[Cultivation of a permanent fish cell line in serum-free media special experiences with a cytotoxicity test for waste water samples]

The use of fetal calf serum (FCS) as standard medium additive for the cell cultivation must be regarded critically from the point of view of animal welfare as well as for scientific reasons and makes it necessary to look for alternatives. In the last years an in vitro cytotoxicity assay for the testing of industrial waste waters with the permanent fish cell line RTG-2 was established and pre-validated as an alternative to the fish test with the golden orfe. The application of FCS is also a special problem with regard to the testing of waste waters in a cytotoxicity test so that FCS-alternatives were tested. The RTG-2 cells were successfully adapted to the two solvents Basal Medium Supplement (BMS) and Ultroser-G (U-G) that are used to replace serum. The characterisation of these adapted cell lines showed no significant differences in growth rate, adhesion rate, viability and sensitivity to chemicals in comparison to the original RTG-2 cells. On the determination of the cytotoxicity of industrial waste waters the RTG-2 cells adapted to the BMS medium indicated a clearly higher toxicity of the waste water samples than the original RTG-2 cells. This result confirms the thesis that serum components react with waste water elements and thus change the bio-availability of toxic compounds.

[Toxicity assessment of waste water samples with fish cell lines]

An in vitro cytotoxicity assay with fish cell lines was established in order to reduce the number of animals used in fish tests with the golden orfe (leuciscus idus melanotus). In three co-ordinated working laboratories the toxicity of seven chemicals was determined according to a standardized protocol using the permanent cell lines D11 and RTG-2. There was an almost linear correlation (r=0.99) between the IC50 values after neutral red uptake and crystal violet staining of the chemically treated RTG-2 cells. These cells were more sensitive than D11 cells (r=0.93) with regard to chemically induced cytotoxicity. The test protocol was adapted for the testing of waste water (ten samples). Two samples induced cytotoxic effects in RTG-2 cells while in none of the three laboratories the same samples were found cytotoxic in D11 cells. The data with the seven chemicals and the still limited number of waste water samples (10) lend strong support to the assumption that the fish cell line RTG-2 can be regarded as well suited for detecting toxic properties of waste water.