Variation in isotopic niche, digestive tract morphology, and mercury concentrations in two sympatric waterfowl species wintering in Atlantic Canada

Seventy-four American black ducks (38 males, 36 females) were collected for this study, with 37 collected opportunistically by hunters in coastal saltmarsh habitat of Nova Scotia and New Brunswick, Canada, from 26 November 2013 to 27 March 2014, and 37 collected and euthanized by cervical dislocation by Environment and Climate Change Canada employees in an urban freshwater pond in St. John’s, Newfoundland and Labrador, from 11 February 2014 to 15 April 2014. Thirteen mallards (six males, seven females) were collected opportunistically by hunters in coastal saltmarsh habitat of Nova Scotia from 30 January 2014 to 25 March 2014. All ducks were sent to Acadia University, where they were stored frozen at −18 °C.

The collection of black ducks and mallards for this project was pre-approved by Acadia University (Animal Care Permit No. 02-14) and Environment and Climate Change Canada (Scientific Permit No. ST2785).

Blood samples were taken from the heart of each frozen carcass, and stored whole at −18 °C. Additional blood samples were collected during winter banding operations in 2015. Live-trapped ducks were caught in a pond in Grand Manan, New Brunswick, using a baited single-wire funnel trap, and in a coastal saltmarsh in Cole Harbour, Nova Scotia, using a cannon net. Prior to drawing blood, the wing surface was sterilized with 95% ethanol, and then approximately 50 μL of blood were drawn from the brachial vein using a capillary tube. The sampled ducks were released where they were caught, and blood samples were stored whole at −18 °C.

Whole blood samples were dried at 60 °C for 48 h, ground into a powder, and sent to the Stable Isotopes in Nature Laboratory (SINLAB, University of New Brunswick, Fredericton, New Brunswick, Canada) for analysis of isotopes of carbon and nitrogen. Samples were combusted in an elemental analyzer, and gases were sent to the isotope-ratio mass spectrometer using a continuous flow interface. Data are reported as differences in isotopic ratios, for which the units are parts per thousand (or per mil; ‰), compared with Pee Dee Belemnite, for carbon, and atmospheric nitrogen, for nitrogen, according to the following equation:where δX is the isotope of interest (either δ¹⁵N or δ¹³C, in ‰), R is the ratio of the abundance of the heavy to the light isotope (¹⁵N/¹⁴N or ¹³C/¹²C), with R_sample being the ratio within the sample, and R_std the ratio of heavy to light isotope within the international standard (Hobson and Clark 1992).

Due to the known effects of species and location on isotopic ratios in blood of the birds in this study (English et al. 2017), samples were divided in to three groups: coastal black ducks, urban black ducks, and coastal mallards. We used stable isotope Bayesian ellipses in R (SIBER) from the package “SIBER” and used a probabilistic method (Jackson et al. 2011) to assess differences in isotopic niches among the three groups of ducks following the posterior draws of Bayesian-simulated standard ellipse areas (SEA_b; represented in ‰²). The SEA_b values were compared among the three groups using Bayesian inference as described in Jackson et al. (2011) (see Pettitt-Wade et al. 2015; Karlson et al. 2018 for examples). A Bayesian probability (p) of SEA_b size difference >0.6 was considered to be significant (e.g., Pettitt-Wade et al. 2015). Layman’s metrics (Layman et al. 2007) were used to further quantify the trophic structure of the three groups of birds.

All 74 black ducks and three of the 13 mallards were sent to the Avian Energetics Laboratory (Long Point, Ontario), where full carcass processing on thawed birds occurred using standardized methods (e.g., Reinecke et al. 1982; Morton et al. 1990). To account for the effect of body size on digestive tract morphology, we extracted the first principal component (PC1) score of the correlation matrix of ln-transformed tarsus length, culmen length, head length, wing chord length, and plucked body length as an index of overall body size (as per Kehoe et al. 1988; Jónsson and Afton 2017). The PC1 accounted for 58% of the total original variance. Four gut measurements were examined: empty gizzard mass, small intestine length, large intestine length, and the combined length of the first and second segments of the caeca. Gut morphology data for each variable were regressed on PC1 to adjust them for body size (all regressions were positive and significant (p < 0.05)). The residuals from each regression were used to calculate a new variable (y_i ) corrected for body size of each digestive tract component as per the following formula:where y_obs is the observed measurement of the particular digestive tract component, and a and b are the values from the regression of each digestive tract component on PC1.

Waterfowl gut morphology is strongly affected by the annual cycle (Drobney 1984; Moorman et al. 1992) so we only examined ducks collected during overlapping time periods. We excluded 24 coastal ducks collected at the end of November 2013 due to a lack of urban ducks collected near this time. For the black ducks, we used a multivariate analysis of covariance (ANCOVA) to assess the effects of the factors “sex” and “location” (coastal vs. urban) on each digestive tract component, with date and isotope (either δ¹⁵N or δ¹³C) as the covariate. The gut morphology of the small number of mallards (n = 3) was compared with black ducks using a separate multivariate analysis of variance (ANOVA).

From carcasses, we removed whole blood from clots in the heart, veins, or arteries. Samples (generally ∼1–2 g) were freeze-dried, homogenized to a powder using a clean mortar and pestle, and transferred to 1.5 mL Eppendorf tubes and frozen until analysis. We analyzed for total Hg at the Center for Analytical Research on the Environment at Acadia University. Samples were analyzed on a Nippon Instruments MA-3000 Mercury Analysis System using thermal pyrolysis, gold amalgamation preconcentration, and atomic absorption spectroscopy detection. Samples were not blank corrected, as mean blanks were −0.01 ± 0.01 ng/g (n = 11) with the method detection limit (MDL) calculated as 3× the standard deviation of the blanks (MDL = 0.03 ng/g). All samples were well above detection limits, and repeated measurements on seven samples yielded similar values (mean difference 1.8%). Internal quality control included analytical blanks and certified reference material (DORM-4, National Research Council of Canada). The mean recovery for the certified reference material (n = 13) was 95.4% ± 4.4% (SD) for total mercury (THg), so we did not recovery-correct THg values. THg concentrations are reported in parts per billion (ppb) dry weight.

Data on THg concentrations were not normally distributed (Shapiro–Wilk normality test: W = 0.86; p < 0.001), and were thus log₁₀-transformed. Only black ducks (n = 74) were analysed using ANCOVA. For black ducks, the factorial effects of sex and habitat (coastal vs. urban), and the covariates of date of sampling and isotope (either δ¹⁵N or δ¹³C), on blood Hg concentrations were investigated using ANCOVA. The data from mallards (n = 3) were compared with the coastal and urban black ducks using a separate ANOVA. All statistical analyses were performed using R version 3.4.3 (R Development Core Team 2015).

Total convex hull area (as described by Layman et al. 2007) was greatest in coastal black ducks (48.22 ‰²), followed by coastal mallards (15.00 ‰²), and was the smallest in urban black ducks (8.26 ‰²). When the SEA_b were compared, isotopic niche varied significantly among the three groups (Fig. 1). Estimated SEA_b values for coastal black ducks were wider (mean SEA_b; 95% credible interval =12.80; 9.41–16.12 ‰²) than urban black ducks (1.72; 1.18–2.28 ‰²; Bayesian p = 1.00, where p > 0.6 indicates a difference) and coastal mallards (8.18; 3.68–13.18 ‰²; Bayesian p = 0.94). Coastal mallards occupied a wider isotopic niche than urban black ducks (Bayesian p = 1.00; Fig. 1). Coastal black ducks and mallards overlapped in their isotopic niches, but urban black ducks were isolated.

Fifty-three black ducks (31 males and 22 females) and three mallards (all males) were used in the digestive tract analysis. Means and standard deviations of the gut measurements are shown in Table 1. Sex and date had no effect on any of the gut measurements (all p > 0.05), so both sexes were pooled and only the effects of isotope and location are reported.

In black ducks, small intestine length and gizzard mass were significantly, positively correlated with δ¹⁵N, and all gut measurements except gizzard mass were affected by location (Table 2; Fig. 2). For the gizzard mass, the interaction between location and δ¹⁵N was significant (F_1,51 = 10.46; p = 0.002). Gizzard mass in coastal black ducks tended to increase with increasing δ¹⁵N (β = 5.14 ± 2.41; F_1,14 = 4.56, p = 0.051), whereas gizzard mass in urban black ducks decreased (β = −3.08 ± 1.24; F_1,35 = 6.21, p = 0.017) with increasing δ¹⁵N (Fig. 2). Tukey’s Honest Significant Difference (HSD) testing on the effect of location showed significant differences in the lengths of the small intestines but no differences in the lengths of the large intestines or caeca (Table 1).

The only gut measurement related to δ¹³C was gizzard mass, and all gut measurements except gizzard mass were affected by location (Table 2). None of the interactions between δ¹³C and location were significant (all p > 0.05). Tukey’s HSD testing on the effect of location showed significant differences in the lengths of the small intestines, large intestines, and caeca between coastal and urban black ducks (Table 2).

When the covariates were removed and the effect of location alone was measured against gut morphology, location had a significant effect on small intestine, large intestine, and total caeca length (multivariate ANOVA; all p < 0.05), but did not affect gizzard mass (p = 0.132). In the cases where location had a significant effect on gut morphology, coastal black ducks had consistently longer guts than urban black ducks (Tukey’s HSD: all p < 0.05). Gizzard mass of coastal black ducks was almost 3× more variable than urban black ducks (Table 1).

In the separate multivariate ANOVA that included the mallards, the only significant difference for mallards was found in the small intestine length (F_2,53 = 10.04, p < 0.001), where coastal mallards had shorter small intestines than the coastal black ducks (Tukey’s HSD: p < 0.001). The gut morphologies of coastal mallards and urban black ducks did not differ (all p > 0.05).

THg concentrations in blood samples did not vary between sexes of black ducks (F_1,73 = 0.15; p = 0.702), so both sexes were pooled for the analysis. Both δ¹⁵N (F_1,73 = 138.04; p < 0.001) and location (F_1,73 = 26.73; p < 0.001; Fig. 3) had a strong effect on THg concentrations, with coastal black ducks containing higher and a wider range of blood THg concentrations than urban black ducks (Table 3; Tukey’s HSD: p < 0.001). THg was positively correlated with δ¹⁵N in coastal (β = 0.12, F_1,38 = 25.36, p < 0.001) and urban (β = 0.27, F_1,35 = 7.63, p = 0.009) black ducks (Fig. 3). The interaction between δ¹⁵N and location was not significant (F_1,74 = 3.13; p = 0.081), and δ¹³C (F_1,73 = 0.536; p = 0.467) and date (F_1,73 = 0.133, p = 0.716) did not have any effect on THg concentrations.

In the ANOVA that compared THg concentrations among the three groups of ducks in this study, the group variable was significant (F_2,74 = 10.57, p < 0.001). Coastal mallards and urban black ducks had similar blood THg concentrations (Table 3; Tukey’s HSD: p = 0.751) and coastal mallards had significantly lower blood THg concentrations than coastal black ducks (p = 0.004).

Coastal and urban black ducks exploit different ecological niches at the northern limit of their winter range. When using isotopic niche as a proxy for measuring ecological niche, our data showed that coastal black ducks occupy a wider ecological niche than urban black ducks, and are generalists foraging on foods higher in δ¹⁵N values compared with urban black ducks. While birds occupying wide isotopic niches generally have diverse diets, it should be noted that this may not be the case if the consumer is a specialist on prey with a high variance in isotopic signatures (Yeakel et al. 2016). Based on our previous work on winter black duck diets (English et al. 2017), isotopic research on wintering black ducks in Maine and New Jersey (Barboza and Jorde 2018), and our current study in which coastal black ducks had the greatest total convex hull area (an indicator of trophic diversity, Layman et al. 2007), coastal black ducks are not specialists. The urban black ducks in our study are likely not specialists in the classic sense, but rather have a limited diet due to exploiting a human-altered habitat in which a small variety of food items are available to them (English et al. 2017).

The isotopic niches of coastal and urban black ducks did not overlap, which may suggest this species can avoid intraspecific competition for wintering habitat by exploiting different ecological niches. Coastal saltmarsh is the traditional wintering habitat for black ducks (Longcore et al. 2000), and during winter at higher latitudes this habitat becomes restricted by ice cover (Barboza and Jorde 2018). By also wintering in urban parks, black ducks can avoid competing for saltmarsh habitat and can exploit an ample supply of anthropogenic food resources and survive winter in Atlantic Canada (English et al. 2017).

Coastal black ducks and coastal mallards overlapped in their isotopic niches, which may suggest that these ducks are competing for food resources in coastal areas. However, coastal black ducks occupied a wider isotopic niche that contained more marine signatures (i.e., higher δ¹⁵N or δ¹³C values; Hobson and Clark 1992) than the coastal mallards; we suspect that despite sharing wintering habitat in coastal areas, some black ducks and mallards are not competing for the same food resources. Black ducks evolved in coastal marine habitats (Longcore et al. 2000) and therefore may be better suited to exploit these habitats (or more resistant to competitive exclusion, but see Morton 1998) than mallards, whose traditional habitat is inland plains and wetlands where they may outcompete black ducks for habitat (Ankney et al. 1987; Drilling et al. 2002).

Gut morphology varied with isotopic niche. In general, coastal black ducks had longer guts than urban black ducks, and the small sample of mallards had gut morphologies similar to urban black ducks. Diet affects gut morphology in waterfowl in various ways (Miller 1975; Kehoe and Ankney 1985; Kehoe et al. 1988; Jónsson and Afton 2017), and the different gut morphologies of coastal and urban black ducks are likely a result of the different diets of these two groups (English et al. 2017). Longer guts are often associated with a higher diversity or quality of diet in waterfowl (van Gils et al. 2008). Consequently, we suspect the longer guts of the coastal black ducks reflect their more natural and diverse diets, as opposed to the shorter guts of the urban black ducks that mainly digest anthropogenic foods.

The urban black ducks in this study occupied a narrow isotopic niche and had relatively short guts, but there was still a high amount of variation in their gut length. Habitat explained much of the variation in digestive tract morphology in our study, but there is likely another factor contributing to the variation that we did not measure. We did not have reliable age data on the birds in this study, as age determination for black ducks in winter can be challenging (Ashley et al. 2006). Age is known to affect gut morphology in Lesser Snow Geese (Anser caerulescens caerulescens (Linnaeus, 1758); Jónsson and Afton 2017) and male mallards (Olsen et al. 2011), but the variation in gut morphology in these studies was better explained by differences in diet and habitat. Intermittent disruptions in food availability and cold stress may also affect gut morphology in black ducks (Barboza and Jorde 2002).

There was no difference in gizzard mass between coastal and urban black ducks. We predicted gizzard mass would be highest in coastal black ducks due to the need to digest hard-shelled prey but instead found their gizzard masses were more variable. Coastal black ducks exhibited the widest isotopic niche and had the most diverse diets of the groups of ducks in this study (English et al. 2017), and differences in gizzard mass may reflect individual differences in diets of coastal black ducks. Jónsson and Afton (2017) obtained a similar result by observing more variability in the gizzard masses of Snow Geese collected from coastal areas than from rice-prairies. Olsen et al. (2011) found that gizzard mass in male mallards was unresponsive to changes in diet through winter, despite these authors predicting the opposite. Waterfowl digestive tracts have a high degree of plasticity, and attributing differences in digestive tracts between groups to one factor is often challenging (Karasov and McWilliams 2005; Olsen et al. 2011).

The gut morphology of the coastal mallards we studied was similar to that of urban black ducks and, despite being sampled in very different winter habitats, is likely to be attributable to a similar reliance on vegetation in their diet through winter (English et al. 2017). A larger sample size of mallards may have introduced more variability in lengths of the digestive tracts, and the results from our small sample size should be cautiously interpreted.

Blood Hg concentrations varied with isotopic niche and δ¹⁵N levels. In birds, Hg and δ¹⁵N are often positively correlated in the blood, as Hg bioaccumulates through a food chain (Wolfe et al. 1998; Sebastiano et al. 2017), and this was the case in our study. As predicted, the coastal black ducks that occupied a wider isotopic niche had higher concentrations of blood Hg than the urban black ducks that occupied a narrow isotopic niche. Occupying a wide isotopic niche may increase an organism’s exposure to contaminants in its environment. However, it should be noted that any consumer that feeds principally on a certain type of contaminated prey, irrespective of its trophic position, will be relatively more contaminated than conspecifics feeding on less contaminated food (e.g., Hindmarch and Elliott 2015).

Hg has been detected in marine biota and in coastal areas of the Bay of Fundy in Atlantic Canada (Elliott et al. 1992; Goodale et al. 2008; Sunderland et al. 2012), and concentrations of Hg ranging from 0.01 to 0.04 ppm have been found in black duck prey from this region (English et al. 2015). Since the coastal black ducks in this study contained various marine invertebrates in their gizzards (English et al. 2017) it is likely the Hg accumulation in their blood was a result of eating more contaminated prey, while the urban black ducks were presumably consuming much less contaminated food items (anthropogenic foods such as bread or grains). Blood Hg concentrations in coastal black ducks were highly variable, and may again reflect individual differences in diet. Despite coastal black ducks having a mean Hg blood concentration almost 6× higher than the urban black ducks, body condition was similar (English et al. 2018). The differences in gut morphology may also be contributing to the differences in the blood Hg concentrations, as the longer guts of coastal black ducks may provide more time for the uptake of contaminants (Kleinow and James 2001).

Our small, localized sample of mallards precludes any broad prediction of Hg concentrations in mallards across Atlantic Canada, but our results aligned with our prediction of lower blood Hg concentrations in coastal mallards when compared with coastal black ducks. Mallory et al. (2018) recently found ∼4× higher blood Hg in mallards and one black duck collected in October in this region than each species had in the winter (this study), suggesting that both species may experience even higher Hg exposure in summer or premigration diets (or habitats). Moreover, the black duck had approximately 5× higher blood Hg than the mallards (Mallory et al. 2018), consistent with our findings. The coastal mallards and black ducks in this study were collected in the same habitats, and despite overlapping isotopic niches, had very different blood Hg concentrations. Even though coastal mallards in Atlantic Canada forage on marine invertebrates (Drilling et al. 2002; English et al. 2017) it is possible that the three mallards in our study were foraging at a lower trophic level, not on the same invertebrate prey as the black ducks, or were foraging on species with atypical δ¹⁵N/Hg relationships (e.g., polychaetes in the Bay of Fundy; Sizmur et al. 2013). The bioavailability of methylmercury is not consistent through saltmarshes in the Bay of Fundy (O’Driscoll et al. 2011), and depending where these mallards were foraging in the saltmarsh they may have been exposed to less Hg than the black ducks.

Recently, high Hg concentrations in the feathers and breast meat of black ducks were reported along the Penobscot River in Maine, an area contaminated by a chemical plant that operated along that river from 1967 to 2000 (Sullivan and Kopec 2018). The Hg concentrations in breast meat (mean 0.82 ± 0.21 ppm wet weight) were high enough for the State of Maine to prompt a warning against the consumption of black ducks harvested along the Penobscot River. Black ducks are one of the main species of waterfowl hunted in Atlantic Canada (Longcore et al. 2000), and the Canadian Food Inspection Agency does not list a Hg limit for the consumption of poultry or waterfowl (the only one listed is a 0.5 ppm limit for Hg in seafood products suitable for human consumption, CFIA 2018). Provencher et al. (2014) called for the continued monitoring of Hg contamination of marine birds because of the potential health implications on the wildlife and the people who consume these birds. Our research on two hunted and consumed species indicates the need for this continued monitoring, and it should be extended to other species of wildlife hunted and consumed in Atlantic Canada.