2775 Environmental Toxicology and Chemistry, Vol. 22, No. 11, pp. 2775–2782, 2003 Printed in the USA 0730-7268/04 $12.00 + .00 USING REPRODUCTIVE ENDPOINTS IN SMALL FORAGE FISH SPECIES TO EVALUATE THE EFFECTS OF ATHABASCA OIL SANDS ACTIVITIES GERALD R. TETREAULT,*²‡ M ARK E. MCMASTER,‡ D. GEORGE DIXON,² and J OANNE L. PARROTT‡ ²University of Waterloo, Department of Biology, Waterloo, Ontario N2L 3G1, Canada ‡National Water Research Institute, Environment Canada, Canada Center for Inland Waters, Burlington, Ontario L7R 4A6 ( Received 7 January 2003; Accepted 27 March 2003) Abstract—The main objective of this study was to evaluate the influence of naturally occurring oil sands–related compounds (OSRC) on reproductive function in fish in order to assess the impacts of anthropogenic point-source inputs. The health of slimy sculpin (Cottus cognatus) and pearl dace (Semotilus margarita) collected from the Alberta Athabasca Oil Sands (Canada) watershed were examined. Two rivers were selected for study: the Steepbank and the Ells. These rivers originate outside the oil sands formation, where fish are unexposed (Ref), exposed to naturally occurring oil sands–related compounds (Nat), or exposed to naturally occurring compounds as well as adjacent to surface mining activity (Dev). Assessment endpoints included gonadosomatic indices (GSI), fecundity, and in vitro gonadal steroid production. In vitro gonadal incubations demonstrated lower levels of steroid production at sites along the Steepbank River within the oil sands deposit. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity, an indicator of exposure to OSRC, was elevated twofold at the site with natural compounds and up to 10-fold at the site adjacent to development compared to EROD activity in fish from the reference site. Fish collected in the Ells River had a threefold induction in EROD activity but no significant reduction in steroid production when compared to reference fish. No consistent alterations in gonadal development were seen in fish collected from sites within the oil sands deposit. This research in the Athabasca River basin provides baseline information of the health of fish populations within the oil sands deposit prior to further development in the area. Keywords—Oil sands Forage fish Endocrine disruption Monitoring INTRODUCTION The Athabasca oil sands deposit in northern Alberta (Can- ada) covers more than 42,000 km 2 and represents the largest of the four oil sands areas in Alberta [1]. Syncrude Canada Ltd. and Suncor Oil Sands Group Inc. conduct surface mining and extraction of bitumen adjacent to the Athabasca River, downstream of Fort McMurray. Organic naphthenic acids (NA) and polycyclic aromatic hydrocarbon (PAH) compounds derived from bitumen occur naturally within the Athabasca basin at measurable concentrations [2]. The main chemical constituents in semipermeable membrane device (SPMD) di- alysates from surface water sites in the oil sands area were aromatic PAHs, alkyl-PAHs, naphthenic acids, benzothio- phenes, and methyl carbozles [2]. Measurable levels of 7- ethoxyresorufin-O-deethylase (EROD) activity in fish cell lines on exposure to SPMD extracts deployed at sites within the oil sands deposit suggest that fish are exposed to naturally oc- curring oil sands–related compounds (OSRC) that are capable of inducing mixed function oxygenase enzyme (MFO) activity [2]. An assessment of the contribution of hydrocarbon contam- ination released to northern rivers from natural oil sands de- posits quantified levels of PAHs and their alkylated analogues in sediments taken from tributaries of the Athabasca River [3]. In general, PAH levels in the Ells and Steepbank Rivers sed- iments ranged from 0.01 to 1.13 g/g. Phenanthrene, pyrene, and chrysene were found at high concentrations in the Ells River sediment, while acenaphthene and benzo[ghi]-perylene were detected in Steepbank River sediments [3,4]. Previous studies of fish exposed to fossil fuel–contaminated * To whom correspondence may be addressed (gerald.tetreault@ec.gc.ca). sediments have demonstrated reduced ovarian growth accom- panied by a significant decline in circulating 17-estradiol [5,6], testosterone, and 11-ketotestosterone [7,8] in Atlantic croaker (Micropogonias undulates). A few studies have also evaluated reproductive function in freshwater fish species ex- posed to PAH-contaminated sediments and waters [9–12]. Mc- Master et al. [9] documented reduced plasma testosterone and 11-ketotestosterone levels in male brown bullhead (Ictalurus nebulosus) exposed to PAH-contaminated sediments. In a study conducted within the oils sands area, significant reduc- tions in reproductive sex steroid levels were seen in yellow perch (Perca flavescens) captured from reference sites and exposed to fine-tailings wastewater from the oils sands activity [11,12]. However, these reductions in circulating sex steroid concentrations were not accompanied by corresponding re- ductions in gonadal development. The objective of this study was to evaluate the potential of OSRC (both naturally occurring and those released because of mining activity) to alter reproductive endocrine function in wild fish from the Athabasca oil sands area. Fish were collected from unexposed reference sites, sites exposed OSRC through natural weathering, and sites adjacent to potential anthropo- genic inputs associated with mining activity. Impacts on fish were assessed in terms of changes in condition factor, gona- dosomatic index (GSI), liver somatic index, fecundity, in vitro gonadal steroid productive capacity, and hepatic EROD activ- ity. A unique feature of this project is its use of small-bodied sentinel fish species (slimy sculpin, Cottus cognatus, and pearl dace, Semotilus margarita). The in vitro gonadal incubation procedure has been used in small-bodied fish (such as the fathead minnow, Pimephales pormelas [13]; spoonhead scul- pin, Cottus ricei [14]; trout-perch, Perscopsis omiscomaycus [15]; and the mummichog, Fundulus heteroclitus [16]) with