Effects of different organic substrate compositions on the decontamination of aged PAH-polluted soils through outdoor co-composting.

The effects of different organic substrate compositions on the efficiency of outdoor co-composting as a bioremediation technology for decontaminating soil polluted by polycyclic aromatic hydrocarbons (PAHs) was investigated. Four different substrate mixtures and two different aged PAH-contaminated soils were used in a semi-pilot-scale experiment that lasted nearly 700 days. The two soils (A and B) differed concerning both the initial concentrations of the Æ©16 US EPA PAHs (5926 vs. 369 mg kg-1, respectively) and the type of predominant PAH group by molecular weight. The experiments revealed that while the composition of the organic substrate had an impact on the rate of PAH degradation, it did not significantly influence the final extent of PAH degradation. Notably, the organic substrate consisting of green waste and wood chips (GW) was found to facilitate the most rapid rate of PAH degradation (first-order rate constant k = 0.033±0.000 d-1 with soil A over the initial 42 days of the experiment and k = 0.036±0.000 d-1 with soil B over the initial 56 days). Despite the differences in organic substrate compositions and types of soil being treated, PAH degradation levels exceeded at least 95% in all the treatments after more than 680 days of co-composting. Regardless of the composition, the removal of low- and medium- (2-4 rings) molecular-weight PAHs was nearly complete by the end of the experiment. Furthermore, high-molecular-weight PAHs (5 rings and more) were significantly degraded during co-composting, with reductions ranging from 54% to 79% in soil A and from 59% to 68% in soil B. All composts were dominated by Proteobacteria, Firmicutes, and Actinobacteria, with significant differences in abundance between soils. Genera with PAH degradation potentials were detected in all samples. The results of a battery of toxicity tests showed that there was almost no toxicity associated with the final composts.

New insights into vermiremediation of sewage sludge: The effect of earthworms on micropollutants and vice versa.

Vermicomposting represents an environmentally friendly method for the treatment of various types of biowastes, including sewage sludge (SS), as documented in numerous studies. However, there are few papers providing insights into the mechanisms and toxicity effects involved in SS vermicomposting to present a comprehensive overview of the process. In this work, the vermiremediation of SS containing various micropollutants, including pharmaceuticals, personal care products, endocrine disruptors, and per/polyfluoroalkyl substances, was studied. Two SSs originating from different wastewater treatment plants (WWTP1 and WWTP2) were mixed with a bulking agent, moistened straw, at ratios of 0, 25, 50, and 75% SS. Eisenia andrei earthworms were introduced into the mixtures, and after six weeks, the resulting materials were subjected to various types of chemical and toxicological analyses, including conventional assays (mortality, weight) as well as tissue- and cell-level assays, such as malondialdehyde production, cytotoxicity tests and gene expression assays. Through the vermiremediation process significant removal of diclofenac (90%), metoprolol (88%), telmisartan (62%), and triclosan (81%) was achieved. Although the concentrations of micropollutants were substantially different in the original SS samples, the micropollutants vermiaccumulated to a similar extent over the incubation period. The earthworms substantially eliminated the present bacterial populations, especially in the 75% SS treatments, in which the average declines were 90 and 79% for WWTP1 and WWTP2, respectively. To the best of our knowledge, this is the first study to investigate the vermiremediation of such a large group of micropollutants in real SS samples and provide a thorough evaluation of the effect of SS on earthworms at tissue and cellular level.

Species phylogeny, ecology, and root traits as predictors of root exudate composition.

Root traits including root exudates are key factors affecting plant interactions with soil and thus play an important role in determining ecosystem processes. The drivers of their variation, however, remain poorly understood. We determined the relative importance of phylogeny and species ecology in determining root traits and analyzed the extent to which root exudate composition can be predicted by other root traits. We measured different root morphological and biochemical traits (including exudate profiles) of 65 plant species grown in a controlled system. We tested phylogenetic conservatism in traits and disentangled the individual and overlapping effects of phylogeny and species ecology on traits. We also predicted root exudate composition using other root traits. Phylogenetic signal differed greatly among root traits, with the strongest signal in phenol content in plant tissues. Interspecific variation in root traits was partly explained by species ecology, but phylogeny was more important in most cases. Species exudate composition could be partly predicted by specific root length, root dry matter content, root biomass, and root diameter, but a large part of variation remained unexplained. In conclusion, root exudation cannot be easily predicted based on other root traits and more comparative data on root exudation are needed to understand their diversity.

Remedial trial of sequential anoxic/oxic chemico-biological treatment for decontamination of extreme hexachlorocyclohexane concentrations in polluted soil.

During production of γ-hexachlorocyclohexane (γ-HCH), thousands of tons of other isomers were synthesized as byproducts, and after dumping represent sources of contamination for the environment. Several microbes have the potential for aerobic and anaerobic degradation of HCHs, and zero-valent iron is an effective remediation agent for abiotic dechlorination of HCHs, whereas the combination of the processes has not yet been explored. In this study, a sequence of anoxic/oxic chemico-biological treatments for the degradation of HCHs in a real extremely contaminated soil (10-30 g/kg) was applied. Approximately 1500 kg of the soil was employed, and various combinations of reducing and oxygen-releasing chemicals were used for setting up the aerobic and anaerobic phases. The best results were obtained with mZVI/nZVI, grass cuttings, and oxygen-releasing compounds. In this case, 80 % removal of HCHs was achieved in 129 days, and 98 % degradation was achieved after 1106 days. The analysis of HCHs and their transformation products proved active degradation when slight accumulation of the transformation product during the anaerobic phase was followed by aerobic degradation. The results document that switching between aerobic and anaerobic phases, together with the addition of grass, also created suitable conditions for the biodegradation of HCHs and monochlorobenzene/benzene by microbes.

In vitro toxicity assessment of polyethylene terephthalate and polyvinyl chloride microplastics using three cell lines from rainbow trout (Oncorhynchus mykiss).

The RTgill-W1 (gill), RTG-2 (gonad), and RTL-W1 (liver) cell lines derived from a freshwater fish rainbow trout (Oncorhynchus mykiss), were used to assess the toxicity of polyethylene terephthalate (PET) and two forms of polyvinyl chloride (PVC). Two size fractions (25-µm and 90-µm particles) were tested for all materials. The highest tested concentration was 1 mg/ml, corresponding to from 70 000 ± 9000 to 620 000 ± 57 000 particles/ml for 25-µm particles and from 2300 ± 100 to 11 000 ± 1000 particles/ml for 90-µm particles (depending on the material). Toxicity differences between commercial PVC dry blend powder and secondary microplastics created from a processed PVC were newly described. After a 24-h exposure, the cells were analyzed for changes in viability, 7-ethoxyresorufin-O-deethylase (EROD) activity, and reactive oxygen species (ROS) generation. In addition to the microplastic suspensions, leachates and particles remaining after leaching resuspended in fresh exposure medium were tested. The particles were subjected to leaching for 1, 8, and 15 days. The PVC dry blend (25 µm and 90 µm) and processed PVC (25 µm) increased ROS generation, to which leached chemicals appeared to be the major contributor. PVC dry blend caused substantially higher ROS induction than processed PVC, showing that the former is not suitable for toxicity testing, as it can produce different results from those of secondary PVC. The 90-µm PVC dry blend increased ROS generation only after prolonged leaching. PET did not induce any changes in ROS generation, and none of the tested polymers had any effect on viability or EROD activity. The importance of choosing realistic extraction procedures for microplastic toxicity experiments was emphasized. Conducting long-term experiments is crucial to detect possible environmentally relevant effects. In conclusion, the tested materials showed no acute toxicity to the cell lines.

Growth suppression of bacteria by biofilm deterioration using silver nanoparticles with magnetic doping.

Decades of antibiotic use and misuse have generated selective pressure toward the rise of antibiotic-resistant bacteria, which now contaminate our environment and pose a major threat to humanity. According to the evolutionary "Red queen theory", developing new antimicrobial technologies is both urgent and mandatory. While new antibiotics and antibacterial technologies have been developed, most fail to penetrate the biofilm that protects bacteria against external antimicrobial attacks. Hence, new antimicrobial formulations should combine toxicity for bacteria, biofilm permeation ability, biofilm deterioration capability, and tolerability by the organism without renouncing compatibility with a sustainable, low-cost, and scalable production route as well as an acceptable ecological impact after the ineluctable release of the antibacterial compound in the environment. Here, we report on the use of silver nanoparticles (NPs) doped with magnetic elements (Co and Fe) that allow standard silver antibacterial agents to perforate bacterial biofilms through magnetophoretic migration upon the application of an external magnetic field. The method has been proved to be effective in opening micrometric channels and reducing the thicknesses of models of biofilms containing bacteria such as Enterococcus faecalis, Enterobacter cloacae, and Bacillus subtilis. Besides, the NPs increase the membrane lipid peroxidation biomarkers through the formation of reactive oxygen species in E. faecalis, E. cloacae, B. subtilis, and Pseudomonas putida colonies. The NPs are produced using a one-step, scalable, and environmentally low-cost procedure based on laser ablation in a liquid, allowing easy transfer to real-world applications. The antibacterial effectiveness of these magnetic silver NPs may be further optimized by engineering the external magnetic fields and surface conjugation with specific functionalities for biofilm disruption or bactericidal effectiveness.

Effects of silver sulfide nanoparticles on the earthworm Eisenia andrei.

The massive production and use of silver nanoparticles (Ag NPs) have led to their increasing release into the environment. Even though the antimicrobial and cytotoxic effects of native nanoparticles have been well studied, the environmental impacts of transformation products such as silver sulfide nanoparticles (Ag2S NPs) have not been elucidated. In the present study, we assessed the toxicity of Ag2S NPs and silver nitrate (AgNO3), as a source of Ag, to the earthworm Eisenia andrei using a nominal concentration of 5 mg Ag kg-1 soil. We used the OECD guidelines to assess effects on weight loss and mortality for 14 days. After exposure, we also extracted the immune effector cells (coelomocytes) and conducted a battery of biomarker tests. To ensure the quality of the toxicological results, the structural changes of NPs during the experiment and the uptake of silver by the earthworms were monitored. During the experiment, mortality effects were not detected, but a weight loss was observed in the earthworms exposed to Ag2S NPs. Altough Ag2S NPs were engulfed by E. andrei cells, neither phenoloxidase activity nor lipid peroxidation differed from the untreated control group. Cells from earthworms treated with Ag2S NPs exerted very broad value range of nitric oxide (NO) generation, suggesting an imbalance in the NO metabolism. Overall, this study suggests minimal risks associated with Ag2S NPs exposure to earthworms. However, further studies are needed to assure no immunotoxicological or chronic effects on a wider range of terrestrial organisms.

Resistant Genes and Multidrug-Resistant Bacteria in Wastewater: A Study of Their Transfer to the Water Reservoir in the Czech Republic.

Wastewater is considered the most serious source of the spread of antibiotic resistance in the environment. This work, therefore, focuses on the fate and spread of antibiotic resistance genes (ARGs) in wastewater and the monitoring of multidrug-resistant strains. ARGs were monitored in the nitrification and sedimentation tanks of the wastewater treatment plant (WWTP) and in the dam into which this WWTP flows, at various times. The highest relative abundance was found for the blaTEM > tetW > blaNDM-1 > vanA resistance genes, respectively. An increased concentration of tetracycline (up to 96.00 ng/L) and ampicillin (up to 19.00 ng/L) was found in water samples compared to other antibiotics detected. The increased incidence of seven ARGs and four antibiotics was observed in the November and December sampling times. Isolated ampicillin-resistant strains showed a high degree of resistance to ampicillin (61.2% of the total isolates had a minimum inhibitory concentration (MIC) ≥ 20 mg/mL). In 87.8% of isolates, out of the total number, the occurrence of two or more ARGs was confirmed. These multidrug-resistant strains were most often identified as Aeromonas sp. This strain could represent a significant role in the spread of multidrug resistance through wastewater in the environment.

Evaluation of estrogenic and antiestrogenic activity in sludge and explanation of individual compound contributions.

Mixture toxicity, including agonistic and antagonistic effects, is an unrevealed environmental problem. Estrogenic endocrine disruptors are known to cause adverse effects for aquatic biota, but causative chemicals and their contributions to the total activity in sewage sludge remain unknown. Therefore, advanced analytical methods, a yeast bioassay and mixture toxicity models were concurrently applied for the characterization of 8 selected sludges with delectable estrogenic activity (and 3 sludges with no activity as blanks) out of 25 samples from wastewater treatment plants (WWTPs). The first applied full logistic model adequately explained total activity by considering the concentrations of the monitored compounds. The results showed that the activity was primarily caused by natural estrogens in municipal WWTP sludge. Nevertheless, activity in a sample originating from a car-wash facility was dominantly caused by partial agonists - nonylphenols - and only a model enabling prediction of all dose-response curve parameters of the final mixture curve explained these results. Antiestrogenic effects were negligible, and effect-directed analysis identified the causative chemicals.

The driving factors of per- and polyfluorinated alkyl substance (PFAS) accumulation in selected fish species: The influence of position in river continuum, fish feed composition, and pollutant properties.

Per- and polyfluorinated alkyl substances (PFASs) represent a group of highly recalcitrant micropollutants, that continuously endanger the environment. The present work describes the geographical trends of fish contamination by individual PFASs (including new compounds, e.g., Gen-X) assessed by analyzing the muscle tissues of 5 separate freshwater fish species from 10 locations on the Czech section of the Elbe River and its largest tributary, the Vltava River. The data of this study also showed that the majority of the detected PFASs consisted of long-chain representatives (perfluorooctane sulfonate (PFOS), perfluorononanoic acid, perfluorodecanoic acid, and perfluoroundecanoic acid), whereas short-chain PFASs as well as other compounds such as Gen-X were detected in relatively small quantities. The maximum concentrations of the targeted 32 PFASs in fish were detected in the lower stretches of the Vltava and Elbe Rivers, reaching 289.9 ng/g dw, 140.5 ng/g dw, and 162.7 ng/g dw for chub, roach, and nase, respectively. Moreover, the relationships between the PFAS (PFOS) concentrations in fish muscle tissue and isotopic ratios (δ15N and δ13C) were studied to understand the effect of feed composition and position in the river continuum as a proxy for anthropogenic activity. Redundancy analysis and variation partitioning showed that the largest part of the data variability was explained by the interaction of position in the river continuum and δ15N (δ13C) of the fish. The PFAS concentrations increased downstream and were positively correlated with δ15N and negatively correlated with δ13C. A detailed study at one location also demonstrated the significant relationship between δ15N (estimated trophic position) and PFASs (PFOS) concentrations. From the tested physicochemical properties, the molecular mass and number of fluorine substituents seem to play crucial roles in PFAS bioaccumulation.

Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone.

In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.

Biphenyl 2,3-Dioxygenase in Pseudomonas alcaliphila JAB1 Is Both Induced by Phenolics and Monoterpenes and Involved in Their Transformation.

The involvement of bacterial aromatic ring-hydroxylating dioxygenases (ARHDs) in the degradation of aromatic pollutants, such as polychlorinated biphenyls (PCBs), has been well studied. However, there is considerable speculation as to the origin of this ability. One hypothesis is centered on a connection between the ability to degrade aromatic pollutants and the necessity of soil bacteria to cope with and/or utilize secondary plant metabolites (SPMs). To investigate this connection, we researched the involvement of biphenyl 2,3-dioxygenase (BPDO), an ARHD essential for the degradation of PCBs, in the metabolism of SPMs in the soil bacterium Pseudomonas alcaliphila JAB1, a versatile degrader of PCBs. We demonstrated the ability of the strain JAB1 to transform a variety of SPMs, namely the flavonoids apigenin, flavone, flavanone, naringenin, fisetin, quercetin, morin, and catechin, caffeic acid, trans-cinnamic acid, and the monoterpenes (S)-limonene and (R)-carvone. Of those, the transformation of flavone, flavanone, and (S)-limonene was conditioned by the activity of JAB1-borne BPDO and thus was researched in more detail, and we found evidence for the limonene monooxygenase activity of the BPDO. Furthermore, the bphA gene in the strain JAB1 was demonstrated to be induced by a wide range of SPMs, with monoterpenes being the strongest inducers of the SPMs tested. Thus, our findings contribute to the growing body of evidence that ARHDs not only play a role in the catabolism of aromatic pollutants, but also of natural plant-derived aromatics, and this study supports the hypothesis that ARHDs participate in ecological processes mediated by SPMs.

Beta-lactam resistance development during the treatment processes of municipal wastewater treatment plants.

This work monitored the effect of a municipal and a village wastewater treatment plant (WWTP) technology on the fate of beta-lactam resistance genes in bacterial populations in different phases of the wastewater treatment process. In case of the municipal WWTP1, the bacteria possessing a high ampicillin resistance (minimal inhibitory concentration (MIC) values of 20 mg/mL) accumulated in the sedimentation tank, which was accompanied with a higher concentration of ampicillin in the wastewater samples (28.09 ng/L) and an increase in the relative abundance of the blaTEM gene in the bacterial population. However, an opposite trend was revealed with the blaNDM-1 gene, making the sedimentation processes of WWTP1 crucial only for the accumulation of the blaTEM gene. Similarly, the comparison with the WWTP2 showed that the accumulation of the ampicillin resistance in bacterial population probably depended on the WWTP technology and wastewater composition. Out of the four tested resistance genes (blaTEM, blaKPC, blaNDM-1, and blaOXA-48), blaTEM and blaNDM-1 genes were the only two detected in this study. According to NGS analysis of bacterial 16 S rRNA gene, Gammaproteobacteria dominated the ampicillin-resistant bacteria of the WWTP sedimentation tanks. Their relative abundance in the bacterial population also increased during the sedimentation processes in WWTP1. It could indicate the role of the bacterial taxon in ampicillin resistance accumulation in this WWTP and show that only 9.29% of the original bacterial population from the nitrification tank is involved in the documented shifts in beta-lactam resistance of the bacterial population.

Discovering the potential of an nZVI-biochar composite as a material for the nanobioremediation of chlorinated solvents in groundwater: Degradation efficiency and effect on resident microorganisms.

Abiotic and biotic remediation of chlorinated ethenes (CEs) in groundwater from a real contaminated site was studied using biochar-based composites containing nanoscale zero-valent iron (nZVI/BC) and natural resident microbes/specific CE degraders supported by a whey addition. The material represented by the biochar matrix decorated by isolated iron nanoparticles or their aggregates, along with the added whey, was capable of a stepwise dechlorination of CEs. The tested materials (nZVI/BC and BC) were able to decrease the original TCE concentration by 99% in 30 days. Nevertheless, regarding the transformation products, it was clear that biotic as well as abiotic transformation mechanisms were involved in the transformation process when nonchlorinated volatiles (i.e., methane, ethane, ethene, and acetylene) were detected after the application of nZVI/BC and nZVI/BC with whey. The whey addition caused a massive increase in bacterial biomass in the groundwater samples (monitored by 16S rRNA sequencing and qPCR) that corresponded with the transformation of trichloro- and dichloro-CEs, and this process was accompanied by the formation of less chlorinated products. Moreover, the biostimulation step also eliminated the adverse effect caused by nZVI/BC (decrease in microbial biomass after nZVI/BC addition). The nZVI/BC material or its aging products, and probably together with vinyl chloride-respiring bacteria, were able to continue the further reductive dechlorination of dichlorinated CEs into nonhalogenated volatiles. Overall, the results of the present study demonstrate the potential, feasibility, and environmental safety of this nanobioremediation approach.

The sensitivity of multiple ecotoxicological assays for evaluating Microcystis aeruginosa cellular algal organic matter and contribution of cyanotoxins to the toxicity.

Secondary metabolites of cyanobacteria and algae released during algal blooms often exhibit toxic effects, but only a small number of the metabolites are the subject of routine analytical screenings. Alternatively, ecotoxicological assays offer a better representation of the overall negative effects. The aim of this work was to compare multiple assays in their sensitivity towards cellular algal organic matter (COM) of the toxin-producing cyanobacterium Microcystis aeruginosa. Multiple endpoints were investigated: mortality, growth inhibition, bioluminescence inhibition, genotoxicity, endocrine-disrupting effects, oxidative stress, and the induction of ethoxyresorufin-O-deethylase (EROD). Three rainbow trout (Oncorhynchus mykiss) cell lines as well as representatives of bacteria, yeasts, algae, vascular plants, and crustaceans were employed, and the results were expressed per mg of dissolved organic carbon (DOC) in the COM. M. aeruginosa COM was toxic to the RTgill-W1, RTG-2, and RTL-W1 cell lines (EC50 values ranging from 0.48 ± 0.02 to 1.9 ± 0.1 mgDOC/L), to the crustacean Thamnocephalus platyurus (LC50 = 20 ± 1 mgDOC/L), and to Lepidium sativum (IC50 = 241 ± 13 mgDOC/L). In contrast, no effect was observed for bacteria and yeasts, and the growth of the alga Desmodesmus subspicatus was even stimulated. No genotoxicity, endocrine-disrupting effects or increase in oxidative stress or EROD activity was detected. The content of six microcystins (MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF), anatoxin-a, cylindrospermopsin, and nodularin in the M. aeruginosa COM was determined by liquid chromatography-tandem mass spectrometry. An artificially prepared mixture of the detected cyanotoxins in the corresponding concentrations did not induce response in the O. mykiss cell lines and T. platyurus, suggesting that other cyanobacterial metabolites are responsible for the toxicity of M. aeruginosa.

In Vitro Interactions of TiO2 Nanoparticles with Earthworm Coelomocytes: Immunotoxicity Assessment.

Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide. Once they arrive in the soil environment, they can endanger living organisms. Hence, monitoring and assessing the effects of these nanoparticles is required. We focus on the Eisenia andrei earthworm immune cells exposed to sublethal concentrations of TiO2 NPs (1, 10, and 100 µg/mL) for 2, 6, and 24 h. TiO2 NPs at all concentrations did not affect cell viability. Further, TiO2 NPs did not cause changes in reactive oxygen species (ROS) production, malondialdehyde (MDA) production, and phagocytic activity. Similarly, they did not elicit DNA damage. Overall, we did not detect any toxic effects of TiO2 NPs at the cellular level. At the gene expression level, slight changes were detected. Metallothionein, fetidin/lysenin, lumbricin and MEK kinase I were upregulated in coelomocytes after exposure to 10 µg/mL TiO2 NPs for 6 h. Antioxidant enzyme expression was similar in exposed and control cells. TiO2 NPs were detected on coelomocyte membranes. However, our results do not show any strong effects of these nanoparticles on coelomocytes at both the cellular and molecular levels.

In Vitro Study of the Toxicity Mechanisms of Nanoscale Zero-Valent Iron (nZVI) and Released Iron Ions Using Earthworm Cells.

During the last two decades, nanomaterials based on nanoscale zero-valent iron (nZVI) have ranked among the most utilized remediation technologies for soil and groundwater cleanup. The high reduction capacity of elemental iron (Fe0) allows for the rapid and cost-efficient degradation or transformation of many organic and inorganic pollutants. Although worldwide real and pilot applications show promising results, the effects of nZVI on exposed living organisms are still not well explored. The majority of the recent studies examined toxicity to microbes and to a lesser extent to other organisms that could also be exposed to nZVI via nanoremediation applications. In this work, a novel approach using amoebocytes, the immune effector cells of the earthworm Eisenia andrei, was applied to study the toxicity mechanisms of nZVI. The toxicity of the dissolved iron released during exposure was studied to evaluate the effect of nZVI aging with regard to toxicity and to assess the true environmental risks. The impact of nZVI and associated iron ions was studied in vitro on the subcellular level using different toxicological approaches, such as short-term immunological responses and oxidative stress. The results revealed an increase in reactive oxygen species production following nZVI exposure, as well as a dose-dependent increase in lipid peroxidation. Programmed cell death (apoptosis) and necrosis were detected upon exposure to ferric and ferrous ions, although no lethal effects were observed at environmentally relevant nZVI concentrations. The decreased phagocytic activity further confirmed sublethal adverse effects, even after short-term exposure to ferric and ferrous iron. Detection of sublethal effects, including changes in oxidative stress-related markers such as reactive oxygen species and malondialdehyde production revealed that nZVI had minimal impacts on exposed earthworm cells. In comparison to other works, this study provides more details regarding the effects of the individual iron forms associated with nZVI aging and the cell toxicity effects on the specific earthworms' immune cells that represent a suitable model for nanomaterial testing.

Screening for 32 per- and polyfluoroalkyl substances (PFAS) including GenX in sludges from 43 WWTPs located in the Czech Republic - Evaluation of potential accumulation in vegetables after application of biosolids.

Highly persistent, toxic and bioaccumulative per - and polyfluoroalkyl substances (PFAS) represents a serious problem for the environment and their concentrations and fate remain largely unknown. The present study consists of a PFAS screening in sludges originating from 43 wastewater treatment plants (WWTPs) in the Czech Republic. To analyze an extended group of PFAS consisting of 32 PFAS, including GenX and other new replacements of older and restricted PFAS in sludge, a new method was optimized and validated using pressurized solvent extraction, followed by the SPE clean-up step to eliminate the observed matrix effects and LC-MS/MS. The results revealed high PFAS contamination of sewage sludge, reaching values from 5.6 to 963.2 ng g-1. The results showed that in the majority of the samples (about 60%), PFOS was the most abundant among the targeted PFAS, reaching 932.9 ng g-1. Approximately 20% of the analyzed samples contained more short-chain PFAS, suggesting the replacement of long-chain PFAS (especially restricted PFOA and PFOS). GenX was detected in 9 samples, confirming the trend in the use of new PFAS. The results revealed that significantly higher contamination was detected in the samples from large WWTPs (population equivalent > 50,000; p-value <0.05). Concerning the application of sludge in agriculture, our prediction using the respective PFAS bioconcentration factors, the observed concentrations, and the legislatively permitted management of biosolids in Czech Republic agriculture revealed that PFAS can cause serious contamination of cereals and vegetables (oat, celery shoots and lettuce leaves), as well as general secondary contamination of the environment.

Oxidative stress in microbes after exposure to iron nanoparticles: analysis of aldehydes as oxidative damage products of lipids and proteins.

Due to their enhanced reactivity, metal and metal-oxide nanoscale zero-valent iron (nZVI) nanomaterials have been introduced into remediation practice. To ensure that environmental applications of nanomaterials are safe, their possible toxic effects should be described. However, there is still a lack of suitable toxicity tests that address the specific mode of action of nanoparticles, especially for nZVI. This contribution presents a novel approach for monitoring one of the most discussed adverse effects of nanoparticles, i.e., oxidative stress (OS). We optimized and developed an assay based on headspace-SPME-GC-MS analysis that enables the direct determination of volatile oxidative damage products (aldehydes) of lipids and proteins in microbial cultures after exposure to commercial types of nZVI. The method employs PDMS/DVB SPME fibers and pentafluorobenzyl derivatization, and the protocol was successfully tested using representatives of bacteria, fungi, and algae. Six aldehydes, namely, formaldehyde, acrolein, methional, benzaldehyde, glyoxal, and methylglyoxal, were detected in the cultures, and all of them exhibited dose-dependent sigmoidal responses. The presence of methional, which was detected in all cultures except those including an algal strain, documents that nZVI also caused oxidative damage to proteins in addition to lipids. The most sensitive toward nZVI exposure in terms of aldehyde production was the yeast strain Saccharomyces cerevisiae, which had an EC50 value of 0.08 g/L nZVI. To the best of our knowledge, this paper is the first to document the production of aldehydes resulting from lipids and proteins as a result of OS in microorganisms from different kingdoms after exposure to iron nanoparticles.

Novel assay for the toxicity evaluation of nanoscale zero-valent iron and derived nanomaterials based on lipid peroxidation in bacterial species.

Nano-scale zero-valent iron (nZVI) began attracting research attention in remediation practice in recent decades as a prospective nanomaterial applicable to various contaminated matrices. Despite concerns about the negative effects of nanomaterials on ecosystems, the number of reliable toxicity tests is limited. We have developed a test based on the evaluation of oxidative stress (OS). The test employed the analysis of a typical OS marker (malondialdehyde, MDA), after exposure of six bacterial strains to the tested nanomaterial. We also attempted to use other OS and cell membrane damage assays, including the determination of glutathione and lactate dehydrogenase, respectively. However, we found that the components of these assays interfered with nZVI; therefore, these tests were not applicable. The MDA assay was tested using nZVI and three newly engineered oxide shell nZVI materials with different oxide thicknesses. Six different bacterial species were employed, and the results showed that the test was fully applicable for the concentrations of nanomaterials used in remediation practice (0.1-10 g/L). MDA was produced in a dose-response manner, and the bacteria showed a similar response toward pure pyrophoric nZVI, reaching EC50 values of 0.3-1.1 g/L. We observed different responses in the absolute production of MDA; however, the MDA concentrations were correlated with the cell membrane surfaces of the individual strains (R > 0.75; P < 0.09). Additionally, the EC50 values correlated with the thickness of the oxide shells (except for Escherichia coli: R > 0.95; P < 0.05), documenting the reliability of the assay, where reactivity was confirmed to be an important factor for reactive oxygen species production.