Supporting Information to the Manuscript AECT-D-12-03757

Supporting information to the manuscript AECT-D-12-03757

Understanding the chronic impacts of oil refinery wastewater requires consideration of sediment contributions to toxicity

Jennifer R. Lougherya, Tim J. Arciszewskia, Karen A. Kidda, Angella Mercera, L. Mark Hewittb, Deborah L. MacLatchyc, and Kelly R. Munkittricka

a Canadian Rivers Institute and Department of Biology University of New Brunswick, 100 Tucker Park Road, Saint John, NB, E2L 4L5, email:

b Environment Canada, Burlington, ON L7R 4A6

c Current address: Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo, Ontario, Canada N2L 3C5

SI Figure 1: Total number of fish caught with 10 minnow traps per month in Little River upstream of the current effluent outfall (LRU) and in Little River below the current effluent outfall (LRD).

SI Table 1: Summary table of laboratory bioassays, effluent concentrations employed and endpoints assessed.

Date / Salinity (ppt) / Effluent Concentrations* / Endpoints / Duration
May 2006 / 16 / Control, 25%, 50% / Fish and organ weights and indices, Steroid production, EROD induction / 7d
October 2006 / 0 / Control, 25%, 50%, 100% / Fish and organ weights and indices, EROD induction / 14d
April 2007 / 16 / Control-Control, Control-25%, Control-50%, Exposed-Control, Exposed-25%, Exposed 50% / Fish and organ weights and indices, Steroid production, EROD induction / 14d
July 2007 / 0 / Control-Control, Control-25%, Control-50%, Exposed-Control, Exposed-25%, Exposed 50% / Fish and organ weights and indices, Steroid production, EROD induction / 14d
February 2009 / 0 / Control, 50% / Fish and organ weights and indices, Steroid production, EROD induction, egg production / 35d

*Concentrations presented as sediment-water in 2007 bioassays

SI Table 2: Summary table of sediment collection dates, number of samples collected, sites used and collection method by year.

Year / Months / Samples/Site/Month / Sites / Collection method
2008 / May, July, November / 1 / Little River Reference;
10m below outfall; 800m below outfall. / Ekman Dredge
2009 / August, September, October, November / 3 / Little River Reference;
10m below outfall; 800m below outfall. / Ekman Dredge
2010 / May, June, July, August, September, October / 3 / Little River Reference;
10m below outfall; 800m below outfall; Older outfall site above current outfall. / Ekman Dredge
2010 / September / 1 / Little River Reference;
10m below outfall; Older outfall site above current outfall / Sediment Corer

SI Table 3: Concentrations of plasma (August 2006) and in vitro produced (June 2007) 17-β estradiol (E2), testosterone (T), and 11- ketotestosterone (11-KT) in mummichog caged at the upstream (R ), 10 m below the current outfall (10m) and 800 mbelow the current outfall(800m) sites. Values are mean ± SE and values sharing a letter are not statistically different.

Source / Date / Sex / Site / E2 (ng/mL) / n / T(ng/mL) / n
Plasma / August 2006 / F / R / 8.64 ± 5.66 A / 17 / 0.63 ± 0.09 A / 18
10m / 3.40 ± 0.55 A / 12 / 0.71 ± 0.17 A / 12
800m / 8.78 ± 3.66 A / 6 / 1.28 ± 0.26 A / 6
11-KT / T
M / R / 0.32 ± 0.10 A / 6 / 0.68 ± 0.15 A / 6
10m / 0.63 ± 0.18 A / 7 / 0.58 ± 0.17 A / 7
800m / 0.77 ± 0.23 A / 6 / 0.81 ± 0.36 A / 6
In vitro / June 2007 / F / R / 462 ± 102 A / 13
10m / 595 ± 235 A / 5
800m / 399 ± 56 A / 9

SI Figure 2: Ethyoxyresorufin-o-deethylase activity (pmol/mg/min) in female mummichog caged in Little River for 14 d at an upstream reference site (R), 10 m below thecurrent outfall (10m) and 800 m below thecurrent outfall (800m) (June 2007).

SI Table 4: Summary statistics of the plasma sex steroid levels estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT) in adult mummichog exposed to oil refinery effluent in 16-ppt saline water for 7 d in May 2006. Values are mean ± SE.

Sex / Effluent (%) / E2 (ng/mL) / N / T(ng/mL) / N
F / 0 / 2.72 ± 0.38 A / 12 / 0.63 ± 0.09 A / 13
25 / 4.00 ± 0.80 A / 12 / 0.71 ± 0.14 A / 12
50 / 2.14 ± 0.42 A / 9 / 0.25 ± 0.06 B / 9
11KT / T
M / 0 / 0.95 ± 0.29 A / 11 / 1.27 ± 0.44 A / 10
25 / 0.52 ± 0.16 A / 11 / 0.76 ± 0.19 A / 11
50 / 0.97 ± 0.22 A / 9 / 0.69 ± 0.14 A / 9

SI Figure 3: Ethyoxyresorufin-o-deethylase (EROD) activity (pmol/mg/min) in female and male mummichog exposed for 14 d to 0%, 25%, 50% and 100% oil refinery effluent in freshwater (October 2006).* indicate extreme values. Different letters indicate statistical significance (P<0.05).

SI Table 5: Gonadal testosterone levels in mummichog exposed for 14 d to 0%, 25%, and 50% oil refinery effluent and to Little River sediment (E) or Hazen Creek sediment (C) in 16-ppt saline water (April 2007) or fresh water (July 2007). Values are mean ± SE and numbers sharing a letter are not statistically different.

Date / Sex / Treatment
Effluent(%) Sediment / n / Testosterone (ng/mL) / Min / Max
April 2007 / F / 0 / C / 9 / 56 ± 20 A / 0.7 / 179
25 / C / 8 / 110 ± 33 A / 35 / 307
50 / C / 9 / 295 ± 79 B / 32 / 753
0 / E / 9 / 88 ± 29 A / 15 / 281
25 / E / 8 / 113 ± 55 A / 1 / 371
50 / E / 7 / 97 ± 47 A / 14 / 361
M / 0 / C / 9 / 157 ± 22 A / 70 / 289
25 / C / 7 / 133 ± 15 A / 74 / 185
50 / C / 9 / 174 ± 28 A / 65 / 285
0 / E / 9 / 184 ± 23 A / 111 / 330
25 / E / 7 / 148 ± 17 A / 69 / 196
50 / E / 7 / 143 ± 8 A / 110 / 171
July 2007 / F / 0 / C / 8 / 194 ± 43 B / 98 / 476
25 / C / 7 / 434 ± 65 A / 199 / 646
50 / C / 8 / 276 ± 52 AB / 99 / 517
0 / E / 7 / 684 ± 159 A / 223 / 1453
25 / E / 9 / 390 ± 60 A / 193 / 664
50 / E / 9 / 504 ± 148 AB / 56 / 1478
M / 0 / C / 7 / 249 ± 37 A / 104 / 359
25 / C / 8 / 300 ± 49 A / 117 / 514
50 / C / 9 / 251 ± 56 A / 81 / 612
0 / E / 7 / 393 ± 33 A / 290 / 538
25 / E / 8 / 298 ± 22 A / 240 / 407
50 / E / 8 / 333 ± 61 A / 124 / 602

SI Table 6: Summary statistics for mummichog exposed to oil refinery effluent, 0% and 50%, in freshwater, February 2009. Values are mean ± SEM numbers sharing a letter are not statistically different.

Sex / Treatment / N / Length / Weight / LSI / GSI / Condition
F / 0% / 14 / 85.2 ± 2.2 B / 7.75 ± 0.49 B / 3.42 ± 0.20 A / 7.38 ± 1.31 A / 1.24 ± 0.05 A
50% / 15 / 81.8 ± 1.3 A / 6.36 ± 0.31 A / 3.79 ± 0.28 A / 5.37 ± 0.55 A / 1.16 ± 0.03 A
M / 0% / 15 / 86.33 ± 2.3 A / 7.27 ± 0.59 B / 2.14 ± 0.19 A / 0.91 ± 0.06 A / 1.10 ± 0.03 A
50% / 12 / 78.50 ± 2.2 A / 5.40 ± 0.49 A / 2.30 ± 0.22 A / 0.94 ± 0.12 A / 1.09 ± 0.04 A

Table 7: Gonadal reproductive steroid production in mummichog after exposure to oil refinery effluent, 0%, and 50%, in freshwater, in February 2009.

Sex / Concentration / N / Hormone (ng/ml/hr)
Estradiol / Testosterone
F / 0% / 13 / 16.05 ± 3.11 A / 3.11 ± 0.89 A
50% / 15 / 25.82 ± 4.58 A / 7.79 ± 2.32 B
11-ketotestosterone / Testosterone
M / 0% / 15 / 22.17 ± 2.01 A / 16.66 ± 1.76 B
50% / 12 / 21.41 ± 2.25 A / 11.28 ± 0.81 A

SI Table 8: Liver EROD activity in mummichog after exposure to oil refinery effluent at 0%, and 50% effluent in February 2009.

Sex / Concentration / N / EROD
F / 0% / 13 / 11.93 ± 5.65 A
50% / 15 / 9.83 ± 3.97 A
M / 0% / 15 / 16.49 ± 5.05 A
50% / 12 / 12.50 ± 11.5 A

SI Figure 4: Cumulative egg production for mummichog exposed to 50% oil refinery effluent (black diamonds) and reference mummichog (white diamonds) in freshwater with a pre-exposure phase (February 2009).

SI Figure 5: Linear regression of April 2007 average EROD activity per aquaria to total aquaria sediment PAH concentrations.

SI Figure 6: 2008 Total sediment PAH concentrations by site (R, reference site; 10m, 10 m below the current outfall; 800m, 800 mbelow the current outfall) and collection month (May, white bars; July, vertical bars; November, horizontal bars).

SI Figure 7: Average total PAHs across August, September, October and November per site (R, upstream reference site; 10m, 10 m below the current outfall; 800m, 800 m below the current outfall) in 2009.

SI Table 9: Perlyene concentrations and proportion of 5–ring PAHs by depth within the sediment cores taken at the current and old effluent outfall locations.

Core section (cm) / Perlyene concentration (mg/kg dw) / Perlyene-sum of 5-ring PAHs (%)
Current outfall / Old outfall / Current outfall / Old outfall
0-5 / - / 0.40 / 26 / 10
5-10 / 0.42 / 0.13 / 37 / 13
10-15 / 0.16 / 0.04 / 32 / 10
15-20 / 0.28 / 0.25 / 10 / 59
20-25 / 0.25 / 0.35 / 10 / 111

SI Figure 8: Polycyclic aromatic hydrocarbon (PAH) levels in effluent (parts-per trillion; ppt) samples taken in December 2006 and in sediments (parts-per-million; ppm) used in the April 2007 bioassay. Y-axis displays PAH concentration on a log scale.