Weathered Hydrocarbon Biotransformation: Implications for Bioremediation, Analysis, and Risk Assessment
Weathered petroleum hydrocarbons are highly complex and important soil contaminants, which, despite 40 years of petroleum research, are still not sufficiently understood or appropriately characterized for informing contaminated land risk assessments. Improved insights into biotransformation of these contaminants and their residual toxicity are essential for improving risk assessments, bioremediation strategies, and effective regeneration of previously contaminated land. The remediation of land contaminated with weathered hydrocarbons has long been limited by inappropriate analytical methodology, the absence from risk assessment frameworks, reduced stakeholder confidence, lack of ecotoxicological analysis in risk assessments, and a distinct paucity of information regarding weathered hydrocarbon toxicity, distribution, transport, and availability in the environment. Recent research has resulted in the development of a robust analytical method for identification of hydrocarbon residues (weathered hydrocarbons) which are the principal source of the organic carcinogens or suspected carcinogens that drive quantitative risk assessment (e.g., benzo[a]pyrene), development of a tool kit for contaminated sites incorporating ecotoxicological consideration, and an improved understanding of weathered hydrocarbon toxicity and biotransformation chemistry. However, knowledge gaps still remain, and additional implications for bioremediation practitioners have been identified concerning remedial methodology at previously remediated sites.
Petroleum hydrocarbons are common environmental contaminants. They are components of crude oil and products derived from it and are consequently found on a variety of sites including refineries, chemical materials and by-products storage sites, and manufactured gas production sites. They may also be present as a result of spills and leaks during transportation. They are a highly complex mixture of aliphatic and aromatic hydrocarbons with minor amounts of other heterogenic compounds such as nitrogen, sulfur, and oxygen (Farrell-Jones 2003). Once released to the environment, they are subject to physical, chemical, and biological processes that further change their composition, toxicity, availability, and distribution (partitioning) within the environment. Such degradation processes (weathering processes) include adsorption, volatilization, dissolution, biotransformation, photolysis, oxidation, and hydrolysis (Brassington et al. 2007; Pollard et al. 1994, 2005). The extent of weathering experienced is particularly important when characterizing petroleum contamination prior to remediation (Wang et al. 1998), especially the heavy oils, which have high viscosity (ca. 50–360 mPa s), high boiling point (ca. 300– > 600 °C), and carbon number ranges in excess of C20. These weathering processes shift their chemical composition toward recalcitrant, asphaltenic products of increased hydrophobicity.
Petroleum hydrocarbon fractions (based on equivalent carbon numbera) for use in UK human health risk assessment. Hydrocarbon fractions usually identified for weathered oils are in bold (Environment Agency 2005)
EC > 5–6
EC > 5–7
EC > 6–8
EC > 7–8
EC > 8–10
EC > 8–10
EC > 10–12
EC > 10–12
EC > 12–16
EC > 12–16
EC > 16–35
EC > 16–21
EC > 35–44
EC > 21–35
EC > 35–44
EC > 44–70
Although these important qualitative and quantitative differences between weathered and non-weathered petroleum hydrocarbons are widely acknowledged (see, for review, Brassington et al. 2007), weathered hydrocarbons are not sufficiently understood or appropriately characterized for assessing risk at contaminated sites.
Measuring the total concentration of petroleum hydrocarbons (TPH) in soil does not give a useful basis for the evaluation of the potential risks to human and the environment. The variety of physical-chemical properties, and thus differences in the migration and fate of individual compounds, and the toxicity of different fractions and compounds in oil products must be taken into account in human health risk assessments .
2 Weathered Petroleum Hydrocarbon Analysis in Soil
Summary of different analytical methods developed for risk assessment frameworks (Modified from Brassington et al. (2007 ))
Massachusetts Department of Environmental Protection (MaDEP 1994)
Canadian Council of Ministers of the Environment (CCME 2000 )
New Zealand (Ministry for the Environment 1999 )
New South Wales (National Environment Protection Council 1999 )
Risdon et al. (2008)
Use of two methods. Volatile petroleum hydrocarbon (VPH) method (MaDEP 2004b) and extractable petroleum hydrocarbon (EPH) method developed by MaDEP. VPH method uses purge and trap with methanol. EPH method uses DCM for extraction and solvent exchanges into hexane. Using one of several US EPA solvent extraction methods (MaDEP 2004a)
Vortex or shaker method using n-pentane
Purge and trap for C6 to C10 range using methanol. Soxhlet for the C10 to C50 range
Purge and trap is used for the C6 to C9 range. For the C10 to C36 range, any methods that meet set performance criteria are used
US EPA methods 3540B (US EPA 2005) or 3540C (US EPA 1996a) (Soxhlet extraction), 3550B (US EPA 1996b) (sonication extraction) or sequential bath sonication and agitation described by NEPC (National Environment Protection Council 1999)
Sequential ultrasonic solvent extraction for the nC8 to nC40 using 1:1 acetone/hexane mixture
The EPH method uses those specified by the US EPA. However, after fractionation the use of a gentle stream of air or nitrogen is recommended to bring the sample to the required volume. Evaporation is not applicable to the VPH method
Uses an evaporation vessel after extraction for the C10 to C50 range. After silica gel cleanup, rotary evaporator is used to reach the required sample volume
Use of any method that meets set of performance criteria
US EPA methods specified for extraction using Kuderna-Danish (K-D) evaporation
Not required but can be used to achieve lower limits
Silica gel cleanup for EPH method. Not applicable to VPH method
Extract fractionation using alumina or silica
One of two specified cleanup steps for C10 to C50 range, not fractionated
Cleanup steps and fractionation are optional as this may not be required for each sample/analytical approach
Not necessary; however is used to fractionate samples. Uses microscale silica gel column chromatography after a water partition step
EPH uses GC-FIDa. VPH may use either GC/FIDa or GC/PIDb
For the C10 to C36 range, GC-FIDa is used, and for the C6 to C9 range, GC-MSc is used
GC-MSc, or GC-FIDa; however the use of GC/MSc to identify unusual mixtures is noted as being necessary when analyzing by GC-FIDa
Combination of GC-FID and GC-MS depending upon level of analysis
Various extraction techniques for petroleum hydrocarbons exist within the open literature; however many suffer from inter-method variation and both positive and negative bias (Buddhadasa et al. 2002; Environment Agency 2003; Whittaker et al. 1995). Historically, Soxhlet extraction has been the benchmarked method, due to its exhaustive nature, high recoveries, and ability to be easily standardized (Risdon et al. 2008; Shu et al. 2003). However Soxhlet suffers from long extraction times, a need to concentrate samples, high solvent use, and the degradation of thermally liable compounds (Risdon et al. 2008; Shu et al. 2003). Consequently this has resulted in investigations into and development of the alternative methods (Hawthorne et al. 2000; Hollender et al. 2003; Risdon et al. 2008; Whittaker et al. 1995). Alternative methods to Soxhlet include ultrasonication (Banjoo and Nelson 2005; Risdon et al. 2008; Sanz-Landaluze et al. 2006), pressurized liquid extraction (Hawthorne et al. 2000), supercritical fluid extraction (Hawthorne et al. 2000), subcritical water extraction (Hawthorne et al. 2000), and microwave-assisted extraction (Saifuddin and Chua 2003). Although some of the alternative methods offer improvements over Soxhlet extraction, most of these methods need further refinement and optimization, as there have been conflicting results from different investigations into the same method. For example, Heemken et al. (1997), Sun et al. (1998), Banjoo and Nelson (2005), and Sporring et al. (2005) demonstrated ultrasonic methods that had equivalent or better extraction efficiencies than Soxhlet, whereas investigations by Song et al. (2002) and Hollender et al. (2003) gave worse extraction efficiencies.
Recently, a novel and robust ultrasonic extraction method for contaminated soils with weathered hydrocarbons has been developed and optimized (Risdon et al. 2008). The method covers the determination of TPH between nC8 and nC40 and subranges of hydrocarbons including diesel range organic compounds , kerosene range organic compounds, and mineral oil range organic compounds in soils. Further modifications to the TPH carbon banding may be made as requested for risk assessment including ranges known as Texas Risk banding (TPH C8–C10, C10–C12, C12–C16, C16–C21, and C21–C5) as well as separation of the aliphatic and aromatic fractions as defined in the UK regulatory framework (Environment Agency 2005). The method can be routinely used for measuring hydrocarbons down to 10 mg kg−1 in soil. Detection limits may vary for individual carbon ranges calculated on the percentage of the full range they cover. With an extraction efficiency and recovery between 95% and 99%, this method can be easily positioned as a good alternative to Soxhlet extraction and shows a good potential for implementation as a standard method potentially providing further insight to the contaminated land sector. The method has been accredited ISO17025 for TPH analysis, banding, and class separation.
3 Risk Assessment for Weathered Hydrocarbons
Risk assessment is an established requirement for the management of contaminated land (ARCADIS Geraghty and Miller International Inc. 2004) and now a widely used support tool for environmental management decisions. It is employed as a means of assessing and managing potential impacts to human and ecosystem health (Vegter et al. 2002). Several risk-based frameworks for petroleum hydrocarbons in soil have been published under the auspices of the Total Petroleum Hydrocarbon Criteria Working Group (TPHCWG 1999), the American Society for Testing and Materials (ASTM 1994), the Massachusetts Department of Environmental Protection (MADEP 2002), the Environment Agency of England and Wales (Environment Agency 2005), the American Petroleum Institute (API 2001), and the Canadian Council of Ministers of the Environment (CCME 2000), each reflecting national legislation, a range of expert judgments, and socioeconomic issues. Typically these frameworks adopt a three-tiered approach with increasingly sophisticated levels of data collection and analysis, as an assessor moves through the tiers. However, these frameworks, and the exposure assessment methods embedded within them, do not specifically address weathered hydrocarbons, although some acknowledge that petroleum products released to the environment will have undergone some degree of degradation (API 2001; Environment Agency 2005; MADEP 2002; TPHCWG 1998a).
Assessing the risk of weathered hydrocarbons that they pose at contaminated sites is complicated because the profile of compounds present in weathered oil can be very different from the composition of fresh oil. However, many hydrocarbon compounds would be sufficiently similar in structure to expect that they might have similar toxicities and endpoints. In view of these factors, the UK approach to human health risk assessment of petroleum hydrocarbons favored the adoption of a combined indicator and fraction approach within a tiered risk-based framework (Environment Agency 2005). Specific indicator compounds (genotoxic carcinogens and noncarcinogens) should be assessed because these are often the key risk drivers at petroleum-contaminated sites. Genotoxic carcinogens are assumed not to have a threshold concentration as even very small concentrations (or doses) are assumed to pose some (albeit small) risk of cancer. There are cases in which carcinogenicity can be assumed to occur only after some dose or threshold concentration is reached, depending on the mode of action by which the contaminant is thought to cause cancer. The assessment of fractions should facilitate a more representative picture of risk at sites where the origin of the petroleum hydrocarbons contamination may be unclear. The fractions are typically used to consider threshold health effects (Environment Agency 2005).
List of potential indicator compounds for weathered hydrocarbons
Potential indicator compounds for weathered hydrocarbon-contaminated soils
Aromatic > EC10–EC12
Aromatic > EC12–EC16
Aromatic > EC16–EC21
Aromatic > EC21–EC35
As shown in Tables 1 and 3, use of the combined indicator and fraction approach instead of measuring the total concentration of TPH in weathered oils provides a better insight of the carbon range compounds and the key risk drivers within each fraction. Access to this detailed information is important for assessing human and environmental risks and effective remediation at contaminated sites.
4 Use of Fugacity Modeling to Parametrize and Conceptualize Fate and Behavior of Petroleum Hydrocarbons
Understanding the environmental fate of a contaminant is a key requirement when estimating potential risks to human health. To achieve this, meaningful information on a substance’s behavior and distribution, toxicity, concentration, and potential exposure at a site is essential (Allan et al. 2006; Environment Agency 2003).
The overriding dominance of the nonaqueous phase liquid (NAPL) for hydrophobic contaminants is theoretically established but rarely incorporated into the exposure assessment tools used to derive soil screening levels and guideline values (Pollard et al. 2008). This oversight is likely to have a marked influence on soil assessment criteria at hydrocarbon-contaminated sites. Its significance comes into play when one considers the residual risk posed by posttreatment residues. Level I and II fugacity models were developed comprising four phases within the soil matrix, namely, air, water, mineral soil, and NAPL. The implications of the fugacity modeling developed by Pollard et al. 2008 are important for risk analysts and remediation engineers. The fugacity modeling confirmed the propensity for risk critical compounds to be preferentially partitioned to the NAPL and soil phases. However, modeled depletion times for contaminants in the context of authentic soils are immaterial, and thus research efforts should be focused on the likely exposures of humans and other receptors to residual saturation at hydrocarbon-contaminated sites. The results indicate clearly the need for modifications to the exposure assessment models used to generate soil screening guidelines or guideline values, so as to better represent contaminant fate in the multimedia systems.
5 Bioavailability Complexity
Given the multiple variables affecting the availability of chemicals in the soil, we should look at bioavailability not as a fixed value (concentration), but as a dynamic process between an organism and the chemical uptake over time (Lanno et al. 2004). However the evaluation of contaminant-soil matrix interactions, that might partially be the cause of the non-accessibility, is still a challenge (Wu et al. 2013, 2014 ). Research should focus on understanding and accurately representing the bioavailable fraction and ensuring that this parameter is correctly quantified in the risk assessment.
6 Bioremediation of Weathered Hydrocarbons
There is a plethora of approaches for the remediation of contaminated land, encompassing physical, chemical, and biological methods which can contain, destroy, or separate the contaminants. Implementation of the EU Landfill Directive (The Council of the European Union 1999) encourages the development and implementation of alternative remediation techniques such as bioremediation in a move away from the mainstay method of excavation and disposal. The organic nature of petroleum hydrocarbons makes these contaminants highly suited to bioremediation techniques as such, and due to the widespread, health, and ecological hazards posed by petroleum hydrocarbons, greater interest has been directed at these contaminants. Bioremediation is a well-established method that works well for remediating petroleum hydrocarbon-contaminated soil (Flathman et al. 1994; Hyman and Dupont 2001). Bioremediation methods are often optimized, using biostimulation and bioaugmentation to enhance biotransformation, reduce cost, and process duration.
Typically, biotransformation is rapid in the initial stages of bioremediation, with rates seen to asymptote toward the end of remediation treatments (Ellis 1994; Fogel 1994; Wood 1997) as the proportion of less bioavailable and recalcitrant compounds increase. Weathered hydrocarbons generally display relatively low bioavailability and are more recalcitrant than their non-weathered counterparts. As such optimization of bioremediation can prove essential to the successful remediation of weathered hydrocarbons (Giles et al. 2001; Guerin 2000).
Further, on the basis of physicochemical parameters, the study suggested that the success of bioremediation considering both biostimulation and bioaugmentation approaches was largely dependant of the oil contaminant and soil structure characteristics. For the majority of the contaminated soils investigated, mineral nutrients played an essential role without which in some cases bioremediation could not occur. Slow-release fertilizers were shown to be an important alternative to liquid fertilizers, in mitigating issues arising from the addition of liquid fertilizers. Combining bioaugmentation strategies with biostimulation may improve the rate and extent of weathered hydrocarbon degradation, while the potential benefit of bioaugmentation still needs further evidence. Ex situ bioremediation for treatment will allow greater control over soil temperature, water holding capacity, and leaching.
The design of an efficient bioremediation system always requires a careful site assessment. Consideration of the physical, chemical, and biological properties of the contaminated sites is essential in establishing appropriate response and recovery methods. Despite the ability of indigenous microorganisms to degrade petroleum hydrocarbons, there are still situations where the use of a microbial inoculum might enhance petroleum hydrocarbon biodegradation.
Ecotoxicological tests used for weathered hydrocarbons (selected species are in bold)
Eisenia fetida, Lumbricus terrestris, Lumbricus rubellus
Brassica alba mustard white, Triticum aestivum (Consort) wheat, Pisum sativum, pea
Luminescence-based bacterial biosensors
Metabolic: Vibrio fischeri, Escherichia coli HB101, Pseudomonas putida F1 Tn5
Catabolic: Escherichia coli HMS174, Escherichia coli DH5α, Pseudomonas putida TVA8
The research demonstrated that a gross reduction of the hydrocarbons does not represent environmental nor sustainable improvement, as reported by the biological response. In order to integrate and rank the effectiveness of remediation treatments, the biological indicator data were transformed into a soil quality index (SQI) (Dawson et al. 2007). The results highlighted that biological receptor specificity defined the risk-based endpoint and reinforced the concepts of risk reduction within a dynamic and degrading oil matrix. Furthermore, the changes in toxicity and to some extent the bioavailability of fractions within the matrix reflect that some pollutants may be mobilized during remedial activities .
7 Research Needs/Knowledge Gaps
It is clear that successful bioremediation of land contaminated with weathered petroleum hydrocarbons depends upon the successful integration of effective analysis techniques, informed optimization of bioremediation, and an appropriate risk assessment protocol. Remediation of contaminated soils is limited by several factors including a lack of toxicological and environmental fate and behavior data, inadequate and variable chemical analyses, negative stakeholders’ perception of bioremediation techniques, and variable risk assessments that do not always consider weathered hydrocarbons. While the work of researchers has taken important steps toward addressing key knowledge gaps and methodological limitations within analysis and risk protocols, there is still work to be done.
Further investigation is still required to increase our knowledge on weathered hydrocarbon chemical, toxicological, and biological diagnostics as well as environmental fate and behavior. This combined diagnostic approach will significantly help to identify optimal remediation strategies and contribute to change the overconservative nature of the current risk assessments.
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