1
Toxicology of Chemical Dispersants in Alaskan Whales
A PWSRCAC-funded project by The Wise Laboratory of Environmental and Genetic Toxicology
Maine Center for Toxicology and Environmental Health University of Southern Maine
Oil Spill Exposure Modes for Large Marine Mammals
- On the surface or - Within the water column
Photo: Ron Wooten/NMFS Photo: Dan Lawn ADEC
2
Dispersants ® Exposure mode within the water column
Goal of spreading the oil 3-dimensionally Used Corexit® EC9500A and Corexit® EC9527A
(U.S. Air Force, Tech. Sgt. Adrian Cadiz)
Dispersants move oil into the water column and to bottom and it also resurfaces... - Issues with how long it remains in the water column and on the bottom Surface oil continues to move to inshore waters & onto beaches or offshore
Dispersant Outcomes
® Key Issue: ® Few toxicological evaluations have been completed beyond the
large marine animals that died from immediate exposure
3
Study Objectives
® Objective 1. Measure the Cytotoxicity of Chemical Dispersants and Dispersed Oil on Whale Cells. Ø Cytotoxicity is the ability of an agent to induce cell death. This
objective will determine the cytotoxic effects of these agents in whale cells using assays for cell survival.
® Objective 2. Determine the Genotoxicity of Chemical Dispersants and Dispersed Oil on Whale DNA. Ø Genotoxicity is the ability of an agent to damage DNA. This
objective will determine the genotoxic effects of these agents in whale cells by considering chromosomal aberrations.
® Large toothed whale (30-50 tons; 40-60 feet)
® Global distribution ® Lifespan ~ 100 years
Alaskan Whales
Photo Courtesy of Ocean Alliance
Sperm Whale (Physeter macrocephalus)
® Large baleen whale (~36 tons; 40-55 feet)
® Global distribution, highly migratory
® Lifespan 50+ years
The Humpback Whale (Megaptera novaeangliae)
4
Why Study Chromosomes?
® Chromosomes package the DNA for proper cell division
® Damaged chromosomes can lead to disease states and reproductive problems
® Chromosome aberration studies are required tests by the FDA and EPA
http://www.ncbi.nlm.nih.gov
Marine Mammal Cell Culture
Tissue Piece
Cells
Biopsy
5
Normal Mitotic Cell with 2 Centrosomes
Controlled Toxicology Experiments in Wild Species
Cell Culture
Aberrant Mitotic Cell
with 4 Centrosomes
Mitotic Cell With 2 Broken Chromosomes
Normal Mitotic Cell With Chromosomes
DNA
Microtubules
Centrosomes
Chemical Dispersants
® Corexit 9500
® Corexit 9527
® Compositions (from EPA): Ø 1,2-Propanediol Ø Ethanol, 2-butoxy- (Corexit 9527 only) Ø Butanedioic acid, 2-sulfo-, 1,4-bis(2-
ethylhexyl) ester, sodium salt (1:1) Ø Sorbitan, mono-(9Z)-9-octadecenoate Ø Sorbitan, mono-(9Z)-9-octadecenoate,
poly(oxy-1,2-ethanediyl) derivs Ø Sorbitan, tri-(9Z)-9-octadecenoate,
poly(oxy-1,2-ethanediyl) derivs Ø 2-Propanol, 1-(2-butoxy-1-methylethoxy)- Ø Distillates (petroleum), hydrotreated light
6
Cytotoxicity Assay
® Seed plates with monolayer of cells
® Treat for 24 hours
® Harvest, re-plate, and allow cells to grow for ~2 weeks Ø Change media every 5 days
® Stain and count colonies
® Compare growth of treated cells with that of untreated controls
Control
Middle
High
Corexit 9500 and 9527 Cytotoxicity in Sperm Whale Skin Cells
0
20
40
60
80
100
120
0 0.02 0.04 0.06 0.08 0.1 0.12
Rel
ativ
e S
urvi
val (
Per
cent
of C
ontro
l)
Dispersant Treatment (% of total volume)
Sperm Whale - 9500
Sperm Whale - 9527
7
Corexit 9500 and 9527 Cytotoxicity in Humpback Whale Skin Cells
0
20
40
60
80
100
120
0 0.02 0.04 0.06 0.08 0.1 0.12
Rel
ativ
e S
urvi
val (
Per
cent
of C
ontro
l)
Dispersant Treatment (% of total volume)
Humpback Whale - 9500
Humpback Whale - 9527
Sperm Whale Cells Were Most Sensitive to Corexit 9500
0
20
40
60
80
100
120
0 0.02 0.04 0.06 0.08 0.1 0.12
Rel
ativ
e S
urvi
val (
Per
cent
of C
ontro
l)
Dispersant Treatment (% of total volume)
Humpback Whale - 9500 Humpback Whale - 9527 Sperm Whale - 9500 Sperm Whale - 9527
8
Chromosome Instability Assay (indicates structural abnormalities)
Chromatid lesion
Chromatid exchange
Isochromatid lesion
® Seed plates with layer of cells
® Treat for 24 hours
® Add colchicine for final hour to arrest cells in metaphase
® Harvest and prepare metaphase cells
® Stain and count metaphases
® A minimum of 100 metaphases analyzed per concentration
Basic Experimental Protocol
Corexit 9500 Induces Genotoxicity in Sperm Whale Skin Cells
0
10
20
30
0 0.005 0.025 0.05
Am
ount
of D
amag
e
9500 Concentration (ppm)
9500 Percent Damage 9500 Total Damage 9500+S9 Percent Damage 9500+S9 Total Damage
9
Dispersants are Genotoxic to Sperm Whale Cells but not Humpback Whale Cells
0
5
10
15
20
25
30
0 0.01 0.02 0.03 0.04 0.05 0.06
Per
cent
of D
amag
ed M
etap
hase
s
Dispersant Concentration (% of total volume)
Humpback Whale - 9500 Humpback Whale - 9527 Sperm Whale - 9500 Sperm Whale- 9527
Oil Mixtures ® WAF – Water Accommodated
Fraction of oil Ø Oil is stirred in water overnight Ø The water portion of the mixture is
used for treatments ® CEWAF – Chemically Enhanced
Water Accommodated Fraction of oil Ø Oil is stirred in water overnight Ø Dispersant added, stirred 1h more Ø The water portion of the mixture is
used for treatments http://www.usm.edu/gcrl/toxicology/toxicology.oil.research.php
10
PAH Contents of WAF and CEWAF
Compound WAF
Concentrations ng/L
CEWAF Concentrations
Ng/L 1,4,6,7-Tetramethylnaphthalene 7305 ± 615 309857 ± 29277 1-Methylnaphthalene 65900 ± 4100 2981429 ± 253409 1-Methylphenanthrene 10730 ± 1970 475714 ± 42110 2,3,5-Trimethylnaphthalene 20250 ± 3250 1140000 ± 106077 2,3,6-Trimethylnaphthalene 28150 ± 4250 1592857 ± 142590 2,6-Dimethylnaphthalene 26600 ± 5600 2015714 ± 161759 2-Methylanthracene 1006 ± 324 12791 ± 1784 2-Methylnaphthalene 69600 ± 7400 3915714 ± 332636 Acenaphthene 1345 ± 155 42243 ± 4007 Acenaphthylene 613 ± 89 5669 ± 781 Anthracene 609 ± 9 14414 ± 1292 Benz[a]anthracene 547 ± 141.5 13754 ± 1691 Benzo[a]pyrene 720 ± 96 13336 ± 1218 Benzo[b]fluoranthene 495 ± 83 11806 ± 1203 Benzo[e]pyrene 798 ± 26 27529 ± 2929 Benzo[j,k]fluoranthenes 535 ± 85.5 10531 ± 1078 Biphenyl 12000 ± 1100 503714 ± 39511 C1-Benzo[a]anthracenes/Chrysenes 5935 ± 945 224714 ± 21089 C1-Fluoranthenes/Pyrenes 15050 ± 1350 470286 ± 42803 C2 Phenanthrenes/Anthracenes 45400 ± 4800 1875714 ± 168634 C2-Benzo[a]anthracenes/Chrysenes 6740 ± 1330 239571 ± 25775 C2-Biphenyls 32800 ± 200 450000 ± 39019 C2-Dibenzothiophenes 49800 ± 9000 1870000 ± 161629
Compound (cont’d) WAF
Concentrations ng/L
CEWAF Concentrations
Ng/L C2-Dibenzothiophenes 49800 ± 9000 1870000 ± 161629 C2-Fluoranthenes/Pyrenes 23600 ± 4700 747857 ± 64394 C2-Fluorenes 26850 ± 4850 1039429 ± 86076 C3-Dibenzothiophenes 42050 ± 9250 1417143 ± 110899 C3-Fluoranthenes/Pyrenes 11545 ± 1855 380429 ± 31620 C3-Fluorenes 32200 ± 3700 1187143 ± 73799 C3-Naphthalenes 114500 ± 12500 5885714 ± 519839 C3-Phenanthrenes/Anthracenes 36600 ± 3600 1345714 ± 113155 C4-Dibenzothiophenes 24700 ± 3400 669429 ± 36708 C4-Naphthalenes 62300 ± 4200 2478571 ± 232394 C4-Phenanthrenes/Anthracenes 69300 ± 8300 1891429 ± 166239 Chrysene 2450 ± 790 111114 ± 10845 Dibenz[a,h]anthracene 243 ± 11 6956 ± 556 Dibenzothiophene 11685 ± 2015 522000 ± 43224 Fluoranthene 955 ± 3.5 11327 ± 1200 Fluorene 5260 ± 820 227571 ± 18178 Indeno[1,2,3-cd]pyrene 632 ± 53 6059 ± 580 Naphthalene 100500 ± 500 2288571 ± 187812 Perylene 743 ± 94.5 14314 ± 1392 Phenanthrene 16900 ± 3000 740714 ± 64736 Pyrene 1079 ± 161 29286 ± 2435 Total 987018 ± 108952 39218129 ± 3287878
40x more PAHs present in dispersed oil!
CEWAF is Cytotoxic in Sperm Whale Skin Cells but WAF is Not
0
20
40
60
80
100
120
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Rel
ativ
e S
urvi
val (
Per
cent
of C
ontro
l)
Total PAH level ug/L
WAF WAF + S9 CEWAF CEWAF + S9
11
CEWAF is Cytotoxic in Humpback Whale Skin Cells but WAF is Not
0
20
40
60
80
100
120
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Rel
ativ
e S
urvi
val (
Per
cent
of C
ontro
l)
Total PAH Level (ug/L)
WAF
WAF + S9
CEWAF
CEWAF + S9
WAF is Slightly Genotoxic to Sperm Whale Cells but Not Humpback Whale Cells
0
5
10
15
20
25
30
0 20 40 60 80 100 120
Per
cent
of D
amag
e
Total PAH Level (ug/L)
Sperm whale: WAF Sperm whale: WAF + S9 Humpback whale: WAF Humpback whale: WAF + S9
12
CEWAF is Genotoxic in Sperm Whale Cells but not Humpback Whale Cells
0
5
10
15
20
25
30
0 500 1000 1500 2000 2500
Per
cent
of D
amge
Total PAH Level (ug/L)
Sperm whale: CEWAF Sperm whale: CEWAF + S9 Humpback whale: CEWAF Humpback whale: CEWAF + S9
Conclusions
® Data indicate toxicity is likely Ø The dispersant concentrations experienced in spills are
high enough to kill whale cells and damage DNA Ø Dispersant mixed with oil increases the levels of toxic
PAHs Ø Dispersant mixed with oil is more cytotoxic and genotoxic
to whale cells Ø Further studies into the mechanisms of toxicity and with
additional tissues and species is warranted