ACCESS NUMBER: 30660-19914 

STUDY TITLE: The Effects of Simultaneous Exposure to Petroleum Hydrocarbons, 

Hypoxia and Prior exposure on the Tolerance and Sublethal Responses of Marine 

Animals 

REPORT TITLE: Effects of Simultaneous Exposure to Petroleum Hydrocarbons, 

Hypoxia and Prior exposure on the Tolerance and Sublethal Responses of Marine 

Animals 

CONTRACT NUMBER: 14-35-0001-30660-19914 

SPONSORING OCS REGION: Gulf of Mexico OCS Region 

APPLICABLE PLANNING AREA: Central Gulf of Mexico 

FISCAL YEARS OF PROJECT FUNDING: 1993-1995 

COMPLETION DATE OF REPORT: September 1998 

COSTS: FY 1993-FY 1995: $176,268 

CUMULATIVE PROJECT COST: $176,268 

PROJECT MANAGER: William B. Stickle Jr. 

AFFILIATION: Louisiana State University 

ADDRESS: Department of Biological Sciences, 202 Life Science Building, Baton 

Rouge, LA 70803-1715 

PRINCIPAL INVESTIGATOR: William B. Stickle Jr. 

KEYWORDS: Water soluble fraction, crude oil, hypoxia, Gulf killifish, blue crab, 

tolerance, growth, EROD, RNA:DNA ratio, synergism

BACKGROUND: Coastal Louisiana has been impacted by oil field activities for the last 

half century and the waters over the continental shelf west of the Mississippi River delta, 

are also subject to extensive periods of hypoxia during the summer months. Marine 

and estuarine fish are sensitive to exposure to the water-soluble fraction (WSF) of crude 

oil but their tolerance doesn’t change much after 12-24h exposure because of the 

presence of an inducible cytochrome P-450 enzyme system for metabolizing aromatic 

hydrocarbons. In contrast, invertebrates tolerate high concentrations of the WSF for 

short time periods, but their tolerance declines during long-term exposure. The 

determination of long term tolerance and sublethal biomarkers have not been used to 

assess the effects of gradients of oxygen tension and the WSF of crude oil on marine 

and estuarine fauna. 
ACCESS NUMBER: 30660-19914 

OBJECTIVES: The intent of this project was to study the combined effects of the WSF

of South Louisiana crude oil and hypoxia on the tolerance of the Gulf killifish, and 

juvenile blue crabs and lesser blue crabs. In addition, the affects of chronic exposure of 

these species to sublethal concentrations of the WSF of crude oil on the tolerance, 

RNA:DNA ratios and killifish condition and induction of cytochrome P-450 enzymes 

were compared under hypoxic conditions with the responses of these species from 

more pristine locales. There have been no studies investigating these interactions to 

date.

DESCRIPTION: Tolerance was determined over 28 days for killifish, juvenile blue crabs 

and juvenile lesser blue crabs exposed to five levels of hypoxia and six concentrations 

of the WSF of South Louisiana crude oil. Sublethal responses were determined on 

eight animals per time-concentration combination at three WSF concentrations on days 

0,1, 7, 14, and 28 of exposure. RNA:DNA analyses were determined on all animals and 

EROD analysis of cytochrome P-450 activation as well as condition index and liversomatic index was also determined on Gulf killifish.

SIGNIFICANT CONCLUSIONS: The hypoxia tolerance of the Gulf killifish was higher 

than that of the lesser blue crab and the blue crab. Minimal site differences existed in 

hypoxia tolerance. There was no difference in the 28-day tolerance of blue crabs to the 

WSF of South Louisiana crude oil exposed to normoxia or hypoxia. The Gulf killifish 

from the produced water site in Louisiana was more tolerant of the WSF than those 

from Florida and fish exposed to the WSF under normoxia were more tolerant than 

those exposed under hypoxia. Induction of EROD activity occurred in a dose related 

manner in Fundulus grandis from both locations exposed to the WSF under hypoxia (75 

Torr O2

)

, peaked on the seventh day of exposure and was significantly higher in fish 

exposed to 35?g oil/liter from the produced water site than in killifish from Florida. 

RNA:DNA ratios are not a particularly sensitive indicator of sublethal WSF stress in 

either Gulf killifish or the blue crab. EROD activity, an indicator of cytochrome p-450 

induction, increases in a dose related manner in the liver of Fundulus grandis from both 

locations, peaks on the 7th day of exposure and is higher in fish from the produced 

water site. WSF exposure induced hepatic tissue growth in both populations. Weight 

loss was greatest in fish from Florida. Increases in liver size and RNA:DNA ratios were 

noted with EROD induction. Stimulation of growth in white muscle tissue by WSF 

exposure was observed in the Louisiana population. The condition factor (K) of the 

Florida population decreased more than the Louisiana population. Differences in (K) 

between hypoxia and hypoxia + WSF treatments were minor, suggesting small changes 

in condition would be sufficient to affect survival in F. grandis. Therefore, Fundulus 

grandis, from the chronically polluted site are more tolerant of exposure to the WSF of 

South Louisiana crude oil and exhibit an increased rate of EROD activity compared with 

the Florida population. However, these biological differences in response patterns are 

minor. 

STUDY RESULTS: Chronic exposure to sublethal concentrations of the WSF of crude 

oil leads to increased tolerance and condition of the Gulf killifish when exposed to the 

WSF of South Louisiana crude oil. Exposure to the WSF under hypoxia produces slight 
ACCESS NUMBER: 30660-19914 

increases in sensitivity to crude oil in comparison with exposure under normoxia. Blue 

crabs and lesser blue crabs are not sensitive to the additive effects of hypoxia and the 

WSF of South Louisiana crude oil.

STUDY PRODUCTS: Stickle, W.B. The Effects of Simultaneous Exposure to 

Petroleum Hydrocarbons, Hypoxia and Prior Exposure on the Tolerance and Sublethal 

Responses of Marine Animals: Final Report. OCS Study MMS 2002-009. U.S. Dept. of 

the Interior, Minerals Management Service, Gulf of Mexico OCS Region, New Orleans, 

Louisiana, 26 pp.