2.2.1 Sample sites
The mallards studied were collected from Southland and Waikato in New Zealand (Figure 2.1). The first study area was located in the Southland Plains, centred on the Lochiel community within the Southland Region of the South Island (46°12´18.68´´S, 168°19´46.19´´E). Predominately flat, early maps (c.a. 1850) indicate a mosaic of tussock grassland, forest and bogs, while land cover now is dominated by pasture and forestry. While agricultural expansion has barely increased since 1985, transition in land use is still occurring with the expansion of dairy land into previously sheep and beef pastoral and arable land (Ledgard, 2013). The study area was made up primarily of private land with part of the Oreti River, numerous streams and man-made ponds created either for effluent settling or as waterfowl habitat. The Southland region, known as a hotspot for mallards, sells just over 15% of the game-bird hunting licences sold nation-wide (Garrick et al., 2017). Long-term population trends indicate that this population is stable (E. Garrick, Southland Fish and Game, pers. comm.).
The second study site was located in the Waipa District, centred around the Ohaupo community within the Waikato Region of the central North Island (37°55´39.36´´S, 175°17´16.98´´E). Historically, this area was dominated by large peat-swamps, alluvial flats, and vast native forest. Since human arrival this area has been highly modified and is now dominated by pastoral farming (predominantly dairy) and forestry, with only 18% of fragmented pre-European vegetation left in the lowlands of the region. With the removal of farming subsidies in the 1980s, farmers mitigated their economic loss by intensifying land use and increased stocking rates (MacLeod and Moller, 2006b). Within the boundaries of this site are both the Waikato and Waipa
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Rivers, 14 peat lakes, numerous streams, drainage ditches, and man-made effluent ponds. The mallard population within this region, once renowned for being abundant, appears to be unstable with fluctuating annual counts (Sheppard, 2017). Despite this, the region is still responsible for about 20% of the game-bird hunting licences sold nation-wide (D. Klee, Auckland/Waikato Fish and Game, pers. comm.).
2.2.2 Sample collection
Bloods
A total of 171 female pre-breeding season blood samples were collected in June–November 2014 (n = 77) and 2015 (n = 94), in collaboration with another study (Sheppard, 2017). Birds were caught using baited funnel traps (Bub, 1991), mist-nests (Bacon and Evrard, 1990), automatic nest traps (Weller, 1957), long-handled dip nets (Loos and Rohwer, 2002), net-gun, or walk-in traps (Dietz et al., 1994). Females were implanted with a 22-g intra-abdominal VHF transmitter (Model IMP/150, Telonics, Mesa, Arizona, Rotella et al., 1993, Paquette et al., 1997) to be tracked for the subsequent breeding season. While birds were anesthetised for implant surgery, < 3 ml of blood was collected from the right jugular vein and transferred into 5 ml lithium heparin microtainers and kept on ice until samples could be stored at -20oC until processed. Pre-breeding season females were aged by the perceived depth of the Bursa of Fabricus from cloaca examination, then verified by the characteristics of the greater secondary coverts, primaries, and general wing plumage. Two age cohorts were identified; adult (after-second year) and juvenile (after-hatch year). Procedures were approved under University of Auckland Animal Ethics Permit 001331.
Post-breeding season bloods were collected from Waikato birds in late January 2016 (n =
70), and from Southland birds in February 2017 (n = 70). Birds were trapped using modified walk- in funnel traps (McDougall, 2012). Birds were removed from holding pens and restrained by hand while blood samples were collected from the right jugular or the metatarsal vein using a 25-gauge needle and 5 ml syringe (Figure 2.2). Pressure was applied to the sample site for at least 30 seconds or until bleeding stopped. Birds were placed in an observation pen for 10 minutes after sampling, prior to release back into wild habitat. A volume of 0.5–2 ml of blood was collected in 5 ml lithium heparin microtainers and kept on ice until samples could be stored at -20oC until processed. Age was established by the perceived depth of the Bursa of Fabricus from cloaca examination (Siegel- Causey, 1990), with juveniles (hatch-year birds) able to be distinguished from adults (post-hatch year birds). Research and procedures were approved under Massey University Animal Ethics approval 16/04.
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Figure 2.1 Field sites. Map created from “Vegetative cover of New Zealand” shapefile imported from LRIS
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All blood samples were analysed at Hills Laboratory (Food & Bioanalytical Division, Waikato Innovation Park, Ruakura Lane, Hamilton, New Zealand). A detailed description of the methodology used can be found in American Public Health Association (APHA) “Standard Methods for the Examination of Water and Wastewater” (22nd Edition), Methods 3030Fa and 3125. In summary, an average of 1.69 g of blood (range 0.044–2.730 g) was digested with 2.5 ml of nitric and 0.5 ml of hydrochloric acid at 85oC for one hour. Blood concentrations of Cd, Cu, and Pb were then determined by inductively coupled plasma mass spectrometry (ICP-MS). Blanks and standard reference material were processed and analysed to ensure the quality of the methodology. Limits of detection (LOD) were as follows; Cd = 0.0002 mg.kg-1, Cu = 0.0050 mg.kg-1, and Pb = 0.0010 mg.kg-1. All results reported are in relation to wet weight (w.w.).
In this study, birds with blood Cd and Pb concentrations above 0.2 mg.kg-1 were regarded as being elevated or above background levels (White and Finley, 1978, Pain, 1996), with Pb levels above 0.5 mg.kg-1 considered to be consistent with acute clinical toxicity (Pain, 1996). Thresholds for toxicity based on Cu levels in blood are unknown.
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Eggshells
Eggshells were collected from the nests of tracked females (n = 57) throughout the 2015 breeding season, August–December (Figure 2.3). Data concerning females’ nesting attempts and clutch numbers were recorded in collaboration with a research programme investigating female reproduction (Sheppard, 2017). Full clutches, both hatched shell-fragments and abandoned eggs, were collected together in plastic bags and frozen at -20oc until processed. Once eggs were thawed at room temperature, egg contents and membranes were separated from the shell. External debris, nesting material, and organic matter was gently wiped or washed off. Shells from multiple eggs were pooled by clutch, and sent to Hills Laboratory for analysis, following similar methods as blood samples. Shell samples weighting at least 0.5 g (range 0.501–0.519 g) were ground and homogenised, then digested following the Nitric Acid-Hydrochloric Acid Digestion Method 3030F (Eaton et al., 2005) as for bloods. Concentrations of the metals in the shells were established by ICP-MS with the following limit of detection (LOD); Cd = 0.002 mg.kg-1, Cu = 0.050 mg.kg-1, and Pb = 0.010 mg.kg-1.
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2.2.3 Statistical analysis
Data were analysed using the statistical software R studio (R Core Team, 2015). Values below the reported LOD were considered as one-half of the relevant LOD and included for statistical analysis, to minimise nominal type 1 error rates (Clarke, 1998). Data were tested for normality (Shapiro-Wilk), and all distributions of the data significantly differed from normal. After log transformations only Cu levels in eggshells established normality, while log-log (+10) transformation established normality in Cd and Pb in pre-breeding blood samples and Pb in post- breeding bloods. For non-normal data, nonparametric tests were used. A Mann-Whitney test was used for two independent samples to compare two groups (Waikato vs Southland; adult vs juvenile; male vs female; year 2014 vs 2015). Log-log-transformed Cd and Pb values comparing site, age cohort, year (pre-breeding), and sex (post-breeding) were analysed using an analysis of variance (ANOVA), including assessment of interactions between factors. Log-transformed shell Cu levels were analysed using a two-sample t-test for comparison between sites, but due to the small sample size of known female ages at lay a Mann-Whitney test was used for the comparison between ages (of females). Interactions between sample types were established by two different tests. Two- sample t-tests were used to establish differences between sample types: pre- vs post-breeding season bloods, and pre-breeding season bloods vs eggshells. A Spearman correlation test was used to assess the correlation between heavy metal values in pre-breeding season bloods compared to that within eggshells. Results are presented as mean ± standard deviation, median, and range for whole samples. The results are grouped by site (Waikato or Southland), age (adult or juvenile) and sex (male or female) where appropriate. Results were considered significant at p < 0.05.