Effects of tetracycline on the soil microflora: function,diversity, resistance

Background  Tetracycline is a widely used antibiotic in animal production. Significant amounts of the substance reach the soil via feces, urine and manure application. As tetracycline is a persistent compound with antibacterial activity, its presence in soil may have undesired direct and indirect effects. These have been investigated so far focusing on effects on selected microbial functions. Objectives  The aim of the present study was to obtain comprehensive information on potential effects of tetracycline on the soil microflora under environmentally relevant conditions. The investigations included function and structure of the microbial biocoenosis and the distribution of resistance genes. Methods  Pig manure rich in tetracycline resistance genes was applied to a sandy soil. This soil as well as an unamended soil were additionally treated with several concentrations of tetracycline. The spiked soils were incubated in outdoor lysimeters for several months. Substrate induced respiration, PLFAs, ten selected resistance genes, and the concentrations of tetracycline were determined. Results  The test concentrations, though far exceeding environmental relevance, caused only small effects. An establishment of resistance could not be detected. Applied resistance genes were not detectable at the end of the study even in the presence of added tetracycline. Conclusion  Due to the high sorption capacity of the antibiotic, environmentally relevant concentrations of tetracycline do not seem to cause undesired effects on the soil microflora.     

Terrestrial Ecotoxicity of Eight Chemicals in a Systematic Approach (7 pp)

Background and Objective   Terrestrial ecotoxicity data are required for many research purposes. The data are derived either from the literature or elaborated by own investigations. As the terrestrial toxicity tests are usually time-consuming and labour intensive, the experiments are performed with a limited number of test organisms and soils. In the context of a project sponsored by CEFIC-LRI (European Chemical Industry Council – Long-Range Research Initiative), EC50-values were systematically elaborated for eight chemicals with a wide range of logKow-values (CdCl2, Trinitrotoluene, 3,4-dichloroaniline, 2,4-dichlorophenol, Tributyltinchloride, Pentachlorophenol, Benzo(a)pyrene, p,p-dichloro-2,2-diphenyl-1,1,1-trichloroethane). The substances were selected covering a broad range of physico-chemical and ecotoxicological properties. As toxicity endpoints, microbial activities, plant germination and growth as well as reproduction of earthworms and collembola were determined. As such systematic investigations are rarely performed and for some substances no data existed, the data pool is made available to the scientific community.Methods   All toxicity tests were conducted on three different soil types (sandy soil, silty soil, loamy soil), according to ISO and OECD guidelines Results, Discussion, Conclusion and Outlook   The different toxicities of the chemicals, the influence of soil properties on bioavailability as well as different sensitivities of test organisms and test parameters are reflected by the EC50-values. The results showed that the EC50-values calculated on the basis of nominal concentrations can significantly vary from EC50-values derived from analytical concentrations for some substances. To avoid false conclusions, this has to be considered especially when concentrations determined in the field are compared with toxicity data obtained from the literature or calculated on the basis of nominal concentrations. Moreover, the results indicate that terrestrial ecotoxicological tests should be accompanied by chemical analyses, to be sure to derive sound EC50-values. As the tests were performed according to international guidelines, the comprehensive data pool relevant for the terrestrial ecotoxicity of eight chemicals is considered to be of great value for further research and discussions dealing with the habitat function of soils.     

Bioassays for the ecotoxicological and genotoxicological assessment of contaminated soils (results of a round robin test)

For the assessment of contaminated or remediated soils, in addition to chemical analyses, ecotoxicological tests are performed which focus on the retention function of soils (determined by tests with aqueous soil extracts) and on the habitat function (determined by tests with soil). While numerous standardized test guidelines exist to investigate effects by chemical substances, respective test procedures for soil assessments are lacking. To close this gap, a round robin test was performed to support standardization. Four contaminated soils were tested using a total of fifteen test systems including ecotoxicological and genotoxicological tests with soil extracts and soil. The results obtained from the tests with soil extracts are presented in this article.     

Validation of Microplate Bioassays for the Assessment of Contaminated and Remediated Sites

Background, Goal and Scope  Bioassays are frequently used to investigate the water extractable ecotoxicological and genotoxicological potential of contaminated soil samples. A laboratory intercomparison study was performed for validation of miniaturised biological test systems for the assessment of contaminated and remediated sites. The successful performance of this study resulted in an optimisation of microplate assays with respect to the testing of chemicals and environmental samples. Methods  For this purpose, miniaturised bioassays were chosen, which, because of their stage of development, are suitable for routine application in the characterisation of the water extractable ecotoxicological and genotoxicological potential of soils. These ecotoxicological and genotoxicological assays were performed with contaminated soil samples by three institutions at the same time. Results and Discussion  The toxicological assessment of the contaminated and remediated soil samples using LID-values, as a rule, was highly uniform. Some minor deviations could, for the most part, be explained by the heterogeneity of the soil samples and, to a lesser extent, by methodical aspects. The difference in sensitivity towards contaminants of the two bacteria Vibrio fischeri and Pseudomonas putida was pointed out. In the algae test with Desmodesmus subspicatus, the influence of the highest sample concentrations on the growth controls became obvious. It was recommended to modify the experimental setup of the microtitration plate, i.e. to place growth controls located next to both the lowest and the highest dilution steps of the sample. The Ames-test did in some cases provide new information on the genotoxicity of the samples, but is not considered useful in a test battery for the evaluation of the genotoxic potential because of its great expense in time and work. Conclusions  The investigations in this laboratory-intercomparison study for the assessment of the water extractable toxic potential of soil samples show that different bioassays are needed, which, in contrast to chemical-analytical methods, can detect the complete effects of all present pollutants in contaminated and remediated soils and solid substrates path-specifically. Recommendations and Outlook  If the recommended modifications for the performance of the bacterial and algae growth inhibition assays on microplates are taken into consideration, these tests can substitute the tests performed on a macro scale. The usefulness of the umu-test and the NM2009-test for the investigation of the genotoxic potential has been proven. Although the tests performed on microplates require much lower sample amounts, it is recommended that sample amounts be eluted in accordance with current guidelines to ensure representativity of the sample. Further work should focus on toxicity identification studies in the future by combining chemical and toxicological analyses.     

Earthworm avoidance test for soil assessments

For ecotoxicological assessments of contaminated or remediated soils pointing to the habitat function of soils for biocenoses, standardized tests with earthworms (acute test, reproduction test) are available among others. Tests used for routine applications should be sensitive and indicate impacts on test organisms after short test periods. The usually applied earthworm tests do not satisfactorily fulfil these criteria. Therefore, in the present work, a behavioural test with earthworms (test criterion: avoidance) was investigated in detail using uncontaminated, artificially contaminated and originally contaminated soils. It was demonstrated that the avoidance behaviour is primarily determined by pollutants, and not by chemical-physical soil properties. The sensitivity of the presented test reaches the sensitivity of established tests. For waste sites, a considerably higher sensitivity was determined. An avoidance behaviour of at least 80% of the worms leaving the soil to be assessed is proposed as a criterion for toxicity.     

Proposal of a testing strategy and assessment criteria for the ecotoxicological assessment of soil or soil materials

Background and Scope  Information on a potential contamination of soils or soil materials are derived by chemical analysis which takes place specifically for a given substance. For a comprehensive assessment, information on the bioavailable and mobile contaminant fraction, including all metabolites, is desirable. During the last years several research projects were initiated in Germany, to supplement the chemical analyses and to elaborate a suitable testing strategy. The main goal of this contribution is to elucidate the results of these research projects and to summarize the test strategy, which is recommended based on these results. Results and Conclusion  Ecotoxicological tests, which are standardized for the assessment of chemicals, were regarded as a suitable starting basis for a cost effective, pragmatic approach. Aquatic tests (testing of aqueous soil extracts) focus on the retention function of soils and terrestrial tests (testing of soil) on the habitat function. Suitable reference systems for the terrestrial tests and assessment criteria for both test types (terrestrial and aquatic) were elaborated. On the basis of a round robin test and a laboratory comparison test, a minimal test battery was established. This minimal test battery can be supplemented by further tests if more or specific information is required. Outlook  The recommendations should encourage the discussion regarding the application of biological methods for the assessment of soil quality. Such an assessment is or at least can be required by soil protection laws which have been adopted in some European countries within the last years.