Assessing the impact of imidacloprid, glyphosate, and their mixtures on multiple biomarkers in Corbicula largillierti.

Pesticide mixtures are frequently utilized in agriculture, yet their cumulative effects on aquatic organisms remain poorly understood. Aquatic animals can be effective bioindicators and invasive bivalves, owing to their widespread distribution, provide an opportunity to assess these impacts. Glyphosate and imidacloprid, among the most prevalent pesticides globally, are frequently detected in freshwater systems in South America. This study aims to understand the cumulative effects of pesticide mixtures on aquatic organisms, using invasive Corbicula largillierti clams from a natural stream in northwestern Argentina. We conducted 48-hour exposure experiments using two concentrations of imidacloprid (20 and 200 µg L-1 a.i), two concentrations of glyphosate (0.3 and 3 mg L-1 a.i), and two combinations of these pesticides (both at low and high concentrations, respectively), simulating the direct contamination of both pesticides based on their agronomic recipe and observed values in Argentine aquatic environments. Clam metabolism was assessed through the examination of multiple oxidative stress parameters and measuring oxygen consumption rate as a proxy for standard metabolic rate (SMR). Our findings revealed that imidacloprid has a more pronounced effect compared to glyphosate. Imidacloprid significantly decreased clam SMR and cellular levels of reduced glutathione (GSH). However, when both pesticides were present, also cellular glycogen and thiobarbituric acid-reactive substances (TBARS) were affected. Proteins and glutathione S-Transferase (GST) activity were unaffected by either pesticide or their mixture at the assayed concentrations, highlighting the need to test several stress parameters to detect toxicological impacts. Our results indicated additive effects of imidacloprid and glyphosate across all measured parameters. The combination of multiple physiological and cytological biomarkers in invasive bivalves offers significant potential to enhance biomonitoring sensitivity and obtain insights into the origins and cellular mechanisms of chemical impacts. These studies can improve pollution regulatory policies and pesticide management.

Ecotoxicological evaluation of surface waters in Northern Namibia.

The increasing pressure on freshwater systems due to intensive anthropogenic use is a big challenge in central-northern Namibia and its catchment areas, the Kunene and the Kavango Rivers, and the Cuvelai-Etosha Basin, that provide water for more than 1 million people. So far, there is no comprehensive knowledge about the ecological status and only few knowledge about the water quality. Therefore, it is crucial to learn about the state of the ecosystem and the ecological effects of pollutants to ensure the safe use of these resources. The surface waters of the three systems were sampled, and three bioassays were applied on three trophic levels: algae, daphnia, and zebrafish embryos. Additionally, in vitro assays were performed to analyze mutagenicity (Ames fluctuation), dioxin-like potential (micro-EROD), and estrogenicity (YES) by mechanism-specific effects. The results show that acute toxicity to fish embryos and daphnia has mainly been detected at all sites in the three catchment areas. The systems differ significantly from each other, with the sites in the Iishana system showing the highest acute toxicity. At the cellular level, only weak effects were identified, although these were stronger in the Iishana system than in the two perennial systems. Algae growth was not inhibited, and no cytotoxic effects could be detected in any of the samples. Mutagenic effects and an estrogenic potential were detected at three sites in the Iishana system. These findings are critical in water resource management as the effects can adversely impact the health of aquatic ecosystems and the organisms within them.

Status quo report on wastewater treatment plant, receiving water's biocoenosis and quality as basis for evaluation of large-scale ozonation process.

The project DemO3AC (demonstration of large-scale wastewater ozonation at the Aachen-Soers wastewater treatment plant, Germany) of the Eifel-Rur Waterboard contains the construction of a large-scale ozonation plant for advanced treatment of the entire 25 million m³/yr of wastewater passing through its largest wastewater treatment plant (WWTP). In dry periods, up to 70% of the receiving water consists of treated wastewater. Thus, it is expected that effects of ozonation on downstream water biocoenosis will become observable. Extensive monitoring of receiving water and the WWTP shows a severe pollution with micropollutants (already prior to WWTP inlet). (Eco-)Toxicological investigations showed increased toxicity at the inlet of the WWTP for all assays. However, endocrine-disrupting potential was also present at other sampling points at the WWTP and in the river and could not be eliminated sufficiently by the WWTP. Total cell counts at the WWTP are slightly below average. Investigations of antibiotic resistances show no increase after the WWTP outlet in the river. However, cells carrying antibiotic-resistant genes seem to be more stress resistant in general. Comparing investigations after implementation of ozonation should lead to an approximation of the correlation between micropollutants and water quality/biocoenosis and the effects that ozonation has on this matter.

An ecotoxicological view on neurotoxicity assessment.

The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.

Fipronil and two of its transformation products in water and European eel from the river Elbe.

Fipronil is an insecticide which, based on its mode of action, is intended to be predominantly toxic towards insects. Fipronil bioaccumulates and some of its transformation products were reported to be similar or even more stable in the environment and to show an enhanced toxicity against non-target organisms compared to the parent compound. The current study investigated the occurrence of Fipronil and two of its transformation products, Fipronil-desulfinyl and Fipronil-sulfone, in water as well as muscle and liver samples of eels from the river Elbe (Germany). In water samples total concentrations of FIP, FIP-d and FIP-s ranged between 0.5-1.6ngL(-1) with FIP being the main component in all water samples followed by FIP-s and FIP-d. In contrast, FIP-s was the main component in muscle and liver tissues of eels with concentrations of 4.05±3.73ngg(-1) ww and 19.91±9.96ngg(-1) ww, respectively. Using a physiologically based toxicokinetic (PBTK) model for moderately hydrophobic organic chemicals, the different distributions of FIP, FIP-d and FIP-s in water and related tissue samples could be attributed to metabolic processes of eels. The measured concentrations in water of all analytes and their fractional distribution did not reflect the assumed seasonal application of FIP and it seems that the water was constantly contaminated with FIP, FIP-d and FIP-s.

Bioaccumulation and molecular effects of sediment-bound metals in zebrafish embryos.

Predicting the bioavailability and effects of metals in sediments is of major concern in context with sediment risk assessment. This study aimed to investigate the bioavailability and molecular effects of metals spiked into riverine sediments to zebrafish (Danio rerio) embryos. Embryos were exposed to a natural and an artificial sediment spiked with cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) individually or as a mixture at concentrations ranging from 150 to 3000 mg/kg dry weight (dw) over 48 h, and uptake of metals was determined. Furthermore, transcript abundances of the metallothioneins MT1 and MT2, the metal-responsive element-binding transcription factor (MTF) and the genes sod1, hsp70 and hsp90α1 were measured as indicators of metal-induced or general cellular stress. D. rerio embryos accumulated metals from sediments at concentrations up to 100 times greater than those spiked to the sediment with the greatest bioaccumulation factor (BAF) for Cu from artificial sediment (275.4 ± 41.9 (SD)). Embryos accumulated greater concentrations of all metals from artificial than from natural sediment, and accumulation was greater when embryos were exposed to individual metals than when they were exposed to the mixture. Exposure of embryos to Zn or the mixture exhibited up to 30-fold greater transcript abundances of MT1, MT2 and hsp70 compared to controls which is related to significant uptake of Zn from the sediment. Further changes in transcript abundances could not be related to a significant uptake of metals from sediments. These studies reveal that metals from spiked sediments are bioavailable to D. rerio embryos directly exposed to sediments and that the induction of specific genes can be used as biomarkers for the exposure of early life stages of zebrafish to metal-contaminated sediments.

Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy.

The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose-response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet assay and molecular biomarker analysis should prospectively be considered to assess the ecotoxicological potential of sediments allowing for a comprehensive hazard ranking. In order to elucidate mode of action, novel techniques such as flow cytometry have been adopted and proved to be valuable tools for advanced risk assessment and management.

Ecotoxicological effect characterisation of widely used organic UV filters.

Chemical UV filters are used in sun protection and personal care products in order to protect consumers from skin cancer induced by ultraviolet (UV) radiation. The present study aims to evaluate the effects of three common UV filters butyl-methoxydibenzoylmethane (B-MDM) ethylhexyl-methoxycinnamate (EHMC) and octocrylene (OCR) on aquatic organism, focussing particularly on infaunal and epibentic invertebrates (Chironomus riparius, Lumbriculus variegatus, Melanoides tuberculata and Potamopyrgus antipodarum). Due to their life habits, these organism are especially affected by lipophilic substances. Additionally, two direct sediment contact assays utilising zebra fish (Danio rerio) embryos and bacteria (Arthrobacter globiformis) were conducted. EHMC caused a toxic effect on reproduction in both snails with lowest observed effect concentrations (LOEC) of 0.4 mg/kg (Potamopyrgus antipodarum) and 10 mg/kg (Melanoides tuberculata). At high concentrations sublethal effects could be observed for D. rerio after exposure to EHMC (NOEC 100 mg/kg). B-MDM and OCR showed no effects on any of the tested organism.

International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

The Yangtze-Hydro Project: a Chinese-German environmental program.

Water of good quality is one of the basic needs of human life. Worldwide, great efforts are being undertaken for an assured water supply. In this respect, one of the largest water technology projects worldwide is the Yangtze Three Gorges Dam in China. There is a need for extensive scientific and technical understanding of the challenges arising from this large hydrological engineering project. German and Chinese groups from various scientific fields are collaborating to provide knowledge for the sustainable management of the reservoir. In this project description, the Yangtze Three Gorges Dam Project, its goals and challenges, are described in brief, and the contributions of the German research projects are presented.

Developmental toxicity and endocrine disrupting potency of 4-azapyrene, benzo[b]fluorene and retene in the zebrafish Danio rerio.

This study examined the developmental toxicity of the polycyclic aromatic hydrocarbons (PAHs) 11H-benzo(b)fluorene (BBF) and 4-azapyrene (AP) in comparison to the known teratogen retene. Developmental toxicity assays were performed in zebrafish embryos exposed for 120 h. BBF and retene induced a similar dioxin-like phenotype, whereas AP showed distinct effects, particularly craniofacial malformations. Microarray analysis revealed that for BBF and retene, drug metabolism pathways were induced, which were confirmed by subsequent studies of cyp1a gene expression. For AP, microarray analysis revealed the regulation of genes involved in retinoid metabolism and hematological functions. Studies with a panel of CALUX(®) bioassays to screen for endocrine disrupting activity of the compounds also revealed novel antagonistic effects of BBF and retene on androgen and progesterone receptors. Classification analysis revealed distinct gene expression profiles for both individual and combined PAH exposure. This study highlights the potential health risk of non priority PAHs.

Estrogen receptor mediated activity in bankside groundwater, with flood suspended particulate matter and floodplain soil - an approach combining tracer substance, bioassay and target analysis.

Bankside groundwater is widely used as drinking water resource and, therefore, contamination has to be avoided. In the European Union groundwater protection is explicit subject to Water Framework Directive. While groundwater pollution may originate from different sources, this study investigated on impacts via flood events. Groundwater was sampled with increasing distance to the river Rhine near Karlsruhe, Germany. Samples were HPLC-MS-MS analyzed for the river contaminant carbamazepine to indicate river water infiltration, giving permanent presence in 250 m distance to the river (14-47 µg L⁻¹). Following a flood event, concentrations of about 16-20 µg L⁻¹ could also be detected in a distance of 750 m to the river. Furthermore, estrogenic activity as determined with the Yeast Estrogen Screen assay was determined to increase up to a 17ß-ethinylestradiol equivalent concentration (E-EQ)=2.9 ng L⁻¹ near the river, while activity was initially measured following the flood with up to E-EQ=2.6 ng L⁻¹ in 750 m distance. Detections were delayed with increasing distance to the river indicating river water expansion into the aquifer. Flood suspended matter and floodplain soil were fractionated and analyzed for estrogenic activity in parallel giving up to 1.4 ng g⁻¹ and up to 0.7 ng g⁻¹, respectively. Target analysis focusing on known estrogenic active substances only explained < 1% of measured activities. Nevertheless, river water infiltration was shown deep into bankside groundwater, thus, impacting groundwater quality. Therefore, flood events have to be in the focus when aiming for groundwater and drinking water protection as well as for implementation of Water Framework Directive.

Variability of sediment-contact tests in freshwater sediments with low-level anthropogenic contamination--determination of toxicity thresholds.

Freshwater sediments with low levels of anthropogenic contamination and a broad range of geochemical properties were investigated using various sediment-contact tests in order to study the natural variability and to define toxicity thresholds for the various toxicity endpoints. Tests were performed with bacteria (Arthrobacter globiformis), yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), higher plants (Myriophyllum aquaticum), and the eggs of zebrafish (Danio rerio). The variability in the response of some of the contact tests could be explained by particle size distribution and organic content. Only for two native sediments could a pollution effect not be excluded. Based on the minimal detectable difference (MDD) and the maximal tolerable inhibition (MTI), toxicity thresholds (% inhibition compared to the control) were derived for each toxicity parameter: >20% for plant growth and fish-egg survival, >25% for nematode growth and oligochaete reproduction, >50% for nematode reproduction and >60% for bacterial enzyme activity.

Effect-directed analysis of Ah receptor-mediated activities caused by PAHs in suspended particulate matter sampled in flood events.

Suspended particulate matter (SPM) sampled during a flood event in the year 2004 at the rivers Neckar and Rhine (Southwest Germany) was assessed for aryl hydrocarbon receptor (AhR)-mediated activities using EROD induction in the rainbow trout liver cell line RTL-W1. All EROD inductions were normalized to the positive control TCDD and given as bio-TEQ values. Since all samples indicated elevated AhR-mediated toxicities, an effect-directed analysis (EDA) was applied to identify substances causing the effects. In three primary fractions (F1 to F3) non-polar aliphatics, non-polar aromatic substances and more polar substances were separated. Fraction F2, co-eluting with non-polar polyaromatic substances (PACs) including polycyclic aromatic hydrocarbons (PAHs) gave highest AhR-agonistic effects and, thus, were sub-fractionated into seven secondary fractions (F2-1 to F2-7). Fraction F2-1, co-eluting with PCBs and PCDD/Fs, did not cause AhR-agonist activities. F2-2 to F2-4 containing PACs of less than 16 aromatic C-atoms produced minor activities. Highest inductions were detected with fraction F2-5 to F2-7, containing substances of more than 16 aromatic C-atoms (bio-TEQs up to approximately 4500 pg/g). Concentrations and relative potencies (REPs) of priority EPA-PAHs allowed the calculation of chemical toxicity equivalent concentrations (chem-TEQ values). Based on the chem-TEQs, EPA-PAHs explained between 5 and 58% of crude extract bio-TEQs from both rivers. Whereas fractions F2-1 to F2-4 indicated no biological activities, EPA-PAHs in fraction F2-5 to F2-7 accounted for 2 to 137% of AhR-related activities.

Assessing contamination levels of Laguna Lake sediments (Philippines) using a contact assay with zebrafish (Danio rerio) embryos.

The present study investigated the suitability of a sediment contact assay using zebrafish (Danio rerio) embryos to evaluate the degree of lake sediment contamination. As endpoints, developmental parameters (mortality, abnormality, heart rate, and hatching rate) as well as stress protein responses (hsp 70 levels) in the developing embryos were recorded during a 96-h exposure. Fertilized zebrafish eggs were exposed to both the whole as well as organic extract concentrations prepared from collected sediments from 5 sites along Laguna Lake, Philippines. Compared to whole sediment exposure, more severe embryotoxic and teratogenic responses were elicited in embryos exposed to organic extracts. However, since whole sediment-exposed embryos also revealed significant developmental defects, this exposure phase served as the more realistic exposure scenario in our study. Weak to strong upregulation of hsp 70 levels was also registered among embryos exposed to both whole sediments and organic extracts. The observed embryotoxic and proteotoxic responses by zebrafish embryos to Laguna Lake sediment exposures were discussed in relation to the analyzed contaminants in the sediments (heavy metals, PAHs (perylene), solvent). Overall, the present study points out that the sediment contact assay with zebrafish embryos offers a practicable and highly sensitive bioassay for the general assessment of sediment toxicity.

Sequential fractionation procedure for the identification of potentially cytochrome P4501A-inducing compounds.

A multistep fractionation procedure for the separation of nonpolar aromatic compounds with respect to cytochrome P4501A induction is presented. Normal-phase HPLC on nitrophenylpropyl silica and cyanopropyl silica was tested for group-specific separation as a first fractionation step. Subsequent individual compound-specific PAH fractionation was done by means of reversed-phase HPLC. Electron-donor-acceptor HPLC and size-exclusion chromatography were applied to separate PAHs, PCBs, PCNs and PCDD/Fs according to their number of aromatic carbon atoms, their hydrophobicity, their degree of chlorination, their planarity and their molecular size. The method was validated for complex environmental mixtures on the basis of two sediment extracts.

Measurement of vitellogenin-mRNA expression in primary cultures of rainbow trout hepatocytes in a non-radioactive dot blot/RNAse protection-assay.

The induction of vitellogenin synthesis both in vivo and in vitro has proven to be a reliable biomarker for assessing the estrogenic activity of individual substances and the more complex effluents of sewage treatment plants. However, due to the requirement of radioactively labelled nucleotides, the measurement of vitellogenin-mRNA has not been widely used in routine testing--even though this technique promises elevated sensitivity. In order to develop a practicable, reliable and cost-effective bioassay suitable for routine testing, a combined dot-blot/RNAse protection assay, utilising digoxigenin-labelled cRNA transcripts of plasmid psg5Vg1.1 was used for the quantification of vitellogenin-mRNA in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes. By re-cloning the Vg1.1 insert into a pGemZf7(-)-vector, the sense-transcript of Vg1.1 was utilized as a standard for the quantification of vitellogenin-mRNA concentrations. Male rainbow trout hepatocytes were cultured as monolayers in pure M199 medium. The addition of serum supplements did not result in increased expression of vitellogenin-mRNA following 17 beta-estradiol administration. This indicates that for this assay no supplementation of the culture medium is necessary. After addition of 17 beta-estradiol, hepatocytes exhibited an exponential time-dependent expression of vitellogenin-mRNA over a period of 144 h. The dot blot system was sufficiently sensitive to detect vitellogenin-mRNA following addition of 1 microM 17 beta-estradiol after 6 h of incubation. However, the amount of vitellogenin-mRNA expressed was found to be a function of both incubation time and inducer concentration. Prolonged incubation times were therefore required to enhance the sensitivity of the system. After a 96-h incubation, detection limits for 17 beta-estradiol were between 100 pM and 1 nM. Vitellogenin-mRNA could not be detected in untreated hepatocytes. The vitellogenin-mRNA dot blot/RNAse protection assay was further used as a tool for assessing the estrogenic potential of the xenoestrogens nonylphenol and bisphenol A, which exhibited estrogenic activities approximately 2000-fold less than the natural inducer 17 beta-estradiol. The vitellogenin-mRNA response to 17 alpha-ethinylestradiol reached maximum efficacy down to the lowest tested concentration of 10(-9) M. The assay also successfully identified estrogenic activity in selected waste water samples.