Influence of ozonation and UV/H2O2 on the genotoxicity of secondary wastewater effluents.

The implementation of novel wastewater treatment technologies, including Advanced Oxidation Processes (AOPs) such as ozonation and ultraviolet radiation (UV) combined with hydrogen peroxide (H2O2), can be a promising strategy for enhancing the quality of these effluents. However, during effluent oxidation AOPs may produce toxic compounds that can compromise the water reuse and the receiving water body. Given this possibility, the aim of this study was to evaluate the genotoxic potential of secondary effluents from two different Wastewater Treatment Plants (WWTP) that were subjected to ozonation or UV/H2O2 for periods of 20 (T1) and 40 (T2) minutes. The genotoxic potential was carried out with the Comet assay (for clastogenic damage) and the Micronucleus assay (for clastogenic and aneugenic damage) in HepG2/C3A cell culture (metabolizing cell line). The results of the comet assay revealed a significant increase in tail intensity in the Municipal WWTP (dry period) effluents treated with UV/H2O2 (T1 and T2). MN occurrence was noted across all treatments in both Pilot and Municipal WWTP (dry period) effluents, whereas nuclear buds (NBs) were noted for all Pilot WWTP treatments and UV/H2O2 treatments of Municipal WWTP (dry period). Moreover, the UV/H2O2 (T1) treatment of Municipal WWTP (dry period) exhibited a noteworthy incidence of multiple alterations per cell (MN + NBs). These findings imply that UV/H2O2 treatment demonstrates higher genotoxic potential compared to ozonation. Furthermore, seasonal variations can have an impact on the genotoxicity of the samples. Results of the study emphasize the importance of conducting genotoxicological tests using human cell cultures, such as HepG2/C3A, to assess the final effluent quality from WWTP before its discharge or reuse. This precaution is essential to safeguard the integrity of the receiving water body and, by extension, the biotic components it contains.

Assessment of phytotoxic potential and pathogenic bacteria removal from secondary effluents during ozonation and UV/H2O2.

Wastewater reuse is an important strategy for water resource management. For this reason, the disinfection process must be appropriated, eliminating pathogenic microorganisms. Ozonation (O3) and UV/H2O2 treatments can be used for effluent disinfection, but few studies just address the Escherichia coli quantification. In this study, secondary effluents from two wastewater treatment plants with different characteristics were exposed to O3 (5 and 10 mg L-1) or UV/H2O2 (H2O2: 90 mg L-1) treatments and evaluated by BD Phoenix ™ 100 (Becton Dickinson, USA) and MALDI-TOF for the characterization of the indigenous microorganisms in the effluents, before and after treatments. Additionally, all the samples were tested for phytotoxicity by Lactuca sativa bioassay. The results showed that the highest ozone dose and the UV/H2O2 treatment were effective in removing E. coli. UV/H2O2 was more efficient as it eliminated most of the microorganisms. Acinetobacter sp., Aeromonas and Pseudomonas were still found after O3 treatment. Bacillus sp. was found after O3 and UV/H2O2 treatments. The results with L. sativa showed inhibition of root growth for all dry period (low rainfall) samples of one of the WWTP, due to the high concentration of the phytotoxicity compounds. For environmental and human health safety, treated effluents should be evaluated for their toxic and pathogenic potential before being released into the environment. Pathogens evaluation on treated effluents should cover a wider range of pathogenic microorganisms than those routinely required by legislation.

Phyto-genotoxicity assessment of different associations between sludges from Water and Sewage Treatment Plants, before and after the bioremediation process.

Water Treatment Plants (WTP) and Sewage Treatment Plants (STP) generate residues known as sludge (WS and SS, respectively). SS and WS present some positive characteristics for reuse in agriculture. The aim of the present study was to evaluate, using the Allium cepa test, the effectiveness of the bioremediation process in the detoxification of SS and WS sludges. In this study, the phytotoxic, cytotoxic, genotoxic, and mutagenic potentials of pure sludge samples (WS and SS) were evaluated, as well as the association of these two sludges with soil (S), before and after the bioremediation process. In the T0 period (before undergoing bioremediation), the SS, SS + S, and SS + WS samples totally inhibited the germination of A. cepa, proving the high phytotoxic potential of these samples. For the T1 period (after 6 months of bioremediation), phytotoxicity was observed for the SS, SS + S, SS + WS, and SS + WS + S samples, but there was not a complete inhibition of germination and radicles growth, allowing the evaluation of the other parameters (cytogenotoxic and mutagenic potential). No cytotoxicity was observed for any sample, both in T0 and T1. As for the genotoxicity parameter, a significant result was observed for the pure WS sample in T0 and for all samples in T1, when compared to NC. The genotoxic alteration most found in meristematic cells exposed to treatments was of binucleated cells. Mutagenic potential was also observed for samples of WS and WS + S in T0. From this study, we can conclude that, after six months of bioremediation, despite the SS phytotoxicity being reduced, all samples were genotoxic to the A. cepa organism test.

PBAT biodegradable mulch films: Study of ecotoxicological impacts using Allium cepa, Lactuca sativa and HepG2/C3A cell culture.

Biodegradable mulch films are an alternative to polyethylene films used in agriculture for weed control, improving crop productivity. This change could minimize the residue production and costs related to the final disposal. Nevertheless, the environmental safety of these biodegradable products is scarcely investigated. In this work, samples of poly(butylene adipate-co-terephthalate)-PBAT mulch films, with and without UV stabilizer additives, were prepared. Aqueous extracts of soil samples, where mulch films were disposed, were investigated using bioassays with Lactuca sativa, Allium cepa, and cell culture HepG2/C3A. As PBAT is expected to suffer photodegradation and biodegradation, soil samples mixed with films before and after these processes were evaluated. Soil aqueous extracts promoted root grown (mainly hypocotyl) of L. sativa, probably due to presence of nutrients. So, to evaluate toxicity potential, in this case it was necessary to use aqueous extract prepared with soil instead of ultrapure water as the control. After doing this analysis it was observed that no adverse impacts due to PBAT films occurred. No chromosomal abnormalities were observed in A. cepa bioassay for any of tested samples. The absence of genotoxic potential was confirmed by comet assay and micronucleus test using human hepatocarcinoma cell line HepG2/C3A. These results showed that the soil did not induce damage to the tested organisms, before and after degradation of PBAT films.

The applicability of gene sequencing and MALDI-TOF to identify less common gram-negative rods (Advenella, Castellaniella, Kaistia, Pusillimonas and Sphingobacterium) from environmental isolates.

Our aim was to identify less common non-fermenting gram-negative rods during the bioremediation process. Five genera were found: Advenella, Castellaniella, Kaistia, Pusillimonas and Sphingobacterium, for a total of 15 isolates. Therefore, we evaluated the applicability of four methods currently available for bacteria identification: (1) conventional biochemical methods, (2) the VITEK®-2 system, (3) MALDI-TOF mass spectrometry and (4) 16S rRNA gene sequencing. The biochemical methods and the VITEK®-2 system were reliable only for the Sphingobacterium isolate and solely at the genus level. Both MALDI-TOF mass spectrometry platforms (Bruker and VITEK® MS) did not achieve reliable identification results for any of these genera. 16S rRNA gene sequencing identified eight isolates to the species level but not to the subspecies level, when applicable. The remaining seven isolates were reliably identified through 16S rRNA gene sequencing to the genus level only. Our findings suggest that the detection and identification of less common genera (and species) that appeared at certain moments during the bioremediation process can be a challenge to microbiologists considering the most used techniques. In addition, more studies are required to confirm our results.

Evaluation of the potential agricultural use of biostimulated sewage sludge using mammalian cell culture assays.

Among the bioremediation processes, biostimulation is an effective methodology for the decontamination of organic waste by the addition of agents that stimulate the indigenous microbiota development. Rice hull is a biostimulating agent that promotes the aeration of edaphic systems and stimulates the aerobiotic activity of soil microorganisms. The present study aimed to evaluate the efficacy of the bioremediation and biostimulation processes in reducing the toxicity of sewage sludge (SS) and to evaluate its possible application in agriculture using cytotoxic and genotoxic assays in human hepatoma cells (HepG2). SS of domestic origin was tested as both the pure product (PSS) and mixed with soil (S) and with a stimulating agent, such as rice hull (RH), in different proportions (SS + S and SS + S + RH); we also examined different remediation periods (3 months - T1 and 6 months - T2). For the PSS sample, a significant induction of micronucleus (MN) in T2 was observed with nuclear buds in all of the periods assessed, and we observed the presence of more than one alteration per cell (MN and nuclear bud) in T1 and T2. The PSS sample caused genotoxic effects in the HepG2 cells even after being bioremediated. For the samples containing soil and/or rice hull, no toxic effects were observed in the test system used. Therefore, the addition of SS to agricultural soils should be conducted with caution, and it is important that the SS undergoes a remediation process, such as bioremediation and biostimulation treatments.

Ecotoxicological and microbiological assessment of sewage sludge associated with sugarcane bagasse.

Sewage sludge (SS) obtained after sewage treatment process may contain several toxic substances. Bioremediation can decrease the toxicity of the sludge, mainly when it is associated with stimulant agents, such as sugarcane bagasse (B). Samples of pure SS (SSP); SS+B; SS+Soil; and SS+B+Soil were bioremediated for 1, 3, and 6 months (T1, T2, and T3, respectively). After each period, the cytotoxic, genotoxic, and mutagenic potentials of the solid samples and their respective aqueous extracts (aqueous eluate and percolate water) were evaluated by the Allium cepa test. A microbiological analysis of the samples was also performed after each period tested. All solid samples of SS+B (in T1, T2, and T3) and the solid sample of SSP (treatment T3) showed a significant decrease of cell division (cytotoxic effects). The aqueous eluate extracts of SS+B (T1 and T3) and SSP (T2 and T3) induced cytotoxic effect. The solid sample of SS+B (T2 and T3) and aqueous extracts of SSP (T1) were genotoxic, indicating a harmful effect of SS on A. cepa, even after 6 months of bioremediation. There was an alternation in the microbial community both in diversity and in abundance, with the predominance of nonfermenting gram-negative bacilli. The tested bioremediation periods were not sufficient for the complete detoxification of SS, and the use of B did not seem to contribute to the degradation of the pollutants to inert compounds. These data emphasize that a specific relationship should exist between the sludge characteristic and the biostimulating agent used to promote a more efficient bioremediation. These results suggest the necessity to study longer periods of biodegradation and the use of other decomposing agents for greater safety and sustainability for the agricultural use of this residue.