Titanium dioxide is known as a photocatalyst, that may be activated by UV-A light and thus be able to lead to chemical reactions, to mineralize organic compounds, to inactivate biomolecules and to kill microorganisms, respectively. To estimate the capability of the photocatalytic activity a novel experimental setup using photovoltage measurements was studied. A distinct correlation between the photocatalytic activity of the titanium dioxide samples and the measurable photovoltages could be demonstrated. The experimental setups included the construction of different electrochemical cells based on TiO2 and using different liquid and gel electrolytes. The investigations were carried out on titanium dioxide layers as well as on TiO2 particle materials. The electrochemical measurement of the resulting voltage was optimised and the results were compared with conventionally used methods for the determination of the photocatalytic activity like the decolourization of methylene blue solutions. Additionally, the increase of carbon dioxide concentration in the gas phase was taken as a measure for total mineralization. Results indicate strong correlations between the different methods.

Originally designed as a general alternative to acute fish toxicity testing (AFT), the fish embryo toxicity test (FET) has become subject to concerns with respect to neurotoxic substances. Whereas oxygen uptake in the fish embryo primarily occurs via diffusion across the skin, juvenile and adult fish rely on active ventilation of the gills. As a consequence, substances including, e.g., neurotoxicants which prevent appropriate ventilation of gills ("respiratory failure syndrome") might lead to suffocation in juvenile and adult fish, but not in skin-breathing embryos. To investigate if this respiratory failure syndrome might play a role for the higher sensitivity of juvenile and adult fish to neurotoxicants, a modified acute toxicity test using post-embryonic, early gill-breathing life-stages of zebrafish was developed with chlorpyrifos, permethrin, lindane, aldicarb, ziram and aniline as test substances. Additionally, a comparative study into bioaccumulation of lipophilic substances with logKow > 3.5 and swimbladder deflation as potential side effects of the respiratory failure syndrome was performed with 4 d old skin-breathing and 12 d old gill-breathing zebrafish. With respect to acute toxicity, post-embryonic 12 d larvae proved to be more sensitive than both embryos (FET) and adult zebrafish (AFT) to all test substances except for permethrin. Accumulation of chlorpyrifos, lindane and permethrin was 1.3- to 5-fold higher in 4 d old than in 12 d old zebrafish, suggesting that (intermediate) storage of substances in the yolk might reduce bioavailability and prevent metabolization, which could be a further reason for lower toxicity in 4 d than in 12 d old zebrafish. Whereas ziram and aniline showed no significant effect on the swimbladder, zebrafish exposed to chlorpyrifos, lindane and permethrin showed significantly deflated swimbladders in 12 d old larvae; in the case of aldicarb, there was a significant hyperinflation in 4 d old larvae. Swimbladder deflation in post-embryonic 12 d zebrafish larvae might be hypothesized as a reason for a lack of internal oxygen supplies during the respiratory failure syndrome, whereas in 4 d old embryos cholinergic hyperinflation of the swimbladder dominates over other effects. Regarding acute lethality, the study provides further evidence that the switch from transcutaneous to branchial respiration in post-embryonic zebrafish life-stages might be the reason for the higher sensitivity of juvenile and adult fish to neurotoxic substances.

Tamara Grummt passed away on January 26, 2020 in Oelsnitz/Vogtland, Germany. Tamara was one of the scientific pioneers in the field of environmental toxicology, namely genotoxicity and hygiene of drinking and bathing waters. Her passing is not only a great loss to environmental research and to the global environmental toxicology community-we have lost an outstanding personality with the heart in the right place, who has become, for many of us, a wonderful friend.

An amendment to this paper has been published and can be accessed via the original article.

Since only a few studies have investigated effects of microplastics (MPs) by routes other than ingestion, this study was designed to analyze the accumulation patterns and transfer of toxic substances associated with microplastic exposure by simple attachment to (1) adult zebrafish (Danio rerio) gills and (2) zebrafish embryos. Two sizes of fluorescently labelled polymers (1-5 and 10-20 μm) loaded with the model polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) were used to analyze fate, accumulation and transfer of microplastic-associated persistent organic pollutants (POPs) on gills and embryos. Results indicate that microplastics did not permanently accumulate at high amounts in adult zebrafish gills after 6 nor 24 h of incubation: Most particles only superficially adhered to the mucus layer on the filaments, which is constantly being excreted. In contrast, the smaller and heavier MPs (1-5 μm) accumulated in high numbers on the surface of zebrafish egg chorions. In both exposure scenarios, transfer of BaP could be visualized with fluorescence microscopy: A prominent BaP signal was visible both in gill filaments and arches after 6 and 24 h incubation and in zebrafish embryos after exposure to BaP-spiked microplastics. Furthermore, the gill EROD (Ethoxyresorufin-O-deethylase) assay showed a clear trend to CYP 1A (Cytochrom P450 1 A) induction via exposure to BaP-spiked microplastics. However, BaP from spiked microplastics did not reach sufficiently high concentrations to be able to induce morphological effects in the fish embryo toxicity test (FET). In contrast, control exposure to waterborne BaP did induce effects in the FET. As a conclusion, microplastics can also transfer POPs not only via ingestion, but also by simple attachment to epithelia or via the water column. However, further studies are needed to clarify if these interactions are of environmental concern relative to waterborne exposure to toxic substances.

The uptake of microplastic particles and the transfer of potential harmful substances along with microplastics has been studied in a variety of organisms, especially invertebrates. However, the potential accumulation of very small microplastic particles along food webs ending with vertebrate models has not been investigated so far. Therefore, a simple artificial food chain with Artemia sp. nauplii and zebrafish (Danio rerio) was established to analyze the transfer of microplastic particles and associated persistent organic pollutants (POPs) between different trophic levels. Very small (1–20mm) microplastic particles accumulated in Artemia nauplii and were subsequently transferred to fish. Virgin particles not loaded with POPs did not cause any observable physical harm in the intestinal tracts of zebrafish, although parts of the particles were retained within the mucus of intestinal villi and might even have been taken up by epithelial cells. The transfer of associated POPs was tested with the polycyclic aromatic hydrocarbon benzo[a]pyrene and an ethoxyresorufin-O-deethylase (EROD) assay for CYP1A induction in zebrafish liver as well as via fluorescence analyses. Whereas a significant induction in the EROD assay could not be shown, because of high individual variation and low sensitivity regarding substance concentration, the fluorescence tracking of benzo[a]pyrene indicates that food-borne microplastic-associated POPs may actually desorb in the intestine of fish and are thus transferred to the intestinal epithelium and liver. Environ Toxicol Chem 2016;35:1656–1666. # 2016 SETAC

Low level metal contaminations are a prevalent issue with often unknown consequences for health and the environment. Effect-based, multifactorial test systems with zebrafish embryos to assess in particular developmental toxicity are beneficial but rarely used in this context. We therefore exposed wild-type embryos to the metals copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) for 72 h to determine lethal as well as sublethal morphological effects. Motor neuron damage was investigated by immunofluorescence staining of primary motor neurons (PMNs) and secondary motor neurons (SMNs). In vivo stainings using the vital dye DASPEI were used to quantify neuromast development and damage. The consequences of metal toxicity were also assessed functionally, by testing fish behavior following tactile stimulation. The median effective concentration (EC50) values for morphological effects 72 h post fertilization (hpf) were 14.6 mg/L for cadmium and 0.018 mg/L for copper, whereas embryos exposed up to 45.8 mg/L cobalt showed no morphological effects. All three metals caused a concentration-dependent reduction in the numbers of normal PMNs and SMNs, and in the fluorescence intensity of neuromasts. The results for motor neuron damage and behavior were coincident for all three metals. Even the lowest metal concentrations (cadmium 2mg/L, copper 0.01 mg/L and cobalt 0.8 mg/L) resulted in neuromast damage. The results demonstrate that the neuromast cells were more sensitive to metal exposure than morphological traits or the response to tactile stimulation and motor neuron damage.

The fate assessment of nanomaterials in municipal sewage treatment plants (STP) is a crucial step for their environmental risk assessment and may be assessed by monitoring full scale STP, dosage to medium scale pilot STP or by laboratory testing. For regulatory purposes preferably standardised test protocols such as the OECD guidelines for testing of chemicals should be used. However, these test protocols have not yet been specifically designed for nanoparticles. Therefore, the fate and behavior of a TiO2 nanomaterial (P25, average hydrodynamic diameter <250 nm) was investigated in laboratory sewage treatment plants according to the OECD Guideline for the Testing of Chemicals 303 A. It is concluded that this guideline is applicable for the testing of nanomaterials if modifications regarding the dosage, nitrifying conditions, and a characterisation of the nanoparticles in the effluent are applied. A compilation of the cumulative mass balance by comparison of the total dosage added with the amount in the outflow and in the activated sludge is recommended. In this study, the majority of the TiO2 nanomaterial (>95%) was retained in the sewage sludge and only 3–4% was found in the effluent. No effect of the TiO2 nanomaterials on the biodegradation or nitrification was observed.

2,2-bis(chlorophenyl)-1,1,1-trichloroethane (DDT) metabolites, other than those routinely measured [i.e., 2,2-bis(chlorophenyl)-1,1-dichloroethylene (DDE) and 2,2-bis(chlorophenyl)-1,1-dichloroethane (DDD)], have recently been detected in elevated concentrations not only in the surface water of Teltow Canal, Berlin, but also in sediment samples from Elbe tributaries (e.g., Mulde and Havel/Spree). This was paralleled by recent reports that multiple other metabolites could emerge from the degradation of parent DDT by naturally occurring organisms or by interaction with some heavy metals. Nevertheless, only very few data on the biological activities of these metabolites are available to date. The objective of this communication is to evaluate, for the first time, the cytotoxicity, dioxin-like activity, and estrogenicity of the least-studied DDT metabolites.

TiO2 coated surfaces are able to generate highly reactive oxidizing species under mild UV-A light exposure in the presence of water and oxygen. We have demonstrated that these radicals are sufficient to eliminate different pathogenic bacteria, by breaking their cell walls. The photocatalytic activity of surfaces coated with titanium dioxide offers therefore an alternative possibility of disinfection. However, restriction of bacterial growth does not protect surfaces from bacterial derived contaminations, such as endotoxins. Lipopolysaccharides (LPS) and Ribonuclease A (RNAse A) represent the two most abundant contaminations, causing severe problems in biomedical and immunological research as well as in the pharmaceutical industry. Due to their high stability, complete removal of these contaminants is technically challenging. Using irradiated TiO2 coated glass plates, RNAse A and LPS containing contaminations could be completely inactivated. By establishing highly sensitive immuno-based assays, destruction of the contaminants was quantified and shown to be independent of the initial concentrations, following a zero-order reaction. Exposure for 96h resulted in a reduction of 11ng of LPS and 7 units of RNase A cm−2 surface. These amounts are comparable to contamination levels found under standard working conditions. Titanium dioxide coatings provide therefore a powerful tool for auto-disinfection and self-cleaning of surfaces.

Lungs are permanently and simultaneously challenged by airborne microorganisms and airborne pollutants. Temporal increase of airborne particulate matter (APM), a potential carrier for extractable organic matter (EOM), degrades the situation of pulmonary patients. The Ah receptor (AhR) has been described as an important factor influencing the immunological challenge by viral infections. Molecular mechanisms underlying epidemiological observations are not well understood. Cytokine secretion (IL-6, IL-8, and TGF-β) from human bronchial epithelial cells (Beas2B) was determined as an indicator for immune responses upon co-stimulation with an artificial analog of viral dsRNA [polyinosinic/polycytidylic acid, (PIC)] and EOM of Standard Reference Material 1649a (SRM). Since polycyclic aromatic hydrocarbons are major components of APM usually acting via the AhR, particular focus was on AhR involvement.

Goal, Scope and Background. Situated in the transboundary belt between Montenegro and Albania, Lake Skadar is the largest freshwater reservoir in Southeastern Europe. Because of the wide range of endemic, rare or endangered plant and animal species it supports, Lake Skadar and its extensive adjacent wetlands are internationally recognised as a site of significance and importance (Ramsar site). Within the last 10 to 20 years, Lake Skadar was exposed to intensive pollution. For the assessment of the ecotoxic load of the sediments sampled in Lake Skadar, a triad approach was recently applied. Overall, a complex spectrum of ecotoxic loads was elucidated. The aim of the present study was to use plant-based bioassays for assessing the sediment quality of Lake Skadar in order to facilitate and complement the triad test battery. The newly developed sediment contact test with Myriophyllum aquaticum and the aquatic growth inhibition test with Lemna minor were applied to native sediments and pore water, respectively, allowing the investigation of different toxicity-effects caused by particle-bound pollutants as well as pollutants in the interstitial water. This investigation is the first application of the novel sediment contact test with Myriophyllum aquaticum to lake sediments.Methods. Sediment, samples were taken from nine selected sites at Lake Skadar and investigated by the sediment contact assay with Myriophyllum aquaticum. The pore water was extracted from these sediment samples to be analysed in the aquatic growth inhibition test with Lemna minor. The results of the sediment contact tests were compared with each other and with those of the aquatic growth inhibition test.Results and Discussion. Both applied macrophyte biotests revealed distinct changes in the growth behaviour of the two macrophytes subsequent to the exposure to the investigated natural sediments of Lake Skadar. The Myriophyllum sediment contact test revealed significant toxicity in the sediment samples from Radus and Kamenik, whereas the aquatic Lemna test showed inhibition effects for the samples from Sterbeq, Plavnica and Kamice. Data obtained with the newly developed Danio rerio contact test and the Arthrobacter globiformis contact test confirmed the Myriophyllum results.Analyses of the heavy metal content in the sediments revealed low or moderate contamination levels. Correlation analyses between the content of heavy metals in the sediments and growth inhibition of Myriophyllum aquaticum showed a significant correlation between Cr concentrations and growth inhibition. Comparable findings are available for a German river system. In contrast, no significant correlation between inhibition rates and concentration of metals could be observed with Lemna minor.Conclusions. It was shown that the newly developed sediment contact test with Myriophyllum aquaticum is applicable to lake sediments. In both the sediment contact test with Myriophyllum aquaticum on whole sediments and the aquatic growth inhibition test with Lemna minor on pore water, plant growth was influenced by the natural sediments and its components. Therefore, both test systems were found to be suitable for the detection of phytotoxic effects upon exposure to sediments. Myriophyllum aquaticum as test organism of the contact test grows directly in the sediment without an additional water-layer. Thus, it is able to detect toxicity caused by particle-bound phytotoxic substances as well as pore water-related contamination, while the floating Lemna minor can only detect effects emanating from pore water. Significant differences of the results were observed between these two test systems and, accordingly, the two different exposure scenarios. Hence, none of the tests can replace the other one and, as a consequence, both should be included into a test battery for the assessment of sediment toxicity.Recommendations and Perspectives. Both plant assays were shown to be reliable tools for the evaluation of the eco-toxicological risk potentials of pore water and solid-phase sediment. They should become a complement to the standardised test battery generally used for comprehensive hazard assessment.

Goals, Scope and Background Fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River between Sigmaringen and Ulm, despite intensive and continuous stocking and improvement of water quality since the 1970s. Similar problems have been reported for other rivers, e.g. in Switzerland, Great Britain, the United States and Canada. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube River, sediment, suspended matter and waste water samples from sewage treatment plants were collected at selected locations and analyzed in a bioanalytical approach using a battery of bioassays. The results of this pilot study will be used to decide if a comprehensive weight-of-evidence study is needed. Methods Freeze-dried sediments and suspended particulate matters were extracted with acetone in a Soxhlet apparatus. Organic pollutants from sewage water were concentrated using XAD-resins. In order to investigate the ecotoxicological burden, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1 cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenase assay (toxicity to bacteria), (4) yeast estrogen screen assay (endocrine disruption), (5) fish egg assay with the zebrafish (Danio rerio; embryo toxicity) and (6) Ames test with TA98 (mutagenicity). Results and Discussion The results of the in vitro tests elucidated a considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic, embryotoxic and estrogenic burden in the upper Danube River, although with a very inhomogeneous distribution of effects. The samples taken from Riedlingen, for example, induced low embryo toxicity, but the second highest 17β-estradiol equivalent concentration (1.8 ng/L). Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, with clear-cut dose-response relationships for the embryotoxic effects of contaminated sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that – at least for fish eggs – the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locations along the rivers Rhine and Neckar demonstrated similar or even higher ranges of ecotoxicological burdens in the Danube River. Conclusions The complex pattern of ecotoxicological effects caused by environmental samples from the Danube River, when assessed in an in vitro biotest battery using both acute and more specific endpoints, showed that integration of different endpoints is essential for appropriate hazard assessment. Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper Danube River. However, based on the results of this pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline. Recommendations and Perspective . In order to confirm the ecological relevance of the in vitro results for the situation in the field and especially for the decline of the grayling and other fishes, further integrated investigations are required. For linking the weight of evidence obtained by in vitro assays and fish population investigations, the application of additional, more specific biomarkers (e.g. vitellogenin induction, EROD and micronucleus assay) has been initiated in fish taken from the field as well as in situ investigations.

Goal, Scope and Background. Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations.Methods. Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction.Results and Discussion. The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane.Conclusion. Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations.Recommendation and Perspective. Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.

GOAL, SCOPE AND BACKGROUND:Some anthropogenic pollutants posses the capacity to disrupt endogenous control of developmental and reproductive processes in aquatic biota by activating estrogen receptors. Many anthropogenic estrogen receptor agonists (ERAs) are hydrophobic and will therefore readily partition into the abiotic organic carbon phases present in natural waters. This partitioning process effectively reduces the proportion of ERAs readily available for bioconcentration by aquatic biota. Results from some studies have suggested that for many aquatic species, bioconcentration of the freely-dissolved fraction may be the principal route of uptake for hydrophobic pollutants with logarithm n-octanol/water partition coefficient (log Kow) values less than approximately 6.0, which includes the majority of known anthropogenic ERAs. The detection and identification of freely-dissolved readily bioconcentratable ERAs is therefore an important aspect of exposure and risk assessment. However, most studies use conventional techniques to sample total ERA concentrations and in doing so frequently fail to account for bioconcentration of the freely-dissolved fraction. The aim of the current study was to couple the biomimetic sampling properties of semipermeable membrane devices (SPMDs) to a bioassay-directed chemical analysis (BDCA) scheme for the detection and identification of readily bioconcentratable ERAs in surface waters.METHODS:SPMDs were constructed and deployed at a number of sites in Germany and the UK. Following the dialytic recovery of target compounds and size exclusion chromatographic cleanup, SPMD samples were fractionated using a reverse-phase HPLC method calibrated to provide an estimation of target analyte log Kow. A portion of each HPLC fraction was then subjected to the yeast estrogen screen (YES) to determine estrogenic potential. Results were plotted in the form of 'estrograms' which displayed profiles of estrogenic potential as a function of HPLC retention time (i.e. hydrophobicity) for each of the samples. Where significant activity was elicited in the YES, the remaining portion of the respective active fraction was subjected to GC-MS analysis in an attempt to identify the ERAs present.RESULTS AND DISCUSSION:Estrograms from each of the field samples showed that readily bioconcentratable ERAs were present at each of the sampling sites. Estimated log Kow values for the various active fractions ranged from 1.92 to 8.63. For some samples, estrogenic potential was associated with a relatively narrow range of log Kow values whilst in others estrogenic potential was more widely distributed across the respective estrograms. ERAs identified in active fractions included some benzophenones, various nonylphenol isomers, benzyl butyl phthalate, dehydroabietic acid, sitosterol, 3-(4-methylbenzylidine)camphor (4-MBC) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (AHTN). Other tentatively identified compounds which may have contributed to the observed YES activity included various polycyclic aromatic hydrocarbons (PAHs) and their alkylated derivatives, methylated benzylphenols, various alkyl-phenols and dialkylphenols. However, potential ERAs present in some active fractions remain unidentified.CONCLUSIONS AND OUTLOOK:Our results show that SPMD-YES-based BDCA can be used to detect and identify readily bioconcentratable ERAs in surface waters. As such, this biomimetic approach can be employed as an alternative to conventional methodologies to provide investigators with a more environmentally relevant insight into the distribution and identity of ERAs in surface waters. The use of alternative bioassays also has the potential to expand SPMD-based BDCA to include a wide range of toxicological endpoints. Improvements to the analytical methodology used to identify ERAs or other target compounds in active fractions in the current study could greatly enhance the applicability of the methodology to risk assessment and monitoring programmes.