Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole

Three parallel experiments were conducted exposing adult red-legged partridges to seeds treated with one of three pesticide formulations: Score® 25 EC (difenoconazole 25 % w/v), Pormasol® Forte (thiram 80 % w/w), and Escocet® (imidacloprid 35 % w/v). Experiments are designated with the name of the corresponding active ingredient (i.e. difenoconazole, thiram or imidacloprid). Seeds were sprayed to obtain two target concentrations, a low dose corresponding to the one recommended for cereal seed coating, as indicated in product label, and a high dose that was twice the recommended one. Captive-born, 1 year-old red-legged partridges were housed in pairs in outdoor cages and assigned to one of the seven experimental groups (six groups corresponding to the two application doses of each pesticide, plus a control group). Because control group is shared among the three experiment, control records in the dataset appeared by triplicate, with a replicate assigned to each experiment for statistical purposes. Six breeding pairs were exposed to each treatment. On the day of beginning of experiment (March 15th 2010), animals were weighed, tarsus was measured and a blood sample was taken, on which we calculated haematocrit and measured oxidative stress biomarkers (in the cellular fraction), biochemistry, vitamins and carotenoids (in plasma). Body condition was estimated using the scaled mass index of the log-log regression between tarsus length and body weight. During 10 days, partridges were fed exclusively with treated wheat (low and high pesticide exposure groups), or with untreated wheat (control group). At the end of exposure, and 14 days after the end of exposure, we repeated measurements and blood collection and analyses. Prior to and just after pesticide exposure, we took digital photographs under standardized conditions that were analysed with Photoshop to measure the red, carotenoid-based coloration of the beak and eye ring of partridges. We estimated cell-mediated immune responsiveness using the phytohemagglutinin (PHA)-skin test. On the last day of exposure and on day 13 after end of exposure, we measured wing web thickness and injected intradermally the wing web with PHA to stimulate T lymphocyte proliferation and macrophage infiltration. Wing web thickness was measured again 24 h after injection, and the increase in thickness was interpreted as an indicator of cell-mediated immune responsiveness. Humoral immune response was estimated using a haemagglutination test after injecting, 2 days after the end of exposure, an antigen (i.e. sheep red blood cells) that stimulated the synthesis of specific antibodies. Serial dilutions of plasma of blood collected at the end of exposure (before SRBC injection) and 14 days later (12 days after antigen injection) were used in agglutination tests of RBC taken from the same sheep whose RBCs had been injected to partridges. Once exposure ended, partridges were kept in the cages and fed ad libitum with commercial, non-treated food. Because some birds died during the experiments, partridges that were alone in the cages were paired again with mates exposed to the same treatment (when possible). Eggs were picked up, measured (maximum length and width, and mass) identified individually and incubated until hatching. Birth date, as well as tarsus length and body weight of hatchling was recorded. Chicks were individually marked and housed in closed rooms in optimal conditions. We measured (tarsus length) and weighed all chicks upon 8, 16, 24 and 32 days of age, and calculated their body condition as for adults. Unhatched eggs were opened and examined to see whether they were fecundated or not. Shell thickness of hatched and unhatched eggs was measured in the equatorial region after separation of the inner membrane and drying.