Supplementary informationMultilevel responses of adult zebrafish to crude and chemically dispersed oil exposure

Supplementary information

Multilevel responses of adult zebrafish to crude and chemically dispersed oil exposure

Ada Esteban-Sánchez^a,#, Sarah Johann^b,#, Dennis Bilbao^c, Ailette Prieto^c, Henner Hollert^b Thomas-B.

Seiler^d,e, Amaia Orbea^a*.

aCBET research group, Dept. Zoology and Animal Cell Biology; Research Centre for Experimental Marine Biology and Biotechnology (PIE) & Science and Technology Faculty (STF), University of the Basque Country (UPV/EHU), Spain;

bDept. Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Germany

cIBeA research group, Dept. Analytical Chemistry, PIE & STF, UPV/EHU, Spain;

d Dept Ecosystem Analysis, Institute for Environmental Research (Biology V), RWTH Aachen University, Germany;

e Ruhr District Institute of Hygiene, Gelsenkirchen, Germany

#First authors, these authors have contributed equally to this work.

*Corresponding author: amaia.orbea@ehu.eus

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1. Chemical analysis of target PAHs in exposure solution and carcasses

Table S1. List of Abbreviations and limits of detection of target PAHs analyzed in exposure solutions and carcasses.

PAH Abbreviation

Limit of detection – water (ng/L)

Limit of detection – fish (ng/g)

Phenanthrene Phen 1.0 9.0

Anthracene Ant 0.4 1.8

Fluoranthene Flr 1.1 6.0

Pyrene Pyr 1.3 4.2

11H-benzo[a]fluorene 11H-B[a]Flu 1.7 4.6 11H-benzo[b]fluorene 11H-B[b]Flu 1.3 5.1 Benzo[a]anthracene B[a]A

1.8 4.3

Chrysene Chr 1.8

Benzo[b]fluoranthene B[b]Flr 3.2 3.6

Benzo[k]fluoranthene B[k]Flr 2.9 3.0

Benzo[a]pyrene B[a]P 2.3 3.3

Benzo[e]pyrene B[e]P 2.8 3.5

Indeno[1,2,3-cd]pyrene Ind[1,2,3-cd]P 10.5 3.0

Dibenzo[a,h]anthracene D[a,h]A 8.6 7.2

Benzo[g,h,i]perylene B[g,h,i]Per 4.5 5.4

Dibenzo[a,e]pyrene D[a,e]P 5.2 6.1

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Table S2. Concentrations of target PAHs (ng/L) in 5% WAFOIL exposure media over two cycles (exposure media was changed every 72 h) and analyzed using GC-MS. Values below detection limits are expressed as BDL.

Phen Ant Flr Pyr11H-

B[a]Flu

11H- B[b]Flu

B[a]A + Chr

6 h 160.6 BDL 11.3 53.5 11.5 BDL BDL

12 h 91.2 BDL BDL 41.1 BDL BDL BDL

24 h 69.2 BDL BDL 34.5 BDL BDL BDL

36 h 37.5 BDL BDL 9.7 BDL BDL BDL

48 h 19.6 BDL BDL BDL BDL BDL BDL

60 h 20.1 BDL BDL BDL BDL BDL BDL

72 h 16.8 BDL BDL BDL BDL BDL BDL

78 h 135.1 BDL 11.7 52.4 12.7 BDL BDL

84 h 53.5 BDL BDL 22.7 BDL BDL BDL

96 h 57.2 BDL BDL 17.1 BDL BDL BDL

108 h 24.6 BDL BDL BDL BDL BDL BDL

120 h 26.0 BDL BDL BDL BDL BDL BDL

132 h 18.3 BDL BDL BDL BDL BDL BDL

144 h 18.4 BDL BDL BDL BDL BDL BDL

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39 40

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Table S3. Concentrations of target PAHs (ng/L) in 25% WAFOIL exposure media over two cycles (exposure media was changed each 72 h) and analyzed using GC-MS. Values below detection limits are expressed as BDL. Note that the value at 6 h for the first cycle is missing due to technical problems.

Phen Ant Flr Pyr 11H-

B[a]Flu

11H- B[b]Flu

12 h 1480.8 BDL 87.5 271.5 168.1 68.2

24 h 778.9 BDL 15.8 208.9 22.9 12.9

36 h 396.5 BDL BDL 68.4 BDL BDL

48 h 354.3 BDL BDL 46.3 BDL BDL

60 h 256.6 BDL BDL 27.0 BDL BDL

72 h 200.3 BDL BDL 18.1 BDL BDL

78 h 1289.9 BDL 30.4 182.3 47.5 26.5

84 h 1022.5 BDL 25.0 270.1 34.5 20.5

96 h 623.8 BDL 13.5 176.9 18.0 10.2

108 h 338.2 BDL BDL 59.0 BDL BDL

120 h 218.2 BDL BDL 32.9 BDL BDL

132 h 610.7 BDL BDL 46.2 BDL BDL

144 h 29.6 BDL BDL 19.8 BDL BDL

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Table S4. Concentrations of target PAHs (ng/L) in 5% WAFOIL+D exposure media over two cycles (exposure media was changed each 72 h) and analyzed using GC- MS. Values below detection limits are expressed as BDL. Note that the value at 24 h for the first cycle and at 72 h for the second cycle are missing due to technical problems.

Phen Ant Flr Pyr 11H-

B[a]Flu

11H- B[b]Flu

B[a]A

+ Chr B[b]Flr B[k]Flr B[a]P B[e]P Ind[1,2,3-

cd]P D[a,h]A B[g,h,i]Per

6 h 2511.9 BDL 420.0 287.5 1119.9 529.4 719.6 80.2 15.5 107.5 37.4 BDL BDL BDL

12 h 1627.1 BDL 305.7 234.2 742.2 388.9 489.1 68.1 10.5 91.3 14.3 BDL BDL BDL

36 h 397.8 BDL 244.3 143.8 704.9 315.8 308.7 74.9 BDL 85.0 17.1 BDL BDL 16.1

48 h 133.9 BDL 226.1 148.4 647.5 292.3 356.0 67.1 BDL 81.9 14.1 BDL BDL 23.7

60 h BDL BDL 184.4 112.4 586.0 245.3 274.5 58.2 BDL 72.5 8.2 BDL 15.8 54.2

72 h BDL BDL 147.1 88.0 464.4 202.6 201.9 51.1 BDL 59.9 12.0 BDL 22.2 40.3

78 h 2836.9 BDL 408.7 298.4 914.6 515.6 478.9 68.4 BDL 93.8 27.3 BDL BDL 16.9

84 h 2767.8 BDL 396.5 276.8 1035.0 513.2 535.6 85.6 15.8 112.8 32.4 BDL BDL BDL

96 h 2706.1 BDL 376.3 260.5 1011.1 466.1 528.5 105.9 13.9 127.5 29.8 BDL BDL BDL

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h 1542.1 BDL 281.9 190.3 814.8 379.5 462.8 74.7 9.8 92.4 18.0 BDL BDL BDL

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h BDL BDL 288.2 204.1 843.8 376.5 372.7 82.3 BDL 94.1 18.7 BDL BDL 26.5

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h BDL BDL 163.6 111.8 520.2 204.9 262.8 44.4 BDL 48.6 10.1 BDL BDL BDL

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Table S5. Concentrations of PAHs (ng/g) in fish carcass tissue after 21 days of exposure to WAF dilutions of NNS crude oil. Fish were exposed to WAF dilutions of crude oil only (WAFOIL) and chemically dispersed crude oil (FINASOL OSR 52, WAFOIL+D). Values below detection limits are expressed as BDL.

Phen Ant Flr Pyr B[a]Flu B[b]Flu B[a]

A + Chr

B[b]Flr B[k]Flr B[a]P B[e]P Ind[1,2,3-

cd]P B[g,h,i]Per

Control 83.4 BDL 23.0 42.8 BDL BDL 283.5 BDL BDL BDL BDL 0.8 162.2

5% WAFOIL 52.3 BDL 23.5 123.1 BDL BDL 198.0 BDL BDL BDL BDL 1.7 106.4

25% WAFOIL 229.

0 35.8 17.4 103.2 BDL BDL

235.9 BDL BDL BDL BDL

1.3 47.8

5% WAFOIL+D 782.

1 BDL 238.

4 BDL BDL BDL

604.0 BDL BDL BDL BDL

BDL 215.2

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2. Relative induction of biochemical biomarkers of exposure in adult zebrafish tissues

Table S6. Relative biochemical biomarker activity in brain, liver and gills of adult zebrafish after short- (3 d) and long-term (21 d) exposure to WAF dilutions of NNS crude oil. Fish were exposed to WAF dilutions of crude oil only (WAFOIL) and chemically dispersed (FINASOL OSR 52) crude oil (WAFOIL+D). Data represent the mean relative induction (IF) of acetylcholinesterase (AChE), 7-ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST) and catalase (CAT) in tissue homogenates of 4 replicates per treatment (1 replicate = pool of 3 individual tissues) compared to unexposed control with standard deviation.

3 days 21 days

Control 5% WAFOIL 25% WAFOIL 5% WAFOIL+D Control 5% WAFOIL 25% WAFOIL 5% WAFOIL+D

mean IF SD mean IF SD mean IF SD mean IF SD mean IF SD mean IF SD mean IF SD mean IF SD

EROD liver

1.00 0.38 0.98 .056 0.46 0.14 10.18 7.45 1.00 0.18 1.20 1.28 0.49 0.37 5.33 1.88

EROD gills

1.00 0.16 0.89 0.23 0.86 0.35 2.82 0.52 1.00 0.50 0,96 0.13 0.53 0.05 1.64 0.42

GST liver

1.00 0.15 0.81 0.05 1.06 0.30 1.03 0.32 1.00 0.11 1.40 0.16 1.30 0.12 1.30 0.05

GST gills

1.00 0.13 0.67 0.12 0.93 0.19 1.75 0.13 1.00 0.16 1.10 0.08 0.95 0.13 1.90 0.34

CAT liver

1.00 0.37 0.78 0.10 0.91 0.25 0.82 0.30 1.00 0.18 1.99 0.80 0.94 0.24 0.89 0.18

CAT gills

1.00 0.23 0.94 0.14 0.96 0.16 0.96 0.13 1.00 0.28 2.30 1.49 1.93 1.11 2.47 0.21

AChE brain

1.00 0.16 1.21 0.14 0.76 0.13 0.75 0.12 1.00 0.10 1.47 0.47 0.78 0.16 0.66 0.06

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3. Relative induction of micronuclei in peripheral blood samples of adult zebrafish

Table S7. Relative micronucleus frequency (IF) in erythrocytes from adult zebrafish blood smears after short- (3 d) and long-term (21 d) exposure to WAF-dilutions of an NNS crude oil compared to unexposed to control. Data represent the mean IF of 7-10 individual fish exposed to WAF dilutions of crude oil only (WAFOIL) and chemically dispersed crude oil (FINASOL OSR 52, WAFOIL+D) with standard deviation (SD).

3 days 21 days

mean IF SD mean IF SD

5 % WAFOIL 1.21 0.72 0.93 0.55

25 % WAFOIL 0.83 0.46 1.20 0.84

5 % WAFOIL+D 0.82 0.40 2.20 1.06

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