Detection of imidacloprid and metabolites in Northern Leopard frog (Rana pipiens) brains.

Neonicotinoids are a new type of highly water-soluble insecticide used in agricultural practices to eliminate pests. Neonicotinoids bind almost irreversibly to postsynaptic nicotinic acetylcholine receptors in the central nervous system of invertebrates, resulting in overstimulation, paralysis, and death. Imidacloprid, the most commonly used neonicotinoid, is often transported to nearby wetlands through subsurface tile drains and has been identified as a neurotoxin in several aquatic non-target organisms. The aim of the present study was to determine if imidacloprid could cross the blood-brain barrier in adult Northern Leopard frogs (Rana pipiens) following exposure to 0, 0.1, 1, 5, or 10 µg/L for 21 days. Additionally, we quantified the breakdown product of imidacloprid, imidacloprid-olefin, and conducted feeding trials to better understand how imidacloprid affects foraging behavior over time. Exposure groups had 12 to 313 times more imidacloprid in the brain relative to the control and breakdown products showed a dose-response relationship. Moreover, imidacloprid brain concentrations were approximately 14 times higher in the 10 µg/L treatment compared to the water exposure concentration, indicating imidacloprid can bioaccumulate in the amphibian brain. Reaction times to a food stimulus were 1.5 to 3.2 times slower among treatment groups compared to the control. Furthermore, there was a positive relationship between mean response time and log-transformed imidacloprid brain concentration. These results indicate imidacloprid can successfully cross the blood-brain barrier and bioaccumulate in adult amphibians. Our results also provide insights into the relationship between imidacloprid brain concentration and subsequent altered foraging behavior.

Differential effect of trilostane on the progestin milieu in the pregnant mare.

Trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase, was administered intravenously to pregnant mares (n = 3) between day 277 and day 282 of gestation to determine its effect on the progestin milieu. In addition, placental tissue from mares at mid-gestation (150-300 days) (n = 4) were exposed to either deuterium-labelled pregnenolone alone or deuterium-labelled pregnenolone and trilostane to examine the effect of trilostane on placental metabolism of pregnenolone. Blood samples were collected from indwelling jugular catheters at frequent intervals for 48 h after infusion. Both plasma samples and incubation media were quantitatively analysed, after solid phase extraction and steroid derivitization, for concentrations of eight different progestin metabolites using gas chromatography and mass spectrometry. Trilostane infusion differentially affected the progestin milieu in vivo without inducing abortion. Forty-five minutes after infusion, maternal plasma concentrations of pregnenolone, 5-pregnene-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta,20 beta-diol and 3 beta-hydroxy-5 alpha-pregnan-20-one increased (P < 0.05) and remained high for 37 h. Concentrations of 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one and 5 alpha-pregnane-3 beta,20 alpha-diol decreased 15 min after infusion (P < 0.05), yet 1.5 h after infusion, concentrations had increased and remained high until 37 h after infusion. Trilostane inhibited the conversion of pregnenolone to progesterone in vitro (P < 0.001) while mediating an increase (P < 0.05) in concentrations of 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one. These observations demonstrate that the pregnant mare possesses unique steroid hormone metabolic activity and suggests that another steroid, perhaps 5 alpha-pregnane-3,20-dione and not progesterone, is the important steroid precursor for the other progestin metabolites found in circulating plasma.