Incorporation of Deepwater Horizon oil in a terrestrial bird

The 2010 Deepwater Horizon (DWH) oil spill released up to 700 000 m³ of oil (Crone and Tolstoy 2010) and 500 000 t of gaseous hydrocarbons (Joye et al 2011) into the northern Gulf of Mexico over 87 days. It is estimated that 10%–29% of the spilled oil was chemically dispersed, 20%–25% evaporated or dissolved, 23%–27% was recovered at the well head, burned or skimmed at the surface, while 12%–13% naturally dispersed (Lehr et al 2010). The remaining oil was at least partly deposited on the seafloor (Chanton et al 2015), or reached coastal marshes and beaches of the northern Gulf of Mexico (Joye 2015).

Several studies have since attempted to trace the ecological fate of spilled DWH oil. Fossil carbon, such as oil, completely lacks radiocarbon (¹⁴C) because of its geologically short 5730-yr half-life. Oil carbon is also depleted in stable carbon (¹³C) compared to surface production in the open ocean. The incorporation of oil carbon into an ecosystem, therefore, may be tracked by measuring the depletion in ¹⁴C and/or ¹³C that results from the admixture of oil carbon and carbon derived from surface water primary production (White et al 2005).

A metagenomic analysis of microbial communities demonstrated that they had responded to the release of oil (Redmond and Valentine 2012), thus making it potentially available to higher trophic levels. A stable carbon (δ¹³C) analysis has demonstrated that subsurface DWH oil carbon entered zooplanktonic communities soon after the spill (Graham et al 2010). Other radiocarbon analyses confirmed this finding in the planktonic food web of the open waters of the Gulf of Mexico (Chanton et al 2012). However, the extent to which DWH oil was incorporated into the coastal food web is less clear. Depleted radiocarbon values in the tissues of coastal invertebrates and fishes indicated that DWH oil was assimilated by coastal organisms (Wilson et al 2016). Yet only a minimal trace of DWH oil seemed to have reached estuarine filter-feeding barnacles (Balanus sp.) and marsh mussels (Geukensia demissa) (Fry and Anderson 2014). Moreover, oysters from coastal waters of Mississippi contaminated by the DWH oil did not show any evidence of oil incorporation into their shells or soft tissues (Carmichael et al 2012).

We know of no reports of DWH oil in tissues of entirely terrestrial animals; such a link would demonstrate contamination into higher trophic levels beyond direct contact with oil. We screened for oil carbon assimilation at higher trophic levels by investigating hydrocarbon-related changes in carbon isotopic composition in a terrestrial bird, the seaside sparrow (Ammodramus maritimus). We measured radiocarbon and stable carbon content in feathers and crop contents collected from birds in 2011 from sites that were oiled or left uncontaminated by the DWH oil spill. We also analyzed coastal sediments collected in 2011 in nearby locations that were reached by Macondo 252 (MC252) oil, and conducted oil source-fingerprinting.

Seaside sparrow sample collection

The seaside sparrow is a year-round resident of Louisiana marshes requiring highly specific habitat for foraging and nesting (Post et al 1983). The seaside sparrow is socially monogamous, and both parents feed their chicks during the nine days from hatching to fledgling, as well as for a few days after fledging (Post et al 1983).

Adults and juveniles both feed on a variety of prey, including terrestrial as well as marine invertebrates (Post and Greenlaw 2006), thus exposing this species to a variety of contaminants through a number of potential routes. Consistent with this, the seaside sparrow was found to be a sensitive indicator species for mercury contamination in salt marshes (Warner et al 2010, Winder 2012).

We collected ten seaside sparrows (eight juveniles, two adults) in August 2011 with a shotgun loaded with 22 dust (collection permits: USFWS MB679782; LDWF LNHP11-068) on two sites (four juveniles and one adult bird each; figure 1) that we classified as oiled or uncontaminated (control) based on Shoreline Cleanup and Assessment Technique (SCAT) surveys maps (http://gomex.erma.noaa.gov/erma.html#/x=-89.37870&y=29.14486&z=7&layers=16+6770+15879+19872+19897) (Santner et al 2011). In order to control for the potential confounding effect of sex, we molecularly sexed all birds according to established protocols. All birds were male, with the exception of two females from the control site. Crop contents were flash-frozen using liquid nitrogen in the field following terminal collection. One or two primary or secondary feathers were collected from each bird.

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Sample preparation and carbon isotopic analyses

Sediment sample collection and oil source-fingerprinting

The average radiocarbon value (Δ¹⁴C) was 18.4‰ (3.7 SE) in feathers, and 25.1‰ (2.0 SE) in crop contents. The radiocarbon content was lower in the feathers as well as in the crop contents of birds from oiled compared to control plots (figures 2(a) and (b)), which is consistent with the incorporation of DWH oil into the tissues of exposed birds.

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The average stable carbon (δ¹³C) value was −12.6‰ (0.7 SE) in feathers and −15.0‰ (0.7) in crop contents. Sparrow feathers from the oiled site had lower δ¹³C values than those from unexposed birds (δ¹³C control: −11.2‰ (0.7 SE); oiled: −13.7‰ (0.7 SE); F_1,7 = 5.6; p = 0.0499, N = 9). The δ¹³C values for crop contents did not differ between birds from control and oiled sites (δ¹³C control: −13.8 (0.8 SE); oiled: −16.1 (1.1 SE); F_1,8 = 2.92; p = 0.126, N = 10).

When we restricted the analyses to the juvenile birds, the radiocarbon values were significantly lower in birds from the oiled site, in the feathers (control site: 28.7‰ (2.9 SE), oiled site: 9.4‰ (2.5 SE); F_1,6 = 25.22, P = 0.004, N = 7) as well as the crop contents (control site: 30.18‰ (1.45 SE), oiled site: 18.73‰ (1.45 SE), F_1,7 = 31.19, P = 0.0014, N = 8). δ¹³C values, however, did not differ between the two groups for either feathers (control site: −11.1‰ (1.0 SE), oiled site: −14.0‰ (0.8 SE); F_1,6 = 5.17, P = 0.072, N = 7) or crop contents (control site: −14.1‰ (0.7 SE), oiled site: −15.1‰ (0.7 SE); F_1,7 = 0.94, P = 0.371, N = 8). Results were qualitatively unchanged when we excluded the two females in order to check for the potential confounding effect of sex on the difference between the oiled and control sites (details not shown).

The Δ¹⁴C and δ¹³C values were positively correlated in feathers (slope (SE) = 4.12 (1.49); R² = 0.45, p = 0.028, N = 9; figure 3), but not in crop content samples (slope (SE) = 1.19 (0.89); R² = 0.08, p = 0.216, N = 10). Irrespective of the location, Δ¹⁴C values for feathers and crop contents were significantly and positively correlated to each other (slope (SE) = 1.74 (0.27); R² = 0.83, p = 0.0004, N = 9), while this was not the case for stable carbon values (slope (SE) = 0.35 (0.19); R² = 0.23, p = 0.11, N = 9). Finally, there was a strong positive relationship between δ¹³C values in feathers and Δ¹⁴C values in the crop contents (slope (SE) = 0.27 (0.07); R² = 0.61, p = 0.0083, N = 9), which is somewhat puzzling given the different timescales that are indicated by the two tissues.

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The sediments from control compared to oiled locations did not differ in either stable carbon isotopes (δ¹³C control: −18.4‰ (2.2 SE); oiled: −14.5‰ (0.4 SE); F_1,8 = .3.03; p = 0.120, N = 10), or radiocarbon content (Δ¹⁴C control: 19.4‰ (29.5 SE); oiled: 120.0‰ (65.3 SE); F_1,8 = 1.97; p = 0.198, N = 10). The stable carbon values of sediments were positively associated with the radiocarbon values, although the relationship was marginally non significant (slope (ES) = 16.99 (8.91); R² = 0.31, p = 0.093).

In three of five sediment samples from the oiled site the diagnostic ratios of the eleven biomarkers confirmed the presence of MC252 oil. In the other two samples, our analyses yielded inconclusive or negative evidence, with only ten or eight out of eleven biomarkers matching MC252 oil, respectively. In none of the five sediment samples from the control site did the diagnostic ratios indicate the presence of MC252 oil.

Our analyses of carbon isotopic composition of seaside sparrow tissue indicated incorporation of oil carbon into the tissues of birds collected from an area reached by DWH oil. To our knowledge, this is the first demonstration that we know of where DWH oil was incorporated into a terrestrial vertebrate species. Importantly, data from the same population of seaside sparrows examined here indicated that reproductive success was reduced in birds from oiled plots in the early years after the spill (Bergeon Burns et al 2014), although reproductive success seemed to recover in subsequent years. In principle, such reduction of population fitness could be due to the indirect effects of oil contamination on the salt marsh ecosystem. Such indirect effects include a reduction in habitat quality as well as in the abundance of insects and other invertebrates that are part of the seaside sparrow diet. Consistent with this interpretation, several studies have shown population declines in insects, spiders and other invertebrates in sites oiled as a result of the DWH accident (McCall and Pennings 2012, Zengel et al 2015, Husseneder et al 2016, Zengel et al 2016). DWH oil also impacted vegetation cover (Zengel et al 2015), including plant species (particularly Spartina alterniflora, Distichlis spicata and Juncus roemerianus) used by seaside sparrows as nesting habitat, thus potentially affecting the outcome of reproductive attempts. Because we could trace the direct incorporation of DWH oil into the tissues and prey of seaside sparrows, the present results suggest that direct toxicological effects, not only habitat degradation or trophic interactions, might be responsible for the demonstrated reduction in seaside sparrow reproductive success.

Furthermore, the oil incorporation and direct toxic effects are consistent with our finding of increased expression of cytochrome P-4501A (CYP1A), a marker of exposure to PAHs. CYP1A expression in 2011 was higher in birds from sites reached by DWH oil versus unoiled sites (Bergeon-Burns, Stouffer, Taylor, Woltmann, in review), echoing findings from marine organisms (Whitehead et al 2012).

Importantly, the direct (i.e. toxicological) and indirect (i.e. ecological) pathways of effect of DWH oil on seaside sparrow populations are not mutually exclusive. Rather, they are expected to compound in additive or synergistic ways to ultimately determine fitness reductions in exposed organisms (Whitehead 2013). Future studies might characterize how oil-derived stress propagated through the coastal food web, and impacted higher trophic levels. In seaside sparrows, this will imply analyzing reproductive success while also controlling for food abundance as well as vegetation composition and cover in both oiled and control sites. Since our study only analyzed ten individual birds, we could not examine multiple locations from a wider region or explicitly analyze variation in exposure due to age and sex, a task that is left to future studies with larger sample sizes. Following seaside sparrows of known sex through reproduction will also clarify whether offspring of exposed parents are at risk due to maternal transfer of contaminants through the egg.

Interestingly, the depletion of radiocarbon in birds from oiled sites was greater for feathers than for crop contents (figure 2). As for many songbirds, feather development in juvenile seaside sparrows continues for a few days after fledging (9–10 days of age). The feathers, therefore, incorporate carbon from food fed by seaside sparrow parents, as well as from items that the fledglings obtained independently. The information provided by feathers, therefore, encompasses more life stages, locations and time compared to information from an analysis of crop contents. In spite of this, our results indicate that the carbon isotopic composition of feathers provides a sensitive, integrated measure of oil carbon assimilation by birds over longer timescales than from crop contents alone, with the additional benefit that feathers can be collected without the need to euthanize the birds.

Oil source fingerprinting in sediments from the same site where we sampled the exposed birds indicated the presence of DWH oil. Alternative explanations for the depletion of radiocarbon in seaside sparrow tissues are therefore unlikely. Wilson et al (2016) suggested that the decline in ¹⁴C values that they detected in the Eastern Gulf of Mexico in a variety of fish and invertebrate tissue and shell samples could be due to the input of radiocarbon-depleted dissolved inorganic carbon from the karst system of Western Florida. Given the different geology of our study sites, this explanation does not apply to our findings. Moreover, the demonstrated positive relationship between stable carbon values and radiocarbon values in feathers from seaside sparrows indicated that radiocarbon depletion was due to oil carbon contribution, rather than to freshwater input (Chanton et al 2012).

We detected a small, but statistically significant depletion in stable carbon values in feathers (∼2‰) of exposed seaside sparrows compared to unexposed birds. This difference is one order of magnitude smaller than the one detected using radiocarbon (∼20‰). The difference in stable carbon values in crop contents, instead, was not statistically significant. Thus, our results confirm that the higher sensitivity of radiocarbon analyses compared to stable carbon analyses might be needed to detect oil incorporation into biological matrices, particularly in organisms that come in contact with oil through their diet. Given the absence of radiocarbon from oil, the organic production in the ocean differs from DWH oil by 1000‰ for radiocarbon (Fry and Anderson 2014), as opposed to only 5–7‰ for stable carbon (Graham et al 2010).

We documented statistically significant differences in radiocarbon values between the tissues of exposed and control birds following the DWH oil spill. These results are consistent with the incorporation of DWH oil into the tissues of the exposed birds, and provide the first evidence that the oil made it into the terrestrial food web. The documented differences are admittedly small, corresponding to ∼2% oil carbon incorporation in feathers and ∼1% in crop contents. These small contributions are in line with other findings in organisms that are taxonomically and ecologically distant from the seaside sparrow (Chanton et al 2012, Fry and Anderson 2014). Our data indicating a decline in reproductive success of exposed birds in 2012 and 2013 (Bergeon Burns et al 2014) suggest that the toxicological significance of such small incorporation should not be underestimated.

We thank Fred Sheldon at the LSU Museum of Natural Science for collecting the birds. Brian Fry helped with radiocarbon and stable carbon analyses. Ed Overton and Buffy Meyer at the LSU College of Coast and Environment conducted oil source-fingerprinting, and greatly helped with the interpretation of the results. We are grateful to the personnel of the Rafter Radiocarbon lab (Lower Hutt, NZ) for assistance in the clarification of the methods, and interpretation of the results. Ivan Frigerio helped with geo-referencing the map of the sampling sites. This research was made possible by a grant from The Gulf of Mexico Research Initiative. Data are publicly available through the Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org (doi: 10.7266/N7D21VN2).