Chemical and toxicological assessment of leachates from UV-degraded plastic materials using in-vitro bioassays.

The broad use of plastics and the persistence of the material results in plastic residues being found practically everywhere in the environment. If plastics remain in the (aquatic) environment, natural weathering leads to degradation processes and compounds may leach from plastic into the environment. To investigate the impact of degradation process on toxicity of leachates, different types of UV irradiation (UV-C, UV-A/B) were used to simulate weathering processes of different plastic material containing virgin as well as recyclate material and biodegradable polymers. The leached substances were investigated toxicologically using in-vitro bioassays. Cytotoxicity was determined by the MTT-assay, genotoxicity by using the p53-CALUX and Umu-assay, and estrogenic effects by the ERα-CALUX. Genotoxic as well as estrogenic effects were detected in different samples depending on the material and the irradiation type. In four leachates of 12 plastic species estrogenic effects were detected above the recommended safety level of 0.4 ng 17ß-estradiol equivalents/L for surface water samples. In the p53-CALUX and in the Umu-assay leachates from three and two, respectively, of 12 plastic species were found to be genotoxic. The results of the chemical analysis show that plastic material releases a variety of known and unknown substances especially under UV radiation, leading to a complex mixture with potentially harmful effects. In order to investigate these aspects further and to be able to give recommendations for the use of additives in plastics, further effect-related investigations are advisable.

Photolytic degradation of novel polymeric and monomeric brominated flame retardants: Investigation of endocrine disruption, physiological and ecotoxicological effects.

Ecotoxicological effects of photolytic degradation mixtures of the two brominated flame retardants PolymericFR and Tetrabromobisphenol A-bis (2,3-dibrom-2-methyl-propyl) Ether (TBBPA-BDBMPE) have been studied in vitro and in vivo. Both substances were experimentally degraded separately by exposure to artificial UV-light and the resulting degradation mixtures from different time points during the UV-exposure were applied in ecotoxicological tests. The in vitro investigation showed no effects of the degraded flame retardants on the estrogenic and androgenic receptors via the CALUX (chemically activated luciferase gene expression) assay. Short-term exposures (up to 96 h) of Lumbriculus variegatus lead to temporary physiological reactions of the annelid. The exposure to degraded PolymericFR lead to an increased activity of Catalase, while the degradation mixture of TBBPA-BDBMPE caused increases of Glutathione-S-transferase and Acetylcholine esterase activities. Following a chronic exposure (28 d) of L. variegatus, no effects on the growth, reproduction, fragmentation and energy storage of the annelid were detected. The results indicate that the experimental degradation of the two flame retardants causes changes in their ecotoxicological potential. This might lead to acute physiological effects on aquatic annelids, which, however, do not affect the animals chronically according to our results.

Effect-based evaluation of ozone treatment for removal of micropollutants and their transformation products in waste water.

The aim of this interdisciplinary research project in North Rhine-Westphalia (NRW), Germany, entitled "Elimination of pharmaceuticals and organic micropollutants from waste water" involved the conception of cost-effective and innovative waste-water cleaning methods. In this project in vitro assays, in vivo assays and chemical analyses were performed on three municipal waste-water treatment plants (WWTP). This publication focuses on the study of the in vitro bioassays. Cytotoxic, estrogenic, genotoxic and mutagenic effects of the original as well as enriched water samples were monitored before and after wastewater treatment steps using MTT and PAN I, ER Calux and A-YES, micronucleus and Comet assays as well as AMES test. In most cases, the measured effects were reduced after ozonation, but in general, the biological response depended upon the water composition of the WWTP, in particular on the formed by-products and concentration of micropollutants. In order to be able to assess the genotoxic and/or mutagenic potential of waste-water samples using bioassays like Ames test, Comet assay or micronucleus test an enrichment of the water sample via solid-phase extraction is recommended. This is in agreement with previous studies such as the "ToxBox"-Project of the Environmental Agency in Germany.

Evaluation of mixture effects of endocrine active substances in wastewater using CALUX reporter-gene assays.

Endocrine active substances (EAS), which are commonly used in pharmaceuticals and personal care products, are released into surface water mainly through WWTP effluents and have been shown to cause adverse effects in aquatic organisms. In wastewater, a variety of EAS with different hormonal activities is present, which can lead to additive effects or mask an endocrine activity. To investigate hormonal combination effects, with a focus on estrogen and androgen-modulators, influent samples from municipal and hospital wastewater treatmenr plants were spiked with 17α-ethinylestradiol, toremifene, 17α-methyltestosterone and bicalutamide and analyzed using in vitro reporter gene CALUX assays. All wastewaters caused endocrine activities in human cells, which were modified by adding one or several endocrine active substances. As expected, estrogenic activity was reduced in presence of the anti-estrogenic toremifene and androgenic activity decreased with the anti-androgen bicalutamide. In general, substance addition caused a similar trend in altered endocrine activities; however, their intensities differed between the wastewaters. Our results indicate that masking effects, leading to a suppressed biological signal, are of significant importance in the assessment of complex water samples, and combination effects rather than single substances determine the final biological effect. This emphasizes the need of effect-based tools in the assessment of water samples.

Correction to: (Anti-)estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: comparison of three in vitro bioassays.

Linda Gehrmann and Helena Bielak contributed equally to this work and are regarded as joint first authors.

Effect-based methods in combination with state-of-the-art chemical analysis for assessment of water quality as integrated approach.

Complex mixtures of chemicals in waste and finally in surface water may pose a risk to the environment and also to human health. This contamination of surface water cannot be addressed with chemical analysis alone. Tools are required to detect and assess these micropollutants which might cause adverse effects. Effect-directed analysis (EDA) with effect-based methods in combination with state-of-the-art chemical analysis can meet this challenge. The present paper summarizes and outlines current experiences with analytical tools and bioassays as integrated approach for assessment of water quality. The need for a holistic and solution-oriented procedure of water quality monitoring is described. To integrate and evaluate existing information about toxicity pathways, which are essential for the EDA approach, the adverse outcome pathway (AOP) concept is useful and recommended. An integration of AOP concept in water quality assessment and further requirements are discussed.

Toxicological and ecotoxicological evaluation of the water quality in a large and eutrophic freshwater lake of China.

Effect-based methods (EBMs) are recommended as holistic approach for diagnosis and monitoring of water quality; however, the application of EBMs is still scare in China. In the present study, water quality of the freshwater lake Taihu (China) was investigated by EBMs. Different types of water samples were collected from three bays of the lake during 2015, 2016 and 2017. A battery of seven effect-based bioassays, including both specific and non-specific toxicity assays, was used. The bioassay battery was recently suggested based on joint activities of the EU project SOLUTIONS and the NORMAN network on emerging pollutants and is also under discussion for being implemented into monitoring activities in the context of the European Water Framework Directive (WFD). Adverse effects were observed towards the primary producer, primary consumer and fish, indicating the potential ecotoxicity of water in Taihu Lake. Mutagenic and estrogenic effects were found in the Ames fluctuation assay and ERα CALUX (Chemically Activated Luciferase Gene-eXpression) assay, respectively, highlighting the potential risks on human health. Algal growth inhibition and mutagenic effects can be observed during each of the three years. Acute toxicity towards Daphnia magna and estrogen receptor agonistic effects were found in at least one of the samples collected in 2016 and 2017, but not in 2015. The endpoints for fish toxicity in the Danio rerio fish embryo test included both lethal and additionally several sublethal effects (only for samples from 2017) and were not compared between years. Algal growth inhibition, fish embryo toxicity, mutagenic effect and estrogenicity were observed in each of the three bays, while Daphnia acute toxicity was only found in Zhushan Bay. Taking together, this study provides a big picture on the water quality of Taihu Lake. The battery of effect-based tools is promising to be a routine for water quality monitoring in China.

(Anti-)estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: comparison of three in vitro bioassays.

Endocrine-disrupting chemicals are mainly discharged into the environment by wastewater treatment plants (WWTPs) and are known to induce adverse effects in aquatic life. Advanced treatment with ozone successfully removes such organic micropollutants, but an increase of estrogenic effects after the ozonation of hospital wastewater was observed in previous studies. In order to investigate this effect, estrogenic and androgenic as well as anti-estrogenic and anti-androgenic activities were observed during treatment of hospital wastewater using three different effect-based reporter gene bioassays. Despite different matrix influences, sensitivities, and test-specific properties, all assays used obtained comparable results. Estrogenic and androgenic activities were mainly reduced during the biological treatment and further removed during ozonation and sand filtration, resulting in non-detectable agonistic activities in the final effluent. An increased estrogenic activity after ozonation could not be observed in this study. Antagonistic effects were removed in the biological treatment by up to 50 % without further reduction in the advanced treatment. Due to the presence of antagonistic substances within the wastewater, masking effects were probable. Therefore, this study showed the relevance of antagonistic activities at hospital WWTPs and illustrates the need for a better understanding about antagonistic effects.

Influence of the copper-induced viable but non-culturable state on the toxicity of Pseudomonas aeruginosa towards human bronchial epithelial cells in vitro.

The viable but non-culturable (VBNC) state of the opportunistic bacterium Pseudomonas aeruginosa was previously shown to be induced by copper ions in concentrations relevant to those in drinking water plumbing systems. This decrease of bacterial culturability without loss of viability might have an influence on human health due to an underestimation of the actual contamination in drinking water systems. The aim of this study was to investigate the influence of culturable P. aeruginosa, viable but not culturable as well as culturable again after resuscitation from the VBNC state on human bronchial epithelial cells (BEAS-2B) in vitro. Cyto- and genotoxic effects of P. aeruginosa at different states were studied using trypan blue, MTT, xCELLigence as well as the micronucleus assay. While P. aeruginosa in the VBNC state did not have any cytotoxic or genotoxic effect on BEAS-2B cells, untreated (culturable) and resuscitated P. aeruginosa did show cell damage, including disruption of cell membranes, inhibition of mitochondrial activity and cell proliferation as well as DNA-damaging effects. We conclude from our study that P. aeruginosa after resuscitation from the VBNC state regains its viability and cyto-/genotoxicity and therefore might influence human health.

Investigation of full-scale ozonation at a municipal wastewater treatment plant using a toxicity-based evaluation concept.

Effluents from municipal wastewater treatment plants (WWTPs) are known to be point sources of micropollutants for surface waters. The aim of this study was to examine a reconstructed full-scale ozonation equipped with a pump-injector system for ozone (O3) dosage and a fluidized moving-bed reactor as biological posttreatment at a municipal WWTP utilizing an effect-directed approach. This approach consists of chemical analysis in combination with toxicological tests for the assessment of treatment efficiency of the plant. Chemical analysis showed elimination rates > 80% for pharmaceuticals and industrial chemicals. Analysis of endocrine disruptors was limited due to substance concentrations below the limit of detection (LOD). Estrogenic activity was detected by the Arxula Adeninivorans yeast estrogen screen (A-YES) at low concentrations (pg to ng EEQ/l range). Estrogenic activity was reduced by more than 90% after ozonation. In contrast, androgenic activity (measured in the Adeninivorans yeast androgen screen, A-YAS) was still found after O3 treatment and after biological posttreatment, which is consistent with the data obtained by chemical analysis. Furthermore, no marked genotoxic or cytotoxic effects were observed after ozonation using the alkaline comet and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assays, respectively. Results suggest that the applied specific O3 dose of 0.4 mgO3/mgDOC is a safe operation setup in terms of toxicologically relevant transformation products. In addition, no adverse effects on primary producers, as evidenced by algae growth inhibition tests, were detected. The monitored biofilm growth in the biological posttreatment exhibited a steady state after one month. Based on computational fluid dynamics (CFD) simulations and biomass, one might conclude that O3 did not apparently enter biological posttreatment to a great extent and that hydraulic retention time in the O3 reactor was sufficient. Our data demonstrate the effectiveness of a full-scale O3 treatment in combination with a fluidized moving-bed reactor as biological posttreatment for the reduction of a majority of micropollutants without the release of relevant toxic transformation products as assessed by a chemical and toxicity-based approach.

Since 2015 the SinoGerman research project SIGN supports water quality improvement in the Taihu region, China.

The Taihu (Tai lake) region is one of the most economically prospering areas of China. Due to its location within this district of high anthropogenic activities, Taihu represents a drastic example of water pollution with nutrients (nitrogen, phosphate), organic contaminants and heavy metals. High nutrient levels combined with very shallow water create large eutrophication problems, threatening the drinking water supply of the surrounding cities. Within the international research project SIGN (SinoGerman Water Supply Network, www.water-sign.de), funded by the German Federal Ministry of Education and Research (BMBF), a powerful consortium of fifteen German partners is working on the overall aim of assuring good water quality from the source to the tap by taking the whole water cycle into account: The diverse research topics range from future proof strategies for urban catchment, innovative monitoring and early warning approaches for lake and drinking water, control and use of biological degradation processes, efficient water treatment technologies, adapted water distribution up to promoting sector policy by good governance. The implementation in China is warranted, since the leading Chinese research institutes as well as the most important local stakeholders, e.g. water suppliers, are involved.

Effect of sulfide on the cytotoxicity of arsenite and arsenate in human hepatocytes (HepG2) and human urothelial cells (UROtsa).

Arsenic, a common poison, is known to react with sulfide in vivo, forming thioarsenates. The acute toxicity of the inorganic thioarsenates is currently unknown. Our experiments showed that a fourfold sulfide excess reduced acute arsenite cytotoxicity in human hepatocytes (HepG2) and urothelial cells (UROtsa) significantly, but had little effect on arsenate toxicity. Speciation analysis showed immediate formation of thioarsenates (up to 73 % of total arsenic) in case of arsenite, but no speciation changes for arsenate. Testing acute toxicity of mono- and trithioarsenate individually, both thioarsenates were found to be more toxic than their structural analogue arsenate, but less toxic than arsenite. Toxicity increased with the number of thio groups. The amount of cellular arsenic uptake after 24 h corresponded to the order of toxicity of the four compounds tested. The dominant to almost exclusive intracellular arsenic species was arsenite. The results imply that thiolation is a detoxification process for arsenite in sulfidic milieus. The mechanism could either be that thioarsenates regulate the amount of free arsenite available for cellular uptake without entering the cells themselves, or, based on their chemical similarity to arsenate, they could be taken up by similar transporters and reduced rapidly intracellularly to arsenite.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Induction of altered mRNA expression profiles caused by fibrous and granular dust.

Natural and synthetic fibres and particles are being introduced into the workplace and environment daily. Comparative analyses of the induced signalling pathways are essential in order to understand the potential hazards of these particles. To identify the molecular characteristics of particles and fibres, we selected crocidolite and chrysotile asbestos as representatives for fibered dust and titanium dioxide (TiO2) (100-200 nm), zirconium dioxide (ZrO2) (50-100 nm) and hematite (Fe2O3) (20 nm) as representatives for bio-persistent granular dust. SV-40 virus-transformed human bronchial epithelial cells (BEAS-2B) were exposed to well-defined fibres and particles. RT2 Profiler™ PCR Array Human Stress & Toxicity PathwayFinder was used to compare the relative mRNA expression of 84 genes. A detailed characterization of the dust samples used in this study was accomplished to ensure comparability to other studies. Investigation of mRNA expression of 84 signalling molecules attributed to pathways such as DNA damage and repair; oxidative/metabolic stress; growth arrest and senescence; inflammation, proliferation and carcinogenesis; and heat shock and apoptosis revealed that crocidolite and chrysotile asbestos induced mRNA expression of pathway molecules involved in proliferation and carcinogenesis, as well as inflammation. Titanium dioxide, zirconium dioxide and hematite mainly induced pathway molecules responsible for oxidative/metabolic stress and inflammation. Our findings suggest that the hazards of fibered dust mainly include the induction of direct toxicity by altering signalling pathways such as carcinogenesis and proliferation, while granular dust shows indirect toxicity by altering signalling pathways involved in inflammatory processes. PCR arrays, therefore, may be a helpful tool to estimate the hazard risk of new materials.

Reaction of gadolinium chelates with ozone and hydroxyl radicals.

Gadolinium chelates are used in increasing amounts as contrast agents in magnetic resonance imaging, and their fate in wastewater treatment has recently become the focus of research. Oxidative processes, in particular the application of ozone, are currently discussed or even implemented for advanced wastewater treatment. However, reactions of the gadolinium chelates with ozone are not yet characterized. In this study, therefore, rate constants with ozone were determined for the three commonly used chelates Gd-DTPA, Gd-DTPA-BMA, and Gd-BT-DO3A, which were found to be 4.8 ± 0.88, 46 ± 2.5, and 24 ± 1.5 M(-1) s(-1), respectively. These low rate constants indicate that a direct reaction with ozone in wastewater is negligible. However, application of ozone in wastewater leads to substantial yields of (•)OH. Different methods have been applied and compared for determination of k((•)OH+Gd chelate). From rate constants determined by pulse radiolysis experiments (k((•)OH+Gd-DTPA) = 2.6 ± 0.2 × 10(9) M(-1) s(-1), k((•)OH+Gd-DTPA-BMA) = 1.9 ± 0.7 × 10(9) M(-1) s(-1), k((•)OH+Gd-BT-DO3A) = 4.3 ± 0.2 × 10(9) M(-1) s(-1)), it is concluded that a reaction in wastewater via (•)OH radicals is feasible. Toxicity has been tested for educt and product mixtures of both reactions. Cytotoxicity (MTT test) and genotoxicity (micronuclei assay) were not detectable.

Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

BACKGROUND: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. METHODS: UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. RESULTS: All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. CONCLUSIONS: It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

Ozonation products of triclosan in advanced wastewater treatment.

Triclosan is an antimicrobial agent widely used in many household and personal care products. Widespread use of this compound has led to the elevated concentrations of triclosan in wastewater, wastewater treatment plants and receiving waters. In this study removal of triclosan by aqueous ozone was investigated and the degradation products formed during ozonation of an aqueous solution of triclosan were analyzed by GC-MS and HPLC-MS/MS. The following transformation products have been identified: 2,4-dichlorophenol, chloro-catecol, mono-hydroxy-triclosan and di-hydroxy-triclosan during treatment process. Cytotoxicity and genotoxicity of pure triclosan and 2,4-dichlorophenol have been investigated and the results showed reduced genotoxic effects after ozonation, though the respective chlorophenol is harmful to aquatic organisms.

Comparison of micro- and nanoscale Fe⁺³-containing (Hematite) particles for their toxicological properties in human lung cells in vitro.

The specific properties of nanoscale particles, large surface-to-mass ratios and highly reactive surfaces, have increased their commercial application in many fields. However, the same properties are also important for the interaction and bioaccumulation of the nonbiodegradable nanoscale particles in a biological system and are a cause for concern. Hematite (α-Fe2O3), being a mineral form of Fe(III) oxide, is one of the most used iron oxides besides magnetite. The aim of our study was the characterization and comparison of biophysical reactivity and toxicological effects of α-Fe2O3 nano- (d < 100 nm) and microscale (d < 5 µm) particles in human lung cells. Our study demonstrates that the surface reactivity of nanoscale α-Fe2O3 differs from that of microscale particles with respect to the state of agglomeration, radical formation potential, and cellular toxicity. The presence of proteins in culture medium and agglomeration were found to affect the catalytic properties of the hematite nano- and microscale particles. Both the nano- and microscale α-Fe2O3 particles were actively taken up by human lung cells in vitro, although they were not found in the nuclei and mitochondria. Significant genotoxic effects were only found at very high particle concentrations (> 50 µg/ml). The nanoscale particles were slightly more potent in causing cyto- and genotoxicity as compared with their microscale counterparts. Both types of particles induced intracellular generation of reactive oxygen species. This study underlines that α-Fe2O3 nanoscale particles trigger different toxicological reaction pathways than microscale particles. However, the immediate environment of the particles (biomolecules, physiological properties of medium) modulates their toxicity on the basis of agglomeration rather than their actual size.

Transformation products and reaction kinetics of fragrances in advanced wastewater treatment with ozone.

The reaction of the fragrance compounds 4,6,6,7,8,8-hexamethyl-1,3,4,7-tetrahydrocyclopenta[g]isochromene (HHCB), 1-(3,5,5,6,8,8-hexamethyl-6,7-dihydronaphthalen-2-yl)ethanone (AHTN), 1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene (musk xylene/MX), 1-(4-tert-butyl-2,6-dimethyl-3,5-dinitrophenyl)ethanone (musk ketone/MK), and 1-(2,3,8,8-tetramethyl-1,3,4,5,6,7-hexahydronaphthalen-2-yl)ethanone (OTNE) with ozone in tap water as well as waste water treatment plant (WWTP) effluents is described. Several transformation products are characterized by means of gas chromatography coupled to mass spectrometry. One transformation product (HHCB-Lactone) was confirmed by means of a true standard. Musk xylene and musk ketone do not react with ozone under the conditions used in this study. AHTN and HHCB reacted slowly to a multitude of transformation products, while OTNE reacted quickly to several stable transformation products. The reaction constants and half lives are used to predict removal efficiencies for full scale reactors.