The CPCAT as a novel tool to overcome the shortcomings of NOEC/LOEC statistics in ecotoxicology: a simulation study to evaluate the statistical power.

Species reproduction is an important determinant of population dynamics. As such, this is an important parameter in environmental risk assessment. The closure principle computational approach test (CPCAT) was recently proposed as a method to derive a NOEC/LOEC for reproduction count data such as the number of juvenile Daphnia. The Poisson distribution used by CPCAT can be too restrictive as a model of the data-generating process. In practice, the generalized Poisson distribution could be more appropriate, as it allows for inequality of the population mean µ and the population variance σ 2 . It is of fundamental interest to explore the statistical power of CPCAT and the probability of determining a regulatory relevant effect correctly. Using a simulation, we varied between Poisson distribution ( µ = σ 2 ) and generalized Poisson distribution allowing for over-dispersion ( µ < σ 2 ) and under-dispersion ( µ > σ 2 ). The results indicated that the probability of detecting the LOEC/NOEC correctly was ≥ 0.8 provided the effect was at least 20% above or below the mean level of the control group and mean reproduction of the control was at least 50 individuals while over-dispersion was missing. Specifically, under-dispersion increased, whereas over-dispersion reduced the statistical power of the CPCAT. Using the well-known Hampel identifier, we propose a simple and straight forward method to assess whether the data-generating process of real data could be over- or under-dispersed.

Generalized concentration addition accurately predicts estrogenic potentials of mixtures and environmental samples containing partial agonists.

Cell-based bioanalytical tools are considered one alternative to overcome limitations of sensitivities of instrumental, analytical chemistry for monitoring estrogenic chemicals in the environment. Because these tools also reflect non-additive interactions of chemicals in mixtures, their outcomes often deviate from outcomes of chemical analytical approaches that assume additivity, e.g. the concentration addition (CA) model. Often this is because CA is unable to adequately represent effects of partial agonists, i.e. estrogens with lesser efficacies compared to 17ß-estradiol. A generalized concentration addition (GCA) model has been proposed to address this shortcoming. In the present study, we investigated effects of mixtures of isomers of nonylphenol as partial model agonists in a cell-based estrogenicity assay. Whether the GCA model was able to more accurately predict the outcomes of these and previously published mixture experiments was evaluated, as well as the potency of a set of comprehensively characterized sewage effluent samples, compared to CA. If samples contained partial agonists, the GCA model consistently predicted potencies of mixtures and extracts of environmental samples more accurately than did the CA model. These findings enable more accurate estimations of potencies of estrogenicity explained by concentrations of agonists and partial agonists, thus significantly improving the ability to identify causative chemicals.

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.

Uptake, elimination, and biotransformation of 17α-ethinylestradiol by the freshwater alga Desmodesmus subspicatus.

Bioconcentration and transformation of the potent and persistent xeno-estrogen 17α-ethinylestradiol (EE2) by organisms at the basis of the food web have received only little research attention. In this study, uptake, elimination, and biotransformation of radiolabeled EE2 ((14)C-EE2) by the freshwater green alga Desmodesmus subspicatus were investigated. The alga highly incorporated radioactivity following (14)C-EE2 exposure. Up to 68% of the test compound was removed from the medium by D. subspicatus within a rather short time period (72 h C(algae)/C(water): 2200 L/kg wet weight). When the algae were transported to clear medium, a two-stage release pattern was observed with an initially quick elimination phase following slower clearance afterward. Interestingly, D. subspicatus brominated EE2 when bromide was available in the medium, a transformation process demonstrated to occur abiotically but not by algae. The consequence of the presence of more hydrophobic mono- and dibrominated EE2 in the environment remains to be further investigated, as these products were shown to have a lower estrogenic potency but are expected to have a higher bioaccumulation potential and to be more toxic than the mother compound.

A list of fish species that are potentially exposed to pesticides in edge-of-field water bodies in the European Union--a first step towards identifying vulnerable representatives for risk assessment.

Surrogate species are used in standard toxicity tests for the environmental risk assessment of chemicals. Test results are then extrapolated to the situation in the field, which is often associated with a large degree of uncertainty. Since a vulnerable species in the field is not only characterised by its intrinsic sensitivity to a stressor but also by its potential for exposure and its population resilience, the identification of focal species based on these three components of vulnerability is needed for a more ecologically relevant risk assessment. This study listed European fish species that are susceptible to pesticide exposure in the field and thus achieved the first step towards identifying focal species for the risk assessment of pesticides for fish in Europe. A step-wise filtering approach was applied to list freshwater fish species that are native to Europe and widespread in the European Union, which inhabit streams, ditches or ponds in agricultural landscapes and therefore, are at an elevated risk of being exposed to pesticides. Out of the 579 fish species occurring in European freshwater, 27 species met the filtering criteria. The resulting list was verified based on monitoring studies that were conducted in agricultural landscapes over the past 20 years. Focal fish species that can be used for a more ecologically relevant environmental risk assessment of pesticides in Europe can be identified from the produced list of species by further assessing their ecological (life history and dispersal characteristics) and intrinsic sensitivities.

A plea for the use of copepods in freshwater ecotoxicology.

Standard species used in ecological risk assessment are chosen based on their sensitivity to various toxicants and the ease of rearing them for laboratory experiments. However, this mostly overlooks the fact that species in the field that may employ variable life-history strategies, which may have consequences concerning the vulnerability of such species to exposure with contaminants. We aimed to highlight the importance of copepods in ecology and to underline the need to include freshwater copepods in ecotoxicology. We carried out a literature search on copepods and Daphnia in ecology and ecotoxicology to compare the recognition given to these two taxa in these respective fields. We also conducted a detailed analysis of the literature on copepods and their current role in ecotoxicology to characterize the scale and depth of the studies and the ecotoxicological information therein. The literature on the ecology of copepods outweighed that in ecotoxicology when compared with daphnids. Copepods, like other zooplankton, were found to be sensitive to toxicants and important organisms in aquatic ecosystems. The few studies that were conducted on the ecotoxicology of copepods mainly focused on marine copepods. However, very little is known about the ecotoxicology of freshwater copepods. To enable a more realistic risk higher tier environmental risk assessment, we recommend considering freshwater copepods as part of the hazard assessment process. This could include the establishment of laboratory experiments to analyse the effects of toxicants on copepods and the development of individual-based models to extrapolate effects across species and scenarios.

Promoting effects on reproduction increase population vulnerability of Daphnia magna.

Environmental risk assessment of chemicals is based on single species tests at the individual level with single compounds. However, the protection goal is the sustainability of a population, which faces several natural stressors and mixtures of chemicals in the environment. Therefore, experiments were undertaken to quantify the combined effects of chemicals with different modes of action on Daphnia magna populations. Populations continuously exposed to dispersogen A and at abundance equilibrium were treated with a 2-d pulse of p353-nonylphenol. In previous studies, dispersogen A was shown to act as a natural info-chemical, promoting the reproduction of daphnids (higher offspring quantity) coupled with reduced offspring fitness, whereas nonylphenol in pulsed-exposure caused size-selective mortality. Dispersogen A caused accelerated population growth to maximum abundance, shifted the population structure towards smaller individuals, and increased the population sensitivity to nonylphenol. The authors showed that a positive effect observed at the individual level can be transposed to a negative effect when monitored at the population level. So far, positive effects are not addressed in environmental risk assessment, and even in higher-tier testing, population structure is not quantified. Both factors indicate a potential mismatch between protection aim and risk assessment practice.

Interlaboratory comparison of a standardized toxicity test using the nematode Caenorhabditis elegans (ISO 10872).

A ring test was carried out within the standardization process of ISO 10872 to evaluate the precision of the toxicity test for the nematode Caenorhabditis elegans. Eight different laboratories tested aqueous solutions of the reference substance benzylcetyldimethylammonium chloride as well as native sediments and soils for toxic effects on the growth and reproduction of C. elegans. Validity criteria were met in all laboratories. Average median- and low-effect concentrations were determined to be 15.1 mg L(-1) (EC50) and 8.7 mg L(-1) (EC10) for growth and 7.5 mg L(-1) (EC50) and 3.8 mg L(-1) (EC10) for reproduction of C. elegans, with ECx values showing a high degree of reproducibility (CV(R) : <21% and <11% for EC10 and EC50, respectively) and repeatability (CV(r) : <20% and <7% for EC10 and EC50, respectively). The toxic effects of the sediments and soils revealed by the different laboratories were well related to each samples' degree of chemical contamination. Moreover, the effects showed an acceptable reproducibility (CV(R) : 5-33% and 0-28% for growth and reproduction, respectively) and repeatability (CV(r) : 3-13% and 0-12% for growth and reproduction, respectively). The present study confirms that the toxicity test with C. elegans according to ISO 10872 is a reliable and precise tool to assess the toxicity of aqueous media, freshwater sediments, and soils.

Combination of a higher-tier flow-through system and population modeling to assess the effects of time-variable exposure of isoproturon on the green algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata.

A flow-through system was developed to investigate the effects of time-variable exposure of pesticides on algae. A recently developed algae population model was used for simulations supported and verified by laboratory experiments. Flow-through studies with Desmodesmus subspicatus and Pseudokirchneriella subcapitata under time-variable exposure to isoproturon were performed, in which the exposure patterns were based on the results of FOrum for Co-ordination of pesticide fate models and their USe (FOCUS) model calculations for typical exposure situations via runoff or drain flow. Different types of pulsed exposure events were realized, including a whole range of repeated pulsed and steep peaks as well as periods of constant exposure. Both species recovered quickly in terms of growth from short-term exposure and according to substance dissipation from the system. Even at a peak 10 times the maximum predicted environmental concentration of isoproturon, only transient effects occurred on algae populations. No modified sensitivity or reduced growth was observed after repeated exposure. Model predictions of algal growth in the flow-through tests agreed well with the experimental data. The experimental boundary conditions and the physiological properties of the algae were used as the only model input. No calibration or parameter fitting was necessary. The combination of the flow-through experiments with the algae population model was revealed to be a powerful tool for the assessment of pulsed exposure on algae. It allowed investigating the growth reduction and recovery potential of algae after complex exposure, which is not possible with standard laboratory experiments alone. The results of the combined approach confirm the beneficial use of population models as supporting tools in higher-tier risk assessments of pesticides.

Diurnal and nocturnal functional response of juvenile Notonecta maculata considered as a consequence of shifting predation behaviour.

Presence or absence of light is considered to significantly affect predation within freshwater communities. In relation to light regime, the scope of the present study is to identify proximate factors accounting for different rates of predation in juvenile Notonecta maculata. It has been shown that foraging of juvenile N. maculata on Daphnia magna is reduced under dark conditions. These differences are accounted for by the presence/absence of light and are not regulated by any endogenous circadian rhythm. Direct observation of predation components revealed that in the dark, daphnid prey is detected at shorter distances, thus lowering the rate of encounter which finally results in a decreasing number of prey items eaten. Handling time was found to significantly increase during dark periods. Since the amount of food ingested per prey item increases to a certain extent with increasing handling time, it is suggested that the decreasing number of daphnids consumed is compensated by gaining a higher amount of food per item during a lengthier, more thorough, handling of the prey. A mechanistic model that describes the foraging process along a general predation cycle was parameterized based on the results of direct observations, instead of using classic functional response curves. This allows the comparison of model output to independent functional response data. A good correlation between observed and predicted data confirms the assumption that the reduction of the nocturnal predation rate is caused by shifting predation behaviour and indicates that the crucial light/dark differences in the foraging of N. maculata are considered in the approach.

Some nonylphenol isomers show antiestrogenic potency in the MVLN cell assay.

It has been shown that nonylphenol (NP) isomers vary in their estrogenic potency. These differences may be due to varieties in receptor affinity, receptor activation, or activation/deactivation of non-receptor mediated side paths of reporter gene translation. In the present study we investigated the underlying mechanism of the different estrogenic potency of seven nonylphenol isomers. An estrogen receptor binding assay was conducted with the human estrogen receptor alpha (hERalpha). Additionally we co-incubated the nonylphenol isomers with two concentrations of 17beta-estradiol (E2) in the MVLN cell assay to measure the potency of the isomers to compete with E2. No significant differences were found between the nonylphenol isomer binding affinities for the hERalpha. The IC(50) were in the range of 2.1-8.1x10(-6)M and the binding affinity relative to estradiol (set to 1) were between 2.6 and 6.7x10(-3). Only two test items (p353-NP and p-NP) were able to increase the estrogenic response of 100pM estradiol. The response of the other isomers co-incubated with 100pM E2 showed varying degrees of inhibition of the response in the MVLN reporter gene assay. Thus, it appears that all isomers bind to the ER but some are partial agonists while others are antagonists in the MVLN reporter gene assay.

Thresholds of toxicological concern for endocrine active substances in the aquatic environment.

The threshold of toxicological concern (TTC) concept proposes that an exposure threshold value can be derived for chemicals, below which no significant risk to human health or the environment is expected. This concept goes further than setting acceptable exposure levels for individual chemicals, because it attempts to set a de minimis value for chemicals, including those of unknown toxicity, by taking the chemical's structure or mode of action (MOA) into consideration. This study examines the use of the TTC concern concept for endocrine active substances (EAS) with an estrogenic MOA. A case study formed the basis for a workshop of regulatory, industry and academic scientists held to discuss the use of the TTC in aquatic environmental risk assessment. The feasibility and acceptability, general advantages and disadvantages, and the specific issues that need to be considered when applying the TTC concept for EAS in risk assessment were addressed. Issues surrounding the statistical approaches used to derive TTCs were also discussed. This study presents discussion points and consensus findings of the workshop.

An indispensable asset at risk: merits and needs of chemicals-related environmental sciences.

BACKGROUND: Modern societies depend on environmental sustainability and on new generations of individuals well-trained by environmental research and teaching institutions. In the past, significant contributions to the identification, assessment, and management of chemical stressors with legal consequences have been made. MAIN FEATURES: Within this article, we intend to elucidate the merits and the emerging challenges of chemicals-related environmental sciences. The manuscript is supported by more than 70 professors and university academics of leading institutions in Germany, Switzerland, Austria, and other countries in Europe, but addresses topics of global concern. RESULTS AND DISCUSSION: Many environmental problems of pollutants remain to be addresses, since new chemical compounds or classes of new compounds are continuously developed and brought to the market and sooner or later "emerge" in the environment. Further issues are the inclusion of transformation products and chemical mixtures in environmental risk assessment, the long-term presence of xenobiotics bound to soils and sediments, as well as an understanding of the ecological relevance of ecotoxicological end points. CONCLUSION AND PERSPECTIVES: We point out the need for a strong academic research and education system in chemicals-related environmental sciences to ministries, politicians, and research funding institutions and we propose to create specific units in the national funding bodies that address basic and interdisciplinary research in this field.

Life stage- dependent bioconcentration of a nonylphenol isomer in Daphnia magna.

Bioaccumulation is an important aspect for the fate and effects of xenobiotics in the environment. In this study we used a radiolabeled nonylphenol isomer to investigate the bioconcentration in Daphnia magna at different ages. Apart from the total radioactivity we measured the metabolism of p353-NP in D. magna, to calculate the amount of p353-NP compared to total radioactivity found within the daphnids. Bioconcentration factors, based on wet weight, calculated from the rate constants for total radioactivity in neonates and adults were 4271 kg/l and 760 kg/l respectively, leading to a 5.6 deviance in bioconcentration. This deviance was even more pronounced, nearly one order of magnitude, for the p353-NP concentration with bioconcentration factors of 302 kg/l for neonates and 31 kg/l for adults. We were able to describe the bioconcentration for all daphnids by a weight-dependent one- compartment model. These results pointed out that it is not possible to compare bioconcentration experiments conducted with different substances and different sized daphnids. Additionally it was shown that it is not possible to describe the bioconcentration by measuring the total radioactivity. Metabolism of nonylphenol occurs at a very fast rate and bioconcentration is not triggered by the partition between two phases, but by metabolism. Discrimination between the two mechanisms was achieved using radiolabeled substances and a pseudo two-compartment model to describe metabolism and elimination by two rate constants which afterwards can be compared between different substances.

Nonylphenol isomers differ in estrogenic activity.

Nonylphenol belongs to the most investigated xenohormones acting at the estrogen receptor. Technical nonylphenol contains approximately 20 para-substituted isomers. Because of limitations in testing and quantifying all 20 isomers in the mixture, the linear form, 4n-NP, is often used as a reference substance, even though it is not present in the technical mixture. Here, we report on the synthesis and estrogenic potency of six nonylphenol isomers that occur at different proportions in technical nonylphenol mixtures. The relative potency of each isomer was determined by use of the MVLN transcriptional activation cell assay. As well, a subset of isomers was tested in the E-screen assay. One isomer, p353-NP, exhibited the same relative potency as the nonylphenol mixture, whereas the other isomers were found to be less potent. Two isomers, p22-NP and p262 NP, and the linear 4n-NP were found to be weak ER agonists with responses near the detection limit in the MVLN assay. Two isomers, p262-NP and 4n-NP, exhibited measurable activity in the E-screen. Our results demonstrate that defined p-NP isomers are most suitable for reflecting the estrogenic potency of technical NP mixtures. Among other applications, they should be used in the future to explain differences in estrogenic potency due to NPs as detected by various in vitro assays.

Adaptation to environmental stress in Daphnia magna simultaneously exposed to a xenobiotic.

In standardized ecotoxicological testing chemicals are investigated under optimal conditions for the test organisms despite the fact that environmental factors such as predation pressure and food availability are important parameters regulating natural populations. Food limitation and predator presence can induce shifts in life-history traits in various Daphnia species, especially trade-offs in reproductive biomass allocation. These adaptive responses are thought to ensure survival of the population in a highly variable environment. A xenobiotic dispersant (used in textile dyeing processes) also shifted the biomass allocation of Daphnia magna. To assess whether the dispersant could hinder D. magna adaptation to varying environmental conditions, we conducted experiments with food level and presence of Chaoborus larvae as environmental factors and simultaneous exposure to the dispersant. At low food level and in presence of the predator, D. magna produced fewer but larger sized neonates, regardless of dispersant exposure. The dispersant shifted biomass allocation towards more but smaller sized offspring in all experiments. However, the adaptive response to the environmental factors and the dispersant effect cancelled each other out in that they induced independently from each other opposite shifts in biomass allocation. In summary, the dispersant exposure resulted not in an inhibition of the adaptive response but in a reduction of the value of the response. Our study with this model substance demonstrates that xenobiotics can affect the adaptation of organisms to environmental stress which can result in effects likely to be overlooked in standardized testing.

Removal of estrogenic activity from municipal waste landfill leachate assessed with a bioassay based on reporter gene expression.

The leachate of a municipal waste landfill was tested for estrogenic activity using a reporter-gene-based bioassay with a human breast-cancer-derived cell line (MVLN cells). The leachate was processed with two different membrane-employing processes operating in parallel. One process consists of aerobic biological degradation, ultrafiltration, and subsequent adsorption to activated carbon. The second process was a reverse osmosis treatment of the raw leachate. Both processes are common in the treatment of landfill leachate. Here, the efficacy of the two processes to remove "estrogenicity" was compared. Both treatment processes removed more than 97% of the estrogenic activity, calculated as estradiol equivalents (EEQs), but they were not equally effective. After adsorption to activated carbon, no estrogenicity was detected, whereas concentrated effluent of the reverse osmosis treatment still elicited an estrogenic response in the bioassay. On the basis of chemical analysis, it is proposed that bisphenol A was responsible for the majority of estrogenic activity in the raw and treated leachate. Although the contribution of treated leachate to the estrogenic load on the aquatic environment seems to be low compared to that of sewage treatment works, the high estrogenic activity in raw landfill leachate stresses the necessity for the appropriate treatment of these leachates.

Phytotoxicity of coloured substances: is Lemna duckweed an alternative to the algal growth inhibition test?

Coloured substances cause problems when interpreting algal tests, because effects due to light absorption can interact with potential toxicity. The Lemna Duckweed growth inhibition test can complement the algal test, on condition that the test is performed on a black, not reflecting surface. On white surfaces, test solution colour can strongly impact Lemna growth. For example, average control sample growth rate of is much higher on white surfaces (0.362 d(-1)) than on black surfaces (0.284 d(-1)). We found that 10 mg l(-1) of the dyestuff "Brilliant Blue R spezial" inhibited average Lemna growth rate about 22% on white surfaces but did not inhibit growth on black surfaces. The reason for this difference stems from the difference in amount of light reflected from below the test beakers. With Brilliant Blue on white surfaces, the test solution colour reduces utilizable light and causes a deterioration of light conditions, whereas on a black surfaces, reflected light is absent a priori, and thus no inhibiting effect was measured. Of particular importance is the choice of test parameter. With Brilliant Blue, a LOEC for average growth rate, based on frond numbers, of 320 mg l(-1) was determined. However, when average growth rate was calculated using dry weights of the plants, the LOEC decreased clearly to 1.0 mg l(-1). In this study, the Lemna test was much more sensitive than the algal test. We recommend Lemna tests be used in addition to algal tests, because doing so may significantly improve the assessment of phytotoxicity of chemicals and sewage.

The importance of light intensity in algal tests with coloured substances.

Current algal test protocols do not distinguish between chemical toxicity and growth inhibition due to physical shading when coloured substances like dyes are evaluated. To eliminate the interfering physical effect, we investigated the importance of light intensity and culture volume in the algal growth inhibition tests with Desmodesmus subspicatus and three selected dyestuffs. A photon flux density above light saturation is needed to prevent inhibition of algal growth solely due to the light absorption by dyes. Furthermore, a smaller culture volume and moderate shaking of the test flasks is advantageous in that these conditions decrease the influence of the light absorption. At 120 microE s(-1) m(-2), the shading effect of three tested dyes could be eliminated, up to a concentration of 100 mg l(-1). In a mixture with a blue dyestuff, the model toxicant potassium dichromate showed nearly the same EC50 as if applied alone, indicating that the impact of the dye was negligible. Also a small culture volume decreases the inhibition of the average growth rate of Desmodesmus. Thus, by increasing the light intensity and optionally also reducing the culture volume, this method allows a more direct measure of a potential inherent chemical toxicity of coloured substances to algae. Additionally, high light intensities and lower culture volumes result in lower coefficients of variation, which improves the sensitivity of the test for the statistical calculation of toxicity data.