Identification of freeze-thaw artifact in fresh and decomposed black rockfish (Sebastes melanops) and steelhead trout (Oncorhynchus mykiss).

Identification of freeze-thaw artifact in fish can help to determine whether they have been harvested within the appropriate season and monitor adherence to fishing regulations. Recognition of freeze-specific changes will also prevent potential misinterpretation due to decomposition, disease, injury, or species variation. An initial survey using black rockfish (Sebastes melanops) identified which tissues reliably exhibit freeze artifact. Tissues were exposed to different treatments: immediate formalin fixation; refrigeration or storage at room temperature for 24, 48, or 72 hours; or freezing for 1, 8, or 28 days. Three fish underwent a combination of treatments. Tissue changes in each treatment group were compared macroscopically and microscopically. Macroscopic changes in frozen-thawed and never-frozen fish overlapped somewhat; however, microscopic findings of skeletal myocyte cavitation, lens liquefaction, and brain tissue fractures were consistent findings only in frozen-thawed tissues. A validation study was then done to establish the accuracy of microscopic analysis. Brain and paired ocular and skeletal muscle samples from 61 steelhead trout (Oncorhynchus mykiss) were fixed in formalin either fresh or after being frozen for 4 weeks. Weighted kappa values showed both high observer accuracy and interobserver agreement in the identification of freeze-thaw status. Based on these findings, microscopic changes in the skeletal muscle, eye, and brain are considered consistent and easily identifiable indicators of a previous freeze-thaw cycle and should not be confused with a pathologic process.

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

Golden Eagles (Aquila chrysaetos) are susceptible to anthropogenic mortality factors, including toxic compounds in the environment such as anticoagulant rodenticides (AR) and sources of man-made energy. The physical and behavioral effects of some toxins may predispose eagles to certain causes of death (COD). To investigate the influence of ARs on mortality of Golden Eagles at wind turbine farms, we randomly tested liver samples from 31 eagles found dead on wind farms and submitted to the National Fish and Wildlife Forensic Laboratory from 2013-20. The comparison group was composed of 31 Golden Eagles sampled during the same time frame with a COD of power line electrocution as a proxy for a relatively lower effort and altitude activity. Associations between COD, AR exposure, sex, and life stage were assessed. In each group, 12 birds (35%) were found to have been exposed to brodifacoum or bromadiolone prior to death. Logistic regression showed no significant association between COD and sex (P=0.194) or life stage (P=0.895). Across both mortality types, life stage was not a significant predictor of AR exposure (P=0.725), but males were more likely to have been exposed to ARs (P=0.032). These findings suggest that there is no difference in the influence of anticoagulant exposure on higher and lower altitude activity in Golden Eagles.

Qualitative identification of imidacloprid in postmortem animal tissue by gas chromatography-tandem mass spectrometry.

During an avian mass mortality event investigation at the National Fish and Wildlife Forensic Laboratory in Ashland, OR, imidacloprid became an insecticide of concern. A qualitative analytical toxicology screen of seeds, plucks (tongue, esophagus, and trachea), and ventricular contents was requested. A method for the extraction and qualitative analysis of the insecticide in animal tissues was therefore developed. The procedure relies on a combined Food Emergency Response Network (FERN) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach to sample extraction followed by qualitative analysis by gas chromatography-tandem mass spectrometry. Since imidacloprid is not amenable to the conditions of gas chromatography, a trimethylsilyl derivative was created and characterized. Proposed mechanisms for the creation of this derivative and its mass spectrum are described. The imidacloprid-trimethylsilyl (TMS) derivative was detected in all samples submitted.

Using an alternate light source to detect electrically singed feathers and hair in a forensic setting.

Mortality due to electrical injury in wildlife may occur in the form of lightning strike or power line contact. Evidence of electrical contact may be grossly obvious, with extensive singeing, curling, and blackening of feathers, fur, or skin. Occasionally, changes may be subtle, owing to lower current or reduced conductivity, making a definitive diagnosis of electrocution more difficult. We describe the use of an alternate light source in the examination of cases of lightning strike and power line contact in wildlife, and the enhanced detection of changes due to electrical currents in the hair and feathers of affected animals. Subtle changes in the wing feathers of 12 snow geese and 1 wolf that were struck by separate lightning events were made obvious by the use of an alternate light source. Similarly, this technique can be used to strengthen the evidence for power line exposure in birds.