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.

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.

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.

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.

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.

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.

Assessment of bacterial biodetoxification of herbicide atrazine using Aliivibrio fischeri cytotoxicity assay with prolonged contact time.

In our study, we determined and compared the atrazine-biodetoxification ability of 41 bacterial strains and 21 consortia created of those with over 50% degradation rate in pure cultures. Biodegradation capacity was measured with GC-MS. Detoxification was assessed based on the cytotoxic effect of end-products to Aliivibrio fischeri in chronic bioluminescence inhibition assay with 25 h contact time. Chronic A. fischeri assay adapted to a microplate, which is suitable for examine numerous residues simultaneously, also appeared to be significantly more sensitive to atrazine compared to the standard acute (30 min) test. Due to its sensitivity, the chronic assay could be a valuable tool to provide a more comprehensive view of the ecological risks of atrazine and other chemicals. Thirteen strains were able to degrade more than 50% of 50 ppm atrazine. Four of these belong to Rhodococcus aetherivorans, R. qingshengii, Serratia fonticola and Olivibacter oleidegradans which species' atrazine degrading ability has never been reported before. Four consortia degrading ability was more effective than that of the creating individual strains; moreover, their residues did not show cytotoxic effects to A. fischeri. However, in several cases, the degradation products of sole strains and consortia resulted in significant bioluminescence inhibition. Thus high biodegradation (>90%) does not certainly mean the reduction or cessation of toxicity highlighting the importance of the evaluation of biological effects of degradation residues to improve the efficiency and abate the ecological risks of bioremediation techniques.

Development of a combined method to assess the complex effect of atrazine on sex steroid synthesis in H295R cells.

The aim of the study was to develop a rapid, cost-effective combined testing method to assess the indirect effect of compounds interfering with sex steroid synthesis and to determine complex effects of atrazine on estrogen and androgen synthesis in vitro on H295R human cell line. Steroidogenic assay was performed on H295R human adrenocortical carcinoma cell line. Instead of standard analytical methods, bioluminescence bioreporter assays (Saccharomyces cerevisiae BLYES and BLYAS) were used to measure estrogenic and androgenic effects of sex steroid hormones released by human cells in response to atrazine. Atrazine resulted in elevated estrogen production presumably due to its well documented inductive effect on aromatase on H295R cell line, detected by BLYES. Interestingly, results of BLYAS test showed concentration-dependent increase of androgen production in H295R cells. That indicates that atrazine can not only increase estrogen level via aromatase induction, but may interfere in androgen synthesis as well. The combined method allows us to assess the androgenic and estrogenic effect of sex steroids produced by human cells in increased or decreased quantity as a result of the different chemicals, without determining specific analytical measurement endpoints, by using the yeast based bioluminescent bioreporter test.

Takotsubo Cardiomyopathy: What we have Learned in the Last 25 Years? (A Comparative Literature Review).

We performed a comparative literature review, to elucidate the major features of the Takotsubo (stress) cardiomyopathy (TCM) collected in last 25 years. TCM is characterized by left- or biventricular apical ballooning with a clinical presentation, electrocardiographic abnormalities, and biomarker profils similar to those seen in acute myocardial infarction. Epidemiological studies have shown that TCM is more common in postmenopausal women; however exact figures are not available. The underlying aetiology is still largely undetermined. Elevated catecholamine levels, lack of estrogen, disturbed myocardial fatty acid metabolism and plaque rupture with spontaneous thrombolysis are potentially discussed mechanisms responsible for inducing a prolonged stunned myocardium. Strong emotional or physical stress is the most frequently described trigger in the literature. Therapy recommendations include appropriate antiplatelet treatment, ß-blockers and ACE inhibitors. The abnormal kinetics usually resolve or improve within a month and carry a favorable prognosis in most cases. However, all the suspected complications of an acute myocardial infarction, including cardiogenic shock or lethal arrhythmias, may still occur.

Influence of oral antiplatelet therapy on hemorrhagic complications of pacemaker implantation.

OBJECTIVE: We retrospectively assessed the incidence of hemorrhagic complications associated with pacemaker implantation in patients receiving one or more antiplatelet agents. DESIGN AND SETTING: Retrospective multicenter case-control study. Data were collected from three cardiac units in Germany from 2006 to 2010. METHODS: A total of 495 pacemaker patients were enrolled. 99 patients received dual antiplatelet therapy (aspirin and clopidogrel), 198 were given only aspirin and 198 had no antiplatelet therapy (control). Patients were matched for age and sex. Implant-related bleeding complications were defined as major bleeding, if surgical pocket exploration or blood transfusion were needed. Minor bleeding complications were defined as one or more of the following conditions: fall of hemoglobin content >1.5 g/dl not requiring blood transfusion; pocket hematoma; pocket effusion not requiring surgical revision. MEASUREMENTS AND RESULTS: Hemorrhagic complications were seen in 4/198 control patients and 6/198 aspirin-only patients [2 vs. 3 %, p = 0.5, OR = 1.52 (0.42-5.46)]. Patients undergoing dual antiplatelet therapy had significantly more bleeding complications than patients in the aspirin-only group [11/99: 11.1 vs. 3 %, p = 0.005, OR = 3.95 (1.43-11.16)]. Major complications occurred in 1/198 control patients and 2/198 aspirin-only patients [0.5 vs. 1 %, p = 0.6, OR = 2.01 (0.18-22.35)]. In contrast, 7/99 patients undergoing dual antiplatelet therapy exhibited major complications [7.1 vs. 1 %, p = 0.004, OR = 7.46 (1.52-36.50)]. CONCLUSIONS: Although in patients undergoing pacemaker implantation dual antiplatelet therapy with aspirin and clopidogrel caused a significant increase of bleeding complications, the use of aspirin alone was not associated with a significant increase in bleeding complications.

Olivibacter oleidegradans sp. nov., a hydrocarbon-degrading bacterium isolated from a biofilter clean-up facility on a hydrocarbon-contaminated site.

A novel hydrocarbon-degrading, Gram-negative, obligately aerobic, non-motile, non-sporulating, rod-shaped bacterium, designated strain TBF2/20.2(T), was isolated from a biofilter clean-up facility set up on a hydrocarbon-contaminated site in Hungary. It was characterized by using a polyphasic approach to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate is affiliated with the genus Olivibacter in the family Sphingobacteriaceae. It was found to be related most closely to Olivibacter ginsengisoli Gsoil 060(T) (93.3% 16S rRNA gene sequence similarity). Strain TBF2/20.2(T) grew at pH 6-9 (optimally at pH 6.5-7.0) and at 15-42 °C (optimally at 30-37 °C). The major fatty acids were iso-C(15:0) (39.4%), summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1)ω7c; 26.0%), iso-C(17:0) 3-OH (14.5%) and C(16:0) (4.5%). The major menaquinone was MK-7 and the predominant polar lipid was phosphatidylethanolamine. The DNA G+C content of strain TBF2/20.2(T) was 41.2 mol%. Physiological and chemotaxonomic data further confirmed the distinctiveness of strain TBF2/20.2(T) from recognized members of the genus Olivibacter. Thus, strain TBF2/20.2(T) is considered to represent a novel species of the genus Olivibacter, for which the name Olivibacter oleidegradans sp. nov. is proposed. The type strain is TBF2/20.2(T) (=NCAIM B 02393(T) =CCM 7765(T)).

Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from composts.

The aim of our work was to determine the presence of Pseudomonas aeruginosa in compost raw materials, immature and mature compost, and compost-treated soil. Twenty-five strains of P. aeruginosa were isolated from a raw material (plant straw), immature and mature compost and compost-treated soil samples. The strains were identified using the PCR method for the detection of species specific variable regions of 16S rDNA. Strains were examined for the presence of five different virulence-related gene sequences (exoA, exoU, exoT, exoS and exoY) and their antibiotic resistance profiles were determined. Based on our results, species P. aeruginosa can reach significant numbers (up to 10(6) MPN/g sample) during composting and 92.0% of the isolated strains carrying at least two gene sequences encoding toxic proteins. Various types of drug resistance were detected among compost originating strains, mainly against third generation Cephalosporins and Carbapenems. Six isolates were able to resist two different classes of antibiotics (third generation Cephalosporins and Carbapenems, wide spectrum Penicillins or Aminoglycosides, respectively). Based on our results, composts can be a source of P. aeruginosa and might be a concern to individuals susceptible to this opportunistic pathogen.

[Takotsubo syndrome from original description up to now]. / Das Takotsubo-Syndrom von der Erstbeschreibung bis heute.

Stress-induced cardiomyopathy, also known as takotsubo syndrome, imitates an acute ST elevation myocardial infarction or an acute coronary syndrome, but without concomitant coronary artery disease. It mainly affects postmenopausal women, but no established epidemiologic data of this syndrome are available to date. Furthermore, the underlying etiologies are still largely unknown. The most frequently described trigger is strong emotional stress. Supportive therapy with aspirin, beta-blockers and angiotensin-converting enzyme inhibitors is recommended. The abnormal kinetics usually reverse or improve within 4-5 weeks. Compared with acute myocardial infarction, takotsubo cardiomyopathy carries a favorable prognosis. However, severe complications, including ventricular fibrillation and cardiogenic shock, may still occur.