Study species, capture and captive housing

White-crowned sparrows can migrate thousands of kilometers between their wintering and breeding grounds, and are frequently used as models in migration research (e.g.45,50). They are granivores, and have the potential to be directly exposed to insecticides through consumption of treated seeds or granules. These characteristics make them a valuable test species for providing insight into the potential effects of neurotoxic insecticides on migratory birds occupying agricultural fields during migration. In May 2016, A total of 57 Gambel’s white-crowned sparrows (Z. l. gambelii) were captured in mist nets or sparrow traps near Saskatoon, Saskatchewan (SK) (51° 58’ 27” N 106° 42’ 6” W) using playbacks or baited seed feeders as attractants. Birds were transported to the Facility for Applied Avian Research at the University of Saskatchewan, where they were held in cages (24”L × 24”W × 24”H) in an outdoor pen, with 2 to 3 birds per cage. Birds were provided with water and a mixture of millet, black oil sunflower seeds, and poultry starter crumbles (Proform 26%) ad libitum. Prior to dosing, birds were held for ~2 weeks to allow for acclimation to captivity and to increase the likelihood that they were in a migratory state, which was assessed through video monitoring of nighttime activity, increasing fat scores, and weight gain. At the end of the experiment, all birds were humanely euthanized by CO 2 asphyxiation, gonadal sex was determined, and tissues were collected for future analysis. Research protocols were in compliance with the Canadian Council on Animal Care guidelines and approved by the University of Saskatchewan Animal Care Committee (AUP 20110043), and conducted under Canadian Wildlife Service Scientific Permit 15SKSC005. The timeline for experimental procedures (Emlen funnel trials, dosing, and body mass monitoring) is illustrated in Figure S1.

Emlen funnel trials

Migratory behaviour was monitored with nocturnal Emlen funnel trials51, using flower pots as modified Emlen funnels as previously described27 (Fig. S2). Emlen funnels are circular in shape with sloped sides that force the bird to return to the center after an activity bout, while being wide enough so that outward movement can be detected by overhead cameras. Trials were conducted in an open field without any visual cues on evenings with clear skies.

Funnels were placed upright with birds having a clear view of the sky at least 35 minutes prior to sunset, and were left undisturbed for at least 5 minutes before recording data. Video of funnel trials was recorded from approximately 30 minutes before to 30 minutes after sunset using digital video cameras (ADS-180, Swann Communications) suspended 10 ft above the funnels. Each camera recorded movements from six funnels simultaneously onto a digital video recorder (DVR8-2550, Swann Communications). Each bird was tested in four trials over the course of the experiment: 1) pre-dosing: after the acclimation period to confirm birds are in a state of zugunruhe (migratory restlessness), 2) post-dosing: ~10 hours after the final dose, 3) 3 d recovery: 3 days after the post-dosing Emlen trial to test acute recovery, 4) 14 d recovery: 14 days after the post-dosing Emlen trial to test long-term recovery.

Dosing

51 sparrows that were confirmed to be in a state of zugunruhe were randomly assigned to one of four dose groups or the control groups. Dosing started the morning after the first (pre-dosing) Emlen trial. Birds were orally dosed between 09:00 and 11:00 by gavage with 4.1 µg imidacloprid/g bw/day (IMI low; n = 11; 7 male, 4 female), 10.25 µg imidacloprid/g bw/day (IMI high; n = 12, 6 male, 6 female), 2.9 µg chlorpyrifos/g bw/day (CPF low; n = 9; 6 male, 3 female), 7.4 µg chlorpyrifos/g bw/day (CPF high; n = 9; 4 male, 5 female) (nominal concentrations), or a vehicle control (food-grade organic sunflower oil, Compliments brand, Sobeys Canada; n = 10; 4 male, 6 female) once per day for 3 days (acute 72 hour exposure) at a volume of 10 µl dosing solution/g bw. Dosing concentrations were selected through pilot studies conducted the previous year, and based on published values for the median lethal dose (LD50) for house sparrows (Passer domesticus) for each compound28,52,53. Therefore, these doses correspond to 10% or 25% of the predicted house sparrow LD50. Dosing solutions were made by dissolving technical grade imidacloprid (Sigma Aldrich 37894) or chlorpyrifos (Dursban, Sigma Aldrich 442573) in a small volume of acetone, then diluting with sunflower oil. Solutions were stirred overnight to evaporate off acetone, and stored in amber glass bottles in the dark for the duration of the study.

Monitoring

Body mass (g) and fat scores (0 to 5) were measured between 09:00 and 11:00 at capture, before each dose, the morning after the last dose, the morning after the 3 day recovery Emlen trial, and the morning after the 14 day recovery Emlen trial. At capture and at the completion of the experiment, structural measures (tarsus, wing length, tail length, bill length, head-bill length) were taken.

Chemical Analysis

Concentrations of imidacloprid and chlorpyrifos in dosing solutions were confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS) analyses at the National Hydrology Research Centre, Environment and Climate Change Canada, Saskatoon, SK.

Dosing solution samples (500 µL) were transferred to a 50 ml centrifuge tube containing 10 ml Milli-Q water. An additional 4.5 ml Milli-Q water was added along with 15 ml of acetonitrile containing 1% acetic acid, then briefly vortexed. AOAC QuEChERS extraction salts (Agilent-#5982-6755) were added and the tube shaken vigorously for 2 minutes. After mixing, the solution was centrifuged (5 min @ 5000 rpm). An EMR Lipid dSPE tube (Agilent-#5982-1010) was prepared by adding 5 ml Milli-Q water and briefly vortexing. 5 mL of the QuEChERS extract (acetonitrile layer) from above was transferred to the EMR lipid dSPE tube, vortexed thoroughly, shaken for 2 minutes followed by 5 minutes of centrifuging at 5000 rpm. 5 mL of this supernatant was transferred to an EMR-Lipid polish tube (Agilent-#5982-0101) and immediately vortexed then centrifuged for 5 min @5000 rpm. 200 µL of the acetonitrile layer was transferred into a 1.8 ml amber glass LC vial containing 800 µL of Milli-Q water followed by subsequent dilutions to bring the concentration within the calibration curve. 20uL of a 2.5 mg/L solution of internal standard (d4-imidacloprid, CDN Isotopes, Pointe-Claire, Quebec, CA) was added to the LC vial and vortex mixed immediately prior to instrumental analysis.

For the LC/MS/MS analysis, a Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA), consisting of a solvent degassing unit, pump and autosampler, was used with a Waters XTerra MS-C18 (3.5 µm dia. particle size) column (2.1–100 mm) (Waters Corp., Milford, MA) at 30 °C. Isocratic elution of the analytes was achieved with an 25:75 (v/v) mix of solvent A (100% water and 0.1% formic acid) and solvent B (100% acetonitrile and 0.1% formic acid) respectively at a flow rate of 200 µL/minute. The run time was 10 min and the sample injection volume was 20 µL. Chlorpyrifos and imidacloprid were quantified (internal standard method) and their concentrations confirmed using the Micromass Quattro Ultima triple quadrupole mass spectrometer (Waters Corp., Milford, MA) equipped with an electrospray ionization interface set to positive ion mode. Ionization and MS/ MS conditions were optimized by infusing a 0.5 mg/L solution of each insecticide into the ion source in a 50:50 (v/v) acetonitrile:water solution containing 0.1% formic acid. MRM transitions, selected from the product ion scan and optimal cone voltages and collision energies for each analyte are provided in Table S1. Other instrumental settings were as follows: source temperature, 90 °C; capillary voltage, 3.95 kV; desolvation temperature, 220 °C; nitrogen desolvation gas flow rate, 487 L/h; nitrogen cone gas flow rate, 153 L/h; nitrogen nebulizer gas flow rate was at maximum flow; and multiplier voltage, 700 V. Argon was used as the collision gas at a pressure which increased the Pirani gauge reading to 2.10 × 10E-4 torr. Resolution was set to achieve unit mass resolution for quadrupole 1 and approximately 2 amu resolution for quadrupole 3.

Analytical standards of imidacloprid and chlorpyrifos were purchased from Chem Service (West Chester,PA, USA). A five-level calibration curve (5 to 100 µg/L) was analyzed before and after each batch of samples which also contained a laboratory or field blank and a fortified sample. Intermediate (1.0 mg/L) and working calibration standards were prepared fresh daily in Milli-Q water by serially diluting a substock containing each analyte at 100 mg/L in pure acetonitrile. This was necessary as it was observed that chlorpyrifos standards made in water were subject to degradation but were found stable in acetonitrile. All dosing solutions, blanks, and QC samples were run in triplicate. Measured concentrations of imidacloprid were 1.07 µg/µL ± 2%RSD and 0.40 µg/µL ± 11.8%RSD (nominal concentrations 1.03 µg/µL and 0.41 µg/µL). Measured concentrations of chlorpyrifos were 0.65 µg/µL ± 12.7%RSD and 0.23 µg/µL ± 5.7%RSD (nominal 0.74 and 0.29 µg/µL). Mean recoveries from sunflower oil fortified with chlorpyrifos at 0.51 µg/µL (n = 3) was 62.5% ± 3.1%RSD while sunflower oil fortified with imidacloprid at 0.72 µg/µL (n = 3) was 80.9% ± 2.7%RSD. Vehicle control oil and all blanks had no detectable levels of chlorpyrifos or imidacloprid.

Statistical analysis

Statistical analysis for effects on body mass and activity was completed using SAS 9.4. Activity was square root-transformed to meet assumptions of normality. Comparisons between dose groups over time were made using linear mixed models (proc MIXED) with bird ID as a repeated subject effect, and fixed effects of dose, time and dose*time. Sex and the sex*treatment interaction were included as fixed factors, but non-significant terms were removed from the final model. Tests for differences between means were adjusted for multiple comparisons using the Tukey-Kramer method. Fat scores were compared among dose groups using the Kruskal-Wallis test. Fisher’s Exact Test was used to assess effects of dose on survival. Significance level was set at α = 0.05

BirdOriTrack software54 was used to analyze videos and determine activity (distance each bird moved every 30 seconds during valid hops, relative to the radius of the funnel [radius = 1]) and mean orientation for each bird in each trial. A hop was considered valid when the movement was ≥ 0.5 of the radius from the centre of the funnel. Videos were reviewed immediately following the pre-dosing Emlen funnel trial to qualitatively determine bird activity, as only active birds that appeared to be in a state of zugunruhe continued on in the experiment for dosing and further trials. Circular statistics were analyzed using Oriana 4.02 (Kovach Computing Services). The strength of orientation was assessed using Rayleigh’s uniformity test (Rayleigh Z statistic). Significant directional (vs. uniform) orientation was tested within each treatment group at each time point. The difference in the mean orientation direction of birds between pre-dosing and later time points within each dose group was compared using Hotelling’s paired test.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.