Seasonal dynamics of the standard test species Lemna sp. in outdoor microcosms.

Lemna L. sp. is a free-floating aquatic macrophyte that plays a key role as a standard test species in aquatic risk assessment for herbicides and other contaminants. Population modeling can be used to extrapolate from laboratory to field conditions. However, there are insufficient data on longer-term seasonal dynamics of this species to evaluate such models. Therefore, several long-term growth experiments were conducted in outdoor microcosms (surface area 0.174 m2). Monitoring parameters included biomass, frond numbers, water parameters, and weather data. Three different datasets were generated: frond numbers and biomass from weekly to monthly destructively sampled microcosms; a year-round dataset of frond numbers from five continuously monitored microcosms; and seasonal growth rates without the effect of density dependence over 1-2 weeks in freshly inoculated microcosms. Lemna sp. reached a maximum of approximately 500 000 fronds m-2 and 190 g dry weight m-2. During the first winter, the microcosms were covered by ice for approximately four weeks, and Lemna sp. populations collapsed. The second winter was warmer, without any ice cover, and Lemna sp. populations maintained high abundance throughout the winter. Dry weight per frond was not constant throughout the year but was highest in autumn and winter. Growth rates without density dependence under outdoor environmental conditions reached 0.29 day-1 for frond number, 0.43 day-1 for fresh weight, and 0.39 day-1 for dry weight. In linear regressions, these growth rates were best explained by water temperature. For the populations continuously monitored throughout a year, the nitrogen-to-phosphorus ratio best explained the growth rate of frond numbers. This study yielded a relevant dataset for testing and refining Lemna population models used in chemical risk assessment as well as for managing ecosystems and combating the effects of eutrophication. Integr Environ Assess Manag 2024;00:1-14. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Mixture Toxicity of Herbicides with Dissimilar Modes of Action to Myriophyllum spicatum.

Considering the vital role of rooted macrophytes in the aquatic ecosystem, validating assumptions on the interactive effects of herbicides with different modes of action at an environmentally relevant mixture ratio is necessary. We investigated the effects of diflufenican (a carotenoid biosynthesis inhibitor) and iodosulfuron-methyl-sodium (IMS; an acetolactate synthase inhibitor) in a 14-day growth inhibition experiment with Myriophyllum spicatum, wherein single compounds and their combination were tested in parallel (n = 84). The assessment was done using three different methods: significance testing, model deviation ratio (MDR), and mixture interaction factor (MIF). Interactions relative to both concentration addition and independent action were assessed via significance testing. This revealed that diflufenican and IMS acted antagonistically relative to both models for fresh weight and total shoot length (p < 0.05) and that there was slight synergism for the number of side shoots (p < 0.001) relative to concentration addition. The MDR and MIF can only assess interactions relative to the concentration addition model. According to MDR, the mixture appeared to show no interaction (neither antagonistic nor synergistic), whereas the MIF method revealed that the compounds acted antagonistically for fresh weight and that there was a slight synergism for total shoot length and number of side shoots. We conclude that inferences about mixture toxicity interactions are method- and endpoint-dependent, which can have implications for regulatory mixtures assessment. Environ Toxicol Chem 2022;41:2209-2220. © 2022 SETAC.

Seasonal dynamics of the macrophyte test species Myriophyllum spicatum over two years in experimental ditches for population modeling application in risk assessment.

Myriophyllum spicatum is a sediment-rooted, aquatic macrophyte growing submerged, with a wide geographical distribution and high ecological relevance in freshwater ecosystems. It is used in testing and risk assessment for pesticides in water and sediment. Population models enable effects measured under laboratory conditions to be extrapolated to effects expected in the field with time-variable environmental factors including exposure. These models are a promising tool in higher-tier risk assessments. However, there is a lack of data on the seasonal dynamics of M. spicatum, which is needed to test model predictions of typical population dynamics in the field. To generate such data, a two-year study was set up in outdoor experimental systems from May 2017 to May 2019. The growth of M. spicatum was monitored in 0.2025 m2 plant baskets installed in an experimental ditch. Parameters monitored included biomass (fresh weight [FW] and dry weight [DW]), shoot length, seasonal short-term growth rates of shoots, relevant environmental parameters, and weather data. The results showed a clear seasonal pattern of biomass and shoot length and their variability. M. spicatum reached a maximum total shoot length (TSL) of 279 m m-2 and a maximum standing crop above-ground DW of 262 g m-2 . Periodical growth rates reached up to 0.072, 0.095, and 0.085 day-1 for total length, FW, and DW, respectively. Multivariate regression revealed that pH (as a surrogate for the availability of carbon species) and water temperature could explain a significant proportion of the variability in M. spicatum growth rates (p < 0.05). This study has provided an ecologically relevant data set on seasonal population dynamics representative of shallow freshwater ecosystems, which can be used to test and refine population models for use in chemical risk assessment and ecosystem management. Integr Environ Assess Manag 2022;18:1375-1386. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Fungicides: An Overlooked Pesticide Class?

Fungicides are indispensable to global food security and their use is forecasted to intensify. Fungicides can reach aquatic ecosystems and occur in surface water bodies in agricultural catchments throughout the entire growing season due to their frequent, prophylactic application. However, in comparison to herbicides and insecticides, the exposure to and effects of fungicides have received less attention. We provide an overview of the risk of fungicides to aquatic ecosystems covering fungicide exposure (i.e., environmental fate, exposure modeling, and mitigation measures) as well as direct and indirect effects of fungicides on microorganisms, macrophytes, invertebrates, and vertebrates. We show that fungicides occur widely in aquatic systems, that the accuracy of predicted environmental concentrations is debatable, and that fungicide exposure can be effectively mitigated. We additionally demonstrate that fungicides can be highly toxic to a broad range of organisms and can pose a risk to aquatic biota. Finally, we outline central research gaps that currently challenge our ability to predict fungicide exposure and effects, promising research avenues, and shortcomings of the current environmental risk assessment for fungicides.

Is the tier-1 effect assessment for herbicides protective for aquatic algae and vascular plant communities?

In the aquatic tier-1 effect assessment for plant protection products with an herbicidal mode of action in Europe, it is usually algae and/or vascular plants that determine the environmental risks. This tier includes tests with at least 2 algae and 1 macrophyte (Lemna). Although such tests are considered to be of a chronic nature (based on the duration of the test in relation to the life cycle of the organism), the measurement endpoints derived from the laboratory tests with plants (including algae) and used in the first-tier effect assessment for herbicides are acute effect concentrations affecting 50% of the test organisms (EC50 values) and not no-observed-effect concentrations (NOECs) or effect concentrations affecting 10% of the test organisms (EC10) values. Other European legislative frameworks (e.g., the Water Framework Directive) use EC10 values. The present study contributes to a validation of the tiered herbicide risk assessment approach by comparing the standard first-tier effect assessment with results of microcosm and mesocosm studies. We evaluated EC50 and EC10 values for standard test algae and macrophytes based on either the growth rate endpoint (Er C50) or the lowest available endpoint for growth rate or biomass/yield (Er /Ey C50). These values were compared with the regulatory acceptable concentrations for the threshold option as derived from microcosm and mesocosm studies. For these studies, protection is maintained if growth rate is taken as the regulatory endpoint instead of the lowest value of either growth rate or biomass/yield in conjunction with the standard assessment factor of 10. Based on a limited data set of 14 herbicides, we did not identify a need to change the current practice. Environ Toxicol Chem 2018;37:175-183. © 2017 SETAC.

How TK-TD and population models for aquatic macrophytes could support the risk assessment for plant protection products.

This case study of the Society of Environmental Toxicology and Chemistry (SETAC) workshop MODELINK demonstrates the potential use of mechanistic effects models for macrophytes to extrapolate from effects of a plant protection product observed in laboratory tests to effects resulting from dynamic exposure on macrophyte populations in edge-of-field water bodies. A standard European Union (EU) risk assessment for an example herbicide based on macrophyte laboratory tests indicated risks for several exposure scenarios. Three of these scenarios are further analyzed using effect models for 2 aquatic macrophytes, the free-floating standard test species Lemna sp., and the sediment-rooted submerged additional standard test species Myriophyllum spicatum. Both models include a toxicokinetic (TK) part, describing uptake and elimination of the toxicant, a toxicodynamic (TD) part, describing the internal concentration-response function for growth inhibition, and a description of biomass growth as a function of environmental factors to allow simulating seasonal dynamics. The TK-TD models are calibrated and tested using laboratory tests, whereas the growth models were assumed to be fit for purpose based on comparisons of predictions with typical growth patterns observed in the field. For the risk assessment, biomass dynamics are predicted for the control situation and for several exposure levels. Based on specific protection goals for macrophytes, preliminary example decision criteria are suggested for evaluating the model outputs. The models refined the risk indicated by lower tier testing for 2 exposure scenarios, while confirming the risk associated for the third. Uncertainties related to the experimental and the modeling approaches and their application in the risk assessment are discussed. Based on this case study and the assumption that the models prove suitable for risk assessment once fully evaluated, we recommend that 1) ecological scenarios be developed that are also linked to the exposure scenarios, and 2) quantitative protection goals be set to facilitate the interpretation of model results for risk assessment.

An ecosystem services approach to pesticide risk assessment and risk management of non-target terrestrial plants: recommendations from a SETAC Europe workshop.

The registration of plant protection products (PPPs) in the EU is under Regulation 1107/2009, which recommends a tiered approach to assessing the risk to non-target terrestrial plants (NTTPs). However, little information is provided on how to perform and implement higher tier studies or how to use them to refine the risk assessments. Therefore, a stakeholder workshop was organized to consolidate current knowledge and expertise to aid the further development of testing and assessment procedures for NTTPs. This brief communication highlights the agreed recommendations of the workshop, which relate to the three main themes, i.e. specific protection goals, risk assessment and mitigation. The participants of the workshop adopted the European Food Safety Authority (EFSA) approach of using an ecosystem services framework for identifying specific protection goals. First, delivery and protection of ecosystem services were discussed for in-crop, in-field and off-crop, and off-field areas. Second, lower and higher tier risk assessment methods, including modelling approaches, were evaluated. Third, options for risk mitigation of spray drift and run-off were discussed and evaluated. Several important knowledge gaps were identified, and specific data collation and literature-based tasks were actioned to begin to address them. A full workshop report is planned for the fall of 2014.

Uptake, translocation, and elimination in sediment-rooted macrophytes: a model-supported analysis of whole sediment test data.

Understanding bioaccumulation in sediment-rooted macrophytes is crucial for the development of sediment toxicity tests using macrophytes. Here, we explore bioaccumulation in sediment-rooted macrophytes by tracking and modeling chemical flows of chlorpyrifos, linuron, and six PCBs in water-sediment-macrophyte systems. Chemical fluxes across the interfaces between pore water, overlying water, shoots, and roots were modeled using a novel multicompartment model. The modeling yielded the first mass-transfer parameter set reported for bioaccumulation by sediment-rooted macrophytes, with satisfactory narrow confidence limits for more than half of the estimated parameters. Exposure via the water column led to rapid uptake by Elodea canadensis and Myriophyllum spicatum shoots, followed by transport to the roots within 1-3 days, after which tissue concentrations gradually declined. Translocation played an important role in the exchange between shoots and roots. Exposure via spiked sediment led to gradual uptake by the roots, but subsequent transport to the shoots and overlying water remained limited for the chemicals studied. These contrasting patterns show that exposure is sensitive to test set up, chemical properties, and species traits. Although field-concentrations in water and sediment will differ from those in the tests, the model parameters can be assumed applicable for modeling exposure to macrophytes in the field.

Chronic aquatic effect assessment for the fungicide azoxystrobin.

The present study examined the ecological effects of a range of chronic exposure concentrations of the fungicide azoxystrobin in freshwater experimental systems (1270-L outdoor microcosms). Intended and environmentally relevant test concentrations of azoxystrobin were 0 µg active ingredient (a.i.)/L, 0.33 µg a.i./L, 1 µg a.i./L, 3.3 µg a.i./L, 10 µg a.i./L, and 33 µg a.i./L, kept at constant values. Responses of freshwater populations and community parameters were studied. During the 42-d experimental period, the time-weighted average concentrations of azoxystrobin ranged from 93.5% to 99.3% of intended values. Zooplankton, especially copepods and the Daphnia longispina group, were the most sensitive groups. At the population level, a consistent no-observed-effect concentration (NOEC) of 1 µg a.i./L was calculated for Copepoda. The NOEC at the zooplankton community level was 10 µg azoxystrobin/L. The principle of the European Union pesticide directive is that lower-tier regulatory acceptable concentrations (RACs) are protective of higher-tier RACs. This was tested for chronic risks from azoxystrobin. With the exception of the microcosm community chronic RAC (highest tier), all other chronic RAC values were similar to each other (0.5-1 µg a.i./L). The new and stricter first-tier species requirements of the European Union pesticide regulation (1107/2009/EC) are not protective for the most sensitive populations in the microcosm study, when based on the higher tier population RAC. In comparison, the Water Framework Directive generates environmental quality standards that are 5 to 10 times lower than the derived chronic RACs.

Inter-laboratory trial of a standardized sediment contact test with the aquatic plant Myriophyllum aquaticum (ISO 16191).

A whole-sediment toxicity test with Myriophyllum aquaticum has been developed by the German Federal Institute of Hydrology and standardized within the International Organization for Standardization (ISO; ISO 16191). An international ring-test was performed to evaluate the precision of the test method. Four sediments (artificial, natural) were tested. Test duration was 10 d, and test endpoint was inhibition of growth rate (r) based on fresh weight data. Eighteen of 21 laboratories met the validity criterion of r ≥ 0.09 d(-1) in the control. Results from 4 tests that did not conform to test-performance criteria were excluded from statistical evaluation. The inter-laboratory variability of growth rates (20.6%-25.0%) and inhibition (26.6%-39.9%) was comparable with the variability of other standardized bioassays. The mean test-internal variability of the controls was low (7% [control], 9.7% [solvent control]), yielding a high discriminatory power of the given test design (median minimum detectable differences [MDD] 13% to 15%). To ensure these MDDs, an additional validity criterion of CV ≤ 15% of the growth rate in the controls was recommended. As a positive control, 90 mg 3,5-dichlorophenol/kg sediment dry mass was tested. The range of the expected growth inhibition was proposed to be 35 ± 15%. The ring test results demonstrated the reliability of the ISO 16191 toxicity test and its suitability as a tool to assess the toxicity of sediment and dredged material.

Effects of linuron on a rooted aquatic macrophyte in sediment-dosed test systems.

Effects of linuron on the sediment-rooted aquatic macrophyte Myriophyllum spicatum L. were studied in sediment-dosed test systems following a proposed guideline with extended test duration. Sediment, pore water, overlying water and macrophyte shoots were sampled weekly for chemical analyses. Linuron was stable in the sediments. Sediment and pore water concentrations were in equilibrium after 48 h. Overlying water concentrations increased over time, but did not reach equilibrium with pore water concentrations and were 100 times lower. Mass balances showed a rapid uptake of linuron by macrophyte roots. Known pathways and the compound's properties support the conclusion that Myriophyllum takes up linuron from pore water directly through the roots. Hence, effects on macrophytes in this type of sediment toxicity test should be expressed in terms of pore water concentrations. Pore water concentration is the most relevant parameter for describing effects on macrophytes.

The relative sensitivity of macrophyte and algal species to herbicides and fungicides: an analysis using species sensitivity distributions.

Lemna spp. are the standard test species representing aquatic macrophytes in the current risk assessment schemes for herbicides and plant growth regulators in the European Union and North America. At a Society of Environmental Toxicology and Chemistry (SETAC) 2008 workshop on Aquatic Macrophyte Risk Assessment for Pesticides (AMRAP), a Species Sensitivity Distribution (SSD) working group was formed to address uncertainties about the sensitivity of Lemna spp. relative to other aquatic macrophyte species. For 11 herbicides and 3 fungicides for which relevant and reliable data were found for at least 6 macrophyte species, SSDs were fitted using lognormal regression. The positions of L. gibba (the most commonly tested Lemna species) and Myriophyllum spicatum (for which standardized test methods are under development) in each SSD were determined where data were available. The sensitivity of standard algal test species required for pesticide registration in the United States under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) relative to the macrophytes in each SSD was also examined (algae were not included in the SSD). L. gibba was among the most sensitive macrophyte species for approximately 50% of the chemicals examined. M. spicatum was among the most sensitive macrophytes for approximately 25% of the chemicals. In most cases, the lowest FIFRA algal species endpoint was lower than the most sensitive macrophyte endpoint. Although no single species consistently represented the most sensitive aquatic plant species, for 12 of 14 chemicals L. gibba and the FIFRA algae included an endpoint near or below the 5th percentile of the macrophyte SSD. For the other compounds, M. spicatum was the most sensitive species of all aquatic plants considered.

Towards a renewed research agenda in ecotoxicology.

New concerns about biodiversity, ecosystem services and human health triggered several new regulations increasing the need for sound ecotoxicological risk assessment. The PEER network aims to share its view on the research issues that this challenges. PEER scientists call for an improved biologically relevant exposure assessment. They promote comprehensive effect assessment at several biological levels. Biological traits should be used for Environmental risk assessment (ERA) as promising tools to better understand relationships between structure and functioning of ecosystems. The use of modern high throughput methods could also enhance the amount of data for a better risk assessment. Improved models coping with multiple stressors or biological levels are necessary to answer for a more scientifically based risk assessment. Those methods must be embedded within life cycle analysis or economical models for efficient regulations. Joint research programmes involving humanities with ecological sciences should be developed for a sound risk management.

AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology.

INTRODUCTION AND BACKGROUND: Primary producers play critical structural and functional roles in aquatic ecosystems; therefore, it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals. The standard required macrophyte test species is the floating (non-sediment-rooted) duckweed Lemna spp. This macrophyte species might not be representative of all floating, rooted, emergent, and submerged macrophyte species because of differences in the duration and mode of exposure; sensitivity to the specific toxic mode of action of the chemical; and species-specific traits (e.g., duckweed's very short generation time). DISCUSSION AND PERSPECTIVES: These topics were addressed during the workshop entitled "Aquatic Macrophyte Risk Assessment for Pesticides" (AMRAP) where a risk assessment scheme for aquatic macrophytes was proposed. Four working groups evolved from this workshop and were charged with the task of developing Tier 1 and higher-tier aquatic macrophyte risk assessment procedures. Subsequently, a SETAC Advisory Group, the Macrophyte Ecotoxicology Group (AMEG) was formed as an umbrella organization for various macrophyte working groups. The purpose of AMEG is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field, including prospective as well as retrospective risk assessments for chemicals. As AMEG expands, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services.

Sensitivity of submersed freshwater macrophytes and endpoints in laboratory toxicity tests.

The toxicological sensitivity and variability of a range of macrophyte endpoints were statistically tested with data from chronic, non-axenic, macrophyte toxicity tests. Five submersed freshwater macrophytes, four pesticides/biocides and 13 endpoints were included in the statistical analyses. Root endpoints, reflecting root growth, were most sensitive in the toxicity tests, while endpoints relating to biomass, growth and shoot length were less sensitive. The endpoints with the lowest coefficients of variation were not necessarily the endpoints, which were toxicologically most sensitive. Differences in sensitivity were in the range of 10-1000 for different macrophyte-specific endpoints. No macrophyte species was consistently the most sensitive. Criteria to select endpoints in macrophyte toxicity tests should include toxicological sensitivity, variance and ecological relevance. Hence, macrophyte toxicity tests should comprise an array of endpoints, including very sensitive endpoints like those relating to root growth.

Ecological impact in ditch mesocosms of simulated spray drift from a crop protection program for potatoes.

Outdoor aquatic ditch mesocosms were treated with a range of pesticides to simulate various spray drift rates resulting from a typical crop protection program used in the cultivation of potatoes in The Netherlands. The main experimental aims of the present study were to provide information on the fate and ecological effects of drift of the pesticides into surface water and to evaluate the effectiveness of drift-reduction measures in mitigating risks. The pesticides selected and the dosage, frequency, and timing of application were based on normal agricultural practices in the potato crop. Applications of prosulfocarb, metribuzin (both herbicides), lambda-cyhalothrin (insecticide), chlorothalonil, and fluazinam (both fungicides) were made in the sequence typical of the spray calendar for potatoes. A total of 15 treatments with the various compounds were made by spray application to the water surface at 0.2%, 1%, and 5% of the recommended label rates. Chemical fate and effects on ecosystem function and structure (phytoplankton, zooplankton, chlorophyll-a, macroinvertebrates, macrophytes, breakdown of plant litter) were investigated. To interpret the observed effects, treatment concentrations were also expressed in toxic units (TU), which describe the relative toxicity of the compounds with standard toxicity test organisms (Daphnia and algae). After treatment, each compound disappeared from the water phase within 2 d, with the exception of prosulfocarb, for which 50% dissipation time (DT50) values ranged between 6 and 7 d. At the 5% treatment level, an exposure peak of 0.9 TUalgae was observed, which resulted in short-term responses of pH, oxygen, and phytoplankton. At the 5% treatment level, exposure concentrations also exceeded 0.1 TUDaphnia, and this resulted in long-term effects on zooplankton and macroinvertebrates, some of which did not fully recover by the end of the present study. At the 1% treatment level, only slight transient effects were observed on a limited number of zooplankton and macro-invertebrate species and on pH. At the 0.2% level, no consistent treatment-related effects were observed. Most of the observed effects were consistent with the results from higher-tier and mesocosm studies with the individual compounds. Multi and repeated stress played a small role within the applied pesticide package, because of rapid dissipation of most substances and the absence of many simultaneous applications. This suggests that risk assessments based on the individual compounds would in this case have been sufficiently protective for their uses in a crop protection program.

Effects of lambda-cyhalothrin in two ditch microcosm systems of different trophic status.

The fate and effects of the pyrethroid insecticide lambda-cyhalothrin were compared in mesotrophic (macrophyte-dominated) and eutrophic (phytoplankton-dominated) ditch microcosms (approximately 0.5 m3). Lambda-cyhalothrin was applied three times at one-week intervals at concentrations of 10, 25, 50, 100, and 250 ng/L. The rate of dissipation of lambda-cyhalothrin in the water column of the two types of test systems was similar. After 1 d, only 30% of the amount applied remained in the water phase. Initial, direct effects were observed primarily on arthropod taxa. The most sensitive species was the phantom midge (Chaoborus obscuripes). Threshold levels for slight and transient direct toxic effects were similar (10 ng/L) between types of test systems. At treatment levels of 25 ng/L and higher, apparent population and community responses occurred. At treatments of 100 and 250 ng/L, the rate of recovery of the macroinvertebrate community was lower in the macrophyte-dominated systems, primarily because of a prolonged decline of the amphipod Gammarus pulex. This species occurred at high densities only in the macrophyte-dominated enclosures. Indirect effects (e.g., increase of rotifers and microcrustaceans) were more pronounced in the plankton-dominated test systems, particularly at treatment levels of 25 ng/L and higher.