Ecological Recovery Potential of Freshwater Organisms: Consequences for Environmental Risk Assessment of Chemicals.

Chemical contaminants released into the in the environment may have adverse effects on (non-target) species, populations and communities. The return of a stressed system to its pre-disturbance or other reference state, i.e. the ecological recovery, may depend on various factors related to the affected taxon, the ecosystem of concern and the type of stressor with consequences for the assessment and management of risks associated with chemical contaminants. Whereas the effects caused by short-term exposure might be acceptable to some extent, the conditions under which ecological recovery can serve as a decision criterion in the environmental risk assessment of chemical stressors remains to be evaluated. For a generic consideration of recovery in the risk assessment of chemicals, we reviewed case studies of natural and artificial aquatic systems and evaluate five aspects that might cause variability in population recovery time: (1) taxonomic differences and life-history variability, (2) factors related to ecosystem type and community processes, (3) type of disturbance, (4) comparison of field and semi-field studies, and (5) effect magnitude, i.e., the decline in population size following disturbance. We discuss our findings with regard to both retrospective assessments and prospective risk assessment.

ARAapp: filling gaps in the ecological knowledge of spiders using an automated and dynamic approach to analyze systematically collected community data.

The ARAMOB data repository compiles meticulously curated spider community datasets from systematical collections, ensuring a high standard of data quality. These datasets are enriched with crucial methodological data that enable the datasets to be aligned in time and space, facilitating data synthesis across studies, respectively, collections. To streamline the analysis of these datasets in a species-specific context, a suite of tailored ecological analysis tools named ARAapp has been developed. By harnessing the capabilities of ARAapp, users can systematically evaluate the spider species data housed within the ARAMOB repository, elucidating intricate relationships with a range of parameters such as vertical stratification, habitat occurrence, ecological niche parameters (moisture and shading) and phenological patterns. Database URL: ARAapp is available at  www.aramob.de/en.

Getting more out of the zebrafish light dark transition test.

In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.

Fate of weathered multi-walled carbon nanotubes in an aquatic sediment system.

The widespread application of carbon nanotubes (CNT) in various consumer products leads to their inevitable release into aquatic systems. But only little is known about their distribution among aquatic compartments. In this study, we investigated the partitioning of radiolabeled, weathered multi-walled CNT (14C-wMWCNT) in an aquatic sediment system over a period of 180 days (d). The applied nanomaterial concentration in water phase was 100 µg L-1. Over time, the wMWCNT disappeared exponentially from the water phase and simultaneously accumulated in the sediment phase. After 2 h incubation just 77%, after seven days 30% and after 180 d only 0.03% of applied radioactivity (AR) remained in the water phase. The respective values for the disappearance times DT50 and DT90 were 3.2 d and 10.7 d. Further, minor mineralization of 14C-wMWCNT to 14CO2 was observed with values below 0.06% of AR. In addition, a study was carried out to estimate the deposition of wMWCNT in the water phase with and without sediment in the test system for 28 d. We found no influence of a sediment phase on the sedimentation behavior of wMWCNT in the water phase: After 6.5 d and 7.3 d 50% of the applied wMWCNT subsided in the presence and absence of sediment, respectively. The slow removal of wMWCNT from the water body by deposition into sediment implies that in addition to sediment-dwelling organisms, pelagic organisms are also at risk of exposure to nanomaterials and prone for their take-up.

A trophic transfer study: accumulation of multi-walled carbon nanotubes associated to green algae in water flea Daphnia magna.

Carbon nanotubes (CNT) are promising nanomaterials in modern nanotechnology and their use in many different applications leads to an inevitable release into the aquatic environment. In this study, we quantified trophic transfer of weathered multi-walled carbon nanotubes (wMWCNT) from green algae to primary consumer Daphnia magna in a concentration of 100 µg L-1 using radioactive labeling of the carbon backbone (14C-wMWCNT). Trophic transfer of wMWCNT was compared to the uptake by daphnids exposed to nanomaterials in the water phase without algae. Due to the rather long observed CNT sedimentation times (DT) from the water phase (DT50: 3.9 days (d), DT90: 12.8 d) wMWCNT interact with aquatic organisms and associated to the green algae Chlamydomonas reinhardtii and Raphidocelis subcapitata. After the exposition of algae, the nanotubes accumulated to a maximum of 1.6 ± 0.4 µg 14C-wMWCNT mg-1 dry weight-1 (dw-1) and 0.7 ± 0.3 µg 14C-wMWCNT mg-1 dw-1 after 24 h and 48 h, respectively. To study trophic transfer, R. subcapitata was loaded with 14C-wMWCNT and subsequently fed to D. magna. A maximum body burden of 0.07 ± 0.01 µg 14C-wMWCNT mg-1 dw-1 and 7.1 ± 1.5 µg 14C-wMWCNT mg-1 dw-1 for D. magna after trophic transfer and waterborne exposure was measured, respectively, indicating no CNT accumulation after short-term exposure via trophic transfer. Additionally, the animals eliminated nanomaterials from their guts, while feeding algae facilitated their excretion. Further, accumulation of 14C-wMWCNT in a growing population of D. magna revealed a maximum uptake of 0.7 ± 0.2 µg mg-1 dw-1. Therefore, the calculated bioaccumulation factor (BAF) after 28 d of 6700 ± 2900 L kg-1 is above the limit that indicates a chemical is bioaccumulative in the European Union Regulation REACH. Although wMWCNT did not bioaccumulate in neonate D. magna after trophic transfer, wMWCNT enriched in a 28 d growing D. magna population regardless of daily feeding, which increases the risk of CNT accumulation along the aquatic food chain.

Application of the Closure Principle Computational Approach Test to Assess Ecotoxicological Field Studies: Comparative Analysis Using Earthworm Field Test Abundance Data.

Field studies to determine the effects of chemicals on earthworm communities are generally conducted according to International Organization for Standardization standard 11268-3 (and later comments). However, statistical test procedures suggested in the guideline are frequently criticized, mainly for 2 reasons: 1) Earthworm abundances are count data and often do not fulfill requirements for multiple t tests (normal distribution and homogeneity of variance), and 2) the resulting toxicity metrics of multiple testing procedures (no/lowest-observed-effect concentrations [NOEC/LOEC]) fail to adequately detect the actual level of effects. Recently, a new method to overcome these shortcomings was presented by the introduction of the closure principle computational approach test (CPCAT). We applied this statistical method to assess chemical effects on abundance in a large dataset of 26 earthworm field studies (with up to 3 test chemical application rates) and an additional extended study with 6 application rates. A comparative analysis was provided considering results of well-established multiple testing approaches (Dunnett's test) with particular consideration of the degree of overdispersion found in these data. It was shown that the CPCAT detects substantially more effects in earthworm field tests as statistically significant than standard t test approaches. This lowered the LOEC/NOEC for many chemical treatments to control comparisons. As a consequence, the statistically detected NOECs/LOECs were often set at lower percentage deviations between control and chemical treatment. This is the first time the performance of the CPCAT has been assessed within a comprehensive analysis of earthworm field study data. Environ Toxicol Chem 2021;40:1750-1760. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A "plan bee" for cities: Pollinator diversity and plant-pollinator interactions in urban green spaces.

Green infrastructure in cities is considered to serve as a refuge for insect pollinators, especially in the light of an ongoing global decline of insects in agricultural landscapes. The design and maintenance of urban green spaces as key components of green infrastructure play a crucial role in case of nesting opportunities and for foraging insects. However, only few research has explored the impact of urban green space design on flower visitor communities, plant-pollinator interaction and the provision of the ecosystem service of pollination in cities. We investigated the abundance and diversity of pollinator communities in different urban park types in designed, standardized vegetation units, linked the visitation rates to the structural composition of the park types and derived indices for implemented pollination performances. The study was performed in two different structural park elements, flower beds and insect-pollinating trees. To gain a comprehensive understanding of the interaction between plants and pollinators, we calculated a plant-pollinator network of the recorded community in the investigation area. Visitation rates at different park types clearly showed, that the urban community gardens in comparison to other urban park types had a significantly higher abundance of pollinator groups, comparable to results found on a rural reference site. Tilia trees contributed significantly to the ecosystem service of pollination in investigated green spaces with a high supply of nectar and pollen during their flowering period. Calculations of pollination performances showed that recreational parks had comparably low visitation rates of pollinators and a high potential to improve conditions for the ecosystem service of pollination. The results indicated the strong potential of cities to provide a habitat for different groups of pollinators. In order to access this refuge, it is necessary to rely on near-natural concepts in design and maintenance, to create a wide range of flower diversity and to use even small green patches. Based on the findings, we encourage an integrated management of urban free spaces to consider parks as key habitats for pollinators in anthropogenic dominated, urban environments.

MITAS: A model for assessing the time-dependent risk of sequential applications of pesticides for soil organisms by consideration of exposure, degradation and mixture toxicity.

In agricultural landscapes it is common practice to apply pesticides as a spray series. Within a vegetation period multiple applications result in a mixture of different pesticides in the soil and other environmental compartments. •A model named MITAS (MIxture Toxicity of Application Spray series) has been developed to calculate the soil concentration of pesticides and the resulting time-dependent mixture risk for earthworms. MITAS creates tables and graphs representing the mixture risk for an applied spray series time-dependently.•A simulation of the impact of application series for a period of up to three years is possible with MITAS. Calculation of the soil concentration is based on the assumptions of the German pesticide registration. Mixture risk for each day within the entire modelling period is calculated.•Mixtures of pesticides from former and multiple applications are the rule in agricultural soils as various studies have shown. Thus, considering the impact of pesticide mixtures is necessary to protect soil organisms. MITAS can assess not only the maximum mixture risk for soil organisms, but also how long a certain risk threshold may be exceeded, above which unacceptable effects on exposed organisms may occur.

Short exposure to cadmium disrupts the olfactory system of zebrafish (Danio rerio) - Relating altered gene expression in the olfactory organ to behavioral deficits.

Fish strongly rely on olfaction as a variety of essential behaviors such as foraging and predator avoidance are mediated by the olfactory system. Cadmium (Cd) is known to impair olfaction and accumulate in the olfactory epithelium (OE) and bulb (OB) of fishes. In the present study, the acute toxicity of Cd on olfaction in zebrafish (Danio rerio) was characterized on the molecular and behavioral level. To this end, quantitative real-time PCR was performed in order to analyze the expression of selected genes in both the OE and OB. Moreover, the response of zebrafish to an alarm cue was investigated. Following 24 h of exposure to Cd, the expression of genes associated with olfactory sensory neurons was reduced in the OE. Furthermore, the antioxidant genes peroxiredoxin 1 (prdx1) and heme oxygenase 1 (hmox1), as well as the metallothionein 2 gene (mt2) were upregulated in the OE, whereas hmox1 and the stress-inducible heat shock protein 70 gene (hsp70) were upregulated in the OB upon exposure to Cd. Following stimulation with a conspecific skin extract, zebrafish displayed a considerable disruption of the antipredator behavior with increasing Cd concentration. Taken together, Cd impaired olfaction in zebrafish, thereby disrupting the antipredator response, which is crucial for the survival of individuals. Cellular stress followed by disruption of olfactory sensory neurons may have contributed to the observed behavioral deficits.

Do you smell the danger? Effects of three commonly used pesticides on the olfactory-mediated antipredator response of zebrafish (Danio rerio).

Fish are warned about the presence of predators via an alarm cue released from the skin of injured conspecifics. The detection of this odor inherently initiates an antipredator response, which increases the chance of survival for the individual. In the present study, we assessed the effect of three commonly used pesticides on the antipredator response of zebrafish (Danio rerio). For this, we analyzed the behavioral response of zebrafish to a conspecific skin extract following 24 h of exposure to the respective contaminants. Results demonstrate that fish exposed to 20 µg/L of the organophosphate insecticide chlorpyrifos significantly reduced bottom-dwelling and freezing behavior, suggesting an impairment of the antipredator response. For the urea-herbicide linuron and the pyrethroid insecticide permethrin, no statistically significant effects could be detected. However, linuron-exposed fish appeared to respond in an altered manner to the skin extract; some individuals failed to perform the inherent behaviors such as erratic movements and instead merely increased their velocity. Furthermore, we determined whether zebrafish would avoid the pesticides in a choice maze. While fish avoided permethrin, they behaved indifferently to chlorpyrifos and linuron. The study demonstrates that pesticides may alter the olfactory-mediated antipredator response of zebrafish in distinct ways, revealing that particularly fish exposed to chlorpyrifos may be more prone to predation.

Modification and quantification of in vivo EROD live-imaging with zebrafish (Danio rerio) embryos to detect both induction and inhibition of CYP1A.

The visualization of specific activation of the aryl hydrocarbon receptor (AhR) directly in the zebrafish embryo (Danio rerio) via live-imaging is a reliable tool to investigate the presence of dioxin-like substances in environmental samples. The co-existence of inducers and inhibitors of cytochrome P450-dependent monooxygenases (CYP1A) is typical of complex environmental mixtures and requires modifications of the in vivo EROD assay: For this end, zebrafish embryos were used to evaluate the EROD-modifying potentials of common single-compound exposures as well as binary mixtures with the PAH-type Ah-receptor agonist ß-naphthoflavone. For chemical testing, chlorpyrifos and Aroclor 1254 were selected; ß-naphthoflavone served as maximum EROD induction control. Chlorpyrifos (≤EC10) could be documented to be a strong CYP1A inhibitor causing characteristic edema-related toxicity. Aroclor 1254 resulted in inhibition of CYP1A catalytic activity in a concentration- and specific time-dependent manner. Next to a fast CYP1A induction, CYP1A inhibition could also be detected after 3h short-term exposure of zebrafish embryos to chlorpyrifos. This communication also describes techniques for the quantification of fluorescence signals via densitometry as a basis for subsequent statistical assessment. The co-exposure approach with zebrafish embryos accounts for the nature of potential interaction between CYP1A inducers and inhibitors and thus pays tribute to the complexity of environmental mixtures. The co-exposure EROD live-imaging assay thus facilitates a better understanding of mixture effects and allows a better assessment and interpretation of (embryo) toxic potentials.

Morphological dataset of aboveground macrofungal communities within different forest conversion stages in the Eifel National Park in Germany.

This dataset includes 235 aboveground macrofungal species observed at 15 sampling sites, which are associated with five different forest conversion stages. We used a space-for-time substitution approach to represent a forest conversion from Norway spruce (Picea abies) to European beech (Fagus sylvatica) through three different, widely used management practices. In addition to the results of 75 macrofungal field surveys, this data article includes information about site characteristics, vegetation structure, and observation frequencies. A multivariate statistic and myco-ecological interpretation of the macrofungal dataset is presented in an associated research article entitled "Forest conversion from Norway spruce to European beech increases species richness and functional structure of aboveground macrofungal communities" (Heine et al., 2019) [1].

Assessment of urban green space structures and their quality from a multidimensional perspective.

Facing the growing amount of people living in cities and, at the same time, the need for a compact and sustainable urban development to mitigate urban sprawl, it becomes increasingly important that green spaces in compact cities are designed to meet the various needs within an urban environment. Urban green spaces have a multitude of functions: Maintaining ecological processes and resulting services, e.g. providing habitat for animals and plants, providing a beneficial city microclimate as well as recreational space for citizens. Regarding these requirements, currently existing assessment procedures for green spaces have some major shortcomings, which are discussed in this paper. It is argued why a more detailed spatial level as well as a distinction between natural and artificial varieties of structural elements is justified and needed and how the assessment of urban green spaces benefits from the multidimensional perspective that is applied. By analyzing a selection of structural elements from an ecological, microclimatic and social perspective, indicator values are derived and a new, holistic metrics1 is proposed. The results of the integrated analysis led to two major findings: first, that for some elements, the evaluation differs to a great extent between the different perspectives (disciplines) and second, that natural and artificial varieties are, in most cases, evaluated considerably different from each other. The differences between the perspectives call for an integrative planning policy which acknowledges the varying contribution of a structural element to different purposes (ecological, microclimatic, social) as well as a discussion about the prioritization of those purposes. The differences in the evaluation of natural vs. artificial elements verify the assumption that indicators which consider only generic elements fail to account for those refinements and are thus less suitable for planning and assessment purposes. Implications, challenges and scenarios for the application of such a metrics are finally discussed.

Fishing for contaminants: identification of three mechanism specific transcriptome signatures using Danio rerio embryos.

In ecotoxicology, transcriptomics is an effective way to detect gene expression changes in response to environmental pollutants. Such changes can be used to identify contaminants or contaminant classes and can be applied as early warning signals for pollution. To do so, it is important to distinguish contaminant-specific transcriptomic changes from genetic alterations due to general stress. Here we present a first step in the identification of contaminant class-specific transcriptome signatures. Embryos of zebrafish (Danio rerio) were exposed to three substances (methylmercury, chlorpyrifos and Aroclor 1254, each from 24 to 48 hpf exposed) representing sediment typical contaminant classes. We analyzed the altered transcriptome to detect discriminative genes significantly regulated in reaction to the three applied contaminants. By comparison of the results of the three contaminants, we identified transcriptome signatures and biologically important pathways (using Cytoscape/ClueGO software) that react significantly to the contaminant classes. This approach increases the chance of finding genes that play an important role in contaminant class-specific pathways rather than more general processes.

Simulation-based assessment of the impact of fertiliser and herbicide application on freshwater ecosystems at the Three Gorges Reservoir in China.

Dams have profound impacts on river ecosystems, amongst them inundation of land, altered dynamics of the water body or uprising reservoir backwaters influencing tributary or upstream river sections. Along the outstandingly ecologically important Yangtze River in China, the Three Gorges Reservoir (TGR) is the largest project, covering an area of 1080 km2. From the beginning, the dam-project came in for criticism on increasing environmental risks due to sub-merging former industrial and urban areas. We simulated dynamics of biotic and abiotic components of the TGR ecosystem (trophic guilds of aquatic organisms, hydrodynamics, nutrients), as well as the behaviour of the herbicidal substance propanil and its metabolites 3,4-Dichloroaniline (DCA) and 3,3',4,4'-tetrachloroazoxybenzene (TCAB). A modelling environment, provided by the AQUATOX software, was adapted to the specific situation at a tributary reach to the Yangtze river 'Daning River'. As the simulated food web contained several interconnected trophic levels, a significant biomagnification of metabolites was demonstrated by our simulation studies. In particular, newly emerging stagnant downstream sections of tributaries exhibited high probabilities due to accumulating pesticides from upstream sources. The common problem of algal blooms in the TGR-region was addressed by dose-response simulation experiments with essential nutrients. Impacts on structure and abundance of populations of aquatic organisms were shown. However, even high nutrient loads resulted in only slight changes of densities of organisms of all trophic levels. Nevertheless, the probabilities for large-scale algal blooms affecting drinking water quality were considered low because of high flow velocities and discharge rates towards the Yangtze River. We see high potential of simulation-based assessments that provide information for risk managers dealing with whole catchment areas. They are put in the position to differentiate the magnitude of impacts of various factors and decide about the most effective remediation measures.

Looking back - Looking forward: A novel multi-time slice weight-of-evidence approach for defining reference conditions to assess the impact of human activities on lake systems.

Lake ecosystems are sensitive recorders of environmental changes that provide continuous archives at annual to decadal resolution over thousands of years. The systematic investigation of land use changes and emission of pollutants archived in Holocene lake sediments as well as the reconstruction of contamination, background conditions, and sensitivity of lake systems offer an ideal opportunity to study environmental dynamics and consequences of anthropogenic impact that increasingly pose risks to human well-being. This paper discusses the use of sediment and other lines of evidence in providing a record of historical and current contamination in lake ecosystems. We present a novel approach to investigate impacts from human activities using chemical-analytical, bioanalytical, ecological, paleolimnological, paleoecotoxicological, archeological as well as modeling techniques. This multi-time slice weight-of-evidence (WOE) approach will generate knowledge on conditions prior to anthropogenic influence and provide knowledge to (i) create a better understanding of the effects of anthropogenic disturbances on biodiversity, (ii) assess water quality by using quantitative data on historical pollution and persistence of pollutants archived over thousands of years in sediments, and (iii) define environmental threshold values using modeling methods. This technique may be applied in order to gain insights into reference conditions of surface and ground waters in catchments with a long history of land use and human impact, which is still a major need that is currently not yet addressed within the context of the European Water Framework Directive.

Edaphostat: interactive ecological analysis of soil organism occurrences and preferences from the Edaphobase data warehouse.

Database URL: Edaphostat is part of the Edaphobase Web Application available at https://portal.edaphobase.org.

The impact of chemical pollution on the resilience of soils under multiple stresses: A conceptual framework for future research.

Soils are faced with man-made chemical stress factors, such as the input of organic or metal-containing pesticides, in combination with non-chemical stressors like soil compaction and natural disturbance like drought. Although multiple stress factors are typically co-occurring in soil ecosystems, research in soil sciences on this aspect is limited and focuses mostly on single structural or functional endpoints. A mechanistic understanding of the reaction of soils to multiple stressors is currently lacking. Based on a review of resilience theory, we introduce a new concept for research on the ability of polluted soil (xenobiotics or other chemical pollutants as one stressor) to resist further natural or anthropogenic stress and to retain its functions and structure. There is strong indication that pollution as a primary stressor will change the system reaction of soil, i.e., its resilience, stability and resistance. It can be expected that pollution affects the physiological adaption of organisms and the functional redundancy of the soil to further stress. We hypothesize that the recovery of organisms and chemical-physical properties after impact of a follow-up stressor is faster in polluted soil than in non-polluted soil, i.e., polluted soil has a higher dynamical stability (dynamical stability=1/recovery time), whereas resilience of the contaminated soil is lower compared to that of not or less contaminated soil. Thus, a polluted soil might be more prone to change into another system regime after occurrence of further stress. We highlight this issue by compiling the literature exemplarily for the effects of Cu contamination and compaction on soil functions and structure. We propose to intensify research on effects of combined stresses involving a multidisciplinary team of experts and provide suggestions for corresponding experiments. Our concept offers thus a framework for system level analysis of soils paving the way to enhance ecological theory.

Bioassay battery interlaboratory investigation of emerging contaminants in spiked water extracts - Towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring.

Bioassays are particularly useful tools to link the chemical and ecological assessments in water quality monitoring. Different methods cover a broad range of toxicity mechanisms in diverse organisms, and account for risks posed by non-target compounds and mixtures. Many tests are already applied in chemical and waste assessments, and stakeholders from the science-police interface have recommended their integration in regulatory water quality monitoring. Still, there is a need to address bioassay suitability to evaluate water samples containing emerging pollutants, which are a current priority in water quality monitoring. The presented interlaboratory study (ILS) verified whether a battery of miniaturized bioassays, conducted in 11 different laboratories following their own protocols, would produce comparable results when applied to evaluate blinded samples consisting of a pristine water extract spiked with four emerging pollutants as single chemicals or mixtures, i.e. triclosan, acridine, 17α-ethinylestradiol (EE2) and 3-nitrobenzanthrone (3-NBA). Assays evaluated effects on aquatic organisms from three different trophic levels (algae, daphnids, zebrafish embryos) and mechanism-specific effects using in vitro estrogenicity (ER-Luc, YES) and mutagenicity (Ames fluctuation) assays. The test battery presented complementary sensitivity and specificity to evaluate the different blinded water extract spikes. Aquatic organisms differed in terms of sensitivity to triclosan (algae > daphnids > fish) and acridine (fish > daphnids > algae) spikes, confirming the complementary role of the three taxa for water quality assessment. Estrogenicity and mutagenicity assays identified with high precision the respective mechanism-specific effects of spikes even when non-specific toxicity occurred in mixture. For estrogenicity, although differences were observed between assays and models, EE2 spike relative induction EC50 values were comparable to the literature, and E2/EE2 equivalency factors reliably reflected the sample content. In the Ames, strong revertant induction occurred following 3-NBA spike incubation with the TA98 strain, which was of lower magnitude after metabolic transformation and when compared to TA100. Differences in experimental protocols, model organisms, and data analysis can be sources of variation, indicating that respective harmonized standard procedures should be followed when implementing bioassays in water monitoring. Together with other ongoing activities for the validation of a basic bioassay battery, the present study is an important step towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring.