Science of the Total Environment 527–528 (2015) 306–312

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Persistent organic pollutants (POPs) in blubber of common bottlenose dolphins (Tursiops truncatus) along the northern Gulf of Mexico coast, USA Brian C. Balmer a,b,⁎, Gina M. Ylitalo c, Lauren E. McGeorge a, Keri L. Baugh c, Daryle Boyd c, Keith D. Mullin d, Patricia E. Rosel e, Carrie Sinclair d, Randall S. Wells b, Eric S. Zolman a, Lori H. Schwacke a a

National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science, 331 Fort Johnson Road, Charleston, SC 29412, USA Chicago Zoological Society, c/o Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA c National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, WA 98112, USA d National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Southeast Fisheries Science Center, 3209 Frederic Street, MS 39567, USA e National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Southeast Fisheries Science Center, 646 Cajundome Boulevard, Lafayette, LA 70506, USA b

H I G H L I G H T S • • • •

Health concerns have been documented in NGoM dolphins following the DWH oil spill. We examine POP exposure as a potential factor in dolphin mortality following DWH. POP levels were in the lower range as compared to other southeastern U.S. sites. POPs are not likely to be a primary factor for dolphin health issues following DWH.

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Article history: Received 19 February 2015 Received in revised form 4 May 2015 Accepted 4 May 2015 Available online xxxx Editor: D. Barcelo Keywords: Common bottlenose dolphin Deepwater Horizon oil spill Gulf of Mexico Persistent organic pollutants Tursiops truncatus

a b s t r a c t A number of studies were initiated in response to the Deepwater Horizon (DWH) oil spill to understand potential injuries to bottlenose dolphins (Tursiops truncatus) that inhabit the northern Gulf of Mexico (NGoM) estuarine waters. As part of these studies, remote biopsy skin and blubber samples were collected from dolphins at six field sites that received varying degrees of oiling: Barataria Bay (BB), Chandeleur Sound West (CSW), Chandeleur Sound East (CSE), Mississippi Sound South (MSS), Mississippi Sound North (MSN), and St. Joseph Bay (SJ). Blubber samples from 108 male dolphins were analyzed for persistent organic pollutant (POP) concentrations, as high levels of POPs have been previously reported in other southeastern U.S. dolphins and the potential contribution of these compounds to adverse health effects in NGoM dolphins must be considered. Dolphin blubber levels of summed POPs (ΣPOPs) did not differ significantly across sites (F-test, P = 0.9119) [μg/g lipid; geometric mean and 95% CI]; CSW [65.9 (51.4–84.6)], SJ [74.1 (53.0–104)], MSN [74.3 (58.7–93.9)], BB [75.3 (56.4–101)], CSE [80.5 (57.8–112)], and MSS [82.5 (65.9–103)]. Overall, POP concentrations were in the lower half of the range compared to previously reported concentrations from other southeastern U.S. sites. Increased dolphin mortalities have been ongoing in the NGoM and have been suggested to be linked with the DWH oil spill. In addition, lung disease, impaired adrenal function, and serum biochemical abnormalities have been reported in dolphins from BB, an area that was heavily oiled. The results of this study suggest that POPs are likely not a primary contributor to the poor health conditions and increased mortality observed in some populations of NGoM dolphins following the DWH oil spill. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Following the explosion and collapse of the Deepwater Horizon (DWH) drilling platform in April 2010 and the uncontrolled release of ⁎ Corresponding author at: NOAA/NCCOS Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC 29412, USA. E-mail address: [email protected] (B.C. Balmer).

http://dx.doi.org/10.1016/j.scitotenv.2015.05.016 0048-9697/© 2015 Elsevier B.V. All rights reserved.

oil from the wellhead for several months, oil reached northern Gulf of Mexico (NGoM) bays, sounds, and estuaries (BSE) (Michel et al., 2013; OSAT, 2013), that served as habitat for common bottlenose dolphins (Tursiops truncatus) (Waring et al., 2013). A number of studies were initiated to understand potential injuries to BSE dolphins from the oil spill, including capture-release health assessments of dolphins that were conducted in heavily oiled Barataria Bay, Louisiana (BB). The BB dolphin capture-release study found significant health effects including poor

B.C. Balmer et al. / Science of the Total Environment 527–528 (2015) 306–312

body condition, a high prevalence of lung disease, and abnormally low adrenal hormone levels that are consistent with previous studies of petroleum toxicity (Schwacke et al., 2014). In addition to the documented health concerns in live dolphins, increased strandings of dead dolphins along coastal areas that received oiling following the DWH spill have also been observed, and DWH oil has been suggested as a contributing factor (Venn-Watson et al., 2015). While this evidence of increased morbidity and mortality is suggestive of petroleum-related toxicity, other potential contributing factors, such as exposure to persistent organic pollutants (POPs), must also be considered. POPs, lipophilic chemicals that bioaccumulate in fatty tissues, are of particular concern for dolphins as these chemicals are biomagnified in higher trophic level organisms (J. Yordy et al., 2010; Yordy et al., 2010a). In fact, the bottlenose dolphin has been suggested as a sentinel species for examining regional trends of POPs for estuarine waters (Kucklick et al., 2011) due to the species' year-round residency and top trophic position in estuarine ecosystems. In general, polychlorinated biphenyls (PCBs) have been found at the highest concentration as compared to other measured POP classes in dolphin tissues, and have been detected at concentrations ranging from 33.1– 450 μg/g lipid in all sites along the southeastern U.S. where dolphins have been sampled (Kucklick et al., 2011). High levels of POPs in marine mammals have been associated with endocrine disruption and immune suppression (Ross et al., 1996; Schwacke et al., 2012), and have been suggested to increase susceptibility to disease epidemics (Aguilar and Borrell, 1994). To understand whether POPs were likely a contributing factor for the poor health and increased mortality of dolphins along the NGoM coast, biopsy sampling was conducted and dolphin blubber was analyzed for concentrations of POPs as part of the DWH Natural Resource Damage

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Assessment (NRDA). Here we report POP concentrations measured in remote biopsy blubber samples from dolphins collected in cold and warm seasons during 2010–2012 at six different NGoM field sites.

2. Materials and methods 2.1. Sampling region During 2010–2012, bottlenose dolphin remote biopsy samples were collected from six NGoM sampling sites that experienced varying degrees of oiling: Barataria Bay (BB), Chandeleur Sound West (CSW), Chandeleur Sound East (CSE), Mississippi Sound South (MSS), Mississippi Sound North (MSN), and St. Joseph Bay (SJ) (Fig. 1). BB, an estuary approximately 24 km long and 19 km wide, is located in southeastern Louisiana and separated from the Gulf of Mexico by a series of barrier islands including Grand Isle and Isle Grand Terre. BB was classified as receiving prolonged and heavy oiling in 2010 and a classification of heavy to moderate residual DWH oiling in 2011 (Michel et al., 2013). Chandeleur Sound (CS) is a large, shallow bay located north of the Mississippi River delta and east of New Orleans, Louisiana. The Chandeleur Islands, which stretch approximately 80 km north to south and comprise the eastern boundary of the Sound, received heavy oiling in 2010 followed by a classification of heavy to moderate residual DWH oiling in 2011 (Michel et al., 2013). The salt marsh estuaries along the eastern shore of Louisiana, which constitute the western boundary of CS, received heavy to moderate oiling in 2010 followed by residual DWH moderate to light oiling in 2011 (Michel et al., 2013). Based upon genetic analyses, dolphins located in the western mainland and eastern barrier island regions of CS may represent separate stocks

Fig. 1. Dolphin sampling sites in the Northern Gulf of Mexico (NGoM) [Barataria Bay (BB), Chandeleur Sound West (CSW), Chandeleur Sound East (CSE), Mississippi Sound South (MSS), Mississippi Sound North (MSN), and St. Joseph Bay (SJ)].

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(Rosel, pers. comm.). Thus, CS samples were grouped into two sites: Chandeleur Sound West (CSW) and Chandeleur Sound East (CSE). Mississippi Sound (MS) has a surface area of 2129 km2 (Higgins and Eleutrius, 1978) and extends eastward from Half Moon Island, Louisiana to Cedar Point, Alabama (Eleuterius, 1978). The northern boundary of MS, the mainland coastlines of Mississippi and Alabama, received moderate to light oiling in 2010 followed by residual DWH light to no oiling in 2011 (Michel et al., 2013). The southern boundary of MS is formed by a series of barrier islands (Cat, Ship, Horn, Petit Bois, and Dauphin Islands) and received heavy to moderate oiling in 2010 followed by residual DWH light oiling in 2011 (Michel et al., 2013). Based on photoidentification surveys (Hubard et al., 2004) and satellite-linked telemetry (Mullin, pers. comm.), dolphins along the northern mainland and those associated with the southern barrier islands may be separate stocks. Thus, MS samples were grouped into one of two sites: Mississippi Sound North (MSN) and Mississippi Sound South (MSS). SJ, which is approximately 24 km long and 10 km wide, is located along the Florida Panhandle between Panama City and Apalachicola. The Gulf-side of St. Joseph Peninsula, received some very light oiling in 2010, otherwise no DWH-associated oil was observed in the region (Michel et al., 2013). 2.2. Remote biopsy sample collection Remote biopsy samples were collected using a modified rifle or a Barnett Panzer V crossbow (Barnett Outdoors, LLC, Tarpon Springs, FL, USA). The methodology for sample collection and in-field processing has been described in detail in Sinclair et al. (2015). Briefly, samples were collected from individual dolphins at a distance of 3–10 m, targeting the flank of the animal below the dorsal fin and above the midline (Gorgone et al., 2008). The remote biopsy sample that was obtained consisted of skin and a full-thickness section of blubber approximately 0.7–0.8 g in weight. The skin sample for genetic analyses was stored at room temperature in 20% DMSO saturated with NaCl, and was used to determine sex using the polymerase chain reaction (PCR) methods described by Rosel (2003). The blubber sample used for contaminant analyses was stored in a pre-cleaned Teflon vial (Savillex, Eden Prairie, MN, USA), frozen in a liquid N2 dry shipper in the field, and stored at −80 °C in the lab prior to sample analysis. Digital photographs and in some instances digital video, were obtained to identify sampled individuals using dorsal fin identification (reviewed in Urian et al., 1999). The remote biopsy sampling was conducted under two NOAA Scientific Research Permits and IACUC approvals issued to KDM and RSW. 2.3. Remote biopsy sample analysis Adult female cetaceans transfer the majority of their body burden of lipophilic contaminants (approximately 80%) to their calves through lactation (Cockcroft et al., 1989; Yordy et al., 2010b). In contrast, male cetaceans have no substantive mechanism for offloading contaminants, thus body burdens increase over their lifespan (Wells et al., 2005; Yordy et al., 2010b). Accordingly, only blubber samples from male dolphins were analyzed to provide an indicator of overall POP concentrations at a given field site and sampling period (Kucklick et al., 2011). Full-depth blubber samples were extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) for POPs as described previously (Schwacke et al., 2014; Sloan et al., 2014). Briefly, approximately 0.4 g of minced blubber was dried with sodium or magnesium sulfate and extracted with dichloromethane on an accelerated solvent extractor (ASE). The extract was cleaned up on a gravity flow column containing alumina/silica to remove polar compounds. The precleaned sample extract was further cleaned up using size exclusion chromatography to remove lipids prior to GC/MS analysis. Percent lipid was determined gravimetrically as described in Sloan et al. (2014) and lipid classes (i.e. sterol esters/wax esters, triglycerides, free

fatty acids, cholesterol, phospholipids) were determined in a 1 mL subsample of pre-cleaned sample extract using thin-layer chromatography with flame ionization detection (TLC-FID) (Sloan et al., 2014; Ylitalo et al., 2005). Duplicate TLC-FID analyses were conducted for each sample extract and the percent contribution of each lipid class to the sum of the five classes was determined for each sample run. Then, for each lipid class, the average percent contribution between the two sample runs was reported. Concentrations of POPs are reported as μg/g (lipid weight). In total 77 compounds were analyzed including 45 PCB congeners (ΣPCBs) (IUPAC PCB numbers 17, 18, 28, 31, 33, 44, 49, 52, 66, 70, 74, 82, 87, 95, 99, 101, 105, 110, 118, 128, 138, 149, 151, 153, 156, 158, 170, 171, 177, 180, 183, 187, 191, 194, 195, 196, 199, 200, 201, 202, 205, 206, 207, 208, and 209), 15 polybrominated diphenyl ether congeners (ΣPBDEs) (28, 47, 49, 66, 85, 99, 100, 153, 154, 155, 183, two unidentified pentabrominated diphenyl ethers, one unidentified hexabrominated diphenyl ether and one unidentified heptabrominated diphenyl ether), 6 dichlorodiphenyl-dichloroethanes (ΣDDTs) (o,p′-DDD, DDE, and DDT; and p,p′-DDD, DDE, and DDT), 8 chlordanes (ΣCHLs) (alpha chlordane, cis-nonachlor, beta chlordane, heptachlor, heptachlor epoxide, nonachlor III, oxychlordane, and trans-nonachlor), hexachlorobenzene (HCB), dieldrin, and mirex. Total POPs (ΣPOPs) included all 77 analyzed compounds and total organochlorine pesticides (ΣOCPs) included the sum of ΣDDTs, ΣCHLs, HCB, dieldrin, and mirex. Samples were extracted, cleaned, and analyzed by GC/MS in groups of 10–20 with one method blank and National Institute of Standards and Technology (NIST) standard reference material (SRM) 1945 Organics in Whale Blubber. Individual analyte concentrations measured in NIST SRM 1945 were in excellent agreement with reference values published by NIST. The concentration of each analyte that was measured in the NIST SRM 1945 was, on average, within 16% of the published NIST certified value. The limit of detection (LOD) for each analyte was defined as the greater of either the analyte mass in the lowest detectable calibration solution divided by the sample mass, or the analyte's average mass detected in blanks plus three times the standard deviation (Sloan et al., 2014). The LOD values for PCB congeners ranged from b 0.00067 μg/g (wet weight) to b 0.008 μg/g (wet weight). For chlorinated pesticides and PBDEs, the LOD values ranged from b 0.00066 μg/g, wet weight to b0.0084 μg/g, wet weight and b 0.00068 μg/g, wet weight to b0.0084 μg/g, wet weight, respectively. 2.4. Statistical analyses POP concentrations from male dolphin blubber samples were lipid normalized to reduce the variations in lipid content associated with different field sites and other contributing factors such as nutritional health (Struntz et al., 2004), and log transformed to meet the statistical assumptions of equal variance and normality. Prior to statistical analyses, concentration values below the LOD were replaced with ½ of the LOD and analytes with a detection rate of b 75% were removed (Kucklick et al., 2011). Data were first analyzed to assess the effect of sampling season. Samples were available for cold months (December, January, or February) and warm months (May, June, August, or September) for four field sites: BB, CSW, CSE, and MSN. However, the CSE site was not included in this analysis because there was only a single cold season sample. Percent lipid data were arcsine transformed to meet the statistical assumptions of normality. A two-way analysis of variance (ANOVA; JMP 11, SAS Institute, Cary, NC) was performed to compare percent lipid between sampling seasons (warm versus cold) and among field sites (BB, CSW, and MSN). Similarly, a two-way ANOVA was performed to compare ΣPOPs between sampling seasons and among field sites. An additional effect was included to examine the interaction between sampling site and season in both ANOVAs. When the interaction effect was not significant at or below the α = 0.05 threshold, it was removed and the ANOVA was run again. A Tukey's Honestly Significant Difference

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(HSD) test for unequal sample sizes was used to identify pairwise statistical differences. A second analysis was then conducted using only samples collected during the warm season. One-way ANOVAs were performed to compare percent lipid, ΣOCPs, and ΣPOPs among the entire collection of field sites (BB, CSW, CSE, MSS, MSN, and SJ). A multivariate analysis of variance (MANOVA) was performed to compare concentrations of the various POP classes (ΣPCB, ΣDDT, ΣCHL, ΣPBDE, dieldrin, mirex, and HCB) among all field sites. When the MANOVA indicated a significant multivariate effect, an univariate ANOVA was conducted for each POP class. When an univariate ANOVA showed a significant effect, a Tukey's HSD test for unequal sample size was used to identify pairwise statistical differences among field sites and percent lipid, ΣOCPs, ΣPOPs, and POP classes. PCB compounds were summed based upon their number of chlorines into three classes: tri-/tetra-/penta-; hexa-/hepta-; and octa-/nona-/deca-. A MANOVA was performed to compare PCB compound classes among field sites. When the MANOVA indicated a significant multivariate effect, an univariate ANOVA was conducted for each PCB compound class. When an univariate ANOVA showed a significant effect, a Tukey's HSD test for unequal sample size was used to identify pairwise statistical differences among field sites and PCB compound classes. 3. Results From 2010–2012, 169 remote biopsy samples were collected from NGoM male dolphins: BB (N = 38), CSW (N = 41), CSE (N = 15), MSS (N = 16), MSN (N = 43), and SJ (N = 16) (Fig. 1). Blubber percent lipid was significantly higher in CSW and MSN during the cold season but no difference was observed between seasons in BB (F-test, P b 0.0001 for interaction between season and field site) (Fig. 2). Lipid-normalized ΣPOPs were significantly higher in all three field sites during the warm season (F-test, P b 0.0001 for interaction between season and field site) (Fig. 3). Based upon the significant differences in lipid and ΣPOPs between seasons, only samples collected during the warm season were used to compare POP concentrations across the broader collection of field sites. From the 169 remote samples collected during 2010–2012, 108 were collected from NGoM dolphins during the warm seasons in 2010 and 2011: BB (N = 19), CSW (N = 19), CSE (N = 14), MSS (N = 16), MSN (N = 24), and SJ (N = 16) (Table 1). No warm season samples were collected in 2012. Blubber percent lipid content ranged from 15– 27% and was significantly affected by site (F-test, P = 0.0003). Most

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Fig. 3. Concentrations (geometric mean, 95% CI) of summed persistent organic pollutants (Σ POPs) measured in remote biopsy blubber samples of Northern Gulf of Mexico (NGoM) male bottlenose dolphins based on field site and collection season. Cold season includes December, January, and February, and warm season includes May, June, August, and September. Field site abbreviations: BB—Barataria Bay, CSW—Chandeleur Sound West, CSE—Chandeleur Sound East, MSS—Mississippi Sound South, MSN—Mississippi Sound North, and SJ—St. Joseph Bay. Statistically homogeneous groups are indicated by the same letter subscripts.

field sites had similar percent lipid, but percent lipid for SJ was lower than CSW, CSE, MSS, and MSN (Table 1). There was no significant difference in ΣPOPs (F-test, P = 0.9119) or ΣOCPs (F-test, P = 0.4199) across field sites (Table 1). POP classes differed significantly across field sites (Wilks' lambda = 0.0879, P b 0.0001). The univariate ANOVAs indicated significant differences for the four POP classes with the lowest measured concentrations; ΣPBDEs, dieldrin, mirex, and HCB (Table 1, Fig. 4). ΣPBDE concentrations were significantly lower in SJ compared to CSE and MSN (F-test, P = 0.0301). Dieldrin was significantly lower in SJ compared to all other sites (F-test, P b 0.0001). Lower mirex concentrations were observed in BB compared to MSS, MSN, and SJ (F-test, P = 0.0018). HCB levels were significantly lower in SJ compared to BB (F-test, P = 0.0115). POP classes with the highest concentrations measured, ΣPCBs, ΣDDTs, and ΣCHLs, did not differ significantly across field sites (F-test, P = 0.8239, P = 0.2913, and P = 0.2976, respectively). PCB compound classes differed significantly across field sites (Wilks' lambda = 0.7134, P = 0.0001). The univariate ANOVAs indicated a significant difference in the tri-/tetra-/penta- compound class, with PCB concentrations lower in SJ compared to BB (F-test, P = 0.0121) (Fig. 5). PCB concentrations did not differ significantly for the hexa-/ hepta-, and octa-/nona-/deca-compound classes among field sites (F-test, P = 0.8146 and P = 0.6119). 4. Discussion

Fig. 2. Percent lipid values (geometric mean, 95% CI) determined in remote biopsy blubber samples of Northern Gulf of Mexico (NGoM) male bottlenose dolphins based on field site and collection season. Cold season includes December, January, and February, and warm season includes May, June, August, and September. Field site abbreviations: BB—Barataria Bay, CSW—Chandeleur Sound West, and MSN—Mississippi Sound North. Statistically homogeneous groups are indicated by the same letter subscripts.

Based upon previous studies in which high levels of POPs were identified as a likely cause of health effects in dolphins (e.g. Schwacke et al., 2012), background POP exposure was considered as part of the investigation of bottlenose dolphin morbidity and mortality in the NGoM following the DWH oil spill. Both POPs and petroleum constituents have been shown to produce toxic effects through the aryl hydrocarbon receptor (AhR) pathway, and similar types of effects such as immune suppression and reproductive impairment have been reported for these chemical classes (Kannan et al., 2000; Ross et al., 1996; Schwartz et al., 2004a,b). Endocrine disruption has also been associated with both contaminant classes, although interference with reproductive and thyroid hormone receptors or binding proteins have been more commonly reported for POPs (e.g. Boas et al., 2012; Cheek et al., 1999; Meerts et al., 2000; Tabuchi et al., 2006), while the hypothalamic–pituitary–adrenal

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Table 1 POP concentrations (μg/g lipid; geometric mean, 95% CI) and proportion of lipid (geometric mean, 95% CI) measured in remote biopsy blubber samples of Northern Gulf of Mexico (NGoM) male bottlenose dolphins collected during the warm season at six field sites. Statistically homogeneous groups are indicated by the same letter subscripts.

Sampling years sample size Lipid ΣPCBs1 ΣDDTs2 ΣCHLs3 ΣPBDEs4 Dieldrin Mirex HCB5 ΣOCPs6 ΣPOPs7 1 2 3 4 5 6 7

Barataria Bay

Chandeleur Sound West

Chandeleur Sound East

Mississippi Sound South

Mississippi Sound North

St. Joseph Bay

2010 19 0.22 (0.18–0.25)AB 51.4 (38.5–68.6)A 16.2 (11.8–22.4)A 3.69 (2.76–4.94)A 2.92 (2.46–3.47)AB 0.52 (0.45–0.60)A 0.17 (0.12–0.24)B 0.07 (0.06–0.07)A 20.8 (15.2–28.3)A 75.3 (56.4–101)A

2010 and 2011 19 0.25 (0.23–0.28)A 42.1 (32.7–54.3)A 15.9 (12.1–20.9)A 3.97 (3.17–4.97)A 2.78 (2.35–3.28)AB 0.49 (0.43–0.55)A 0.28 (0.21–0.38)AB 0.05 (0.05–0.06)AB 20.8 (16.1–26.9)A 65.9 (51.4–84.6)A

2010 and 2011 14 0.27 (0.22–0.34)A 52.8 (38.1–73.4)A 19.0 (13.3–27.3)A 4.08 (2.90–5.74)A 3.31 (2.56–4.28)A 0.51 (0.37–0.71)A 0.34 (0.24–0.50)AB 0.06 (0.05–0.08)AB 24.2 (17.0–34.3)A 80.5 (57.8–112)A

2010 and 2011 16 0.27 (0.23–0.33)A 48.3 (39.2–59.4)A 25.5 (19.3–33.9)A 3.87 (3.14–4.77)A 3.01 (2.57–3.52)AB 0.41 (0.31–0.55)A 0.46 (0.36–0.59)A 0.05 (0.03–0.08)AB 30.6 (23.5–40.0)A 82.5 (65.9–103)A

2010 and 2011 24 0.25 (0.21–0.29)A 43.4 (34.7–54.2)A 21.9 (16.1–29.7)A 3.87 (3.14–4.78)A 2.99 (2.45–3.64)A 0.47 (0.39–0.56)A 0.38 (0.27–0.53)A 0.05 (0.04–0.06)AB 27.2 (20.7–35.8)A 74.3 (58.7–93.9)A

2010 16 0.15 (0.12–0.20)B 48.1 (34.4–67.2)A 19.9 (14.0–28.3)A 2.66 (1.91–3.72)A 1.88 (1.28–2.77)B 0.14 (0.12–0.18)B 0.36 (0.24–0.54)A 0.03 (0.03–0.04)B 23.3 (16.5–32.9)A 74.1 (53.0–104)A

∑PCBs include 45 PCB congeners. See Section 2.3, Remote biopsy sample analysis for full list. ∑DDTs include o,p′-DDD, DDE, and DDT; and p,p′-DDD, DDE, and DDT. ∑CHLs include alpha chlordane, cis-nonachlor, beta chlordane, heptachlor, heptachlor epoxide, nonachlor III, oxychlordane, and trans-nonachlor. ∑PBDEs include 28, 47, 49, 66, 85, 99, 100, 153, 154, 155, 183, Br5DE04, BrDE05, Br6DE01, and Br7DE01. Hexachlorobenzene. ∑OCPs include ∑DDTs, ∑CHLs, HCB, mirex and dieldrin. ∑POPs are the sum of all measured compounds.

(HPA) axis is a suggested target for petroleum-related chemicals (Burns et al., 2014; Mohr et al., 2008). Nevertheless, the potential for POPs as a contributing factor, which combined with petroleum exposure could cause additive or even synergistic toxic effects, required investigation. In 2010 and 2011, a cluster of stranded dead dolphins was identified near BB, an area that was exposed to a high degree of oiling over a prolonged period, and the highest annual dolphin stranding numbers for Louisiana, since approximately 1990, occurred during this period (Litz et al., 2014; Venn-Watson et al., 2015). In addition, significant health concerns, including evidence of disruption of the HPA axis, were reported for live dolphins in BB (Schwacke et al., 2014). There were no statistical differences between NGoM sites with respect to ΣPOP concentrations. Furthermore, we found that concentrations of POPs measured in dolphins from all six NGoM sites were comparable to, or lower than, those previously measured among dolphins in other southeastern U.S. sites. When compared to concentrations reported by Kucklick et al. (2011), these NGoM dolphins rank overall in the lower half as compared to the other 11 previously reported southeastern U.S. sites. These results confirm previously reported findings from Schwacke et al. (2014) that found relatively low POP levels in the BB dolphins that were sampled during the capture-release health assessments and found to be in poor health and with impaired adrenal response. Trends with regard to patterns of various POP classes were also similar in the NGoM sites to those previously reported in other dolphins across the southeastern U.S. (e.g. Balmer et al., 2011; Hansen et al., 2004; Litz et al., 2007; Wilson et al., 2012) and in other regions of the world (reviewed in Aguilar et al., 2002) with the highest concentrations

being found for ΣPCBs followed by ΣDDTs, ΣCHLs, and ΣPBDEs. Therefore, it is unlikely that POPs significantly contributed to the observed disease conditions in BB, which had not been previously observed in other populations (Schwacke et al., 2014), and/or to unusually high rates of stranded dead dolphins that occurred following the DWH oil spill near BB or near the other sites in the NGoM (Venn-Watson et al., 2015). While the POP levels measured in blubber of dolphins from the NGoM sites were comparable to or lower than in dolphins from previously sampled sites, the potential additive and/or synergistic effects of POPs and oil exposure must still be considered. For PCBs, the POP class measured at the highest concentrations, a study of stranded porpoises demonstrated a 50% increase in risk of infectious disease at blubber concentrations ~45.00 μg/g lipid, and a 2-fold increase in risk at ~80.00 μg/g lipid (Hall et al., 2006). In dolphins, PCBs in blubber were associated with reduced circulating thyroid hormones and reduced functional immune response (Schwacke et al., 2012), and the lowest thyroid hormone concentrations and immune responses were observed with blubber PCB concentrations that exceeded ~70.00 μg/g lipid. The range of geometric mean concentrations reported here (65.9–82.5 μg/g lipid; Table 1) overlaps with the lower of these values, indicating that some portion of the sampled dolphins had PCB concentrations that could be expected to produce adverse health effects. While under ordinary conditions, the population-level effects of PCBs in BB and other NGoM sites would be expected to be low in comparison to other southeastern U.S. sites, additional stress from DWH oil exposure in addition to the background POP exposure could have exacerbated toxicological effects.

Fig. 4. Concentrations (geometric mean, 95% CI) of seven classes of persistent organic pollutants (POPs) measured in remote biopsy blubber samples of Northern Gulf of Mexico (NGoM) male bottlenose dolphins collected during the warm season across six field sites. Field site abbreviations: BB—Barataria Bay, CSW—Chandeleur Sound West, CSE—Chandeleur Sound East, MSS—Mississippi Sound South, MSN—Mississippi Sound North, and SJ—St. Joseph Bay. Statistically homogeneous groups are indicated by the same letter subscripts.

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Fig. 5. Concentrations (geometric mean, 95% CI) of polychlorinated biphenyl (PCB) compounds grouped into three classes (tri-/tetra-/penta-; hexa-/hepta-; and octa-/nona-/ deca-) measured in remote biopsy blubber samples of Northern Gulf of Mexico (NGoM) male bottlenose dolphins collected during the warm season across six field sites. BB—Barataria Bay, CSW—Chandeleur Sound West, CSE—Chandeleur Sound East, MSS—Mississippi Sound South, MSN—Mississippi Sound North, and SJ—St. Joseph Bay. Statistically homogeneous groups are indicated by the same letter subscripts.

POP levels in BB dolphins were significantly lower in the cold season compared to those collected in the warm season (Fig. 3). However, unlike CSW and MSN in which blubber percent lipid was higher in the cold season, the blubber percent lipid in BB dolphins was similar between seasons (Fig. 2). Although measurements of body condition [e.g. parameters measured by Schwacke et al. (2014)] were not taken in this study, one hypothesis for these results may be that the similar blubber percent lipid between seasons reflects poor body condition in BB dolphins. If abundance and/or quality of prey species in BB were reduced following the DWH oil spill, this could potentially have affected the dolphins' ability to build winter lipid stores. Alternatively, poor body condition could have been related to other conditions such as impaired adrenal function or chronic lung disease as previously described for BB dolphins (Schwacke et al., 2014). Regardless, these results suggest that seasonal differences in POP concentrations were not solely dependent on the lipid content of the blubber. Additional blubber sampling in BB during different seasons, in future years, could help better understand this phenomenon. Although there were very few statistical differences in POP concentrations across field sites, some interesting variations were seen among the NGoM sites, with the highest ΣPOP concentrations measured in dolphins around the barrier islands of Mississippi and adjacent eastern Louisiana (MSS and CSE). A major route of exposure and accumulation of POPs in dolphins is from consumption of contaminated prey, and contaminant levels of dolphin prey species vary depending on numerous factors including trophic level, distribution patterns, and proximity to contaminant sources (e.g. Storelli et al., 2006; Wirth et al., 2014). The higher levels of POPs measured in dolphins sampled around the barrier islands may be a result of differences in dolphin prey selection and variations in prey species' contaminant levels between the estuarine and coastal waters. Several studies in the southeastern U.S. have identified differences in prey selection between coastal and estuarine bottlenose dolphins. Along the east coast of the U.S., Gannon and Waples (2004) determined that prey species of estuarine bottlenose dolphins were primarily Atlantic croaker (Micropogonias undulatus), and coastal dolphins primarily preyed upon weakfish (Cynosicon regalis) and some inshore squid species (Loligo sp.). Along the west coast of Florida, both estuarine and coastal dolphins feed primarily upon soniferous fish species (e.g. family Sciaenidae); however, coastal dolphins also had up to 20% of their diet composed of cephalopods (Barros and Odell, 1990; Barros and Wells, 1998). Future research measuring POP levels in NGoM dolphin prey species within both coastal and estuarine habitats

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and analysis of stomach contents from stranded dolphins would provide insight into the differences in contaminant profiles identified in this study. Contaminant profiles have been suggested as a potential tool to use in collaboration with genetic analyses for marine mammal stock identification (e.g. Balmer et al., 2011; Hansen et al., 2004). The variation between samples collected surrounding the barrier islands to those collected from more inland waters of the NGoM supports this hypothesis. However, in this study, there was a complicating factor in potentially using POP profiles to differentiate dolphin groups related to the effect of sampling season on POP concentrations (see Figs. 2 and 3). Marine mammal blubber thickness and lipid content fluctuate across seasons (Pabst et al., 1999) and lipid normalization of POP concentrations reduces some of the variability associated with seasonal effects (reviewed in Kucklick et al., 2011). However, Yordy et al. (2010a) determined that as lipid is mobilized from dolphin blubber to other tissues, the distribution of contaminants is not solely lipid-dependent. In California sea lions (Zalophus californianus), increases in POP concentrations within the blubber were observed as an animal's weight decreased, and lower chlorinated PCB congeners were lost at higher rates than other POP classes (Hall et al., 2008). Thus, POP concentrations do not necessarily decrease as a constant function of lipid. This may be because as lipid is mobilized and depleted, POPs are mobilized but rather than being eliminated, they are re-sequestered in the remaining blubber. The mainland (CSW and MSN) field sites had significant changes in lipid content across seasons (Fig. 2) and lipid-normalized POP levels were significantly higher in the warm season (Fig. 3). Thus, seasonal differences in the distribution and mobilization of POP concentrations in the blubber could influence the interpretation of different POP profiles within a given region. Additional studies that would increase the sample size during the cold season and repeated samples from the same individuals across seasons would provide further insight into fine-scale variations in POP profile patterns. Although Yordy et al. (2010a) provided the first insights into the distribution and mobilization of POPs in different tissues, the study was limited to a sample size of four individual dolphins. A more in-depth study comparing POP levels within dolphins across seasons would identify the mobilization of contaminants among tissue types and provide insight into differences in POP levels observed in remote biopsy samples. NOAA disclaimer This publication does not constitute an endorsement of any commercial product or intend to be an opinion beyond scientific or other results obtained by the National Oceanic and Atmospheric Administration (NOAA). No reference shall be made to NOAA, or this publication furnished by NOAA, to any advertising or sales promotion which would indicate or imply that NOAA recommends or endorses any proprietary product mentioned herein, or which has as its purpose an interest to cause the advertised product to be used or purchased because of this publication. Acknowledgments This work was part of the DWH NRDA being conducted cooperatively among NOAA, other Federal and State Trustees, Chicago Zoological Society, and BP. Remote biopsy sampling was conducted in Louisiana and Mississippi under NMFS Scientific Research Permit No. 779-163302 and in St. Joseph Bay under NMFS Scientific Research Permit No. 15543. We would like to thank the following researchers for their support in field work operations (Jason Allen, Aaron Barleycorn, Kevin Barry, Penn Clarke, Annie Gorgone, Mary Gryzbek, Michael Hendon, Suzanne Lane, Tony Martinez, Paula Olsen, Errol Ronje, J. C. Salinas, Michelle Savoie, Jen Sinclair, Todd Speakman, Kate Sprogis, Angie Stiles, and Jesse Wicker), and laboratory and data analyses (Bernadita Anulacion, Jennie Bolton, Ron Pearce, and Lynsey Wilcox). We would

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