Detection of SARS-CoV-2 RNA in the Danube River in Serbia associated with the discharge of untreated wastewaters.

In Serbia less than 13% of collected municipal wastewaters is being treated before their release in the environment. This includes all municipal wastewater discharges from Belgrade (capital city of Serbia; population 1,700,000). Previous research has identified the impacts of raw wastewater discharges from Belgrade on the Danube River, and this study investigated if such discharges also provided a pathway for SARS-CoV-2 RNA material. Samples were collected during the most critical circumstances that occurred so far within the COVID-19 pandemics in Serbia. Grab and composite samples were collected in December 2020, during the peak of the third wave (in terms of reported cases) at the site which receives the wastewater loads in Belgrade. Grab samples collected upstream and downstream of Belgrade were also analyzed. RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. SARS-CoV-2 RNA (5.97 × 103 to 1.32 × 104 copies/L) was detected only in samples collected at the site strongly impacted by the wastewaters where all three applied primer sets gave positive signals. Determined concentrations correspond to those reported in wastewater influents sampled at treatment plants in other countries indicating an epidemiological indicator function of used approach for rivers with high pollution loads in countries with poor wastewater treatment.

Loss of heterozygosity of selected tumor suppressor genes in human testicular germ cell tumors.

Human testicular germ cell tumors (TGCTs) are histologically heterogenous neoplasms with a variable malignant potential. Two main groups of germ cell tumors occur in men: seminomas and nonseminomas. In the present study, a set of four tumor suppressor genes was investigated in testicular cancers. CDH1, APC, p53, and nm23-H1 genes were tested for loss of heterozygosity (LOH). Thirty-eight testicular germ cell tumors (17 seminomas and 21 nonseminomas) were analyzed by PCR using restriction fragment length polymorphism or the dinucleotide/tetranucleotide repeat polymorphism method. An allelic loss of p53 at exon 4 was detected in five nonseminomas, whereas LOH of p53 at intron 6 occurred in one of the seminoma and two of the nonseminoma samples. Allelic losses of the APC gene were present in three seminomas and one nonseminoma, whereas one seminoma and three nonseminomas showed LOH of CDH1. The analysis of allelic losses showed no common structural genetic alterations in tumor tissues, although a different pattern of LOH was observed between the two main histological groups of TGCTs.

Citotoxicity status of electroplating wastewater prior/after neutralization/purification with alkaline solid residue of electric arc furnace dust.

Toxicological safety of new procedure for the neutralisation/purification of wastewater originated from zinc plating facility was investigated. Wastewater was treated with alkaline solid residue-by-product of zinc recovery from electric arc furnace dust. For determination of cytotoxic potential of untreated and purified wastewater MTT test on HEp2 (human laryngeal carcinoma) and HeLa (human cervical carcinoma) cells lines and alkaline comet assay on human leukocytes were used. Then 100% of the sample as well as different dilutions were tested. Compared to negative control 100, 75 and 50% of the sample of untreated wastewater significantly decreased survival of both HEp2 and HeLa cell lines. In the presence of undiluted sample survival percentage of HeLa and HEp2 cells were only 2.3 and 0.3% respectively. Only undiluted purified wastewater showed slight but insignificant decrease of the survival of both cell lines. Even 0.5% of the sample of original electroplating wastewater exhibited significantly higher value of all comet assay parameters compared to negative control. There was no significant difference between negative control and purified wastewater for any of comet assay parameters. Significantly lower level of primary DNA damage recorded after treatment with purified water, even comparable with negative control, confirmed effectiveness of the purification process.

The assessment of genotoxic effects of wastewater from a fertilizer factory.

In this study we investigated cytotoxic, mutagenic and genotoxic effects of different concentrations of wastewater from the phosphoric gypsum depot near the factory for fertilizing agents 'INA Petrokemija' (Kutina, Croatia). The Ames test was performed on Salmonella typhimurium TA98 and TA100 strains, in the presence of S9 mix, glutathione and buffer, respectively. Cytotoxicity was studied on human laryngeal carcinoma cells (HEp2) and human cervical cells (HeLa). The level of lipid peroxidation in these two cell lines was evaluated in parallel. To establish the levels of primary DNA damage, the alkaline comet assay was performed on treated human peripheral blood leukocytes. No mutagenic effects of phosphoric gypsum on Salmonella typhimurium strains in the presence of S9 mix, GSH or PBS were observed. However, strong cytotoxic effect was observed on both human cell lines when they were treated with different concentrations of wastewater. Lipid peroxidation was induced and increased by prolonged time of incubation, highlighting that the damage was not repaired, but increased with the time of incubation. The results of the alkaline comet assay indicate significant DNA damaging potential of wastewater for human leukocytes. Since phosphoric gypsum transport water in its present composition and acidity is highly toxic and acts as prooxidant, causing free radicals formation and DNA damage, urgent neutralization/purification of the wastewater to a level acceptable for disposal into the environment is mandatory.

Multidrug efflux transporters limit accumulation of inorganic, but not organic, mercury in sea urchin embryos.

Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.

Cytotoxicity and mutagenicity study of waste and purified water samples from electroplating industries prepared by use of ferrous sulfate and wood fly ash.

Toxicological safety of the new purification method for electroplating wastewaters (EWW) has been assessed. Method utilizes waste by-product ferrous sulfate and wood fly ash to scavenge heavy metals from EWW. Energy dispersive X-ray fluorescence (EDXRF) analysis of all samples met the law requirements for safe depositing of waste into the environment. The toxicity study consisted of determination of frequency of bacterial and human cell lines survival and the Ames assay (plate incorporation assay and the preincubation assay). Unexpectedly, data obtained indicate cytotoxicity and slight mutagenic potential of purified water. Ames assay showed that combination of alkalis and heavy metals present in the water purified with original fly ash and eluates of fly and waste ash were metabolically activated and conjugated with glutathione, resulting in new metabolites active and toxic for the cell. In order to reduce this effect pretreatment of fly ash (partial removal of highly soluble compounds) are necessary prior to its usage for neutralization /coagulation/flocculation processes.

Toxicological characterization of the new water cleaning product and its waste by-product.

Water extracts genotoxicity of the coagulant produced from industrial waste (red mud and waste base) and its waste mud remained after waste water treatment by the coagulation/flocculation process were studied. Tests were conducted in order to confirm nontoxicity of this new product prior to commercial production and usage and also to recommend a safe way for a handling and disposal of the remaining waste material. The toxicity investigation included (i) determining frequency of the cell survival, (ii) the Ames assays, (iii) micronucleus assay, and (iv) cell proliferation kinetics (expressed as mitotic index). These techniques were also employed in toxicity testing of the different concentrations of metal salts, zinc chloride, and lead nitrate in this case since these two elements were present in high concentrations in the waste water intended for the purification with the new coagulant. Mixture of metal salts in the concentrations that represent maximum allowed values for water extracts of technological waste was also tested. Two strains of Salmonella typhimurium, TA98, and TA100 were used for determining cytotoxicity and for the Ames test, while the cytogenetic investigations were performed on human peripheral blood lymphocytes. Water extracts of the coagulant and its waste mud did not induce a significant increase of the micronuclei in human peripheral blood lymphocytes. They also did not disturb lymphocyte proliferation kinetics in vitro. As regards lead nitrate it proved not to be cytotoxic on bacterial strains in the tested concentration range (1-100 mg/L), whereas zinc chloride showed cytotoxic effect for the concentrations above 25 mg/L. The Ames test results for the noncytotoxic concentrations of these metals were negative. Comparing to the negative control a significantly higher number of the micronuclei was determined after the treatment of the whole blood with 100 mg/L of zinc chloride, as well as with 10 and 100 mg/L of lead nitrate. A linear, dose dependent increase was obtained for both salts. Similar results were obtained on the basis of the mitotic index.