Scientific Opinion of the Scientific Panel on Plant Protection Products and their Residues (PPR Panel) on the genotoxic potential of triazine amine (metabolite common to several sulfonylurea active substances).

The Panel received a mandate from the European Commission to assess the genotoxic potential of triazine amine based on available information submitted by the applicants. Available information includes experimental genotoxicity data on triazine amine, Quantitative Structure-Activity Relationship (QSAR) analysis and read across with structurally similar compounds. Based on the overall weight of evidence, the Panel, in agreement with the cross-cutting Working Group Genotoxicity, concluded that there is no concern for the potential of triazine amine to induce gene mutations and clastogenicity; however, the potential to induce aneugenicity was not adequately investigated. For a conclusion, an in vitro micronucleus assay performed with triazine amine would be needed.

Scientific Opinion on the setting of health-based reference values for metabolites of the active substance terbuthylazine.

The EFSA Panel on Plant Protection Products and their Residues was requested to establish health-based reference values for groundwater metabolites (LM2, LM3, LM4, LM5 and LM6) of the active substance terbuthylazine based on the available evidence, unless the evidence was considered insufficient to do so. The request was accepted under the explicit circumstance that the reassessment would be made according to a different methodology than the routine methodology currently applied for the assessment of metabolites in groundwater. While for metabolites LM2, LM4 and LM5, it was concluded that the reference values for terbuthylazine are applicable, substance-specific reference values could not be derived for metabolites LM3 and LM6. The applied threshold of toxicological concern (TTC) approach has shown that metabolites LM3 and LM6 are of potential concern for consumer health, since at least one representative groundwater leaching scenario results in exposure above the relevant threshold. Moreover, other sources of exposure to LM3 and LM6 could not be excluded with certainty. It is therefore recommended to address the specific toxicities of metabolites LM3 and LM6.

Scientific statement on the coverage of bats by the current pesticide risk assessment for birds and mammals.

Bats are an important group of mammals, frequently foraging in farmland and potentially exposed to pesticides. This statement considers whether the current risk assessment performed for birds and ground dwelling mammals exposed to pesticides is also protective of bats. Three main issues were addressed. Firstly, whether bats are toxicologically more or less sensitive than the most sensitive birds and mammals. Secondly, whether oral exposure of bats to pesticides is greater or lower than in ground dwelling mammals and birds. Thirdly, whether there are other important exposure routes relevant to bats. A large variation in toxicological sensitivity and no relationship between sensitivity of bats and bird or mammal test-species to pesticides could be found. In addition, bats have unique traits, such as echolocation and torpor which can be adversely affected by exposure to pesticides and which are not covered by the endpoints currently selected for wild mammal risk assessment. The current exposure assessment methodology was used for oral exposure and adapted to bats using bat-specific parameters. For oral exposure, it was concluded that for most standard risk assessment scenarios the current approach did not cover exposure of bats to pesticide residues in food. Calculations of potential dermal exposure for bats foraging during spraying operations suggest that this may be a very important exposure route. Dermal routes of exposure should be combined with inhalation and oral exposure. Based on the evidence compiled, the Panel concludes that bats are not adequately covered by the current risk assessment approach, and that there is a need to develop a bat-specific risk assessment scheme. In general, there was scarcity of data to assess the risks for bat exposed to pesticides. Recommendations for research are made, including identification of alternatives to laboratory testing of bats to assess toxicological effects.

Scientific Opinion about the Guidance of the Chemical Regulation Directorate (UK) on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessments.

The EFSA Panel on Plant Protection Products and their Residues reviewed the guidance on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessment. The inclusion of aged sorption is a higher tier in the groundwater leaching assessment. The Panel based its review on a test with three substances taken from a data set provided by the European Crop Protection Association. Particular points of attention were the quality of the data provided, the proposed fitting procedure of aged sorption experiments and the proposed method for combining results obtained from aged sorption studies and lower-tier studies on degradation and adsorption. Aged sorption was a relevant process in all cases studied. The test revealed that the guidance could generally be well applied and resulted in robust and plausible results. The Panel considers the guidance suitable for use in the groundwater leaching assessment after the recommendations in this Scientific Opinion have been implemented, with the exception of the use of field data to derive aged sorption parameters. The Panel noted that the draft guidance could only be used by experienced users because there is no software tool that fully supports the work flow in the guidance document. It is therefore recommended that a user-friendly software tool be developed. Aged sorption lowered the predicted concentration in groundwater. However, because aged sorption experiments may be conducted in different soils than lower-tier degradation and adsorption experiments, it cannot be guaranteed that the higher tier predicts lower concentrations than the lower tier, while lower tiers should be more conservative than higher tiers. To mitigate this problem, the Panel recommends using all available higher- and lower-tier data in the leaching assessment. The Panel further recommends that aged sorption parameters for metabolites be derived only from metabolite-dosed studies. The formation fraction can be derived from parent-dosed degradation studies, provided that the parent and metabolite are fitted with the best-fit model, which is the double first-order in parallel model in the case of aged sorption.

The toxic effects of benzyl glucosinolate and its hydrolysis product, the biofumigant benzyl isothiocyanate, to Folsomia fimetaria.

Natural isothiocyanates (ITCs) are toxic to a range of pathogenic soil-living species, including nematodes and fungi, and can thus be used as natural fumigants called biofumigants. Natural isothiocyanates are hydrolysis products of glucosinolates (GSLs) released from plants after cell rupture. The study investigated the toxic effects of benzyl-GSL and its hydrolysis product benzyl-ITC on the springtail Folsomia fimetaria, a beneficial nontarget soil-dwelling micro-arthropod. The soil used was a sandy agricultural soil. Half-lives for benzyl-ITC in the soil depended on the initial soil concentration, ranging from 0.2 h for 67 nmol/g to 13.2 h for 3,351 nmol/g. For benzyl-ITC, the concentration resulting in 50% lethality (LC50) value for F. fimetaria adult mortality was 110 nmol/g (16.4 mg/kg) and the concentration resulting in 50% effect (EC50) value for juvenile production was 65 nmol/g (9.7 mg/kg). Benzyl-GSL proved to be less toxic and consequently an LC50 value for mortality could not be estimated for springtails exposed to benzyl-GSL. For reproduction, an EC50 value was estimated to approximately 690 nmol/g. The study indicates that natural soil concentrations of ITCs may be toxic to beneficial nontarget soil-dwelling arthropods such as springtails.

Sorgoleone.

Sorgoleone, a major component of the hydrophobic root exudate of sorghum [Sorghum bicolor (L.) Moench], is one of the most studied allelochemicals. The exudate also contains an equivalent amount of a lipid resorcinol analog as well as a number of minor sorgoleone congeners. Synthesis of sorgoleone is constitutive and compartmentalized within root hairs, which can accumulate up to 20 microg of exudate/mg root dry weight. The biosynthesis pathway involves unique fatty acid desaturases which produce an atypical 16:3 fatty acyl-CoA starter unit for an alkylresorcinol synthase that catalyzes the formation of a pentadecatrienylresorcinol intermediate. This intermediate is then methylated by SAM-dependent O-methyltransferases and dihydroxylated by cytochrome P450 monooxygenases. An EST data set derived from a S. bicolor root hair-specific cDNA library contained all the candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Sorgoleone interferes with several molecular target sites, including inhibition of photosynthesis in germinating seedlings. Sorgoleone is not translocated acropetally in older plants, but can be absorbed through the hypocotyl and cotyledonary tissues. Therefore, the mode of action of sorgoleone may be the result of inhibition of photosynthesis in young seedlings in concert with inhibition of its other molecular target sites in older plants. Due to its hydrophobic nature, sorgoleone is strongly sorbed in soil which increases its persistence, but experiments show that it is mineralized by microorganisms over time.

In situ silicone tube microextraction: a new method for undisturbed sampling of root-exuded thiophenes from marigold (Tagetes erecta L.) in soil.

The difficulties of monitoring allelochemical concentrations in soil and their dynamics over time have been a major barrier to testing hypotheses of allelopathic effects. Here, we evaluate three diffusive sampling strategies that employ polydimethylsiloxane (PDMS) sorbents to map the spatial distribution and temporal dynamics of root-exuded thiophenes from the African marigold, Tagetes erecta. Solid phase root zone extraction (SPRE) probes constructed by inserting stainless steel wire into PDMS tubing were used to monitor thiophene concentrations at various depths beneath marigolds growing in PVC pipes. PDMS sheets were used to map the distribution of thiophenes beneath marigolds grown in thin glass boxes. Concentrations of the two major marigold thiophenes measured by these two methods were extremely variable in both space and time. Dissection and analysis of roots indicated that distribution of thiophenes in marigold roots also was quite variable. A third approach used 1 m lengths of PDMS microtubing placed in marigold soil for repeated sampling of soil without disturbance of the roots. The two ends of the tubing remained out of the soil so that solvent could be washed through the tubing to collect samples for HPLC analysis. Unlike the other two methods, initial experiments with this approach show more uniformity of response, and suggest that soil concentrations of marigold thiophenes are affected greatly even by minimal disturbance of the soil. Silicone tube microextraction gave a linear response for alpha-terthienyl when maintained in soils spiked with 0-10 ppm of this thiophene. This method, which is experimentally simple and uses inexpensive materials, should be broadly applicable to the measurement of non-polar root exudates, and thus provides a means to test hypotheses about the role of root exudates in plant-plant and other interactions.

Mineralization of the allelochemical sorgoleone in soil.

The allelochemical sorgoleone is produced in and released from the root hairs of sorghum (Sorghum bicolor). Studies have confirmed that it is the release of sorgoleone that causes the phytotoxic properties of sorghum, and sorgoleone has a potential to become a new natural herbicide, or the weed suppressive activity of sorghum can be utilized in integrated weed management. Since sorgoleone is released into soil, knowledge of the fate of sorgoleone in soil is essential if it is to be utilized as an herbicide. Fate studies will characterize the persistence and mobility of the compound. Three types of radioactively labelled sorgoleone were produced and used to study mineralization (complete degradation to CO(2)) of this lipid benzoquinone in four soils, two from the United States of America (Mississippi) and two from Denmark. The studies showed that sorgoleone was mineralized in all soils tested. The methoxy group of sorgoleone was readily mineralized, whereas mineralization of the remaining molecule was slower. Mineralization kinetics indicated that microorganisms in American soils were able to use sorgoleone as a source of energy.

Fate of glyphosate in soil and the possibility of leaching to ground and surface waters: a review.

The very wide use of glyphosate to control weeds in agricultural, silvicultural and urban areas throughout the world requires that special attention be paid to its possible transport from terrestrial to aquatic environments. The aim of this review is to present and discuss the state of knowledge on sorption, degradation and leachability of glyphosate in soils. Difficulties of drawing clear and unambiguous conclusions because of strong soil dependency and limited conclusive investigations are pointed out. Nevertheless, the risk of ground and surface water pollution by glyphosate seems limited because of sorption onto variable-charge soil minerals, e.g. aluminium and iron oxides, and because of microbial degradation. Although sorption and degradation are affected by many factors that might be expected to affect glyphosate mobility in soils, glyphosate leaching seems mainly determined by soil structure and rainfall. Limited leaching has been observed in non-structured sandy soils, while subsurface leaching to drainage systems was observed in a structured soil with preferential flow in macropores, but only when high rainfall followed glyphosate application. Glyphosate in drainage water runs into surface waters but not necessarily to groundwater because it may be sorbed and degraded in deeper soil layers before reaching the groundwater. Although the transport of glyphosate from land to water environments seems very limited, knowledge about subsurface leaching and surface runoff of glyphosate as well as the importance of this transport as related to ground and surface water quality is scarce.

Formation and degradation kinetics of the biofumigant benzyl isothiocyanate in soil.

Glucosinolates (GSLs) are produced by plants of the Capparales order. Upon enzymatic hydrolysis the GSLs can be transformed to the toxic isothiocyanates (ITCs), which can be used as biofumigants for the control of soil-borne pests. The rates of ITC formation and degradation are critical to both biofumigation and the toxicity and leaching of GSLs and ITCs in soil. Degradation kinetics of benzyl GSL and benzyl ITC in a sandy and clayey surface and subsoil at 8-9 degrees C at natural moisture contents were investigated, as was the rate of formation of ITC from the GSL. Degradation of GSL followed logistic kinetics with t 1/2 = 0.7-9.1 days. Degradation was faster in clayey soil compared to sandy soil, and faster in surface soil compared to subsoil. In surface soils, up to 25% of added GSL was detected as ITC, while only 1-6% were detected in the subsoils. ITC degradation followed first-order kinetics with t 1/2 = 0.3-1.7 days, with faster degradation in subsoils than in surface soils. Based on the data for GSL hydrolysis and ITC degradation, the concentration of ITC following GSL application was successfully modeled assuming complete conversion of glucosinolate to isothiocyanate and first-order degradation of isothiocyanate.

Degradation kinetics of glucosinolates in soil.

Glucosinolates are compounds produced by all cruciferous plants. They can be hydrolyzed to several biologically active compounds and, as such, may serve as naturally produced pesticides. To optimize the pesticidal (biofumigation) effect and to assess the risk of glucosinolate leaching and spread in the environment, the degradation in soil of glucosinolates has been studied. The kinetics of degradation of four glucosinolates, two aliphatic (but-3-enyl and 2-hydroxy-but-3-enyl) and two aromatic (benzyl and phenethyl), in four soils was largely independent of the specific glucosinolate structure. Degradation followed logistic kinetics. Degradation was much faster in a clayey soil (half-life, 3.5-6.8 h) than in a sandy soil (half-life, 9.2-15.5 h). Degradation was much slower or nonexistent in the subsoil (<25 cm soil depth). The glucosinolates are not sorbed in the soil, and the degradation potential is, to a large extent, associated with the clay fraction. Measured activity in the soils of the enzyme myrosinase, which can catalyze the hydrolysis of glucosinolates, correlated well with the glucosinolate degradation kinetics. Autoclaving, but not sodium azide or gamma-irradiation, effectively blocked glucosinolate degradation, indicating that extracellular myrosinase is important for glucosinolate degradation.

Extraction and determination of glucosinolates from soil.

The use of glucosinolate-containing plants as soil-incorporated biofumigants for pest and disease control has raised questions regarding the fate of glucosinolates in soil; however, no method for routine analysis of glucosinolates in soil has been reported. A simple method to extract glucosinolates from soil with quantification as desulfoglucosinolates by HPLC is presented. The method involves two extractions with 70% methanol at room temperature, centrifugation, and filtration prior to the desulfation step. The desulfoglucosinolates are then quantified by HPLC using established protocols for plant tissue analysis. There were no significant interfering peaks from the soil extracts, and the method provided high extraction efficiencies (around 100%) for both aromatic (benzyl) and aliphatic (2-propenyl) glucosinolates when amended at a wide range of realistic field soil concentrations (1.6-120 nmol/g of soil). The method was equally effective in three diverse Australian soils that varied in organic matter, clay content, and pH. The method was effective in air-dried or field-moist soil, although evidence for rapid glucosinolate degradation in field-moist soil indicates that extraction of moist soils should be performed as soon as possible after sampling. The method is compatible with field soil sampling at remote sites and utilizes the same equipment and protocols already established for plant tissue analysis. Extraction of glucosinolates in the field following incorporation of Indian mustard (Brassica juncea) and rape (Brassica napus) green manure crops was also tested. Eight different glucosinolates contained in the plant tissues were identified and quantified in soil extracts at concentrations ranging from 0.11 to 21.7 nmol/g of soil.

Modeling the kinetics of the competitive adsorption and desorption of glyphosate and phosphate on goethite and gibbsite and in soils.

The herbicide glyphosate and inorganic phosphate compete for adsorption sites in soil and on oxides. This competition may have consequences for the transport of both compounds in soil and hence for the contamination of groundwater. We present and evaluate six simple, kinetic models that only take time and concentrations into account. Three of the models were found suitable to describe the competition in soil. These three models all assumed both competitive and additive adsorption, but with different equations used to describe the adsorption. For the oxides, three additional models assuming only competitive adsorption were also found suitable. This is in accordance with the observation that the adsorption in soil is both competitive and additive, whereas the adsorption on oxides is competitive. All models can be incorporated in transport models such as the convection-dispersion equation.