Methods: House sparrows eggs will be gathered from 70 established nest boxes at 3 farms near UMBS. Methods are largely based on a paper by Dr. Gary Heinz et al. (2009), a large scale LC50 study on many bird species (but only a few songbirds). Expected developmental timeline is based on Dr. Ted Anderson’s work. As recommended by Dr. Anderson and Rob Aldredge (who have previously worked with these populations), all eggs from the first clutch will be collected to synchronize egg-laying for future clutches and establish when new eggs are laid. A sample of eggs from this first clutch will be used to establish a baseline for mercury in these populations of house sparrows. This step may be unnecessary, depending on the timing of my arrival and the laying of the first clutch. The first clutch is often fairly well-synchronized, and if I arrive before the first clutch is laid I may not need to synchronize it.
Seventy (70) newly laid eggs from three subsequent clutches will be collected (one egg per nest, with a mode clutch size of 5), and injected with methylmercury dissolved in corn oil using an established method of “air cell injection,” with which I am quite familiar). Doses will include 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 µg of mercury per g of egg weight (µg/g). A control group will be injected with solely corn oil. A non-injected control will also be used, to control for possible mortality from the injection procedure. All eggs will then be placed in an incubator (37.5 C, humidity 70 %,) until 2 days before hatch (day 10, mode hatch day of 12), and will be checked daily for mortality. On day 10, the embryos will be euthanized and dissected for brain, heart, liver, kidney, and gastrocnemius muscle tissues. A sample of feathers, the entire egg shell, and remaining yolk will also be kept for analysis. All samples, along with remaining carcasses, will be frozen immediately for analysis upon returning to Ann Arbor. Samples of each tissue will be analyzed using a DMA-80 (Direct Mercury Analyzer, Milestone Productivity Tools) in Dr. Niladri Basu’s University of Michigan lab. Neurochemical assays will also be conducted; N-methyl-D-aspartic acid (NMDA), g-aminobutyric acid (GABA), glutamine synthetase (GS), and glutamic acid decarboxylase (GAD) assays are possible, depending on the weight of brain tissue obtained. Discussions about which are most relevant are currently ongoing, but this has little bearing on how my work will be conducted at UMBS.