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).

A field spray drift study to determine the downwind effects of isoxaflutole herbicide to nontarget plants.

Spray drift buffers are often required on herbicide labels to prevent potential drift effects to nontarget plants. Buffers are typically derived by determining the distance at which predicted exposure from spray drift equals the ecotoxicology threshold for sensitive plant species determined in greenhouse tests. Field studies performed under realistic conditions have demonstrated, however, that this approach is far more conservative than necessary. In 2016, the US Environmental Protection Agency estimated that isoxaflutole (IFT), a herbicide used to control grass and broadleaf weeds, could adversely affect downwind nontarget dicot plants at distances of ≥304 m from the edge of the treated field due to spray drift. This prediction implies that a buffer of at least 304 m is required to protect nontarget plants. To refine the predicted buffer distance for IFT, we conducted a field study in which sensitive nontarget plants (lettuce and navy bean, two to four leaf stage) were placed at various distances downwind from previously harvested soybean fields sprayed with Balance® Flexx Herbicide. The test plants were then transported to a greenhouse for grow out following the standard vegetative vigor test protocol. There were three trials. One had vegetation in the downwind deposition area (i.e., test plants placed in mowed grass; typical exposure scenario) and two had bare ground deposition areas (worst-case exposure scenario). For both plant species in bare ground deposition areas, effects on shoot height and weight were observed at 1.52 m but not at downwind distances of ≥9.14 m from the edge of the treated area. No effects were observed at any distance for plants placed in the vegetated deposition area. The field study demonstrated that a buffer of 9.14 m protects nontarget terrestrial plants exposed to IFT via spray drift even under worst-case conditions. Integr Environ Assess Manag 2022;18:757-769. © 2021 Bayer. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

A Field Study Method as a Potential Higher Tier Option to Refine Herbicide Risk Assessment for Nontarget Terrestrial Plants.

During herbicide spray application, nontarget terrestrial plants (NTTPs) growing in the off-field area need to be protected from unacceptable effects of herbicide drift. The risk of such unintended effects is assessed in order to establish whether a particular use can be approved, possibly in combination with mitigation measures. In Europe, the risk of herbicide treatment to NTTPs is assessed on the basis of tier 2 studies done under controlled conditions in greenhouses. Following the concept of a tiered testing approach, higher tier field studies under more realistic conditions could be used to refine the risk assessment. No current guideline for conducting higher tier NTTP field studies is available. We developed an NTTP higher tier field study method done on an experimental plant community established by sowing of a seed mixture. The setup was optimized in 3 pilot field studies and subsequently used for a definitive study testing effects of the herbicide iofensulfuron-sodium. Results show that the method can be regarded as a suitable higher tier option for assessing effects of herbicides on NTTPs. Growth of species from the soil seed bank cannot be avoided and has to be carefully considered when evaluating results. Adaptations of the study design may be necessary when testing different herbicides. Community-level endpoints were at the same level as single-species endpoints. Results of the field study were compared to standard greenhouse study results for the same herbicide. No observed effect rates (NOERs) in the field were about a factor of 10 higher and show that the current tier 2 risk assessment for NTTPs can be regarded as protective in this case. Whether the present field study design and the assessed endpoints can be used in higher tier risk assessment of NTTPs depends on selection of the specific protection goal and requires further discussion. Integr Environ Assess Manag 2020;16:691-705. © 2020 Bayer AG. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Plastid Located WHIRLY1 Enhances the Responsiveness of Arabidopsis Seedlings Toward Abscisic Acid.

WHIRLY1 is a protein that can be translocated from the plastids to the nucleus, making it an ideal candidate for communicating information between these two compartments. Mutants of Arabidopsis thaliana lacking WHIRLY1 (why1) were shown to have a reduced sensitivity toward salicylic acid (SA) and abscisic acid (ABA) during germination. Germination assays in the presence of abamine, an inhibitor of ABA biosynthesis, revealed that the effect of SA on germination was in fact caused by a concomitant stimulation of ABA biosynthesis. In order to distinguish whether the plastid or the nuclear isoform of WHIRLY1 is adjusting the responsiveness toward ABA, sequences encoding either the complete WHIRLY1 protein or a truncated form lacking the plastid transit peptide were overexpressed in the why1 mutant background. In plants overexpressing the full-length sequence, WHIRLY1 accumulated in both plastids and the nucleus, whereas in plants overexpressing the truncated sequence, WHIRLY1 accumulated exclusively in the nucleus. Seedlings containing recombinant WHIRLY1 in both compartments were hypersensitive toward ABA. In contrast, seedlings possessing only the nuclear form of WHIRLY1 were as insensitive toward ABA as the why1 mutants. ABA was furthermore shown to lower the rate of germination of wildtype seeds even in the presence of abamine which is known to inhibit the formation of xanthoxin, the plastid located precursor of ABA. From this we conclude that plastid located WHIRLY1 enhances the responsiveness of seeds toward ABA even when ABA is supplied exogenously.

Recombinant Whirly1 translocates from transplastomic chloroplasts to the nucleus.

Whirly1 was shown to be dually located in chloroplasts and nucleus of the same cell. To investigate whether the protein translocates from chloroplasts to the nucleus, we inserted a construct encoding an HA-tagged Whirly1 into the plastid genome of tobacco. Although the tagged protein was synthesized in plastids, it was detected in nuclei. Dual location of the protein was confirmed by immunocytological analyses. These results indicate that the plastidial Whirly1 is translocated from the plastid to the nucleus where it affects expression of target genes such as PR1. Our results support a role of Whirly1 in plastid-nucleus communication.