Monitoring methaemoglobinaemia in birds using 5 μL of whole blood

Methaemoglobin (MetHb) forming compounds such as para-aminopropiophenone (PAPP) and sodium nitrite (NaNO(2)) have recently been adopted for the lethal control of a range of invasive carnivores and mustelids. Determining the relative hazard of these compounds to non-target bird species is an important component of ecological risks evaluation. Problematically, some potential non-target bird species may be as small as 10 g in body mass, thus placing limitations on blood volumes that can be routinely sampled. Accordingly, we developed methods to quantify markers of increasing methaemoglobinaemia at their point of collection that required only 5 μL of whole blood. A 3 μL blood aliquot is pipetted into a plastic micro-cuvette and placed in a custom made holder optically coupled to the Ocean Optics spectrometer, enabling absorbance for oxyhaemoglobin (HbO: 575 nm) and MetHb (630 nm) to be determined. Haemoglobin (HbFe(2+)), packed cell volume (PCV) and lactate (LAC) data were generated from the remaining 2 μL aliquot apportioned to biosensor strips for the Cera-Check(®) and Lactate Scout(®) point-of-care devices. After oral doses of PAPP, a methaemoglobinaemia absorbance index (MAI = absorbance 575 nm–absorbance 630 nm) was strongly and significantly associated with dose-dependent declines in HbFe(2+) in 9 bird species. Quantifying dose-dependent responses to MetHb-forming agents at the point of sample collection avoids analytical and storage artifacts arising from sample degradation that appears to be a much greater problem in avian blood compared to mammalian blood.