Reproducibility, reliability, and regulatory relevance of plant visual injury assessments.

The registration of herbicides in the European Union requires an assessment of risks to nontarget terrestrial plants (NTTPs). Regulatory plant studies are performed to determine risk-assessment-relevant endpoints (50% effect rate) for quantitative parameters, mostly biomass and survival. Recently, the European Food Safety Authority stated that endpoints for qualitatively assessed plant visual injuries (PVIs) such as necrosis, chlorosis, and so forth should be considered for the risk assessment as equal to endpoints derived from quantitatively determined parameters. However, the lack of guidance in the NTTP test guidelines on how to assess PVI and how to derive a statistically meaningful endpoint for PVI makes their use in risk assessments challenging. To evaluate and improve the reliability, reproducibility, and regulatory relevance of PVI assessments in NTTP studies, the PVI Working Group was formed in 2022 within the SETAC Plant Interest Group. In a first exercise, research needs, guidance gaps, and shortcomings in current methodologies were identified and are presented together with recommendations for a future, validated, and harmonized method for the assessment of PVI. Survey results revealed a high variability in how PVI are currently assessed, and that the reliability of these data is unclear. Under current conditions, the PVI data can rather be seen as supportive information instead of using the data for the statistically sound determination of a regulatory endpoint. Consequently, standardization and harmonization of procedures for the assessment of PVI are needed. An improved scoring methodology should be developed that allows for a precise, statistically sound endpoint determination. Regarding the regulatory relevance of PVI, further research is required to assess the biological meaning of PVI data and how this is connected to the regulatory requirements and protection goals. Last but not least, guidance is required on how to evaluate the historically available PVI data that are based on various assessment methodologies. Integr Environ Assess Manag 2023;00:1-9. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Ecological impact of repeated applications of chlorpyrifos on zooplankton community in mesocosms under Mediterranean conditions.

The effects of the insecticide chlorpyrifos were studied in plankton-dominated mesocosms under Mediterranean conditions. Chlorpyrifos was applied four times at 1 week intervals at nominal concentrations of 0.033, 0.1, 0.33, and 1 microg/l simulating repeated agricultural applications. The lowest 7 days time weighted averaged concentrations (TWAC) during the 28 days exposure period were calculated using the FOCUS equation to express the no observed effect concentration (NOEC) values. At population level the lowest NOEC calculated was 0.012 microg/l (treatment concentration 0.033 microg/l). The most affected taxon was Cladocera (Daphnia group galeata) followed by Copepoda (cyclopoids and nauplii). No effects were observed on phytoplankton (chlorophyll-a biomass) at any treatment level. The smallest NOEC(community) calculated by means of multivariate techniques was 0.1 microg/l when expressed in terms of the nominal treatment level and 0.074 microg/l when based on the lowest 7 days TWA concentration during the 28 days application period. Indirect effects on zooplankton populations were observed due to shifts in competition and predation between populations. Compared with previous micro/mesocosm experiments simulating a single application exposure regime, results from our study revealed a lower threshold level for the most sensitive measurement endpoint (difference a factor of three (in terms of nominal treatment level), more severe indirect effects and longer recovery periods of the affected populations (> 13 weeks in the test systems treated with 1 microg/l). These differences could be attributed to the repeated pulse exposure scenario approach designed for our studies together with the particular climatic conditions involving our Mediterranean mesocosms (i.e., temperature, cladocerans life history, and algae blooms).

Responses of zooplankton in lufenuron-stressed experimental ditches in the presence or absence of uncontaminated refuges.

Outdoor experimental ditches were used to evaluate the influence of untreated refuges on the recovery of zooplankton communities following treatment with the fast-dissipating insecticide lufenuron. Each experimental ditch was divided into three sections of the same surface area. The treatments differed in the proportion of ditch (0, 33, 67, and 100% of the surface area) to which the insecticide was applied at the same nominal treatment (3 mug/L). During the first week postapplication, a barrier was placed between treated and untreated ditch sections. The untreated sections were included to provide a source of organisms for recovery of affected zooplankton populations in the treated sections of the ditch after the removal of the barrier. Cyclopoida were the most affected by lufenuron treatment, followed by Daphnia gr. galeata. These and other direct effects of treatment on larvae of the phantom midge Chaoborus spp. resulted in clear indirect effects on populations of Calanoida, Ceriodaphnia, and Rotifera. Overall, faster recovery of the zooplankton community was observed in the treated sections of ditches that were sprayed for a smaller proportion of their surface area. Nevertheless, individual zooplankton populations showed considerable differences in rate of recovery. Cyclopoida showed a relatively slow rate of recovery even in the partially treated ditches. Daphnia gr. galeata recovered more rapidly in treated ditch sections in the presence of unsprayed ditch sections, illustrating the potential influence of unexposed refuges. Furthermore, the presence of refuges most likely dampened the magnitude and duration of indirect effects in the ditches treated with lufenuron.

Zooplankton community responses to chlorpyrifos in mesocosms under Mediterranean conditions.

The effects of chlorpyrifos (organophosphate insecticide) on zooplankton were studied in outdoor experimental tanks (mesocosms) sited in the Mediterranean Region (Madrid, Spain) at two nominal concentrations of chlorpyrifos (0.1 and 1 microg a.s./L applied as Chas 48) and control were used. Five tanks were used as control and the treatments were performed in quintuplicate. A single chlorpyrifos application simulating spray-drift was conducted. The population and community effects were analyzed by means of univariate statistics and through the multivariate principal response curves (PRC) technique. The most affected zooplankton taxa were cladocerans (Daphnia group galeata), copepods (cyclopoids and copepod nauplii) and rotifers (Keratella cochlearis) showing in all the cases significant decreases in abundance at 1 microg chlorpyrifos/L. The calculated NOEC was 0.1 microg/L for these taxa as well as for the community. The zooplankton community was considered to be recovered after 99 days post-application. The results of this experiment were similar to those derived from mesocosm/microcosm studies performed in temperate regions. This indicates that a chlorpyrifos concentration of 0.1 microg chlorpyrifos/L could be the appropriate safe level for zooplankton community in different climatic regions. However, at treatment level of 1.0 microg/L the time required for full recovery of the affected populations (particularly Cladocera) was longer than in the other experiments performed in temperate regions.