René P. A. Van Wijngaarden

Ordination techniques for analysing response of biological communities to toxic stress in experimental ecosystems

The ordination techniques principal component analysis (PCA) and redundancy analysis (RDA) are considered to be useful tools for evaluating community responses in experimental ecotoxicology. Concepts and interpretation of these techniques are summarized. Application of PCA and RDA is illustrated in a case study. In this study, the effects of a single application of the insecticide Dursban® 4E (a.i. chlorpyrifos) on an aquatic macroinvertebrate community in microcosms were analysed. Four treatment (nominal chlorpyrifos concentration: 35 μg l-1) and four control microcosms were used. PCA visualized a change in species composition with time. Immediately after treatment, a major shift in species composition occurred in treated microcosms. RDA demonstrated that this shift was due to the treatment. RDA also showed that non-arthropods were generally insusceptible to chlorpyrifos; most arthropods were affected. Dynamics of separate taxa were visualized, giving indications of possible primary and secondary effects for these taxa. A Monte Carlo permutation test was used to decide whether treatment had a significant effect on the species composition and to investigate the state of recovery in time. In general, the RDA results gave an adequate condensation of detailed information on abundance and effects obtained by more conventional univariate statistical analysis for some individual taxa of the community. In combination with toxicity and ecological data, ordination techniques can provide insight into effects of toxic substances in complex biological communities.

Aquatic risk assessment of a realistic exposure to pesticides used in bulb crops: a microcosm study.

The fungicide fluazinam, the insecticide lambda-cyhalothrin, and the herbicides asulam and metamitron were applied to indoor freshwater microcosms (water volume approximately 0.6 m3). The treatment regime was based on a realistic application scenario in tulip cultivation. Concentrations of each pesticide were equal to 0%, 0.2%, 0.5%, 2%, and 5% spray drift emission of label-recommended rates. Contribution of compounds to the toxicity of the pesticide package was established by expressing their concentrations as fractions of toxic units. The fate of the compounds in the water, and responses of phytoplankton, zooplankton, periphyton, macroinvertebrates, macrophytes, decomposition, and water quality were followed for 13 weeks. The half-lives of lambda-cyhalothrin, metamitron, and fluazinam were 1 to 2 d; that of asulam was >30 d. No consistent effects could be demonstrated for the 0.2% treatment regime that was therefore considered the no-observed-effect concentration community (NOEC). The macroinvertebrate populations of Gammarus pulex, Asellus aquaticus, and Proasellus meridianus were the most sensitive end points, followed by species of copepods and cladocerans. Responses mainly were due to lambda-cyhalothrin. The 0.5% treatment regime resulted in short-term effects. Pronounced effects were observed at the 2% and 5% treatment levels. At the end of the experiment, the macrophyte biomass that consisted of Elodea nuttallii, showed a decline at the two highest treatment levels, asulam being the causal factor (NOEC: 0.5% treatment level). Primary production was reduced at the 5% treatment level only. In our experiment, the first-tier risk assessment procedure for individual compounds was adequate for protecting sensitive populations exposed to realistic combinations of pesticides. Spray drift reduction measures seem to be efficient in protecting aquatic ecosystems in agricultural areas.

Acute toxicity tests with Daphnia magna, Americamysis bahia, Chironomus riparius and Gammarus pulex and implications of new EU requirements for the aquatic effect assessment of insecticides

Threshold concentrations for treatment related effects of 31 insecticides, as derived from aquatic micro-/mesocosm tests, were used to calibrate the predictive value of the European Tier-1 acute effect assessment on basis of laboratory toxicity tests with Daphnia magna, Chironomus spp., Americamysis bahia and Gammarus pulex. The acute Tier-1 effect assessment on basis of Daphnia (EC50/100) overall was protective for organophosphates, carbamates and most pyrethroids but not for neonicotinoids and the majority of insect growth regulators (IGRs) in the database. By including the 28-day water-spiked Chironomus riparius test, the effect assessment improves but selecting the lowest value on basis of the 48-h Daphnia test (EC50/100) and the 28-day Chironomus test (NOEC/10) is not fully protective for 4 out of 23 insecticide cases. An assessment on basis of G. pulex (EC50/100) is sufficiently protective for 15 out of 19 insecticide cases. The Tier-1 procedure on basis of acute toxicity data (EC50/100) for the combination of Daphnia and A. bahia and/or Chironomus (new EU dossier requirements currently under discussion) overall is protective to pulsed insecticide exposures in micro-/mesocosms. For IGRs that affect moulting, the effect assessment on basis of the 48-h Chironomus test (EC50/100) may not always be protective enough to replace that of the water-spiked 28-day C. riparius test (NOEC/10) because of latency of effects.

Effects of the antibiotic enrofloxacin on the ecology of tropical eutrophic freshwater microcosms

The main objective of the present study was to assess the ecological impacts of the fluoroquinolone antibiotic enrofloxacin on the structure and functioning of tropical freshwater ecosystems. Enrofloxacin was applied at a concentration of 1, 10, 100 and 1,000 μg/L for 7 consecutive days in 600-L outdoor microcosms in Thailand. The ecosystem-level effects of enrofloxacin were monitored on five structural (macroinvertebrates, zooplankton, phytoplankton, periphyton and bacteria) and two functional (organic matter decomposition and nitrogen cycling) endpoint groups for 4 weeks after the last antibiotic application. Enrofloxacin was found to dissipate relatively fast from the water column (half-dissipation time: 11.7h), and about 11% of the applied dose was transformed into its main by-product ciprofloxacin after 24h. Consistent treatment-related effects on the invertebrate and primary producer communities and on organic matter decomposition could not be demonstrated. Enrofloxacin significantly affected the structure of leaf-associated bacterial communities at the highest treatment level, and reduced the abundance of ammonia-oxidizing bacteria and ammonia-oxidizing archaea in the sediments, with calculated NOECs of 10 and <1 μg/L, respectively. The ammonia concentration in the microcosm water significantly increased in the highest treatment level, and nitrate production was decreased, indicating a potential impairment of the nitrification function at concentrations above 100 μg/L. The results of this study suggest that environmentally relevant concentrations of enrofloxacin are not likely to result in direct or indirect toxic effects on the invertebrate and primary producer communities, nor on important microbially mediated functions such as nitrification.

Acute tier-1 and tier-2 effect assessment approaches in the EFSA Aquatic Guidance Document: are they sufficiently protective for insecticides?

BACKGROUND The objective of this paper is to evaluate whether the acute tier-1 and tier-2 methods as proposed by the Aquatic Guidance Document recently published by the European Food Safety Authority (EFSA) are appropriate for deriving regulatory acceptable concentrations (RACs) for insecticides. The tier-1 and tier-2 RACs were compared with RACs based on threshold concentrations from micro/mesocosm studies (ETO-RAC). A lower-tier RAC was considered as sufficiently protective, if less than the corresponding ETO-RAC. RESULTS ETO-RACs were calculated for repeated (n = 13) and/or single pulsed applications (n = 17) of 26 insecticides to micro/mesocosms, giving a maximum of 30 insecticide × application combinations (i.e. cases) for comparison. Acute tier-1 RACs (for 24 insecticides) were lower than the corresponding ETO-RACs in 27 out of 29 cases, while tier-2 Geom-RACs (for 23 insecticides) were lower in 24 out of 26 cases. The tier-2 SSD-RAC (for 21 insecticides) using HC5 /3 was lower than the ETO-RAC in 23 out of 27 cases, whereas the tier-2 SSD-RAC using HC5 /6 was protective in 25 out of 27 cases. CONCLUSION The tier-1 and tier-2 approaches proposed by EFSA for acute effect assessment are sufficiently protective for the majority of insecticides evaluated. Further evaluation may be needed for insecticides with more novel chemistries (neonicotinoids, biopesticides) and compounds that show delayed effects (insect growth regulators).

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.

Effects of time-variable exposure regimes of the insecticide chlorpyrifos on freshwater invertebrate communities in microcosms

The present study compared the effects of different time-variable exposure regimes having the same time-weighted average (TWA) concentration of the organophosphate insecticide chlorpyrifos on freshwater invertebrate communities to enable extrapolation of effects across exposure regimes. The experiment was performed in outdoor microcosms by introducing three different regimes: a single application of 0.9 µg active ingredients (a.i.)/L; three applications of 0.3 µg a.i./L, with a time interval of 7 d; and continuous exposure to 0.1 µg a.i./L for 21 d. Measurements showed that the TWA(21d) concentration in the continuous-exposure treatment (0.098 µg/L) was slightly lower than in the three-application (0.116 µg/L) and single-application (0.126 µg/L) treatments. The application of chlorpyrifos resulted in decreased abundances in the arthropod community, with the largest adverse effects reported for the mayfly Cloeon dipterum and cladocerans Daphnia gr. longispina and Alona sp., while smaller effects were observed for other insects, copepods, and amphipods. At the population level, however, the mayfly C. dipterum only responded to the single-application treatment, which could be explained by the toxicokinetics of chlorpyrifos in this species. At the end of the experimental period the invertebrate community showed approximately the same effect magnitude for all treatment regimes. These results suggest that for this combination of concentrations and duration of the TWA, the TWA concentration is more important for most species than the peak concentration for the assessment of long-term risks of chlorpyrifos.

Is the chronic Tier-1 effect assessment approach for insecticides protective for aquatic ecosystems?

We investigated the appropriateness of several methods, including those recommended in the Aquatic Guidance Document of the European Food Safety Authority (EFSA), for the derivation of chronic Tier-1 regulatory acceptable concentrations (RACs) for insecticides and aquatic organisms. The insecticides represented different chemical classes (organophosphates, pyrethroids, benzoylureas, insect growth regulators, biopesticides, carbamates, neonicotinoids, and miscellaneous). Chronic Tier-1 RACs derived using toxicity data for the standard species Daphnia magna, Chironomus spp., and/or Americamysis bahia, were compared with Tier-3 RACs derived from micro- and mesocosm studies on basis of the ecological threshold option (ETO-RACs). ETO-RACs could be derived for 31 insecticides applied to micro- and mesocosms in single or multiple applications, yielding a total number of 36 cases for comparison. The chronic Tier-1 RACs calculated according to the EFSA approach resulted in a sufficient protection level, except for 1 neonicotinoid (slightly underprotective) and for several pyrethroids if toxicity data for A. bahia were not included. This latter observation can be explained by 1) the fact that A. bahia is the most sensitive standard test species for pyrethroids, 2) the hydrophobic properties of pyrethroids, and 3) the fact that long-term effects observed in (epi) benthic arthropods may be better explained by exposure via the sediment than via overlying water. Besides including toxicity data for A. bahia, the protection level for pyrethroids can be improved by selecting both D. magna and Chironomus spp. as standard test species for chronic Tier-1 derivation. Although protective in the majority of cases, the conservativeness of the recommended chronic Tier-1 RACs appears to be less than an order of magnitude for a relatively large proportion of insecticides when compared with their Tier-3 ETO-RACs. This may leave limited options for refinement of the chronic effect assessment using laboratory toxicity data for additional species. Integr Environ Assess Manag 2016;12:747-758.

Exposure and effects of sediment-spiked fludioxonil on macroinvertebrates and zooplankton in outdoor aquatic microcosms

Information from effects of pesticides in sediments at an ecosystem level, to validate current and proposed risk assessment procedures, is scarce. A sediment-spiked outdoor freshwater microcosm experiment was conducted with fludioxonil (lipophilic, non-systemic fungicide) to study exposure dynamics and treatment-related responses of benthic and pelagic macroinvertebrates and zooplankton. Besides blank control and solvent control systems the experiment had six different treatment levels (1.7-614mga.s./kg dry sediment) based around the reported 28-d No Observed Effect Concentration (NOEC) for Chironomus riparius (40mga.s./kg dry sediment). Twelve systems were available per treatment of which four were sacrificed on each of days 28, 56 and 84 after microcosm construction. Fludioxonil persisted in the sediment and mean measured concentrations were 53-82% of the initial concentration after 84days. The dissipation rate increased with the treatment level. Also exposure concentrations in overlying water were long-term, with highest concentrations 28days after initiation of the experiment. Sediment-dwelling Oligochaeta and pelagic Rotifera and Cladocera showed the most pronounced treatment-related declines. The most sensitive sediment-dwelling oligochaete was Dero digitata (population NOEC 14.2mga.s./kg dry sediment). The same NOEC was calculated for the sediment-dwelling macroinvertebrate community. The most sensitive zooplankton species was the cladoceran Diaphanosoma brachyurum (NOEC of 1.6μga.s./L in overlying water corresponding to 5.0mga.s./kg dry sediment). At the two highest treatments several rotifer taxa showed a pronounced decrease, while the zooplankton community-level NOEC was 5.6μga.s./L (corresponding to 14.2mga.s./kg dry sediment). Zooplankton taxa calanoid Copepoda and Daphnia gr. longispina showed a pronounced treatment-related increase (indirect effects). Consequently, an assessment factor of 10 to the chronic laboratory NOECs of Chironomus riparius (sediment) and Daphnia magna (water) results in a regulatory acceptable concentration that is sufficiently protective for both the sediment-dwelling and pelagic organisms in the microcosms.

Effects of the Herbicide Metsulfuron-Methyl on a Plant Community, Including Seed Germination Success in the F1 Generation

A field trial was set up to simulate a field margin environment to analyze sub-lethal effects of the herbicide metsulfuron-methyl on several endpoints of non-target terrestrial plants (NTTPs). Both vegetative and reproductive endpoints were evaluated. The experiment was conducted in an experimentally established field strip with sown species. The treatments consisted of 5 dosages and a control: 0, 0.0097, 0.0193, 0.058, 0.174 and 0.348 gram active ingredient per hectare (g a.i./ha). The plant cover, number of (flowering) individuals per species and fruit collection were performed and estimated weekly for a period of 4 months. At the end of the growing season, the total dry biomass per species was obtained and the collected fruits were weighted, counted and sieved to obtain the seeds. The seeds were counted and weighted as well, before they were used in a germination experiment to test the seed emergence of the F1 generation. The herbicide only affected the biomass of Matricaria recutita at the treatment levels tested (0.058 g a.i./ha and higher). Field dosages of 0.174 and 0.348 g a.i./ha differed significantly in the endpoint “plant cover” compared to lower dosages and controls. The F1 generations of Sinapis alba, Centaurea cyanus and Phacelia tanacetifolia were particularly affected at field dosages of 0.0193 g a.i./ha and higher, showing significantly lower seed germination rates. This would imply that spray drift of metsulfuron-methyl might lead to shifts in species compositions and succession in vegetation in off-crop areas adjacent to arable fields. Conducting germination experiments is necessary to investigate a herbicides effect on the full life cycle of plants.