ACCESS NUMBER: 30660-19933 

STUDY TITLE: Studying and Verifying the Use of Chemical Biomarkers for Identifying 

and Quantitating Oil Residues in the Environment 

REPORT TITLE: Studying and Verifying the Use of Chemical Biomarkers for 

Identifying and Quantitating Oil Residues in the Environment 

CONTRACT NUMBER: 14-35-0001-30660-19933 

SPONSORING OCS REGION: Gulf of Mexico 

APPLICABLE PLANNING AREA: Central Gulf of Mexico 

FISCAL YEARS OF PROJECT FUNDING: 1997 - 2000 

COMPLETION DATE OF REPORT: November 28, 2000 

COSTS: FY 1997: $16,892; FY 1998: $64,949; FY 1999: $16,420; FY 2000: 

$102,127; CUMULATIVE PROJECT COST: $200,388 

PROJECT MANAGERS: M.S. Miles, B.M. Ashton, R.S. East 

AFFILIATION: Louisiana State University, Institute for Environmental Studies, 

Response and Chemical Assessment Team 

ADDRESS: 42 Atkinson Hall, Baton Rouge, LA 70803 

PRINCIPAL INVESTIGATOR*: Edward B. Overton, Ph.D 

KEY WORDS: Biomarkers; oil weathering; self-normalizing fingerprint indexes; oil 

degradation; tarballs; bioaccumulation; GC/MS 

BACKGROUND: Petroleum released into the marine environment undergoes varying 

degrees of environmental weathering that affect its composition. As weathering 

proceeds, certain groups of oil constituents are lost in a predictable sequence. 

However, biomarkers are petroleum components that remain detectable and relatively 

unchanged in oil residues even after natural environmental weathering. They are also 

typically resistant to biodegradation and are therefore, useful as chemical markers. 

Biomarker fingerprinting by gas chromatography/mass spectrometry (GC/MS) and the 

calculation of eight different source-normalizing fingerprint indexes (SFI) may be 

necessary for environmental samples in which identification of spilled oil is difficult due 

to weathering of target compounds. 

OBJECTIVE(S): The objective of this research is to refine the use of biomarkers as 

tools for oil spill identification and assessment, and to verify the analytical approach 

utilized by the Louisiana State University, Institute for Environmental Studies, Response 
ACCESS NUMBER: 30660-19933 

and Chemical Assessment Team (LSU IES/RCAT) with both laboratory and field 

evaluation studies. 

DESCRIPTION: The research was divided into 4 sub-divisions: preliminary stages; a 

water degradation experiment; a bioaccumulation study; and classification and 

identification of tarballs collected from three areas of southwest Louisiana. The 

preliminary stages included a literature search and GC/MS method development. A 

comprehensive literature review of environmental and petroleum industry/geological 

journals was conducted; and a total of 180 pertinent articles out of over 300 titles were 

acquired and reviewed. The instrument methodology involved the development of an 

improved GC/MS analytical method that included newly identified biomarker families in 

addition to those compounds currently used for oil spill response. 

The water degradation experiment evaluated the changes in the composition of Eugene 

Island crude oil, after exposure to nine natural waters collected across the country, with 

or without additional nutrients. BiologTM analyses were performed in conjunction with 

the water degradation experiment to determine whether significantly different bacterial 

communities were present in the various waters. The final stage of the water 

degradation experiment examined the stability of the SFI for the pre- and postweathered reference oil after 28-days of laboratory-simulated weathering. 

The bioaccumulation experiment examined biomarker up-take in bivalves exposed to oil 

of known biomarker composition from samples collected recently from Prince William 

Sound, Alaska. Theoretically, biomarkers are not significantly altered by microbial 

degradation; hence, there is a possibility that these lipid soluble materials will 

accumulate in marine organisms and could be used as an indicator of possible 

exposure during oil spills. The results of this task will establish a time frame in which 

biomarker compounds may be detected in bivalves since the samples were collected 

ten years after the T/V Exxon Valdez incident. 

Classification and identification of tarballs collected from three areas along the 

southwest coast of Louisiana was carried out at several different levels. A total of 88 

tarballs were collected and described based on seven different morphological 

descriptions and eight weathering classifications. Visual comparisons were also carried 

out for each biomarker fingerprint. As a result of the visual comparison, three of the SFI 

were chosen for scatterplot analysis due to the fact that the indexes selected were 

relatively unaffected by weathering and laboratory variations. 

SIGNIFICANT CONCLUSIONS: The biomarker compounds that provide the best tool 

for oil spill identification, both visually and statistically, are C-3 dibenzothiophene, C-3 

phenanthrene, C-3 DBT peak area/C-3 Phen peak area, and Norhopane/Hopane. 

These compounds, as demonstrated by the water degradation experiment, remain 

stable even after weathering and will distinguish one oil source from another when other 

biomarkers are lost (i.e. nC-17/pristane and nC-18/phytane). GC/MS analysis and 

visual examination should still include all eight compounds because, when combined, 

they provide information about the extent of weathering and biodegradation from their 
ACCESS NUMBER: 30660-19933 

respective fingerprints. Visual examinations of the fingerprints can easily distinguish 

one type of oil or weathering stage from another based on the pattern of the fingerprint 

alone. 

STUDY RESULTS: One part of the water degradation experiment was to assess the 

effects of microorganism populations present in different water sources in two ways: 

BiologTM microplate analyses and percent reduction of total target aromatic 

hydrocarbons (TTAH). The organisms in the different water samples had widely varying 

metabolic capabilities; however, the results from the Biolog analyses and the %TTAH 

calculated from the GC/MS data did not correlate. This is due to several factors: 

differing carbon sources; differing nutrient substrates; and differing dominant microbial 

communities. 

The uptake of biomarkers, and in general polycyclic aromatic hydrocarbons (PAHs), 

was examined in bivalves exposed to oil from the T/V Exxon Valdez. The analysis 

provided important information about the uptake and utilization of petroleum 

hydrocarbons by filter feeding organisms after 10 years of exposure. Overall, the 

biomarker and PAH concentration for both mussel and clam tissues were low. 

Unfortunately, a baseline concentration of biomarker compounds has not been 

established for biological samples from the beginning of the T/V Exxon Valdez incident. 

The classification and identification of tarball samples collected in 1998 suggests that 

the majority of the tarballs collected were from mid- to heavy-range crude oil sources. 

Of the 70 tarballs extracted and analyzed, four matches were found through visual 

comparison and double SFI scatterplots. Since the distribution of sources was 

extensive, it is believed that the tarballs collected represent oil contamination from small 

petroleum and/or bilge waste discharge. 

STUDY PRODUCT(S): Ashton, B.M.; East, R.S.; Walsh, M.M.; Miles, M.S., Overton, 

E.B. 2000. Studying and Verifying the Use of Chemical Biomarkers for Identifying and 

Quantitating Oil Residues in the Environment. A final report for the U.S. Department of 

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

Contract No. 14-35-0001-30660-19933. OCS Study MMS 2000-086. 77 pp. 

Ashton, B.M.; East, R.S.; Walsh, M.M.; Miles, M.S. 2000. Studying and Verifying the 

Use of Chemical Biomarkers for Identifying and Quantitating Oil Residues in the 

Environment. Poster presentation at the Clean Gulf Conference, November 13-15, New 

Orleans, LA. 

Ashton, B.M.; East, R.S.; Walsh, M.M.; Miles, M.S. 2000. Studying and Verifying the 

Use of Chemical Biomarkers for Identifying and Quantitating Oil Residues in the 

Environment. Poster presentation at the Environmental State of the State V 

Conference, November 16-17, Baton Rouge, LA. 

*P.I.’s affiliation may be different than that listed for Project Manager(s)