In the European Union (EU), anticoagulant rodenticides (AR) represent more than 90% of the commercially available products for use against commensal rodents. The only other active ingredients (CO2, chloralose, corn cob) represent minor alternatives. A major issue in the EU is the resistance level of rat and mice populations, as well as potential non-target species exposure. This study presents results of surveys of anticoagulant resistance in Norway rats based on the sequencing of the VKORC1 gene, the major gene involved in AR and an investigation of the presence of AR residues detected in rodents trapped alive in urban and rural areas in order to investigate the potential risk of secondary poisoning of predators and scavengers. For resistance monitoring, rats were either trapped alive in the city of Lyon or its surroundings, or alternatively rat tails were obtained from pest control operators from France. Specific DNA primers were used for DNA sequencing and mutation identifications. AR residues were monitored by LC-MS-MS (for the 8 ARs marketed in Europe), with a limit of quantification of 1.0 µg/kg in liver samples. AR resistance appears to be extremely common (45-70% of all rats tested, depending on the part of France), with the notable exception of downtown Lyon where all rats are susceptible to AR. AR residues are detected in almost 100% of the rats trapped and tested (>200 individuals in/around Lyon). These results show that resistance is common in France, and evidence from neighboring countries suggests that this is an EU-wide problem. More surprising is the fact that all rodents tested contain detectable residues of AR, which could potentially result in secondary poisoning.

Zinc phosphide has been recently approved in Europe as a vole control product. Currently, only one formulation (lentils/pellets) is marketed with 0.8% Zn3P2. It is applied with a delivery device for burrow baiting. In many instances, zinc phosphide poisoning has been confirmed in non-target species (primary poisoning). In order to be prepared for potential non-target poisoning incidents in wildlife, the SAGIR network, FREDON Franche-Comté, and University of Franche-Comté conducted a field study on common voles to test the sampling method and storage impact under realistic field conditions on the detection of zinc phosphide. The toxicology laboratory of Vetagro Sup, member of SAGIR, worked on the improvement of the World Health Organization WHO technique in order to lower the Limit of Quantification (LOQ) and to validate the technique for the correct identification of field cases. The specificity was tested on 20 gastric content samples (100%), and the LOQ was established at 0.01 g/l (i.e., a 100-fold decrease as compared with the 1995 WHO technique). Zinc content was measured by Flame Atomic Absorption Spectroscopy and non-poisoned animals were tested to check baseline values and to estimate recovery of spiked samples (94-102%). Quantification of zinc in the liver of poisoned versus control animals was also performed. A total of 30 voles were collected in treated and control fields and submitted for analysis. Technicians were not aware of the poisoning status of the animals when performing analytical investigations. Twenty-one individuals were trapped in the control area and nine in the treated fields. Phosphine and high levels of Zn could be detected in eight of the nine intoxicated individuals. Phosphine was not detected in the control voles. Zinc concentrations in the gastric content were significantly different between negative control and exposed animals, but liver concentrations of zinc were similar.