Growth of Chironomus dilutus Exposed to Ozone-Treated and Untreated Oil Sands Process Water
J. Anderson 1, S.B. Wiseman 1, E. Franz 1, M. Gamal El-Din 2, J. Martin 3, P. Jones 1,4, K. Liber 1,4, and J.P. Giesy 1, 5, 6, 7
1. Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada
2. Department of Civil and Environmental Engineering, University of Alberta, Edmonton, AB, Canada
3. Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Chemistry, University of Alberta, Edmonton, AB, Canada
4. School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada
5. Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
6. State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, Peoples Republic of China
7. Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
• Canada is home to the second largest proven oil reserves, mostly in Alberta’s
Athabasca region
• Extraction of usable material (bitumen) from oil sands requires vast quantities
of fresh water and produces wastewater - “oil sands process water” (OSPW)
• Approximately 4 m3 of OSPW are produced per 1 m3 of oil sands processed,
but more efficient water recycling has reduced freshwater needs
• OSPW has been shown to be toxic to a number of different species;
naphthenic acids (NAs) are believed to be responsible for much of the toxicity
• Companies are currently held to a zero-discharge policy so treatment methods
are required to target NAs and effectively eliminate toxicity of OSPW
• One potential treatment method is ozonation, a method commonly used in
municipal water treatment and shown to target more persistent and chronically
toxic NAs (Fig. 1)
OSPW After Ozonation with
OSPW NAs Before Ozonation
80 mg O3/L
Relative Intensity
O3
6
4
2
Relative Intensity
8
8
• Ozonation reduced the growth inhibition observed in untreated OSPW and
may be a treatment option to reduce OSPW toxicity towards aquatic
invertebrates
• No significant differences in survival between any exposure group and the freshwater controls (Figs.
2a and 3a)
• Untreated (100%) OSPW significantly inhibited C. dilutus growth (Figs. 2b and 3b)
• Need to identify an optimal dose of ozonation and investigate whether a
dose effect of ozonation is present
• Low ozonation study (50 mg/L)
100% OSPW-exposed larvae were 64% smaller than freshwater controls (Fig. 2b)
Exposure to full strength ozone-treated OSPW  no difference in mean wet mass from
controls
1. Transcriptome responses to OSPW and ozonated OSPW
• Illumina RNAseq transcriptome sequencing of C. dilutus larvae exposed
to freshwater, untreated OSPW, and ozone-treated OSPW to determine
gene expression profiles
 Biomarkers of OSPW exposure
 Biomarkers of effect
 Molecular basis of toxicity
• High ozonation study (80 mg/L)
100% OSPW-exposed larvae were 77% smaller than freshwater controls (Fig. 3b)
Exposure to full strength ozone-treated OSPW  only 40% smaller than controls
Survival
a
Growth
• Over 250 million sequence reads returned by Illumina
• Contigs (>300,000) created using CLC Workbench
• Scaffolds will be built and sequences will be annotated using BLAST2GO
• Gene expression profiles will be generated for the different treatments to
determine exposure specific transcriptome responses
b
6
4
2
12
0
7 8 9
10 11 12
13 14 15
16 17 18
19 20 21
22
Carbon number
6
0 Rings
2. Behavioral Responses in C. dilutus exposed to OSPW and
Ozonated OSPW
• Preliminary studies suggest OSPW exposure drastically alters larvae
burrowing and tube-building behaviours (Fig. 4)
• Effects appear to be attenuated by ozonation of OSPW
12
0
7 8 9
10 11 12
13 14 15
16 17 18
19 20 21
22
6
0
*
Rings
Carbon number
Fig. 1 Naphthenic acid profile of OSPW prior to ozonation and after treatment with
80 mg/L O3.
a
Study Objectives:
1.To characterize the effects of exposure to OSPW in a model benthic
invertebrate, Chironomus dilutus
2. To determine whether remediation of OSPW using ozonation effectively
reduces its toxicity to aquatic invertebrates
c
Fig. 2a and 2b Effects of low dose ozonation (50 mg/L) on survival and growth (individual wet mass) of C.
dilutus larvae following a 10-day exposure. Differences from freshwater controls in survival were determined by
Kruskal-Wallis one-way ANOVAs and differences in mass were determined by Student-Newman-Keuls
comparisons across treatments (p=0.05). Significant differences designated by an asterisk (*).
Survival
a
Growth
b
A
• Test organism – Chironomus dilutus larvae (formerly C. tentans), started 1314 days post-oviposition
• Exposure waters – Freshwater control, saltwater control, ozonated OSPW,
untreated OSPW
• Two degrees of ozonation – Low (50 mg/L)
and high (80mg/L)
• Exposure conditions – Continuous aeration,
50% water change every 2 days, daily
feeding, 10 larvae per rep, 4 reps per
treatment, 10 day exposure period
• End points – Survival, growth
(measured as wet mass)
• Parallel assay terminated at Day 4, total
RNA collected from pooled samples,
sequencing by Illumina RNASeq
b
B
Fig. 4 Burrowing behaviours observed in a) fresh water, b) OSPW, and c) ozonated
OSPW. In the freshwater control, all individuals are within tubes, while the ozonated
group has a few individuals not burrowing and the untreated OSPW group displays very
little burrowing and tube-building activity. These preliminary results will be investigated
further in upcoming studies.
B
A
B
B
B
B
B
C
Figs. 3a and 3b Effects of high dose ozonation (80 mg/L) on survival and growth (individual wet mass) of C.
dilutus larvae following a 10-day exposure. Differences from freshwater controls in survival were determined by
Kruskal-Wallis one-way ANOVAs and differences in mass were determined by Student-Newman-Keuls comparisons
across treatments (p=0.05). Significant differences designated by different letters.
• This research was supported by research grants from the Alberta Water
Research Institute and Western Economic Diversification Canada to J.P.G.
• J.P.G. is supported by the Canada Research Chairs Program
• J.A. was supported by an NSERC Alexander Graham Bell Canada
Graduate Scholarship
• Thanks to Syncrude Canada Ltd.
for support and assistance