The effect of oil sands process-affected water and model naphthenic acids on photosynthesis and growth in Emiliania huxleyi and Chlorella vulgaris

(cid:1) Emiliania huxleyi was generally more sensitive than Chlorella vulgaris to surrogate NAs


Introduction
The Athabasca oil sands deposit in Alberta, Canada is one of the largest reservoirs of bitumen in the world, covering an area over 100,000 km 2 .Oil sands mining operations currently generate 1.9 million barrels of oil per day and production is expected to increase to 4.8 million barrels by 2030 (CAPP, 2011).Such large-scale industrial operations inevitably have severe environmental impacts.During oil sands mining, large quantities of oil sands process water (OSPW) are generated which are stored in vast tailings ponds.These ponds contribute to the contamination of local aquatic ecosystems (Headley and McMartin, 2004) and pose a threat to environmental and human health (Siddique et al., 2011).The OSPW hydrocarbons comprise mainly asphaltenes, aromatic compounds (typically high molecular weight), alkanes and naphthenic acids (Whitby, 2010;Strausz et al., 2011).Naphthenic acids (NAs) comprise of mixtures of aliphatic, alicyclic and aromatic carboxylic acids, which demonstrate acute and chronic toxicity to several organisms including fish (Young et al., 2007), plants (Kamaluddin and Zwiazek, 2002;Armstrong et al., 2008), bacteria (Frank et al., 2008;Johnson et al., 2011Johnson et al., , 2012) ) and phytoplankton (Leung et al., 2003).Establishing the environmental impact of NA contamination presents a considerable challenge, since NAs may enter marine and freshwater environments through natural seepages and anthropogenic sources such as discharge from oil refineries and oil spillage events (Brient et al., 1995;Yergeau et al., 2012).
Over the last decade, measurements of chlorophyll fluorescence have become a routine technique for monitoring photosynthetic performance in both higher plants and algae (Baker, 2008).The dark-adapted parameter F V /F M is a measure of the maximum efficiency of photosystem II (PSII) photochemistry.Changes in the value of F V /F M provide a simple and rapid way to monitor abiotic and biotic stress in photosynthetic organisms (Baker, 2008;Murchie and Lawson, 2013).Chlorophyll fluorescence measurements (e.g.F V /F M ) have previously been used in several plant and algal studies investigating the toxicity of heavy metals (Lu et al., 2000) and polycyclic aromatic hydrocarbons (PAHs) (Huang et al., 1997).However, to our knowledge there are no studies that have investigated the effects of OSPW and NAs on F V /F M .
In the present study, the marine alga Emiliania huxleyi and the freshwater alga Chlorella vulgaris were selected as representative phytoplanktonic organisms, since both are biogeographically widespread in their respective environments.The present study aimed to investigate the effects of the acid extractable organic fraction (AEO) of OSPW and individual surrogate NAs, on maximum photosynthetic efficiency of PSII (F V /F M ) and cell growth in E. huxleyi and C. vulgaris.Such information is crucial, as it will provide a better understanding of the physiological responses of phytoplankton to OSPW and NAs, thus enabling improved monitoring of NA pollution in aquatic ecosystems.

Sources of OSPW and NAs
Experiments were conducted with surrogate NAs associated with petroleum acids or OSPW and the AEO fraction of OSPW.Two surrogate NAs used in this study were (4 0 -n-butylphenyl)-4butanoic acid (n-BPBA) and (4 0 -tert-butylphenyl)-4-butanoic acid (tert-BPBA) and were synthesized using a modified Haworth synthesis (Smith et al., 2008).OSPW was collected at a 2 m depth from a Suncor tailings pond (courtesy of L. Gieg, University of Calgary, Canada).The AEO fraction of OSPW was extracted from 1 L OSPW using an ethyl acetate liquideliquid extraction procedure and the total acid concentration determined by GCeMS as described previously (Johnson et al., 2011).NA and AEO stock solutions were prepared using 0.1 M NaOH to final concentrations of 1, 10, 50 or 100 mg L À1 (media pH was adjusted to 7.5 for enrichment solution with artificial water (ESAW) media or 7.1 for BG11 freshwater media immediately following addition).NA concentrations were selected to include the highest concentration generally observed in OSPW (Holowenko et al., 2002).

Media and growth conditions
Stock cultures of E. huxleyi (strain CCMP 370 e a non-coccolith producing strain) and C. vulgaris (strain CCAP/211/12) were obtained from the University of Essex culture collection.Both strains were cultured using axenic practices in low light using cool white fluorescent tubes with a light dark cycle of 14:10 at a photon flux density of 150 mmol m 2 s À1 in a controlled environment growth room (Fitotron PG660, Sanyo).E. huxleyi cultures were grown in 1 L of 0.2 mm filtered ESAW media, pH 7.5 (Berges et al., 2001(Berges et al., , 2004)), and C. vulgaris cultures were grown in 1 L of 0.2 mm filtered BG11 freshwater media, pH 7.1 (Berges et al., 2004).Cultures were incubated at 16 C (within the range for growth of E. huxleyi (https://NCMA.bigelow.org)and C. vulgaris (Nowack et al., 2005;Schluter et al., 2006), for a total of eight days and harvested for experimental treatments during exponential growth.Triplicate 100 mL sterile serum bottles (SigmaeAldrich) containing 75 mL filtered media were inoculated simultaneously with either E. huxleyi or C. vulgaris at an initial cell density of 6 Â 10 4 cells mL À1 .Cells were acclimated to experimental conditions for 24 h prior to the addition of NAs.Day zero measurements were taken immediately prior to NA addition.Control cultures of E. huxleyi and C. vulgaris were inoculated into filtered EASW or BG11 media respectively, containing no NAs.Procedural controls containing 75 mL of 0.1 M NaOH (Fisher Scientific) were also established (with media pH adjusted to 7.5 for ESAW or 7.1 for BG11 immediately after addition).Killed controls for all treatments were prepared by heating cultures of E. huxleyi and C. vulgaris to 60 C for 1 h before NA addition and incubation.

Maximum photosynthetic efficiency (F V /F M ) measurements
Sub-samples (2 mL) were removed daily over the eight day exposure period and dark adapted for 30 min before measuring F V / F M , using a Fast tracka II Fast Repetition Rate Fluorometer with a Fast act system (Chelsea Instruments, Molesey, UK).

Cell abundance and light microscopy
Cell density and cell volume measurements were calculated daily using a Z2 Coulter Particle and Size Analyser (Beckman Coulter, CA, USA).Media blanks were used to account for nonbiological particles in the media.Cell fragments were excluded from coulter counter analysis by including a lower size limit for detection.Growth rates were calculated between days zero and three, during the exponential growth phase of both algae.All cultures were examined by light microscopy on day six using an Olympus BX41 brightfield microscope fitted with a Colorview camera and imaging system (Colorview II).

NA extraction and gas chromatography mass spectrometry analysis
The cultures that demonstrated significant growth were analysed further for NA degradation as follows: sub-samples (15 mL) were removed at day eight and replicates were pooled together in order to obtain sufficient volume for NA extraction.Killed controls were also extracted for comparison.NAs were extracted using ethyl acetate as described previously (Johnson et al., 2011).Samples were analysed on a 7890A GC system connected to a 5975 VL MS (triple axis detector) and a 120 model autosampler (Agilent Technologies).
Samples (1 ml) were injected by splitless injection (270 C injection temperature) onto a 50 m Â 320 m Â 0.52 mm 19091Z-115E column (Agilent Technologies) using helium as the carrier gas.Oven temperature was set at 50 C for 5 min with an increase to 250 C at a rate of 8 C min À1 and a final hold for 15 min.Data was analysed using Chemstation software (Agilent Technologies).

Statistical analysis
Statistical analysis was performed using PASW statistics version 18.0.0.Repeated measures ANOVA was used to determine if significant differences in F V /F M occurred throughout the time course of the experiment.If the assumption of sphericity of the data was violated, a Greenhouse-Geisser correction was applied to produce a more conservative F-statistic by reducing the degrees of freedom.Growth parameters and degradation data were analysed using oneway ANOVA with post hoc Tukey test.

Effect of the AEO fraction of OSPW and surrogate NAs on maximum photosynthetic efficiency (F V /F M )
The F V /F M of E. huxleyi was reduced to zero by day six when incubated with n-BPBA at !10 mg L À1 (Fig. 1a).When incubated with tert-BPBA, greater concentrations (!50 mg L À1 ) were required to cause complete reduction of F V /F M in E. huxleyi (Fig. 1c).In contrast to the surrogate NAs, the AEO fraction did not inhibit F V /F M in E. huxleyi and the F V /F M remained between 0.39 and 0.45 throughout the eight-day incubation period (Fig. 1e).When C. vulgaris cells were incubated with n-BPBA, tert-BPBA or AEO, no significant differences in F V /F M were found in comparison to controls (F 13, 41 ¼ 2.32, p ¼ 0.22).The F V /F M parameter remained within the range of 0.43e0.67 for all treatments.This indicates that the surrogate NAs and the AEO fraction of OSPW had no effect on the maximum photosynthetic efficiency of C. vulgaris up to 100 mg L À1 (Fig. 1b, d and f).The F V /F M for all procedural and killed controls also remained constant throughout (Fig. 1), suggesting that any effects observed were not due to the addition of sodium hydroxide.
Whilst 1 mg L À1 of the AEO fraction of OSPW had no significant impact on E. huxleyi growth, greater concentrations (i.e.!10 mg L À1 ) resulted in significantly increased growth rates (in the range of m ¼ 0.64e0.77)compared to controls (p 0.002 in all cases) (Table 1).Cell abundances for E. huxleyi exposed to !10 mg L À1 of the AEO fraction of OSPW (3.62e5.56Â 10 6 cells mL À1 ) were also significantly greater at day eight than for controls (p < 0.001 in all cases) (Fig. 2e).The growth of the procedural controls was consistent with the no-NA controls throughout, suggesting that any observed effect was not due to addition of sodium hydroxide (Table 1, Fig. 2a, c and e).
Although concentrations of 50 mg L À1 n-BPBA had no significant effect on the growth rate of C. vulgaris, cell densities were significantly reduced (to 9.06 Â 10 6 cells mL À1 ) by day eight with 50 mg L À1 n-BPBA, compared to controls (1.42 Â 10 7 cells mL À1 ) (p 0.002).The growth rate for C. vulgaris cultures incubated with 100 mg L À1 n-BPBA was significantly reduced (m ¼ 0.73) compared to controls (m ¼ 1.15), and cell abundance was almost four-fold lower than controls by day eight (3.60 Â 10 6 cells mL À1 , p < 0.001) (Fig. 2b, Table 2).Whilst the growth rate of C. vulgaris did not appear to be significantly affected by tert-BPBA up to 100 mg L À1 compared to controls, by day eight, cell densities were significantly lower in the cultures incubated with 50 and 100 mg L À1 tert-BPBA, (8.95 Â 10 6 and 5.85 Â 10 6 cells mL À1 respectively, p 0.001 in both cases) (Fig. 2b and d).Exposure to the AEO fraction of OSPW (up to 100 mg L À1 ) had no significant effect on C. vulgaris growth rate or cell density (Fig. 2f, Table 2).Growth from procedural controls was consistent with no-NA controls throughout, suggesting that there was no effect of sodium hydroxide addition (Table 2, Fig. 2b, d and f).
By day eight, E. huxleyi cell volumes differed significantly between treatments (F 13, 41 ¼ 104.69, p < 0.001) (Table 1).Specifically, cells incubated with 50 mg L À1 n-BPBA were significantly larger (94.89 mm 3 ) than controls (82.51 mm 3 ) (p ¼ 0.003) as were cells incubated with 10 and 50 mg L À1 tert-BPBA (94.06e101.56mm 3 ) (p ¼ 0.003).In contrast to n-and tert-BPBA, when E. huxleyi cells were incubated with the AEO fraction of OSPW at !10 mg L À1 , cells were significantly reduced in size (40.95e57.35mm 3 , p < 0.001 in all cases) compared to controls (Table 1).The cell volume of procedural controls was consistent with no-NA controls at day eight, confirming that there was no effect of sodium hydroxide addition on cell volume (Table 1).C. vulgaris cells incubated with !50 mg L À1 n-BPBA, and 100 mg L À1 tert-BPBA had significantly larger cell volumes (between 57.29 and 79.41 mm 3 ) compared to controls (50.57mm 3 ) (p < 0.010 in all cases) (Table 2).The cell volume of C. vulgaris cells was not significantly affected by the AEO fraction of OSPW, (up to 100 mg L À1 ) (Table 2).The cell volume of procedural controls was consistent with no-NA controls at day eight, confirming that there was no effect of NaOH on cell volume (Table 2).

Effect of the AEO fraction of OSPW and surrogate NAs on cell morphology
The effect of the AEO fraction of OSPW and surrogate NAs on cell morphology of E. huxleyi and C. vulgaris was investigated using light microscopy (Fig. 3).When E. huxleyi cells were exposed to 1 mg L À1 n-or tert-BPBA, there was little difference in cell morphology compared to controls (Fig. 3c and e).However, when E. huxleyi cells were exposed to 10 mg L À1 tert-BPBA, cells underwent extensive changes in morphology, becoming irregular in appearance.Cell wall damage was apparent and the appearance of several small, round inclusions inside and around cells was noted (Fig. 3d).It was not possible to image cells incubated with !10 mg L À1 n-BPBA or !50 mg L À1 tert-BPBA due to the toxicity of the NAs resulting in low cell abundances.Image analysis confirmed the observed reduction in the cell size of E. huxleyi when exposed to 100 mg L À1 AEO fraction of OSPW (Fig. 3f).Microscopy analysis also confirmed the presence of larger C. vulgaris cells when incubated with 100 mg L À1 n-and tert-BPBA compared to controls, although no dark inclusions were observed in C. vulgaris cells incubated with n-and tert-BPBA as seen in E. huxleyi (Fig. 3j and k).

Biodegradation of the AEO fraction of OSPW and surrogate NAs
Since there were observed differences in NA sensitivity between E. huxleyi and C. vulgaris, it was hypothesised that this was due to differential biodegradation of the BPBA isomers by the two algae.Therefore, the algal cultures that clearly demonstrated growth were further analysed against killed and abiotic controls to determine whether NA biodegradation had occurred (Supplementary Fig. S1).It was found that whilst C. vulgaris cultures partially degraded n-BPBA (at 1 and 10 mg L À1 ) and tert-BPBA (at 1 mg L À1 ), tert-BPBA (at 10 mg L À1 ) and the AEO fraction of OSPW remained.In contrast, E. huxleyi cultures almost completely removed tert-BPBA (at 1 mg L À1 ) and partially degraded tert-BPBA (at 10 mg L À1 ) but were unable to degrade either n-BPBA (at 1 mg L À1 ) or the AEO fraction of OSPW (Supplementary Fig. S1).All controls demonstrated no abiotic loss of NAs by photodegradation (data not shown).

Discussion
This is the first report to describe the effects of the AEO fraction of OSPW and surrogate NA compounds on maximum photosynthetic efficiency of PSII (F V /F M ) and cell growth in E. huxleyi and C. vulgaris.Such information is important as it provides a better understanding of the physiological responses of photosynthetic microorganisms to NAs and may enable improved monitoring of NA pollution in aquatic ecosystems.
Here, we demonstrated that the marine alga E. huxleyi was highly sensitive to the surrogate NAs n-and tert-BPBA at !10 mg L À1 , in terms of photosynthetic efficiency, cell growth and morphology, compared to the freshwater alga C. vulgaris, which was more tolerant.Differential sensitivity to the two surrogate BPBA isomers was also observed, whereby n-BPBA was generally more toxic than tert-BPBA.Similar findings were previously obtained with n-and tert-butylcyclohexylbutanoic acid isomers using oyster embryos (Smith et al., 2008).In contrast to the results of our study, tert-BPBA was previously shown to be more toxic to a bacterial enrichment culture than n-BPBA (Johnson et al., 2011).It is well known that NA toxicity can be structure specific, with lower molecular weight acids often demonstrating acute toxicity (Holowenko et al., 2002;Frank et al., 2008).Although the exact mechanism of NA toxicity to algae is unknown, NAs are anionic surfactants (Roberts, 1991) and their acute toxicity is thought to be related to these properties.More specifically, NAs acting as surfactants can disrupt the lipid bilayer of membranes and change membrane properties via polar narcosis (Roberts, 1991;Frank et al., 2008).There is also evidence to suggest surfactants interact with and denature cell wall proteins in algae, altering cell permeability and the potential to take in other nutrients and chemicals (Lewis, 1990;Goff et al., 2013).
Although differential sensitivity between algal species may be expected (Fairchild et al., 2009), one may hypothesise that the difference observed herein was due to the ability of C. vulgaris cultures to more readily biodegrade the BPBA isomers to less toxic metabolites compared to E. huxleyi.Indeed, it has been previously shown that biodegradation of the BPBA isomers by a bacterial culture produces ethanoic acid metabolites that are less toxic than the butanoic acid parent compounds (Johnson et al., 2011).In the present study, C. vulgaris partially degraded both n-and tert-BPBA, whilst only tert-BPBA was partially degraded by E. huxleyi.Previous studies have shown that phytoplankton such as Selenastrum sp., Navicula sp. and Dunaliella sp. may also degrade certain NAs (Headley and McMartin, 2004;Quesnel et al., 2011).It was also possible that the surrogate NAs were susceptible to photodegradation under UV light, thus reducing their toxicity (McMartin et al., 2004;Mishra et al., 2010).However, in the present study, relatively low levels of artificial light were used with no UV element and abiotic controls showed that photodegradation had not occurred (data not shown).
In contrast to the toxic effects of surrogate NAs observed herein, the AEO fraction of OSPW at concentrations up to 100 mg L À1 (i.e.within the top range found in tailings ponds) had no impact on F V / F M in either algae species studied.Furthermore, the AEO fraction appeared to have a stimulatory effect on the growth of E. huxleyi (but no apparent effect on C. vulgaris).Whilst ESAW media is well known to support high growth rates in E. huxleyi (Berges et al., 2001(Berges et al., , 2004)), the apparent stimulation of E. huxleyi cells incubated with the AEO fraction of OSPW herein may have been due to the presence of other acid-extractable constituents (Grewer et al., 2010) such as metals and salts, which provided additional nutrients or co-factors for E. huxleyi, but not for C. vulgaris.NAs have previously been shown to have a stimulatory effect on root and shoot growth in Arabidopsis thaliana, which may be due to the broad structural similarity of some NAs to plant growth regulators such as auxins (Leishman et al., 2013).In addition, NAs from OSPW have been shown to stimulate plant growth (as measured by CO 2 uptake) in cattails (Typha latifolia) (Wort, 1976;Bendell-Young et al., 2000).Further work is required to determine whether a direct stimulatory effect of the AEO of OSPW occurs in photosynthetic organisms such as the algae studied herein, or whether other, indirect factors such as increased CO 2 uptake also play a role.
In single celled microorganisms it is not uncommon for changes in cell size to occur in response to stress (Li, 1979;Fisher et al., 1981;Goff et al., 2013).In this study, the presence of both n-and tert-BPBA resulted in an increased cell size for both E. huxleyi and C. vulgaris, compared to controls.It is likely that this increase in cell size was in response to toxic stress, whereby a decrease in surface area to volume ratio reduced NA uptake into the cell.Indeed, previous studies have shown that phytoplankton species with a smaller cell size accumulate higher amounts of contaminants such as atrazine (Tang, 1997) and dichlorodiphenyltrichloroethane (Rice and Sitka, 1973) relative to species with a larger cell size, due in part to their larger surface area to volume ratio.Alternatively, increased cell sizes could be due to arrested cell growth cycle prior to cell division or the cells have increased vacuolization, following NA exposure.A similar increase in cell size to that observed in this study has also been noted in other phytoplankton species in the presence of NAs (Goff et al., 2013) and metal contaminants (Li, 1979;Fisher et al., 1981).
In addition to changes in cell size, E. huxleyi also underwent changes in morphology following exposure to tert-BPBA.Specifically, cells changed from rounded to irregular shape; showed signs of cell wall damage and there was the appearance of several small, round inclusions inside and surrounding cells which may be nuclear fragments resulting from apoptosis.Goff et al. (2013) reported changes to algae morphology following exposure to the NA fraction of OSPW.Specifically, Goff et al. (2013) noted that Chlamydomonas reinhardtii cells experienced increased roundness and increased diameter with exposure to NAs.In addition, Goff et al. (2013) described the formation of palmelloids (groups of cells remaining in the remnants of the mother cell wall) when C. reinhardtii were exposed to OSPW NAs.
Overall, there was a clear and opposite difference in the sensitivity of the two algae towards surrogate NAs (a toxic response was observed) compared to the AEO fraction of OSPW (a stimulatory response was observed), highlighting a need for caution when extrapolating toxicity data from surrogate NAs, as they may be poor predictors of the response to NAs found in OSPW.The marine alga E. huxleyi was highly sensitive to the surrogate NAs, in terms of photosynthetic efficiency, cell growth and morphology, compared to the freshwater alga C. vulgaris, which was more tolerant.This report provides a better understanding of the physiological responses of marine and freshwater phytoplankton to surrogate NAs and the AEO fraction of OSPW and will enable improved monitoring of NA pollution in aquatic ecosystems in the future.

Table 1
Growth rates and cell volumes of Emiliana huxleyi cultures incubated for eight days with NAs.Values represent means of triplicate samples with standard deviation in parentheses.Growth rates (m) were calculated over days 0e3.Stars (*) represent results that are statistically different from no-NA controls (p < 0.05).

Table 2
Growth rates and cell volumes of Chlorella vulgaris cultures incubated for eight days with NAs.Values represent means of triplicate samples with standard deviation in parentheses.Growth rates (m) were calculated over days 0e3.Stars (*) represent results that are statistically different from no-NA controls (p < 0.05).