Data obtained from various government and industry sources has been graphed, and significant trends in average concentrations of dissolved chloride, dissolved sodium, and sulphate both across the Athabasca River and over time (1966–2006) were computed 8. Using this data, we know that the highest average concentrations (across the years 1996–2006) of these parameters in the Athabasca River are 21.09 mg/L dissolved sodium, 22.71 mg/L dissolved chloride, and 45.27 mg/L sulphate. The concentrations of dissolved sodium and dissolved chloride in the 100% sodium chloride treatment (25.43 and 38.90 mg/L, respectively) and the concentration of sulphate in the 100% treatment of the sulphate experiment (101.37 mg/L) were chosen to imitate these levels (Table 1). Although the concentrations of dissolved sodium and chloride used in the present study were slightly higher than was seen in the Athabasca River by Squires et al. 8, they are still representative of the average concentrations of these parameters in the Athabasca River. The concentration of sulphate in the 100%, however, was approximately twice the amount of average sulphate measured in the Athabasca River. The concentration of sulphate in the 50% treatment (57.13 mg/L) was much closer to the average concentrations; therefore, these levels are still representative of the actually in-river concentrations of these parameters.
We also observed an unrepresentative spike in total organic carbon to 14.80 mg/L in the 12.5% treatment in the dissolved sodium experiment. However, because this is based on a single measurement, it is unlikely to be representative of the average conditions in this treatment during the entire experiment; therefore, it is not considered to be of particular concern. This is further realized when this level is outside of the averaged levels seen throughout all treatments in the mouth water experiment. There was an increase in TOC in the 100% treatment of the dissolved sulphate experiment to 6.90 mg/L. While this was also based on a single measurement, unlike the spike seen in the 12.5% treatment in the dissolved sodium, this is still within the averaged range seen in the mouth-water treatments and could therefore reasonably be considered representative of average conditions for this experiment.
Fish reproduction and gill histology
NaCl experiment. All treatments except the 12.5% treatment had higher cumulative egg production than the 0% control, although not statistically significant. This result is similar to what is seen in the mean/eggs/female/d endpoint, where all treatments had higher egg production than the control except for the 25% treatment. Low levels of salinity are often added in aquaculture practices to help reduce fish stress (due to handling, crowding, etc.) 22. It is known that the toxicity of other freshwater constituents (such as ammonia and nitrate) depends on salinity levels, decreasing with higher salinity 23. The induction of anti-stress reactions in algae, amphipods, and fish includes the induction of certain stress proteins, which can induce multiple stress resistance 24. At the lowest concentrations used in the present study (6.25%), it is possible that we are seeing this protective effect.
In the sodium chloride experiment, the only negative percentage of change from control for total eggs/female/day was found in the 6.25 (3.67 mg/L) and 12.5% (3.23 mg/L) treatments. This result differs from the cumulative eggs/female and the mean eggs/female/day endpoints, where increases in egg production occurred at these levels. Total eggs/female/day was calculated by totaling the number of eggs each replicate breeding trio produced across the entire exposure period, then dividing this by the number of days of exposure (21) and the number of females per treatment (two per replicate, three replicates per treatment, equalling six females per treatment). Because this is a total number and is based on 21 d of exposure, we cannot necessarily extrapolate this value past the 21 d of this specific experiment. The cumulative and mean endpoints are not static totals and instead illustrate the general trend of reproductive output during the experiment. This is considered more realistic of what can be expected in a natural population and can predict the potential population-level effects of these constituents more effectively.
Gills are the major site of respiration and are in close, constant contact with the water. As such, they are particularly vulnerable to contaminants such as salinity 25. In similarly conducted experiments in the laboratory using dissolved sodium, the SLW/SLL was found to be significantly higher than the 0% control in the 6.25% (30.33 mg/L) and the 100% (88 mg/L) treatments 15. In these same experiments, the greatest decreases in the percentage of change from control for total eggs/female/d in the laboratory dissolved sodium experiment was also in the 6.25 and 100% treatments. These results demonstrate a possible link between increasing gill diffusion distance and decreasing reproductive output in the fathead minnow.
In the present study, there was a significant increase in SLW/SLL in the 12.5% (3.23 mg/L Na) treatment (p < 0.001), which may have contributed to a non significant decrease in reproductive ability as measured by cumulative egg production. If the duration of the exposure period in this experiment had been increased past 21 d, it is possible that the decrease in cumulative egg production in the 12.5% treatment would become significant and reflect the changes seen in the gill epithelium as has been demonstrated by laboratory studies 15.
The greatest increases in reproductive output (cumulative eggs/female, mean eggs/female/day, and percentage of change from control for total eggs/female/day) in the sodium chloride experiment occurred in the highest treatments (50 and 100%). At these higher concentrations, it is possible that we are seeing a different type of salinity effect. A study conducted by Cataldi et al. 26 tested whether the presence of certain levels of salinity (freshwater, iso-osmotic water, and seawater) would affect the osmolarity capabilities of fish after being exposed to stress. They found that fish held in freshwater had a significant decrease in serum osmolality, whereas fish held in seawater had a significant increase in serum osmolality.
In the present study, the 100% treatment had a significantly larger diffusion distance (SLW/SLL). Although not significant, however, the 100% treatment also had a greater cumulative egg production than the control and a positive change from control for total eggs/female/day. In this treatment, it is possible that despite the larger diffusion distance, the increase in osmolality, which occurs with exposure to higher levels of salinity over a longer period of time (such as the 21-d exposure period used in the present study), could have reduced stress, allowing fish to allot more energy to reproduction.
Additionally, the dissolved chloride concentration in the 100% treatment was 38.90 mg/L. In previous laboratory studies 15, the ideal amount of dissolved chloride for fish reproduction was found to be between 22.22 and 49.56 mg/L. The only treatment to fall within this range in the present experiment was the 100% treatment. It is possible that the effects of the presence of this higher level of dissolved chloride (through competitive binding) would offset the effects of dissolved sodium, allowing for an increase in reproduction ion this treatment.
The presence of turbidity and/or organic carbon in water collected from the main stem of the Athabasca River downstream of the oil sands operation and its potential effect on fathead minnow reproduction has not been studied previously. There was an increase of 175% in the 50% treatment (8.20 mg/L TOC). The highest level of mean egg production was significantly greater (p < 0.001) than the 0% control treatment and occurred in the 50% mouth-water treatment. The 50% mouth-water treatment also had the highest level of turbidity at 5.58 NTU. Turbidity consists of many aspects including sediment, algal cells, dissolved humic substances, dissolved minerals, and detrital organic matter 27. Humic substances are very complex organic molecules that can make up to most (80%) of the dissolved organic matter present in freshwater ecosystems 28. The presence of humic substances can evoke anti-stress reactions in exposed organisms in their effort to try to remove these easily accumulated substances 24. Low levels of humic substances have been shown to induce phase I and II enzymes, providing a protective effect; in the case of some amphipods, it also increases the number of offspring produced 24. Therefore, exposure to low concentrations of humic substances can help train the defense system and lead to stress resistance, thereby allowing for more energy to be diverted to other activities such as reproduction.
Located immediately upstream of the collection site for the mouth water used in the present study are several large oil sands mining and extraction sites. A study conducted by Tetreault et al. 29 sampled two small forage fish species (slimy sculpin and pearl dace) from several sites along the Athabasca River near the oil sands operations and compared results with fish sampled upstream of the oil sands operations. No significant differences in length, weight, condition factor, LSI, or GSI were found in either species. However, some differences in levels of steroid production at sites downstream of the oil sands operations were demonstrated. A more recent study by Kavanagh et al. 5 assessed the potential of aged OSPW on fathead minnow reproduction. These authors demonstrated a significant decrease in cumulative number of eggs for fathead minnows exposed to aged OSPW compared to reference water. The Kavanagh study also demonstrated a decrease in plasma steroid levels (testosterone, 11-ketotestosterone, and 17β-estradiol) in some cases. These studies focused on the impact of aged OSPW, which is currently held on site and not released into the Athabasca River adjacent to the oil sands operations.
Presently, no study has yet assessed the impact of Athabasca River water downstream of the oil sands operations on the reproductive potential of the fathead minnow. In the present study, a negative percentage of change from control for total eggs/female/d was seen in the 25% treatment, and a positive percentage of change from control was seen in the 50% treatment. It is unclear from the present study what factors might be attributed to these different changes. However, due to the complex nature of the oil sands process water and the natural abundance of oil sands in the surrounding area, it is important to clarify the potential impacts of actual in-river concentrations of oil sands related water quality parameters on the reproduction of local fish species.
A study conducted using aged process-affected water from the oil sands operations near the mouth of the Athabasca River showed that although the water was not acutely toxic to yellow perch or goldfish, there were impacts indicated on gills 30. These included changes in mucous cell proliferation; the consequence of these changes was an increase in the distance for gas exchange along the secondary lamellae, potentially reducing the efficiency of gas exchange. In contrast, all treatments in the present study had significantly lower diffusion distances (SLW/SLL) than the 0% control. This result would imply that increasing amounts of mouth water have a positive effect (lower diffusion distance) on the gill epithelium. One reason for this could be that the ionic potential of the mouth water is closer to equilibrium with the fish versus the ionic potential of the headwater (0% control). A greater difference in electric potential causes increase stress and damage to the animal due to the need to allocate greater energy to osmoregulation 31, 27. This is further reflected in the cumulative egg production, where all treatments (except the 25%) had higher cumulative egg production than the 0% control. This trend is also similar to what was observed in the laboratory experiments conducted with dissolved sodium, in which a link between decreased diffusion distance and increased reproductive output was demonstrated 15. Because the trends in cumulative egg production in the present study mirrored those seen in this previous laboratory work, it can be reasonably assumed that if the duration of exposure was increased, these trends may become significant.
The primary role of the gill epithelium in aquatic organisms is to support life through gas exchange, acid-base regulation, nitrogenous waste excretion, immunity, and ion transport 31. The fish gills are considered one of the most sensitive organs to external pollution exposure due to their direct contact with the water. As such, several methods have been developed to assess morphological and physical changes to this area 22. Previous research has shown a link between decreasing diffusion distance (SLW/SLL) and increasing reproductive output and has demonstrated that changes in the gill functions can impact the reproductive performance of aquatic biota 15.
There were higher levels of TOC in the 12.5 (4.27 mg/L), 25 (4.80 mg/L), 50 (8.20 mg/L), and 100% (10.73 mg/L) treatments, which can also contribute to the trends in gill damage and consequently reproductive output. Organic matter has been shown to influence the flux of Na+ across gills of freshwater organisms such as Daphnia and fish 31. In past decades, growing recognition that organic matter can affect the physiology of organisms through several mechanisms including activation of glutathione S-transferase, induction of heat shock proteins, and CYP1A enzymes, as well as changes in behavior 31. The presence of organic matter at concentrations of 10 mg/L can also alter the fundamental physiological properties of fish gills by hyperpolarizing gill membranes 31. This happens when humic substances complex with biologically active ions, such as free Ca2 + , from the water. Free Ca2+ is an important constituent of epithelial tight junctions, and reductions in this ion to very low levels are known to cause a general increase in diffusive ion losses, as well as selectively enhance the permeability of the gills to Na+ relative to Cl–, resulting in hyperpolarization 31, 32.
There was no difference among treatments for cumulative eggs/female, total eggs/female/d and mean eggs/female/d in the dissolved sulphate experiment compared to the 0% control. However, at the end of the experiment (day 21 of exposure), all treatments had lower cumulative egg production than the 0% control. Although not significant, there was also an increase in mean egg production in the 25% treatment in the dissolved sulphate experiment.
There have been a few studies on the toxicity of sulphate in the aquatic environment. The water flea, Hyalella azteca, was found to have an LC50 of 512 mg/L SO4 33. Toxicity associated with excess SO4 can be related to indirect effects on calcium availability rather than direct impacts from SO4 11. The toxicity of SO4 to aquatic biota was found to decrease with increasing hardness and chloride concentrations 33. The presence of calcium and chloride can aid in the biota's ability to osmoregulate and therefore tolerate higher ionic solutions 33, 11.
The province of British Columbia, Canada has established a water quality guideline to protect aquatic life for sulphate of 100 mg/L 34. In the dissolved sulphate experiment, three treatments had lower percentage of change from control for total eggs/female/d than the control, that is, 6.25 (18.57 mg/L), 12.5 (23.33 mg/L) and 100% (101.37 mg/L). The highest of these treatments had a concentration comparable to this guideline set by British Columbia. This guideline was based on the acute toxicity data generated from several species of invertebrates, fish, algae, moss and amphibians with a twofold safety margin. Our results are based on chronic exposures (21 d) to sublethal levels of sulphate to assess the impacts on the reproductive output on fathead minnows. Because this water quality guideline is based on short-term exposures (<24 h) on lower trophic-level species (C. dubia), it is unlikely that our lowest concentration in which an effect is observed would correlate with this guideline value. It would therefore be worthwhile to conduct further studies on the sublethal effects of sulphate on the population of higher order species.
In the dissolved sulphate experiment, there was a significant increase in BET in the 50% treatment (p = 0.039) and significant decrease in SLW/SLL in the 6.25, 12.5, and 100% treatments (p < 0.05) compared with the 0% control. Both BET and SLW/SLL are measures of gill epithelium thickness. Decreases in either of these measurements correspond to decreases in the diffusion distance across the gill. These treatments also all had a negative change from the control for total eggs/female/d. Therefore, we saw decreased gill diffusion distances in the treatments that had the lowest cumulative egg production. This is opposite from what was observed in the sodium chloride experiment in the present study, as well as was observed in previous laboratory studies 15.
One explanation for these conflicting results is the presence of higher turbidity in the 6.25 (5.13 NTU) and 12.5% (5.07 NTU) treatments and higher TOC (6.90 mg/L) in the 100% treatment relative to the rest of the treatments in the present study (Table 1). While we saw increased reproduction of fathead minnow likely in response to increased levels of TOC and turbidity in the mouth-water experiment, there are thresholds beyond which adverse effects on fish populations are observed. It is possible that the presence of higher levels of organic matter in these treatments can have a negative effect on fish reproduction.
The reproductive success of salmonid fish are known to be especially sensitive to suspended organic matter since matter depositing on the stream beds will block pores in the gravel, which are ideal for depositing eggs 27. In general, though, high enough levels of suspended organic matter are known to clog fish gills, which decreases the ability of the fish for oxygen exchange and osmoregulation 35. Also, suspended sediments can cause stress by suppressing the immune system, which can then lead to susceptibility to disease 27. Therefore, sublethal thresholds that account for the effects of turbidity on reproductive potential are necessary and important when considering the impacts of contaminants on the aquatic ecosystem.