Application of biological early warning systems in wastewater treatment plants: Introducing a promising approach to monitor changing wastewater composition.

Wastewater treatment plants (WWTPs) are a major source of micropollutants to surface waters. Currently, their chemical or biological monitoring is realized by using grab or composite samples, which provides only snapshots of the current wastewater composition. Especially in WWTPs with industrial input, the wastewater composition can be highly variable and a continuous assessment would be advantageous, but very labor and cost intensive. A promising concept are automated real-time biological early warning systems (BEWS), where living organisms are constantly exposed to the water and an alarm is triggered if the organism's responses exceed a harmful threshold of acute toxicity. Currently, BEWS are established for drinking water and surface water but are seldom applied to monitor wastewater. This study demonstrates that a battery of BEWS using algae (Chlorella vulgaris in the Algae Toximeter, bbe Moldaenke), water flea (Daphnia magna in the DaphTox II, bbe Moldaenke) and gammarids (Gammarus pulex in the Sensaguard, REMONDIS Aqua) can be adapted for wastewater surveillance. For continuous low-maintenance operation, a back-washable membrane filtration system is indispensable for adequate preparation of treated wastewater. Only minor deviations in the reaction of the organisms towards treated and filtered wastewater compared to surface waters were detected. After spiking treated wastewater with two concentrations of the model compounds diuron, chlorpyrifos methyl, and sertraline, the organisms in the different BEWS showed clear responses depending on the respective compound, concentration and mode of action. Immediate effects on photosynthetic activity of algae were detected for diuron exposure, and strong behavioral changes in water flea and gammarids after exposure to chlorpyrifos methyl or sertraline were observed, which triggered automated alarms. Different types of data analysis were applied to extract more information out of the specific behavioral traits, than only provided by the vendors algorithms. To investigate, whether behavioral movement changes can be linked to impact other endpoints, the effects on feeding activity of G. pulex were evaluated and results indicated significant differences between the exposures. Overall, these findings provide an important basis indicating that BEWS have the potential to act as alarm systems for pollution events in the wastewater sector.

Evaluation of a full-scale wastewater treatment plant with ozonation and different post-treatments using a broad range of in vitro and in vivo bioassays.

Micropollutants present in the effluent of wastewater treatment plants (WWTPs) after biological treatment are largely eliminated by effective advanced technologies such as ozonation. Discharge of contaminants into freshwater ecosystems can thus be minimized, while simultaneously protecting drinking water resources. However, ozonation can lead to reactive and potentially toxic transformation products. To remove these, the Swiss Federal Office for the Environment recommends additional "post-treatment" of ozonated WWTP effluent using sand filtration, but other treatments may be similarly effective. In this study, 48 h composite wastewater samples were collected before and after full-scale ozonation, and after post-treatments (full-scale sand filtration, pilot-scale fresh and pre-loaded granular activated carbon, and fixed and moving beds). Ecotoxicological tests were performed to quantify the changes in water quality following different treatment steps. These included standard in vitro bioassays for the detection of endocrine, genotoxic and mutagenic effects, as well as toxicity to green algae and bacteria, and flow-through in vivo bioassays using oligochaetes and early life stages of rainbow trout. Results show that ozonation reduced a number of ecotoxicological effects of biologically treated wastewater by 66 - 93%: It improved growth and photosynthesis of green algae, decreased toxicity to luminescent bacteria, reduced concentrations of hormonally active contaminants and significantly changed expression of biomarker genes in rainbow trout liver. Bioassay results showed that ozonation did not produce problematic levels of reaction products overall. Small increases in toxicity observed in a few samples were reduced or eliminated by post-treatments. However, only relatively fresh granular activated carbon (analyzed at 13,000 - 20,000 bed volumes) significantly reduced effects additionally (by up to 66%) compared to ozonation alone. Inhibition of algal photosynthesis, rainbow trout liver histopathology and biomarker gene expression proved to be sufficiently sensitive endpoints to detect the change in water quality achieved by post-treatment.

Effects of treated wastewater on the ecotoxicity of small streams - Unravelling the contribution of chemicals causing effects.

Wastewater treatment plant effluents are important point sources of micropollutants. To assess how the discharge of treated wastewater affects the ecotoxicity of small to medium-sized streams we collected water samples up- and downstream of 24 wastewater treatment plants across the Swiss Plateau and the Jura regions of Switzerland. We investigated estrogenicity, inhibition of algal photosynthetic activity (photosystem II, PSII) and growth, and acetylcholinesterase (AChE) inhibition. At four sites, we measured feeding activity of amphipods (Gammarus fossarum) in situ as well as water flea (Ceriodaphnia dubia) reproduction in water samples. Ecotoxicological endpoints were compared with results from analyses of general water quality parameters as well as a target screening of a wide range of organic micropollutants with a focus on pesticides and pharmaceuticals using liquid chromatography high-resolution tandem mass spectrometry. Measured ecotoxicological effects in stream water varied substantially among sites: 17ß-estradiol equivalent concentrations (EEQbio, indicating the degree of estrogenicity) were relatively low and ranged from 0.04 to 0.85 ng/L, never exceeding a proposed effect-based trigger (EBT) value of 0.88 ng/L. Diuron equivalent (DEQbio) concentrations (indicating the degree of photosystem II inhibition in algae) ranged from 2.4 to 1576 ng/L and exceeded the EBT value (70 ng/L) in one third of the rivers studied, sometimes even upstream of the WWTP. Parathion equivalent (PtEQbio) concentrations (indicating the degree of AChE inhibition) reached relatively high values (37 to 1278 ng/L) mostly exceeding the corresponding EBT (196 ng/L PtEQbio). Decreased feeding activity by amphipods or decreased water flea reproduction downstream compared to the upstream site was observed at one of four investigated sites only. Results of the combined algae assay (PSII inhibition) correlated best with results of chemical analysis for PSII inhibiting herbicides. Estrogenicity was partly and AChE inhibition strongly underestimated based on measured steroidal estrogens respectively organophosphate and carbamate insecticides. An impact of dissolved organic carbon on results of the AChE inhibition assay was obvious. For this assay more work is required to further explore the missing correlation of bioassay data with chemical analytical data. Overall, the discharge of WWTP effluent led to increased estrogenicity, PSII and AChE inhibition downstream, irrespective of upstream land use.

Wastewater alters feeding rate but not vitellogenin level of Gammarus fossarum (Amphipoda).

Wastewater treatment plant (WWTP) effluents release complex mixtures of organic and inorganic micropollutants, including endocrine disrupting compounds, into receiving water bodies. These substances may cause adverse effects in aquatic communities as well as in ecosystem functions they provide. The aim of this study was to determine the potential impact of secondary treated wastewater released into a small Swiss stream on leaf litter decomposition based on feeding rates of the amphipod shredder Gammarus fossarum measured in situ. Additionally, endocrine disrupting effects downstream of the WWTP were investigated by measuring vitellogenin (vg) induction in male gammarids exposed in situ, as well as estrogen receptor activation using the Yeast Estrogen Screen (YES) involving passive sampler and grab water sample extracts. Extracts were also analysed for 424 organic micropollutants and selected transformation products. Gammarid feeding rate was significantly reduced 100, 200 and 400 m downstream of the WWTP effluent relative to the upstream site. While YES results showed significantly elevated estrogenicity at downstream sites, vg production in male gammarids was not induced. A laboratory experiment, in which gammarids were exposed to WWTP effluent, supported this observation. These results, hence, suggest that treated wastewater released into aquatic ecosystems impairs the ecosystem function of leaf litter decomposition. Vg levels in male gammarids measured by UPLC-MS/MS did, however, not alter.

Effect-based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European Water Framework Directive.

Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.

Effect of operational and water quality parameters on conventional ozonation and the advanced oxidation process O3/H2O2: Kinetics of micropollutant abatement, transformation product and bromate formation in a surface water.

The efficiency of ozone-based processes under various conditions was studied for the treatment of a surface water (Lake Zürich water, Switzerland) spiked with 19 micropollutants (pharmaceuticals, pesticides, industrial chemical, X-ray contrast medium, sweetener) each at 1 µg L-1. Two pilot-scale ozonation reactors (4-5 m3 h-1), a 4-chamber reactor and a tubular reactor, were investigated by either conventional ozonation and/or the advanced oxidation process (AOP) O3/H2O2. The effects of selected operational parameters, such as ozone dose (0.5-3 mg L-1) and H2O2 dose (O3:H2O2 = 1:3-3:1 (mass ratio)), and selected water quality parameters, such as pH (6.5-8.5) and initial bromide concentration (15-200 µg L-1), on micropollutant abatement and bromate formation were investigated. Under the studied conditions, compounds with high second-order rate constants kO3>104 M-1 s-1 for their reaction with ozone were well abated (>90%) even for the lowest ozone dose of 0.5 mg L-1. Conversely, the abatement efficiency of sucralose, which only reacts with hydroxyl radicals (OH), varied between 19 and 90%. Generally, the abatement efficiency increased with higher ozone doses and higher pH and lower bromide concentrations. H2O2 addition accelerated the ozone conversion to OH, which enables a faster abatement of ozone-resistant micropollutants. Interestingly, the abatement of micropollutants decreased with higher bromide concentrations during conventional ozonation due to competitive ozone-consuming reactions, except for lamotrigine, due to the suspected reaction of HOBr/OBr- with the primary amine moieties. In addition to the abatement of micropollutants, the evolution of the two main transformation products (TPs) of hydrochlorothiazide (HCTZ) and tramadol (TRA), chlorothiazide (CTZ) and tramadol N-oxide (TRA-NOX), respectively, was assessed by chemical analysis and kinetic modeling. Both selected TPs were quickly formed initially to reach a maximum concentration followed by a decrease of their concentrations for longer contact times. For the studied conditions, the TP's concentrations at the outlet of the reactors ranged from 0 to 61% of the initial parent compound concentration, CTZ being a more persistent TP against further oxidation than TRA-NOX. Finally, it was demonstrated in both reactors that the formation of bromate (BrO3-), a potentially carcinogenic oxidation by-product, could be controlled by H2O2 addition with a general improvement on micropollutant abatement. Post-treatment by granular activated carbon (GAC) filtration enabled the reduction of micropollutants and TPs concentrations but no changes in bromate were observed. The combined algae assay showed that water quality was significantly improved after oxidation and GAC post-treatment, driven by the abatement of the spiked pesticides (diuron and atrazine).

Endocrine Disruption and In Vitro Ecotoxicology: Recent Advances and Approaches.

Endocrine-disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling. Omnipresent in the environment, they can cause adverse effects in a wide range of wildlife. Accordingly, Endocrine Disruption is one focal area of ecotoxicology. Because EDCs induce complex response patterns in vivo via a wide range of mechanisms of action, in vitro techniques have been developed to reduce and understand endocrine toxicity. In this review we revisit the evidence for endocrine disruption in diverse species and the underlying molecular mechanisms. Based on this, we examine the battery of in vitro bioassays currently in use in ecotoxicological research and discuss the following key questions. Why do we use in vitro techniques? What endpoints are we looking at? Which applications are we using in vitro bioassays for? How can we put in vitro data into a broader context? And finally, what is the practical relevance of in vitro data? In critically examining these questions, we review the current state-of-the-art of in vitro (eco)toxicology, highlight important limitations and challenges, and discuss emerging trends and future research needs.

Micropollutant-induced tolerance of in situ periphyton: Establishing causality in wastewater-impacted streams.

The overarching aim of this field study was to examine causal links between in-situ exposure to complex mixtures of micropollutants from wastewater treatment plants and effects on freshwater microbial communities in the receiving streams. To reach this goal, we assessed the toxicity of serial dilutions of micropollutant mixtures, extracted from deployed passive samplers at the discharge sites of four Swiss wastewater treatment plants, to in situ periphyton from upstream and downstream of the effluents. On the one hand, comparison of the sensitivities of upstream and downstream periphyton to the micropollutant mixtures indicated that algal and bacterial communities composing the periphyton displayed higher tolerance towards these micropollutants downstream than upstream. On the other hand, molecular analyses of the algal and bacterial structure showed a clear separation between upstream and downstream periphyton across the sites. This finding provides an additional line of evidence that micropollutants from the wastewater discharges were directly responsible for the change in the community structure at the sampling sites by eliminating the micropollutant-sensitive species and favouring the tolerant ones. What is more, the fold increase of algal and bacterial tolerance from upstream to downstream locations was variable among sampling sites and was strongly correlated to the intensity of contamination by micropollutants at the respective sites. Overall, our study highlights the sensitivity of the proposed approach to disentangle effects of micropollutant mixtures from other environmental factors occurring in the field and, thus, establishing a causal link between exposure and the observed ecological effects on freshwater microbial communities.

Effect-based tools for monitoring estrogenic mixtures: Evaluation of five in vitro bioassays.

In vitro estrogen receptor transactivation assays (ERTAs) are increasingly used to measure the overall estrogenic activity of environmental water samples, which may serve as an indicator of exposure of fish or other aquatic organisms to (xeno)estrogens. Another potential area of application of ERTAs is to assist the monitoring of the potent steroids 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) under the Water Framework Directive (WFD) watch-list mechanism. Chemical analysis of E2 and EE2 is currently hampered by limits of quantification being mostly above the proposed annual average Environmental Quality Standards (AA-EQS) of 0.4 and 0.035 ng/L, respectively. Sensitive ERTAs could circumvent current detection challenges by measuring total estrogenic activity expressed as E2-equivalent (EEQ) concentrations. However, the use of different ERTAs results in different EEQ concentrations for the same sample. Reasons for these differences are known, but it remains unclear how to use and interpret bioassay results in a harmonised way. The aim of this study was to compare the intra- and inter-day variability of EEQ measurements using five different ERTAs (YES, ERα-CALUX, MELN, T47D-KBluc and GeneBLAzer-ERα) with regard to their applicability as effect-based tools in environmental monitoring. Environmentally relevant artificial mixtures of (xeno)estrogens were prepared to represent samples with higher (i.e. multiple times the AA-EQS for E2) or lower pollution levels (i.e. around the AA-EQS for E2). Mixtures were tested either directly or following solid phase extraction (SPE). The SPE step was included, as environmental samples typically require enrichment before analysis. Samples were analysed repeatedly to test intra-day and inter-day variability. Estrogenicity was quantified using the 10% effect level (PC10) of the positive control (E2) and expressed as EEQ concentrations. The average coefficient of variation (CV) of EEQ concentrations for the five ERTAs and all samples was 32%. CV was lower for intra-day experiments (30%) compared to inter-day experiments (37%). Sample extraction using SPE did not lead to additional variability; the intra-day CV for SPE extracted samples was 28%. Of the five ERTAs, ERα-CALUX had the best precision and repeatability (overall CV of 13%).

Integrating chemical analysis and bioanalysis to evaluate the contribution of wastewater effluent on the micropollutant burden in small streams.

Surface waters can contain a range of micropollutants from point sources, such as wastewater effluent, and diffuse sources, such as agriculture. Characterizing the source of micropollutants is important for reducing their burden and thus mitigating adverse effects on aquatic ecosystems. In this study, chemical analysis and bioanalysis were applied to assess the micropollutant burden during low flow conditions upstream and downstream of three wastewater treatment plants (WWTPs) discharging into small streams in the Swiss Plateau. The upstream sites had no input of wastewater effluent, allowing a direct comparison of the observed effects with and without the contribution of wastewater. Four hundred and five chemicals were analyzed, while the applied bioassays included activation of the aryl hydrocarbon receptor, activation of the androgen receptor, activation of the estrogen receptor, photosystem II inhibition, acetylcholinesterase inhibition and adaptive stress responses for oxidative stress, genotoxicity and inflammation, as well as assays indicative of estrogenic activity and developmental toxicity in zebrafish embryos. Chemical analysis and bioanalysis showed higher chemical concentrations and effects for the effluent samples, with the lowest chemical concentrations and effects in most assays for the upstream sites. Mixture toxicity modeling was applied to assess the contribution of detected chemicals to the observed effect. For most bioassays, very little of the observed effects could be explained by the detected chemicals, with the exception of photosystem II inhibition, where herbicides explained the majority of the effect. This emphasizes the importance of combining bioanalysis with chemical analysis to provide a more complete picture of the micropollutant burden. While the wastewater effluents had a significant contribution to micropollutant burden downstream, both chemical analysis and bioanalysis showed a relevant contribution of diffuse sources from upstream during low flow conditions, suggesting that upgrading WWTPs will not completely reduce the micropollutant burden, but further source control measures will be required.

The 2015 Annual Meeting of SETAC German Language Branch in Zurich (7-10 September, 2015): Ecotoxicology and environmental chemistry-from research to application.

This report provides a brief review of the 20th annual meeting of the German Language Branch of the Society of Environmental Toxicology and Chemistry (SETAC GLB) held from September 7th to 10th 2015 at ETH (Swiss Technical University) in Zurich, Switzerland. The event was chaired by Inge Werner, Director of the Swiss Centre for Applied Ecotoxicology (Ecotox Centre) Eawag-EPFL, and organized by a team from Ecotox Centre, Eawag, Federal Office of the Environment, Federal Office of Agriculture, and Mesocosm GmbH (Germany). Over 200 delegates from academia, public agencies and private industry of Germany, Switzerland and Austria attended and discussed the current state of science and its application presented in 75 talks and 83 posters. In addition, three invited keynote speakers provided new insights into scientific knowledge 'brokering', and-as it was the International Year of Soil-the important role of healthy soil ecosystems. Awards were presented to young scientists for best oral and poster presentations, and for best 2014 master and doctoral theses. Program and abstracts of the meeting (mostly in German) are provided as Additional file 1.

Oxidation of cetirizine, fexofenadine and hydrochlorothiazide during ozonation: Kinetics and formation of transformation products.

The efficiency of wastewater ozonation for the abatement of three nitrogen-containing pharmaceuticals, two antihistamine drugs, cetirizine (CTR) and fexofenadine (FXF), and the diuretic drug, hydrochlorothiazide (HCTZ), was investigated. Species-specific second-order rate constants for the reactions of the molecular, protonated (CTR, FXF) or deprotonated (HCTZ) forms of these compounds with ozone were determined. All three compounds are very reactive with ozone (apparent second order rate constants at pH 7: kO3,pH7 = 1.7·10(5) M(-1)s(-1), 8.5·10(4) M(-1)s(-1) and 9.0·10(3) M(-1)s(-1) for CTR, HCTZ and FXF, respectively). Transformation product (TP) structures were elucidated using liquid chromatography coupled with high-resolution tandem mass spectrometry, including isotope-labeled standards. For cetirizine and hydrochlorothiazide 8 TPs each and for fexofenadine 7 TPs were identified. The main TPs of cetirizine and fexofenadine are their respective N-oxides, whereas chlorothiazide forms to almost 100% from hydrochlorothiazide. In the bacteria bioluminescence assay the toxicity was slightly increased only during the ozonation of cetirizine at very high cetirizine concentrations. The main TPs detected in bench-scale experiments were also detected in full-scale ozonation of a municipal wastewater, for >90% elimination of the parent compounds.

In vitro bioassays to screen for endocrine active pharmaceuticals in surface and waste waters.

In the context of the European Water Framework Directive (WFD) it is fully recognized that pharmaceuticals can represent a relevant issue for the achievement of the good chemical and ecological status of European surface water bodies. The recent European Directive on the review of priority substances in surface water bodies has included three pharmaceuticals of widespread use (diclofenac, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2)) in the European monitoring list, the so-called watch list. Endocrine active pharmaceuticals such as EE2 and E2 (also occurring as natural hormone) can cause adverse effects on aquatic ecosystems at very low levels. However, monitoring of these pharmaceuticals within the watch list mechanism of the WFD and national monitoring programs can be difficult because of detection problems of most routine analytical methods. With proposed annual average Environmental Quality Standards (AA-EQS) of 0.035 ng/L and 0.4 ng/L, respectively, the estrogenic pharmaceutical EE2 and the natural hormone E2 are among those substances. Sensitive in vitro bioassays could reduce the current detection problems by measuring the estrogenic activity of environmental samples. In a short review article the application of this approach to screen and assess the risks of endocrine active pharmaceuticals with a focus on estrogenic pharmaceuticals in environmental waters is discussed.

Treatment of micropollutants in municipal wastewater: ozone or powdered activated carbon?

Many organic micropollutants present in wastewater, such as pharmaceuticals and pesticides, are poorly removed in conventional wastewater treatment plants (WWTPs). To reduce the release of these substances into the aquatic environment, advanced wastewater treatments are necessary. In this context, two large-scale pilot advanced treatments were tested in parallel over more than one year at the municipal WWTP of Lausanne, Switzerland. The treatments were: i) oxidation by ozone followed by sand filtration (SF) and ii) powdered activated carbon (PAC) adsorption followed by either ultrafiltration (UF) or sand filtration. More than 70 potentially problematic substances (pharmaceuticals, pesticides, endocrine disruptors, drug metabolites and other common chemicals) were regularly measured at different stages of treatment. Additionally, several ecotoxicological tests such as the Yeast Estrogen Screen, a combined algae bioassay and a fish early life stage test were performed to evaluate effluent toxicity. Both treatments significantly improved the effluent quality. Micropollutants were removed on average over 80% compared with raw wastewater, with an average ozone dose of 5.7 mg O3 l(-1) or a PAC dose between 10 and 20 mg l(-1). Depending on the chemical properties of the substances (presence of electron-rich moieties, charge and hydrophobicity), either ozone or PAC performed better. Both advanced treatments led to a clear reduction in toxicity of the effluents, with PAC-UF performing slightly better overall. As both treatments had, on average, relatively similar efficiency, further criteria relevant to their implementation were considered, including local constraints (e.g., safety, sludge disposal, disinfection), operational feasibility and cost. For sensitive receiving waters (drinking water resources or recreational waters), the PAC-UF treatment, despite its current higher cost, was considered to be the most suitable option, enabling good removal of most micropollutants and macropollutants without forming problematic by-products, the strongest decrease in toxicity and a total disinfection of the effluent.

Impairment of trophic interactions between zebrafish (Danio rerio) and midge larvae (Chironomus riparius) by chlorpyrifos.

The effects of chemicals on biotic interactions, such as competition and predation, have rarely been investigated in aquatic ecotoxicology. This study presents a new approach for the investigation of predator-prey interactions between zebrafish (Danio rerio) and midge larvae (Chironomus riparius) impaired by chlorpyrifos (CHP), a neurotoxic insecticide. With a simple experimental design including four different treatments: (1) control, (2) predator exposed, (3) prey exposed and (4) both, predator and prey, exposed, we were able to detect by visual observation an increase in the feeding rate of zebrafish preying on exposed chironomids after acute (2 h) exposure to 6 µg/l CHP. Previously, a decrease in the burrowing behaviour of exposed chironomid larvae was observed. However, when pre-exposing simultaneously both predators and prey, no significant differences in the feeding rate of zebrafish were observed. This suggests an impairment in prey recognition of the exposed zebrafish. At a lower CHP concentration (1 µg/l), no differences in feeding rate of zebrafish were observed. We therefore propose the use of trophic interactions as parameters in higher tier studies for chemical testing and evaluation of ecotoxicological risk assessment.

Gammarus spp. in aquatic ecotoxicology and water quality assessment: toward integrated multilevel tests.

The amphipod genus Gammarus is widespread and is structurally and functionally important in epigean freshwaters of the Northern Hemisphere. Its presence is crucial, because macroinvertebrate feeding is a major rate-limiting step in the processing of stream detrius. In addition, Gammarus interacts with multiple trophic levels bu functioning as prey, predator, herbivore, detritivore, and shredder. Such a broad span of ecosystem participation underlines the importance of Gammarus to pollutants and other disturbances may render it a valuable indicator for ecosystem health. This review summarizes the vast number of studies conducted with Gammarus spp. for evaluating aquatic ecotoxicology endpoints and examines the suitability of this native invertabrate species for the assessment of stream ecosystem health in the Northern Hemisphere. Numerous papers have been published on how pollutants affect gammarind behavior (i.e., mating, predator avoidance), reproduction, development, feeding activity, population structure, as well as the consequences of pollution on host-parasite, predator-prey, or native-invasive species interactions. Some biochemical and molecular biomarkers have already been established, such as the measurement of vitellogenin-like proteins, metallothioneins, alkali-labile phosphates (in proteins), and lipogenic enzyme activities for assessing endocrine distribution and detoxification mechanisms.

Interactions between effects of environmental chemicals and natural stressors: a review.

Ecotoxicological effect studies often expose test organisms under optimal environmental conditions. However, organisms in their natural settings rarely experience optimal conditions. On the contrary, during most of their lifetime they are forced to cope with sub-optimal conditions and occasionally with severe environmental stress. Interactions between the effects of a natural stressor and a toxicant can sometimes result in greater effects than expected from either of the stress types alone. The aim of the present review is to provide a synthesis of existing knowledge on the interactions between effects of "natural" and chemical (anthropogenic) stressors. More than 150 studies were evaluated covering stressors including heat, cold, desiccation, oxygen depletion, pathogens and immunomodulatory factors combined with a variety of environmental pollutants. This evaluation revealed that synergistic interactions between the effects of various natural stressors and toxicants are not uncommon phenomena. Thus, synergistic interactions were reported in more than 50% of the available studies on these interactions. Antagonistic interactions were also detected, but in fewer cases. Interestingly, about 70% of the tested chemicals were found to compromise the immune system of humans as judged from studies on human cell lines. The challenge for future studies will therefore be to include aspects of combined stressors in effect and risk assessment of chemicals in the environment.

Behavioural and developmental toxicity of chlorpyrifos and nickel chloride to zebrafish (Danio rerio) embryos and larvae.

In order to assess the combined toxicity of environmental chemicals with different modes of action in acute (2 h) and subchronic (11 d) exposures, embryos and larvae of Danio rerio were exposed to a heavy metal salt, nickel chloride (NiCl2), the insecticide chlorpyrifos (CHP) and their binary mixtures. Chlorpyrifos is an acetylcholine esterase inhibitor, which is likely to affect behaviour of the organism. NiCl2 targets the active sites of enzymes and is regarded as an unspecific toxicant for aquatic organisms. Several endpoints, such as locomotor activity, morphological abnormalities, and mortality of D. rerio embryos and larvae were studied. During acute exposures to > or =0.25 mg/L of chlorpyrifos, locomotor activity tended to increase. However, this activity decreased significantly at > or =7.5 mg Ni/L. Subchronic exposures to CHP resulted in behavioural changes at much lower concentrations (> or =0.01 mg/L) and considerably earlier than the observed increase in morphological abnormalities and mortality (LC(50) (10 d): 0.43 mg/L). Combined CHP and NiCl2 mixtures led to an antagonistic deviation from the concept of independent action, in the case of locomotor activity. Compared to developmental or survival parameters, behaviour was the most sensitive endpoint for CHP exposure in this study; therefore we recommend this parameter to complement already established endpoints.

Effects of 3,4-dichloroaniline and diazinon on different biological organisation levels of zebrafish (Danio rerio) embryos and larvae.

In this study the effects of 3,4-dichloroaniline (3,4-DCA), a decomposition product of the herbicides propanil and diuron (and other pesticides), and diazinon, a neurotoxic insecticide, on early life stages of zebrafish Danio rerio were assessed. The toxicity of these substances with different modes of action (acetylcholine esterase inhibitor vs. polar narcosis) was tested for single substances as well as in binary mixtures. To study effects on different biological organisation levels (from the molecular up to the whole organism level) the molecular stress response regarding Hsp70, the embryonic and larval development and the locomotor activity were investigated as integrative biomarkers. In single substance tests 3,4-dichloroaniline elicited deformations at > or = 0.25 mg/l during the 11 days subchronic test, whereas locomotor activity and mortality were impaired at > or = 0.5 mg/l. Diazinon effects on those parameters were obvious at > or = 2 mg/l, except for the deformation rate (11 days:1 mg/l). In equitoxic mixtures of both substances concentration additivity was observed for deformation rate and mortality (11 days). An increase in the Hsp70 content occurred in zebrafish exposed to 0.25 mg 3,4-DCA/l as well as to 0.05 mg diazinon/l; in mixtures concentration additivity could be shown. The investigated endpoints varied in respect to their sensitivity, with deformations and Hsp70 levels as most sensitive parameters concerning 3,4-DCA and Hsp70 as most sensitive parameter concerning diazinon. Accordingly, for an integrated understanding of the effects of chemicals and their mixtures on fish, a battery of different test methods should be applied.

Behavior of Corophium volutator (Crustacea, Amphipoda) exposed to the water-accommodated fraction of oil in water and sediment.

We investigated the short-term effects of the water accommodated fraction (WAF) of weathered Forties crude oil on the behavior of Corophium volutator in the Multispecies Freshwater Biomonitor (MFB). When exposing C. volutator to 25 and 50% WAF in aqueous exposures, hyperactivity with an additional increase in ventilation was detected, whereas exposure to 100% WAF led to hypoactivity (narcosis). In a sediment exposure with 100% WAF, there was an increased tendency toward hyperactivity. In a pulse experiment, hyperactivity appeared at and after a 130-min exposure to 50% WAF in a majority of cases. Our experiments suggest that the behavior of C. volutator as measured in the MFB may be an appropriate parameter for coastal monitoring.