Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus)

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mrežnica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.