Marine Environmental Research 28 (1989) 303-307

Assessment of the Genotoxicity of Produced Water Discharges Associated with Oil and Gas Production

Using a Fish Embryo and Larval Test

Carol B. Daniels & Jay C. Means

Institute for Environmental Studies, Louisiana State University, Baton Rouge, Louisiana 70803, USA

A B S T R A C T

Widespread discharge of produced waters (PW) into brackish and marine waters of the northwest Gulf oflMexico, which serve as spawning groundsJbr man), commercially important .species offish, has raised concern over the potential impact that these complex mixtures may have on aquatic life. In the present stud)', the results of investigations which assess the clastogenic effects of produetion water discharged as the result o f o i l and gas production activiO, at two coastal sites are reported. In order to assess the impact of the P W discharges on spawning populations t~l" aquatic organisms, chromosome aberration analysis was performed on Cypr inodon variegatus embryos 4- 7 days old, which were exposed directly to varying dilutions o~ P W collected at Pass Fourchon and Timbalier Island .for a period of 5 days, Metaphase chromosomes were scored (percentage aberration per 100 metaphases) jbr aberrations at a magn(fication of 1000 x . When compared to the control treatment, higher levels of chromosomal aberrations were observed Jor treatments (2 and 4 % P W) in which embryos were expose d to P W from Pass Fourchon or Timbalier Island. A maximum frequency off 13% was found to occur at the highest test concentration (4% PW), with most of these expressed as breaks ( chromatid and chromosome). In contrast, exposure to water from the enclosed canal at Fourchon resulted in the ,formation ~/ chromosome breaks and changes in ploidy.

L a r g e v o l u m e s o f p r o d u c e d w a t e r s t (1.79 mi l l ion bar re ls per day , 1 bar re l =

159 liters) are r o u t i n e l y d i s c h a r g e d in to n e a r - s h o r e a n d e s tua r ine wa te r s o f

t The terms produced water and formation water are used to describe the water released to the surface from permeable petroleum-bearing sedimentary rocks during offshore production of oil and gas. 1 Because produced water typically has salinity levels in excess of modern seawater, the term oilfield brine is often used to describe it.

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304 Carol B. Daniels, Jay C. Means

the Louisiana coast. 2 Because of the complex nature of these formation waters (produced water may contain elevated levels of petroleum hydrocarbons, partially oxidized organics, trace metals, sulfide and elemental sulfur), it has been suggested that the impact of production water in shallow semi-enclosed canals and natural bayous may be heightened because of depressed flushing rates, high particulate retention (turbidity), and the relatively high organic matter content of the particles; all conditions that facilitate the persistence of hydrocarbons and other organic compounds in the water column. Since estuaries represent some of the most productive spawning grounds for many commercially important species of fish, shellfish and crustaceans, it is critical that any potential impacts that these complex mixtures may have upon aquatic life be accurately assessed. In the present study, we report the results ofcytogenetic investigations using embryos of an estuarine fish (Cyprinodon variegatus) to determine the potential mutagenic and clastogenic threat posed to developing organisms exposed to production water.

Produced waters (PW) were collected directly from the outfall pipe from the product ion facilities at Pass Fourchon and Timbalier Island. Additionally, sediment and water column samples were taken and characterized for hydrocarbons and trace metals. 2 A 1:25 dilution (4% PW) of field-collected produced water was performed to reduce the salinity of the samples to an acceptable level (10%o). Following the initial dilution, water samples underwent further dilution 2% PW to achieve a concentration gradient for use in the genotoxicity bioassays. Ten parts per thousand artificial seawater (Instant Ocean) constituted the control treatment.

Thirty 4 7 days old Cyprinodon embryos were placed in 2 oz glass jars with Teflon-lined caps containing 50 ml of oxygenated treatment water. Three exposure chambers per treatment were maintained in the dark at 25°C for a total of 5 days. Colchicine was added to a final concentration of 0.05% 4 h prior to the end of the 5 day exposure period. Following treatment with colchicine, embryos were subjected to a 90min hypotonic treatment and three 30rain treatments with fixative (3:1 methanol-acetic acid). Whole embryos were macerated and slides prepared according to the technique of Baksi & Means.1 Vital stain was used to insure embryo viability during the preceding steps and non-viable embryos were discarded prior to slide preparation. Average values of 17% mortality and 50% hatch have been recorded for control treatments for a 5 day exposure period.

Slides were scored for chromosome aberrations using a Zeiss Axioplan microscope equipped with a 35 mm camera at a magnification of × 1000. Photographs were taken on Kodak Technical Ektachrome film (ASA 160). A total of 100 metaphases per treatment were scored whenever possible. Aberration frequencies were recorded both as the percentage of metaphases

Genotoxicity ~?[ produced water discharges 305

TABLE 1

Site Dilution of Percentage Types 0/ (volume) original sample (%) aberration aberration

Fourchon 0 1 Breaks (2-4 x 107 liters/day) 2 5 Breaks

4 20 Ploidy

Timbalier 0 1 Breaks l l ' l x 10 7 liters/day) 2 12 Gap, breaks,

rings, exchanges 4 13 Gap, breaks, ploidy

having at least one aberration and also as the frequency of individual types of aberrations (e.g. breaks, gaps, exchanges, etc.). Changes in chromosome number (ploidy) were also measured.

Data presented here (Table 1) support the hypothesis that production water discharged into semi-enclosed areas may pose long-term ecological hazard for estuarine fishes inhabiting these waters. Produced water discharged at both sites, Pass Fourchon and Timbalier Island, were found to induce high frequencies of clastogenic effects, i.e. chromosome aberrations, in developing embryos of C. variegatus (Table 1). An elevation in the number of chromosome aberrations was noted in the Pass Fourchon treatment group, when the level of PW was increased from 2% and 4% PW (5% and 20% aberration, respectively) suggesting that the genetic effect is concentration-dependent (control = 1% aberration).

In embryos exposed to produced water from the Timbalier Island site, a maximum frequency of 13 % aberrations was found to occur at the highest test concentration 4% PW). Although induction of chromosome aber- rations was noted at both treatment levels, 4% and 2% PW, induction did not appear to be concentration-dependent. Although all classes of aberrations were formed as a consequence of exposure to Timbalier produced water (Fig. 1), most of the aberrations formed were expressed as breaks (chromotid and chromosome). In contrast, exposure of embryos to the Pass Fourchon PW resulted most frequently in changes in ploidy. The wide spectra of aberrations formed suggest differences in the mode of action of the mutagenic components in the two formation waters. Changes in the concentration of the individual compounds in the effluent water might lead to differences in sensitivities of the organism to mutagenic agents. Such differences will affect the extent to which various mutagens enter the cell, ultimately determining which compounds will interact with cellular DNA.

Although the variability in total toxic organic and metal loads of different formation waters limits conclusions about the toxicity of produced water

306 Carol B. Daniels, Jay C. Means

Fig. 1. Chromosome aberrations formed in C. variegatus embryos as the result of exposure to produced water. (a) Control treatment (2n=48, maximum chromosome length at

1000 × = 5mM). (b) Exchange. (c) Gap.

Genotoxicity of produced water discharges 307

discharged from other sites, these results document the potential effects at two typical sites in Louisiana. Based on available information, we calculated that for a 1 km impact zone within the bayou, based on sediment analyses, 2 it takes between 2 and 4 days for the volume (---5 x 107 liters) to be totally replaced by produced water from these discharges. Since the frequency of spontaneous aberrations noted I (,,~ 1 in 105 metaphases)was conservatively 5000 × lower than the minimum response observed here, it would take more than 1000 volume exchanges/day of water to reduce the 1'1 × 107 liters/day load at Timbalier Island to a level of no effect and 2400 volume exchanges/ day at the Bayou Rigaud site. Note that these estimates do not take into account bioconcentration, sorption, changes in bioavailability or changes in concentration due to degradation, volatilization, etc. However, the data presented here suggest the need for continued research on the effects of chronic operat ional discharges of formation waters into estuarine environments upon aquatic species.

A C K N O W L E D G E M ENTS

The genotoxicity studies were supported in part by a cooperative agreement between LSU and the US Environmental Protection Agency, Environ- mental Research Laboratory, Pacific Division, Newport, OR and the Louisiana Depar tment of Environmental Quality. Produced water characterizations were conducted with support from the US Department of the Interior, Minerals Management Service. The assistance of Mr Charles Henry, analytical chemist, and Mr John Collins, fish culturist, is acknowledged.

REFERENCES

1. Baksi, S. M. & Means, J. C., J. Fish Biology, 32 (1988) 321 5. 2. Boesch, D. F., Rabalais, N. N., Milan, C. S., Henry, C. B., Means, J. C., Gambrell,

R. P. & Overton, E. B., In Impacts of Outer Continental Shelf(OCS) Related Activities on Sensitive Coastal Habitats, Vol. II, Produced Waters. Report to Minerals Management Service, Baton Rouge, LA, 1988, 167 pp.