Evaluation of Genotoxic Pressure along the Sava River

<div><p>In this study we have performed a comprehensive genotoxicological survey along the 900 rkm of the Sava River. In total, 12 sites were chosen in compliance with the goals of GLOBAQUA project dealing with the effects of multiple stressors on biodiversity and functioning of aquatic ecosystems. The genotoxic potential was assessed using a complex battery of bioassays performed in prokaryotes and aquatic eukaryotes (freshwater fish). Battery comprised evaluation of mutagenicity by SOS/<i>umuC</i> test in <i>Salmonella typhimurium</i> TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg—modified comet assay) was studied in blood cells of bleak and spirlin (<i>Alburnus alburnus/Alburnoides bipunctatus</i> respectively). Result indicated differential sensitivity of applied bioassays in detection of genotoxic pressure. The standard and Fpg—modified comet assay showed higher potential in differentiation of the sites based on genotoxic potential in comparison with micronucleus assay and SOS/<i>umuC</i> test. Our data represent snapshot of the current status of the river which indicates the presence of genotoxic potential along the river which can be traced to the deterioration of quality of the Sava River by communal and industrial wastewaters. The major highlight of the study is that we have provided complex set of data obtained from a single source (homogeneity of analyses for all samples).</p></div

SOS induction rate (mean ± SE) in SOS/<i>umuC</i> assay.

<p>Red line represents threshold induction value (1.5); PC—positive controls: 4NQO (0.5 μg/mL) in experiments without metabolic activation and benzo(a)pyrene (10 μg/mL) in experiments with metabolic activation.</p

Cell viability, frequency of hedgehogs and frequency of micronuclei in fish blood samples (mean ± SE).

<p>Cell viability, frequency of hedgehogs and frequency of micronuclei in fish blood samples (mean ± SE).</p

Overview of the parameters analyzed within the Sava survey 2015.

<p>Overview of the parameters analyzed within the Sava survey 2015.</p

The average element concentrations in whole body composite (wbc) samples of <i>A</i>.<i>alburnus</i> and <i>A</i>. <i>bipunctatus</i> (marked with *) in mgkg^-1 ww, and MPI values for each sampling station; different letters denote significant differences among studied sites (p < 0.05).

<p>The average element concentrations in whole body composite (wbc) samples of <i>A</i>.<i>alburnus</i> and <i>A</i>. <i>bipunctatus</i> (marked with *) in mgkg^-1 ww, and MPI values for each sampling station; different letters denote significant differences among studied sites (p < 0.05).</p

Sampling sites along the Sava River.

<p>Sampling sites along the Sava River.</p

Correlation between the levels of DNA damage obtained in different assays.

<p>Correlation of the values obtained in standard comet assay and (A) buffer exposed slides, (B) Fpg exposed slides and (C) net 8-oxoG sites; full line—regression line; dashed line—95 confidence level.</p

The values of tail intensity % obtained in standard alkaline comet assay in fish blood cells; values are represented as mean ± SE; different letters denote significant differences among studied sites (p < 0.05).

<p>The values of tail intensity % obtained in standard alkaline comet assay in fish blood cells; values are represented as mean ± SE; different letters denote significant differences among studied sites (p < 0.05).</p

Fpg—modified comet assay, tail intensity values for buffer and Fpg—exposed slides and net contribution of 8-oxoG sites (mean ± SE); different letters denote significant differences among studied sites (p < 0.05).

<p>Fpg—modified comet assay, tail intensity values for buffer and Fpg—exposed slides and net contribution of 8-oxoG sites (mean ± SE); different letters denote significant differences among studied sites (p < 0.05).</p

Cryopreservation effects on the cell viability and DNA damage level.

<p>*statistical significance in comparison with fresh sample (p < 0.05).</p