Toxicological assessment of agrochemicals on bees using machine learning tools.

Machine learning (ML) is a branch of artificial intelligence (AI) that enables the analysis of complex multivariate data. ML has significant potential in risk assessments of non-target insects for modeling the multiple factors affecting insect health, including the adverse effects of agrochemicals. Here, the potential of ML for risk assessments of glyphosate (herbicide; formulation) and imidacloprid (insecticide, neonicotinoid; formulation) on the stingless bee Melipona quadrifasciata was explored. The collective behavior of forager bees was analyzed after in vitro exposure to agrochemicals. ML algorithms were applied to identify the agrochemicals that the bees have been exposed to based on multivariate behavioral features. Changes in the in situ detection of different proteins in the midgut were also studied. Imidacloprid exposure leads to the greatest changes in behavior. The ML algorithms achieved a higher accuracy (up to 91%) in identifying agrochemical contamination. The two agrochemicals altered the detection of cells positive for different proteins, which can be detrimental to midgut physiology. This study provides a holistic assessment of the sublethal effects of glyphosate and imidacloprid on a key pollinator. The procedures used here can be applied in future studies to monitor and predict multiple environmental factors affecting insect health in the field.

Synergistic effects of glyphosate formulation herbicide and tank-mixing adjuvants on Pardosa spiders.

Glyphosate-based herbicides are the world's most consumed agrochemicals, and they are commonly used in various agroecosystems, including forests, as well as in urban zones and gardens. These herbicides are sold as formulations containing adjuvants. Other tank-mixing adjuvants (most often surfactants) are commonly added to these formulations prior to application. According to the manufacturers of agrochemicals, such tank mixes (as these are known in agronomic and horticultural practice) have modified properties and perform better than do the herbicides as used alone. The effects of these tank mixes on the environment and on beneficial arthropods are almost unknown. Therefore, we studied whether a herbicide formulation mixed with adjuvant has modified effects on one of the most common genera of ground-dwelling wolf spiders vis-à-vis the herbicide formulation and adjuvants themselves. Specifically, we studied the synergistic effect in the laboratory on the predatory activity (represented by the number of killed flies) of wolf spiders in the genus Pardosa after direct treatment using the glyphosate-based herbicide formulation Roundup klasik Pro®, Roundup klasik Pro® in a mixture with the surfactant Wetcit®, Roundup klasik Pro® in a mixture with the surfactant Agrovital®, and the surfactants alone. We found that pure surfactants as well as herbicide-and-surfactants tank mixes significantly decrease the predatory activity of Pardosa spiders in the short term even as Roundup klasik Pro® did not itself have any such effect. Our results support the hypothesis that plant protection tank mixes may have modified effect on beneficial arthropods as compared to herbicide formulations alone. Therefore, testing of pesticide tank mixes is highly important, because it is these tank mixes that are actually applied to the environment.

Setal-epidermal, muscular and enzymatic anomalies induced by certain agrochemicals in the earthworm Eudrilus eugeniae (Kinberg).

Eudrilus eugeniae, the vermicomposing worm, is found in considerable numbers in agricultural fields in India due to their eventual transfer through vermimanure. These worms are very often exposed to pesticides, herbicides, chemical fertilisers and other soil amendments. This paper reports the effects of variable concentrations of urea, phosphogypsum (PG), paper mill sludge (PMS) and two organophosphorus agrochemicals, monocrotophos and glyphosate, on certain morphological, histological and biochemical parameters of E. eugeniae. Results indicated setal anomalies, epidermal lesions, clitellar swelling and constriction of the body. Disintegration of connective tissue, vacuolation of dermis and significant alterations in protein, lipid peroxidation levels and activities of lactate dehydrogenase, acetylcholinesterase and catalase have also been observed in the treated worms. It is proposed that setae, connective tissue, protein and enzymes in E. eugeniae could be useful markers to evaluate toxicity due to the test chemicals.

Impact of agrochemicals on non-target species: Calathus fuscipes Goeze 1777 (Coleoptera: Carabidae) as model.

Carabid beetles are important in the biological control of arable crop pests. Agricultural practices can produce over time a delayed toxic effect at the organismal and population levels and can compromise the survival on these species. In this research, we quantified the cumulative sublethal effect on body size, Malpighian tubules and immune responses in Calathus fuscipes adults living in the potato field and exposed to lambda-cyhalothrin and cymoxanil-based commercial formulates. Reductions of morphological parameters such as body, pronotum and elytron in both males and females from the potato field indicated that the pre-imaginal stages (larvae and pupae) suffer the sublethal effects of exposure to the larvicide control action of lambda-cyhalothrin. Ultrastructural alterations recorded in Malpighian tubules at the level of plasma membrane, mitochondria and nucleus indicated the reduction of the detoxification capability. The basal phenoloxidase and lysozyme-like enzyme activities have measured as markers of immune competence. Spectrophometric analyses showed that the chronic exposure in field causes an increase of basal phenoloxidase enzyme activity, while the lytic activity of haemolymph was not affected. As a result, the use of larvicides and fungicides have a harmful effect on beneficial species such C. fuscipes living in the soil of potato fields. These morphological and physiological results recorded at the organismal level can provide useful information of effects at the population and community levels to preserve the biodiversity of agroecosystem.

Application of a novel integrated toxicity testing strategy incorporating "3R" principles of animal research to evaluate the safety of a new agrochemical sulfoxaflor.

Plant protection products (PPPs) and the active substance(s) contained within them are rigorously and comprehensively tested prior to registration to ensure that human health is not impacted by their use. In recent years, there has been a widespread drive to have more relevant testing strategies (e.g., ILSI/HESI-ACSA and new EU Directives), which also take account of animal welfare, including the 3R (replacement, refinement, and reduction) principles. The toxicity potential of one such new active substance, sulfoxaflor, a sulfoximine insecticide (CAS #946578-00-3), was evaluated utilizing innovative testing strategies comprising: (1) an integrated testing scheme to optimize information obtained from as few animals as possible (i.e., 3R principles) through modifications of standard protocols, such as enhanced palatability study design, to include molecular endpoints, additional neurotoxicity and immunotoxicity parameters in a subchronic toxicity study, and combining multiple test guidelines into one study protocol; (2) generation of toxicokinetic data across dose levels, sexes, study durations, species, strains and life stages, without using satellite animals, which was a first for PPP development, and (3) addition of prospective mode of action (MoA) endpoints within repeat dose toxicity studies as well as proactive inclusion of specific MoA studies as an integral part of the development program. These novel approaches to generate key data early in the safety evaluation program facilitated informed decision-making on the need for additional studies and contributed to a more relevant human health risk assessment. This supplement also contains papers which describe in more detail the approach taken to establish the MoA and human relevance framework related to toxicities elicited by sulfoxaflor in the mammalian toxicology studies: developmental toxicity in rats mediated via the fetal muscle nicotinic acetylcholine receptor (nAChR) ( Ellis-Hutchings et al. 2014 ); liver tumors in rodents mediated via CAR/PXR ( LeBaron et al. 2014 ); and Leydig cell tumors in Fischer 344 rats ( Rasoulpour et al. 2014 ).

The sensitivity of metabolomics versus classical regulatory toxicology from a NOAEL perspective.

The identification of the no observed adverse effect level (NOAEL) is the key regulatory outcome of toxicity studies. With the introduction of "omics" technologies into toxicological research, the question arises as to how sensitive these technologies are relative to classical regulatory toxicity parameters. BASF SE and metanomics developed the in vivo metabolome database MetaMap®Tox containing metabolome data for more than 500 reference compounds. For several years metabolome analysis has been routinely performed in regulatory toxicity studies (REACH mandated testing or new compound development), mostly in the context of 28 day studies in rats (OECD 407 guideline). For those chemicals for which a toxicological NOAEL level was obtained at either high or mid-dose level, we evaluated the associated metabolome to investigate the sensitivity of metabolomics versus classical toxicology with respect to the NOAEL. For the definition of a metabolomics NOAEL the ECETOC criteria (ECETOC, 2007) were used. In this context we evaluated 104 cases. Comparable sensitivity was noted in 75% of the cases, increased sensitivity of metabolomics in 8%, and decreased sensitivity in 18% of the cases. In conclusion, these data suggest that metabolomics profiling has a similar sensitivity to the classical toxicological study (e.g. OECD 407) design.

Caged Gammarus fossarum (Crustacea) as a robust tool for the characterization of bioavailable contamination levels in continental waters: towards the determination of threshold values.

We investigated the suitability of an active biomonitoring approach, using the ecologically relevant species Gammarus fossarum, to assess trends of bioavailable contamination in continental waters. Gammarids were translocated into cages at 27 sites, in the Rhône-Alpes region (France) during early autumn 2009. Study sites were chosen to represent different physico-chemical characteristics and various anthropic pressures. Biotic factors such as sex, weight and food availability were controlled in order to provide robust and comparable results. After one week of exposure, concentrations of 11 metals/metalloids (Cd, Pb, Hg, Ni, Zn, Cr, Co, Cu, As, Se and Ag) and 38 hydrophobic organic substances including polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyles (PCBs), pentabromodiphenylethers (PBDEs) and organochlorine pesticides, were measured in gammarids. All metals except Ag, and 33 organic substances among 38 were quantified in G. fossarum, showing that this species is relevant for chemical biomonitoring. The control of biotic factors allowed a robust and direct inter-site comparison of the bioavailable contamination levels. Overall, our results show the interest and robustness of the proposed methodological approach for assessing trends of bioavailable contamination, notably for metals and hydrophobic organic contaminants, in continental waters. Furthermore, we built threshold values of bioavailable contamination in gammarids, above which measured concentrations are expected to reveal a bioavailable contamination at the sampling site. Two ways to define such values were investigated, a statistical approach and a model fit. Threshold values were determined for almost all the substances investigated in this study and similar values were generally derived from the two approaches. Then, levels of contaminants measured in G. fossarum at the 27 study sites were compared to the threshold values obtained using the model fit. These threshold values could serve as a basis for further implementation of quality grids to rank sites according to the extent of the bioavailable contamination, with regard to the applied methodology.

Gossypol--a polyphenolic compound from cotton plant.

Gossypol (C(30)H(30)O(8)) is a polyphenolic compound derived from the cotton plant (genus Gossypium, family Malvaceae). The presence of six phenolic hydroxyl groups and two aldehydic groups makes gossypol chemically reactive. Gossypol can undergo Schiff base formation, ozonolysis, oxidation, and methylation to form gossypol derivatives. Gossypol and its derivatives have been the target of much research due to their multifaceted biological activities including antifertility, antivirus, anticancer, antioxidant, antitrypanosomal, antimicrobial, and antimalarial activities. Because of restricted rotation of the internaphthyl bond, gossypol is a chiral compound, which has two atropisomers (i.e., (+)- and (-)-gossypol) that exhibit different levels of biological activities. This chapter covers the physiochemical properties, analyses, biological properties, and agricultural and clinical implications of gossypol.

[Criteria for evaluating deviations from good laboratory practice]. / Criteri per la valutazione delle deviazioni dai principi di buona pratica di laboratorio.

The assessment of compliance with the principles of good laboratory practice (GLP) requires that different observations be integrated in a comprehensive assessment of the adequacy of the test facility inspected. The major types of deviations are examined in detail, since they might jeopardize the scientific quality of data. These are: a) deviations, even though seemingly minor ones, which could signal an inadequate understanding of the GLP principles (e.g., insufficient attention to personnel training); b) inadequate application of the GLP principles to basic procedures and/or structures (e.g., clean-dirty interfaces in the laboratory animal unit); c) inadequacies concerning specific study types. The elaboration of new guidelines leads to the definition of new quality requirements and critical points. One such case is the development of tests to identify endocrine effects which in turn triggers the need of structures and personnel adequate to examine seminological or hormonal parameters.

[Chemical compound safety: typology of competency accreditation for assay centers and analytical laboratories]. / La sicurezza dei composti chimici: tipologie di riconoscimento della competenza per i Centri di saggio e i laboratori di analisi.

The use of chemicals warrants many benefits on which modern society is entirely dependent. On the other hand, the lack of reliable information about the impact of the use of chemicals raises increasing concern. In order to guarantee the safety of chemicals it is mandatory to proceed to risk assessment, which in turn consists of hazard evaluation and exposure estimation. These activities are strictly dependent upon the availability of reliable data and information, produced by, e.g., test facilities, test laboratories and clinical laboratories, the specific competence of which has been properly recognised. All this applies in the pre-marketing phase as well as during the use of chemical substances. In this latter phase it is necessary to carry out an appropriate monitoring of environment, food and, in specific situations, human beings (biological monitoring). In the field of chemical safety, standards, legal instruments and operative instruments are nowadays available. These tools make it possible to assess both the quality of data and the competence of the entities involved in the production of the data themselves.