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A comparative survey of the anaerobic survival potential of four different bivalve species and the interference of associated bacteria has been carried out. Individuals from both subtidal and intertidal environments were considered by selecting the following species: Mytilus edulis (subtidal epifaunal), Spisula subtruncata (subtidal infaunal), Macoma balthica (intertidal infaunal) and Cerastoderma edule (intertidal infaunal). Anaerobiosis was simulated in the laboratory by subjecting individuals to the following conditions: nitrogen atmosphere, air atmosphere and anoxic seawater incubation. Moreover, the effect of the antibiotic CA (chloramphenicol) was investigated, either as a pre-treatment of individuals kept under normoxic conditions for a week or directly added to the anoxic incubation media. According to survival performances of the individuals, intertidal animals that use to cope with tidal fluctuations in the coastline (emersion processes) had an extraordinary greater capacity to survive aerial exposure as compared to both nitrogen gas and anoxic seawater incubations most likely due to their capacity to perform aerobiosis at certain rate from atmospheric oxygen availability. Specifically, Macoma balthica enlarged its survival potential up to 24.8 days (LT(50)) under air exposure at 12 degrees C as compared to other specific treatments used here (4.9 days). The latter pattern was also observed, although in a much lower magnitude, for the other intertidal species Cerastoderma edule that survived 3.7 and 4.6 days (LT(50)) under nitrogen atmosphere and anoxic seawater incubation, respectively as compared to 9.5 days for emersed individuals. In contrast to the subtidal species, aerial exposure of both intertidal species led to a much higher survival performances than incubation of individuals in anoxic media with the presence of antibiotic. Survival capacity of the subtidal species Mytilus edulis and Spisula subtruncata was statistically similar under air and nitrogen atmospheres and anoxic seawater incubation. Then, subtidal species have a limited ability to air breathing as a conclusion of a similar survival in atmospheric and anoxic seawater incubations. Remarkably, M. edulis represented the only exception when considering longer-term survival capacity compared to the LT(50) values. Indeed, differences in LT(90) values for M. edulis were statistically different, values decreasing significantly from 19.7-19.9 days (under both nitrogen and air atmospheres) to 16.7 days when individuals are incubated in anoxic seawater. This may be due to the adverse effects of anaerobic bacteria that spontaneously proliferate within the static seawater incubations. As well as for S. subtruncata, possible aerobic processes under aerial exposure of mussels seemed to be not significant for the enlargement of its survival potential, since results obtained for both air and nitrogen atmospheres are similar. Pre-treatment with the antibiotic chloramphenicol caused survival capacity to increase by a factor of approx. 2 (M. edulis) and 34-44% (S. subtruncata). In contrast to intertidal species, the direct addition of the antibiotic to the incubation media caused the highest survival performances in both subtidal species. Habitat differences and species-dependent variability must be considered as significant sources of variation when studying the anaerobic performance of individuals using the most common experimental anaerobic techniques to test survival potential.

Composition of free amino acids (FAA) in juveniles of Mytilus galloprovincialis was analysed along a large geographical coastline area in Galicia (NW Spain). Individuals were sampled in February 2003, three months after the Prestige oil spill. Pollution values at sampling time were reported as polycyclic aromatic hydrocarbons (PAHs) concentrations in soft tissues of individuals and varied between the highest amount observed in Carrumeiro mussels (502 ng/g dw) and the lowest in Pindo mussels (196 ng/g dw), both locations being close to each other in the centre of the geographical area under study. Pollution values in the other populations varied within the range of 241-347 ng/g dw. Total free amino acids (TFAA) were highest in Aguiño-Pindo-Carrumeiro juveniles at the centre of the Coastline area studied (420-462 micromol/g dw) as compared to the other populations at North and South of Galicia (312-347 micromol/g dw). TFAA results were based on the variability observed in protein free amino acids (PFAA micromol/g dw) among populations (214-249 micromol/g dw for Aguiño-Pindo-Carrumeiro mussels and 98-149 micromol/g dw for the other populations) whereas non-protein free amino acids (NPFAA) taurine and ornithine did not show any significant spatial pattern of variation. Glycine and alanine represented the most abundant PFAA (16-29% and 2.7-11.9% of TPFAA, respectively) and significant correlations between PFAA and both the protein content of soft tissues (r=-0.82) and the condition index of juveniles (r=0.86) were observed. No significant relationships were detected, however, between pollution values in soft tissues as PAHs and FAA profiles with the exception of alanine concentrations as percentage of TFAA (r=0.88; P<0.01). The latter seemed to be an "all or nothing" effect likely due to the influence of other abiotic factors at one of the sampling sites. Such relationship was found not significant when the outlier represented by Carrumeiro mussels was removed from the analysis. The most abundant free amino acid taurine (43.2-68.5%TFAA) followed an inverse variability of that of glycine and by extension of the group PFAA most likely as a compensatory decrease in mussel populations with low protein content (and high condition index). Accordingly, taurine:glycine (t:g) ratio varied between 1 and 2 in most mussel populations but increased up to 3.2-4.2 in Miranda and Bueu mussels at both ends of the geographical interval studied with a corresponding PAHs concentrations of 261 and 304 ng/g dw, respectively. These mussel populations with the highest t:g ratios were characterised by the lowest PFAA contents (below 40%) and condition index values (below 10%). Results of the present study established a significant link between energetic status of growing juveniles and FAA concentrations in environments with different pollution degrees. Variability of the free amino acids profiles in soft tissues were related to endogenous factors of juveniles (protein content, condition index) whereas no relationship with contamination values could be observed. The utility of t:g ratio as general condition factor for M. galloprovincialis is also corroborated for in situ growing juveniles.

The anoxic survival time of the bivalves Chamelea gallina, Cerastoderma edule and Scapharca inaequivalvis from two different ecosystems and differing anoxia tolerances was studied in static (closed) and flow-through systems. The antibiotics chloramphenicol, penicillin and polymyxin were added, and molybdate (specific inhibitor of the process of sulfate reduction). Survival in (near) anoxic seawater of Chamelea was studied in a static system by comparing untreated seawater with autoclaved seawater and untreated clams with clams incubated in well-aerated seawater, containing the broad-spectrum antibiotic chloramphenicol, prior to the anoxic survival test. With untreated clams and natural seawater (median mortality time 2.4 days) a decrease in pH and exponential accumulation of sulfide and ammonium was observed in the anoxic medium, indicating excessive growth of (sulfate reducing) bacteria. In sterilized seawater LT50 (2.1 days) was not significantly different and again considerable amounts of ammonium and sulfide accumulated. However, pre-treatment of clams with chloramphenicol resulted in an increase of LT50 (11.0 days) by approximately fivefold. Accumulation of ammonium and sulfide was retarded, but was finally even stronger than in the medium containing untreated clams. Median mortality times were 2.5 and 2.4 days for Chamelea and 2.7 and 2.9 days for Cerastoderma for static and flow-through incubations, respectively. Addition of chloramphenicol increased strongly survival time in both systems with corresponding values of 11.0 and 16.3 days for Chamelea, and 6.4 and 6.5 days for Cerastoderma. LT50 of Scapharca in anoxic seawater was 14.4 days. Chloramphenicol and penicillin increased median survival time to 28.5 and 28.7 days, respectively, whereas polymyxin displayed no effect (LT50=13.6 days). Molybdate added to artificial sulfate free seawater blocked biotic sulfide formation, but did not improve survival time (LT50=13.7 days). Overall the results indicate that proliferation of anaerobic pathogenic bacteria, firmly associated with the bivalves, is a main cause of death besides lack of oxygen. Bacterial damage is probably caused by injury of the tissues of the clams and not by the release of noxious compounds to the medium.