Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.

The large-scale use of neonicotinoid insecticides has raised growing concerns about their potential adverse effects on farmland birds, and more generally on biodiversity. Imidacloprid, the first neonicotinoid commercialized, has been identified as posing a risk for seed-eating birds when it is used as seed treatment of some crops since the consumption of a few dressed seeds could cause mortality. But evidence of direct effects in the field is lacking. Here, we reviewed the 103 wildlife mortality incidents reported by the French SAGIR Network from 1995 to 2014, for which toxicological analyses detected imidacloprid residues. One hundred and one incidents totalling at least 734 dead animals were consistent with an agricultural use as seed treatment. Grey partridges (Perdix perdix) and “pigeons” (Columba palumbus, Columba livia and Columba oenas) were the main species found. More than 70% of incidents occurred during autumn cereal sowings. Furthermore, since there is no biomarker for diagnosing neonicotinoid poisonings, we developed a diagnostic approach to estimate the degree of certainty that these mortalities were due to imidacloprid poisoning. By this way, the probability that mortality was due to poisoning by imidacloprid-treated seeds was ranked as at least “likely” in 70% of incidents. As a result, this work provides clear evidence to risk managers that lethal effects due to the consumption by birds of imidacloprid-treated seeds regularly occur in the field. This in turn raises the question of the effectiveness of the two main factors (seed burying and imidacloprid-treated seeds avoidance) that are supposed to make the risk to birds negligible. Risk factors and the relevance of mitigation measures are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s11356-016-8272-y) contains supplementary material, which is available to authorized users.

Estimating exposure of wild birds to plant protection products is of key importance in the risk assessment process evaluating their harmful potential. In this paper, we propose an ecologically-relevant methodology to estimate potential exposure to active substances (ASs) of a farmland focal bird, the gray partridge Perdix perdix. It is based on bird habitat use of fields at the time of pesticide applications. It accounts for spatio-temporal heterogeneity at population and landscape scales. We identify and quantify the potential exposure to 179 ASs of 140 clutches during pre-laying, laying, and incubation phases, and of 75 coveys. The data come from a large scale field study combining radiotelemetry and a farmer survey. They were collected in 12 different representative sites. The proportion of clutches potentially exposed to a given chemical was ≥5% for 32 ASs; prothioconazole and epoxiconazole ranking first. 71% of clutches were potentially exposed to ≥1 AS and 67% to ≥2 ASs. Mixtures involved 2 to 22 ASs. They emerged from commercial formulations, tank mixtures, bird habitat use, and combinations. ASs were fungicides (53%), herbicides (25%), and insecticides (16%) used on a variety of crops in April-June, when ground-nesting birds are breeding. The European Food Safety Authority conclusions report a long-term first-tier toxicity-to-exposure ratio (TERlt) <5 for 11 out of 19 documented ASs, and higher-tier TERlt <5 for 5 out of 10 ASs. This suggests a potential risk for bird reproduction in farmlands. Globally 13% of coveys were potentially exposed to 18 ASs during the first month (1-4 coveys per AS). The use of our field data in future research and risk assessment is discussed.

The contamination of the eggs of farmland birds by currently used plant protection products (PPPs) is poorly documented despite a potential to adversely impact their breeding performance. In this context, 139 eggs of 52 grey partridge Perdix perdix clutches, collected on 12 intensively cultivated farmlands in France in 2010–2011, were analysed. Given the great diversity of PPPs applied on agricultural fields, we used exploratory GC/MS-MS and LC/MS-MS screenings measuring ca. 500 compounds. The limit of quantification was 0.01 mg/kg, a statutory reference. A total of 15 different compounds were detected in 24 clutches. Nine of them have been used by farmers to protect crops against fungi (difenoconazole, tebuconazole, cyproconazole, fenpropidin and prochloraz), insects (lambda-cyhalothrin and thiamethoxam/clothianidin) and weeds (bromoxynil and diflufenican). Some old PPPs were also detected (fipronil(+sulfone), HCH(α,β,δ isomers), diphenylamine, heptachlor(+epoxyde), DDT(Σisomers)), as well as PCBs(153, 180). Concentrations ranged between <0.01 and 0.05 mg/kg but reached 0.067 (thiamethoxam/clothianidin), 0.11 (heptachlor + epoxyde) and 0.34 (fenpropidin) mg/kg in some cases. These results testify an actual exposure of females and/or their eggs to PPPs in operational conditions, as well as to organochlorine pollutants or their residues, banned in France since several years if not several decades, that persistently contaminate the environment.Routes of exposure, probability to detect a contamination in the eggs, and effects on egg/embryo characteristics are discussed with regard to the scientific literature.Electronic supplementary materialThe online version of this article (doi:10.1007/s11356-016-6093-7) contains supplementary material, which is available to authorized users.

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Numerous manmade chemicals released into the environment can interfere with normal, hormonally regulated biological processes to adversely affect the development and reproductive functions of living species. Various in vivo and in vitro tests have been designed for detecting endocrine disruptors, but the number of chemicals to test is so high that to save time and money, (quantitative) structure-activity relationship ((Q)SAR) models are increasingly used as a surrogate for these laboratory assays. However, most of them focus only on a specific target (e.g. estrogenic or androgenic receptor) while, to be more efficient, endocrine disruption modelling should preferentially consider profiles of activities to better gauge this complex phenomenon. In this context, an attempt was made to evaluate the endocrine disruption profile of 220 structurally diverse pesticides using the Endocrine Disruptome simulation (EDS) tool, which simultaneously predicts the probability of binding of chemicals on 12 nuclear receptors. In a first step, the EDS web-based system was successfully applied to 16 pharmaceutical compounds known to target at least one of the studied receptors. About 13% of the studied pesticides were estimated to be potential disruptors of the endocrine system due to their high predicted affinity for at least one receptor. In contrast, about 55% of them were unlikely to be endocrine disruptors. The simulation results are discussed and some comments on the use of the EDS tool are made.

Botulism in wild birds is a widespread and potentially lethal disease raising major conservation issues. Botulism is also of public health concern. Due to the action of botulinum neurotoxins, mostly produced by Clostridium botulinum, botulism can affect wild birds, livestock, and humans. This study is part of a project aimed at improving our understanding of the pathogenesis of botulism in wild avifauna, which is still poorly understood. Indeed, the prevalence and dynamics of C. botulinum in the digestive tract or in bird tissue, whether as intermittent carriage related to environmental contamination or as part of the normal avian microbiota, is still unknown. In this study, we specifically addressed the presence of a healthy carrier status of wild birds, and its role in outbreaks. To answer this question, we monitored the estimated prevalence of C. botulinum in wild birds through samples from banded and swabbed birds as well as from hunted bird organs. Our results do not support the hypothesis of a healthy carriage outside of outbreaks, which raises the question of the bioavailability of the bacterium and toxin in the environment. Finally, the gene encoding botulinum neurotoxin type E was detected in keel muscle from a hunted bird, showing that recommendations on the consumption of wild bird meat are needed following a botulism outbreak.

A suite of models is proposed for estimating the risk of pesticides against the grey partridge (Perdix perdix) and their clutches. Radio-tracked data of females, description and location of the clutches, and data on the pesticide treatments during the laying periods of the partridges were used as basic information. Quantitative structure-activity relationship (QSAR) and quantitative structure-property relationship (QSPR) modelling allowed us to characterize the pesticides by their 1-octanol/water partition coefficient (log P), vapour pressure, primary and ultimate biodegradation potential, acute toxicity (LD) on P. perdix, and endocrine disruption potential. From these physicochemical and toxicological data, the system of integration of risk with interaction of scores (SIRIS) method was used to design scores of risk for pesticides, alone or in mixture. A program, written in R (version 3.1.1), called Simulation of Toxicity in Perdix perdix (SimToxPP), was designed for estimating the risk of substances, considered alone or in mixture, against the grey partridge during breeding. The software tool is flexible enough to simulate realistic in situ scenarios. Different examples of applications are shown. The advantages and limitations of the approach are briefly discussed.