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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
In a preliminary experiment we compared controls with clams treated with strep- tomycin sulphate by incubating three replicates n 5 50 for each condition. LT
values
50
were 4.7060.12 and 9.3160.21, respectively. Since variability between replicates of 50 clams each was small, we have not used replicates in our studies to the effect of different
chemicals on survival time. Fig. 1A shows the anoxic survival curves of clams incubated in anoxic seawater
compared to the incubations in the presence of different antibiotics. Median survival time LT of Macoma in the flask without addition of antibiotics was 4.8 days. A
50
positive effect on survival time was observed due to the presence of all combinations of antibiotics. Most favourable was Provasoli’s antibiotic solution that increased LT
50
from 4.8 to 14.9 days, closely followed by penicillin plus streptomycin LT 5 13.4
50
days, chloramphenicol LT 5 13.3 days and 5-oxytetracycline LT
5 13.0 days.
50 50
The curves of these antibiotics were mutually not significantly different all comparisons
1
P . 0.05. Except for 5-oxytetracycline sulphide 17 mM, NH 0.3 mM there was
4
in these incubations virtually no biotic formation of sulphide 2 mM and small
1
increases of ammonium NH range: 0.02–0.12 mM in the medium Fig. 1B and C. In
4
the medium with penicillin there was some sulphide 4 mM and ammonium
1
formation NH 0.14 mM, but much less as compared to the medium with
4
5-oxytetracycline. However, the survival time was significantly shorter as compared to this and previous media P , 0.001. Least favourable on survival time was addition of
1
streptomycin. Accumulation of sulphide 36 mM and ammonium NH 0.80 mM
4
was in this incubation medium by far highest of all media containing antibiotics Fig. 1B and C. The LT
value of medium with penicillin and streptomycin was 10.2 and 8.9
50
days, respectively Fig. 1A, and the survival curves differed significantly P , 0.05. In
1
the control, sulphide 83 mM and ammonium NH 1.5 mM accumulation was
4
highest Fig. 2B. In general ammonium preceded sulphide accumulation see e.g. 5-oxytetracycline and control; Fig. 1B and C.
No significant difference in survival time was found between the control medium and the medium containing molybdate P . 0.05, Fig. 2A. The median mortality times were
4.8 and 4.4 days, respectively. However, when chloramphenicol was also added, molybdate showed a strongly and significantly negative effect P , 0.001, LT
7.8
50
days; Fig. 2A. Molybdate and chloramphenicol were both able to fully suppress
A . de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
245
Fig. 1. Survival curves A and changes in concentration of sulphide B and ammonium C under semi-static anoxic incubation of Macoma balthica without additions Control and in the presence of several antibiotics.
Arrows indicate when incubation medium was exchanged. Sulphide values in the presence of chloramphenicol, Provasoli’s antibiotic solution see Materials and methods for composition, and penicillin plus streptomycin
PEN STREP were below 2 mM not visible. Abbreviations: CA, chloramphenicol; OTC, 5-oxytetracycline hydrochloride; PEN, penicillin-G; Provasoli, Provasoli’s antibiotic solution; STREP, streptomycin sulphate.
246 A
. de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
Fig. 2. Survival curves A and changes in concentration of sulphide and ammonium B under semi-static anoxic incubation of Macoma balthica without additions Control and in the presence of chloramphenicol
CA, molybdate Mo and molybdate 1 chloramphenicol Mo 1 CA. Arrows indicate when incubation medium was exchanged. Sulphide values in the presence of chloramphenicol and in the presence of molybdate
with and without chloramphenicol were below the detection limit , 1 mM.
1
sulphide formation, but ammonium accumulation NH 1.3 mM appeared hardly to
4
be affected by molybdate Fig. 2B. In the Fig. 3, the results for the bacterial counts are shown. Compared to the flask with
seawater without animals, roughly on average 14 times more bacteria were observed in the water of the flask with animals. In the medium containing molybdate a nine-fold
increase of bacteria was observed. In the presence of chloramphenicol the bacterial count were much lower compared to flask with animals without any addition, and only 3–5
times more than in the flask with only seawater.
In the presence of chloramphenicol and Tris 25 mM survival time was comparable to the survival time of the presence of chloramphenicol alone LT
13.3 days. Without
50
sulphide addition the survival time was significantly P , 0.01 higher at pH 6.8 LT
50
14.8 days than at pH 8.2 LT 12.4 days; Fig. 4A. The antibiotic was used to avoid
50
uncontrolled biotic sulphide formation. The presence of abiotically added sulphide 200
A . de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
247
Fig. 3. Bacterial counts in anoxic seawater without clams SW and anoxic incubations of Macoma balthica without additions control and in the presence of chloramphenicol CA, molybdate Mo and molybdate 1
chloramphenicol Mo 1 CA. Bacterial counts represent means6S.D. n 5 3 of the average value of all daily counts until complete mortality.
Fig. 4. Survival curves of Macoma balthica A and concentrations of sulphide B in anoxic seawater adjusted with 25 mM Tris to pH 6.8 and 8.2 to which chloramphenicol CA and 200 mM sulphide was added.
H S refers to total sulphide.
2
248 A
. de Zwaan et al. J. Exp. Mar. Biol. Ecol. 256 2001 241 –251
mM decreased survival time. The effect was more pronounced at a lower pH. At pH 8.2, the LT
decreased by 20.8 from 12.4 to 9.9 days and at pH 6.8 by 35.2 from
50
14.8 to 9.6 days Fig. 4. However, the relative decreases were an order of magnitude smaller than the positive effect of the simultaneously added chloramphenicol. Therefore,
survival in the presence of sulphide was still much longer about 200 overall increase as for the control without antibiotic LT
4.8 days.
50
During the first 5 days between medium exchange, the added sulphide concentration decreased considerably. In the second interval of 5 days this occurred at a lower extend
and from day 10 onward the sulphide concentration maintained at the initial value Fig. 4B. Fortunately, the changes in sulphide concentrations at both pH values were
identical.
4. Discussion
