Sanja Babić

Impact of treated wastewater on organismic biosensors at various levels of biological organization

Relating the treated wastewater quality and its impact on organismic biosensors (Prussian carp, Carassius gibelio and earthworm, Eisenia fetida) was the main objective of the study. The impact on health status of fish living downstream, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry, oxidative stress, genotoxic effects, as well as multixenobiotic resistance mechanism (MXR) was assessed. Treated wastewater discharged from the WWTP modified the environmental parameters and xenobiotic concentrations of the receiving surface waters. Potential bacterial pathogens from fish and respective waters were found in relatively low numbers, although they comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of effluent fish. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. Micronucleus test did not reveal significant differences between the examined groups, but a higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Earthworms indicated to the presence of MXR inhibitors in water and sludge samples, thus proving as a sensitive sentinel organism for environmental pollutants. The integrative approach of this study could serve as a guiding principle in conducting evaluations of the aquatic habitat health in complex bio-monitoring studies.

Multilevel ecotoxicity assessment of environmentally relevant bisphenol A concentrations using the soil invertebrate Eisenia fetida.

Bisphenol A (BPA) presents a serious threat to soil ecosystems, yet its effects on soil-inhabiting organisms are mostly unexplored. Therefore, the impact of environmentally relevant BPA concentrations on a terrestrial model organism, the earthworm Eisenia fetida, was assessed. Animals were cutaneously exposed to 100nM and 10μM BPA up to 10days (10-d). Next, a battery of biomarkers was used for ecotoxicological evaluation on a cellular, tissue and behavioural level. HPLC analysis showed that after a 10-d exposure, BPA accumulation reached a maximum of 2.50μg BPA per g of wet tissue weight. On the cellular level, up to 3-d BPA exposure caused increased lipid oxidation indicating oxidative stress. Histopathological assessment of cell wall and ovaries after 7- and 10-d BPA exposure showed multiple abnormalities, i.e. hyperplasia of epidermis, increased body wall thickness and ovarian atrophy. Detection of these changes was facilitated by a newly proposed semi-quantitative scoring system. Finally, behavioural changes were detected after only 3days of exposure to 100nM BPA. Altogether, the presented multilevel toxicity evaluation indicates high sensitivity of earthworms to low BPA doses.

Embryotoxic and genotoxic effects of sewage effluents in zebrafish embryo using multiple endpoint testing

Wastewater treatment plant (WWTP) effluents are often complex mixtures of various organic and inorganic substances. Quality control of wastewaters and sludges has been regulated with measuring several physico-chemical parameters and sometimes using biological methods with non-specific responses, while synergistic action mechanisms of contaminants in such complex mixtures is still unknown. Toxic effects of wastewaters within and downstream of the WWTP in City of Virovitica, Croatia, were tested on zebrafish Danio rerio using a set of biomarkers that enabled an insight in wastewaters toxic potential on embryos at the cellular, tissue and the whole organism level during an early ontogenesis (24 and 48 hpf). Exposure of embryos to the wastewater samples from WWTP Virovitica increased mortality and abnormality rate. Heart rate, spontaneous movements and pigmentation formation were also markedly affected. Biochemical markers confirmed the presence of MXR inhibitors in all tested wastewater samples, indicating the increase of pollutant accumulation in the cell/organism. Also, a tendency of DNA damage decrease measured with Comet assay was evident in wastewater samples downstream from WWTP although control levels were not reached in any environmental sample. Histopathological analysis showed that exposure to tested samples resulted in impaired muscle organization, notochord malformation and retardation in eye and brain development at embryos 48 hpf. Furthermore, semi-quantitative histopathology assessment indicated increased percentage of embryo defects in river water sampled several kilometers downstream from the WWTP, confirming toxic potential of WWTP effluents. Extension of the zebrafish embryotoxicity test (ZET) with biochemical and histopathological biomarkers could serve as a guiding principle in biomonitoring of wastewater contamination.

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO2 stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.

Effect of magnesium ions on dielectric relaxation in semidilute DNA aqueous solutions

The effect of magnesium ion Mg(2+) on the dielectric relaxation of semidilute DNA aqueous solutions has been studied by means of dielectric spectroscopy in the 100 Hz-100 MHz frequency range. de Gennes-Pfeuty-Dobrynin semidilute solution correlation length is the pertinent fundamental length scale for sufficiently low concentration of added salt, describing the collective properties of Mg-DNA solutions. No relaxation fingerprint of the DNA denaturation bubbles, leading to exposed hydrophobic core scaling, was detected at low DNA concentrations, thus indicating an increased stability of the double-stranded conformation in Mg-DNA solutions as compared to the case of Na-DNA solutions. Some changes are detected in the behavior of the fundamental length scale pertaining to the single molecule DNA properties, reflecting modified electrostatic screening effects of the Odijk-Skolnick-Fixman type. All results consistently demonstrate that Mg(2+) ions interact with DNA in a similar way as Na(1+) ions do, their effect being mostly describable through an enhanced screening.

Indication of metal homeostasis disturbance in earthworm Eisenia fetida after exposure to semi-solid depot sludge.

Treated sewage sludge is commonly used in agriculture as fertilizer. It is, therefore, necessary to determine possible detrimental influences of sludge application on soil contamination and accumulation of contaminants in tissues of terrestrial animals, which in the long run could also have undesirable effects on humans. With that aim, the study was performed using earthworm Eisenia fetida as test organism and semi-solid depot sludge from a wastewater treatment plant as exposure media. The concentrations of 26 metals/metalloids were determined in depot sludge, and their bioaccumulation was estimated in whole tissue of E. fetida, and for the first time in the soluble tissue fraction, which represents metal fraction available for metabolic requirements and toxic effects. Obtained results have revealed acceptable levels of several elements (Cd, Cr, Cu, Ni, Pb, Zn) in depot sludge, when compared to currently valid regulations, and only moderate accumulation of some elements (e.g. As, Ba, Cd, Co, Fe, Tl, V, and Zn) in earthworms, as a consequence of exposure to depot sludge. However, a concentration increase after exposure to depot sludge was observed in E. fetida for several elements (Cd, Mo, and Zn), which were present in lower concentrations in the exposure mixtures than in soil. Contrary, a concentration decrease was observed for Cs, Mn, and Rb, although they were present in higher concentrations in depot sludge than in soil. It was an indication of disturbance in metal homeostasis in earthworms, possibly caused by exposure to complex mixture of contaminants present in depot sludge. The cumulative effect of exposure to a number of various contaminants (inorganic, organic, microbiological and pharmaceutical), even if each of them was not present in very high concentrations, could have caused distress in earthworms exposed to depot sludge.

Assessment of river sediment toxicity: Combining empirical zebrafish embryotoxicity testing with in silico toxicity characterization

Quantitative chemical analyses of 428 organic contaminants (OCs) indicated the presence of 313 OCs in the sediment extracts from Sava River, Croatia. Pharmaceuticals were present in higher concentrations than pesticides thus confirming their increasing threat to freshwater ecosystems. Toxicity evaluation of the sediment extracts from four locations (Jesenice, Rugvica, Galdovo and Lukavec) using zebrafish embryotoxicity test (ZET) accompanied with semi-quantitative histopathological analyses exhibited correlation with cumulative number and concentrations of OCs at the investigated sites (10.05, 15.22, 1.25, and 9.13 μg/g respectively). Toxicity of sediment extracts and sediment was predicted using toxic unit (TU) approach and persistence, bioaccumulation and toxicity (PBT) ranking. Additionally, influential OCs and genes were identified by graph mining of the prior knowledge informed, site-specific chemical-gene interaction models. Predicted toxicity of sediment extracts (TUext) was similar to the results obtained by ZET and associated histopathology with Rugvica sediment being the most toxic, followed by Jesenice, Lukavec and Galdovo. Sediment TU (TUsed) favoured OCs with low octanol-water partition coefficients like herbicide glyphosate and antibiotics ciprofloxacin and sulfamethazine thus indicating locations containing higher concentrations of these OCs (Galdovo and Rugvica) as the most toxic. Results suggest that comprehensive in silico sediment toxicity predictions advocate providing equal attention to organic contaminants with either very low or very high log Kow.