Aquatic Toxicology 142–143 (2013) 185–194 Contents lists available at ScienceDirect Aquatic Toxicology jou rn al hom ep age: www.elsevier.com/locate/aquatox The immunological effects of oil sands surface waters and naphthenic acids on rainbow trout (Oncorhynchus mykiss) Liane A. Leclair a,∗ , Gillian Z. MacDonald a , Laura J. Phalen a , Bernd Köllner b , Natacha S. Hogan c,d , Michael R. van den Heuvel a a Canadian Rivers Institute, Department of Biology, University of Prince Edward Island, Charlottetown, Canada b Friedrich Loeffler Institute, Federal Research Institute of Animal Health, Institute of Immunology, Greifswald, Germany c Toxicology Centre, University of Saskatchewan, Saskatoon, Canada d Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada a r t i c l e i n f o Article history: Received 15 May 2013 Received in revised form 15 August 2013 Accepted 17 August 2013 Keywords: Oil sands Naphthenic acids Polycyclic aromatic hydrocarbons Fish Immunotoxicity a b s t r a c t There is concern surrounding the immunotoxic potential of naphthenic acids (NAs), a major organic con- stituent in waters influenced by oil sands contamination. To assess the immunological response to NAs, rainbow trout (Oncorhynchus mykiss) waterborne exposures were conducted with oil sands-influenced waters, NAs extracted and purified from oil sands tailings waters, and benzo[a]pyrene (BaP) as a pos- itive control. After a 7 d exposure, blood, spleen, head kidney, and gill samples were removed from a subset of fish in order to evaluate the distribution of thrombocytes, B-lymphocytes, myeloid cells, and T-lymphocytes using fluorescent antibodies specific for those cell types coupled with flow cytometry. The remaining trout in each experimental tank were injected with inactivated Aeromonas salmonicida and held in laboratory water for 21 d and subjected to similar lymphatic cell evaluation in addition to evaluation of antibody production. Fluorescent metabolites in bile as well as liver CYP1A induction were also determined after the 7 and 21 d exposure. Oil sands waters and extracted NAs exposures resulted in an increase in bile fluorescence at phenanthrene wavelengths, though liver CYP1A was not induced in those treatments as it was with the BaP positive control. Trout in the oil sands-influenced water expo- sure showed a decrease in B- and T-lymphocytes in blood as well as B-lymphocytes and myeloid cells in spleen and an increase in B-lymphocytes in head kidney. The extracted NAs exposure showed a decrease in thrombocytes in spleen at 8 mg/L and an increase in T-lymphocytes at 1 mg/L in head kidney after 7 d. There was a significant decrease in antibody production against A. salmonicida in both oil sands- influenced water exposures. Because oil sands-influenced waters affected multiple immune parameters, while extracted NAs impacts were limited, the NAs tested here are likely not the cause of immunotoxicity found in the oil sands-influenced water. © 2013 Elsevier B.V. All rights reserved. 1. Introduction In 2009, Canadian bitumen productionaveraged 1.49 million barrels/day from the oil sands industry and is projected to produce 3.2 million barrels/day by 2019 (Government of Alberta, 2011). The Clarke hot water extraction method used to recover bitumen from oil sands requires about 3 m 3 of water for about 4 m 3 of fluid tailings (Holowenko et al., 2002). Industry is required to safely incorporate resultant tailings and tailings water back into the reclaimed landscape. However, there is concern that substances associated with oil sands mining will enter the wider environment ∗ Corresponding author at: Canadian Rivers Institute, Department of Biology, Uni- versity of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada. Tel.: +19025666072. E-mail address: liane.leclair@gmail.com (L.A. Leclair). and cause detrimental effects on biota such as teleost fish (Kelly et al., 2009). The tailings and wastewaters contain clay, sand, dissolved metals, and organic compounds, including polycyclic aromatic hydrocarbons (PAHs) and naphthenic acid (NAs). NAs are a diverse group of compounds found in petroleum- derived materials such as crude oil. NAs are composed of acyclic, monocyclic, and polycyclic carboxylic acids, with the general for- mula of C n H 2n + z O 2 , where n represents the carbon number and Z specifies the hydrogen deficiency resulting from ring formation or double bonds (Clemente and Fedorak, 2005). Recent research suggests that NAs mixtures contain tricyclic diamondoid acids that differ from what has been generally conceived for NAs structure (Rowland et al., 2011, 2012). The complexity of NAs is further increased as they become transformed in the environment. The more saturated and lower molecular weight NAs tend to be elim- inated entirely through rapid biodegradation, whereas the larger more complex NAs may not be mineralized, but can be oxidized 0166-445X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.08.009