The Cocktail Effects on the Acute Cytotoxicity of Pesticides and Pharmaceuticals Frequently Detected in the Environment.

Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient. In this study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects. Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures. Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor. S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures. Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations. Several combinations displayed synergistic effects at environmentally relevant concentrations. The application of PERMANOVA was proven to be unique and successful in determining the roles of compounds in synergistic, additive, and antagonistic effects in mixtures at different effective concentrations.

In vivo estrogenicity of glyphosate, its formulations, and AMPA on transgenic zebrafish (Danio rerio) embryos.

In this study, the disrupting effects of glyphosate (GLY), aminomethylphosphonic acid (AMPA), and three glyphosate-based herbicides (GBHs) on vitellogenesis in a non-concentration-dependent manner are reported for the first time in 120 h of acute exposure of zebrafish at environmentally relevant concentrations. GBHs are commonly used worldwide in weed control management. Due to their extensive application, they frequently occur in aquatic ecosystems and may affect various organisms. The active substance GLY and its major by-product, AMPA, are the most thoroughly studied chemicals; however, the adverse effects of the complex formulas of GBHs with diverse and unknown content of co-formulants are still not sufficiently researched. This study focused on the embryotoxicity, sublethal malformations, and estrogenic potency of GLY, AMPA, and four commonly used GBHs on zebrafish embryos using a wild type and an estrogen-sensitive, transgenic zebrafish line (Tg(vtg1:mCherry)). After 120 h of exposition, AMPA did not cause acute toxicity, while the LC50 of GLY was 160 mg/L. The GBHs were more toxic with LC50 values ranging from 31 to 111 GLY active equivalent (a.e.) mg/L. Exposure to 0.35-2.8 mg/L GBHs led to sublethal abnormalities: typical symptoms were structural deformation of the lower jaw and anomalies in the olfactory region. Deformity rates were 10-30% in the treated groups. In vivo, fluorescently expressed vtg1 mCherry protein in embryonic liver was detected by a non-invasive microscopic method indicating estrogenic action through vitellogenin production by GLY, AMPA, and GBHs. To confirm the in vivo findings, RT-qPCR method was performed to determine the levels of the estrogenicity-related vtg1 mRNA. After 120 h of exposure to GLY, AMPA, and three GBHs at a concentration of 0.35 mg/L, the expression of vtg1 gene was significantly up-regulated. Our results highlight the risk that short-term GLY and GBH exposure can cause developmental malformations and disrupt the hormonal balance in zebrafish embryos.

Evaluating the In Vivo Virulence of Environmental Pseudomonas aeruginosa Using Microinjection Model of Zebrafish (Danio rerio).

(1) Background: Microinjection of zebrafish (Danio rerio) embryos offers a promising model for studying the virulence and potential environmental risks associated with Pseudomonas aeruginosa. (2) Methods: This work aimed to develop a P. aeruginosa infection model using two parallel exposition pathways on zebrafish larvae with microinjection into the yolk and the perivitelline space to simultaneously detect the invasive and cytotoxic features of the examined strains. The microinjection infection model was validated with 15 environmental and clinical strains of P. aeruginosa of various origins, antibiotic resistance profiles, genotypes and phenotypes: both exposition pathways were optimized with a series of bacterial dilutions, different drop sizes (injection volumes) and incubation periods. Besides mortality, sublethal symptoms of the treated embryos were detected and analyzed. (3) Results: According to the statistical evaluation of our results, the optimal parameters (dilution, drop size and incubation period) were determined. (4) Conclusions: The tested zebrafish embryo microinjection infection model is now ready for use to determine the in vivo virulence and ecological risk of environmental P. aeruginosa.

Presence, variation, and potential ecological impact of microplastics in the largest shallow lake of Central Europe.

The presence of microplastics (MPs) in the global ecosystem has generated a rapidly growing concern worldwide. Although their presence in the marine environment has been well-studied, much less data are available on their abundance in freshwaters. MPs alone and in combination with different chemicals has been shown to cause acute and chronic effects on algae and aquatic invertebrate and vertebrate species at different biological levels. However, the combined ecotoxicological effects of MPs with different chemicals on aquatic organisms are still understudied in many species and the reported data are often controversial. In the present study, we investigated, for the first time, the presence of MPs in Lake Balaton, which is the largest shallow lake of Central Europe and an important summer holiday destination. Moreover, we exposed neonates of the well-established ecotoxicological model organism Daphnia magna to different MPs (polystyrene [3 µm] or polyethylene [≤ 100 µm]) alone and in combination with three progestogen compounds (progesterone, drospirenone, levonorgestrel) at an environmentally relevant concentration (10 ng L-1) for 21 days. The presence of 7 polymer types of MPs in the size range of 50-100 µm was detected in Lake Balaton. Similarly to the global trends, polypropylene and polyethylene MPs were the most common types of polymer. The calculated polymer-independent average particle number was 5.5 particles m-3 (size range: 50 µm - 100 µm) which represents the values detected in other European lakes. Our ecotoxicological experiments confirmed that MPs and progestogens can affect D. magna at the behavioral (body size and reproduction) and biochemical (detoxification-related enzyme activity) levels. The joint effects were negligible. The presence of MPs may lead to reduced fitness in the aquatic biota in freshwaters such as Lake Balaton, however, the potential threat of MPs as vectors for progestogens may be limited.

Glyphosate and glyphosate-based herbicides (GBHs) induce phenotypic imipenem resistance in Pseudomonas aeruginosa.

GBHs are the most widely used herbicides for weed control worldwide that potentially affect microorganisms, but the role of their sublethal exposure in the development of antibiotic resistance of Pseudomonas aeruginosa is still not fully investigated. Here, the effects of glyphosate acid (GLY), five glyphosate-based herbicides (GBHs), and POE(15), a formerly used co-formulant, on susceptibility to imipenem, a potent carbapenem-type antibiotic, in one clinical and four non-clinical environmental P. aeruginosa isolates were studied. Both pre-exposure in broth culture and co-exposure in solid media of the examined P. aeruginosa strains with 0.5% GBHs resulted in a decreased susceptibility to imipenem, while other carbapenems (doripenem and meropenem) retained their effectiveness. Additionally, the microdilution chequerboard method was used to examine additive/antagonistic/synergistic effects between GLY/POE(15)/GBHs and imipenem by determining the fractional inhibitory concentration (FIC) indexes. Based on the FIC index values, glyphosate acid and Total demonstrated a potent antagonistic effect in all P. aeruginosa strains. Dominator Extra 608 SL and Fozat 480 reduced the activity of imipenem in only one strain (ATCC10145), while POE(15) and three other GBHs did not have any effect on susceptibility to imipenem. Considering the simultaneous presence of GBHs and imipenem in various environmental niches, the detected interactions between these chemicals may affect microbial communities. The mechanisms of the glyphosate and GBH-induced imipenem resistance in P. aeruginosa are yet to be investigated.

Novel members of bacterial community during a short-term chilled storage of common carp (Cyprinus carpio).

This work aimed to identify the key members of the bacterial community growing on common carp (Cyprinus carpio) fillets during chilled storage with next-generation sequencing (NGS) and cultivation-dependent methods. Carp fillets were stored for 96 h at 2 °C and 6 °C with and without a vacuum package, and an additional frozen-thawed storage experiment was set for 120 days. Community profiles of the initial and stored fish samples were determined by amplicon sequencing. Conventional microbial methods were used parallelly for the enumeration and cultivation of the dominant members of the microbial community. Cultivated bacteria were identified with 16S rRNA sequencing and the MALDI-TOF MS method. Based on our results, the vacuum package greatly affected the diversity and composition of the forming microbial community, while temperature influenced the cell counts and consequently the microbiological criteria for shelf-life of the examined raw fish product. Next-generation sequencing revealed novel members of the chilled flesh microbiota such as Vagococcus vulneris or Rouxiella chamberiensis in the vacuum-packed samples. With traditional cultivation, 161 bacterial strains were isolated and identified at the species level, but the identified bacteria overlapped with only 45% of the dominant operational taxonomic units (OTUs) revealed by NGS. Next-generation sequencing is a promising and highly reliable tool recommended to reach a higher resolution of the forming microbial community of stored fish products. Knowledge of the initial microbial community of the flesh enables further optimization and development of processing and storage technology.

Dyadobacter subterraneus sp. nov., isolated from hydrocarbon-polluted groundwater from an oil refinery in Hungary.

A Gram-stain-negative, aerobic, non-spore-forming, rod-shaped bacterial strain (UP-52T) was isolated from hydrocarbon-polluted groundwater located near an oil refinery in Tiszaujvaros, Hungary. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Dyadobacter in the family Cytophagaceae. Its closely related species are Dyadobacter frigoris (98.00 %), Dyadobacter koreensis (97.64 %), Dyadobacter psychrophilus (97.57 %), Dyadobacter ginsengisoli (97.56 %) and Dyadobacter psychrotolerans (97.20 %). The predominant fatty acids are summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω7c/C16 : 1 ω6c), C15 : 0 iso, C16 : 1 ω5c and C17 : 0 iso 3OH. The predominant respiratory quinone detected in strain UP-52T is quinone MK-7. The dominant polar lipids are glycolipid, phosphoaminolipid, phospholipid and aminolipid. The DNA G+C content is 40.0 mol%. Flexirubin-type pigment was present. Based on these phenotypic, chemotaxonomic and phylogenetic results, UP-52T represents a novel species of the genus Dyadobacter, for which the name Dyadobacter subterraneus sp. nov. is proposed. The type strain is UP-52T (=NCAIM B.02653T=CCM 9030T).

Validation of microplastic sample preparation method for freshwater samples.

The global presence of microplastics in the environment is well documented nowadays. Studies already showed the potential risks that microplastic particles might cause to the ecosystem, while potential human health effects are currently under investigation. As one of the main inputs of these crucial researches, the concentration of microplastics in the environment should be measured precisely, confidently and monitored regularly to determine exposure levels of these pollutants. Some study highlights, that the results are usually inconsistent and uncertain, due to different sampling and sample preparation methods and the lack of quality assurance and quality control of these processes. The need for a standardized methodology is an emerging issue, as this would provide the right tools to establish a global monitoring system of microplastics. Validated sample preparation methods of water (especially freshwater) samples for microplastic analysis are rarely described. To fulfil the gap, this study aims to create and validate a special toolset and the related standard operating procedure for enhanced sample preparation. A newly developed equipment, the Small Volume Glass Separator was designed to easily isolate microplastics from freshwater samples and concentrate the treated sample in a small volume, thus reducing the brine solution use and the sample transfer steps. These features enable better prevention of contamination and making sample preparation easy, fast and cost-effective. The Small Volume Glass Separator and the related standard operation procedure was validated on model freshwater and wastewater samples with the use of fluorescently tagged microplastics and environmentally relevant microplastics (fragments, fibres). Recoveries were measured with optical microscopy under UV light and with near-infrared spectroscopy/microscopy. Recovery tests with fluorescently tagged microspheres showed that average recovery with the Small Volume Glass Separator is 12-39% higher than that of a widespread sample preparation method. This procedure was also able to recover on average 64%±29% of all the environmentally relevant particles during the validation process. Results show that size and density have a great influence on potential particle loss. Recovery of smaller particles are less with both methods than that of the larger particles, but Small Volume Glass Separator yielded significantly higher recovery for more dense particles. The results of this study help to better understand particle loss during sample preparation and thus contribute to the establishment of standardised microplastic analysis processes.

Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa.

Validation of pressurized fractionated filtration microplastic sampling in controlled test environment.

In the field of microplastic (MP) research in the environment, a significant amount of the currently reported results is uncertain because of the inappropriate methods of sampling, detection and quantification of MPs. Fortunately, many research groups are aware of these challenges, but validated methods, which are the prerequisite of standardized measurements, are scarce. Recovery tests are especially rare in the field of MP sampling. The aim of our research was to take a step forward and collect data on cascade filtration recoveries by modeling different turbulance conditions and sampling depth applying environmentally relevant MP concentrations while obtaining large sample volumes. As reference materials, different polymer types (polyethylene - PE; polypropylene - PP; poly[ethylene terephthalate] - PET; poly[vinyl chloride] - PVC; polyamide - PA) and shapes (sphere, fragment, fiber) were used, and for detection near-infrared spectroscopy/microscopy was applied. The developed method provides information not only on system based MP losses, but on sampling efficiency in a model environment as well. Based on the results, the highest recovery rate of all polymers was 31.4% on average, sampled from the water surface during continuous stirring. In these conditions, 92.4% of the PE spheres and 31.9% of the PE fragments were recovered. This indicates, particles reported in environmental monitoring studies might be less than the real environmental concentration. We can conclude, that surface sampling is more efficient than sampling in a deeper layer of the water column. Our research revealed, that the widespread application of microspheres as reference materials might lead to too optimistic recovery values. The application of reference particles (fragments, fibers) with higher environmental relevance shows much lower recovery rates. Our results highlight, that validating the efficiency of the whole sampling process from the environment is more important than measuring only the filtration device's recovery. This study helps us to better understand the relationship and the possible gaps between the reported MP results and the real-life concentrations in the environment.

Parvularcula mediterranea sp. nov., isolated from marine plastic debris from Zakynthos Island, Greece.

A Gram-negative, dark orange-pigmented, aerobic, non-spore-forming, coccoid-shaped bacterium designated as ZS-1/3T was isolated from a floating plastic litter (polypropylene straw) sample, collected from shallow seawater near the public beach of Laganas on Zakynthos island, Greece. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate is affiliated with the genus Parvularcula in the family Parvularculaceae. Its closest relatives are Parvularcula lutaonensis (98.09  %) and Parvularcula oceanus (95.89  %). The pH and temperature ranges for growth are pH 5-10 and 20-38 °C (optima, pH 7.0 and 28 °C). The predominant fatty acids are C18 : 1 ω7c (56.84  %), C16 : 0 (27.51  %), C18 : 0 (2.25  %) and C12 : 0 (1.42  %). The predominant respiratory quinone detected in strain ZS-1/3T is quinone-10 (Q10); the majority of detected polar lipids are glycolipid. The DNA G+C content is 62.5  mol%. Physiological and chemotaxonomic data further confirmed the distinctiveness of strain ZS-1/3T from other members of the genus Parvularcula. Thus, strain ZS-1/3T is considered to represent a novel species of the genus, for which the name Parvularcula mediterranea. sp. nov. is proposed. The type strain is ZS-1/3T (=NCAIM B 02654T=CCM 9032T).

Effects of Single and Repeated Oral Doses of Ochratoxin A on the Lipid Peroxidation and Antioxidant Defense Systems in Mouse Kidneys.

Ochratoxin-A (OTA) is a carcinogenic and nephrotoxic mycotoxin, which may cause health problems in humans and animals, and it is a contaminant in foods and feeds. The purpose of the present study is to evaluate the effect of oral OTA exposure on the antioxidant defense and lipid peroxidation in the kidney. In vivo administration of OTA in CD1, male mice (1 or 10 mg/kg body weight in a single oral dose for 24 h and repeated daily oral dose for 72 h or repeated daily oral dose of 0.5 mg/kg bodyweight for 21 days) resulted in a significant elevation of OTA levels in blood plasma. Some histopathological alterations, transcriptional changes in the glutathione system, and oxidative stress response-related genes were also found. In the renal cortex, the activity of the glutathione-system-related enzymes and certain metabolites of the lipid peroxidation (conjugated dienes, trienes, and thiobarbituric reactive substances) also changed.

Planktonic and Benthic Bacterial Communities of the Largest Central European Shallow Lake, Lake Balaton and Its Main Inflow Zala River.

Lake Balaton is the largest European shallow lake, which underwent cultural eutrophication in the '70-80s. Therefore, strict pollution control measures were introduced and the water quality has become meso-eutrophic since the millennium. Due to the touristic significance and change in trophic levels of the lake, numerous ecological studies were carried out, but none of them was focused on both benthic and planktonic microbial communities at the same time. In our study, an attempt was made to reveal the spatial bacterial heterogeneity of the Lake Balaton and Zala River by 16S rDNA terminal restriction fragment length polymorphism fingerprinting and Illumina amplicon sequencing methods in the summer of 2017. According to the molecular biology results, mostly well-known freshwater microorganisms, adapted to nutrient-poor conditions were found in the pelagic water column. The LD12 subclade member Fonsibacter ubiquis, the cyanobacterial Synechococcus sp. and unknown Verrucomicrobia species were abundant in the less nutrient-dense basins, while the hgcI clade members showed various distribution. In the estuary and in the nutrient-dense western part of the lake, some eutrophic conditions preferring cyanobacteria (filamentous Anabaena and Aphanizomenon species) were also detectable. The benthic microbial community showed higher diversity, according to the observed appearance of microorganisms adapted to the deeper, less aerated layers (e.g. members of Desulfobacteraceae, Nitrosomonadaceae).

Cytotoxicity and hormonal activity of glyphosate-based herbicides.

Glyphosate-based herbicides (GBHs) are the most widely used pesticides for weed control. In parallel with the renewal of the active ingredient, polyethoxylated POE(15) containing GBHs were banned in the EU in 2016. Since then, co-formulants were changed and numerous GBHs are marketed with different excipients declared as inert substances. In our study, we focused to determine acute and chronic cytotoxicity (by Aliivibrio fischeri assay) and direct hormonal activity (estrogenic and androgenic effects measured by Saccharomyces cerevisiae BLYES/BLYAS strains, respectively) of glyphosate, AMPA, polyethoxylated POE(15) and 13 GBHs from which 11 formulations do not contain polyethoxylated POE(15). Among the pure substances, neither glyphosate nor AMPA had any effects, while polyethoxylated POE(15) exhibited pronounced toxicity and was also estrogenic but not androgenic. Regarding the acute and chronic cytotoxicity and hormonal activity of GBHs, dilution percentages calculated from EC50 values were in the most cases by one or two order of magnitude lower than the minimum recommended dilution for agricultural and household use. Relation could not be observed between the biological effects and type of glyphosate-salts; hence toxicity could be linked to the co-formulants, which are not even declared in 3 GBHs. Toxicological evaluation must focus on these substances and free accessibility of GBHs should be reconsidered.

Effect of oxygen limitation on the enrichment of bacteria degrading either benzene or toluene and the identification of Malikia spinosa (Comamonadaceae) as prominent aerobic benzene-, toluene-, and ethylbenzene-degrading bacterium: enrichment, isolation and whole-genome analysis.

The primary aims of this present study were to evaluate the effect of oxygen limitation on the bacterial community structure of enrichment cultures degrading either benzene or toluene and to clarify the role of Malikia-related bacteria in the aerobic degradation of BTEX compounds. Accordingly, parallel aerobic and microaerobic enrichment cultures were set up and the bacterial communities were investigated through cultivation and 16S rDNA Illumina amplicon sequencing. In the aerobic benzene-degrading enrichment cultures, the overwhelming dominance of Malikia spinosa was observed and it was abundant in the aerobic toluene-degrading enrichment cultures as well. Successful isolation of a Malikia spinosa strain shed light on the fact that this bacterium harbours a catechol 2,3-dioxygenase (C23O) gene encoding a subfamily I.2.C-type extradiol dioxygenase and it is able to degrade benzene, toluene and ethylbenzene under clear aerobic conditions. While quick degradation of the aromatic substrates was observable in the case of the aerobic enrichments, no significant benzene degradation, and the slow degradation of toluene was observed in the microaerobic enrichments. Despite harbouring a subfamily I.2.C-type C23O gene, Malikia spinosa was not found in the microaerobic enrichments; instead, members of the Pseudomonas veronii/extremaustralis lineage dominated these communities. Whole-genome analysis of M. spinosa strain AB6 revealed that the C23O gene was part of a phenol-degrading gene cluster, which was acquired by the strain through a horizontal gene transfer event. Results of the present study revealed that bacteria, which encode subfamily I.2.C-type extradiol dioxygenase enzyme, will not be automatically able to degrade monoaromatic hydrocarbons under microaerobic conditions.

Microaerobic conditions caused the overwhelming dominance of Acinetobacter spp. and the marginalization of Rhodococcus spp. in diesel fuel/crude oil mixture-amended enrichment cultures.

The aim of the present study was to reveal how different microbial communities evolve in diesel fuel/crude oil-contaminated environments under aerobic and microaerobic conditions. To investigate this question, aerobic and microaerobic bacterial enrichments amended with a diesel fuel/crude oil mixture were established and analysed. The representative aerobic enrichment community was dominated by Gammaproteobacteria (64.5%) with high an abundance of Betaproteobacteriales (36.5%), followed by Alphaproteobacteria (8.7%), Actinobacteria (5.6%), and Candidatus Saccharibacteria (4.5%). The most abundant alkane monooxygenase (alkB) genotypes in this enrichment could be linked to members of the genus Rhodococcus and to a novel Gammaproteobacterium, for which we generated a high-quality draft genome using genome-resolved metagenomics of the enrichment culture. Contrarily, in the microaerobic enrichment, Gammaproteobacteria (99%) overwhelmingly dominated the microbial community with a high abundance of the genera Acinetobacter (66.3%), Pseudomonas (11%) and Acidovorax (11%). Under microaerobic conditions, the vast majority of alkB gene sequences could be linked to Pseudomonas veronii. Consequently, results shed light on the fact that the excellent aliphatic hydrocarbon degrading Rhodococcus species favour clear aerobic conditions, while oxygen-limited conditions can facilitate the high abundance of Acinetobacter species in aliphatic hydrocarbon-contaminated subsurface environments.

Acute and chronic toxicity of herbicides and their mixtures measured by Aliivibrio fischeri ecotoxicological assay.

The purpose of our work was to determine the acute and chronic toxicity of three of the EU's most common herbicides - mesotrione, S-metolachlor, terbuthylazine - and their mixtures by Aliivibrio fischeri ecotoxicological assays. While comparing the sensitivity of the acute (30 min) Microtox® standard assay with the chronic (25 h) test adapted to microtiter plate, joint effects (antagonism, additive effect and synergism) to the bioluminescence inhibition (consequently the metabolic damage) in A. fischeri were also determined by Combination Index (CI) method. 30 min of exposure to mesotrione and S-metolachlor resulted in a relatively low acute toxicity (EC50 values were 118 and 265 mg/L), while terbuthylazine did not cause bioluminescence inhibition at all. Results showed that the chronic toxicity of S-metolachlor and terbuthylazine to A. fischeri (EC5010h = 59.2 and 4.9 mg/L and EC5015h = 54.0 and 9.6 mg/L, respectively) is larger by at least one order of magnitude than that after 30 min of contact time. Considering mesotrione no significant difference was experienced in toxicity. Regarding the EC50 values, all of the mixtures had synergistic joint effects in the acute assay. However, in the chronic test all the mixtures showed antagonistic responses with the exception of mesotrione and S-metolachlor (ratio 1:1) combination, which also had additive and synergistic effects after 10 and 15 h of exposure, similarly to the short-term test. This is also the first report of the joint effects of these herbicides. The chronic test is a more sensitive indicator to the active ingredients; both acute and chronic assays supply valuable data of the toxic properties of the pesticides. Moreover, the short- and long-term joint effects of their mixtures supporting a more accurate and reliable risk assessment.

First isolation of carbapenem-resistant Acinetobacter beijerinckii from an environmental sample.

The emergence of opportunistic Acinetobacter spp. in healthcare settings poses a significant threat to public health. The major reasons for nosocomial spread of these species are their abilities to develop and transfer drug resistance against various classes of antibiotics. Considering that Acinetobacter spp. are ubiquitous in nature, can utilize several carbon sources, and reach humans via various pathways, our aim was to obtain information about the environmental strains of this genus. Our first step was to develop and test a multistep isolation procedure based on traditional scientific methods. Antibiotic resistance patterns of the isolated strains were determined, as susceptibility to 12 antibiotics of 7 classes was tested by MIC Test Strip method. Altogether 366 samples (groundwater, surface water, and soil) of 24 sites were investigated and a collection of 37 Acinetobacter isolates was obtained. Among others, clinically important human pathogen Acinetobacter spp., such as A. baumannii, A. johnsonii, and A. gyllenbergii were identified. Three environmental strains were determined as multidrug-resistant including a carbapenem-resistant, hemolytic Acinetobacter beijerinckii strain isolated from a hydrocarbon-contaminated groundwater sample. In summary, it has been found that the applied multistep isolation procedure is applicable to isolate various species of Acinetobacter genus. Based on the antibiotic resistance assay, we can conclude that environmental representatives of Acinetobacter spp. are able to develop multidrug resistance, but at a lower rate than their clinical counterparts.

Identification of microplastics in fish ponds and natural freshwater environments of the Carpathian basin, Europe.

In the past few years, there has been a significant development in freshwater microplastic research. Pollution has been detected in lakes and rivers of several continents, but the number of papers is still marginal compared to the ones investigating marine environments. In this study, we present the first detection of microplastics (MPs) in Central and Eastern European (CEE) surface waters and, globally, the first detection in fish ponds. Samples were taken from different types of fish ponds and natural water bodies along a novel concept down to a particle size of 100 µm, then, after sample preparation, MPs were characterized using an FTIR microscope. 92% of the water samples contained MPs ranging from 3.52 to 32.05 particles/m3. MPs were detected in 69% of the sediment samples ranging from 0.46 to 1.62 particles/kg. Dominant abundance of polypropylene (PP) and polyethylene was shown in water and PP and polystyrene in sediment samples. First results also indicate that fish ponds may act as a deposition area for MPs.

Stable isotope probing of hypoxic toluene degradation at the Siklós aquifer reveals prominent role of Rhodocyclaceae.

The availability of oxygen is often a limiting factor for the degradation of aromatic hydrocarbons in subsurface environments. However, while both aerobic and anaerobic degraders have been intensively studied, degradation betwixt, under micro- or hypoxic conditions has rarely been addressed. It is speculated that in environments with limited, but sustained oxygen supply, such as in the vicinity of groundwater monitoring wells, hypoxic degradation may take place. A large diversity of subfamily I.2.C extradiol dioxygenase genes has been previously detected in a BTEX-contaminated aquifer in Hungary. Older literature suggests that such catabolic potentials could be associated to hypoxic degradation. Bacterial communities dominated by members of the Rhodocyclaceae were found, but the majority of the detected C23O genotypes could not be affiliated to any known bacterial degrader lineages. To address this, a stable isotope probing (SIP) incubation of site sediments with 13C7-toluene was performed under microoxic conditions. A combination of 16S rRNA gene amplicon sequencing and T-RFLP fingerprinting of C23O genes from SIP gradient fractions revealed the central role of degraders within the Rhodocyclaceae in hypoxic toluene degradation. The main assimilators of 13C were identified as members of the genera Quatrionicoccus and Zoogloea, and a yet uncultured group of the Rhodocyclaceae.