ANTICOAGULANT EXPOSURE IN GOLDEN EAGLE (AQUILA CHRYSAETOS) POWER LINE ELECTROCUTION AND WIND TURBINE MORTALITIES

Toxicants to which Golden Eagles (Aquila chrysaetos) may be exposed in the environment may have a direct or indirect effect on behavior and flight capability. Lead, for example, may initiate or enhance neurologic disease, resulting in abnormal feeding and flight behaviors, leading in turn to negative physical impacts (Waterman et al. 1994; Herring et al. 2017). Studies have suggested that exposure to lead predisposes birds to collision trauma (O'Halloran et al. 1989; Ecke et al. 2017). Exposure to this toxicant, however, was shown to not be a predisposing factor in wind turbine–related mortality in Golden Eagles (Viner and Kagan 2021). The association between pesticides or other environmental contaminants and traumatic death has not been extensively explored. It is possible that the subclinical effects of pesticide intoxication alter the natural behavior of birds and predispose eagles to certain anthropogenic mortality factors. Adverse impacts may include a lack of avoidance behavior in proximity to spinning wind turbine blades. If toxicants are influential in avian-wind interactions, these factors would need to be addressed when siting wind farms.

Wind-derived power has had a significant effect on bird populations. The wind was first harnessed for energy generation in 1888 (Kaldellis and Zafirakis 2011) but remained primarily a local utility for decades. During the oil crisis of the early 1970s, wind-generated power expanded in the US and Europe to serve the electricity needs of municipalities. Wind turbine farms were placed where wind speed and air density were optimal and today can commonly be found on ridge tops with good updraft potential (US Energy Information Administration 2021). An early estimate of the effects of wind energy on wildlife in the US suggested avian mortality at wind turbine sites as 10,000 to 40,000 birds per year (Erickson et al. 2001). Merely 12 yr later, Loss et al. (2013) raised that estimate to between 140,000 and 328,000 avian mortalities per annum. Between these time points (2001–13), wind energy generation made a somewhat commensurate increase in capacity from 4,232 megawatts to 61,107 megawatts (US Office of Energy Efficiency and Renewable Energy 2019). By 2018, wind farms provided 96,433 megawatts of cumulative installed wind capacity and delivered 6.5% of the energy consumed by the US populace (American Wind Energy Association 2019).

Our project aimed to explore the relationship between anticoagulant rodenticide (AR) exposure and Golden Eagle mortalities at wind turbine sites in the US. Birds expend less energy at rest than during flight, when they are required to achieve and maintain altitudes where they may interact with wind turbine blades (Bairlein et al. 2015). Thus, in contrast to this higher altitude, higher energy activity, comparison was made to AR exposure in birds dying from power line electrocution at lower altitudes during presumptively lower energy activities.

All birds included in the study were submitted to the National Fish and Wildlife Forensic Laboratory (NFWFL) by US Fish and Wildlife Service law enforcement agents between 2013 and 2020. Forensic postmortem examinations were performed by board-certified veterinary pathologists (T.C.V., R.A.K.) to determine the cause of death. No eagles in this study exhibited gross evidence of anticoagulant toxicity. Samples of liver were taken opportunistically during postmortem examinations and stored in a freezer at –6 C for 24 h to 31 mo until submission for anticoagulant testing.

All eagles in the wind turbine (WT) group were determined to have died due to the impact of a spinning wind turbine blade. Evidence for this diagnosis included characteristic injuries (Pagel et al. 2013) and the location of the carcass when found. Comparison animals were selected from a subset of eagles that died due to electrocution on power lines (PL) as evidenced by singeing of feathers and/or skin and carcass location. The two causes of death were chosen to contrast the behaviors of low-flying and perching activity (PL) with high-flying and hunting activity (WT). Life stage and region of origin of PL eagles were aligned with the WT population as much as possible. Eagles within the study set were from the states of Arizona, California, Colorado, Idaho, Montana, Nevada, North Dakota, Oregon, Utah, and Wyoming (all US).

Anticoagulant rodenticide analysis was performed by the Michigan State University Veterinary Diagnostic Laboratory. In brief, approximately 2 g of tissue was extracted with acetonitrile followed by solid-phase cleanup. Anticoagulants were identified and quantitated in tissues by gradient high performance liquid chromatography with ultraviolet photodiode array and fluorescence (FL) detection using a modified method of Chalermchaikit et al. (1993) that has been previously described (Williams et al. 2014). The panel includes brodifacoum, bromadiolone, chlorophacinone, difenacoum, difethialone, diphacinone, and warfarin. Brodifacoum and difenacoum were screened and quantitated by FL detection in a basic mobile phase system and confirmed by an acidic mobile phase system. Chlorphacinone, difethialone, and diphacinone were screened, confirmed, and quantitated by photodiode array in basic mobile phase. Bromadiolone and warfarin were screened and quantitated by FL in an acidic mobile phase system; bromadiolone was confirmed by FL detection in a basic mobile phase system, whereas warfarin was confirmed by high performance liquid chromatography–electrospray-(+)-tandem quadrupole mass spectrometry. This confirmation was also available for the remaining analytes in difficult situations, as may arise with interfering chromatographic peaks. As an alternative to liquid chromatography tandem mass spectrometry, gas chromatography tandem mass spectrometry was also available for warfarin confirmation if needed. Detection limits for the analytes on gradient high performance liquid chromatography are warfarin 0.02 parts per million (ppm), bromadiolone 0.02 ppm, brodifacoum 0.002 ppm, chlorphacinone 0.2 ppm, diphacinone 0.2 ppm, difethialone 0.07 ppm, and difenacoum 0.02 ppm.

Statistical analysis was performed in R version 4.0.3 (R Core Team 2020). Due to the skewed nature of the results, the Mann-Whitney U-test was used to compare the mean ranks of liver brodifacoum concentrations between the independent WT and PL groups. To capture and analyze for the ARs detected, and account for changes in AR concentration during the postmortem interval (Wyman et al. 2011), AR results were modified into the binary factors of positive and negative exposure based on measurements above and below the detection limits, respectively. Logistic regression was applied to explore the relationships between life stage, sex, AR exposure, and cause of death. A significance level of P=0.05 was applied to the analyses.

Only brodifacoum and bromadiolone were detected in these eagles. Of the 62 Golden Eagles tested, 24 (39%) were positive for at least one AR. Brodifacoum was detected in 11 eagles in the PL group (35%) and in 11 eagles in the WT group (35%; Table 1). Bromadiolone was detected alone in one bird in each group and in combination with brodifacoum in one PL eagle. Bromadiolone concentrations were 0.04 ppm and 0.53 ppm in the PL eagles and 3.46 ppm in the WT eagle. The ranked sums of brodifacoum concentration in the WT and PL eagles were comparable (W=500.5; P=0.751).

The WT group contained 19 subadults and 12 adults, while the PL group contained 20 subadults and 11 adults. The distribution of life stage and sex across the two causes of mortality and AR status is described in Table 2. Across both the WT and PL groups, the difference in concentrations of brodifacoum in the livers of adults and subadults was not statistically significant (W=500; P=0.380). There was a significant difference when considering only sex and AR status within these groupings: female birds were significantly less likely than males to test positive for any anticoagulant (P=0.032).

The prevalence of anticoagulant exposure did not differ between the high and low altitude proxy groups of Golden Eagles in our study (Fig. 1), suggesting that these toxins have similar effects—or lack thereof—on soaring and perching behaviors. Indeed, this background exposure may be indicative of that in the greater population of Golden Eagles submitted to NFWFL. Similarity in AR exposure prevalence across life stages also indicates that the opportunity for exposure is constant. Though only two anticoagulants were detected in the eagles in our study (brodifacoum and bromadiolone), other first- and second-generation ARs remain toxins of concern for raptors.

Anticoagulant rodenticides are commonly classified as “first-generation” and “second-generation” (FGAR and SGAR; Nakayama et al. 2019). The FGAR, which were developed earlier, require multiple feedings for rodenticide effectiveness, while SGAR may kill rodents after a single feeding. The use of rodenticides in the US is regulated by the US Environmental Protection Agency and authorized State agencies (Witmer and Eisemann 2007). The US Environmental Protection Agency lists warfarin, chlorphacinone, and diphacinone as FGAR and difenacoum, brodifacoum, bromadiolone, and difenthialone as SGAR. In 2008, the US Environmental Protection Agency issued a decision to restrict the use of SGARs to agricultural and professional applications, allowing consumers to use only chlorphacinone and diphacinone, both FGAR, in small amounts in bait stations (Bradbury 2008). The ARs registered for use by rodent-control professionals included brodifacoum, bromadiolone, difenacoum, and difethialone. California has recently adopted further restrictions on the use of SGARs in wildlife habitat (California Code, Food and Agricultural Code 2020). Thus, the eagles in our study were probably exposed to anticoagulants used by professional pesticide applicators.

The mechanism of action shared between FGAR and SGAR involves inhibition of the clotting cascade by blocking the reactivation of vitamin K (Rattner et al. 2014). The toxicants are metabolized to varying degrees by cytochrome p450 enzymes in the liver (Horak et al. 2018). The FGAR diphacinone is processed rapidly in the liver of mice and excreted as both the unchanged compound and its metabolites for up to 8 d, while SGARs pass through the body much more slowly, remaining stable for up to several months. This persistent quality widens the opportunity for secondary toxicity in predator species (Eason et al. 2002). Koivisto et al. (2018) found that the presence of hepatic ARs correlated more with diet than with proximity to agricultural centers where these pesticides may be used. Specifically, birds and mammals with a diet dominated by rodents had a high prevalence of AR detected in the liver, regardless of distance to industrial areas or farms. These findings have been repeated in locations as diverse as New Zealand, Spain, and Denmark (Eason et al. 2002; Christensen et al. 2012; Ruiz-Suárez et al. 2014). Thus, while some eagles may choose to hunt in and near wind farms, the prey species in these areas, not the industries in or near the area itself, influence the birds' exposure to ARs.

The diagnosis of AR toxicosis in raptors is fraught with challenges, as there is a dearth of rigorous studies evaluating vitamin K metabolism and AR susceptibility in raptors (Rattner and Harvey 2021). Additionally, studies have shown inconsistency between measured AR concentrations in the liver and clinical signs or gross observations (Stone et al. 2003; Lohr 2018). Few studies exist documenting AR exposure specifically in eagles. Stone et al. (1999) outlined one Bald Eagle (Haliaeetus leucocephalus) and one Golden Eagle exposed to warfarin and brodifacoum, respectively, although the total number of eagles tested was not reported. A subsequent survey of raptors in New York, US (Stone et al. 2003) included five Bald Eagles, one of which had detectable levels of an unspecified AR, and one Golden Eagle, which was also exposed. The anticoagulant was not considered to have been a factor in either eagle's death. A survey of Bald and Golden Eagles submitted to one laboratory for postmortem examination over a 5-yr period found that 13 of the 17 Golden Eagles tested had measurable levels of ARs (Niedringhaus et al. 2021). For only one of these eagles was the AR compound considered to be the cause of mortality. Similarly, Russell and Franson (2014) found a solitary Golden Eagle AR mortality in a review of necropsy results over a 39-yr period.

For hunting, Golden Eagles prefer open, shrubby habitat with sparse vegetation, avoiding grassland, and perching and nesting on high rocks and conifers (Marzluff et al. 1997). Preferred topographic features include steep ridges and south-facing slopes (Singh et al. 2016). Within this low altitude flight zone, Golden Eagles select areas with high updraft potential along ridgelines (Miller et al. 2014). Hedfors (2014) found that Golden Eagles in Sweden fly at lower heights above ground level as the altitude of the topography increases, but flew higher when within 300 m of a wind turbine. The average flight height found for all habitats and eagles in the study was 144 m above ground level. The extension of a US wind turbine blade tip may be up to 146 m above ground level (American Wind Energy Association 2019), easily within the eagle flight zone. Preferred Golden Eagle habitat often overlaps fields of wind energy generation farms, and the highest likelihood for eagle-wind turbine interaction is along the edges of slopes and narrow ridgetops. An extensive review by Thaxter et al. (2017) showed that, in the US, birds of 308 different species have been killed by wind turbine blades. Species represented in the highest numbers in this data set included the Red-tailed Hawk (Buteo jamaicensis), European Starling (Sternus vulgaris), Horned Lark (Eremophila alpestris), Rock Dove (Columba livia), and Golden Eagle. In the northeastern US, where Bald and Golden Eagles are more sparsely distributed, raptor mortalities at wind farms are relatively infrequent and the more abundant passerines are more heavily impacted (Choi et al. 2020).

During periods of rest or feeding, eagles perch most often on cliffs, trees, and hills, resting on electrical systems (distribution lines and transmission lines) only 10.8% of the time (Dwyer et al. 2020). These power line perching events occurred at approximately the same rate on 12 m poles as on 20+ m poles. In each year in the US, approximately 500 Golden Eagles are electrocuted on power lines (Millsap et al. 2016). Electrocution occurs when an eagle comes in contact with two separate wires, or a wire and a ground line, either at a perch or in flight.

The differing behaviors associated with each mortality type in this study (WT=active hunting vs. PL=resting/feeding) offered a hypothetical contrast for anticoagulant exposure, which ultimately did not materialize on examination of the data. Viner and Kagan (2021) explored liver lead concentrations in Golden Eagles struck by wind turbine blades and those with other mortality types (e.g., gunshot, nonlead poisoning) and found that wind turbine birds had both a lower lead exposure prevalence and a lower mean lead concentration than did birds dying of other causes. This discounted the hypothesis that lead burden encourages fatal interactions with wind turbine blades, but rather suggested that the deleterious effects of lead in the body inhibited soaring and hunting behavior. In contrast, the anticoagulant exposure prevalence in the present report was consistent across both mortality types examined and may simply be indicative of background exposure in the general Golden Eagle population.

Though brodifacoum and bromadiolone were the only ARs detected in this study, chlorophacinone, diphacinone (both FGARs), and difethialone (a SGAR) have been found in other birds examined at NFWFL. In their surveyed population, Niedringhaus et al. (2021) found all the aforementioned ARs plus difenacoum in Golden Eagles they tested. Bromadiolone was found in 8/17 Golden Eagles tested, while this AR was found in only 3/62 eagles in our study set. This difference may be related to the geographic provenance of the birds, as many birds tested by Niedringhaus were submitted from eastern states of the US and all our birds came from the US west.

In our study, male Golden Eagles were more likely to have been exposed to ARs than were females (P=0.032). Few studies have explored sex differences in eagles exposed to environmental contaminants. Niedringhaus et al. (2021) described mortality due to AR exposure in 11 Bald Eagles (Haliaeetus leucocephalus), only two of which were male. The solitary Golden Eagle AR mortality in their data was female. In contrast, Wiens et al. (2019) found no variation in AR exposure between sexes or life stages in owls in the US Pacific northwest. Review of NFWFL postmortem records from 2000–21 reveal only three Golden Eagles diagnosed as anticoagulant (brodifacoum or diphacinone) mortalities, of which two were male and one was female. Though this distribution seems similar to our study set, this group is too small for statistically meaningful comparison. The differences seen in our study set may reflect dietary preferences or physiologic differences between the sexes, or the low sample size of the exposed cohort (n=24).

One WT bird had a highly elevated concentration of bromadiolone in the liver (3.46 ppm; limit of detection 0.02 ppm). The effects of this AR on the bird could not be effectively evaluated due to the extent of postmortem decomposition. At the time of discovery, this eagle's body had been populated by large fly larvae and the internal organs were soft and darkly discolored. Fat stores and muscle development, however, were appropriate for the age of the bird, findings that are inconsistent with chronic AR toxicosis. During the postmortem period, the detected concentration of drugs in the liver may increase many fold (Wyman et al. 2011). Thus, while the presence of bromadiolone indicates that this decomposed eagle had been exposed to the toxin, the measured AR concentration is probably not reflective of the level at the time of the bird's death.

This study offers insight into the prevalence of anticoagulant exposure in Golden Eagles with wind farm-associated and electrocution mortality in the US. The differing behaviors and mechanisms of each mortality factor were not reflected in differences in AR exposure. Further studies could explore whether AR exposure prevalence in Golden Eagles is consistent across all causes of death or is isolated to eagles with anthropogenic mortality. Human-made energy creation and pest control continue to negatively impact wild Golden Eagle populations, and conservation and mitigation efforts may logically focus on each factor individually.

The authors would like to thank Margaret Johnson and Alaina Covert for their technical toxicology expertise and Colleen K. Wilson for necropsy assistance. We also thank Ena Gillette and Mary Burnham Curtis for genetic analysis. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the US Fish and Wildlife Service.