Association between chronic organochlorine exposure and immunotoxicity in the round stingray (Urobatis halleri)

Environmental Pollution xxx (2016) 1e9

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Association between chronic organochlorine exposure and immunotoxicity in the round stingray (Urobatis halleri)* Jillian M. Sawyna*, Weston R. Spivia, Kelly Radecki, Deborah A. Fraser, Christopher G. Lowe California State University, Long Beach, 1250 Bellflower Boulevard, Long Beach, CA 90840, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 September 2016 Received in revised form 11 December 2016 Accepted 12 December 2016 Available online xxx

Chronic organochlorine (OC) exposure has been shown to cause immune impairment in numerous vertebrate species. To determine if elasmobranchs exhibited compromised immunity due to high OC contamination along the coastal mainland of southern California, innate immune function was compared in round stingrays (Urobatis halleri) collected from the mainland and Santa Catalina Island. Proliferation and phagocytosis of peripheral blood, splenic, and epigonal leukocytes were assessed. Percent phagocytosis and mean fluorescence intensity (MFI) were evaluated by quantifying % leukocytes positive for, and relative amounts of ingested fluorescent E. coli BioParticles. Total cell proliferation differed between P sites, with mainland rays having a higher cell concentration in whole blood. PCB load explained P P significantly higher % phagocytosis in blood of mainland rays, while PCB and pesticide loads described increased splenic % phagocytosis and MFI in the mainland population. Data provides evidence of strong OC-correlated immunostimulation; however, other site-specific environmental variables may be contributing to the observed effects. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Elasmobranch immunology Phagocytosis Leukocyte proliferation PCBs Pesticides

1. Introduction Historically, large quantities of organic contaminants, such as DDT, PCBs, and chlordanes, were introduced into the Southern California Bight (SCB) via runoff from the urbanized mainland or through wastewater discharge from point sources (Chen et al., 2012). Despite the 1970's ban on the disposal of organochlorines (OCs) into the ocean and remediation efforts, these legacy contaminants remain bound to marine sediments, where they are continuously reintroduced into the ecosystem through biological or physical disturbances (Evans et al., 1991; Calamari et al., 2000). Environmentally persistent, many of these hydrophobic and lipophilic compounds are highly resistant to metabolism in vertebrates. Consequentially, bioaccumulation occurs through food web transfer, resulting in upper trophic level organisms obtaining high OC concentrations, particularly in their fatty tissues (e.g. liver and blubber). OCs are acquired via ingestion or through maternal offloading processes. In mammals, OCs are transferred to offspring during lactation, as a result of lipid mobilization from blubber

*

This paper has been recommended for acceptance by Dr. Chen Da. * Corresponding author. E-mail address: [email protected] (J.M. Sawyna).

during milk production (Addison and Brodie, 1987). In elasmobranch fishes, OC transference occurs during the process of vitellogenesis or the through the production of histotroph during gestation (Lyons et al., 2013; Lyons and Lowe, 2013). For these reasons, numerous marine top predators associated with the SCB, including pinnipeds and elasmobranchs, harbor high contaminant loads (Kajiwara et al., 2001; Blasius and Goodmanlowe, 2008; Lyons et al., 2013; Lyons and Lowe, 2015). Previous studies have shown that OCs can be carcinogenic, cause reproductive impairment, endocrine disruption, and immunotoxicity in numerous vertebrate species (Fry and Toone, 1981; Guillette et al., 1995; Nebert and Dalton, 2006). Marine mammal studies have shown correlative and causative relationships between OC exposure and immunotoxic consequences, often characterized by a change in cell proliferation and/or phagocytosis (Lahvis et al., 1995; Van Loveren et al., 2000; Levin et al., 2004, 2005; Schwacke et al., 2012). Furthermore, studies have associated fatal epizootics in marine mammals with high OC tissue concentrations (Hall et al., 1992; Aguilar and Borrell, 1996; Jepson et al., 1999). Though the exact mechanism involving OC induced immunotoxicity has yet to be completely identified, many studies attribute their activation of the aryl hydrocarbon receptor, AhR, pathway, with the most potent of immunotoxic consequences in mammals (Duffy and Zelikoff, 2006). Activation of the AhR results in the upregulation of genes,

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such as the cytochrome (CYP) P450s (Silkworth et al., 1986). Induction of CYP1A1, a member of the cytochrome P450 superfamily, has been correlated with the formation of DNA adducts, cancer, and leukocyte proliferation and functional abnormalities (Reynaud and Deschaux, 2006). Like other vertebrates, elasmobranchs rely primarily on their innate immune response, using the same classes of leukocytes (granulocytes, monocytes, dendritic cells), to combat acute infection. Moreover, the process of phagocytosis serves as the first line of defense against foreign antigens and for clearance of damaged tissue (Luer et al., 2004). Any deviation from baseline phagocytosis is considered a marker for immune impairment. As in mammals, granulocytes (neutrophils/heterophils) and circulating monocytes/ tissue macrophages are primarily responsible for phagocytic processes (Walsh and Luer, 2015). The spleen, and two organs unique to this subclass of fish, the epigonal and Leydig organs, serve as major sites for leukocyte hematopoiesis (F€ ange and Mattisson, 1981; Honma et al., 1984). Structurally similar to that of higher vertebrates, the spleen consists of red pulp, the primary site of erythropoiesis, as well as white pulp, which functions as a significant lymphopoietic region (Hamlett, 1999). Moreover, the spleen serves as a major site for phagocytosis and antigen stimulation leading to antibody synthesis (Rumfelt, 2015). The epigonal and Leydig organs, on the other hand, are lymphomyeloid, and serve as bone marrow equivalents (Luer et al., 2015). Compared to splenic tissue, very little antigen is trapped within these organs (Rumfelt, 2015). In elasmobranch fishes, OC levels and acquisition have been assessed (Serrano et al., 1997; Storelli and Marcotrigiano, 2001; Storelli et al., 2005; Lyons et al., 2013, 2014; Mull et al., 2013; Lyons and Lowe, 2015); however, the physiological impacts of high OC concentrations on immune function, in multiple tissues, have not been thoroughly investigated. Because some elasmobranchs occupy a comparable trophic position as that of marine mammals, exhibit similarly high maternal investment in their precocial young, have high lipid storage capacities, via large livers, and comparable innate immunity, it is plausible elasmobranchs with high OC levels will exhibit similar symptoms of immunotoxicity. The round stingray (Urobatis halleri) is a locally abundant, benthic forager found throughout the highly OC contaminated waters of the coastal mainland of southern California (Babel, 1967; Hale, 2005; Hoisington and Lowe, 2005). There are two distinct populations of round stingrays, one located at Santa Catalina Island and one along the mainland (Plank et al., 2010). A 42 km, deep water channel separating the mainland from Catalina Island allows Catalina Island to serve as a toxicology reference site (Dodder et al., 2014) and isolates the two ray populations. Furthermore, mainland and Catalina rays possess significant differences in not only contaminant levels, but contaminant signatures. Studies have shown that adult male rays sampled from the mainland had significantly higher OC concentrations, including PCBs, chlordanes, and DDTs, compared to the Catalina Island stingray population (Lyons et al., 2014). In addition, ethoxyresorufin-O-deethylase (EROD) activity, a biomarker for CYP1A1 induction and exposure to AhR ligands including OCs, was four times higher in mainland rays compared to those from Catalina Island (Lyons et al., 2014). Therefore, the round stingray is a suitable model for investigating the effects of OC contamination on elasmobranch innate immunity. The goal of this study was to determine whether there was a change in innate immune function in stingrays sampled from the coastal southern California mainland compared to those from Catalina Island. This was determined by assessing differences in cell proliferation among rays collected from the two sites, by examining if there was a site difference in phagocytosis among leukocytes

harvested from three immune tissues: blood, spleen, epigonal, and by identifying whether concentrations of specific or total OCs correlated with tissue-specific immune response. 2. Material and methods 2.1. Chemicals and reagents Elasmobranch modified RPMI 1640 (Gibco, Grand Island, NY) þ L e glutamine (E-RPMI) was used for tissue culture and cell isolation procedures as described by Walsh and Luer (1998). Stingray cells were assayed for phagocytosis in elasmobranch Ringer's solution (E-Ringer's) following Forster et al. (1972) and modified to include 72 mM trimethylamine oxide (Walsh and Luer, 1998) (Suppl. 1). Other cell culture media used was elasmobranch modified Phosphate Buffered Saline (E-PBS) as described by Walsh and Luer (2004). 2.2. Sample collection Round stingrays (U. halleri) were collected in the spring (MarcheJune) 2015. Rays were obtained by snorkel and dip net from Ripper's Cove Santa Catalina Island, CA (n ¼ 10), and via otter trawl from Long Beach Harbor, CA (n ¼ 19) (Fig. 1). In order to generate the simplest model possible and eliminate any confounding factors associated with variations in sex, only mature, male rays with a disc width >150 mm and total body weight >100 g were kept for processing (Babel, 1967; Hale, 2005). Rays of this sex and size class likely harbored the greatest OC loads, as well (Lyons et al., 2014). Any incidentally caught species were immediately released. Rays meeting the correct size criteria were transported back to the California State University, Long Beach (CSULB) Shark Lab for processing. Upon return to the Shark Lab, stingrays were dipped in 1 ml formalin per gallon seawater for 1 h, then placed in a quarantine tank for 24 h, for parasite removal. While this formalin treatment may have influenced an immune response, all rays underwent the same treatment procedure. Stingrays were then transferred to a closed-system tank, where they were kept for no more than 30 days prior to being euthanized. Euthanasia was conducted by immersion in a seawater ice slurry for 10 min, followed by spinal pithing, in agreement with approved CSULB Animal Welfare Board Protocol #337. Once euthanized, total body weight, total length, and disc width were measured (nearest 0.1 cm), and approximately 3 ml of blood was obtained via cardiac puncture, and transferred into lithium-heparin vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ), and placed on ice for the remainder of dissection. Total weights were obtained for liver, spleen, and epigonal organs. Spleen and epigonal tissue were then placed in 3 ml E-RPMI on ice and a liver lobe sample was frozen at 80  C for subsequent contaminant analyses. 2.3. Cell quantification Total cell concentration in whole blood was obtained using the Scepter 2.0 Automated Handheld Cytometer (EDM Millipore, Billerica, MA). Whole blood smears, (~50 ml blood from cardiac puncture) were prepared and stained with Giemsa (0.01% w/v) (SigmaAldrich, St. Louis, MO) for 30 min. Complete blood counts (CBC) (leukocytes þ erythrocytes þ thrombocytes) and differential leukocyte counts were performed using an EVOS FL-auto microscope (Life Technologies, Carlsbad, CA) via oil immersion (1,000) (Suppl. 2). Circulating amounts of total white blood cells (WBCs) were calculated by enumerating the number of leukocytes per 1000 erythrocytes and determining the percentage of WBCs from the

Please cite this article in press as: Sawyna, J.M., et al., Association between chronic organochlorine exposure and immunotoxicity in the round stingray (Urobatis halleri), Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.12.019

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Fig. 1. Sampling locations for U. halleri collection in southern California. Mainland stingrays were sampled from Long Beach Harbor and Santa Catalina Island rays were collected from Ripper's Cove.

CBC (Suppl. 3). Each leukocyte subpopulation (i.e., granulocytes, monocytes, lymphocytes) was determined as a percentage of the total number of WBCs counted.

indicator dyes fluoresce in acidic environments, such as in a phagosomal compartment after phagocytosis by leukocytes; thus, pHrodo Red E. coli Bioparticles were kept in the dark during preparation to prevent quenching of fluorescence.

2.4. Isolation of leukocytes 2.6. Phagocytosis assays Whole blood was transferred from lithium-heparin tubes to 1.5 ml tubes and centrifuged at 50g for 20 min at 4  C. Buffy coat and plasma supernatant were then removed, avoiding the erythrocyte pellet, and transferred to a new tube. This was repeated 3e4 times for optimum cell yield and purity. Cells from the supernatant were brought up to 3 ml in E-RPMI, plated into 35 mm wells containing 22  22 mm glass coverslips, and incubated at 25  C, 5% CO2 for 1 h. Non-adherent cells were harvested into 50 ml conical polypropylene tube and coverslips were washed 10 times with 1 ml chilled E-PBS to optimize recovery of non-adherent cells. 10 ml cell suspension were counted using a hemocytometer to obtain the total number of isolated WBCs. Cells were then centrifuged at 300g for 5 min at 4  C, and the pellet was brought up to 1  106 cells/ml in E-Ringer's. A portion of the spleen and the whole epigonal organ were homogenized in 3 ml fresh E-RPMI using 50 ml (spleen) and 15 ml (epigonal) disposable tissue grinders and pipetted through 100 mm cell strainers into 50 ml tubes. Cell suspensions were centrifuged at 50g for 10 min at 4  C. Supernatants were transferred to 15 ml tubes and 10 ml suspensions were counted using a hemocytometer. Cells were centrifuged at 200g for 5 min at 4  C, and the pellet was brought up 1  106 cells/ml in E-Ringer's. 2.5. Preparation of E. coli bioParticles During leukocyte isolation, 2 ml E-Ringer's were added to a thawed 2 mg vial of Molecular Probes pHrodo Red E. coli Bioparticles Conjugate for Phagocytosis (Life Technologies, Carlsbad, CA) and sonicated in a water bath for 5e10 min, until particles were homogenized, then vortexed until ready for plating. PHrodo

100 ml cell suspensions of each tissue type were plated in a 96well tissue culture treated plate with 20 ml BioParticles, or ERinger's, for a total volume of 200 ml, and incubated for 2 h at 25  C, 5% CO2. Relative size (forward scatter, FSC), granularity/complexity (side scatter, SSC), and fluorescence data from approximately 10,000 cells per sample were acquired using the Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA) and analyzed using FloJo 7.6.5 software. A control sample of leukocytes without BioParticles was used to define a leukocyte gate based on size/granularity properties. All further analyses were performed using only cells within the leukocyte gate (to exclude uningested BioParticles) (Suppl. 4). Granular (SSC high) and non-granular cells (SSC low) were identified by selecting the different populations according to their granular/SSC property. Fluorescence of cells excited by a 488 nm laser was measured using filter channel 2 (FL2) with an emission wavelength of 530 nm. Mean fluorescence intensity (MFI), calculated as the mean relative level of fluorescence of the nonBioParticle sample subtracted from the mean level of fluorescence of the BioParticle sample, represented the relative amount of fluorescence per cell, i.e. the relative amount of BioParticles ingested per cell (also called phagocytic index). Percent phagocytosis (% phagocytosis) was evaluated as the percentage of cells that ingested one or more BioParticles, and constituted the portion of cells exhibiting a shift in fluorescence from the non-BioParticle sample. 2.7. Organochlorine analysis Liver samples of a subset of mainland (n ¼ 11) and Catalina

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Island (n ¼ 8) rays were sent to CSULB's Institute for Integrated Research in Materials, Environments, and Society (IIRMES) for OC quantification following the methods described by Lyons et al. (2014). Samples were screened for a panel of PCBs (53 congeners) (Suppl. 5), DDT and its byproducts, and chlordane (CHL) congeners/ isomers (Suppl. 6). Hepatic concentrations of OCs were measured using Environmental Chemstation software (Agilent). Compounds measured were then summed to obtain total PCBs and total pesticides (DDTs þ CHLs) for each stingray sample and reported in ng/g wet weight (ng/g ww). Quality-assurance and quality-control samples were run with each batch of study samples to confirm accuracy and precision of data acquired and included 1 blank, 1 certified reference material (Lake Michigan trout tissue 1947, National Institute of Standards and Technology), 2 duplicate matrix spikes, and 1 study sample replicate. Matrix spikes were prepared by adding spike surrogates to subsamples used for PCB and pesticide analysis. The quality control goal was for a 70e130% recovery of spiked analytes, with 90% of replicates yielding a relative percent difference of <30%. Blanks showed no signs of external contamination and certified reference material analytes were well in range of reference values. The mean percentages (±standard deviation [SD]) of recovery of surrogates were 83 ± 4%, 82 ± 3%, 109 ± 6%, and 90 ± 7% for tetrachloro-m-xylene, PCB 30, PCB 112, and PCB 198, respectively. Mean recoveries of matrix spikes were 95 ± 16% for PCBs and 100 ± 30% for pesticides. Mean relative percent difference between replicates of sample duplicates and matrix spikes were 7 ± 11% and 1 ± 6%. 2.8. Statistical analyses A generalized linear model (GLM) with a quasibinomial distribution was used to compare leukocyte differentials and flow cytometry data for % phagocytosis that failed to meet the criteria of the Shapiro-Wilk Normality Test and F Test of Equality of Variance. Because total cell concentration data and MFI data did not meet the assumptions for the Shapiro-Wilk Normality Test and Levene's Test of Equality of Variances, a Mann-Whitney-Wilcoxon Test was used. Total PCB and total pesticide loads were compared within and across sites using a Mann-Whitney-Wilcoxon Test, since data could not be normalized. To understand which factors contributed to changes in MFI and % phagocytosis, generalized linear models were produced that included total weight, liver weight, liver lipid conP P tent, pesticides (DDT þ CHLs), and PCBs. Model selection was based on stepwise removal of model terms, with model comparison determined by AIC. Significances of predictors in candidate model were then evaluated using an ANOVA. All data values were reported as mean (±SE) and all statistical tests were performed using R statistical package (R Development Core Team). 3. Results 3.1. Cell proliferation Leukocyte differentials for Catalina rays were in the range of those for healthy elasmobranchs as described by Walsh and Luer (2004), and thus, served as a suitable baseline. Total circulating cell concentration differed significantly between mainland (1,652,200 ± 55,591 cells/ml) and Catalina Island rays (790,000 ± 105,055 cells/ml; Mann-Whitney-Wilcoxon W ¼ 8, p < 0.001; Fig. 2). No difference in % total WBCs (granulocytes, monocytes, lymphocytes) per 1000 erythrocytes in whole blood was detected between mainland and Catalina Island populations (GLM t ¼ 0.38, p ¼ 0.704; Suppl. 7). Furthermore, leukocyte differentials out of total WBCs did not differ between mainland and

Fig. 2. Total concentrations of cells in Urobatis halleri whole blood. Mainland ray (n ¼ 15) concentrations were significantly higher compared to Catalina Island (n ¼ 10). Boxes represent first and third quartiles, with the dark lines indicating group medians. Whiskers denote variability outside upper and lower quartiles and open circles indicate the outliers. Asterisk denotes p  0.05.

Catalina Island rays, in regards to % granulocytes (33.86 ± 3.94% and 28.89 ± 3.42%; GLM t ¼ 0.83, p ¼ 0.413), % monocytes (1.46 ± 0.36% and 1.15 ± 0.49%; GLM t ¼ 0.49, p ¼ 0.625), and % lymphocytes (64.69 ± 3.86% and 69.96 ± 3.38%; GLM t ¼ - 0.90, p ¼ 0.376; Suppl. 8). Because no difference was found in leukocyte proportions between sites, the increase in total cell proliferation observed in the mainland rays occurred equally across all cell subpopulations. 3.2. Phagocytosis Mainland stingrays had a significantly higher % phagocytosis (23.44 ± 2.89%) in the blood compared to Catalina Island rays (12.98 ± 1.58%; GLM t ¼ 2.57, p ¼ 0.016; Fig. 3A), yet no difference was found in MFI (7610.32 ± 1666.61 and 9531.20 ± 1660.87; Mann-Whitney-Wilcoxon W ¼ 135, 0.069; Fig. 3D). Splenic % phagocytosis was significantly higher in mainland rays (24.7 ± 4.98%) compared to Catalina Island (9.96 ± 1.18%; GLM t ¼ 2.20, p ¼ 0.036; Fig. 3B), as was MFI (2681.74 ± 410.06% and 1197.1 ± 146.97%; Mann-Whitney-Wilcoxon W ¼ 37, p ¼ 0.007; Fig. 3E). Unlike the other tissues, no difference in epigonal % phagocytosis was observed between mainland (12.11 ± 1.73%) and Catalina Island rays (14.11 ± 3.91%; GLM t ¼ - 0.55, p ¼ 0.588; Fig. 3C), or in MFI (3149.10 ± 433.08 and 3415.5 ± 483.76; MannWhitney-Wilcoxon W ¼ 106, p ¼ 0.636; Fig. 3F). 3.3. OC quantification Contaminant quantification analyses showed mainland rays had P significantly higher hepatic PCBs (3732.74 ± 610.01 ng/g ww; P Mann-Whitney-Wilcoxon W ¼ 0, p < 0.001) and pesticides (314.54 ± 110.32 ng/g ww; Mann-Whitney-Wilcoxon W ¼ 2, p < 0.001), compared to the Catalina Island rays (249.34 ± 30.3 ng/g ww) and (4.42 ± 2.95 ng/g ww) (Fig. 4). Both mainland and Catalina Island populations had significantly greater concentrations of P P PCBs than pesticides (W ¼ 120, p < 0.001 and W ¼ 64,

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Fig. 3. Leukocyte percent phagocytosis of E. coli BioParticles in (A) blood, (B) spleen, and (C) epigonal tissue. Mainland rays had a significantly higher % phagocytosis in blood and splenic tissue. Leukocyte mean fluorescence intensity (MFI) for ingested E. coli BioParticles in (D) blood, (E) spleen, and (F) epigonal tissue. Splenic MFI in mainland rays was significantly higher than that of Catalina Island. Whiskers indicate variability outside upper and lower quartiles, dark lines denote group medians, and open circles display outliers. Asterisks show significant difference among groups p  0.05.

Fig. 4. Stingray (A) total PCB and (B) total pesticide loads. Boxes represent fist and third quartiles. Whiskers indicate variability outside upper and lower quartiles, with dark lines denoting group medians. The open circle depicts the outlier and asterisks illustrate p  0.05. Mainland rays had greater concentrations of PCBs and pesticides in their tissues compared to Catalina Island rays.

p < 0.001). Of the pesticides screened, only hexachlorobenzene, chlordane-gamma, chlordane-alpha, trans-nonachlor, cis-nonachlor, and 4,40 -DDE were detected in mainland rays, whereas in the Catalina Island population, only trans-nonachlor levels were found. Of the 53 PCB congeners, PCBs 118, 153, 138, 180 were the most prevalent, comprising 63% of the mean total PCBs from the

mainland and 97% of mean total PCBs from Catalina Island. 3.4. OC-correlated immunotoxicity The best candidate model for explaining % phagocytosis in blood P was PCB load (AIC 85.05; F ¼ 13.57, p ¼ 0.002; Fig. 5A). For MFI,

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Fig. 5. Relationship between stingray immune response and contaminant loads. Based on AIC model selection, the best candidate model for explaining (A) % phagocytosis in blood P P P was PCBs. Increasing PCB and pesticide load significantly predicted increasing (B) splenic % phagocytosis and increasing (C) splenic MFI. Models showed a correlation P P between OC load ( PCB and pesticide) and immunostimulation among individual rays (n ¼ 19).

P P the best model did not include PCB or pesticide loads. It contained total weight, liver weight, and lipid concentration, with only lipid concentration having significance (AIC ¼ 280.41, F ¼ 9.29, p ¼ 0.009; Suppl. 9). The greatest OC-correlated effect was found in splenic tissue, characterized by not only an increase in the number of actively phagocytic cells, but a more robust response from individual P phagocytes as contaminant levels increased. Both increasing PCB P and pesticide loads significantly increased individual rays’ splenic % phagocytosis (AIC ¼ 93.78; F ¼ 9.00, p ¼ 0.009 and F ¼ 8.65, p ¼ 0.010; Fig. 5B). When the outlier was removed, P however, the best model contained only PCBs (AIC ¼ 83.51; F ¼ 25.3, p < 0.001). Results were similar for splenic MFI, whereby P P increasing PCB and pesticide loads increased ray MFI (AIC ¼ 263.09; F ¼ 7.05, p ¼ 0.018 and F ¼ 5.28, p ¼ 0.036; Fig. 5C). P Again, with removal of the outlier ray, only PCBs served as the predictor for increasing MFI (AIC ¼ 242.8; F ¼ 20.23, p < 0.001). Possibly due to the epigonal organ's primary role of

hematopoiesis and its seclusion from antigen interaction, a sitespecific difference in overall % phagocytosis and MFI was not detected in epigonal tissue, and consequentially, no associations between individuals' contaminant profiles and their immune responses were found. The model explaining epigonal % phagocytosis P included PBCs, liver weight, and lipid concentration; however, none of these variables were significant (AIC ¼ 85.45; p > 0.05). For P epigonal MFI, the best model contained pesticides, total weight, liver weight, and lipid concentration (AIC ¼ 260.16), of which only lipid concentration was significant (F ¼ 13.43, p ¼ 0.003). 4. Discussion This study was the first to provide evidence of OC-correlated immunostimulation, primary driven by PCBs, in multiple elasmobranch tissues. This was explained not only by the models, but by the large variation in the mainland population's immune response. The mainland ray population is known to move considerably,

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especially during seasonal migrations (Vaudo and Lowe, 2006). Therefore, the variation in mainland immune response likely corresponds to the variable accumulation of OCs individuals acquire when they access areas of differing contamination levels. Our results indicated both an increase in cell count and phagocytic function in the mainland rays. This proliferation of cells can likely be attributed to a more systemic OC-correlated effect on organ function and hematopoiesis, beyond the activation of individual phagocytes. Similarly, Gelsleichter et al. (2006) found evidence of immunostimulation, via increased proliferation of blood leukocytes in Atlantic stingrays (Dasyatis sabina) with high pesticide concentrations. This primed functional alteration of immunocompetent cells from baseline parameters, is not only indicative of an immuno-challenging environment, but evidence of chemical associated immunotoxicity (Shelley et al., 2009). Xenobiotics have been shown to elicit histopathologic and cellular pathological effects, ranging from abnormal proliferation of stem cells, to altered cell maturation, to changes in cell populations and function (Blakley et al., 1999). The spleen is a specialized organ that functions in the phagocytosis of old erythrocytes for the recycling of iron, the induction of the adaptive immune response, and the destruction of pathogens by numerous tissue-resident macrophages fixed into the walls of the sinusoids of the organ (Mebius and Kraal, 2005). As blood flows through this area, foreign antigens, blood-borne pathogens, and debris are removed by these macrophages (which undergo many rounds of phagocytosis) in an extremely effective manner (Cesta, 2006; Fox, 2011). Therefore, it seems likely that more comprehensive OC-correlated immunostimulation, via activation of more phagocytes (increase % phagocytosis) and increase in individual macrophage phagocytic activity (MFI), would manifest in splenic tissue, a site abundant in continuously activated phagocytes. Overall, resident splenic phagocytes had a lower MFI compared to those in circulation, possibly because large quantities of phagocytes in circulation are likely naïve when they encounter an antigen. This enables them to respond more robustly compared to resident splenic phagocytes, which are chronically undergoing rounds of phagocytosis. Therefore, their capacity to ingest new material (measured by MFI levels) are higher than those of the spleen, which suggests a tissue-specific phagocytic capacity. Interestingly, the blood from mainland rays had an increased proportion of cells that were phagocytic (% phagocytosis, Fig. 3A), but the average number of targets ingested per cell, measured by MFI, was similar to blood cells from Catalina rays (Fig. 3D). When administered an antigenic target, the naïve phagocytes in circulation may quickly reach a maximum individual phagocytic capacity (MFI, Fig. 3D). Thus, an OC-correlated effect may only be manifested in the stimulation of new, previously inactivated blood phagocytes (% phagocytosis, Fig. 3A). Since resident splenic phagocytes undergo chronic stimulation, they may not be reaching their maximum level of phagocytic capability, so an OC-correlated effect may be able to increase their tissue-specific phagocytic abilities, as well as activate new splenic phagocytes. The outlier ray used in the models was very interesting, since it P P had the highest PCB (7442.9 ng/g ww) and pesticide loads (1314.6 ng/g ww) compared to all other sampled rays, yet, had a splenic MFI and % phagocytosis similar to rays with low OC loads. While it is possible this outlier ray had impaired splenic phagocytic function due to some other unknown cause, no other metrics suggested this animal differed from the other sampled rays. When removed from the models, only a PCB correlation was found to be significantly related with immune response. The model suggests that perhaps an individual's pesticide load must reach a certain level in order to indirectly or directly influence immune response in a specific tissue. Furthermore, perhaps an OC-correlated effect

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triggers immunostimulation, until a certain threshold is reached, from which immunosuppression then follows. Rehana and Rao (1992) found similar immunomodulation was indeed dosedependent. Swiss albino mice administered feed containing DDT concentrations of 0.032 mg/kg for 4e16 weeks showed no significant alternations in the primary IgM plaque-forming cell response to sheep red blood cells and lymphoproliferative response. An increased dose of 0.32 and 3.2 mg/kg for 4e16 weeks, however, elicited immunostimulation, which was then followed by drastic immunosuppression up to 24 weeks. To support this dosedependent effect in round stingrays, a larger sample size of older rays, with comparably high contaminant loads as the outlier, would need to be analyzed. The most prevalent PCB congeners detected in our sampled rays, PCB 118, 153, 138, 180, are classified as non-coplanar and are nonAhR-active, with the exception of mono-ortho PCB 118, which displays minimal AhR activity (Van den Berg et al., 2006). Despite their dissociation with the AhR pathway, studies have shown noncoplanars do indeed elicit immunotoxic effects. PCB 138, 153, and 180 were documented to significantly reduce phagocytosis of peripheral blood granular and non-granular leukocytes in vitro in bottlenose dolphins (Tursiops truncatus) and beluga whales (Delphinapterus leucas) (Levin et al., 2004). Furthermore, an additive effect was observed when multiple non-coplanars were administered together. Levin et al. (2005) demonstrated causative OC induced modulation of neutrophil and monocyte phagocytosis in dose response studies for seven marine mammal species, ranging from cetaceans, to pinnipeds, to otters. Non-coplanars PCB 138, 153, 180, coplanar PCB 169, and TCDD were administered in different mixtures, which resulted in synergistic or antagonistic interactions depending on species and cell type. For all species tested, monocyte phagocytosis was significantly modulated only by mixtures that contained at least two non-coplanar PCBs, while PCB 169 and TCDD, individually or as the mixture of the two alone, did not affect monocyte phagocytosis in any species. The exact immune effects correlated with OCs are difficult to predict due to high species, tissue, and cell specificity. Moreover, differences in affinities for individual PCBs and pesticides to their receptor (s) and the existence of different receptor (s) for particular congeners/isomers plays a significant role in the magnitude and variation of OC-correlated immunotoxicity. One study found a decreased generation of leukotriene B4, an inflammatory mediator that recruits neutrophils to regions of tissue damage and promotes the production of inflammatory cytokines, when human neutrophils were pretreated with PCB (3,30 ,4,40 -tetrachlorobiphenyl) or simultaneously incubated with sodium fluoride and PCB (Raulf and €nig, 1991). Interestingly, in the same study, an increase in Ko leukotriene B4 formation was reported when neutrophils were pretreated with sodium fluoride and then exposed to this same PCB congener. Thus, the manner in which OCs inhibit or augment immune cell function is dependent on a complex array of biochemical stimuli and the timing in which those stimuli are initiated. Despite the strong correlation between OC levels and immunostimulation, stressors associated with the mainland, such as extensive microbial contamination, may be causing or acting synergistically with OCs to elicit the observed effects. The SCB is subjected to an influx of opportunistic pathogenic bacteria, including Escherichia coli, enterococci, and Vibrio species, particularly during storm events. During such instances, 90% of sites near urban runoff outlets fail State of California water quality standards for total coliforms, fecal coliforms and enterococci (Noble et al., 2003). Such bacterial challenges could therefore be responsible for modulation of the immune system. Regardless of causative agent, enhancement of phagocytosis can lead to the premature release of cytosolic lysosomes and reactive oxygen species, resulting in inflammation

Please cite this article in press as: Sawyna, J.M., et al., Association between chronic organochlorine exposure and immunotoxicity in the round stingray (Urobatis halleri), Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.12.019

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J.M. Sawyna et al. / Environmental Pollution xxx (2016) 1e9

and tissue damage. Furthermore, chronic immunostimulation can elicit hypersensitivity reactions, autoimmune disease, and inflammatory disease (Blakley et al., 1999; Levin et al., 2005). The impact immunostimulation has on the mainland population of round stingrays remains difficult to determine. While no population assessment has been documented to date, annual lifeguard reports of human injuries related to round stingrays showed a steady increase from 1997 to 2007, suggesting a historically stable mainland ray population (Beck et al., unpubl. data). With that said, it is possible the population suffered a decline due to hindered immune performance several decades ago when environmental contaminant concentrations were higher. Round stingrays, along with other local shark species harboring high contaminant loads, such as white shark (Carchardon carcharis), shortfin mako (Isurus oxyrinchus), and Pacific angel shark (Squatina californica), may still be at risk to normally non-threatening diseases if their immune systems are operating at sub-optimal levels. Such risk could be further exasperated by warmer sea temperatures associated with El ~ o events, which could trigger aggressive proliferation of bacteNin ria (Long et al., 2005). This is especially of concern for species, such as shortfin mako, that are IUCN Red listed as ‘Vulnerable’ because of targeted and by catch associated fishing pressure (Lyons et al., 2013; Lyons and Lowe, 2015). It is well documented that stress induces immune dysregulation, ranging from delayed wound healing, susceptibility to disease, to increased occurrence of cancer (Godbout and Glaser, 2006). Compounded with an already taxed immune system potentially from OCs and an increased susceptibility to infection, the likelihood of survival during or after a stressful capture event is greatly diminished, and could ultimately lead to long-term population declines. It is clear, identification of the molecular pathways involving binding site/receptor (s) for specific OC compounds needs elucidation to fully understand OC effects and interactions. To provide direct evidence of OC toxicity, future long-term dose-response studies are recommended, that incorporate multiple elasmobranch tissues and include the PCB congeners:118, 153, 138, 180; those found to be most prevalent in rays exhibiting an altered immune response in this study. Due to its high abundance in coastal waters, the round stingray may serve as a useful sentinel species for understanding the effects of contaminants on marine life. Moreover, because its life history, behavior, and ecology have been well studied, it may also serve as an ideal model for understanding the physiological mechanisms by which contaminants affect lower vertebrates. Acknowledgements The authors thank Southern California Marine Institute staff and volunteers for assistance in the collection of animals. We are also grateful to Arthur Barraza and Varenka Lorenzi (IIRMES) for performing contaminant analyses. Many thanks to Marika Gotschall, Sachin Neupane, and Brianna Maloney for their laboratory support, Minh-Minh Ho for her artwork, and Connor White for his statistical advice. This work was supported by funding from SCTC Marine Biology Foundation, CSULB Undergraduate Research Opportunities Program, and Donald J. Reish Research Grant. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2016.12.019. References Addison, R., Brodie, P., 1987. Transfer of organochlorine residues from blubber

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Please cite this article in press as: Sawyna, J.M., et al., Association between chronic organochlorine exposure and immunotoxicity in the round stingray (Urobatis halleri), Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.12.019