Oncorhynchus mykiss (rainbow trout) eyed-eggs were purchased from TroutLodge Ltd. (Bonney Lake, WA, USA) and reared at the Aquatic Toxicology Research Facility (ATRF), Toxicology Centre, University of Saskatchewan. Once the larvae hatched, they were transferred to 20-L tanks in an environmental chamber in the Toxicology Centre. The temperature in the environmental chamber was 15 + 1 ºC, with a photoperiod of 16 h light: 8 h dark (light intensity was approximately 400 lux). A ratio of 70% bio-filtered, C-filtered municipal Saskatoon city water (DeCl), and 30% reverse osmosis (RO) water served as culture water. This ratio was slightly adjusted periodically (DeCl:RO ratio modified by less than 25%) to cope with small seasonal changes in the chemistry of the source water. This culture water was used to simulate Athabasca River water (ARW). The simulated ARW was prepared in 50-L plastic carboys and aerated for at least 24 h before being used in culturing or for toxicity tests to remove any chlorine from the water. The ranges in the water chemistry variables between the ARW and the OSPW (Table 3.1) chosen for investigation were based on previous research conducted on D. pulex by Gillio Meina et al. (2019; Chapter 2). Briefly, the ions of interest were added to the simulated ARW to obtain the ranges in water chemistry conditions for acclimation and toxicity tests using their specific salts
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(Table A1-A2). When the effect of pH on V toxicity was tested, glacial-acetic acid (C2H4O2) or
sodium hydroxide (NaOH) was used to reach the desired pH. The pH in the fish aquaria was maintained at 7.5 – 8.5, except for two groups of larvae that were adjusted to pH 6 and pH 9. Fish larvae were acclimated (without V) until they reached the swim-up stage (approximately 12 d). During acclimation and through the toxicity test, larvae were not fed, following Environment and Climate Change (ECCC) protocols (ECCC, 1998). Water changes were periodically conducted to maintain low levels of ammonia (<0.02 mg/L) and high dissolved oxygen (≥ 8.0 mg/L). The V stock solutions were prepared the day before a toxicity test began in 50-L plastic carboys and then diluted with culture water to reach the desired V concentrations via serial dilution (a factor of two was used). Fish holding and testing was done in accordance to Animal Use Protocol #20140036 (University of Saskatchewan).
3.3.3. Toxicity testing procedures
The O. mykiss toxicity tests were initiated when more than 50% of the larvae reached the swim-up stage. Following (ECCC, 1990b) protocols and the recent work of Schiffer and Liber (2017), O. mykiss larvae were exposed for 96 h to a control (untreated) and four geometrically increasing concentrations of V ranging from 2.18 to 35 mg V /L, with five replicates per level and 10 larvae per experimental unit. The pH of the test water was adjusted before each test initiation Table 3.1: Nominal test levels for water chemistry variables and major ions (mg/L) representative of the Athabasca River and oil sands process-affected water (OSPW) used in acute toxicity tests.
Test /Variable Salt used Nominal levels
pH - 6-7-8-9
Alkalinity NaHCO3 70-100-200-300
Na CHO2Na 100-200-300-400
SO4 MgSO4 100-200-300-380
Numbers in bold indicate the water chemistry of the in-house prepared Athabasca River water that corresponds to approximate environmental concentrations.
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with C2H4O2 or NaOH to minimize unintended confounding effects of pH on the influence of an
ETMF on V toxicity. Test vessels consisted of 4-L acid-washed glass jars with polypropylene lids. Except when the effect of pH on V toxicity was tested, a homemade biofilter containing cotton batten imbibed with Stability® water conditioner (Seachem Laboratories, Madison, GA, USA) and an internal air stone was installed inside the jars to help control ammonia levels (Figure B1). The jars were filled to maximum capacity and tightly sealed to reduce the presence of air bubbles and avoid pH drift. When the influence of pH was tested, the biofilter was installed without aeration, because bubbling was accidentally creating an overhead space between the lid and the test water. Mortality was defined as a lack of movement following gentle prodding and was recorded at 96 h. A test was considered acceptable when mortality in the control group was < 10%. All tests met the requirements outlined earlier.
3.3.4. Chemical analysis
Water samples (20 mL) were randomly collected from each treatment at the beginning and end of the test (96 h) in triplicates for analysis of temperature, dissolved oxygen (DO), pH, alkalinity, water hardness, and ammonia (Table B1). Measurements of pH were recorded using an OPRIM PerpHect Log R meter model 370. Temperature and DO concentrations were measured using an ORION DO meter model 835 (ORION Research, Beverly, MA, USA). Alkalinity and hardness were measured using a Hach digital titrator model 16900 (Hach Company, Loveland, CO, USA). Ammonia was measured using an Orion Aquafast®II meter (Thermo Electron, Waltham, MA, USA).
Before any major ion and V analyses were performed, samples were filtered using a syringe filter with a 0.45-µm polyethersulfone membrane (VWR International, Mississauga, ON, Canada).
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Additionally, samples for V analysis were acidified to 2% acid with high purity HNO3 (Fisher
Scientific, Ottawa, ON, Canada). The dissolved concentrations of Na and SO4 were measured in-
house using ion chromatography (Dionex ICS-3000, Sunnyvale, CA, USA). Samples collected for V analysis were measured in-house using an inductively coupled plasma mass spectrometer (ICP- MS) equipped with collision cell technology (Agilent Technologies 8800 ICP-MS Triple Quad, Mississauga, ON, Canada). The limit of detection for V was 0.05 µg V/L. River-water standard reference material (SLRS-5; National Research Council, Ottawa, ON, Canada) and natural water standard reference material (1640a, National Institute of Standard and Technology, Gaithersburg, MD, USA) were used in QA/QC for ICP-MS measurements. The accuracy of SLRS-5 and 1640a analyses were always > 85%, and the precision of duplicates exceeded 90% in all cases.