Use of read-across to simplify the toxicological assessment of a complex mixture of lysimeter leachate metabolites on the basis of chemical similarity and ADME behavior.

This paper describes the further development of a read-across approach applicable to the toxicological assessment of structurally-related xenobiotic metabolites. The approach, which can be applied in the absence of definitive identification of all the individual metabolites, draws on the use of chemical descriptors and multi-variate statistical analysis to define a composite "chemical space" and to classify and characterize closely-related subgroups within this. In this example, consideration of the descriptors driving grouping, combined with empirical evidence for lack of significant further biotransformation of metabolites, leads to the conclusion that, in the absence of any specific structural alerts, the relative toxicity of metabolites within a single grouping will be determined by their relative systemic exposure as described by their ADME characteristics. The in vivo testing of a smaller number of exemplars, selected to have representative ADME properties for each grouping, is sufficient, therefore, to evaluate the toxicity of the remainder. The approach is exemplified using the metabolites of the herbicide S-metolachlor, detected in the leachate of a soil lysimeter.

Building a Conceptual Model for the Environmental Fate of the Fungicide Benzovindiflupyr.

Degradation of the fungicide benzovindiflupyr was slow in standard regulatory laboratory studies in soil and aquatic systems, suggesting it is a persistent molecule. However, the conditions in these studies differed significantly from actual environmental conditions, particularly the exclusion of light, which prevents potential contributions from the phototrophic microorganisms that are ubiquitous in both aquatic and terrestrial environments. Higher tier laboratory studies that include a more comprehensive range of degradation processes can more accurately describe environmental fate under field conditions. Indirect aqueous photolysis studies with benzovindiflupyr showed that the photolytic half-life in natural surface water can be as short as 10 days, compared with 94 days in pure buffered water. Inclusion of a light-dark cycle in higher tier aquatic metabolism studies, to include the contribution of phototrophic organisms, reduced the total system half-life from >1 year in dark test systems to as little as 23 days. The relevance of these additional processes was confirmed in an outdoor aquatic microcosm study in which the half-life of benzovindiflupyr was 13-58 days. In laboratory soil degradation studies, the degradation rate of benzovindiflupyr was significantly faster in cores with an undisturbed surface microbiotic crust, incubated in a light-dark cycle (half-life of 35 days), than in regulatory studies with sieved soil in the dark (half-life >1 year). A radiolabeled field study validated these observations, showing residue decline with a half-life of approximately 25 days over the initial 4 weeks. Conceptual models of environmental fate based on standard regulatory studies may be incomplete, and additional higher tier laboratory studies can be valuable in elucidating degradation processes and improving the prediction of persistence under actual use conditions. Environ Toxicol Chem 2023;42:995-1009. © 2023 SETAC.

Water flow plays a key role in determining chemical biodegradation in water-sediment systems.

Before agrochemicals can be registered and sold, the chemical industry is required to perform regulatory tests to assess their environmental persistence, using defined guidelines. Aquatic fate tests (e.g. OECD 308) lack environmental realism as they are conducted under dark conditions and in small-scale static systems, which can affect microbial diversity and functionality. In this study, water-sediment microflumes were used to investigate the impact of these deficiencies in environmental realism on the fate of the fungicide, isopyrazam. Although on a large-scale, these systems aimed to retain the key aspects of OECD 308 tests. Tests were carried out under both a non-UV light-dark cycle and continuous darkness and under both static and flowing water conditions, to investigate how light and water flow affect isopyrazam biodegradation pathways. In static systems, light treatment played a significant role, with faster dissipation in illuminated compared to dark microflumes (DT50s = 20.6 vs. 47.7 days). In flowing systems (DT50s = 16.8 and 15.3 days), light did not play a significant role in dissipation, which was comparable between the two light treatments, and faster than in dark static microflumes. Microbial phototroph biomass was significantly reduced by water flow in the illuminated systems, thereby reducing their contribution to dissipation. Comprehensive analysis of bacterial and eukaryotic community composition identified treatment specific changes following incubation, with light promoting relative abundance of Cyanobacteria and eukaryotic algae, and flow increasing relative abundance of fungi. We conclude that both water velocity and non-UV light increased isopyrazam dissipation, but the contribution of light depended on the flow conditions. These differences may have resulted from impacts on microbial communities and via mixing processes, particularly hyporheic exchange. Inclusion of both light and flow in studies could improve the extent they mimic natural environments and predict chemical environmental persistence, thus bridging the gap between laboratory and field studies.

Development of a Harmonized Protocol for Measurement of Foliar Wash-off Parameters: First Results.

The foliar wash-off coefficient is a parameter used by environmental fate models to estimate the amount of chemical removed from leaf surfaces by rainfall. In the European Union it is used by FOCUS surface water models to estimate soil loadings following rainfall after leaf surfaces have been treated with plant protection products. Currently, a default value of 0.5/cm is assumed for this parameter, although there is provision to provide experimental data to replace this default. The European Food Safety Authority proposed to increase the default parameter value to 1.0/cm. This increases the need for experimental refinement studies. However, no guidance for a harmonized protocol exists to estimate this parameter. We describe the results of a ring-test conducted to start a process of developing a harmonized experimental protocol to measure the foliar wash-off parameters, conducted by several laboratories across Europe. The proposed design uses whole plants (rather than individual leaves) to retain as much realism as possible. The extent of wash-off is then determined by comparison of compound residues in two sets of plants (with and without a defined rainfall event) measured using a fully validated crop residue method. This initial ring test used tebuconazole (Folicur EW 250) sprayed at 100 g ai/ha onto tomato plants at BBCH25. Each laboratory measured the residues before and after a rainfall event of 20 mm/h for 1 h and calculated the percentage of wash-off from these data. There was good agreement across the eight participating laboratories with a mean percentage of wash-off of 66.8% and a 95% confidence interval of ±11.8%. Determination of robust wash-off parameters was therefore considered feasible using the proposed test design. Environ Toxicol Chem 2023;42:535-541. © 2022 SETAC.

The Impact of Disturbed Soil Structure on the Degradation of 2 Fungicides Under Constant and Variable Moisture.

Degradation of agrochemicals in soil is frequently faster under field conditions than in laboratory studies. Field studies are carried out on relatively undisturbed soil, whereas laboratory studies typically use sieved soil, which can have a significant impact on the physical and microbial nature of the soil and may contribute to differences in degradation between laboratory and field studies. A laboratory study was therefore conducted to determine the importance of soil structure and variable soil moisture on the degradation of 2 fungicides (azoxystrobin and paclobutrazol) that show significant differences between laboratory and field degradation rates in regulatory studies. Degradation rates were measured in undisturbed cores of a sandy clay loam soil (under constant or variable moisture contents) and in sieved soil. For azoxystrobin, degradation rates under all conditions were similar (median degradation time [DegT50] 34-37 d). However, for paclobutrazol, degradation was significantly faster in undisturbed cores (DegT50 255 d in sieved soil and 63 d in undisturbed cores). Varying the moisture content did not further enhance degradation of either fungicide. Further examination into the impact of soil structure on paclobutrazol degradation, comparing undisturbed and sieved/repacked cores, revealed that the impact of sieving could not be mitigated by repacking the soil to a realistic bulk density. Examination of fungal and bacterial community structure using automated ribosomal spacer analysis showed significant initial differences between sieved/repacked and intact soil cores, although such differences were reduced at the end of the study (70 d). The present study demonstrates that disruption of soil structure significantly impacts microbial community structure, and for some compounds this may explain the differences between laboratory and field degradation rates. Environ Toxicol Chem 2021;40:2715-2725. © 2021 SETAC.

The role of indirect photolysis in limiting the persistence of crop protection products in surface waters.

The photodegradation of six crop protection products (CPPs) was studied in 16 natural waters collected from across the midwest of the United States under simulated sunlight to determine the significance of indirect photolysis. The rate of degradation of five of the CPPs was faster in irradiated natural waters than in buffer systems, with the effect particularly significant with the relatively photostable compounds propiconazole and prometryn. Degradation rates were correlated with the concentration of one or more photosensitizers, or ratios thereof, by means of a Pearson's correlation and linear regression analysis. It was found that the photodegradation of chlorotoluron, pinoxaden, propiconazole and prometryn were linked to the concentration of nitrate, pointing to a significant role of hydroxyl radical ((.)OH) as a reactive intermediate. Increased concentrations of dissolved organic carbon (DOC) and bicarbonate relative to nitrate were found to decrease the rate of degradation of these compounds, consistent with a quenching role. Chlorothalonil appeared to be rapidly degraded by means of the carbonate radical ((.)CO(3)(-)), whereas the photodegradation of emamectin was particularly complex. Overall, indirect photolysis significantly enhanced the rate of CPP degradation and fate models based on these experiments appear to offer more realism than those that only take into account direct photolysis.

Evaluation of the Rhizosphere Contribution to the Environmental Fate of the Herbicide Prometryn.

Plant protection products (PPPs) undergo rigorous regulatory assessment to ensure that they do not pose unacceptable risks to the environment. Elucidation of their fate and behavior in soil is an integral part of this environmental risk assessment. The active substance degradation in soil of PPPs is first assessed in laboratory studies (typically following Organisation for Economic Co-operation and Development [OECD] test guideline 307). Conditions in guideline laboratory studies are far removed from those occurring under agricultural use, and the contribution of crop roots has currently not been assessed. We integrated viable plant root systems, representative of 3 different crop types, into the OECD test guideline 307 design to assess their impact on the dissipation of the herbicide prometryn. Significantly faster decline of parent residue and higher formation of nonextractable residues were observed in all 3 planted systems. This led to a reduction in the time required for 50% of the compound to dissipate (DT50) of approximately one-half in the presence of rye grass and hot pepper and of approximately one-third in the presence of red clover. These findings imply that plants and their associated root networks can have a significant influence on PPP dissipation. Based on these data, greater environmental realism could be added to the standardized laboratory study design by the inclusion of plant root systems into higher tier studies, which, in turn, could serve to improve the environmental risk assessment process. Environ Toxicol Chem 2020;39:450-457. © 2019 SETAC.

Inclusion of seasonal variation in river system microbial communities and phototroph activity increases environmental relevance of laboratory chemical persistence tests.

Regulatory tests assess crop protection product environmental fate and toxicity before approval for commercial use. Although globally applied laboratory tests can assess biodegradation, they lack environmental complexity. Microbial communities are subject to temporal and spatial variation, but there is little consideration of these microbial dynamics in the laboratory. Here, we investigated seasonal variation in the microbial composition of water and sediment from a UK river across a two-year time course and determined its effect on the outcome of water-sediment (OECD 308) and water-only (OECD 309) biodegradation tests, using the fungicide isopyrazam. These OECD tests are performed under dark conditions, so test systems incubated under non-UV light:dark cycles were also included to determine the impact on both inoculum characteristics and biodegradation. Isopyrazam degradation was faster when incubated under non-UV light at all collection times in water-sediment microcosms, suggesting that phototrophic communities can metabolise isopyrazam throughout the year. Degradation rate varied seasonally between inoculum collection times only in microcosms incubated in the light, but isopyrazam mineralisation to 14CO2 varied seasonally under both light and dark conditions, suggesting that heterotrophic communities may also play a role in degradation. Bacterial and phototroph communities varied across time, but there was no clear link between water or sediment microbial composition and variation in degradation rate. During the test period, inoculum microbial community composition changed, particularly in non-UV light incubated microcosms. Overall, we show that regulatory test outcome is not influenced by temporal variation in microbial community structure; however, biodegradation rates from higher tier studies with improved environmental realism, e.g. through addition of non-UV light, may be more variable. These data suggest that standardised OECD tests can provide a conservative estimate of pesticide persistence end points and that additional tests including non-UV light could help bridge the gap between standard tests and field studies.

High-resolution accurate mass spectrometry as a technique for characterization of complex lysimeter leachate samples.

Lysimeter studies can be used to identify and quantify soil degradates of agrochemicals (metabolites) that have the potential to leach to groundwater. However, the apparent metabolic profile of such lysimeter leachate samples will often be significantly more complex than would be expected in true groundwater samples. This is particularly true for S-metolachlor, which has an extremely complex metabolic pathway. Consequently, it was not practically possible to apply a conventional analytical approach to identify all metabolites in an S-metolachlor lysimeter study, because there was insufficient mass to enable the use of techniques such as nuclear magnetic resonance. Recent advances in high-resolution accurate mass spectrometry, however, allow innovative screening approaches to characterize leachate samples to a greater extent than previously possible. Leachate from the S-metolachlor study was screened for accurate masses (±5 ppm of the nominal mass) corresponding to more than 400 hypothetical metabolite structures. A refined list of plausible metabolites was constructed from these data to provide a comprehensive description of the most likely metabolites present. The properties of these metabolites were then evaluated using a principal component analysis model, based on molecular descriptors, to visualize the entire chemical space and to cluster the metabolites into a number of subclasses. This characterization and principal component analysis evaluation enabled the selection of suitable representative metabolites that were subsequently used as exemplars to assess the toxicological relevance of the leachate as a whole. Environ Toxicol Chem 2016;35:1401-1412. © 2015 SETAC.

Quantifying soil surface photolysis under conditions simulating water movement in the field: a new laboratory test design.

Soil surface photolysis can be a significant dissipation pathway for agrochemicals under field conditions, although it is assumed that such degradation ceases once the agrochemical is transported away from the surface following rainfall or irrigation and subsequent drainage of soil porewater. However, as both downward and upward water movements occur under field conditions, relatively mobile compounds may return to the surface, prolonging exposure to ultraviolet light and increasing the potential for degradation by photolysis. To test this hypothesis, a novel experimental system was used to quantify the contribution of photolysis to the overall dissipation of a new herbicide, bicyclopyrone, under conditions that mimicked field studies more closely than the standard laboratory test guidance. Soil cores were taken from 3 US field study sites, and the surfaces were treated with [(14) C]-bicyclopyrone. The radioactivity was redistributed throughout the cores using a simulated rainfall event, following which the cores were incubated under a xenon-arc lamp with continuous provision of moisture from below and a wind simulator to induce evaporation. After only 2 d, most of the test compound had returned to the soil surface. Significantly more degradation was observed in the irradiated samples than in a parallel dark control sample. Degradation rates were very similar to those observed in both the thin layer photolysis study and the field dissipation studies and significantly faster than in the soil metabolism studies conducted in the dark. Thus, for highly soluble, mobile agrochemicals, such as bicyclopyrone, photolysis is not terminated permanently by rainfall or irrigation but can resume following transport to the surface in evaporating water.

Fate of the insecticide lambda-cyhalothrin in ditch enclosures differing in vegetation density.

Use of the insecticide lambda-cyhalothrin in agriculture may result in the contamination of water bodies, for example by spray drift. Therefore, the possible exposure of aquatic organisms to this insecticide needs to be evaluated. The exposure of the organisms may be reduced by the strong sorption of the insecticide to organic materials and its susceptibility to hydrolysis at the high pH values in the natural range. In experiments done in May and August, formulated lambda-cyhalothrin was mixed with the water body of enclosures in experimental ditches containing a bottom layer and macrophytes (at different densities) or phytoplankton. Concentrations of lambda-cyhalothrin in the water body and in the sediment layer, and contents in the plant compartment, were measured by gas-liquid chromatography at various times up to 1 week after application. Various water quality parameters were also measured. Concentrations of lambda-cyhalothrin decreased rapidly in the water column: 1 day after application, 24-40% of the dose remained in the water, and by 3 days it had declined to 1.8-6.5%. At the highest plant density, lambda-cyhalothrin residue in the plant compartment reached a maximum of 50% of the dose after 1 day; at intermediate and low plant densities, this maximum was only 3-11% of the dose (after 1-2 days). The percentage of the insecticide in the ditch sediment was 12% or less of the dose and tended to be lower at higher plant densities. Alkaline hydrolysis in the water near the surface of macrophytes and phytoplankton is considered to be the main dissipation process for lambda-cyhalothrin.

Surface water mineralization of isopyrazam according to OECD 309: observations on implementation of the new data requirement within agrochemical regulation.

A surface water mineralization study (according to the Organisation for Economic Co-operation and Development [OECD] guideline OECD 309) is a new requirement in European Union agrochemical regulations; therefore, industry has little experience with this test. The guideline allows for a number of options within the test design, notably the options to conduct the study under diffuse light and to include an inoculum of suspended sediment. The present study was designed to investigate the potential impact of these options on the degradation rate of a representative compound. The fungicide, isopyrazam, was chosen as it was previously shown to be susceptible to metabolism by phototrophic organisms under a fluorescent light-dark cycle. The impact of diffuse light was investigated at light intensities representative of those at depth in large, open water bodies (<7% of the incident intensity), and it was demonstrated that metabolism of isopyrazam by phototrophic microorganisms was rapid (median degradation time for 50% of the test compound [DT50] < 50 d), whereas degradation in continuous darkness was negligible. Furthermore, investigation at 2 different light intensities resulted in similar degradation rates, indicating that this transformation mechanism was not proportional to light intensity, provided that there was sufficient light for photosynthesis to occur. Inclusion of suspended sediment did not have a significant impact on the degradation rate of isopyrazam, except at extremely high sediment concentrations, which were not considered representative of conditions in large, open water bodies.

Assessing the metabolic potential of phototrophic communities in surface water environments: fludioxonil as a model compound.

Differences are often apparent in the observed rates of degradation between laboratory water-sediment studies and outdoor studies in surface water environments. Indeed, previous work has shown that including phototrophic communities in laboratory systems can result in the enhancement of degradation, when compared against systems that exclude phototrophs, incubated in darkness. In phototroph-inclusive systems, a range of metabolic processes and community effects are absent in the standard laboratory systems: metabolism by macrophytes, algae, and periphyton, as well as enhancement of bacterial and fungal communities by macrophyte root structures, algal biofilms, and planktonic algae. Here, the authors demonstrate the metabolic capability of algae and macrophytes in isolation from bacterial and fungal communities. The authors have isolated subcommunities and individual species from complex, phototroph-inclusive test systems, and demonstrated significant degradation of the fungicide fludioxonil in their presence. They have also shown the intrinsic metabolic competence of Elodea canadensis as well as algae from three phyla (Chlorophyta, Cyanophyta, and Bacillariophyta [diatoms]), demonstrating that phototrophic communities have the potential to play a direct role in metabolism in surface water environments. Thus, it seems that current laboratory test systems are failing to consider the role of active, competent organisms that are likely to be involved in the degradation of crop protection products in surface water environments.

Assessing the potential for algae and macrophytes to degrade crop protection products in aquatic ecosystems.

Rates of pesticide degradation in aquatic ecosystems often differ between those observed within laboratory studies and field trials. Under field conditions, a number of additional processes may well have a significant role, yet are excluded from standard laboratory studies, for example, metabolism by aquatic plants, phytoplankton, and periphyton. These constituents of natural aquatic ecosystems have been shown to be capable of metabolizing a range of crop protection products. Here we report the rate of degradation of six crop protection products assessed in parallel in three systems, under reproducible, defined laboratory conditions, designed to compare aquatic sediment systems which exclude macrophytes and algae against those in which macrophytes and/or algae are included. All three systems remained as close as possible to the Organisation for Economic Co-operation and Development (OECD) 308 guidelines, assessing degradation of parent compound in the total system in mass balanced studies using ((14) C) labeled compounds. We observed, in all cases where estimated, significant increases in the rate of degradation in both the algae and macrophyte systems when compared to the standard systems. By assessing total system degradation within closed, mass balanced studies, we have shown that rates of degradation are enhanced in water/sediment systems that include macrophytes and algae. The contribution of these communities should therefore be considered if the aquatic fate of pesticides is to be fully understood.

The behavior of isopyrazam in aquatic ecosystems: implementation of a tiered investigation.

Degradation of a new fungicide, isopyrazam, was slow in water-sediment systems maintained in the dark, with degradation half-life (DegT50) values in the total system (water column and sediment) of greater than one year, and only moderately fast in a photolysis study in buffered pure water (DegT50 > 60 d). This indicated that microbial degradation and direct photolysis are not significant loss mechanisms for this compound. Under more realistic conditions, a number of other processes of natural attenuation occur, such as metabolism by aquatic plants, microalgae, and periphyton and indirect photolysis. A photolysis study in sterile natural water, and water-sediment studies incorporating aquatic macrophytes and microalgae under fluorescent light, were therefore conducted to investigate the contribution of these processes to the fate of isopyrazam. Degradation rates were at least one order of magnitude faster in these higher-tier laboratory studies, indicating that all of these processes may have a role to play in complex natural ecosystems. The fate in an outdoor system, designed to mimic conditions in edge-of-field drainage ditches, also was investigated to provide an integrated picture of the contribution of all the different potential loss mechanisms to the overall fate of isopyrazam. The total system DegT50 in the study was similar to that observed in the higher-tier laboratory studies. Furthermore, the pattern of degradation formation allowed for the contribution of the different degradation processes at work in the microcosm study to be contextualized. The implementation of this tiered approach to investigating the aquatic fate of crop protection products provides a comprehensive explanation of the behavior of isopyrazam and clearly demonstrates that it will not persist in the aquatic environment under natural conditions.