Naphthenic acid mixtures and acid-extractable organics from oil sands

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Juan ManuelSociety. Gutierrez1155 Sixteenth N.W., Washington, Villagomez, KerryStreet M Peru, Connor DC 20036 Published by American


Edington, John V. Headley, Bruce D Pauli, and Vance L. Trudeau is published by the

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Page 1 ofEnvironmental 31 Science & Technology Control Silurana tropicalis larva

Larva exposed to acid Acid Extractible Organics from Oil Sands Process-Affected Water Eye malformation Gut abnormalities

ACS Paragon Plus Environment Face edema

Environmental Science & Technology

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Naphthenic acid mixtures and acid-extractable organics from oil sands process-affected water impair embryonic development of Silurana (Xenopus) tropicalis

5

Juan M. Gutierrez-Villagomez,§ Kerry M. Peru,‡ Connor Edington, § John V. Headley, ‡ Bruce D.

6

Pauli,£ Vance L. Trudeau §*

1 2 3

§

7 ‡

8 9 10

Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.

Watershed Hydrology and Ecology Research Division, Water Science and Technology, Environment and Climate Change Canada, Saskatoon, Saskatchewan, Canada.

£

Ecotoxicology and Wildlife Health Division, Wildlife and Landscape Science Directorate,

11

Science and Technology Branch, Environment and Climate Change Canada, Ottawa, Ontario,

12

Canada.

13 14

KEYWORDS. Naphthenic acids, Acid extractable organics, mixtures, toxicity, Silurana

15

(Xenopus) tropicalis, LC50, EC50, teratogenicity.

16 17

ABSTRACT. Naphthenic acids (NAs) are carboxylic acids naturally occurring in crude oils and

18

bitumen and are suspected to be the primary toxic substances in wastewaters associated with oil

19

refineries and mining of oil sands. Oil sands process-affected water (OSPW) generated by the

20

extraction of bitumen from oil sands are a major source of NAs and are currently stored in

ACS Paragon Plus Environment

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tailings ponds. We report on the acute lethality and teratogenic effects of aquatic exposure of

22

Silurana (Xenopus) tropicalis embryos to commercial NA extracts and from the acid extractable

23

organics (AEOs) fraction of a Canadian OSPW. Using electrospray ionization-high resolution

24

mass spectrometry, we determined that the O2 species proportion were 98.8, 98.9 and 58.6% for

25

commercial mixtures Sigma 1 (S1M) and Sigma 2 (S2M), and AEOs, respectively. The 96h LC50

26

estimates were 10.4, 11.7 and 52.3 mg/L for S1M, S2M, and the AEOs, respectively. The 96h

27

EC50 estimates based on frequencies of developmental abnormalities were 2.1, 2.6 and 14.2

28

mg/L for S1M, S2M, and the AEOs, respectively. The main effects observed were reduced body

29

size, edema, and cranial, heart, gut and ocular abnormalities. Increasing concentrations of the

30

mixtures resulted in increased severity and frequency of abnormalities (p < 0.05). The rank-order

31

potency was S1M > S2M > AEO based on LC50 and EC50 estimates. These data provide insight

32

into the effects NAs in amphibian embryos and can contribute to the development of

33

environmental guidelines for the management of OSPW.

34 35

1. INTRODUCTION.

36

Naphthenic acids (NAs) are carboxylic acid mixtures naturally occurring in crude oil and are

37

classified as contaminants of emerging concern.1 The commercial NA salts are used as wood and

38

fabric preservatives, emulsifiers and surfactants, and paint and ink driers.2 Naphthenic acids have

39

been found in relatively high concentration in some sediments after oil spills.3–5 Considering that

40

there are more than 100 countries that extract oil,6 the potential toxicity that NAs present is of

41

international concern.

42 43

Canada has the largest commercially exploited oil sands deposits in the world.7 The oil sands are non-conventional oil deposits in which bitumen is mixed with sand, clay, and water.


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Approximately 20% of Alberta’s oil sands can be accessed using the surface mining method.8

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The recovery process requires approximately ~2.5 units of water for every 1 unit of bitumen

46

produced.9 This results in a mixture of contaminated water, sand, residual bitumen and various

47

petroleum-based chemicals that are released and retained in nearby tailings ponds.10 The tailings

48

ponds are a storage area for the Oil Sands Process-affected Water (OSPW) that was in contact

49

with bitumen or other contaminants, and additionally dewatered groundwater and storm water

50

runoff. The OSPW contains high concentrations of inorganic salts, heavy metals, polycyclic

51

aromatic compounds (PACs), benzene, toluene, ethylbenzene, and xylene (the so-called BTEX

52

compounds), residual bitumen and NAs.11–13 Some ponds are in close proximity to the Athabasca

53

River,11 raising concerns regarding possible migration of pollutants to the surrounding and

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downstream environments. Frank et al.14 compared NA profiles of samples of groundwater from

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the Athabasca River area and samples of locally produced OSPW and found important

56

similarities, suggesting but not proving that OSPW contaminates groundwater.

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Little is known about the toxicity of NAs nor of the Acid Extractable Organic (AEO) mixtures

58

from OSPW on the development of frogs. Only a few reports regarding the toxicity of NAs in

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frogs have been published focusing on in vitro organotypic cultures15 or potential disruption of

60

metamorphosis.16,17 In our first report, LC50 values of 4.76 mg/L commercial NA preparation

61

(Sigma) were found for tadpoles at an early developmental stage (Gosner stage 28), and even

62

greater toxicity with more developed tadpoles at 96 h, with an LC50 value of 3.04 mg/L in

63

Gosner stage 36 tadpoles.18 We also showed that embryos of Lithobates pipiens and Silurana

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tropicalis exposed to 2-4 mg/l of the Sigma NAs suffered significant reductions (32% and 25%,

65

respectively) in growth and development upon hatching.19 In another study, the same NA

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preparation decreased growth and development, significantly reduced glycogen stores and


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increased triglycerides, indicating disruption to processes associated with energy metabolism and

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hepatic glycolysis.20 None of these studies examined embryogenesis, nor did they asses NAs

69

from OSPW. Vertebrate embryogenesis is probably the most sensitive period to disruption of

70

development21 that can impact long-term phenotypic characteristics essential for survival and

71

population health.22 The aim of this work was to assess the toxicity and potential teratogenic

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effects of the different naphthenic acid mixtures. Toxicity tests were based on measured effects

73

in the Western clawed frog Silurana (Xenopus) tropicalis, an amenable model to identify human

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developmental toxicants. 23 The Oil Sands Industry’s plans for eventual release of OSPW stored

75

in tailings pond back to the environment, development of “wet landscape” remediation

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approaches, and increased use of NAs in numerous other commercial processes begs the question

77

as to the potential effects of exposure of frog embryos and hence the importance of

78

understanding NA toxicity in frogs.

79

2. MATERIAL AND METHODS

80

2.1. Chemicals

81

Human chorionic gonadotropin (hCG) was purchased from Millipore (Burlington, 24

82

Massachusetts, United States). The salts for preparation of the standard FETAX

(Frog

83

Embryo Teratogenesis Assay-Xenopus; pH 7.6-7.9) solution

84

(https://www.astm.org/Standards/E1439.htm) for embryonic exposures (NaCl, NaHCO3, KCl,

85

CaCl2, CaSO4 and MgSO4) were purchased from Fisher Scientific (Waltham, Massachusetts,

86

United States) and Sigma-Aldrich (St. Louis, Missouri, United States). L-cysteine was acquired

87

from Sigma-Aldrich. The two commercial NA mixtures (S1M and S2M) were purchased from

88

Sigma-Aldrich (#70340) with lot number BCBC9959V and BCBK0736V, respectively. A 1000

89

L sample of OSPW from an active settling pond was collected in the Canadian oil sands region


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north of Fort McMurray Alberta in June 2015. The OSPW sample was shipped (Contango

91

Strategies) in a 1000L tote to Saskatoon and then sub divided into a series of 20 L food grade

92

plastic buckets to one of our labs (JVH), and stored at 4ºC (July 2015-Feb 2016) while AEOs

93

were extracted from 600 L following established procedures.25 In brief, after collection, the

94

OSPW was left to settle and the clarified OSPW was acidified to pH 2.5 using sulfuric acid. One

95

L of OSPW was placed in a funnel, 500 mL of dichloromethane was added and the mixture was

96

manually shaken for several minutes. The organic phase was collected and concentrated using a

97

rotary evaporator set to 40 ºC. 150 mL of 0.1 N NaOH was added to the AEO. The pH was

98

reduced to 10 using sulfuric acid. The solution was filtered using 1000 MW cutoff membranes

99

(Millipore). The AEO mixture was stored at 4 ºC, protected from any exposure to light until

100

used for the exposures. The concentration of NAs in the AEOs was 8, 050 mg/L measured using

101

ESI-MS in comparison to a sample of NAs extracted from a different OSPW that was previously

102

characterized.21 The term AEO was adopted to address all the compounds that can be extracted

103

with the acidification and organic extraction of OSPW including NAs. Note that the OSPW

104

extract used here has been submitted to other independent characterizations (see OSPW-1 in

105

Hughes et al. 2017). 26 Apart from classic naphthenic acids, AEO mixtures typically contain

106

other complex structures, such as diamondoid compounds, 27 resin acids, 28 and sulfur- and

107

nitrogen-containing compounds. 29

108

2.2. Chemical analysis

109

The two commercial NA mixtures and the AEO extract were analyzed using electrospray

110

ionization-high resolution mass spectrometry (ESI-HRMS). The conditions were as follows:

111

Samples (5 µL) were introduced into the mass spectrometer by loop injection (flow injection

112

analysis) using a Surveyor MS pump (Thermo Fisher Scientific Inc.) and a mobile phase of


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50:50 acetonitrile/water containing 0.1% NH4OH. Mass spectrometry analysis was carried out

114

using a dual pressure linear ion trap-orbitrap mass spectrometer (LTQ Orbitrap Elite, Thermo

115

Fisher Scientific, Bremen, Germany) equipped with an ESI interface operated in negative ion

116

mode. Data was acquired in full scan mode from m/z 100 to 600 at a setting of 240,000

117

resolution (average mass resolving power (m/Δm50%) was 242,000 at m/z 400). For scan-to-

118

scan mass calibration correction n-butyl benzenesulfonamide was used as lock mass compound.

119

Xcalibur version 2.2 software (Thermo Fisher Scientific San Jose, CA) was used for data

120

acquisition, instrument operation, and semi-quantitative data analysis. Class distributions were

121

established with acquired accurate mass data and Composer version 1.5.3 (Sierra Analytics, Inc.

122

Modesto, CA) with mass accuracies of < 2 ppm.

123

Samples with nominal concentrations of 0, 1, 5, 10, 50 and 100 mg/L (n = 3) were analyzed by

124

ESI-HRMS as described above. The two commercial mixtures were used as standards for the

125

quantification of NAs in their respective water samples. A previously characterized Athabasca

126

Oils Sands OSPW large volume extract 25 was used as a standard for the quantification of NA in

127

the AEO samples. Linear equations forced to zero were built from the data. The coefficient of

128

determination was 0.989, 0.994, and 0.964, for S1M, S2M, and AEOs, respectively. The nominal

129

concentrations of the exposures were corrected to the measured concentrations. There is

130

currently no standard method for the quantification of NAs, therefore the results here are

131

considered semi-quantitative.

132

2.3. Animal husbandry and breeding

133

All the bioassays were performed in accordance with the guidelines of the Canadian Council

134

on Animal Care and approved by Animal Care and Veterinary Services at the University of


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Ottawa. Originally obtained from the US Environmental Protection Agency,30 adult golden strain

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(Silurana (Xenopus) tropicalis frogs from our breeding colony were used for bioassays.

137

The interest in frogs as a test organism is due to their importance in the food web, their

138

susceptibility to pollutants and endocrine disruptors in water, and as a model for vertebrate

139

development. Specifically, the African Western clawed frog S. tropicalis is a diploid model

140

species commonly used in the field of developmental biology and toxicology. 21 A sequenced

141

genome and relatively high number of conserved genes compared to humans, in particular, those

142

controlling development and associated with disease 23 make S. tropicalis an amenable model

143

organism. Moreover, frogs in early developing stages are susceptible to endocrine disruptors, and

144

studies have shown that the exposure of frogs in larval stages to stressors has effects on future

145

morphology, such as body size, locomotor ability, and survivorship. 22

146

Frogs were fed daily with Nasco frog brittle for juvenile Xenopus and kept under a 12 h

147

light/dark cycle and 25 ºC water temperature in a 21020 Tecniplast unit. Embryos of S. tropicalis

148

were obtained by inducing spawning adult frogs by injection of human chorionic gonadotropin

149

(hCG) into the posterior lymph sac.31 Briefly, the frogs were injected with a priming dose of 12.5

150

IU hCG and moved to glass tanks containing FETAX solution at a temperature of 21 ± 1 ºC.

151

After 24 h, the breeding pairs were injected with a boosting dose of 100 IU hCG. Embryos were

152

collected and the jelly coat was removed using a 2% L-cysteine solution during 2 min

153

exposure.24 Fifty mL of test solution was added to the Petri dishes and ten embryos at

154

Nieuwkoop-Faber (NF) stage 9-10 were placed in each petri dish.

155

2.4. Solution preparation

156

Solutions of NAs for the different treatments were prepared daily from a stock that was kept at

157

4 ºC and covered from light. The stock solutions from S1M and S2M were prepared using


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ethanol as a solvent. The final concentration of ethanol in all of the treatments was 0.0025%.

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Concentrations of 0.005% ethanol cause no observable effects on embryos of S. tropicalis.18 The

160

AEO extract contained 8,050 mg/L NAs in a solution of 0.1 N of NaOH. The final concentration

161

of NaOH in all of the treatments was 0.001 N. The pH of the water for all of the treatments was

162

7.8 ± 0.1. The nominal concentrations tested for S1M and S2M were 0, 0.5, 1, 2, 3, 4, 6, 8, 12

163

and 24 mg/L. The nominal concentrations tested for AEOs were 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48,

164

and 96 mg/L. In the exposures, two controls were used, one containing FETAX solution only,

165

and a second control to observe possible effects of the carrier solvent (ethanol or NaOH) on S.

166

tropicalis development.

167

2.5. Toxicity testing

168

Ten cm glass Petri dishes were used as exposure vessels; the use of plastics was avoided since

169

some plastic materials may leach chemicals with estrogenic activity.32 Solutions were therefore

170

prepared using glass cylinders. All glassware was washed as follows: 15 min soaking with hot

171

water and detergent, rinsed twice with reverse osmosis water, later rinsed in a 10% HCl solution,

172

rinsed twice with reverse osmosis water, rinsed with acetone (Fisher Scientific, Certified ACS

173

grade, 99.8%), rinsed twice with reverse osmosis water and kept in an oven at 120 ºC for 3 h.

174

Seven glass Petri dishes containing FETAX solution were used as controls. Seven replicates with

175

ten embryos were used for each treatment. The tests were conducted in a room with a controlled

176

temperature (27 ± 1 ºC) and 12 h light/dark cycle. Each Petri dish was observed every 24 h, data

177

recorded, and dead embryos were removed. At the same time, exposure solutions were replaced

178

with newly prepared solutions. Preliminary dose range-finding experiments were carried out to

179

establish the final test concentrations. At the end of the exposures, digital images of the embryos

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were taken under a light stereomicroscope (Nikon SMZ 1500), with a Nikon DS-Fi1 camera and


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NIS Elements version 3.22.00 software. Images were used for total length (TL), snout-vent

182

length (SVL), tail length (TaL) and interorbital distance (IOD) measurements. The abnormalities

183

were classified according to the FETAX Atlas of abnormalities.33 The frequency of the

184

abnormalities was registered and used for the calculation of the EC50 and EC20. A tadpole was

185

considered affected by the exposure if the presence of at least one type of abnormality was

186

observed. Cranial malformations, edema, and abnormalities of heart, ocular and gut were

187

documented.

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2.6. Data processing and statistical analysis

189

Lethal concentration 50% (LC50), lethal concentration 20% (LC20), effect concentration 50%

190

(EC50), and effect concentration 20% (EC20) were analyzed using a log vs. normalized response-

191

variable slope model (Y = 100/(1+10^((LogEC50-X)*HillSlope))) in GraphPad Prism 6.

192

Morphological effects of the different mixtures on the S. tropicalis embryos were assessed using

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ImageJ™ 1.48 (http://imagej.nih.gov/ij/). Frequencies of abnormalities were analyzed as

194

transformed data using the arcsine square root transformation. The Shapiro-Wilk normality and

195

Levene’s test for equal variances were performed. To analyze possible differences between

196

control and solvent carrier the normally distributed data were analyzed using t-test and the non-

197

normally distributed data were analyzed using the Mann-Whitney Test. Normally distributed

198

data with equal variances of several groups were analyzed using ANOVA and Tukey’s post-hoc

199

test. Normally distributed data with unequal variances of several groups were analyzed using

200

ANOVA Welch and Dunnet T3 as the post-hoc test. Non-normally distributed data with several

201

groups were analyzed using ANOVA on ranks by Kruskal-Wallis, followed by the Student-

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Newman-Keuls (SNK) test. The alpha (α) level was set to 0.05. The statistical analyses were

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performed using SPSS V21 IBM and Sigma Plot 12.0.


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3. RESULTS AND DISCUSSION

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3.1. Chemical characterization

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Analysis by ESI-HRMS showed that the AEO mixture contains a high number of components,

207

being more complex than S1M and SM2 (Figure 1). The two “Sigma” extracts S1M and S2M

208

have similar profiles; however, the proportion of the components between 150-250 m/z is

209

different in the two. Moreover, Sigma and Merichem mixtures have been reported to contain in

210

addition to NAs, ~7% non-polar components with mass spectral characteristics similar to

211

alkanes.34

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The proportion of O2 species was 98.8, 98.9 and 58.6% for S1M, S2M, and AEOs,

213

respectively (Figure 2 a-c). These O2 species are represented by classical NAs with one

214

carboxylic acid group denoted by the formula: CnH2n-zO2 where n indicates the carbon number, z

215

specifies the deficiency of hydrogen from the formation of rings, and groups the NAs in a

216

homologous series.2 The O2 species can therefore be mono carboxylic acids and dihydroxy

217

components. The S1M and S2M are composed largely of the O2 and O4 classes. Other reports

218

show a lower proportion of O2 species in commercial extracts, ranging from 78.5-86.1%,

219

specifically another Sigma extract at 79.9%.35 However, the AEO extract (Figure 2 c) is

220

composed mainly of O2, O3, O4 (89.3%) and other species in a lower proportion (10.7%). The O2

221

species proportion in the AEO extract was estimated as 58.6%, which is in the range of other

222

extracts (50-82.5%) reported in the literature.35,36

223

The double bond equivalences (DBE) were calculated using the equation DBE (CcHhNnOoSs) =

224

c-(h/2) + (n/2) + 1.37 The data obtained by ESI-HRMS in negative-ion mode is principally

225

deprotonated ions [M-H]-, thus, the DBE values are half-integers and 0.5 is subtracted.38 The

226

DBE can be attributed to unsaturated bonds or cyclic structures. The DBE profile for O2 species


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of S1M and S2M are similar. The S1M has a slightly higher proportion of components in the 1.5

228

group with some differences in the proportions of the groups DBE 2.5, 3.5, and 4.5 (Figure 2 d-

229

e). The DBE groups that were more abundant in the AEOs were 3.5 and 4.5. It has been

230

previously reported that the Sigma commercial mixtures are principally composed of aliphatic

231

NAs.34,35 In contrast, the AEOs are mainly composed of 3.5 and 4.5 DBE, presumably double

232

and triple ring NAs. In 2010, Barrow et al.24 reported similar results in the analysis of an OSPW

233

sample using atmospheric pressure photoionization and electrospray ionization Fourier transform

234

ion cyclotron resonance mass spectrometry. In that case, DBE 3, 4, and 7 were the most

235

abundant groups. The most abundant components in S1M and S2M were aliphatic NAs with 19

236

carbons. The differences between S1M and S2M were observed in the proportion of the aliphatic

237

NAs with 9, 10 and 11 carbons (Figure 2 g-h). However, the most abundant components in the

238

AEO mixture are tentatively double ring structures with 13 carbons and triple ring structures

239

with 14 carbons (Figure 2 i).

240

3.2. Lethal toxicity

241

Lethal and sub-lethal effects of the exposure of S. tropicalis to S1M, S2M and the AEOs were

242

assessed with dose-response experiments. The solvent carriers did not show a significant effect

243

on survival, the rate of abnormalities or frequency of teratogenic endpoints measured in embryos

244

of S. tropicalis (Figures S1-S4). However, the exposure to the different extracts tested reduced

245

the survival rate after four days and numerous teratogenic effects were observed (Figure 3).

246

Based on the measured concentrations, the LC50 of S2M was 1.1 times less toxic than S1M, and

247

the AEO was 5 times less toxic than S1M (Table 1). The 96-h LC50 values of S1M and S2M are

248

similar, however, the observed mortality pattern varied. For example, 100% mortality was

249

achieved with the highest S1M concentration after 48h and after 72 h 100% mortality was


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observed in the 12 mg/L treatment. In contrast, nearly 50% mortality was observed after 96 h for

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the 12 mg/L treatment for tadpoles exposed to S2M (Figure 3 a and d). Similar lethal effects

252

have been found in one fish species. The LC50 reported for fathead minnow (Pimephales

253

promelas) is 2.5 mg/L for a Sigma extract and the 96 h LC50 was 7.5-18.5 mg/L for different

254

AEO extracts. 35

255

3.3. Sub-lethal effects

256

Exposure to S1M, S2M, and AEOs significantly increased abnormality rates in embryos of S.

257

tropicalis (Figure 3 b, e and h). Based on measured concentrations, the EC50 of S2M was 1.2

258

times less toxic than S1M, and S1M is 6.7 times more toxic than AEOs (Fig, 3, b, e and h, Table

259

1). In fathead minnow embryos, an EC50 for hatching success of 2.5 and 7.5-18.5 mg/L have

260

been reported for a Sigma extract and AEOs, respectively. 35 These observations indicate that

261

fish and frogs might have a similar sensitivity with respect to embryotoxicity in response to

262

commercial NAs and AEOs, however, direct comparisons between taxa should be conducted to

263

test this hypothesis. The EC20 analysis showed significant differences between S1M and S2M,

264

S1M being the most toxic extract (Table 2). The exposure to S1M, S2M and the AEOs

265

significantly decreased the TL, SVL, TaL, and, IOD (Figure S5-S7, a-d). The main effects

266

observed include edema, cranial, heart, gut and ocular abnormalities. Edema, and gut and heart

267

abnormalities were particularly evident in the 9.3, 11.3 and 55.5 mg/L treatments of the S1M,

268

S2M, and AEO extracts, respectively (Figure 3, c, f and i). There was a positive relationship

269

between increasing concentration of NAs with severity and frequency of abnormalities (Figure

270

S5-S7). Edema and cardiovascular abnormalities have been observed in zebrafish (Danio rerio)

271

larvae exposed over seven days to AEOs extracted from oil sands from the Daqing (China) oil

272

exploration area.39 The results here presented here are also in agreement with Marentette et al. 35


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who reported on cardiovascular deformities in fathead minnows exposed to naphthenic acid

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fraction components (NAFCs) until hatching. Moreover, Marentette et al.40 also suggested

275

oxidative stress as a possible toxic mechanism of naphthenic acid fraction components (NAFC)

276

in walleye (Sander vitreus) embryos. Further research is needed to address the mechanism of NA

277

toxicity, especially in relation to edema, and heart and gut development.

278

The HRMS analysis of the two commercial extracts indicates that there are differences in their

279

carboxylic acid composition. Differences were observed in low MW NAs and in the relative

280

proportions of various components ranging from m/z 157-283 (Figure 1 a-b). Slight differences

281

between S1M and S2M were also observed in the DBE distribution (Figure 2 d-e) and in the

282

distribution of carbon numbers (Figure 2 g-h). The varying chemical composition may be linked

283

to the different effects observed in tadpoles. For instance, based on the EC50 and EC20 of edema,

284

cranial, heart and gut abnormalities, animals exposed to S1M have significantly more cranial

285

defects (p < 0.05) (Table S1). The S2M induced significantly more edema in the tadpoles

286

compared to S1M and AEOs (p < 0.05) (Table S2). However, there were no differences between

287

S1M and S2M for the incidence of heart and gut abnormalities (p > 0.05) (Table S3-S4). The

288

toxicity variation of the two commercial extracts may provide insight for environmental samples

289

that have different origin and weathering history. The trend we observed is that NAs in the AEOs

290

from OSPW were less overtly toxic, but nevertheless produced a similar range of teratogenic

291

effects since the same types of abnormalities were evident after all treatments. As an example,

292

our EC50 estimates for NAs (Table 1) based on total abnormalities were statistically different and

293

respectively, 1.8, 2.8 and 12.3 mg/L for S1M, S2M and AEOs. The main compositional

294

differences (DBE, carbon number ranges; see Figure 2) are likely related to the degree of effect

295

(rank order potency S1M>S2M>AEOs). Marentette et al.35 compared the profile of OSPW


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296

from two different sources, reporting differences in the proportion of 2 and 3 ring structures and

297

small differences in the z family 8, 10, and 12. In the same fashion, Brunswick et al.41 reported

298

differences in the percentage response of individual homologs between lots of Merichem, Acros,

299

and Sigma NA mixtures. Therefore, exposure to different extracts is expected to produce

300

different toxic effects according to distinct chemical profiles. Commercial mixtures have been

301

used previously as standards for concentration measurements of NAs,36, 42 however, these results

302

may be difficult to replicate because of the possibility that commercial extracts can be sourced

303

from different lot numbers. These differences also highlight the challenges with comparative

304

analysis of toxicity of AEO extracts since the origin, history and resulting composition of the

305

OSPW samples vary considerably as reported in publications.

306

A possible solution to remedy issues of characterization of NAs and AEO mixtures would be

307

to adopt standard methods for the extraction and measurement of the NA concentration. Another

308

option may be normalization based on the O2 species concentration. The distribution of O2

309

species according to carbon number and DBE, however, vary significantly for AEO mixtures.

310

The O2 family is represented by classical NAs with just one carboxylic acid group or dihydroxy

311

groups, and published data concur that O2 species are the main toxic and classical NAs in

312

OSPW. 26, 43, 44, 45 Therefore, normalization of the results presented in Figure 2 based on the O2

313

species concentration was performed (See Figure S8; Table 1). The rank order potency with and

314

without normalization to the content of O2 species was the same and was S1M >S2M >AEOs.

315

Based on the measured concentration, S1M is the most toxic mixture being 1.1 times more toxic

316

than S2M and 5 times more toxic than the AEOs. However, when the data was normalized to the

317

O2 family concentration and LC50/EC50 estimates compared, the toxicity of AEOs is only 3

318

times less toxic than the S1M extract (Table 1; Figure S8).


14


319

Page 16 of 31

The LC50, EC50, and EC20 values for the commercial extracts are lower than the corresponding

320

values for AEOs. This trend is in agreement with previous reports where fathead minnows

321

embryos were exposed to fresh and aged OSPW and commercial NAs. 35 Concentrations of NAs

322

ranging from of 80-128 mg/L (high-performance liquid chromatography) or 93-103 mg/L

323

(Orbitrap MS) in OSPW samples collected from tailing ponds. 36 The maximum concentration in

324

groundwater has been reported in some cases up to 48 mg/L and in fresh water up to 0.7

325

mg/L.14,46 As Hughes et al. 45 elegantly discuss, it is difficult to relate actual concentrations

326

between studies because of variations in NA detection and quantification methods. Nevertheless,

327

the LC50, EC50 and EC20 values for S. tropicalis embryos herein with and without O2 species

328

normalization are less than or within the concentration ranges reported in some OSPW and

329

natural groundwater samples in Alberta. Previous species comparisons indicated that rainbow

330

trout fry (Oncorhynchus mykiss) are somewhat more sensitive (96-h median acute lethality) than

331

larval fathead minnows (Pimephales promelas) exposed to the organic fraction of OSPW. 45

332

These previous studies did not assess sublethal, teratogenic effects. Our data clearly indicate that

333

NAs disrupt development at low concentrations.

334

The chemical and morphometric analyses presented here are major steps towards

335

understanding the disruptive effects of NAs on amphibian embryogenesis and can contribute to

336

the development of environmental guidelines and mitigation strategies for the management of

337

NAs and OSPW. This is important given that the Canadian zero-discharge policy for OSPW

338

currently stored in vast ponds in Northern Alberta may change in the near future, thus there is

339

potential for NAs to be released to the aquatic environment. However, significant challenges

340

remain. While, the O2 class of NAs are an important component of toxicity, the significance of

341

other NAs (and variable mixtures) remains to be determined. In a recent fractionation study 43,


15

Page 17 of 31


342

overt lethality in fathead minnow larvae was associated with the O2 class of NAs. Significantly,

343

the potential sublethal effects of the specific subclasses of NAs on vertebrate embryogenesis

344

remain to be investigated in detail.


16


Page 18 of 31

345


17

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346

Figure 1. Electrospray ionization-high resolution mass spectrometry profiles for Sigma 1 (S1M)

347

extract (a), Sigma 2 (S2M) mixture (b), and acid extractible organics (AEOs) (c).

348 349

Figure 2. Electrospray ionization-high resolution mass spectrometry speciation profiles Sigma 1

350

(S1M) extract (a), Sigma 2 (S2M) mixture (b), and acid extractible organics (AEOs) (c). Double

351

bond equivalences (DBE) distribution is given as a function of abundance for O2 for S1M (d),

352

S2M (e), and AEOs (f). Analysis of the distribution of carbon numbers and DBE of NAs in S1M

353

(g), S2M (h), and AEO (i). The values represent the percentage of total signal that accounts for a

354

given carbon number of a given DBE family. The sum of all of the values equals 100%.

355


18


Page 20 of 31

356 357

Figure 3. Dose-response curves for mortality, abnormality rate and main teratogenic effects

358

induced by exposure of S. tropicalis embryos to different mixtures of NAs, S1M (a-c), S2M (d-

359

f), and AEOs (g-i) based on calculated concentrations. Bars represent the standard deviation of

360

the mean (n=7). The most commonly observed abnormalities were: edema, cranial malformation,

361

gut malformation, heart abnormalities, and ocular malformation, as indicated by arrows. Scale

362

bar is equal to 1 mm.


19

Page 21 of 31


363 364

Table 1. Comparison of the LC50 and EC50 of the 2 NA extracts (S1M and S2M) and an AEO

365

extract in S. tropicalis after a 4-day exposure, based on the nominal concentration, measured

366

concentration, and O2 family concentration. Confidence interval 95% indicated between

367

brackets. The coefficient of determination (R2) calculated in GraphPad is also indicated.

368

Different letters indicate values that are significantly different (p < 0.05).

369

Nominal concentration (mg/L)

Measured concentration (mg/L)

Concentration based on the O2 families (mg/L)

S1M LC50

S2M LC50

AEOs LC50

S1M EC50

S2M EC50

AEOs EC50

8.9a

12.4b

45.2c

1.8a

2.8b

12.3c

[7.8, 10.2]

[12.0, 12.8]

[43.6, 46.9]

[1.6, 2.1]

[2.5, 3.0]

[10.8, 14.1]

R2 = 0.987

R2 = 0.961

R2 = 0.966

R2 = 0.859

R2 = 0.875

R2 = 0.846

10.4a

11.7a

52.3b

2.1a

2.6b

14.2c

[9.1, 11.8]

[11.3, 12.1]

[50.4, 54.2]

[1.9, 2.4]

[2.4, 2.9]

[12.4, 16.2]

R2 = 0.987

R2 = 0.961

R2 = 0.966

R2 = 0.860

R2 = 0.908

R2 = 0.844

10.2a

11.6a

30.6b

2.1a

2.6b

8.4c

[8.9, 11.8]

[11.2, 12.0]

[29.5, 31.8]

[1.8, 2.4]

[2.4, 2.8]

[7.4, 9.5]

R2 = 0.987

R2 = 0.961

R2 = 0.966

R2 = 0.856

R2 = 0.907

R2 = 0.854


20


Page 22 of 31

370

Table 2. Comparison of the LC20 and EC20 of the 2 NA extracts (S1M and S2M) and an AEO

371

extract in S. tropicalis after a 4-day exposure, based on the nominal concentration, measured

372

concentration, and O2 family concentration. Confidence interval 95% indicated between

373

brackets. The coefficient of determination (R2) calculated in GraphPad is also indicated.

374

Different letters indicate values that are significantly different (p < 0.05).

Nominal concentration (mg/L)

Measured concentration (mg/L)

Concentration based on the O2 families (mg/L)

S1M LC20

S2M LC20

AEOs LC20

S1M EC20

S2M EC20

AEOs EC20

8.3a

10.2a

35.6b

0.5a

1.4b

4.7c

[8.1, 9.7]

[9.5, 10.7]

[31.4, 39.2]

[0.3, 0.7]

[1.1, 1.7]

[3.9, 5.6]

R2 = 0.987

R2 = 0.961

R2 = 0.966

R2 = 0.859

R2 = 0.875

R2 = 0.846

9.6a

9.5a

41.2b

0.6a

1.3b

5.5c

[9.4, 11.3]

[8.9, 10]

[36.2, 45.3]

[0.4, 0.9]

[1.1, 1.6]

[4.5, 6.5]

R2 = 0.987

R2 = 0.961

R2 = 0.966

R2 = 0.860

R2 = 0.908

R2 = 0.844

9.5a

9.4a

24.2b

0.6a

1.3b

3.2c

[9.3, 11.2]

[8.8, 9.9]

[21.3, 26.6]

[0.3, 0.8]

[1.1, 1.6]

[2.6, 3.8]

R2 = 0.987

R2 = 0.961

R2 = 0.966

R2 = 0.856

R2 = 0.907

R2 = 0.854

375


21

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376

ASSOCIATED CONTENT

377 378 379

Supporting Information. Figure S1, Survival rates of S. tropicalis embryos in control and solvent treatments for 24, 48, 72, and 96 h;

380 381

Figure S2, Effects of the solvent carrier on teratogenic endpoints in S. tropicalis embryos exposed to the Sigma 1 mixture (S1M).

382 383

Figure S3, Effects of the solvent carrier on teratogenic endpoints in S. tropicalis embryos exposed to the Sigma 2 mixture (S2M).

384 385

Figure S4, Effects of the solvent carrier on teratogenic endpoints in S. tropicalis embryos exposed to acid extractible organics (AEOs).

386 387

Figure S5, Teratogenic effects observed in S. tropicalis embryos exposed to the Sigma 1 mixture (S1M).

388 389

Figure S6, Teratogenic effects observed in S. tropicalis embryos exposed to the Sigma 2 mixture (S2M).

390 391

Figure S7, Teratogenic effects observed in S. tropicalis embryos exposed to acid extractible organics (AEOs).

392 393 394 395

Figure S8, Concentration-response curves for mortality, abnormality rate and main teratogenic effects induced by exposure of S. tropicalis embryos to different mixtures of NAs. Concentrations of NAs The Sigma 1 mixture (S1M), Sigma 2 mixture (S2M) and acid extractible organics (AEOs) with concentrations corrected based on O2 family proportion.

396 397

Table S1. Comparison of the EC50 and EC20 based on cranial abnormality rates of the 2 NA extracts and an acid extractible organics (AEOs) in S. tropicalis after a 4-day exposure.

398 399

Table S2. Comparison of the EC50 and EC20 based on edema rates of the 2 NA extracts and an acid extractible organics (AEOs) in S. tropicalis after a 4-day exposure.

400 401

Table S3. Comparison of the EC50 and EC20 based heart abnormality rates of the 2 NA extracts and acid extractible organics (AEOs) in S. tropicalis after a 4-day exposure.

402 403 404 405

Table S4. Comparison of the EC50 and EC20 based on gut abnormality rates of the 2 NA extracts acid extractible organics (AEOs) S. tropicalis after a 4-day exposure.

406

Corresponding Author

407

*E-mail: [email protected]

AUTHOR INFORMATION


22


Page 24 of 31

408

Author Contributions

409

The manuscript was written through contributions of all authors. All authors have given approval

410

to the final version of the manuscript.

411

Notes

412

The authors declare no competing financial interest.

413

ACKNOWLEDGMENT

414

Grant support for JMGV was provided by CONACyT Mexico. The support of NSERC Strategic

415

Grants Program, University of Ottawa Research Chair program and Environment and Climate

416

Change Canada for VLT are also acknowledged with appreciation. This work benefited from the

417

support and ideas of Bruce Maclean, who manages the Mikisew Cree First Nation’s Community-

418

Based Monitoring Program out of Fort Chipewyan, Alberta. We would like to thank Paul Knaga

419

and Dr. Sarah A. Hughes (Shell) for providing the oil sands process-affected water sample from

420

am active settling pond. Note that Oil Sands operations were divested from Shell to CNRL June

421

1, 2017).

422


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Page 25 of 31


423

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