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.

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.

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.

Assessing effects of the fungicide tebuconazole to heterotrophic microbes in aquatic microcosms.

Aquatic ecological risk assessment of fungicides in Europe under Regulation 1107/2009/EC does not currently assess risk to non-target bacteria and fungi. Rather, regulatory acceptable concentrations based on ecotoxicological data obtained from studies with fish, invertebrates and primary producers (including algae) are assumed to be protective to all other aquatic organisms. Here we explore the validity of this assumption by investigating the effects of a fungicide (tebuconazole) applied at its "non-microbial" HC5 concentration (the concentration that is hazardous to 5% of the tested taxa) and derived from acute single species toxicity tests on fish, invertebrates and primary producers (including algae) on the community structure and functioning of heterotrophic microbes (bacteria and aquatic fungi) in a semi-field study, using novel molecular techniques. In our study, a treatment-related effect of tebuconazole (238 µg/L) on either fungal biomass associated with leaf material or leaf decomposition or the composition of the fungal community associated with sediment could not be demonstrated. Moreover, treatment-related effects on bacterial communities associated with sediment and leaf material were not detected. However, tebuconazole exposure did significantly reduce conidia production and altered fungal community composition associated with leaf material. An effect on a higher trophic level was observed when Gammarus pulex were fed tebuconazole-exposed leaves, which caused a significant decrease in their feeding rate. Therefore, tebuconazole may affect aquatic fungi and fungally mediated processes even when applied at its "non-microbial" HC5 concentration.

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.

Effects of the fungicide metiram in outdoor freshwater microcosms: responses of invertebrates, primary producers and microbes.

The ecological impact of the dithiocarbamate fungicide metiram was studied in outdoor freshwater microcosms, consisting of 14 enclosures placed in an experimental ditch. The microcosms were treated three times (interval 7 days) with the formulated product BAS 222 28F (Polyram®). Intended metiram concentrations in the overlying water were 0, 4, 12, 36, 108 and 324 µg a.i./L. Responses of zooplankton, macroinvertebrates, phytoplankton, macrophytes, microbes and community metabolism endpoints were investigated. Dissipation half-life (DT50) of metiram was approximately 1-6 h in the water column of the microcosm test system and the metabolites formed were not persistent. Multivariate analysis indicated treatment-related effects on the zooplankton (NOEC(community) = 36 µg a.i./L). Consistent treatment-related effects on the phytoplankton and macroinvertebrate communities and on the sediment microbial community could not be demonstrated or were minor. There was no evidence that metiram affected the biomass, abundance or functioning of aquatic hyphomycetes on decomposing alder leaves. The most sensitive populations in the microcosms comprised representatives of Rotifera with a NOEC of 12 µg a.i./L on isolated sampling days and a NOEC of 36 µg a.i./L on consecutive samplings. At the highest treatment-level populations of Copepoda (zooplankton) and the blue-green alga Anabaena (phytoplankton) also showed a short-term decline on consecutive sampling days (NOEC = 108 µg a.i./L). Indirect effects in the form of short-term increases in the abundance of a few macroinvertebrate and several phytoplankton taxa were also observed. The overall community and population level no-observed-effect concentration (NOEC(microcosm)) was 12-36 µg a.i./L. At higher treatment levels, including the test systems that received the highest dose, ecological recovery of affected measurement endpoints was fast (effect period < 8 weeks).

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 (EC(50)/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 (EC(50)/100) is sufficiently protective for 15 out of 19 insecticide cases. The Tier-1 procedure on basis of acute toxicity data (EC(50)/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 (EC(50)/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.

Ecological impacts of time-variable exposure regimes to the fungicide azoxystrobin on freshwater communities in outdoor microcosms.

This paper evaluates the effects of different time-varying exposure patterns of the strobilurin fungicide azoxystrobin on freshwater microsocosm communities. These exposure patterns included two treatments with a similar peak but different time-weighted average (TWA) concentrations, and two treatments with similar TWA but different peak concentrations. The experiment was carried out in outdoor microcosms under four different exposure regimes; (1) a continuous application treatment of 10 µg/L (CAT(10)) for 42 days (2), a continuous application treatment of 33 µg/L (CAT(33)) for 42 days (3), a single application treatment of 33 µg/L (SAT(33)) and (4) a four application treatment of 16 µg/L (FAT(16)), with a time interval of 10 days. Mean measured 42-d TWA concentrations in the different treatments were 9.4 µg/L (CAT(10)), 32.8 µg/L (CAT(33)), 14.9 µg/L (SAT(33)) and 14.7 µg/L (FAT(16)). Multivariate analyses demonstrated significant changes in zooplankton community structure in all but the CAT(10) treated microcosms relative to that of controls. The largest adverse effects were reported for zooplankton taxa belonging to Copepoda and Cladocera. By the end of the experimental period (day 42 after treatment), community effects were of similar magnitude for the pulsed treatment regimes, although the magnitude of the initial effect was larger in the SAT(33) treatment. This indicates that for long-term effects the TWA is more important for most zooplankton species in the test system than the peak concentration. Azoxystrobin only slightly affected some species of the macroinvertebrate, phytoplankton and macrophyte assemblages. The overall no observed ecologically adverse effect concentrations (NOEAEC) in this study was 10 µg/L.

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.

Effects of the pyrethroid insecticide gamma-cyhalothrin on aquatic invertebrates in laboratory and outdoor microcosm tests.

The sensitivity of a range of freshwater lentic invertebrates to gamma-cyhalothrin (GCH), a single enantiomer of the synthetic pyrethroid lambda-cyhalothrin, was assessed in single species laboratory tests and an outdoor multi-species ecosystem test. The most sensitive species in the laboratory single species tests with GCH was Chaoborus obscuripes (96 h EC(50): 3.8 ng/l). The species sensitivity distribution curve, based on the laboratory 96 h EC(50) acute toxicity data for eight species, gave a median HC(5) value for GCH of 2.12 ng/l. The NOEC(community) derived from the multi-species ecosystem test was 5 ng/l, and the insects Chaoborus sp. and Caenis sp. were identified as the most sensitive species. The results indicate that the median HC(5), based on eight species selected to include those known to be sensitive to pyrethroids, provided a good estimation of the NOEC(community) for GCH. Furthermore, the results for GCH indicated that the endpoints typically used in higher-tier risk assessments for pesticides in Europe (HC(5) and NOEC(community)) were consistent with expectations when compared to the equivalent endpoints for the racemate LCH.

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.

Threshold levels for effects of insecticides in freshwater ecosystems: a review.

A literature review of freshwater (model) ecosystem studies with neurotoxic insecticides was performed to assess ecological threshold levels, to compare these levels with the first tier approach within European Union (EU) administration procedures, and to evaluate the ecological consequences of exceeding these thresholds. Studies published between 1980 and 2001 were reviewed. Most studies covered organophosphates and synthetic pyrethroids in lentic waters. The most sensitive taxa were representatives of crustaceans, insects and fish. Based on toxic units, threshold values were equivalent for compounds with a similar mode of action. This also accounted for the nature and magnitude of direct effects at higher concentrations. Although laboratory single species toxicity tests may not allow predictions on precise ecological effects, some generalisations on effects and recovery can be made with respect to acute standard laboratory EC50 data. The NOEC(ecosystem) usually is a factor of 10 or more higher than first tier acceptable concentrations, particularly in the case of single applications and acetylcholinesterase inhibitors. Acceptable concentrations, as set by the EU first tier approach, appear to be protective. Recovery of sensitive endpoints usually occurs within 2 months of the (last) application when peak concentrations remain lower than (0.1-1) x EC50 of the most sensitive standard test species. The consistency of response patterns found in model ecosystem studies can be useful when estimating the ecological risks of pesticides. The use of an effect classification system was also helpful in evaluating effects.

Effects of chlorpyrifos in freshwater model ecosystems: the influence of experimental conditions on ecotoxicological thresholds.

Three experiments were conducted to determine the impact of the insecticide chlorpyrifos (single applications of 0.01 to 10 microg AI litre(-1)) in plankton-dominated nutrient-rich microcosms. The microcosms (water volume approximately 14 litres) were established in the laboratory under temperature, light regimes and nutrient levels that simulated cool 'temperate' and warm 'Mediterranean' environmental conditions. The fate of chlorpyrifos in the water column was monitored and the effects on zooplankton, phytoplankton and community metabolism were followed for 4 or 5 weeks. The mean half-life (t1/2) of chlorpyrifos in the water of the test systems was 45 h under 'temperate' conditions and about 30 h under 'Mediterranean' environmental conditions. Microcrustaceans (cladocerans and copepod nauplii) were amongst the most sensitive organisms. All three experiments yielded community NOEC (no observed effect concentrations) of 0.1 microg AI litre(-1), similar to those derived from more complex outdoor studies. Above this threshold level, responses and effect chains, and time spans for recovery, differed between the experiments. For example, algal blooms as an indirect effect from the impact of exposure on grazing organisms were only observed under the 'Mediterranean' experimental conditions. The relatively simple indoor test system seems to be sufficient to provide estimates of safe threshold levels for the acute insecticidal effects of low-persistence compounds such as chlorpyrifos. The robustness of the community NOEC indicates that this threshold level is likely to be representative for many freshwater systems.

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.