Characterizing imidacloprid and metabolites in songbird blood with applications for diagnosing field exposures

Neonicotinoids are the most widely used insecticides globally, but their rapid metabolism in vertebrates makes diagnosing wildlife exposure challenging. More detailed information on the pattern of imidacloprid metabolites over time could be used to better approximate the timing and level of exposure. Here, we applied recently developed sensitive analytical methods to measure imidacloprid (IMI) parent compound along with an expanded suite of metabolites (5-OH-IMI, IMI-olefin, desnitro-IMI, IMI-urea, 6-chloronicotinic acid, 5-AMCP, 6-OH nicotinic acid) and six other neonicotinoids in adult red-winged blackbirds (Agelaius phoeniceus) that were experimentally exposed to one of two field-realistic concentrations of imidacloprid (0.8 or 6.9 mg/kg bw). We measured concentrations in small (25 μL) plasma samples collected pre-exposure and at 1-, 6-, 24- and 48-h post-exposure. Imidacloprid was rapidly absorbed and metabolized within 48 h at both doses, with the largest decrease within 6 h post-exposure. The average proportion of parent IMI decreased from 68% of total detectable residues at 1-h to 34% at 6-h post-exposure. Two primary metabolites in blood were 5-OH-IMI and IMI-olefin, and 5-OH-IMI was the most persistent marker of exposure at 48-h. Desnitro-IMI was consistently detected following very recent (≤ 1-h) IMI exposure, and a higher ratio of parent IMI to metabolites also indicated recent exposure. Other metabolites were only detected in the higher dose group, and could be used as indicators of exposure to higher IMI concentrations. This sensitive analytical method and the observed metabolite patterns could be used to inform a growing body of field studies linking neonicotinoid exposure and effects in free-living birds.