The Interaction of Pesticides with Humin Fractions and Their Potential Impact on Non-Extractable Residue Formation.

The constant influx of pesticides into soils is a key environmental issue in terms of their potential retention in the soil, thus reducing their negative impact on the environment. Soil organic matter (SOM) is an important factor influencing the environmental fate of these substances. Therefore, the aim of this research was to assess the chemical behavior of pesticides (flufenacet, pendimethalin, α-cypermethrin, metazachlor, acetamiprid) toward stable soil humin fractions (HNs) as a main factor affecting the formation of non-extractable residues of agrochemicals in soil. This research was conducted as a batch experiment according to OECD Guideline 106. For this purpose, HNs were isolated from eight soils with different physicochemical properties (clay content = 16-47%, pHKCl = 5.6-7.7, TOC = 13.3-49.7 g·kg-1, TN = 1.06-2.90 g·kg-1, TOC/TN = 11.4-13.7) to reflect the various processes of their formation. The extraction was carried out through the sequential separation of humic acids with 0.1 M NaOH, and then the digestion of the remaining mineral fraction with 10% HF/HCl. The pesticide concentrations were detected using GC-MS/MS. The pesticides were characterized based on the different sorption rates to HNs, according to the overall trend: metazachlor (95% of absorbed compound) > acetamiprid (94% of absorbed compound) > cypermethrin (63% of partitioning compound) > flufenacet (39% of partitioning compound) > pendimethalin (28% of partitioning compound). Cypermethrin and metazachlor exhibited the highest saturation dynamic, while the other agrochemicals were much more slowly attracted by the HNs. The obtained sorption kinetic data were congruous to the pseudo-first-order and pseudo-second-order models related to the surface adsorption and interparticle diffusion isotherm. The conducted research showed that the processes of pesticide sorption, apart from physicochemical phenomena, are also affected by the properties of the pollutants themselves (polarity, KOC) and the soil properties (SOM content, clay content, and pHKCl).

Chloroacetanilides inhibit photosynthesis and disrupt the thylakoid membranes of the dinoflagellate Prorocentrum minimum as revealed with metazachlor treatment.

The chloroacetanilides are among the most commonly used herbicides worldwide, which contaminate aquatic environments and affect aquatic phototrophs. Their sub-lethal toxicity has been evaluated using freshwater algae; however, the modes of cellular toxicity and levels of toxicity to marine organisms are not fully understood. In the present study, we assessed the cellular and molecular effects of chloroacetanilides on marine phototrophs using the dinoflagellate Prorocentrum minimum and the herbicide metazachlor (MZC). The MZC treatment led to a considerable reduction in cell number and pigment, and the EC50 of MZC was calculated to be 0.647 mg/L. The photosynthetic parameters, Fv/Fm and chlorophyll fluorescence significantly decreased with MZC exposure time in a dose-dependent manner. In addition, MZC significantly induced photosynthesis genes, including PmpsbA, PmpsaA, and PmatpB, and the antioxidant PmGST, but not PmKatG. These findings were well matched to reactive oxygen species (ROS) production in MZC-treated cells. Interestingly, we observed inflated vacuoles, undivided chloroplasts, and breakdown of thylakoid membranes in MZC-treated cells. These results support the hypothesis that MZC severely damages chloroplasts, resulting in dysfunction of the dinoflagellate photosynthesis and possibly marine phototrophs in the environment.

Chemical Composition, Antibacterial Activity and Mechanism of Action of Fermentation Products from Aspergillus Niger xj.

Six compounds were isolated and purified from the crude acetone extract of Aspergillus niger xj. Characterization of all compounds was done by NMR and MS. On the basis of chemical and spectral analysis structure, six compounds were elucidated as metazachlor (1), nonacosane (2), palmitic acid (3), 5,5'-oxybis(5-methylene-2-furaldehyde) (4), dimethyl 5-nitroisophthalate (5) and cholesta-3,5-dien-7-one (6), respectively, and compounds 1, 4, 5 and 6 were isolated for the first time from A. niger. To evaluate the antibacterial activity of compounds 1-6 against three plant pathogenic bacteria (Agrobacterium tumefaciens T-37, Erwinia carotovora EC-1 and Ralstonia solanacearum RS-2), and the minimum inhibitory concentrations (MICs) were determined by broth microdilution method in 96-well microtiter plates. Results of the evaluation of the antibacterial activity showed that T-37 strain was more susceptible to metazachlor with the lowest MIC of 31.25 µg/mL. The antibacterial activity of metazachlor has rarely been reported, thus the antibacterial mechanism of metazachlor against T-37 strain were investigated. The permeability of cell membrane demonstrated that cells membranes were broken by metazachlor, which caused leakage of ions in cells. SDS-PAGE of T-37 proteins indicated that metazachlor could damage bacterial cells through the destruction of cellular proteins. Scanning electron microscopy results showed obvious morphological and ultrastructural changes in the T-37 cells, further confirming the cell membrane damages caused by metazachlor. Overall, our findings demonstrated that the ability of metazachlor to suppress the growth of T-37 pathogenic bacteria makes it potential biocontrol agents.

Oxidation of metazachlor herbicide by ozone in the gas and aqueous phases: mechanistic, kinetics, and ecotoxicity studies.

The atmospheric and aqueous ozonolysis of metazachlor (MTZ) is investigated using high-level quantum chemical and kinetic calculations (M06-2X/6-311 + + G(3df,3pd)//M06-2X/6-31 + G(d,p) level of theory). The ozone (O3)-initiated degradation pathways of MTZ under three different mechanisms, namely cycloaddition, oxygen-addition, and single electron transfer (SET), are explored in the temperature range of 283-333 K and 1 atm pressure. As a result, the cycloaddition reaction at the C16C18 double bond of the benzene ring of MTZ is found to be the most dominant channel in the atmosphere with the standard Gibbs free energy of reaction (ΔrG0g) of - 129.13 kJ mol-1 and the highest branching ratio of 95.18%. In the aqueous phase, the main reaction channel turns into the SET mechanism, which owns the lowest Gibbs free energy of activation (ΔG#aq) of 73.8 kJ mol-1 and contributes 87.8% to the ktotal. Over the temperature range of 283-333 K, the total rate constant (ktotal) significantly increases from 8.42 to 5.82 × 101 M-1 s-1 in the atmosphere and from 4.10 × 102 to 2.40 × 104 M-1 s-1 in the aqueous environment. Remarkably, the ecotoxicity assessment shows that MTZ may be harmful to fish and chronically harmful to daphnia. In contrast, its main ozonolysis products exhibit no acute or chronic toxicity or mutagenic effects.

Pesticides and Parabens Contaminating Aquatic Environment: Acute and Sub-Chronic Toxicity towards Early-Life Stages of Freshwater Fish and Amphibians.

Pesticides and personal care products are two very important groups of contaminants posing a threat to the aquatic environment and the organisms living in it.. Therefore, this study aimed to describe the effects of widely used pesticides and parabens on aquatic non-target biota such as fish (using model organisms Danio rerio and Cyprinus carpio) and amphibians (using model organism Xenopus laevis) using a wide range of endpoints. The first part of the experiment was focused on the embryonal toxicity of three widely used pesticides (metazachlor, prochloraz, and 4-chloro-2-methyl phenoxy acetic acid) and three parabens (methylparaben, propylparaben, and butylparaben) with D. rerio, C. carpio, and X. laevis embryos. An emphasis was placed on using mostly sub-lethal concentrations that are partially relevant to the environmental concentrations of the substances studied. In the second part of the study, an embryo-larval toxicity test with C. carpio was carried out with prochloraz using concentrations 0.1, 1, 10, 100, and 1000 µg/L. The results of both parts of the study show that even the low, environmentally relevant concentrations of the chemicals tested are often able to affect the expression of genes that play either a prominent role in detoxification and sex hormone production or indicate cell stress or, in case of prochloraz, to induce genotoxicity.

Modification of the existing maximum residue levels for metazachlor in leeks and honey.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant BASF SE submitted a request to the competent national authority in the Netherlands to modify the existing maximum residue levels (MRLs) for the active substance metazachlor in leeks and honey. The data submitted in support of the request were found to be sufficient to derive MRL proposals. Adequate analytical methods for enforcement are available to control the residues of metazachlor metabolites 479 M04, 479 M08 and 479 M16 in plant matrices and honey at the validated limit of quantification (LOQ) of 0.01 and 0.02 mg/kg for each analyte. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of metazachlor according to the reported agricultural practice is unlikely to present a risk to consumer health.

A novel approach for measuring membrane permeability for organic compounds via surface plasmon resonance detection.

Well-known methods for measuring permeability of membranes include static or flow diffusion chambers. When studying the effects of organic compounds on plants, the use of such model systems allows to investigate xenobiotic behavior at the cuticular barrier level and obtain an understanding of the initial penetration processes of these substances into plant leaves. However, the use of diffusion chambers has disadvantages, including being time-consuming, requiring sampling, or a sufficiently large membrane area, which cannot be obtained from all types of plants. Therefore, we propose a new method based on surface plasmon resonance imaging (SPRi) to enable rapid membrane permeability evaluation. This study presents the methodology for measuring permeability of isolated cuticles for organic compounds via surface plasmon resonance detection, where the selected model analyte was the widely used pesticide metazachlor. Experiments were performed on the cuticles of Ficus elastica, Citrus pyriformis, and an artificial PES membrane, which is used in passive samplers for the detection of xenobiotics in water and soils. The average permeability for metazachlor was 5.23 × 10-14 m2 s-1 for C. pyriformis, 1.34 × 10-13 m2 s-1 for F. elastica, and 7.74 × 10-12 m2 s-1 for the PES membrane. We confirmed that the combination of a flow-through diffusion cell and real-time optical detection of transposed molecules represents a promising method for determining the permeability of membranes to xenobiotics occurring in the environment. This is necessary for determining a pesticide dosage in agriculture, selecting suitable membranes for passive samplers in analytics, testing membranes for water treatment, or studying material use of impregnated membranes.

Hydroxyl radical-initiated decomposition of metazachlor herbicide in the gaseous and aqueous phases: Mechanism, kinetics, and toxicity evaluation.

The oxidation of widely-used herbicide metazachlor (MTZ) by hydroxyl radical (HO•) in the gas and the aqueous phases was investigated in terms of mechanistic and kinetic behaviors using the M06-2X/6-311++G (3df, 3pd)//M06-2X/6-31 + G (d,p) level of theory over the temperature range 250-400 K. The formal hydrogen transfer, HO•-addition, and single electron transfer mechanisms were considered. The overall rate constants in the gas phase range from 8.40 × 1010 to 8.31 × 109 M-1 s-1 at the temperature from 250 to 400 K, respectively, while the ones in the aqueous phase are close to diffusion-controlled rates, with diffusion-corrected rate constants being 1.31 × 109 to 1.27 × 109 M-1 s-1. The formal hydrogen transfer mechanism is the most dominant in the gas phase, whereas the HO•-addition is the most favorable in the aqueous phase. The H-abstraction at two methyl groups and the HO•-addition to C11 and C12 atoms (pyrazole ring), C16 and C18 atoms (benzyl ring) are significant. The short lifetime in the environment, equal to only 4.16 h, requires more attention to this herbicide compound, whereas its lifetime in the aqueous condition varies sharply from half second to several thousand days depending on the HO• concentration. The ecotoxicity estimation of MTZ and its principal transformation products to aquatic organisms suggests that they are harmful or toxic substances. Moreover, the MTZ is a developmental toxicant and mutagenicity-positive, while its decomposed products are developmental toxicants with no mutagenic toxicity. Their bioaccumulation in aquatic organisms is negligible.

Responses of signal crayfish Pacifastacus leniusculus to single short-term pulse exposure of pesticides at environmentally relevant concentrations.

Although pesticides are often discharged into surface waters in pulses as opposed to a sustained release, the effect of episodic pollution events on freshwater crayfish is largely unknown. We monitored change in heart rate and distance moved to assess the response of signal crayfish Pacifastacus leniusculus to short-term exposure to environmentally relevant concentrations of metazachlor (MTZ), terbuthylazine (TER), and thiacloprid (TCL). Crayfish exposed to 20 µg/L of MTZ exhibited a significant increase in mean heart rate and distance moved. Increased heart rate was detected at 118 ± 74 s post-exposure to MTZ. There were no significant differences in mean heart rate and distance moved in crayfish exposed to 6 µg/L of TCL and 4 µg/L of TER. A significant correlation between heart rate and distance moved was found in all exposed groups. These results suggest that pulse exposure to MTZ impact crayfish physiology and behavior during short-term period. With pulse exposure to TCL and TER, crayfish not exhibiting a locomotor response may continue to be exposed to lower, but potentially harmful, levels of pollutants. Evidence of the impacts of pesticide pulse at environmentally relevant concentrations on crayfish is scarce. Further study is required to determine the ecological effects of such events on freshwater crayfish.

Metazachlor effect on poplar - Pioneer plant species for riparian buffers.

Metazachlor belongs to one of the most used herbicides throughout the world. In order to prevent the contamination of water bodies by such herbicides, the riparian buffers are constructed. The selection of appropriate plant species for this purpose is necessary. In our project, we studied the possibility of grey poplar to uptake and biotransform metazachlor, along with the phytotoxic effect of metazachlor and its metabolites. We used two different models - suspension cultures and poplar regenerants cultivated in vitro. Our results show that the herbicide metazachlor is readily metabolized by both suspension cultures and regenerants to 16 detectable metabolites. The detailed scheme of biotransformation pathway in poplar tissue is presented for the first time. The profile of detected metabolites was approximately the same in poplar cell cultures and regenerants, but the ratio and amounts of particular compounds was significantly different. Generally, the highest concentration (peak area/mg of DW) of all metabolites was present in the roots; the only exception was lactate conjugate (deCl-MZCl-Lact), which accumulated in the cultivation media. Although the plants were not visibly affected by metazachlor or its metabolites, they showed changes in activity of antioxidant enzymes and increased content of phenolic substances, the indicators of stress.

The effects of the herbicides terbuthylazine and metazachlor at environmental concentration on the burrowing behaviour of red swamp crayfish.

Despite their low concentrations in many aquatic environments, evidence exists to suggest that herbicides do affect non-target organisms. Given that burrowing is a primary life-history trait in crayfish, herbicides could potentially have serious negative effects on these ecologically important freshwater macroinvertebrates. In this study, we exposed the red swamp crayfish Procambarus clarkii to terbuthylazine (a triazine) and metazachlor (a chloroacetanilide) at an environmental concentration of 2.0 µg/L for 28 days, and then observed their burrowing behaviour for two days. The metazachlor-exposed males excavated a greater number of burrows than the other tested groups, with comparable depths and volumes relative to individual specimen weight. The relative depth and volume of female burrows were identical in all groups. The natural habit of female crayfish of constructing deeper burrows than males was marginally significant in the control and META groups but was not significant for relative volume. The hypothesized adverse effects of chronic exposure to real environmental concentrations of herbicides were not documented in terms of either relative depth or volume. However, the increased number of burrows in metazachlor-exposed animals may mean that this invasive species will cause greater damage to embankments and river banks. The mechanisms behind these effects require closer study.

Omnipresent distribution of herbicides and their transformation products in all water body types of an agricultural landscape in the North German Lowland.

The research of the environmental fate of pesticides has demonstrated that applied compounds are altered in their molecular structure over time and are distributed within the environment. To assess the risk for contamination by transformation products (TP) of the herbicides flufenacet and metazachlor, the following four water body types were sampled in a small-scale catchment of 50 km2 in 2015/2016: tile drainage water, stream water, shallow groundwater, and drinking water of private wells. The TP were omnipresent in every type of water body, more frequently and in concentrations up to 10 times higher than their parent compounds. Especially metazachlor sulfonic acid, metazachlor oxalic acid, and flufenacet oxalic acid were detected in almost every drainage and stream sample. The transformation process leads to more mobile and more persistent molecules resulting in higher detection frequencies and concentrations, which can even occur a year or more after the application of the parent compound. The vulnerability of shallow groundwater and private drinking water wells to leaching compounds is proved by numerous positives of metazachlor-TP with maximum concentrations of 0.7 µg L-1 (drinking water) and 20 µg L-1 (shallow groundwater) of metazachlor sulfonic acid. Rainfall events during the application period cause high discharge of the parent compound and lower release of TP. Later rainfall events lead to high displacement of TP. For an integrated risk assessment of water bodies, the environmental behavior of pesticide-TP has to be included into regular state-of-the-art water quality monitoring.

PLAGA-PEG-PLAGA Terpolymer-Based Carriers of Herbicides for Potential Application in Environment-Friendly, Controlled Release Systems of Agrochemicals.

The present study aimed to develop and prepare new polymer/herbicide formulations for their potential application in environment-friendly, controlled release systems of agrochemicals. Selected biodegradable polymers, including L-Lactide/Glycolide/PEG/Terpolymer (PLAGA-PEG-PLAGA) as well as oligosaccharide-based polymers and their blend with terpolymer, were used to prepare microspheres loaded with two soil-applied herbicides. The degradation process of the obtained polymeric microspheres was evaluated based on (1) their weight loss and surface erosion and (2) the release rate of loaded metazachlor and pendimethalin. The herbicidal effectiveness of the herbicides released to the soil from microspheres was evaluated using the European Weed Research Council (EWRC) rating scale. Moreover, the ecotoxicological effect of herbicide-loaded microspheres buried in soil on the marine bacterial species A. fischeri was assessed. It was found that the gradual degradation rate of microparticles led to the prolonged release of both herbicides that lasted for a few months, i.e., for the entire crop season, which is crucial in terms of agrochemical and environmental protection. Maltodextrin- and dextrin-based microspheres showed higher susceptibility to degradation than terpolymer-based microspheres. The microencapsulation of herbicides protected them from decomposition and excessive leaching into soil and maintained their activity for a longer period than that for non-immobilized herbicides. The ecotoxicological assessment on A. fischeri demonstrated that the proposed microsphere-encapsulated herbicides were less toxic than non-immobilized herbicides.

Biodegradable Blends of Grafted Dextrin with PLGA-block-PEG Copolymer as a Carrier for Controlled Release of Herbicides into Soil.

The presented work aimed to test influence of poly(L-lactide-co-glycolide)-block-poly (ethylene oxide) copolymer modification by blending with grafted dextrin or maltodextrin on the course of degradation in soil and the usefulness of such material as a matrix in the controlled release of herbicides. The modification should be to obtain homogenous blends with better susceptibility to enzymatic degradation. Among all tested blends, which were proposed as a carrier for potential use in the controlled release of plant protection agents, PLGA-block-PEG copolymer blended with grafted dextrin yielded very promising results for their future applications, and what is very importantly proposed formulations provide herbicides in unchanged form into soil within few months of release. The modification PLAGA/PEG copolymer by blending with modificated dextrins affects the improvement of the release profile. The weekly release rates for both selected herbicides (metazachlor and pendimethalin) were constant for a period of 12 weeks. Enzymatic degradation of modified dextrin combined with leaching of the degradation products into medium caused significant erosion of the polymer matrix, thereby leading to acceleration of water diffusion into the polymer matrix and allowing for easier leaching of herbicides outside the matrix.

Toxicity of binary mixtures of pesticides to the marine microalgae Tisochrysis lutea and Skeletonema marinoi: Substance interactions and physiological impacts.

This study screened binary mixtures of pesticides for potential synergistic interaction effects on growth of the marine microalgae Tisochrysis lutea and Skeletonema marinoi. It also examined the single and combined effects of three of the most toxic substances on microalgal physiology. Single substances were first tested on each microalgal species to determine their respective EC50 and concentration-response relationships. The toxicity of six and seven binary mixtures was then evaluated in microplate experiments on the growth of T. lutea and S. marinoi, respectively, using two mixture modelling approaches: isobolograms and the MIXTOX tool, based on Concentration Addition (CA) or Independent Action (IA) models. Significant cases of antagonism (for both species) and synergism (for S. marinoi) were observed for the mixtures of isoproturon and spiroxamine, and isoproturon and metazachlor, respectively. These two mixtures, together with that of isoproturon and diuron, for which additivity was observed, were further studied for their impacts on the physiology of each species. Exposures were thus made in culture flasks at three concentrations, or concentration combinations for mixtures, selected to cause 25%, 50% and 75% growth rate inhibition. The effects of the selected pesticides singly and in combination were evaluated at three perceived effect concentrations on esterase metabolic activity, relative lipid content, cytoplasmic membrane potential and reactive oxygen species (ROS) content by flow cytometry, and on photosynthetic quantum yield (ϕ'M) by PAM-fluorescence. Isoproturon and diuron singly and in mixtures induced 20-40% decreases in ϕ'M which was in turn responsible for a significant decrease in relative lipid content for both species. Spiroxamine and metazachlor were individually responsible for an increase in relative lipid content (up to nearly 300% for metazachlor on S. marinoi), as well as cell depolarization and increased ROS content. The mixture of isoproturon and metazachlor tested on S. marinoi caused a 28-34% decrease in ϕ'M that was significantly higher than levels induced by each of substances when tested alone. This strong decrease in ϕ'M could be due to a combined effect of these substances on the photosynthetic apparatus, which is likely the cause of the synergy found for this mixture.

Evaluation of confirmatory data following the Article 12 MRL review and modification of the existing maximum residue levels for metazachlor in various commodities.

The applicant BASF SE submitted a request to the competent national authority in the Netherlands to evaluate the confirmatory data that were identified for active substance metazachlor in the framework of the review of maximum residue levels (MRLs) under Article 12 of Regulation (EC) No 396/2005 as not available. The data gaps identified during the MRL review were satisfactorily addressed for flowering brassica, head cabbages, kales and kohlrabies. The applicant no longer supported the uses on radishes, turnips, horseradishes and swedes assessed during the MRL review. In addition, in accordance with Article 6 of Regulation (EC) No 396/2005, the applicant submitted a request to modify the existing MRLs in turnips, swedes, horseradishes and leeks to reflect intended new uses. Nevertheless, the residue data submitted were not fully compliant with the currently applicable data requirements. For turnips, horseradishes and swedes, the applicant and the Netherlands requested EFSA to present the data and the deficiencies identified to allow risk managers to take an informed decision on the appropriate MRLs. A change of the existing MRLs for liver of swine and ruminants resulting from the authorised and intended uses of metazachlor is also proposed. EFSA updated the most recent consumer risk assessment for metazachlor and concluded that the long-term and short-term dietary intake is unlikely to present a risk to consumer health.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance.

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7-28 days and 8000 ppm metazachlor for 3-28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3-28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5-50 µM) and NaPB (500 µM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5-25 µM metazachlor or 500 µM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

Modification of the existing maximum residue level for metazachlor in Chinese cabbage.

In accordance with Article 6 of Regulation (EC) No 396/2005, the evaluating Member State (EMS), France, received an application from BASF SE to modify the existing maximum residue level (MRL) for the active substance metazachlor in Chinese cabbage. To accommodate for the intended use of metazachlor, France proposed to raise the existing MRL from the value of 0.2 mg/kg to 0.6 mg/kg. France drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005, which was submitted to the European Commission and forwarded to EFSA. According to EFSA, the data are sufficient to derive a MRL proposal of 0.6 mg/kg for the proposed use on Chinese cabbage. Adequate analytical enforcement methods are available to control the residues of metazachlor on the commodity under consideration. Based on the risk assessment results, EFSA concludes that the proposed use of metazachlor on Chinese cabbage will not result in a consumer exposure exceeding the toxicological reference values and therefore is unlikely to pose a consumer health risk.

Lentic small water bodies: Variability of pesticide transport and transformation patterns.

Lentic small water bodies have a high ecological potential as they fulfill several ecosystem services such as the retention of water and pollutants. They serve as a hot spot of biodiversity. Due to their location in or adjacent to agricultural fields, they can be influenced by inputs of pesticides and their transformation products. Since small water bodies have rarely been part of monitorings/campaigns up to now, their current exposure and processes guiding the pesticide input are not understood, yet. This study presents results of a sampling campaign of 10 lentic small water bodies from 2015 to 2016. They were sampled once after the spring application for a pesticide target screening, before autumn application and three times after rainfall events following the application. The autumn sampling focused on the herbicides metazachlor, flufenacet and their transformation products - oxalic acid and - sulfonic acid as representatives for common pesticides in the study region. The concentrations were associated with rainfall before and after application, characteristics of the site and the water bodies, physicochemical parameters and the applied amount of pesticides. The key results of the pesticide screening in spring indicate positive detections of pesticides which have not been applied for years to the single fields. The autumn sampling showed frequent occurrences of the transformation products, which are formed in soil, from 39% to 94% of all samples (n=71). Discharge patterns were observed for metazachlor with highest concentrations in the first sample after application and then decreasing, but not for flufenacet. The concentrations of the transformation products increased over time and revealed highest values mainly in the last sample. Besides rainfall patterns right after application, the spatial and temporal dissemination of the pesticides to the water bodies seems to play a major role to understand the exposure of lentic small water bodies.

Short-term phytotoxicity in Brassica napus (L.) in response to pre-emergently applied metazachlor: A microcosm study.

In accordance with realistic application approaches, a short-term 1-factorial experiment was set up to investigate the phytotoxic impact of pre-emergent application of the chloroacetamide herbicide metazachlor on Brassica napus. In addition to morphological parameters, the underlying processes that ultimately determine the extent of herbicide-induced phytotoxicity (i.e., herbicide metabolization and cellular antioxidant defense) were examined. The present study demonstrated that metazachlor provoked fasciation of the leaves closely after emergence, which might be linked to its mode of action whereby cell division is impaired through the inhibition of very long chain fatty acid synthesis. The increased activities of antioxidative enzymes and metabolites in leaf tissue indicated the presence of reactive oxygen species under the influence of metazachlor. This resulted in oxidative damage in the form of membrane lipid peroxidation. Simultaneously, the increased activity of glutathione S-transferase and the shift in glutathione redox state suggested activation of the detoxification metabolism. This occurred, however, at the expense of growth, with a temporary reduction in plant height and weight after application. The results indicated that metazachlor disappeared within 3 mo to 4 mo after application, which resulted in the recovery of the crop. In conclusion, metazachlor induces phytotoxicity in the short term, either directly through its mode of action or indirectly through the induction of oxidative stress, which resulted in a temporary reduction in growth. Environ Toxicol Chem 2017;36:59-70. © 2016 SETAC.