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: [email protected] 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 Effluent Concentrations* Endpoints Duration (ppt) May 2006 16 Control, 25%, 50% Fish and organ weights 7d and indices, Steroid production, EROD induction October 2006 0 Control, 25%, 50%, 100% Fish and organ weights 14d and indices, EROD induction April 2007 16 Control-Control, Control-25%, Control- Fish and organ weights 14d 50%, Exposed-Control, Exposed-25%, and indices, Steroid Exposed 50% production, EROD induction July 2007 0 Control-Control, Control-25%, Control- Fish and organ weights 14d 50%, Exposed-Control, Exposed-25%, and indices, Steroid Exposed 50% production, EROD induction February 2009 0 Control, 50% Fish and organ weights 35d and indices, Steroid production, EROD induction, egg production *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/ Sites Collection Month method 2008 May, July, 1 Little River Reference; Ekman Dredge November 10m below outfall; 800m below outfall. 2009 August, 3 Little River Reference; Ekman Dredge September, 10m below outfall; 800m below October, outfall. November 2010 May, June, July, 3 Little River Reference; Ekman Dredge August, 10m below outfall; 800m below September, outfall; Older outfall site above October current outfall. 2010 September 1 Little River Reference; Sediment Corer 10m below outfall; Older outfall site above current outfall 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 m below 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 the current outfall (10m) and 800 m below the current 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 fresh water (October 2006). * indicate extreme values. Different letters indicate statistical significance (P<0.05). SI Table 1: 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 n Testosterone (ng/mL) Min Max Effluent(%) Sediment 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 m below 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 10000.00 effluent (ppt) sediment (ppm)

1000.00 s l e v e l

100.00 H A P

10.00

1.00

PAHs

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.