Response of a nematode community to the fungicide fludioxonil in sediments of outdoor freshwater microcosms compared to a single species toxicity test.

When entering aquatic ecosystems, hydrophobic organic chemicals like the fungicide fludioxonil partition to the sediment compartment where they pose potential risks to benthic invertebrates. To assess the ecological risk for sediment-dwelling invertebrates, nematodes are a suitable organism group, as they are abundantly present and possess key positions in the benthic food web. Therefore, the toxicity of the fungicide fludioxonil to nematodes was assessed in a standardized sediment toxicity test with Caenorhabditis elegans (ISO 10872), as well as in an outdoor sediment-spiked microcosm test system. In the microcosms, effects on the nematode species composition were studied, while exposure concentrations of fludioxonil were monitored in total sediment and pore water. Toxic effects on nematodes were better predicted using concentrations in pore water than total sediment concentrations. In laboratory single species tests, fludioxonil showed considerably lower toxicity in spiked field-collected sediment, compared to artificial ISO-sediments. Applying an assessment factor of 10 to the C. elegans 96-h EC10, a Tier-1 RACNematode of 7.99 mg kg-1 dry artificial sediment (corresponding to 69 µg l-1 in pore water) appeared to be protective for nematode communities in microcosms that showed no response in total abundance and species composition up to 39.9 mg fludioxonil kg-1 dry sediment (corresponding to 392 µg l-1 in pore water).

Toxicity of sediment-bound lufenuron to benthic arthropods in laboratory bioassays.

This paper deals with species sensitivity distributions (SSDs) for the lipophilic insecticide lufenuron and benthic arthropods based on sediment-spiked laboratory toxicity tests. This compound that inhibits chitin synthesis and moulting of arthropods persists in sediment. Using field-collected sediment, toxicity tests were conducted with three macro-crustaceans and six insects. The Hazardous Concentration to 5% of the tested species, the HC5 (and 95% confidence limit), derived from an SSD constructed with 10d-LC50's was 2.2 (1.2-5.7) µg/g organic carbon (OC) in dry sediment. In addition, HC5 values derived from SSDs constructed with 28d-LC10 and 28-d LC50 values were 0.13 (0.02-1.50) µg/g OC and 2.0 (1.3-5.5) µg/g OC, respectively. In 28d toxicity tests with Chironomus riparius and Hyalella azteca, a higher sensitivity was observed when using lufenuron-spiked field-collected sediment than in lufenuron-spiked artificial sediment. Overall, the non-biting midge C. riparius appeared to be a representative and sensitive standard test species to assess effects of lufenuron exposure in sediment. The Tier-1 (based on standard test species), Tier-2 (based on standard and additional test species) and Tier-3 (model ecosystem approach) regulatory acceptable concentrations (RACs) for sediment-spiked lufenuron did not differ substantially. The Tier-2 RAC was the lowest. Since to our knowledge this study is the first in the open literature that evaluates the tiered approach in the sediment effect assessment procedure for pesticides, we advocate that similar evaluations should be conducted for pesticides that differ in toxic mode-of-action.

Effects of sediment-spiked lufenuron on benthic macroinvertebrates in outdoor microcosms and single-species toxicity tests.

Sediment ecotoxicity studies were conducted with lufenuron to (i) complement the results of a water-spiked mesocosm experiment with this lipophilic benzoylurea insecticide, (ii) to explore the predictive value of laboratory single-species tests for population and community-level responses of benthic macroinvertebrates, and (iii) to calibrate the tier-1 effect assessment procedure for sediment organisms. For this purpose the concentration-response relationships for macroinvertebrates between sediment-spiked microcosms and those of 28-d sediment-spiked single-species toxicity tests with Chironomus riparius, Hyalella azteca and Lumbriculus variegatus were compared. Lufenuron persisted in the sediment of the microcosms. On average, 87.7% of the initial lufenuron concentration could still be detected in the sediment after 12 weeks. Overall, benthic insects and crustaceans showed treatment-related declines and oligochaetes treatment-related increases. The lowest population-level NOEC in the microcosms was 0.79µg lufenuron/g organic carbon in dry sediment (µg a.s./g OC) for Tanytarsini, Chironomini and Dero sp. Multivariate analysis of the responses of benthic macroinvertebrates revealed a community-level NOEC of 0.79µg a.s./g OC. The treatment-related responses observed in the microcosms are in accordance with the results of the 28-d laboratory toxicity tests. These tests showed that the insect C. riparius and the crustacean H. azteca were approximately two orders of magnitude more sensitive than the oligochaete L. variegatus. In our laboratory tests, using field-collected sediment, the lowest 28-d EC10 (0.49µg a.s./g OC) was observed for C. riparius (endpoint survival), while for the standard OECD test with this species, using artificial sediment, a NOEC of 2.35µg a.s./g OC (endpoint emergence) is reported. In this particular case, the sediment tier-1 effect assessment using the chronic EC10 (field-collected sediment) or chronic NOEC (artificial sediment) of C. riparius and an assessment factor of 10, seems to be protective for the treatment-related responses observed in the sediment-spiked microcosms.

The minimum detectable difference (MDD) and the interpretation of treatment-related effects of pesticides in experimental ecosystems.

In the European registration procedure for pesticides, microcosm and mesocosm studies are the highest aquatic experimental tier to assess their environmental effects. Evaluations of microcosm/mesocosm studies rely heavily on no observed effect concentrations (NOECs) calculated for different population-level endpoints. Ideally, a power analysis should be reported for the concentration-response relationships underlying these NOECs, as well as for measurement endpoints for which significant effects cannot be demonstrated. An indication of this statistical power can be provided a posteriori by calculated minimum detectable differences (MDDs). The MDD defines the difference between the means of a treatment and the control that must exist to detect a statistically significant effect. The aim of this paper is to expand on the Aquatic Guidance Document recently published by the European Food Safety Authority (EFSA) and to propose a procedure to report and evaluate NOECs and related MDDs in a harmonised way. In addition, decision schemes are provided on how MDDs can be used to assess the reliability of microcosm/mesocosm studies and for the derivation of effect classes used to derive regulatory acceptable concentrations. Furthermore, examples are presented to show how MDDs can be reduced by optimising experimental design and sampling techniques.

Can time-weighted average concentrations be used to assess the risks of metsulfuron-methyl to Myriophyllum spicatum under different time-variable exposure regimes?

We tested the effects of the herbicide metsulfuron-methyl on growth of the submerged macrophyte Myriophyllum spicatum under laboratory conditions using different exposure scenarios. The exposures of each scenario were comparable in the concentration × time factor, viz., the same 21-d time-weighted average (TWA) concentrations but variable in peak exposure concentrations (ranging from 0.1 to 21000 ng ai L⁻¹) and exposure periods (1, 3, 7, 14 or 21 d). To study recovery potential of the exposed M. spicatum plants we continued the observation on shoot and root growth for another 21 d in herbicide-free medium so that the total observation period was 42 d. Non-destructive endpoints, length and number of new shoots and roots, were determined weekly from day 14 onwards. Destructive endpoints, dry-weight (DW) of main shoots, new shoots and new roots, were measured at the end of the experiment (t=42 d). Metsulfuron-methyl exposure in particular inhibited new tissue formation but was not lethal to main shoots. On days 21 and 42 after start exposure, EC10/EC50 values for new tissues expressed in terms of peak concentration (=measured concentration during exposure periods of different length) showed large differences between exposure scenarios in contrast to EC10/EC50 values for days 21 and 42 expressed in terms of 21-d and 42-d TWA concentrations, respectively. At the end of the experiment (day 42), 42-d TWA EC(x) values were remarkably similar between exposure scenarios, while a similar trend could already be observed on day 21 for 21-d TWA EC(x) values. For the macrophyte M. spicatum and exposure to the herbicide metsulfuron-methyl the TWA approach seems to be appropriate to use in the risk assessment. However, the data from the toxicity experiment suggest that on day 21 also the absolute height of the pulse exposure played a (minor) role in the exposure - response relationships observed.

The species sensitivity distribution approach compared to a microcosm study: a case study with the fungicide fluazinam.

We assessed the sensitivity of freshwater organisms (invertebrates and algae) to the fungicide Shirlan (active ingredient fluazinam) in single-species laboratory tests and in microcosms. Species sensitivity distribution (SSD) curves were constructed by means of acute toxicity data for 14 invertebrate species, since algae were much less sensitive. The EC(10)-based SSD gave a median HC(5) value of 0.6microgL(-1) and a 90% confidence interval of 0.1-1.9 microgL(-1). The EC(50)-based SSD gave a median HC(5) value of 3.9 microgL(-1) (90% confidence interval: 0.9-9.9 microgL(-1)). The microcosms were treated four times with Shirlan (concentration range: 0.4-250 microgL(-1)). Responses of the microcosm communities were followed. The 2 microgL(-1) treatment was the no-observed-effect concentration (NOEC(microcosm)). The 10 microgL(-1) treatment resulted in short-term effects on a few zooplankton taxa. Clear effects were observed at 50 and 250 microgL(-1). The responses in the microcosms were in line with the toxicity data for the tested lab species. The median EC(10)-based HC(5) and the lower limit EC(50)-based HC(5) were lower, and the median EC(50)-based HC(5) was slightly higher than the NOEC(microcosm). This is consistent with other studies that compared SSDs with responses in model ecosystems that received repeated applications of pesticides.

Conceptual model for improving the link between exposure and effects in the aquatic risk assessment of pesticides.

Assessment of risks to aquatic organisms is important in the registration procedures for pesticides in industrialised countries. This risk assessment consists of two parts: (i) assessment of effects to these organisms derived from ecotoxicological experiments (=effect assessment), and (ii) assessment of concentration levels in relevant environmental compartments resulting from pesticide application (=exposure assessment). Current procedures lack a clear conceptual basis for the interface between the effect and exposure assessments which may lead to a low overall scientific quality of the risk assessment. This interface is defined here as the type of concentration that gives the best correlation to ecotoxicological effects and is called the ecotoxicologically relevant concentration (ERC). Definition of this ERC allows the design of tiered effect and exposure assessments that can interact flexibly and efficiently. There are two distinctly different exposure estimates required for pesticide risk assessment: that related to exposure in ecotoxicological experiments and that related to exposure in the field. The same type of ERC should be used consistently for both types of exposure estimates. Decisions are made by comparing a regulatory acceptable concentration (=RAC) level or curve (i.e., endpoint of the effect assessment) with predicted environmental concentration (=PEC) levels or curves (endpoint of the exposure assessment). For decision making based on ecotoxicological experiments with time-variable concentrations a tiered approach is proposed that compares (i) in a first step single RAC and PEC levels based on conservative assumptions, (ii) in a second step graphically RAC and PEC curves (describing the time courses of the RAC and PEC), and (iii) in a third step time-weighted average RAC and PEC levels.

Impact of triphenyltin acetate in microcosms simulating floodplain lakes. II. Comparison of species sensitivity distributions between laboratory and semi-field.

The study objectives were to shed light on the types of freshwater organism that are sensitive to triphenyltin acetate (TPT) and to compare the laboratory and microcosm sensitivities of the invertebrate community. The responses of a wide array of freshwater taxa (including invertebrates, phytoplankton and macrophytes) from acute laboratory Single Species Tests (SST) were compared with the concentration-response relationships of aquatic populations in two types of freshwater microcosms. Representatives of several taxonomic groups of invertebrates, and several phytoplankton and vascular plant species proved to be sensitive to TPT, illustrating its diverse modes of toxic action. Statistically calculated ecological risk thresholds (HC5 values) based on 96 h laboratory EC50 values for invertebrates were 1.3 microg/l, while these values on the basis of microcosm-Species Sensitivity Distributions (SSD) for invertebrates in sampling weeks 2-8 after TPT treatment ranged from 0.2 to 0.6 microg/l based on nominal peak concentrations. Responses observed in the microcosms did not differ between system types and sampling dates, indicating that ecological threshold levels are not affected by different community structures including taxa sensitive to TPT. The laboratory-derived invertebrate SSD curve was less sensitive than the curves from the microcosms. Possible explanations for the more sensitive field response are delayed effects and/or additional chronic exposure via the food chain in the microcosms.

Impact of triphenyltin acetate in microcosms simulating floodplain lakes. I. Influence of sediment quality.

Floodplain lakes in the Rhine-Meuse delta of the Netherlands vary considerably in levels of sediment-bound toxicants. Microcosm experiments were done to compare the ecological impact of the fungicide triphenyltin acetate (TPT) between test systems with clean or polluted sediments (10 microcosms each). Differences in sediment quality affected the structure of the aquatic communities that developed in the microcosms. Initially, a faster growth of the macrophyte Elodea nuttallii was observed on the polluted sediments, which contained not only toxicants but also higher organic matter and nutrient levels. Dynamics of TPT concentrations in the overlying water were very similar between the two types of test system. Higher levels of TPT, however, were found in the sediment compartment of the clean sediment systems containing a smaller macrophyte biomass. TPT was very persistent in the sediments. In both test systems representatives of several taxonomic groups showed clear responses to a single application of TPT, although benthic Nematoda were not affected. Although a few differences in the intensity and/or duration of TPT-related population responses were observed between the two types of test system, the background pollutants in the polluted sediment hardly affected the overall sensitivity of the aquatic community to the additional chemical stressor TPT.

Ecological effects of spring and late summer applications of lambda-cyhalothrin on freshwater microcosms.

The aim of the study was to compare the effects of the pyrethroid insecticide lambda-cyhalothrin (treated at 10, 25, 50, 100, 250 ng active ingredient a.i./L) on a drainage ditch ecosystem in spring and late summer. Microcosms (water volume approximately 430 L) were established using enclosures in a 50-cm-deep experimental ditch system containing communities typical of macrophyte-dominated freshwater ecosystems. Effects on macroinvertebrates, zooplankton, phytoplankton, macrophytes, and community metabolism were assessed and evaluated using univariate and multivariate statistical techniques. The macroinvertebrate community responded most clearly to treatment and, as anticipated, insects and crustaceans were among the most sensitive organisms. Statistical analysis showed that the underlying community structure was significantly different between the spring and summer experiments. However, the most sensitive species (Chaoborus obscuripes and Gammarus pulex) were abundant in spring as well as in late summer. In spring and late summer, only slight and transient effects were observed at the community level in the 10-ng/L treatment. Overall, the study did not show substantial differences in the responses of sensitive taxa between spring and late summer treatments, and effects thresholds were similar irrespective of season of treatment.

Comparison of laboratory single species and field population-level effects of the pyrethroid insecticide lambda-cyhalothrin on freshwater invertebrates.

The toxicity of the pyrethroid insecticide lambda-cyhalothrin to freshwater invertebrates has been investigated using data from short-term laboratory toxicity tests and in situ bioassays and population-level effects in field microcosms. In laboratory tests, patterns of toxicity were consistent with previous data on pyrethroids. The midge Chaoborus obscuripes was most sensitive (48- and 96-h EC50 = 2.8 ng/L). Other insect larvae (Hemiptera, Ephemeroptera) and macrocrustacea (Amphipoda, Isopoda) were also relatively sensitive, with 48- and 96-h EC50 values between 10 and 100 ng/L. Generally, microcrustacea (Cladocera, Copepoda) and larvae of certain insect groups (Odonata and Chironomidae) were less sensitive, with 48-h EC50 values higher than 100 ng/L. Mollusca and Plathelminthes were insensitive and were unaffected at concentrations at and above the water solubility (5 microg/L). Generally, the EC50 values based on initial population responses in field enclosures were similar to values derived from laboratory tests with the same taxa. Also, the corresponding fifth and tenth percentile hazard concentrations (HC5 and HC10) were similar (laboratory HC5 = 2.7 ng/L and field HC5 = 4.1 ng/L; laboratory and field HC10 = 5.1 ng/L), at least when based on the same sensitive taxonomic groups (insects and crustaceans) and when a similar concentration range was taken into account. In the three field enclosure experiments and at a treatment level of 10 ng/L, consistent effects were observed for only one population (Chaoborus obscuripes), with recovery taking place within 3 to 6 weeks. The laboratory HC5 (2.7 ng/L) and HC10 (5.1 ng/L) based on acute EC50 values of all aquatic arthropod taxa were both lower than this 10 ng/L, a concentration that might represent the "regulatory acceptable concentration." The HC5 and HC10 values in this study in The Netherlands (based on static laboratory tests with freshwater arthropods) were very similar to those derived from a previous study in the United Kingdom (1.4 and 3.3 ng/L). This suggests that for pesticides like lambda-cyhalothrin, HC5 values based on static laboratory tests may provide a conservative estimate of the potential for community-level effects under field conditions. While these HC5 values are conservative for initial effects, they do not provide information on recovery potential, which may be important for regulatory decision-making.