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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

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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

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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)

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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

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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

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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

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0 0.02 0.04 0.06 0.08 0.1 0.12

Rel

ativ

e S

urvi

val (

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cent

of C

ontro

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Dispersant Treatment (% of total volume)

Sperm Whale - 9500

Sperm Whale - 9527

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Corexit 9500 and 9527 Cytotoxicity in Humpback Whale Skin Cells

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0 0.02 0.04 0.06 0.08 0.1 0.12

Rel

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Dispersant Treatment (% of total volume)

Humpback Whale - 9500

Humpback Whale - 9527

Sperm Whale Cells Were Most Sensitive to Corexit 9500

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0 0.02 0.04 0.06 0.08 0.1 0.12

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Dispersant Treatment (% of total volume)

Humpback Whale - 9500 Humpback Whale - 9527 Sperm Whale - 9500 Sperm Whale - 9527

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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

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10

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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

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Dispersants are Genotoxic to Sperm Whale Cells but not Humpback Whale Cells

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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

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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

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Total PAH level ug/L

WAF WAF + S9 CEWAF CEWAF + S9

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CEWAF is Cytotoxic in Humpback Whale Skin Cells but WAF is Not

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Total PAH Level (ug/L)

WAF

WAF + S9

CEWAF

CEWAF + S9

WAF is Slightly Genotoxic to Sperm Whale Cells but Not Humpback Whale Cells

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0 20 40 60 80 100 120

Per

cent

of D

amag

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Total PAH Level (ug/L)

Sperm whale: WAF Sperm whale: WAF + S9 Humpback whale: WAF Humpback whale: WAF + S9

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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

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Questions?