Characterization of Organic Constituent Patterns at a Produced Water Discharge Site /
Barium Relations to Bioeffects of Produced Water

BACKGROUND:
As produced water (PW) is a very complex mixture of diverse chemical categories consisting of hundreds of organic compounds [NRC, 1985; Boesch and Rabalais, 1987], the principal technical utility of this project is to significantly and systematically reduce the list of PW constituents that must be studied with regards to their fate and transport. There are many scientific works regarding the occurrence and fate of the organic components, but these are mostly limited to the petroleum hydrocarbons, which comprise only a portion of the total organic load of PW [NRC, 1985; Boesch and Rabalais, 1987]. The reasons for this self-imposed restriction are many, but among the likely reasons are the ease of analysis for hydrocarbons and the considerable body of literature regarding their biological effects. However, even with this restriction to just hydrocarbons, PW can harbor hundreds of compounds.
A less arbitrary means to reduce the complexity is to identify the compounds associated with biological effects. For example, we have successfully utilized a bioactivity-based fractionation of PW from a plant at Carpinteria, CA to characterize the constituent responsible for impairment of mussel embryo development [Higashi et al., 1993]; this may also relate to impairment of adult mussel reproductive development [Fan et al., 1993]. The identity of this constituent, Ba, is being verified in a series of laboratory experiments that investigate PW fractions and Ba chemistry as it relates to the symptoms and mechanisms of Ba toxicity in these biological systems; the latter is being conducted in conjunction with the project of Cherr and Fan [Schmitt, 1991]. Unfortunately, the discharge of PW from that source has ceased, making field verification impossible.
In order to study effects of PW in the field, Osenberg, et al. [Schmitt, 1991] have been studying the future PW discharge site near Point Conception, CA ("Gaviota") for the past three years, utilizing the Before-After Control-Impact Pair design (BACIP) for ecological studies. Although this PW plant at Gaviota is currently in operation by reinjection of PW into formations, information from the plant operator suggests that discharge of the PW into marine waters will not occur immediately. Thus, the present situation at Gaviota represents an outstanding opportunity to study organic constituents in the "Before" period of the BACIP study.
 

OBJECTIVES:
The goals of this project are to: (a) identify constituents of produced water (PW) responsible for various bioeffects of interest to SCEI researchers; and (b) estimate their relevance to an environmental situation by surveying the distribution of the bioactive constituents at the Carpinteria site. The scientific utility of goal (a) is to significantly reduce the list of chemicals to be studied, providing foci for other studies and greatly reducing analytical load. Goal (b) will provide basic data of potential interpretability to any case study research currently being conducted at the Carpinteria site.
These goals and research approaches (described in detail in the next section) address two targeted research areas for Environmental Studies in the Southern California Educational Initiative (SCEI). The goals, reformulated in terms of the Studies Framework, are as follows:

Fractionation of PW (addresses Studies Framework Issue #1: Fate and effects of produced water discharges in the nearshore environment)
Produced water will be analyzed in accordance with schemes we have previously applied in order to help establish causal linkages between specific PW constituents (or classes thereof) and bioeffects being studied by SCEI researchers. This will be accomplished by providing such fractions for bioeffects testing by collaborating SCEI researchers (e.g., Cherr and Fan, and Reed et al.).

Toxicant Identification (addresses Studies Framework Issue #2: Long-term effects of drilling discharges)
Our toxicant identification efforts will continue, currently directed by results of toxicity testing, which will expand as other bioeffects of PW are established in the laboratory and field. We will continue to archive PW samples through lyophilization (freeze-drying), and process samples according to our present scheme (Figure IA, and described below). The results of bioeffects testing will direct the choice of the chemical methods to be employed. As such, extensive analytical capability is required on a standby basis, as these substances may be expected to have very diverse chemical structures and properties. A broad range of high-resolution analytical separation (capillary GCMS, HPLC, capillary electrophoresis) and detection systems (NMR, MS, ICP-AES, ICP-MS) will be brought to bear for the identification and early analysis of organic and inorganic substances. Once identified, the bioactive substances will be isolated or synthesized and integrated with toxicity testing (Cherr and Fan) as well as possibly other bioeffects testing (Reed, et al.- kelp zoospore settling tests).
 

DESCRIPTION:
The basic approach in this field study is chemical analysis of PW, site sediment, water column, and outplanted mussels, with the data to undergo BACIP analysis by Osenberg, et al. Due to logistical limitations, the study will focus on organosulfur and hydrophobic compounds, and rely heavily on high-resolution gas chromatographic (GC) and GC-mass spectrometric (GCMS) analyses. All sampling is conducted in coordination with the project of Osenberg, et al., originating from the Gaviota research site, as described elsewhere [Schmitt, 1991]. Samples are collected from field sites (near impact, far impact, and reference) semiannually, each site consisting of water column and outplanted mussels, each at two depths, and sediment sample. In addition, Gaviota PW samples are obtained twice per year in order to track trends in PW composition. Organic solvent extracts of these samples are analyzed by various GC methods. Most of the analytical data will consist of relative quantification of unknown compounds. Thus, an important analytical tool is GCMS, which can catalog structural information on these unknowns for future use and provide running checks on analytical efficiency via analysis of the deuterated standards. For example, mass spectra is used to ensure that all analytical data transferred to Osenberg, et al. consist of the same compounds (based on their GC retention time and mass spectra) throughout the study. Chemical identification of a given peak will commence upon the identification of that peak as related to biological effects in the field, as determined by the BACIP analyses. Another important feature of the project is the preservation of samples by unique means such as freeze-drying. This is vital if subsequent research indicates that certain compounds of biological relevance are not extracted efficiently by the original procedures.

SYNOPSIS OF MAJOR FINDINGS:
Major organic compounds found in PW were the expected hydrocarbons and alkylphenols, but there were also high levels of organosulfur compounds. In particular, thiocarboxylic acids and novel thiopyranones were found in abundance. Organopolysulfides were also identified, as were inorganic forms of sulfur, such as sulfides, thiosulfates, and polysulfides. Sulfide distribution among dissolved, particulate and colloidal phases were also determined. These findings are important to bioeffects due to the strong interaction of both organic and inorganic sulfur compounds with metal ions, as well as the possible bioactivity of the organosulfur compounds themselves.
Also found in Carpinteria PW was polysaccharidic material that is not cellulosic; this material was found in sediments prevailing downcurrent from the outfall, decreasing with distance. These substances may be useful PW markers and may affect microbial communities in the sediment, thereby altering microbial degradation of PW constituents.
Shell Ba content (normalized to Ca) of outplanted mussels showed a decreasing trend with distance from the Carpinteria outfall, but this was not true of all outplant experiments - some outplant experiments showed no clear trend. This data, together with that from the project of Cherr & Fan, is being integrated into and analyzed by the project of Osenberg et al.
This research was completed under MMS Contract No. 14-35-0001-30761, Project #3, Title: Characterization of Organic Constituent Patterns at a Produced Water Site / Barium Relations to Bioeffects of Produced Water, Principal Investigators, Richard Higashi, A. Daniel Jones and Teresa Fan, FY 94-95, 95-96 (no cost).
 

STUDY PRODUCTS:
PUBLICATIONS

1993 Fan, T.W-M., T.D. Colmer, A.N. Lane, and R.M. Higashi. Determination of metabolites by 1H NMR and GC: analysis for organic osmolytes in crude tissue extracts. Analytical Biochemistry 214: 260-271.
1998 Witter, A.E., and A.D. Jones. Comparison of methods for inorganic sulfur speciation in a petroleum production effluent. Environmental Toxicology and Chemistry 17(11):2176-2184.
1998 Witter, A.E., S.A. Mabury, and A.D. Jones. Copper(II) complexation in northern California rice field waters: An investigation using differential pulse anodic and cathodic stripping voltammetry. Science of the Total Environment 212(1):21-37.
 

RESEARCH PRESENTATIONS
1995 Witter, A. E. and A. D. Jones, "A Comparison of Methods for Speciation of Sulfur in a Petroleum Production Effluent" Abstracts of Papers of the American Chemical Society, 211:, n.1-2, (1996): Abstract ENVR 91, 211th American Chemical Society National Meeting, New Orleans, Louisiana, USA, March 24-28, 1996.
1994 Higashi, R.M., T. W-M. Fan, and A.N. Lane. "Pyrolysis GC-MS and NMR studies of humics in contaminated sediments." 15th Annual Meeting of Society of Environmental Toxicology and Chemistry, Denver, CO, USA.
1994 Jones, A.D., A. E. Witter, and R. M. Higashi, "Petroleum-related Contaminants Near a Produced Water Discharge Site in the Santa Barbara Channel," presented as part of the Symposium on Marine Environmental Chemistry, 15th Annual Meeting of the Society for Environmental Toxicology and Chemistry, Denver, CO, October 30-November 4, 1994 (Abstract 394).
1994 Witter, A. E. and A. D. Jones, "Measurement of Complexation Properties of Metal Ions by Polyelectrolytes using Ultrafiltration/GFAAS Methods," 15th Annual Meeting of the Society for Environmental Toxicology and Chemistry, Denver, CO, October 30-November 4, 1994 (Abstract MB14).
1993 Cherr, G.N., G.D. Garman, R.M. Higashi, and M.C. Pillai. Effects of produced water on reproduction and development in marine organisms. 14th Annual Meeting of Society of Environmental Toxicology and Chemistry, Houston, TX, USA.
1993 Witter, A. E., A. D. Jones, and R. M. Higashi, "The Identification of Organosulfur Compounds in Produced Water by Mass Spectrometry," Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, pp. 403a-403b.
1993 Witter, A. E., A. D. Jones, and R. M. Higashi, "Analysis of Organosulfur Compounds in Produced Waters using Mass Spectrometry," 14th Annual Meeting of the Society for Environmental Toxicology and Chemistry, Houston, TX November 14-18, Abstract #444 (p. 126).
1993 Witter, A. E., A. D. Jones, and S. Mabury, "A Comparison of APCI and ESI Methods for the Quantitation of Small Molecules of Environmental Interest using LC/MS," Proceedings of the 10th Montreux LC/MS Symposium at Cornell, July 19-23, 1993, Ithaca, NY.