Prior to testing sediment extracts were prepared using two methods: (i) SS-EN-ISO/TS 21268-2:2007 for soil quality (L/S ratio 10:1; leachant: 0.001 M CaCl2) (ii) SS- EN 12457-2 for waste characterization (L/S ratio 10:1; leachant: ultrapure water).
The test was performed according to standard procedure ISO 13829:2000(E) with and without the addition of an enzymatic complex (S9) to analyze the genotoxicity of both parental compounds and possible metabolites of these. Samples were serially diluted and all concentrations were tested in triplicates, with all samples being tested on three separate plates. A 50 μg/l nitroquinoline-1-oxide (4-NQO) was used as positive control and pure growth medium was used as negative control. Induction of genotoxicity, expressed as ß-galactosidase activity was measured as the absorbance at 420 nm after two hours of exposure, followed by two hours of incubation. Growth was measured as the absorbance at 610 nm. The absorbance was measured with an ELx800 absorbance microplate reader (Biotek, USA). The result was calculated as an induction ratio, inversely proportional to the growth. A sample concentration, at which the induction ratio of the sample was measured to be greater than 1.5, was considered genotoxic. The validity of the tests was assessed according to ISO 13829:2000.