ACCESS NUMBER: 30470 (95-0020) 

STUDY TITLE: University Research Initiative on the Effects of Offshore Petroleum 

Development in the Gulf of Mexico 

REPORT TITLE: Bioavailability and Genotoxicity of Produced Water Discharges 

Associated with Offshore Drilling Operations 

CONTRACT NUMBER: 14-35-0001-30470 

SPONSORING OCS REGION: Gulf of Mexico OCS Region 

APPLICABLE PLANNING AREA: Central 

FISCAL YEARS OF PROJECT FUNDING: 1990-1993 

COMPLETION DATE OF REPORT: July 1994 

COST: FY 1991-1994: $196,000 

PROJECT MANAGER: Dr. Paul Sammarco 

AFFILIATION: Louisiana Universities Marine Consortium 

ADDRESS: 8124 Highway 56, Chauvin, Louisiana 70344 

PRINCIPAL INVESTIGATORS*: G.W. Winston, J.C. Means, L. Riley, S. Dobias, Z. Yan 

KEY WORDS: Central Gulf, Louisiana, biomarkers, mixed function oxidase (MFO), 

cytochromes P450, oxidative stress, polynuclear aromatic hydrocarbons, sediments, 

demersal fish, fish, sea worms, oysters, sea anemones, oxyradicals, EROD, liver, 

contaminants, exposure, pelagic fish, induction, enzymes, biochemical markers, 

benthic, produced waters, oil rigs 

BACKGROUND: The major discharges associated with offshore oil and gas production 

are drilling fluids, drill cuttings, and produced water. During the production of oil and 

gas, water that is trapped within permeable petroleum-bearing sedimentary rocks is 

brought to the surface, carrying with it traces of crude oil, drilling fluids and other 

geological materials (Boesch et al., 1988). This water, called produced water, may 

contain elevated levels of various inorganic (trace metals) and organic (NAH-PAH) 

substances of a potentially toxic nature. This report presents results of laboratory and 

field studies directed by the co-principal investigators. 

OBJECTIVES: The potential impacts of these discharges on several test organisms 

were explore through studies of: 1) the bioavailability of dissolved and sediment-bound 

normal, alkylated and heterocyclic aromatic hydrocarbons to benthic invertebrates and 

demersal fish, eggs and larvae; 2) the availability of both benthic and pelagic organisms 
ACCESS NUMBER: 30470 (95-0020) 

to metabolize these compounds; and 3) the genotoxicity of the compounds and 

metabolites in benthic organisms and demersal fish. 

DESCRIPTION: The study locates at Bayou Rigou, Pass Fourchon (PF) and East 

Timbalier Island (ETI) were used. The daily discharge of OCS produced water at PF 

facility is ca. 18,000 bbl/day (Boesch et al., 1988). Bottom sediments in the dean-end 

portion of PF are typically fine grained. The ETI study focused on a dredged access 

channel from East Timbalier Bay to ETI. Many discharge points are located in this area. 

Total daily discharge of both OCS and inland state produced waters at the ETI site is 

ca. 69,000 bbl/day (Boesch et al., 1988). The dredged channel exhibit slow flow relative 

to the shallow open bay and bottom sediments are typically fine sands. 

Production water discharges from oil-water separation facilities located in nearshore 

estuarine waterways were analyzed for volatile and semi-volatile organic hydrocarbons 

by a purge and trap/gas chromatographic technique. Production water samples were 

extracted for base/neutral, acid extractables by liquid-liquid partitioning (EPA METHOD 

625) and analyzed by GC/MS. 

Sediment samples collected near the discharge sites were analyzed for NAH-PAH by 

methanol-hexane extraction with sonication. Extracts were fractionated into a nonpolar, semi-polar and polar fraction on silica gel. Final fractions were analyzed by 

GC/MS. Field collected produced water and surface water samples were serially diluted 

(0, 25, 50, 75, and 90%) to achieve a concentration gradient for use in the genotoxicity 

bioassays. 

Fish liver microsomes were isolated by differential centrifugation according to standard 

techniques in the literature (Winston et al., 1989). MFO activities were measured in 

0.1M potassium pH phosphate buffer, pH 4, 10 mM glucose-phosphate, 10 mM MgCl2, 

0.4 mM NADP and ~ 1-2 mg of microsomal protein in 1 ml. Specific content of 

cytochrome P450 was measured in dithionite-reduced, CO-liganded microsomes 

(Omura and Sato, 1964) and quantified from the extinction coefficient (e) 91 mM-1 cm1. NAD(P)H-cytochrome c (P450) reductase was measured by monitoring reduction of 

cytochrome c at 550nm (Phillips and Langdon, 1962) and quantified from e = 18.5 mM-1 

cm-1 for ferrocytochrome c. Oxidation of the hydroxyl radical scavenger, KMBA, by 

oxyradicals ethylene production, which can be measured by in the headspace of rubber 

septa-sealed reaction vessels by gas chromatography (Winston et al., 1984). 

P450 contents and reductase activities were determined from field and laboratory 

samples and correlated with levels and types of xenobiotic compounds founds to be 

present in the water, the indigenous sediments, the digestive tract and bile as well as 

the genotoxic endpoints described above. 7-Ethoxyresorufin-o-deethylase (EROD) was 

determined spectrofluorometrically. 

Superoxide dismutase was determined with the SOD-525 Assay of Bioxytech, Cedex, 

France and carried out as described in the manufacturer's instructions. Catalase 

activities were determined as described in Claiborne (1985). 
ACCESS NUMBER: 30470 (95-0020) 

SIGNIFICANT CONCLUSIONS: EROD was induced in Fundulus grandis by 3-

methylcholanthrene (3MC), ethylmethanesulphonate (EMS), and cyclophosphamid 

(CP). Field-exposed organisms yielded much lower EROD values than experimentally 

induced levels reported in microsomes of other fish species. Clear patterns of EROD 

induction were not evident in fish samples from contaminated areas. Liver microsomes 

of all fish, regardless of the area from which they were sampled, exhibited highly 

variable EROD activities, which in some cases showed overlap. Obvious correlations 

were not evident between antioxidant enzyme activities and distance from point sources 

of offshore contamination, contaminated coastal sites vs. reference sites, species, 

collection time, depth of habitat, sediment-dwelling fish vs. demersal fish and between 

offshore site. Microsomal P450 was found to be mostly denatured or highly 

contaminated with other interfering CO-binding chromaphores. EROD, P450 reductase 

and CO-binding data should be reconciled before tenable conclusions of the effects of 

pollutant outfall can be made. Mutagen mobilization into co-solvent extracts were 

detectable by the Salmonella typhimurium assay, with tester strain varying in sensitivity. 

Promutagens were detectable in all extracts, via activation with Arochlor-induced rat 

liver S9. Mutagenicity varied among co-solvent concentrations, perhaps reflecting 

mobilization of different arrays of compounds. The umu gene induction assay, which 

uses S. typhimurium 1535/psk1002, did not detect mutagens in the extracts whether 

assays were conducted in the presence or absence of fish liver S9 or microsomes. 

Isomer specific analyses of bioaccumulated petroleum aromatic hydrocarbons in tissues 

of aquatic organisms yields information on the relative bioavailability of these 

contaminants, which rapidly enter biological foodwebs in aquatic systems. This 

suggests that contaminated sediments remain as bioavailable toxic substances even 

after discharges are suspended. 

STUDY RESULTS: Laboratory studies indicated induction of ethoxyresorufin Odeethylase (EROD) in the marine work, Nereis virens exposed to petroleumcontaminated sediment from Pass Fourchon, and oil production site in coastal 

Louisiana. EROD activity was detected in microsomal fractions extracted from excised 

gut tissue. EROD in worms exposed in 20% sediment was ~14-fold higher than 

controls after 7 days; after 14 days the increase as ~20-fold over controls. In 50% 

sediment as 27-fold induction of EROD was seen after 7 days, and after 14 days 

induction was ~!132-fold. These data could not be reproduced in two subsequent 

experiments, thus, our data needs to be reconciled with those of Fries and Lee (1984), 

who showed EROD induction in gut tissues of N. virens when exposed to 

benzo(a)pyrene via food. Western blots of the worm gut microsomes probed with rabbit 

anti-trout CYP1A1 polyclonal antibody did not indicate the presence of proteins with 

common epitopes of CYP1A1 proteins. Laboratory challenge experiments were 

conducted to evaluate EROD induction in adults, larvae, and embryos of the killifish 

exposed to CP and EMS, two benchmark mutagens with known genotoxic effects on 

Fundulus, and 3MC, a classical MFO inducer and model carcinogen in mammalian 

studies. 3MC exposure induced EROD to 2.8, 1.9, and 2.2 nmol/min/mg in embryos, 

larvae, and adults, respectively. EMS induced EROD to 1.50, 1.60, and 0.57 

nmol/min/mg. CP induced to 1.25, 1.20, and 0.18 nmol/min/mg. 
ACCESS NUMBER: 30470 (95-0020) 

Bottom-dwelling feral fishes were periodically collected from offshore petroleum 

production platforms. S9 and microsomal fractions were prepared from pooled or 

individual livers, and assayed for EROD activity. Field exposed fishes yielded much 

lower EROD values than in microsomes from experimentally induced fish reported in 

the literature. S9 preparations were prone to inhibitory effects and a larger base of field 

data is needed to validate the use of EROD for detecting exposure history and 

qualitatively demonstrating bioavailabity of dissolved and sediment-bound chemicals. 

Collected trips to offshore and coastal marsh areas yielded fish specimens from both 

contaminated and control (pristine) locations. Generally, microsomes from the livers of 

both control fish and fish from contaminated sites showed very low or no detectable 

EROD activity. Clear patterns of EROD activities were not evident in fish sampled from 

contaminated areas. The data point to some recognized problems in biomarker 

research; fish from large areas display temporal and spatial variability; biochemical 

responses of fishes are variable as a function of species, sex, gonadal status, season, 

temperature, and nutrition. Field studies may result in disconcerting data, even though 

controlled laboratory studies are very predictable. 

Oxygen radicals (ROS) are produced as a normal part of all aerobic life. Metals and 

organic chemicals increase ROS production in biological systems with accompanying 

protein degradation, enzyme inactivation, lipid peroxidation, DNA damage and cell 

death. In the few cases where antioxidant enzymes correlate with distance from a 

platform an inverse relationship was noted. This might relate to depletion of antioxidant 

defenses under conditions of high xenobiotic exposure. Obvious correlations were not 

evident between catalase or SOD activity and distance from offshore platforms, 

contaminated coastal vs. reference sites, species, collection time, depth of habitat and 

sediment-dwelling fish vs. demersal fish. Microsomal flavoprotein reductases are ratelimiting components of NAD(P)H-dependent MFO and also active loci of ROS 

production. NAD(P)H-dependent cytochrome c reduction was studied to indicate 

ranges of intra and inter species activities and the effects of field site environments on 

those activities. The specific content of cytochromes P450 were also measured to 

determine the efficacy of microsomal preparations for use in FMO studies. NAD(P)Hcytochrome c reductase activities were measured in liver microsomes of fish sampled 

from various offshore sites. NADH-cytochrome c reductase ranged from below 

detectable limits in Bay Whiff caught at ST 52 to ~1000 nmol/min/mg protein in catfish 

caught at ST 34. NADH-dependent activities were consistently higher in catfish 

(870±170 nmol/min/mg, n=7), irrespective of the distance from the platform at which 

they were caught. Fringed flounder had the next highest activities with NADH (83±44 

nmol/min/mg, n=6). Fringed flounder from ST 36, 37, 52, 53 and 54 had notably lower 

NADH-cytochrome c reductase activities than those caught from ST 34. NADPHdependent activities ranged from 0.2 nmol/min/mg in Bay Whiff caught at ST 53 and 55 

to 300 nmol/min/mg in catfish caught at ST 34. Microsomal P450 was found to be 

mostly denatured or highly contaminated with other interfering CO-binding 

chromaphores. EROD, P450 reductase and CO-binding data must be reconciled before 

tenable conclusions of exposure effects can be made. The large variability among the 

different species, lack of correlation between MFO and field parameters, and 
ACCESS NUMBER: 30470 (95-0020) 

denaturation of MFO catalyst in microsomal preparations are contrary to laboratory 

conducted exposure studies. Additional problems were fish catches were unpredictable 

with respect to species, lack of knowledge of sex and gonadal status of the fish and 

variable conditions of abiotic facts such as fluctuations in oxygen tension. 

Mutagen mobilization from sediment into co-solvent extracts was detectable in the 

Ames assay, with tester strains varying in sensitivity. Promutagens were detectable in 

all extracts, via activiation with Arochlor-induced rat liver S9. Mutagenicity effects varied 

among co-solvent concentrations, perhaps reflecting mobilization of different arrays of 

compounds. Low co-solvent fractions enhanced extraction of water-soluble mutagens 

but suppress extraction of lipophilic mutagen. In high co-solvent fraction appeared to 

suppress the extraction of some water-soluble mutagens. TA 102 was the most 

responsive tester strain with these extracts; TA 98 showed no response to mutagens. 

The umu gene induction assay did not detect mutagens in either the presence or 

absence of fish liver S9 or microsomes. The ability of microsomes from Lake 

Champagne and Pass Fourchon catfish to activate 2AA was compared with that of rate 

liver microsomes. About 2-fold enhancement of umu gene induction with the rat 

microsomes was noted, but no increase was observed over background controls with 

the fish samples. 

Sediment bound contaminants associated with chronic produced water discharges are 

bioavailable to the filter-feeding bivalve Crassostrea virginica. Petroleum-associated 

aromatic hydrocarbons increased in all experiments in a dose- and time-dependent 

fashion. The data suggest that contaminated sediments remain as bioavailable toxic 

substances even after discharges are suspended. 

STUDY PRODUCTS: Winston, G.W., J.C. Means, L. Riley, S. Dobias, and Z. Yan. 

1995. Bioavailabiltiy and genotoxicity of produced water discharges associated with 

offshore drilling operations, final report. Louisiana Universities Marine Consortium for 

U.S. Dept. of the Interior, Minerals Management Service, Gulf of Mexico Region, OCS 

Office, New Orleans, LA. Contract No. 14-35-0001-30470. OCS Study MMS 95-0020. 

110 pp. 

No journal articles have been completed to date; several are in progress. The following 

published abstracts were presented at national meetings. 

1992 Induction of ethoxy-resorufin O-deethlase (EROD) in the killifish Fundulus grandis

by selected mutagens. Means, Reily, Yan and Winston. Presented at the 13th annual 

SETAC meeting, Cincinnati, OH, November 8-12, 1992. 

1992 Induction of EROD in the sandworm Nereis virens exposed to petroleum 

contaminated sediment. Reily, Means, Yan and Winston. Presented at the 13th annual 

SETAC meeting, Cincinnati, OH, November 8-12, 1992. 
ACCESS NUMBER: 30470 (95-0020) 

1994 Field Application of Biomarkers to Petroleum Contaminated Sites in Louisiana. 

L.A. Reily and J.C. Means. Presented at the Annual Society of Toxicology Meeting, 

March 13-17, 1994, Dallas, TX. 

1994 Co-solvent Mobilization and Modeling of Bioaccumulation Potential of PetroleumAssociated Mutagens from sediments. J.C. Means, L.A. Reily and D.J. McMillin. 

Presented at the Annual Society of Toxicology Meeting, March 13-17, 1994, Dallas, TX. 

1994 Methods for Assessing Bioavailability of Petroleum-Associated Mutagens from 

Sediments Using Co-Solvent Mobilization. Presented at the 25th Annual Meeting of the 

Environmental Mutagen Society, Portland, OR, May 7-12, 1994. 

1994 The significance of reactive oxygen species in chemical-contaminated marine 

environments: An overview of studies of the marine mussel, Mytilus edulis. G.W. 

Winston, 15th Conference of the European Society for Comparative Physiology and 

Biochemistry: Biochemical and Physiological Effects of Pollutants and Toxicological 

Assessment of Environmental Quality. Genova, Italy, Sept. 20-24, 1994. 

*P.I.'s affiliation may be different than that listed for Project Manager.