Environmental risks to freshwater organisms from the mycotoxins deoxynivalenol and zearalenone using Species Sensitivity Distributions.

In this study, laboratory experiments have addressed the acute toxicity of two common mycotoxins, deoxynivalenol (DON) and zearalenone (ZON), in a range of freshwater organisms (including rotifers Brachionus calyciflorus, insects Chironomus riparius (larvae), crustaceans Daphnia pulex and Thamnocephalus platyurus, cnidarians Hydra vulgaris, molluscs Lymnaea stagnalis (embryos) and Protozoa Tetrahymena thermophila). Acute EC50 values highlight crustaceans as the most sensitive organisms to DON, with T. platyurus having a 24 h EC50 of 0.14 and D. magna having a 48 h EC50 of 0.13 mg DON/L. During exposures to ZON, H. vulgaris and L. stagnalis embryos showed the highest sensitivity; mortality EC50 values were 1.1 (96 h) and 0.42 mg ZON/L (7 d), respectively. Combining these novel invertebrate toxicity results, along with recent published data for freshwater plant and fish toxicity for analysis of Species Sensitivity Distributions, provides freshwater HC5 values of 5.2 µg DON/L and 43 µg ZON/L, respectively. Using highest reported environmental concentrations and following REACH guidelines, risk ratios calculated here show the risk of ZON to freshwater organisms is low. In contrast, DON may periodically because for concern in streams subject to high agricultural run-off, likely during certain times of year where cereal crops are susceptible to higher fungal infections rates and may pose increased risks due to climate change.

Key Opportunities to Replace, Reduce, and Refine Regulatory Fish Acute Toxicity Tests.

Fish acute toxicity tests are conducted as part of regulatory hazard identification and risk-assessment packages for industrial chemicals and plant protection products. The aim of these tests is to determine the concentration which would be lethal to 50% of the animals treated. These tests are therefore associated with suffering in the test animals, and Organisation for Economic Co-operation and Development test guideline 203 (fish, acute toxicity) studies are the most widely conducted regulatory vertebrate ecotoxicology tests for prospective chemical safety assessment. There is great scope to apply the 3Rs principles-the reduction, refinement, and replacement of animals-in this area of testing. An expert ecotoxicology working group, led by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research, including members from government, academia, and industry, reviewed global fish acute test data requirements for the major chemical sectors. The present study highlights ongoing initiatives and provides an overview of the key challenges and opportunities associated with replacing, reducing, and/or refining fish acute toxicity studies-without compromising environmental protection. Environ Toxicol Chem 2020;39:2076-2089. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Factors Affecting the Growth of Pseudokirchneriella subcapitata in Single-Species Tests: Lessons for the Experimental Design and the Reproducibility of a Multitrophic Laboratory Microcosm.

The need for an integrated risk assessment at an ecologically relevant scale (e.g., at the population/community levels) has been acknowledged. Multispecies systems with increased ecological complexity, however, are difficult if not impossible to reproduce. The laboratory-scale microcosm TriCosm (Pseudokirchneriella subcapitata, Ceriodaphnia dubia, Hydra viridissima) of intermediate complexity was developed for the reproducible assessment of chemical effects at the population/community levels. The system dynamics were repeatable in the short term, but interexperimental variation of algal dynamics in the long term triggered knock-on effects on grazer and predator populations. We present 20 experiments to assess the effects of 12 factors (test medium, vessel type/condition, shaking speed, light intensity/regime, inoculation density, medium preparation components, metal concentration/composition, buffering salt type/concentration) on algal growth in the TriCosm enclosure. Growth rates varied between ≤ 0 and 1.40 (± 0.21) and generally were greatest with increased shaking speed, light exposure, medium buffer, or aeration time. Treatments conducted in dishes with aseptically prepared, lightly buffered, and/or hardly aerated medium resulted in low growth rates. We found that inter-experimental variation of algal dynamics in the TriCosm was caused by a modification of medium preparation (omission of medium aeration) with the aim of reducing microbial contamination. Our findings highlight the facts that consistency in experimental procedures and in-depth understanding of system components are indispensable to achieve repeatability. Environ Toxicol Chem 2019;00:1-13. © 2019 SETAC.

Impacts of the mycotoxin zearalenone on growth and photosynthetic responses in laboratory populations of freshwater macrophytes (Lemna minor) and microalgae (Pseudokirchneriella subcapitata).

Mycotoxins are an important class of chemicals of emerging concern, recently detected in aquatic environments, potentially reflecting the influence of fungicide resistance and climatic factors on fungal diseases in agricultural crops. Zearalenone (ZON) is a mycotoxin formed by Fusarium spp. and is known for its biological activity in animal tissues; both in vitro and in vivo. ZON has been reported in US and Polish surface waters at 0.7 - 96 ng/L, with agricultural run-off and wastewater treatment plants being the likely sources of mycotoxins. As some mycotoxins can induce phytotoxicity, laboratory studies were conducted to evaluate the toxicity of ZON (as measured concentrations) to freshwater algae (Pseudokirchneriella subcapitata) and macrophytes (Lemna minor) following OECD test guidelines 201 and 221, respectively. Zinc sulphate was used as a positive control. In the OECD 201 algal static study (72 h at 24 ±â€¯1 °C), exposure to ZON gave average specific growth rate (cell density) EC50 and yield (cell density) EC50 values of > 3.1 and 0.92 (0.74 - 1.8) mg/L, respectively. ZON was less toxic in the OECD 221 static study and after 7 d at 24 ±â€¯1 °C. L. minor growth was significantly reduced based on frond number and frond area at 11.4 mg ZON/L, showing a higher tolerance than reported for other mycotoxins with Lemna spp. Chlorophyll fluorescence parameters were used as biomarkers of impacts on photosystem II efficiency, with no effect seen in algae but, with responses being observed in L. minor between 5.2 - 14.4 mg ZON/L. ZON toxicity seen here is not of immediate concern in context with environmental levels, but this study highlights that other freshwater organisms including algae are more sensitive to mycotoxins than Lemna sp., the only current source of toxicity data for freshwater plants.

A standardized tritrophic small-scale system (TriCosm) for the assessment of stressor-induced effects on aquatic community dynamics.

Chemical impacts on the environment are routinely assessed in single-species tests. They are employed to measure direct effects on nontarget organisms, but indirect effects on ecological interactions can only be detected in multispecies tests. Micro- and mesocosms are more complex and environmentally realistic, yet they are less frequently used for environmental risk assessment because resource demand is high, whereas repeatability and statistical power are often low. Test systems fulfilling regulatory needs (i.e., standardization, repeatability, and replication) and the assessment of impacts on species interactions and indirect effects are lacking. In the present study we describe the development of the TriCosm, a repeatable aquatic multispecies test with 3 trophic levels and increased statistical power. High repeatability of community dynamics of 3 interacting aquatic populations (algae, Ceriodaphnia, and Hydra) was found with an average coefficient of variation of 19.5% and the ability to determine small effect sizes. The TriCosm combines benefits of both single-species tests (fulfillment of regulatory requirements) and complex multispecies tests (ecological relevance) and can be used, for instance, at an intermediate tier in environmental risk assessment. Furthermore, comparatively quickly generated population and community toxicity data can be useful for the development and testing of mechanistic effect models. Environ Toxicol Chem 2018;37:1051-1060. © 2017 SETAC.

Development and validation of an OECD reproductive toxicity test guideline with the mudsnail Potamopyrgus antipodarum (Mollusca, Gastropoda).

Mollusks are known to be uniquely sensitive to a number of reproductive toxicants including some vertebrate endocrine disrupting chemicals. However, they have widely been ignored in environmental risk assessment procedures for chemicals. This study describes the validation of the Potamopyrgus antipodarum reproduction test within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment. The number of embryos in the brood pouch and adult mortality serve as main endpoints. The experiments are conducted as static systems in beakers filled with artificial medium, which is aerated trough glass pipettes. The test chemical is dispersed into the medium, and adult snails are subsequently introduced into the beakers. After 28 days the reproductive success is determined by opening the brood pouch and embryo counting. This study presents the results of two validation studies of the reproduction test with eleven laboratories and the chemicals tributyltin (TBT) with nominal concentrations ranging from 10 to 1000 ng TBT-Sn/L and cadmium with concentrations from 1.56 to 25 µg/L. The test design could be implemented by all laboratories resulting in comparable effect concentrations for the endpoint number of embryos in the brood pouch. After TBT exposure mean EC10, EC50, NOEC and LOEC were 35.6, 127, 39.2 and 75.7 ng Sn/L, respectively. Mean effect concentrations in cadmium exposed snails were, respectively, 6.53, 14.2, 6.45 and 12.6 µg/L. The effect concentrations are in good accordance with already published data. Both validation studies show that the reproduction test with P. antipodarum is a well-suited tool to assess reproductive effects of chemicals.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

Advancing the 3Rs in regulatory ecotoxicology: A pragmatic cross-sector approach.

The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area in which a high number of vertebrates are used across a variety of industry sectors. Refining, reducing, and replacing the use of animals such as fish, birds, and amphibians for this purpose addresses the ethical concerns and the increasing legislative requirements to consider alternative test methods. Members of the UK-based National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) Ecotoxicology Working Group, consisting of representatives from academia, government organizations, and industry, have worked together over the past 6 y to provide evidence bases to support and advance the application of the 3Rs in regulatory ecotoxicity testing. The group recently held a workshop to identify the areas of testing, demands, and drivers that will have an impact on the future of animal use in regulatory ecotoxicology. As a result of these discussions, we have developed a pragmatic approach to prioritize and realistically address key opportunity areas, to enable progress toward the vision of a reduced reliance on the use of animals in this area of testing. This paper summarizes the findings of this exercise and proposes a pragmatic strategy toward our key long-term goals-the incorporation of reliable alternatives to whole-organism testing into regulations and guidance, and a culture shift toward reduced reliance on vertebrate toxicity testing in routine environmental safety assessment. Integr Environ Assess Manag 2016;12:417-421. © 2015 SETAC.

17ß-Oestradiol may prolong reproduction in seasonally breeding freshwater gastropod molluscs.

Whilst the effects of oestrogenic contaminants in the aquatic environment are well documented in fish, effects in invertebrate species has been subject to debate, possibly due to differences in experimental conditions (temperature, timing and duration of exposure) between studies. It has been suggested that molluscs are only susceptible to oestrogens in periods either following the main spawning or leading up to the maturation of gametes. To investigate this possibility, two temperate, seasonally reproducing gastropods (Planorbarius corneus and Viviparus viviparus) were exposed to two concentrations of 17ß-oestradiol (E2; 10ng/l and 100ng/l nominal) in an outdoor mesocosm (subject to natural seasons). In addition, P. corneus was also exposed to E2 (1, 10 and 100ng/l) in the laboratory at temperatures and photoperiods to simulate summer and autumn. In the mesocosm, both snail species produced similar numbers of eggs/embryos as reference groups in the summer, but the groups exposed to 10ngE2/l (nominal) had significantly higher productivities after the onset of autumn, when entering their quiescent phase, whilst the snails exposed to a higher concentration (100ng/l, nominal) had an increased rate of mortality, and did not experience increased reproduction. In the laboratory, the rate of egg laying in P. corneus was unaffected in simulated summer (20°C, 16h photoperiod), but snails exposed to 10 and 100ng/l (nominal) in simulated autumn (15°C, 12h photoperiod) showed a concentration-dependent inhibition of the natural decline in egg laying observed in the control snails. Overall, rather than an increase in reproductive rate, the response of this species was a perpetuation of summer reproductive rates into autumn. We conclude that exposure to E2 can affect reproduction in the freshwater gastropods studied, but in P. corneus at least, this is dependent on the seasonal conditions (temperature and photoperiod) at which exposures are made.

Estrogenic wastewater treatment works effluents reduce egg production in fish.

Estrogenic chemicals found within wastewater treatment work (WwTW) effluents have been shown individually to inhibit reproduction in fish, but the impact of the WwTW effluents themselves and the complex mixtures of environmental estrogens and other endocrine disrupting chemicals they contain has not been established. In this investigation, the effect of exposure to three WwTW effluents, with differing levels of estrogenic activity, was assessed on egg production in pair-breeding fathead minnow. Exposure to two of the three effluents tested resulted in a reduced egg production (by 28% for effluent I at a dilution of 50% and by 44% for effluent III at full strength), which was proportional to the estrogenic content of the effluents. The test effluents, however, had a greater effect on egg production than might be expected, on the basis of both the response they induced for induction of vitellogenin (an estrogen exposure biomarker) and when compared with an equivalent estrogen exposure to EE2. These data show that reliance on relatively simple biomarker responses for estrogenic activity alone, such as vitellogenin, can significantly underestimate the impacts of estrogenic WwTW effluents on fitness parameters such as reproductive health that are regulated by more complex estrogenic (and other endocrine) signaling mechanisms.

Exposure to treated sewage effluent disrupts reproduction and development in the seasonally breeding Ramshorn snail (subclass: Pulmonata, Planorbarius corneus).

Experiments were conducted to assess the impacts of exposure to sewage treatment works (STW) effluent upon the growth, reproductive function, and sexual development of the European mollusc, Planorbarius corneus under seasonally varying temperatures and photoperiodic conditions. In river water, a clear seasonal change in the number and weight of egg masses (during both 2003 and 2004), and in the number of eggs produced, was found, providing evidence for profound effects of both changing temperature and photoperiod on reproduction. Exposure to STW effluent caused disturbances in this seasonal reproductive cycle at all concentrations tested. The effects included significant dose-dependent increases in fecundity and in the overall length of the reproductive cycle in adult snails exposed to both 50% and 100% effluent relative to river water for a period of up to 14 weeks. Disturbances in the development of both the male and female gametes of the both the adult snails and their developmentally exposed offspring were also seen. These effects were more evident in the offspring than in the adults.

A national risk assessment for intersex in fish arising from steroid estrogens.

The occurrence of intersex fish is widespread in the rivers of England and Wales. The extent of intersex in fish populations is believed to be strongly linked to their exposure to steroid estrogens. The present study presents, to our knowledge, the first national, catchment-based risk assessment for steroid estrogens in the world. A graphical information system-based model predicted the concentrations of estradiol (E2), estrone, and ethinylestradiol, which were combined and compared with known biological effect levels to predict the risk of endocrine disruption for 10,313 individual river reaches (21,452 km) receiving effluent from more than 2000 sewage treatment plants serving more than 29 million people. The large scale of this assessment underlines the usefulness of computer-based risk assessment methods. Overall, 61% [corrected] of the modeled reaches (all percentages are in terms of the total river length modeled) in England and Wales were predicted to be not at risk from endocrine disruption (mean concentrations, <1 ng/L E2 equivalents). A large range existed in the percentage of river reaches at risk in the various regions, from 5% in Wales to 67% in the Thames catchment. Important factors influencing this proportion are the population density, particularly their location, and the available dilution. A very small proportion of reaches (approximately 1-3%) were predicted to be at high risk (>10 ng/L E2 equivalents). Many of these high-risk reaches, however, were ditches, which were composed almost entirely of sewage effluent. The model could be applied equally well to any other chemical of concern emanating from the human population that would be impractical to assess by measurement.

A practicable laboratory flow-through exposure system for assessing the health effects of effluents in fish.

The knowledge that exposure to estrogenic wastewater treatment work (WwTW) effluents induces a range of reproductive abnormalities in fish has highlighted the need to understand the wider health effects of effluents. Access to laboratory-based testing systems for WwTW effluents could greatly facilitate this endeavour. In this investigation, a laboratory-based test system was developed and applied for WwTW effluents using fathead minnows (Pimephales promelas). Sexually maturing fathead minnows were exposed, under flow-through conditions in the laboratory, for up to 21 days to graded concentrations of effluent from three different UK (temperate) WwTWs. The stability of the estrogenic component within the test system was assessed via measurements for estradiol and estrone concentrations in the effluent, and through determining estrogenic responses in an in vitro recombinant yeast estrogen screen (rYES) and in fish (plasma vitellogenin induction). The estrogen component of the effluents was stable within the holding system used (chilled <10 degrees C) for up to 7 days and measured concentrations of estradiol and estrone were shown to differ by less than 20% between the first and final day of use for each batch of effluent. Total estrogenic activity as measured in the rYES was found to be more variable (up to 66% variance between measurements for the two time points) but there was no consistent trend for a reduction in estrogenic activity. Vitellogenin was induced in males in a concentration-dependent manner and the magnitude of the response observed was proportional to the average measured concentrations of estradiol and estrone in the exposure effluent. The system described, thus, provides a robust test method for evaluating the estrogenic effects of temperate WwTW effluents that could be further applied to assess wider health effects, including population-relevant endpoints such as reproduction, using model OECD warm-water fish species such as the fathead minnow.

Associations between altered vitellogenin concentrations and adverse health effects in fathead minnow (Pimephales promelas).

Mechanism specific biomarkers are used in ecotoxicology to identify classes of chemicals and to inform on their presence in the environment, but their use in signalling for adverse effects has been limited by a poor understanding of their associated links with health. In this study an experimental analysis was undertaken to investigate how induction or suppression of an estrogen-dependent biomarker, vitellogenin (VTG), related to health effects in fathead minnow (Pimephales promelas, FHM). Exposure to an oestrogen agonist, estradiol (29 and 60 ng/L), resulted in rapid induction of VTG (elevated plasma concentrations within 2 days of exposure) in male FHM that was subsequently slow to clear from the plasma (concentrations remained elevated 70 days after cessation of exposure). The induction of VTG to concentrations of 0.5 mg/mL, however, and its continued presence in the plasma were not associated with any overt adverse health effects to the males. In contrast, induction of higher concentrations of VTG (>1 mg/mL) in reproductively active FHM exposed to estrone (307 and 781 ng/L), were associated with impacts on male survival (>33% male mortality) and an inhibitory effect on egg production in females (>51% decrease in egg number). Exposure of reproductively active FHM to a chemical that disrupts estrogen biosynthesis (an aromatase inhibitor; fenarimol 497 microg/L) also reduced reproductive success (40% decrease in egg number), and this was associated with a reduction in plasma VTG concentrations in females (36% decrease). These findings show that high level induction or suppression (in females) of plasma VTG are associated with alterations in health status and reproductive fitness. VTG, therefore, has the potential to act as a health measure as well as a biomarker for exposure, for chemicals that alter the oestrogen signalling pathway.

An optimised experimental test procedure for measuring chemical effects on reproduction in the fathead minnow, Pimephales promelas.

The production of viable offspring is fundamental to the survival of any population. Tests that quantify effects on reproduction can, therefore, inform on the potential for long-term health effects of exposure to endocrine active chemicals. Surprisingly little is known, however, about the reproductive capacity of laboratory fish species used for chemical testing. As an example, the fathead minnow, Pimephales promelas, is widely used in chronic assessments of reproductive toxicology, and is readily induced to reproduce in captivity, yet there is little agreement on the reproductive capacity (egg number) of this species. For this species, the notable variation in reported estimates of egg number might relate to differences in the methods of egg collection adopted by many laboratories. To investigate this hypothesis, reproduction was assessed in a total of 200 pair-breeding fathead minnow, using egg collection methods that included the addition of trays placed beneath an inverted U-shaped PVC tile that is conventionally used alone for egg collection. The results demonstrated that the placement of a mesh-screened egg collection tray, beneath the spawning tile, increased estimates of the egg number by 25-67%. In addition, adopting the mesh-screened tray reduced variation in egg number between pairs, within an experiment, from >50% to <30% and variation between experiments was reduced from 53% to 7%. Adoption of the revised system for egg collection shows that egg number in the fathead minnow is considerably more consistent than frequently reported and is a highly robust endpoint against which chemical effects can be challenged effectively.

Bioassay selection, experimental design and quality control/assurance for use in effluent assessment and control.

In the UK Direct Toxicity Assessment Programme, carried out in 1998-2000, a series of internationally recognised short-term toxicity test methods for algae, invertebrates and fishes, and rapid methods (ECLOX and Microtox) were used extensively. Abbreviated versions of conventional tests (algal growth inhibition tests, Daphnia magna immobilisation test and the oyster embryo-larval development test) were valuable for toxicity screening of effluent discharges and the identification of causes and sources of toxicity. Rapid methods based on chemiluminescence and bioluminescence were not generally useful in this programme, but may have a role where the rapid test has been shown to be an acceptable surrogate for a standardised test method. A range of quality assurance and control measures were identified. Requirements for quality control/assurance are most stringent when deriving data for characterising the toxic hazards of effluents and monitoring compliance against a toxicity reduction target. Lower quality control/assurance requirements can be applied to discharge screening and the identification of causes and sources of toxicity.

Estimating spatial patterns of effluent exposure concentrations in direct toxicity assessment studies.

Hydrodynamic models of differing scale and complexity were used to estimate spatial patterns of effluent concentration in discharge plumes in the River Esk and the Lower Tees Estuary. The output from the Tees model was used, in conjunction with measurements of toxicity determined in short-term oyster embryo tests, to predict contours/zones of toxicity in the estuary associated with effluent discharges from four chemical processing sites. One of the discharges also combined the input from a municipal sewage treatment works. The models appeared to be effective in predicting patterns of dilution and dispersion of the effluent discharges in the respective receiving environments. Confirmation of the predictive capabilities of the Tees model was achieved by comparing predicted and measured toxicity in different regions of the plumes associated with the four discharges. Differences between predicted and measured toxicity for two of the four discharges were explicable in terms of failure to take account of the effects of real-time wind conditions when test samples were collected or overlap of adjacent discharge plumes. Suggested refinements to the models and measurement of effluent toxicity would further enhance the utility of this approach for determining the extent and significance of the effects of effluent discharges in receiving environments.

Toxicity reduction evaluation, toxicity identification evaluation and toxicity tracking in direct toxicity assessment.

Toxicity reduction evaluations (TREs) in the River Esk and Lower Tees Estuary were based on the approach described by USEPA, but adapted to tackle the specific problems of the two sites. A combination of toxicity tracking and toxicity identification evaluation (TIE) was used at both locations to enhance the understanding of source and type of toxicants present. The assessment of toxicity at Langholm focussed on pesticides present in the sewerage network. The TIE programme indicated that the most likely toxic agents within the effluent were the organophosphate pesticides diazinon and to lesser extent propetamphos, although these did not account for all of the observed toxicity. The exact source of these toxicants was not clear although toxicity tracking identified two potential candidates. The TRE undertaken on the discharge to the lower Tees utilised high-throughput methods with standard test organisms to generate toxicity information throughout a complex sewerage network. The toxicity tracking information was used in conjunction with TIEs to identify a number of key sources of toxicity. Substantial toxicity was associated with a currently untreated industrial effluent. Chemical analysis and TIE highlighted cyanide as the likely toxicant in this effluent and its possible significance in the final discharge.