Research Articles A New Microbial Contact Assay 9 © 2005 ecomed publishers (Verlagsgruppe Hüthig Jehle Rehm GmbH), D-86899 Landsberg and Tokyo • Mumbai • Seoul • Melbourne • Paris JSS – J Soils & Sediments 5 (1) 9 – 15 (2005) Research Articles A New Microbial Contact Assay for Marine Sediments Dedicated to Prof. Dr. Ulrich Förstner on his 65 th birthday Susanne Heise* and Wolfgang Ahlf Dept. of Environmental Science and Technology, University of Technology, Hamburg-Harburg, D-21071 Hamburg, Germany * Corresponding author ( s.heise@tu-harburg.de) DOI: http://dx.doi.org/10.1065/jss2005.02.131 Abstract Background, Aims and Scope. The number of microbiological contact tests for marine sediments is low, although microorgan- isms enable a rapid screening and monitoring of sediment qual- ity and a high resolution of hazard assessment. As no single biotest can provide reliable answers concerning the potential hazard of environmental samples, a combination of bioassays needs to be applied to serve this purpose. In order to cover as many potential effects as possible, test organisms should have different sensitivi- ties; assays should cover different exposure pathways, and meas- ure the effect on various physiological functions. Methods. 3 different Vibrio species (V. proteolyticus, V. natriegens, V. gazogenes) were tested for their suitability as test organisms in a contact assay on the basis of their activity, sensitivity and their spectrum of salinity tolerance. As a test endpoint, dehydrogenase activity over an incubation time of 2 hours (~ 3 generations) was chosen, quantified by resazurine reduction. The test was mini- aturized to 96 well plates, including a dilution series, and quality criteria were established. The assay was then tested on natural sediments from a contaminated site in the Lübeck Bight. Results and Discussion. Vibrio proteolyticus proved to be the best suited test organism out of those tested for this bioassay. The miniaturized test system revealed a coefficient of variation of positive controls in 16 tests of 17.8%. Its application to con- taminated sediments from the Lübeck Bight showed a good differentiation of samples from different depths and zones, which reflected the general contamination pattern and capping activi- ties in that area. Conclusion. The miniaturized test system that has been devel- oped for V. proteolyticus is suited to assess toxic effects of brack- ish and marine sediments. Due to an increased number of repli- cates and an extensive sediment dilution series, the degree of certainty of hazard assessment is elevated. Recommendations and Outlook. The integration of this sedi- ment contact assay as a complementary test in a microbiologi- cal test battery is recommended Keywords: Contact assay; Lübeck Bight; marine sediments; miniaturization; Vibrio proteolyticus Introduction Bioavailability of contaminants in sediments is a complex result of compound-particle interaction, and goes beyond solubilisation of substances and uptake with food. Evidence that particle-associated contaminants can impact different organisms also on other exposure routes has repeatedly been demonstrated (e.g. Harkey et al. 1994, Liss & Ahlf 1997). Bioavailability of adsorbed compounds has been shown to be influenced for example by geochemical processes (Griscom et al. 2000) and by a number of sediment specific properties such as organic matter, clay content, nanoporosity, and sur- face area (Chung & Alexander 1998). It often decreases with time (Alexander 2000, Kukkonen & Landrum 1998) and compounds can be actively made bioavailable by microor- ganisms (Wick et al. 2001). Due to this complexity, it seems unlikely that chemical data and models alone can predict impacts of sediment-bound compounds to the (benthic) com- munity (Ahlf et al. 2002). Hence, attention is increasingly given to bioassays which examine the effect of sediments in direct exposure to the test organisms. A survey of the Ger- man Environment Agency from 1999 lists 32 organisms (ex- cluding fish species) that were published or mentioned as test organisms for sediments, mainly in direct contact, by experts from OSPARCOM and HELCOM member states. Of these 31 species, 16 are crustacea, 5 are bivalves and 5 are polychaetes (Herbst & Nendza 1999). The only bacte- rium suggested is the bioluminescent inhibition test with Vibrio fischeri, commercially available under names like 'Microtox' or 'LUMIStox'. Although seemingly less common, microbiological test sys- tems have been recommended as efficient methods for rapid screening and continuous monitoring of toxicity of chemicals and wastes (Laake 1984). Ease of handling, short exposure time and good reproducibility are the advantages with the fa- vourable site-effect of low costs. The possibility to increase the number of replicates without straining the workload too much is especially desirable when a heterogeneous matrix like sediment is tested. Their ecological relevance and therewith their importance for the whole process of risk assessment, however, has been questioned (Asante-Duah 1998). The simi- larity of complex biochemical functions with those of higher organisms and the important role of microorganisms in the nutrient cycles of the ecosystem are, in our opinion, strong arguments to expand their application in ecotoxicological test- ing and their integration in biotest batteries. When combinations of biotests are applied to test sediments with the purpose of detecting negative effects, it is desirable to test for acute responses and for those that do not mani- fest in the first generation. Additionally, the test systems within a test battery should reveal different sensitivities and test different endpoints in order to cover a wide range of potential contaminants.