New insights into anticoagulant rodenticide risk assessment in raptors from the southeastern Iberian Peninsula

Abstract

Anticoagulant rodenticides (ARs), used for rodent control, pose a threat to wildlife, especially birds of prey, due to their position in the food chain and sensitivity to these compounds. The main objective of this doctoral thesis is to expand knowledge on the impact of ARs on non-target species by assessing their presence in blood and their effects on four raptor species from the southeastern Iberian Peninsula. Sample analysis using high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer allowed for active monitoring of AR exposure. Additionally, environmental factors and diet were evaluated to understand their potential influence on exposure, as well as physiological biomarkers to analyse possible adverse effects. In the first chapter, AR exposure was investigated in long-eared owl (Asio otus) nestlings from the Region of Murcia. A total of 69 blood samples were analysed, detecting AR residues in 98.6% of them. Second-generation anticoagulant rodenticides (SGARs) predominated, particularly flocoumafen, brodifacoum, difenacoum, and bromadiolone. Most samples contained multiple compounds, reflecting repeated exposure. The diet was dominated by rodents (51.4%) and birds (44.2%), suggesting multiple exposure pathways. Prolongation of prothrombin time (PT) indicated adverse effects on coagulation, although not reaching acute toxicity thresholds. In the second chapter, SGAR exposure was compared between the barn owl (Tyto alba) and the common kestrel (Falco tinnunculus). A total of 136 blood samples were analysed, detecting a prevalence of 50% in barn owl nestlings and 68.6% in kestrel nestlings. Adult barn owls showed 100% prevalence and significantly higher ΣSGAR concentrations than nestlings. Kestrel exposure correlated with urban areas, while barn owl exposure was mainly influenced by human population density and the presence of mixed crops. A higher presence of Rattus spp. in the barn owl diet was associated with increased exposure, and PT was positively correlated with blood ΣSGAR concentrations. The third chapter assessed AR exposure in Eurasian eagle owl (Bubo bubo) nestlings and the influence of environmental and anthropogenic factors, including proximity to riverbeds and landfills. A total of 106 blood samples were collected between 2021 and 2022, with ARs detected in 91.5% of them. SGARs were significantly more prevalent than first-generation anticoagulant rodenticides (FGARs), with high concentrations of flocoumafen, difenacoum, and brodifacoum (up to 57.43 ng mL⁻¹). Brodifacoum and difenacoum exposure was associated with proximity to ephemeral streams, suggesting that these ecosystems may act as contamination pathways. Higher concentrations were also observed near landfills and urbanized areas. In the fourth chapter, the gene expression of vkorc1 and vkorc1l1, molecular targets of ARs, was analysed in 72 blood samples from Eurasian eagle owls. Variations in the expression of both genes were associated with environmental variables and human activity, suggesting possible compensatory mechanisms or physiological adaptations. These results highlight the importance of genetic biomarkers in assessing the toxicological risk of ARs in wildlife species. These findings underscore widespread AR exposure in raptors from southeastern Spain, posing a risk to biodiversity. Variations in diet and habitat use influenced exposure patterns, with evidence of multiple contamination pathways. Coagulation alterations indicate sublethal effects that could compromise avian fitness in the medium to long term. This study emphasizes the urgent need to regulate AR use and develop environmental management strategies to minimize their impact on raptors and other non-target species in semi-arid ecosystems.