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. de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
1. Introduction
Benthic macrofaunal species show great variability in anoxic tolerance, even within closely related taxonomic groups. A comparison of studies is often complicated by
differences in experimental set-up and conditions used to estimate this tolerance Groenendaal, 1980. In a study of four bivalve species from the Mediterranean zone we
showed that the experimental set-up had a great impact on anoxic survival time De Zwaan and Eertman, 1996. In closed systems, a decrease in pH of the incubation
medium and accumulation of ammonium and sulphide occurred. Both a high dose of cadmium and the broad-spectrum antibiotic chloramphenicol increased survival time.
Also a postponed shell blackening and biotic sulphide accumulation was observed.
In the present survey we extend our study to anoxic survival of Macoma balthica, a bivalve from temperate Baltic and Atlantic coastal area. Past reports of mortality rate of
this clam show wide variability in anoxic survival. Brafield 1963 observed that this clam only tolerated anoxia for 2 or 3 days temperature not given. Dries and Theede
1974 reported LT values of specimens collected in the Western Baltic Sea of 22 and
50
20 days at 10 and 158C, respectively. More recent, Jahn and Theede 1997 reported median mortality values of 8–12 days at 108C for clams from the same location. The
large variation in data for M . balthica raises the question whether the results obtained in
closed systems indeed reflect artefacts or tolerance to anoxia. Understanding of the actual mechanisms causing death may help explain the inconsistency in literature data.
Clams Macoma balthica were collected from a mudflat of the Dutch coastal waters. The role of biotic sulphide and the influence of exogenous sulphide were studied in
semi-static systems. We also compared the effect of chloramphenicol and a number of other antibacterial agents applied in aquaculture suppressing bacterial activity. The
activity of bacteria was followed indirectly by estimating the release of sulphide and ammonium and the recording of pH, as well as by counting bacterial numbers in the
systems.
2. Material and methods
2.1. Individuals Infaunal specimens of M
. balthica were sampled at Paulinapolder, a tidal flat of the river Scheldt estuary in the southwestern part of the Netherlands. Average shell length of
the clams was 16.862.1 mm n 5 50. The average weight was 0.47 g and the volume 0.41 ml. Salinity at the sampling site was 2762 psu and the temperature 198C. The
clams were kept in an aquarium with sterilised sandy sediment and well-aerated unfiltered running seawater of 198C and 31 psu, pumped in from an inlet in the Eastern
Scheldt in front of the institute at Yerseke. After 4 days the animals were purged with filtered seawater. The next day the clams were used in the experiments.
2.2. Protocol of experiments Seawater, filtered over natural sand, was prepared anoxic in a 20-l glass reservoir by
A . de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
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vigorously bubbling for 2 h with N . The pH of the anoxic seawater was about 8.2.
2
From this reservoir under continuous flow of N , seawater was siphoned into ten 1-l jars.
2
The oxygen concentration after filling was less than 0.15 mg l Winkler method. To each jar the following antibiotics were added: 5 mg chloramphenicol, 10 mg 5-
oxytetracycline hydrochloride, 100 000 IU penicillin-G sodium salt and 100 mg streptomycin sulphate, 100 000 IU penicillin-G, 100 mg streptomycin sulphate, 20 ml
Provasoli’s antibiotic concentrated solution containing 240 000 IU penicillin-G, 1 mg chloramphenicol, 6000 IU polymyxin B sulphate and 1.2 mg neomycin Sigma. As an
inhibitor of the process of sulphate reduction, 5 g sodium molybdate 20.8 mM final concentration was added to one flask. In order to study the effect of molybdate
separately on survival time also a flask was used in which 5 g sodium molybdate and 5 mg chloramphenicol were both added. Under these conditions bacterial growth should be
inhibited by the activity of chloramphenicol. Also two flasks contained anoxic seawater without additions: one without and one with animals.
In a similar way, four flasks were filled from a reservoir to which 25 mM Tris was added. Two of these flasks were adjusted with 1 M HCl to a pH of 6.8 and two to a pH
of 8.2. From each of these two flasks, to one flask 5 mg chloramphenicol was added and to the other 5 mg chloramphenicol and 200 mM sulphide. Sulphide was added from a
stock solution 5 ml of 40 mM of washed crystals of Na S ? 6–9H O, neutralised with
2 2
HCl. After addition of 50 individuals the bottles were sealed with rubber stoppers and
incubated at 198C. Daily aliquots were taken for the determination of pH, sulphide, ammonium and bacterial count.
All incubation media were exchanged every 5 days in order to prevent decomposition of compounds, especially antibiotics. In such a manner a semi-static system was
obtained. No food was added during anoxic incubations. The clams occupied the bottom of the flasks in one layer. Mortality was controlled daily. Mortality was assessed by the
failure of constriction after the mantle edge of gaping animals was touched. Dead animals were removed from the incubation containers. The experiment was conducted in
autumn 1999.
2.3. Determination of sulphide, ammonium and pH Sulphide was determined after Svenson 1980 and ammonium after Truesdale
1971. The pH was measured using a Radiometer PHM 82 pH meter with a sulphide insensitive electrode.
2.4. Bacterial count Samples for bacterial count were taken in triplicate of the controls seawater with
animals and media containing chloramphenicol and molybdate, separate or in combina- tion. Also a seawater control no animals added was sampled. Samples were fixed in
2 glutaric-dialdehyde final concentration. One ml of sample was diluted in 9 ml Isoton. Cells were counted with a Coulter Multisizer. A counting tube with an orifice of
30 mm diameter was used.
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. de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
2.5. Statistics The non-parametric Kaplan–Meier test was used to estimate log-rank and Wilcoxon
values for comparing the survival curves Kaplan and Meier, 1958. A confidence limit of 95 was used to test the significance of differences between groups. LT
values
50
median survival times were estimated using the trimmed Spearman–Karber method a 5 10 Hamilton et al., 1997.
Throughout the manuscript the term sulphide refers to total sulphide, and the term Tris to the buffer, trishydroxymethylaminomethane.
3. Results
