marine invertebrates, since in the clam Arctica islundica, which is extraordinarily resistant to anoxia and sulphide, no difference in succinate accumulation in the tissue of adductor muscle was found dur-

ing a long-term exposure of 10 days (Oeschger & Storey, 1993). In conclusion, Scrobiculuriu plana is well adapted to withstand anoxic exposure to

hydrogen sulphide. Sulphide exposure caused an elevated oxygen consumption, which was taken as a measure for the influence of this noxious compound on the metabolism

of the clams. Clams are able to maintain an anaerobic metabolism in the presence of sulphide for a substantial period of time. This confirms previous studies and underlines the importance of this pathway for marine invertebrates also under sulphidic conditions

(Oeschger & Vetter, 1992; Volkel & Grieshaber, 1992; Hagermann & Vismann, 1993; Oeschger & Storey, 1993).Whatever detailed disturbances occur in the presence of sulphide, the clams are able to restore pre-incubated rates, i.e. are able to balance those effects which are obviously not detrimental throughout the course of our experimental period. Our results suggest, that the decline of northern European populations of this species in recent years (Reise, 1982; Essink et al., 1991) has to be attributed to other factors than an insufficient adaptability of adult Scrobiculariaplana to oxygen deficiency or exposure to hydrogen sulphide.

Acknowledgements

T.F.P. thanks Prof. J. Hylleberg for generous support and was financed by a grant from the Danish Natural Science Research Council (1 l-8 109). We thank J. Hylleberg for making the visit of R.O. to the University of Aarhus possible. Additional support was provided by a grant-in-aid to R.O. from the Bundesminister fur Forschung und Technologie (DYSMON, 03 F0045A). We are indebted to Prof. H. Theede for support during completion of this study at Bremerhaven, Dr. B. Vismann for a critical

review of the ms. and Mrs. Elsebeth Thomsen and Petra Wencke for technical assis- tance.

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influence of anoxia and hydrogen sulphide on the energy metabolism of scrobicularia plana (da costa)...

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