An investigation of the use of cell culture techniques in ecotoxicity testing

Abstract

The usefulness of cell culture methods in environmental analysis was assessed by direct comparison of a battery of in vitro I in vivo test methods. Criteria for comparison were the ease of handling, sensitivity of the test species, reproducibility, rate of generation of data etc. The experimental work was divided into two main areas, the first related to predictive hazard assessment, the second to environmental monitoring, as follows; 1:- Predictive Hazard Assessment. The ecotoxicity of three chemicals of interest to the fish farming industry (Nuvan [dichlorvos], ivermectin and malachite green) was assessed. In vitro methods used were the cytotoxicity of the chemicals to two cell lines (L929 and RTG-2), each quantified by two dye end-points (crystal violet and neutral red). In vivo methods used were standard toxicity testing (fish acute toxicity, algal growth inhibition) and proposed screening methods (Artemia, Brachionus and Streptocephalus immobilisation). The cell culture methods were found to be rapid, reproducible and versatile. There was no significant difference between the sensitivity of the two end-points. There was no significant difference in the sensitivity of the two cell lines to ivermectin. Sensitivity to Nuvan was largely similar. RTG-2 cells were significantly more sensitive to malachite green. The sensitivity of the cell lines relative to the whole organism trials varied from low (ivermectin), to median (Nuvan), to high (malachite green). Interpretation of the cell culture data in conjunction with Structure Activity Relationship (SAR) data greatly increased this sensitivity. The most sensitive in vivo system was the fish acute toxicity test, the least sensitive was the algal growth inhibition test. Invertebrate screening trials were rapid and reproducible. Invertebrate sensitivity varied between species and toxin. Overall, cell culture methods exhibited a number of advantages over in vivo methods. These included greater statistical strength, versatility, and a wide range of theoretical applicability. Limitations include a lack of systemic factors. However, the use of battery in vitro tests and the interpretation of cytotoxicity data in conjunction with SAR data would decrease these limitations. 2:- Environmental Monitoring. ( a ) Metalliferous effluent study. The toxicity of three fractions of a metalliferous effluent, one low pH, and two neutral, were determined using the L929 cell line (crystal violet end point) and Artemia Experiments on the effects of serum, exposure time and seeding density were carried out on the cell culture system. Effluent fractions were also analysed by AAS. The sensitivity of the cell line to the metalliferous effluents was high. There were no major technical problems with the operation of the cell culture tests. Comparison with AAS data showed that metal concentrations were poor indicators of effluent toxicity. Increasing media serum concentration decreased the indication of toxicity given, at approximately double, the rate seen with the sheep dip. All of the effluent toxicity was expressed within 24 hours. Increased seeding density reduced the toxicity indicated. Artemia sensitivity to metalliferous effluents was low. A high sensitivity to the low pH effluent was shown to be related to pH. v ( b ) Sheep dip study. The toxicity of an organophosphate based sheep dip was assessed using L929 cells (including the effect of serum concentration, seeding density and incubation time on toxicity indicated) and Artemia, as above. A column study to assess the efficacy of a peat based treatment system for the removal of sheep dip from solution was carried out using GC analysis, and toxicity analysis using L929 and Artemia , as above. The sensitivity of the cell line to the sheep dip was found to be relatively low, which limited the amount of data that could be generated on the monitoring of column effluents. Increased media serum concentration reduced the toxicity indicated. Most of the toxic effect occurred within 48 hrs. Higher seeding density reduced toxicity indicated. The sensitivity of Artemia was classed as medium, and was useful for the monitoring of column effluents. Overall, results from the study demonstrated that there are a number of areas of environmental analysis to which cell culture methodologies could make a useful contribution. Analysis of materials in parallel with other ecotoxicity methods demonstrated a number of problems which are a feature of existing methods. Cell culture results improved on a significant proportion of these.