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Accelerated biodegradation of petroleum hydrocarbon waste.

Conventional landfarming approaches to bioremediation of refinery and other petroleum sludges are not acceptable environmentally and are banned in most North American jurisdictions. While initial bioreactor-based systems for treatment of these sludges required batch-cycle process-times of 1-3 months, an accelerated process has now been developed which can be completed in 10-12 days. In this process, up to 99% of total petroleum hydrocarbons are degraded and the sludges are converted from hazardous to non-hazardous according to the United States EPA's toxicity characteristic leachate procedure criteria. Understanding and exploiting mechanisms to improve hydrocarbon accession to the degrading microorganisms was a key development component of the process. Contrasting physiological mechanisms were observed for different component organisms of the mixed culture with respect to their associations with the hydrocarbon substrate; and the beneficial effects of using surfactants were demonstrated. The mixed culture used in the process exhibited a capacity for high-rate degradation of volatile organic carbons and the potential use of the culture as a liquid biofilter was demonstrated. The culture was also effective as an inoculant for the bioaugmentation of total petroleum hydrocarbon-contaminated soil and as a de-emulsifier of oilfield emulsions and could transform some other environmental contaminants which are not predominant components of crude oil.

Biodegradation, Environmental↗

Distribution of PAHs in surface soils from petroleum handling facilities in Calabar.

The level of concentrations of polycyclic aromatic hydrocarbons (PAHs) in surface soils from petroleum handling facilities (kerosene tank, generating plant, petrol stations, mechanic workshops, leaking pipeline and air port fuel dump) from Calabar metropolis southeastern Nigeria was determined by gas chromatography/ mass spectrometry. The results show that total polycyclic aromatic hydrocarbons (PAHs) varied from 1.80 to 334.43 mg/kg with a mean of 50.31 mg/kg. The lowest value of 1.80 mg/kg was obtained from petrol station while the highest value of 334.43 mg/kg was obtained from facility characterised by petrol stations and mechanic workshops. The ratio of phenanthrene/anthracene and fluoranthene/pyrene, varied from 0.43 to 27.72 and from 0.14 to 17.76 respectively. These ratios indicate various sources for the PAH. The two to three ring PAHs are the most abundant. Based on the PAH ratios and content alone it is not possible to distinguish between contribution from motor vehicle exhaust, gasoline spillage, used engine oil or petroleum production. However, considering the area of the study, it is very likely that the major source of soil contamination is originating from petroleum product.

Nigeria↗

Chemical characteristics and source implications of petroleum hydrocarbon contaminants in the sediments near major drainage outfalls along the coastal of Laizhou Bay, Bohai Sea, China.

The associated industrial and urban developments are located to a large extent along the Laizhou Bay, Bohai Sea coastal and raw sewage is often discharged into near shore waters with little treatment. To find out chemical characteristics and pollution source of the petroleum related contaminations in sediments near the major drainage outfalls located in the coastal, in this study, 10 surface sediment samples were collected during June. Sediment samples were extracted by organic solvents, separated by silica gel column chromatography and the concentrations and the profiles of n-alkane, biomarker and PAH in sediments were analyzed by gas chromatography-mass selective detector (GC-MSD). The use of several molecular markers and related indexes derived for n-alkane and PAHs has been proposed for assessing the relative contributions to the environment of hydrocarbon sources. As a result, n-alkanes reflect that the sea area of paper mill (Station ZZ08) is dominated by vascular plant. DY petroleum oil field and outer shore of the paper mill (Station ZZ02) have some degrees of petroleum related hydrocarbon contamination. Whereas the contamination of the sea area of TH River may be ascribed to different sources such as territorial non-point pollution source, domestic sewages, and stormwater runoff. Judged by their PAH ratios, the sediments near the paper mill (Station ZZ02) and the outer station of the oil field (Staion TH2) were pyrolytic. The estuary of Tiao River including the inner Station THX, TH10 and TH05 are petrogenic. The marine sediment near DY drainage outfall may have a mixture source of PAH both pyrolytic and petrogenic.

China↗

Urine mutagenicity of petroleum plant workers.

The mutagenicity of urinary extracts from workers employed in a petroleum plant was analyzed by means of the plate test using S. typhimurium strains TA98 and TA100. Mean urinary mutagenic activities in TA98 were 2-14 times higher in the petroleum plant workers than in the control group. In TA100 these differences were even bigger, the mutagenicity in petroleum plant workers' urine being 3-42 times higher than in the control group. These results suggest that the environmental exposure of people to mutagenic substances is markedly increased in a petrochemical plant.

Environmental Exposure↗

Lack of concordance of the Salmonella/microsome assay with the mouse dermal carcinogenesis bioassay for complex petroleum hydrocarbon mixtures.

Typical petroleum hydrocarbon mixtures were tested directly, without extraction, in the Salmonella/microsome mutagenesis assay in order to determine if the assay would be useful to predict their carcinogenic activity. The carcinogenic activity of each sample had been previously characterized in the in vivo mouse dermal carcinogenesis bioassay. The series of samples evaluated offered several advantages. They spanned a wide boiling point range, were well characterized chemically, had been tested for carcinogenic activity in a single laboratory, and varied in potency in vivo from inactive to highly active. Mutagenicity testing was performed in several well-established contract laboratories that routinely perform the assay. These laboratories were the main contracting laboratories for these assays at the time and had previously tested petroleum samples for clients. Initially, the first laboratory tested 13 samples in five strains of Salmonella typhimurium with and without rat liver S-9 (Arochlor 1254 induced), utilizing both plate and suspension techniques. None of the 13 samples exhibited a mutagenic response, even though 9 of the 13 were slightly to highly dermally carcinogenic in mice. Because of the unexpected results, it was decided to repeat the mutagenicity assays in two other laboratories. Six of the thirteen samples were selected, ranging in carcinogenic potency from negative to highly active. Again, none were mutagenic in the second contract laboratory. In a third facility, only one sample of the six exhibited a definite mutagenic response. However, the response was observed with a sample having only weak carcinogenic activity and, unusual for petroleum hydrocarbons, occurred without activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Improving petroleum contaminated land remediation decision-making through the MCA weighting process.

Internationally petroleum contamination is widespread, posing serious environmental risks including surface and groundwater contamination, thus remediation is essential. The implementation of remediation options is becoming more complex with the increasing influence of stakeholders on the outcome of decision-making processes. Acceptance of remediation schemes during implementation can be increased by involving stakeholders and the public in the decision-making stage. In petroleum remediation involving multiple stakeholders, Multicriteria Analysis has been employed due to its ability to incorporate the preferences of each stakeholder through weighting. The research focused on investigating ways to improve the weighting process. The study demonstrated the utility of SWING, and determined which type of participant and how many participants to include in the decision process, through the application of ELECTRE III and Weighted Summation. It was recommended that a mixture of stakeholders, the public and experts be involved. The total number of participants will be limited by the choice of participatory and weighting methods. The careful selection of participants, as well as the choice of participatory and weighting methods, can minimize the subjectivity involved in MCA weighting, thereby lending decisions in petroleum remediation greater legitimacy.

Adult↗

The effects of perennial ryegrass and alfalfa on microbial abundance and diversity in petroleum contaminated soil.

Enhanced rhizosphere degradation uses plants to stimulate the rhizosphere microbial community to degrade organic contaminants. We measured changes in microbial communities caused by the addition of two species of plants in a soil contaminated with 31,000 ppm of total petroleum hydrocarbons. Perennial ryegrass and/or alfalfa increased the number of rhizosphere bacteria in the hydrocarbon-contaminated soil. These plants also increased the number of bacteria capable of petroleum degradation as estimated by the most probable number (MPN) method. Eco-Biolog plates did not detect changes in metabolic diversity between bulk and rhizosphere samples but denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified partial 16S rDNA sequences indicated a shift in the bacterial community in the rhizosphere samples. Dice coefficient matrices derived from DGGE profiles showed similarities between the rhizospheres of alfalfa and perennial ryegrass/alfalfa mixture in the contaminated soil at week seven. Perennial ryegrass and perennial ryegrass/alfalfa mixture caused the greatest change in the rhizosphere bacterial community as determined by DGGE analysis. We concluded that plants altered the microbial population; these changes were plant-specific and could contribute to degradation of petroleum hydrocarbons in contaminated soil.

Bacteria↗

The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers.

In this study, we evaluated whether the abundance of the functional gene nahAc reflects aerobic naphthalene degradation potential in subsurface and surface samples taken from three petroleum hydrocarbon contaminated sites in southern Finland. The type of the contamination at the sites varied from lightweight diesel oil to high molecular weight residuals of crude oil. Samples were collected from both oxic and anoxic soil layers. The naphthalene dioxygenase gene nahAc was quantified using a replicate limiting dilution-polymerase chain reaction (RLD-PCR) method with a degenerate primer pair. In the non-contaminated samples nahAc genes were not detected. In the petroleum hydrocarbon-contaminated oxic soil samples nahAc gene abundance [range 3 x 10(1)-9 x 10(4) copies (g dry wt soil)(-1)] was correlated (Kendall non-parametric correlation r2=0.459, p<0.01) with the aerobic 14C-naphthalene mineralization potential (range 1 x 10(-5)-0.1 d(-1)) measured in microcosms at in situ temperatures (8 degrees C for subsurface and 20 degrees C for surface soil samples). In these samples nahAc gene abundance was also correlated with total microbial cell counts (r2=0.471, p<0.01), respiration rate (r2=0.401, p<0.01) and organic matter content (r2=0.341, p<0.05). NahAc genes were amplified from anoxic soil layers indicating that, although involved in aerobic biodegradation of naphthalene, these genes or related sequences were also present in the anoxic subsurface. In the samples taken from the anoxic layers, the aerobic 14C-naphthalene mineralization rates were not correlated with nahAc gene abundance. In conclusion, current sequence information provides the basis for a robust tool to estimate the naphthalene degradation potential at oxic zones of different petroleum hydrocarbon-contaminated sites undergoing in situ bioremediation.

Bacteria, Aerobic↗

The 1974 spill of the Bouchard 65 oil barge: petroleum hydrocarbons persist in Winsor Cove salt marsh sediments.

Petroleum hydrocarbons persist in salt marsh sediments in Winsor Cove (Buzzards Bay, Massachusetts) impacted from the 1974 spill of No. 2 fuel oil by the barge Bouchard 65. Intertidal sediment cores were collected from 2001 to 2005 and analyzed for total petroleum hydrocarbons (TPHs). TPHs content was greatest (as high as 8.7 mg g(-1) dry weight) in the surface sediments and decreased with distance landward. Select samples were analyzed for polycyclic aromatic hydrocarbons (PAHs) with values as high as 16.7 microg g(-1) for total naphthalenes and phenanthrenes/anthracenes. These remaining PAHs are mainly C(4)-naphthalenes and C(1)-, C(2)-, and C(3)-phenanthrenes/anthracenes revealing preferential loss of almost all of the naphthalenes and the parent compound phenanthrene. Inspection of the data indicates that biodegradation, water-washing and evaporation were major removal processes for many of the petroleum hydrocarbons in the marsh sediments. In addition, historical data and photographs combined with their recent counterparts indicate that erosion has physically removed these contaminants from this site.

Environmental Monitoring↗

Cytogenetic investigations of human subjects occupationally exposed to chemicals from the petroleum-processing industry.

A cytogenetic investigation was carried out of 55 workers from the petroleum-processing industry and of 30 control subjects. The frequencies of both structural and numerical chromosome aberrations and of sister chromatid exchanges were determined simultaneously and the relationship among these cytogenic indices were analyzed. The incidence of chromosome aberrations and sister chromatid exchanges among the petroleum workers was higher than that among the controls. The cytogenic indices demonstrated a clear dependence on the working environment of the subjects and a correlation with the existence of some reproductive failures in the families of the exposed workers. Our results indicate the presence of some mutagenic risk in the working environment of some of the divisions in the petroleum-processing industry due to the genotoxic influence on the somatic cells of the exposed workers at a cytogenetic level. This potential risk is mainly associated with the presence of some heavy oil-fractions and polycyclic aromatic hydrocarbons in the working environment.

Adult↗

Petroleum and PAH contamination of the Black Sea.

Concern has been expressed regarding the extent of contamination of the Black Sea. Analyses of coastal sediments taken from throughout the region indicate, however, that levels of petroleum hydrocarbons (2-300 microg g(-1) dry wt total hydrocarbons) are generally comparable to those encountered in the Mediterranean and are lower than concentrations reported for highly contaminated areas such as the Gulf, Hong Kong, Taiwan and New York Bight. Highest concentrations of total hydrocarbons (>100 microg g(-1) dry wt) were associated with discharges from Odessa, Sochi and the River Danube. Chronic/degraded petroleum was the major contributor at these sites. Samples from the Ukrainian coastline were comparatively clean (<10 microg g(-1) dry wt total hydrocarbons). Major contributions of fresh oil (as indicated by sigma n-C14-34) occur through the River Danube. Concerning total PAH, concentrations (7-638 ng g(-1) dry wt) compare to relatively unpolluted locations in the Mediterranean and are much lower than levels reported for polluted UK estuaries (e.g. Mersey, Tyne, Thames). Both pyrolytic and petrogenic PAH are present in most samples, although petroleum derived PAH are dominant at Sochi and pyrolytic sources are prevalent in the Bosphorus region. The absence of a correlation between total hydrocarbons and PAH (R2 = 0.04) indicates different primary sources for the two.

Environmental Monitoring↗

Semipermeable membrane devices accumulate conserved ratios of sterane and hopane petroleum biomarkers.

Semipermeable membrane devices (SPMDs) are commonly used as a time-integrated measure of aqueous concentrations of persistent hydrophobic chemicals, including PAH, pesticides, dioxins, and PCBs. Another class of persistent hydrophobic chemicals is petroleum biomarker compounds (hopanes and steranes) that are used for hydrocarbon source identification and allocation. In this study three different passive sampling device designs were exposed to a complex hydrocarbon mixture (Alaska North Slope crude) in a laboratory experiment to determine uptake rates of biomarkers into SPMDs. In addition to the standard triolein filled SPMDs, iso-octane filled SPMDs (ISPMDs) and unfilled low-density polyethylene strips (PESDs) were tested. Uptake rates and effective sampling rates were determined for 53 compounds. There was little variation in sampling rates among the individual biomarkers; average values (ld(-1)) for hopanes were 0.43+/-0.07 (PESD), 0.33+/-0.06 (SPMD), and 0.44+/-0.03 (ISPMD) and average sterane sampling rates were 0.57+/-0.04 (PESD), 0.42+/-0.03 (SPMD), and 0.53+/-0.03 (ISPMD). The primary reason for biomarker analysis is for source discrimination of petroleum. Nineteen different diagnostic ratios were measured, and were found to be remarkably well conserved between the oil, water, and all three devices. This indicates that SPMDs, PESDs, and ISPMDs should each be effective for source discrimination studies of petroleum contamination.

Alaska↗

Effect of alcohol addition on the movement of petroleum hydrocarbon fuels in soil.

Groundwater contamination by fuel spills from aboveground and underground storage tanks has been of growing concern in recent years. This problem has been magnified by the addition of oxygenates, such as ethanol and methyl-tertiary-butyl ether (MTBE) to fuels to reduce vehicular emissions to the atmosphere. These additives, although beneficial in reducing atmospheric pollution, may, however, increase groundwater contamination due to the co-solvency of petroleum hydrocarbons and by the provision of a preferential substrate for microbial utilisation. With the introduction of ethanol to diesel fuel imminent and the move away from MTBE use in many states of the USA, the environmental implications associated with ethanol additive fuels must be thoroughly investigated. Diesel fuel movement was followed in a 1-m soil column and the effect of ethanol addition to diesel fuel on this movement determined. The addition of 5% ethanol to diesel fuel was found to enhance the downward migration of the diesel fuel components, thus increasing the risk of groundwater contamination. A novel method using soil packed HPLC columns allowed the influence of ethanol on individual aromatic hydrocarbon movement to be studied. The levels of ethanol addition investigated were at the current additive level (approx. 25%) for ethanol additive fuels in Brazil and values above (50%) and below (10%) this level. An aqueous ethanol concentration above 10% was required for any movement to occur. At 25% aqueous ethanol, the majority of hydrocarbons were mobilised and the retention behaviour of the soil column lessened. At 50% aqueous ethanol, all the hydrocarbons were found to move unimpeded through the columns. The retention behaviour of the soil was found to change significantly when both organic matter content and silt/clay content was reduced. Unexpectedly, sandy soil with low organic matter and low silt/clay was found to have a retentive behaviour similar to sandy subsoil with moderate silt/clay, but little organic matter. It was concluded that sand grains might have a more important role in the adsorption of petroleum hydrocarbons than first realised. This method has shown that soil packed HPLC columns can be used to provide a quick estimate of petroleum hydrocarbon, and possibly other organic contaminant, movement in a variety of different soil types.

Adsorption↗

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons.

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.

Animals↗

Predicting petroleum phototoxicity.

Phototoxicity to Daphnia magna was studied on 14 polycyclic aromatic hydrocarbons (PAHs) and 22 petroleum products (ranging from diesel to crude oil). The phototoxicity ranking of pure PAHs was about the same as found in another study on fish gill cells in vitro (Toxicology 127 (1998) 143), suggesting that the relative acute phototoxicity does not differ significantly between different species. Most petroleum products were found to be phototoxic, although the results differ somewhat between test methods (preparation of water-accommodated fractions or petroleum ether dissolved oil slurries). The degree of phototoxicity of a sample is related to both the source and the refining process of the crude oil. The best chemical predictors found were the concentrations of eight phototoxic parent PAHs. An even simpler analytical technique suggested for initial screening would be direct measurements of "total PAH," where samples having more than 3% PAH should be studied further.

Animals↗

Photo-oxidation products of petroleum hydrocarbons in the Eastern Red Sea coastal waters.

The level of dissolved oil residues as well as the oxygenated petroleum hydrocarbons in the seawater of the affected area of Jeddah coast, Red Sea was estimated. The dissolved/dispersed petroleum hydrocarbons concentration was found minimal. The minimum level may be attributed to the action of the weathering processes. Selected samples of the dichloromethane extracts of seawater were fractionated, using silica gel columns, into four fractions eluted with solvents of increasing polarity. The fluorescence intensity of these fractions was measured at three different pairs of excitation and emission wavelengths using ultraviolet fluorescence spectrofluorophotometry (UVF). The total fluorescence intensity of the fractions at 280 nm excitation, 327 nm emission wavelengths and 310 nm excitation, 360 nm emission wavelengths exceeds the fluorescence intensity of the whole extract by more than two orders of magnitude. The estimation of higher molecular mass oxygenated compounds in the fourth fractions, read at 380 nm excitation, 430 nm emission wavelengths, indicates rather low concentrations of <1 microg l(-1) hydroxypyrene equivalents. However, the polar fractions (eluted with acetone) account for more than 25% of the total fluorescence intensity of all fractions combined at each pair of wavelengths. The characterization of the products causing the fluorescence particularly the high molecular mass compounds still presents difficulties. Preliminary GC/MS results revealed some evidence for petroleum hydrocarbon oxidation products. Alkyl isobenzofuranones have been found in some seawater extracts. This is an ongoing project to characterize and identify additional compounds from these extracts.

Gas Chromatography-Mass Spectrometry↗

The vanadium isotopic constitution of petroleum asphaltenes: La Luna Formation (Venezuela).

High-resolution mass spectrometry indicates that the isotopic abundance of 50V of the Late Cretaceous La Luna petroleum asphaltenes of marine origin (highly enriched with V > 2000 ppm) is higher by about 3.5% than that of the inorganic source (VOSO4 x 5H2O, Merck). We propose that the difference in the 50V/51V values between the La Luna petroleum asphaltenes and the inorganic source can be best ascribed to the biological processing of seawater vanadium. The fact that the V isotopic compositions of petroleum asphaltenes vary over a very narrow range (2.46-2.52) suggests essentially the same (or similar) and fixed biological source of vanadium.

Hydrocarbons↗

Biochemical changes as early stage systemic biomarkers of petroleum hydrocarbon exposure in rats.

Crude and refined petroleum contain a complex mixture of aliphatic, aromatic, polyaromatic and heterocyclic hydrocarbon compounds. The objective of our research was to investigate early-stage biochemical changes in rats exposed to low dosages of petroleum hydrocarbons. The animals were repeatedly exposed, per oral by gavage, to low dosages (0.5-2.5 ml/kg) of an Alberta crude oil (ACO) and their general health and systemic biochemical parameters were assessed. Rats exposed to these doses of ACO did not show any apparent symptoms of intoxication. Similarly, no significant changes were observed in clinical parameters of systemic impairment. Systemic biochemical assessment has shown that ACO exposure caused marked changes in the activities of several cytochrome P-450 (CYP)-linked polysubstrate monooxygenase enzymes in liver, kidney and lung tissues. Exposure to ACO caused dose-dependent increases in the hepatic activities of ethoxyresorufin-O-deethylase, a CYP 1A1/A2-linked enzyme; pentoxyresorufin-O-dealkylase, a CYP 2B-linked enzyme, and ethoxycoumarin-O-deethylase, a CYP 2B/1A-linked enzyme. Temporal assessment showed that these systemic biochemical changes were reversible in nature. Analysis of biomarker chemicals provided evidence that in exposed animals petroleum hydrocarbons were mainly distributed in the adipose tissues.

Adipose Tissue↗