Implications of bioremediation of polycyclic aromatic hydrocarbon-contaminated soils for human health and cancer risk

Abstract

Bioremediation uses soil microorganisms to degrade polycyclic aromatic hydrocarbons (PAHs) into less toxic compounds and can be performed in situ, without the need for expensive infrastructure or amendments. This review provides insights into the cancer risks associated with PAH-contaminated soils and places bioremediation outcomes in a context relevant to human health. We evaluated which bioremediation strategies were most effective for degrading PAHs and estimated the cancer risks associated with PAH-contaminated soils. Cancer risk was statistically reduced in 89% of treated soils following bioremediation, with a mean percent degradation of 44% across the B2 group PAHs. However, all 180 treated soils had post-bioremediation cancer risk values that exceeded the U.S. Environmental Protection Agency (USEPA) health-based acceptable risk level (by at least a factor of two), with 32% of treated soils exceeding recommended levels by greater than two orders of magnitude. Composting treatments were the most effective at biodegrading PAHs in soils (70% average percent reduction compared with 28–53% for the other treatment types), which was likely due to the combined influence of the rich source of nutrients and microflora introduced with organic compost amendments. Ultimately, bioremediation strategies, in the studies reviewed, were unable to successfully remove carcinogenic PAHs from contaminated soils to concentrations below the target cancer risk levels recommended by the USEPA.

Graphical Abstract

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants formed through the incomplete combustion of organic matter. Their hydrophobic nature and persistence can ultimately lead to their accumulation in soils, particularly at industrial sites where direct contamination occurs.¹ Many PAHs are known or suspected human carcinogens² and, as a result, 16 parent (unsubstituted) PAHs have been classified as ‘priority’ pollutants by the U.S. Environmental Protection Agency (USEPA).³ In 1980, the USEPA initiated the Superfund Program to manage the clean-up of contaminated, hazardous waste sites that pose risks to human health and the environment.⁴ The clean-up process is complex, from preliminary assessment and site investigation to site reuse and redevelopment,⁵ and can use any number of clean-up strategies (or combinations of strategies) depending on the level of contamination, the compounds involved, and site characteristics.^{6, 7}

Bioremediation is a process that uses microorganisms to facilitate the degradation of PAHs into, ideally, less toxic breakdown products. It is generally considered a safer, cleaner, more energy efficient, and more economically viable remediation option than alternative technologies, such as incineration or surfactant flushing, because it uses microorganisms in the soil (treatment can be in situ as in land farming or ex situ as in bioreactors) and requires minimal infrastructure and attention.^6–8 However, in spite of the benefits, bioremediation is the strategy employed at only 6% of U.S. Superfund sites to treat highly contaminated soils.⁹ A number of factors, including soil properties (temperature, pH, organic carbon and mineral contents, nutrient accessibility), environmental conditions, contaminant profiles, initial concentration,¹⁰ microbial populations (number and type), and bioremediation conditions (time, scale, moisture, aeration, and amendments) can influence the bioremediation outcome; these factors have been addressed in numerous studies and previous reviews.^11–13 However, evidence suggests that the toxicity and/or carcinogenicity associated with the contaminated soils may remain following bioremediation treatment^14–16 and in some cases, even increase, despite concentrations of the parent PAHs decreasing.^17–20 The partial degradation of parent PAHs may result in the formation of more polar and toxic byproducts, such as the oxygen-containing PAHs (quinones, ketones, hydroxylated-PAHs etc.).^20–24 However, PAH metabolites are seldom measured and their carcinogenicity and behavior in soils are not well understood.

Current guidelines recommend monitoring the progress and overall success of bioremediation treatments at U.S. Superfund sites through targeted measurements of the 16 priority PAHs.³ Additional estimates, in the form of total excess lifetime cancer risk (ELCR), may also be calculated to determine projected additional ‘incidence’ rates of cancer in adult populations resulting from ingestion or inhalation exposure to PAH-contaminated soils.^25–27 Typically, ELCR estimates are based on the concentrations of only 8 of the 16 priority PAHs classified as probable human carcinogens (these are the B2 group PAHs containing 4- to 6-rings fused benzene rings) and their carcinogenic potencies relative to benzo(a)pyrene (BaP), which is a commonly used reference compound for toxicity assessments.^{28, 29} However, several studies have shown that soil bioremediation strategies often fail to sufficiently degrade these carcinogenic PAHs to levels such that the soils are safe for reuse.^{1, 30–36} Furthermore, few studies extend beyond the reporting of chemical-based outcomes of bioremediation treatments (e.g., PAH concentrations and percent degradation) to place bioremediation outcomes in a human health context, using a metric such as the ELCR. Lemieux et al.³⁷ measured the concentrations of PAHs in ten Swedish soils, from which they calculated ELCR estimates according to Health Canada guidelines.³⁸ When compared to a bioassay approach based on toxicity endpoints for the same soils, the ELCR provided conservative estimates of cancer risk.

To date, many bioremediation studies or culture-based experiments that isolate PAH-degrading bacteria for further development in bioremediation studies are limited because they focus on only one or a handful of parent PAHs.^{10, 22, 39–50} The PAHs of choice in these studies are typically the 2- to 4-ringed priority PAHs,^{10, 22, 40, 41, 47} such as phenanthrene^{39, 43–46, 48, 49, 51, 52} and pyrene,^{44–46, 48–50} while the 4- to 6-ringed carcinogenic PAHs are often ignored. It is noteworthy that PAHs are not present in isolation in the environment, but occur as complex mixtures of both parent PAHs (including, but not limited to, the 16 priority PAHs) and substituted transformation products, such as the oxygenated-PAHs.^{17, 20–22} Thus, many studies focus on only a few PAHs that are less of a human health concern and have limited environmental relevance, particularly when it comes to assessing carcinogenic potential. Previous reviews regarding bioremediation of PAH-contaminated soils have specifically focused on the physico-chemical, biological, and environmental factors influencing bioremediation efficacy,^{1, 11–13, 53} including the microorganisms (bacteria, fungi, and/or algae) and enzymes participating in PAH degradation,^{12, 13} aspects of PAH bioavailability,¹¹ and various bioremediation treatment and amendment types.^{1, 7, 12, 13, 53–59}

Scope of the Review

This review focuses on the cancer risks associated with the bioremediation of PAH-contaminated soils and more specifically, whether bioremediation sufficiently reduces the cancer risks associated with contaminated soils to levels below the target health-based acceptable cancer risk levels recommended by the USEPA.^{60, 61} Our novel approach extends beyond summarizing results previously reported in the literature by performing a metadata analysis to calculate changes in ELCR estimates following bioremediation. We collated PAH concentrations in contaminated soils from the literature, both prior to and following various bioremediation treatments, calculated their associated change in ELCR, and compared both the individual PAH concentrations in soil (for the 16 priority PAHs) and ELCR estimates (which represent the cumulative cancer risk associated with the carcinogenic B2 group PAHs) to current USEPA health-based acceptable risk levels. This is the first review to evaluate which bioremediation strategies are most effective at degrading the carcinogenic B2 group PAHs and furthermore, to summarize bioremediation outcomes in a context relevant to human health and in a manner reflective of current regulatory cancer risk assessment guidelines.

CANCER RISK ESTIMATES FOR BIOREMEDIATION TREATMENTS REPORTED IN THE LITERATURE

Collation of PAH concentrations and percent degradation reported in the literature:

Peer-reviewed manuscripts reporting bioremediation treatments for the detoxification of PAH-contaminated soils were identified in Google Scholar and Web of Science databases using search terms such as “soil PAH (bioremediation)”, “biodegradation”, “composting”, “biostimulation”, and/or “bioaugmentation”. Manuscripts were initially screened for relevance and to assess whether they met our selection criteria: 1) manuscripts were published within the past 20 years (post-1997), 2) data pertained to bioremediation of PAH-contaminated soils (not sediments or sewage sludge), 3) PAHs were extracted from soil using exhaustive approaches with organic solvents (e.g., pressurized liquid extraction, sonication, Soxhlet extraction) and quantified using well-established analytical techniques (e.g., gas or liquid chromatography coupled with mass spectrometry or flame ionization detection), 4) initial soil contamination exceeded a [PAH]_total of 50 mg kg^-1, 5) concentrations in soil were reported for each individual PAH both pre- and post-bioremediation (or a combination of initial concentrations and their associated percent degradation following bioremediation), 6) mean concentrations ± standard deviations (or original replicate data) were reported in tabulated form, and 7) data for at least five of the eight B2 group PAHs were included (benzo(a)anthracene (BaA), chrysene (Chr), BaP, benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), indeno(1,2,3-cd)pyrene (Ind), dibenz(a,h)anthracene (DBahA), and benzo(g,h,i)perylene (BghiP)). There were numerous instances where the data reported in the literature failed to meet one or more of our criteria (e.g., data was displayed in figures, PAH concentrations were combined for 2- to 6-ringed PAHs, standard deviations were omitted etc.), but it was clear that the original (unprocessed) data would meet all the criteria. In these cases, the corresponding authors were contacted if the data had been published more recently than 2010; we explained our objectives and data requirements, and requested their cooperation in providing their original data. If no response was received within two weeks of initial contact, a follow-up request was sent. Of the 12 authors contacted (regarding 15 manuscripts), 3 were willing and able to provide the necessary data. Table S1 in the Supporting Information (SI) lists the 26 references that met our criteria and details the properties of the contaminated soils. For each soil remediation treatment, additional details, such as the bioremediation strategy, time, and amendments were systematically collated (Table S2). The percent degradation was calculated based on the pre- and post-bioremediation PAH concentrations in soil for each soil treatment (Table S3) and for the individual PAHs and treatment types (Table S4).

Calculation of cancer risk for PAH-contaminated soils:

The USEPA defines cancer risk as the “incremental probability of an individual to develop cancer over a lifetime as a result of exposure to a potential carcinogen.⁶²” ELCR values were calculated according to current USEPA guidelines and represent estimated excess ‘incidence rates’ of cancer in adult populations exposed via incidental non-dietary ingestion (e.g., hand-to-mouth transfer).²⁹ This is a chemical-based approach that assumes cancer risk is additive and a function of the exposure scenario,²⁶ the concentrations of individual PAHs in soil, $C_{\text{i}}$(mg kg^-1), and their relative potency factors (${\text{RPF}}_{\text{i}}$)²⁸ (eq 1):

$$\text{ELCR}=(\frac{\text{IR}\cdot \text{EF}\cdot \text{CF}\cdot \text{SF}\cdot {10}^6}{\text{BW}}\sum _{\text{i}=1}^n\left(C_{\text{i}}\cdot {\text{RPF}}_{\text{i}}\right)$$ (eq 1)

where IR is the soil ingestion rate (50 mg d^-1), EF is the exposure factor, CF is a unit conversion factor (10^-6 kg mg^-1), SF is the oral slope factor for BaP (7.3 (mg kg^-1 d^-1)^-1), and BW is body weight (70 kg).⁶³ The EF was calculated based on exposure recommendations, for indoor workers and industrial soil, of 5 days/week and 50 weeks/year over 25 years of exposure, with a life expectancy of 70 years.^{26, 29, 62, 63} The slope factor is expressed as the proportion of the population affected per mg of BaP ingested per kg of body weight per day and represents an upper estimate of the response per unit of contaminant intake over a lifetime.²⁵ The ELCR is expressed as the number of additional cancer cases expected in an exposed population of one million. Under the current risk assessment framework, only the eight B2 group PAHs identified as probable human carcinogens were included in the ELCR calculation, details of which are displayed in Table S5. The USEPA defines health-based acceptable cancer risk levels based on a 10⁻⁶ incidence for individual carcinogens and a 10⁻⁴ incidence for cumulative risk from multiple carcinogens and exposure routes (i.e., the probability of an exposed individual developing cancer during their lifetime ranges between 1 in 10 000 and 1 in 1 million depending on the risk assessment strategy).^{25, 61}

Statistical analyses performed on collated data:

Due to the aggregated nature of the collated data (usually reported as mean concentrations ± standard deviations, based on three measured samples both pre- and post-bioremediation), we used standard simulation methods described in detail below to reconstruct replicate PAH concentrations in soil from the reported mean and standard deviations. Using the reconstructed PAH concentrations allowed us to compute and statistically compare pre- and post-bioremediation ELCR values (see step 2 below for more detail). A simulation for a single literature source involved the following:

1. Using the mean concentration, $C_{\text{i}}$, and standard deviation, ${\text{SD}}_{\text{i}}$, calculate the pre- and post-bioremediation $\alpha$ (shape) and $\beta$ (rate) parameters of a Gamma distribution (eqs 2 and 3).

   $$\alpha ={\left(\frac{C_{\text{i}}}{{\text{SD}}_{\text{i}}}\right)}^2$$ (eq 2)

   $$\beta =\frac{C_{\text{i}}}{{\text{SD}}_{\text{i}}^2}$$ (eq 3)

2. Generate soil concentrations from the resulting Gamma ($\alpha$, rate=$\beta$) distribution, three each for pre- and post-bioremediation samples. A Gamma distribution is an appropriate model for this type of data, because the soil measurements are asymmetrical and bounded by zero. Using the observed mean PAH concentrations and standard deviations (reported in the literature) to compute the Gamma distribution parameters provides us with a statistical model of the PAH concentrations that is consistent with the reported mean and standard deviation.⁶⁴ Thus, data generated from the Gamma ($\alpha$, rate=$\beta$) distribution is a reconstruction of the actual observed data (for which we do not have individual values). Similar approaches have been used in fields such as hydrology.⁶⁵

3. Calculate three pre- and three post-bioremediation ELCR values per soil treatment from the reconstructed data (eq 1).

4. Calculate the mean of the differences between the pre- and post-bioremediation ELCR values.

For each collated reference, the simulation described above was repeated 10 000 times. To determine whether bioremediation was associated with a statistically significant decrease in ELCR, we used our reconstructed data to test the null hypothesis that the mean of the differences in ELCRs was equal to zero. Specifically, bootstrapped 95% confidence intervals on the mean of the differences, taken as the 0.025^th and 0.975^th percentiles of the mean of the differences, were calculated. If the bootstrapped confidence interval contained zero, then we failed to reject the null hypothesis (Figure 1). Our reliance on simulation necessitated the exclusion of data for certain individual PAHs in a handful of the references because the mean concentrations and/or standard deviations were not reported (e.g., mean of <0.5 mg kg^-1, standard deviation of zero) and were unable to be used for calculating $\alpha$ and $\beta$.

Figure 1.

Mean of the differences ± 95% bootstrapped confidence interval in cancer risk pre- and post-bioremediation from reconstructed PAH concentrations for two soil treatments demonstrating a) failure to reject the null hypothesis (no difference in cancer risk pre- and post-bioremediation) and b) rejection of the null hypothesis (decrease in cancer risk following bioremediation). Each of the 10 000 reconstructed datasets provided three differences in ELCR pre- and post-bioremediation; the means of these differences comprise each histogram.

Bioremediation treatment type assignment for collated data:

Comparisons of bioremediation efficiencies and cancer risk contributions were made by classifying different remediation strategies into one of six general treatment types: 1) Killed control – soil was sterilized via autoclaving or addition of mercuric chloride, for example (note that, while killed controls are not strictly bioremediation treatments, we included them as a group to consider abiotic removal processes, such as volatilization and/or photochemical degradation), 2) No additions – growth of indigenous soil microbiota were encouraged through aeration and maintenance of appropriate moisture levels only, 3) Biostimulation – nutrients, such as NH₄NO₃ and KH₂PO₄, and/or organic carbon (e.g., malt extract glucose medium) were added to the soil to stimulate growth of native microorganisms, 4) Bioaugmentation – soil was inoculated with bacterial and/or fungal colonies to enhance its degradation potential, 5) Surfactant – surfactant compounds were added to the soil to improve PAH bioavailability, and 6) Composting – decomposed organic matter (e.g., green waste compost) and its rich source of nutrients and associated microflora, was added to the soil (Table S2). In situations where a combination of strategies were employed, the treatment type was classified as the higher number (1–6) of the two remediation strategies, because these more extensive treatment types likely had a higher degradation potential. For example, where soil was treated with both nutrient additions (3: biostimulation) and fungal inoculations (4: bioaugmentation), the bioremediation strategy was classified as bioaugmentation.⁶⁶ Where comparisons in PAH trends were made between the different treatment types, individual PAHs were only included in the analysis if they had been reported in at least five studies for each bioremediation treatment type (i.e., the 16 priority PAHs). Table S6 provides the number of reported values collated for each of the individual PAHs and each bioremediation treatment group. The killed control group was excluded from comparative analyses because it contained so few soil treatments.^{32, 67, 68}

HOW EFFECTIVE IS BIOREMEDIATION?

Percent degradation of the 16 priority PAHs reported in the literature:

Bioremediation of PAH-contaminated soils aims to reduce the concentrations of PAHs such that the soil can be safely repurposed for future use (be it in residential and/or industrial settings). For all soil treatments in our collated dataset, the summed concentrations of the 16 priority PAHs (${\Sigma }_{16}\text{PAH}$) decreased following bioremediation. The average reduction (± standard error) in soil concentrations of the eight 2- to 4-ringed priority PAHs (acenaphthene, acenaphthylene, anthracene, fluorene, fluoranthene, naphthalene, phenanthrene, and pyrene) (58 ± 1% for all treatments) was greater than for the carcinogenic (4- to 6-ringed) B2 group PAHs (44 ± 1%), irrespective of treatment type (Table S4). Typically, the percent degradation for several of these B2 group PAHs (e.g., BaP and BghiP) was <40% following bioremediation across multiple treatment types (Figure S1). Benzo(g,h,i)perylene showed the lowest average percent degradation (33 ± 3%) of the priority PAHs. Composting treatments were, on average, the most effective at removing the 16 priority PAHs with the highest mean percent degradation of 74 ± 1%. The percent degradation of ${\Sigma }_{16}\text{PAH}$ for the other treatment types generally decreased in the order of surfactant (more effective) (55 ± 1%) > biostimulation (46 ± 2%) > bioaugmentation (37 ± 2%) ~ no additions (36 ± 2%) (least effective) (Table S4).

Based on the literature to date and our present analysis, bioremediation strategies are typically more effective at removing the 2- to 4-ringed PAHs than the B2 group PAHs.^{31, 68–70} These smaller PAHs are more weakly bound within the soil organic matter and more water soluble than the hydrophobic 4- to 6-ringed PAHs, which ensures that they are bioavailable to microorganisms in the soil and are ultimately degraded to a greater extent.⁷¹ However, removal of the 4- to 6-ringed B2 group PAHs is more difficult due to their persistent nature, strong adsorption to organic matter, and limited bioavailability to the microflora responsible for their degradation.^{20, 34, 71} Furthermore, these compounds are listed as probable human carcinogens and have greater relative carcinogenicity than the 2- to 4-ringed PAHs.²⁹ Thus, the focus of bioremediation ‘clean-up’ treatments, including cancer risk estimates and the analysis methods used to assess the overall success of the treatment, should target the carcinogenic PAHs and/or metabolites that significantly contribute to soil carcinogenicity.

Reduction in cancer risk from B2 group PAHs following bioremediation:

Figure 1 illustrates the mean of the differences (n = 10 000) in pre- and post-bioremediation cancer risk, and the associated 95% bootstrapped confidence intervals from reconstructed PAH concentrations and their respective ELCR values, for two soil treatments; one demonstrating no difference in cancer risk post-bioremediation and one illustrating a significant reduction in cancer risk following bioremediation. 160 of the 180 treated soils (89%) exhibited a statistically significant reduction in cancer risk following bioremediation (Table S3). The average percent reduction in ELCR following bioremediation was highest for composting treatments at 70 ± 2% (Figure 2). No reduction in cancer risk was observed in the five killed control treatments (Figure 2), which agrees with expectations that B2 group PAHs do not undergo substantial abiotic degradation or removal.⁶⁷ The other treatment types exhibited reductions in cancer risk following remediation ranging from 28–53%, on average (Table 1), and will be discussed further in the following section. BaP was typically the largest contributor to the total ELCR, both pre- and post-bioremediation, followed by DBahA and BbF (Figure S2). These are B2 group PAHs with RPFs of 1, 10, and 0.8, respectively.

Figure 2.

Average percent reduction in ELCR values following bioremediation for the different treatment types defined within the collated dataset. Error bars represent standard errors where n = 5, 19, 28, 39, 64, 25, and 180 for the killed control, no additions, biostimulation, bioaugmentation, surfactant, composting, and combined treatment groups, respectively.

Table 1.

Mean percent reduction ± standard error (and range) of ELCR values for the B2 group PAHs following bioremediation with the different treatment types. Negative values indicate that ELCR values increased following bioremediation.

Individual PAHsa	No additions	Biostimulation	Bioaugmentation	Surfactant	Composting	All treatments combined
BaA	35 ± 5 (4 – 83)	52 ± 4 (−10 – 81)	35 ± 5 (−10 – 96)	61 ± 3 (14 – 95)	73 ± 5 (19 – 95)	51 ± 2 (−10 – 96)
BaP	29 ± 6 (0 – 84)	26 ± 5 (−42 – 85)	24 ± 5 (−31 – 96)	48 ± 3 (4 – 87)	68 ± 5 (18 – 96)	38 ± 3 (−42 – 96)
BbF	29 ± 5 (2 – 83)	40 ± 5 (−15 – 82)	28 ± 5 (−15 – 98)	52 ± 2 (5 – 87)	71 ± 5 (21 – 96)	45 ± 2 (−15 – 98)
BghiP	21 ± 8 (−33 – 79)	15 ± 4 (−16 – 68)	9 ± 6 (−65 – 71)	37 ± 4 (−4 – 92)	71 ± 6 (0 – 98)	33 ± 3 (−65 – 98)
BkF	41 ± 8 (−1 – 83)	44 ± 4 (−5 – 86)	25 ± 6 (−98 – 92)	59 ± 2 (5 – 87)	49 ± 3 (42 – 57)	45 ± 2 (−98 – 92)
Chr	36 ± 5 (0 – 80)	48 ± 4 (−12 – 83)	39 ± 5 (−23 – 95)	67 ± 3 (19 – 94)	78 ± 3 (32 – 95)	56 ± 2 (−23 – 95)
DBahA	35 ± 5 (4 – 83)	52 ± 4 (−10 – 81)	35 ± 5 (−10 – 96)	61 ± 3 (14 – 95)	73 ± 5 (19 – 95)	51 ± 2 (−10 – 96)
Ind	20 ± 7 (−27 – 78)	−20 ± 10 (−78 – 11)	43 ± 9 (−8 – 98)	30 ± 13 (−3 – 85)	72 ± 6 (11 – 98)	36 ± 5 (−78 – 98)
${\Sigma }_{\text{B}2}\text{PAHs}$	30 ± 2	34 ± 2	28 ± 2	53 ± 1	70 ± 2	44 ± 1

^aBaA = benzo(a)anthracene, BaP = benzo(a)pyrene, BbF = benzo(b)fluoranthene, BghiP = benzo(g,h,i)perylene, BkF = benzo(k)fluoranthene, Chr = chrysene, DBahA = dibenz(a,h)anthracene, Ind = indeno(1,2,3-cd)pyrene.

Comparisons of bioremediation treatment strategies:

Composting techniques were most effective at both degrading PAHs and reducing the ingestion cancer risk associated with contaminated soils. The average reduction in cancer risk was 70 ± 2% for ${\Sigma }_{\text{B}2}\text{PAHs}$ treated with compost; more than double that of the no additions, biostimulation, and bioaugmentation treatments (which averaged 30%, 34%, and 28% reductions, respectively). Composting methods involve applications of previously degraded organic matter to the soil, such as organic green waste (compost). In addition to providing a rich source of carbon and other nutrients that benefit the indigenous microorganisms, compost introduces and helps sustain a diverse, new pool of microflora in the soil.^{53, 59} Once established, these microorganisms can participate in the PAH-degradation process, be it through active metabolism⁷² or production of extracellular enzymes or compounds with surfactant-like properties^{73, 74} that enhance the bioavailability of PAHs for the active degraders in the soil.³⁶ With the combined action of these factors taken into consideration, it is perhaps not surprising that composting techniques appear to be more effective at removing the carcinogenic PAHs from contaminated soils. Moreover, composting constitutes a beneficial and sustainable approach because it improves soil quality⁵⁹, while providing an avenue through which biodegradable organic waste can be reused.⁷⁵

Nevertheless, alternative bioremediation treatment strategies should not be completely discounted. Depending on the age and extent of contamination and the PAHs involved, alternative treatment types may hold several benefits. In the following sections, we delve further into the differences in efficacy between bioremediation treatment types and examine the influence of bioremediation length and multiple bioremediation steps (or sequestration during soil aging).

Influence of bioremediation length

The no additions treatments aim to encourage the growth and subsequent PAH-degrading activity of native microorganisms in the soil, through maintenance of optimal moisture content and aeration alone. This process relies on the microorganisms adapting to the highly-contaminated soil conditions and developing strategies to metabolize or transform the PAHs. While the no additions treatments easily offer the most ‘hands-off’ approach, allowing nature to run its course, they typically exhibited the lowest PAH percent degradation (36 ± 2% for ${\Sigma }_{16}\text{PAHs}$) and reductions in cancer risk (29 ± 2% for ${\Sigma }_{\text{B}2}\text{PAHs}$). Plots of percent degradation against bioremediation time revealed that only acenaphthylene degradation was linearly related to the bioremediation time (p = 0.008, r = 0.72) (Figure S3). Furthermore, degradation of the B2 group PAHs seldom exceeded 50%, irrespective of bioremediation time (Figure S3). Thus, no additions treatments have limited utility except perhaps at isolated site locations, with limited resources, where the need for efficient clean-up is not urgent.

Biostimulation treatments were considered more rigorous than the no additions treatments, because the use of nutrient fertilizers promotes optimal growth conditions for the indigenous microorganisms. In our collated dataset, we found that the percent degradation for the 2- to 4-ringed PAHs were above 50% for the majority of soils (Figure S1) and there were no obvious trends with bioremediation time, except for acenaphthylene, whose percent degradation increased with longer bioremediation times (Figure S4). Conversely, the reduction in concentrations and subsequent cancer risk for the B2 group PAHs was generally lower and did not correlate with bioremediation time, except for Ind and DBahA, which were inversely proportional to bioremediation time (p = 0.009, r = −0.80 and p = 0.037, r = −0.48, respectively). It is unlikely that Ind or DBahA were being formed during bioremediation and as such, these observations could also be explained by: a) our collated dataset combining data from multiple studies with different bioremediation strategies (i.e., various nutrient fertilizers, application regimes, and/or doses), soil types, and indigenous microbial populations, b) transfer of Ind and DBahA from inaccessible (non-extractable) regions of the soil to more bioavailable (extractable) regions, giving observations of relatively higher concentration in soil post-bioremediation (i.e., a physical process rather than microbially driven), or c) increased bioavailability of Ind and DBahA with time causes the activity or diversity of the microbial community to decline over time as a result of increased soil toxicity⁷⁶ and thus, the relative degradation is apparently lower.

With bioaugmentation treatments, soils were inoculated with bacterial or fungal strains known to exhibit PAH-degrading behaviors. Bacterial degradation of PAHs typically involves direct metabolism or mineralization pathways, whereas fungal degradation often results from the production of non-specific extracellular enzymes (particularly in the case of ligninolytic fungi).¹² PAHs In bioaugmentation studies, the average percent degradation for ${\Sigma }_{16}\text{PAHs}$ was typically lower than those observed with biostimulation treatments (37 ± 2% relative to 46 ± 2%, respectively). Saponaro et al. reported that bioaugmentation did not enhance the degradation and removal of PAHs relative to biostimulation treatments in the contaminated manufactured gas plant soil they investigated, suggesting that the indigenous microorganisms were well-adapted to the contaminated soil conditions and/or outcompeted the exotic microorganisms.⁷⁷ Wu et al. also found that bacterial consortia were more successful at degrading PAHs when they were inoculated into soils with similar characteristics to those they were originally isolated from compared with soils exhibiting unrelated characteristics.⁷⁸ In our collated dataset, the percent degradation of a number of PAHs (phenanthrene, anthracene, fluorene, pyrene, BaA, Chr, BaP, BbF, Ind, and DBahA) was linearly correlated (p < 0.05, 0.49 < r < 0.89) with bioremediation time (Figure S5). This may be an indication that the exotic microorganisms introduced into the soil require time to acclimate and begin to thrive in their new environment. As a result, bioremediation efficiency improves over time with bioaugmentation treatments. Alternatively, the bioavailability of the PAHs and/or their metabolic kinetics may be the rate-limiting step preventing degradation from proceeding more efficiently. Regardless, bioaugmentation treatments offer more promising results, particularly for the 4- to 6-ringed carcinogenic PAHs, when longer bioremediation time frames are an acceptable compromise.

Surfactant treatments involve additions or washing of the soil with biosurfactants or synthetic surfactants, such as methyl-$\beta$-cyclodextrin or soybean oil, which act to solubilize PAHs that are strongly sorbed to the soil matrix, thus increasing their bioavailability to PAH-degrading microbial populations. As expected, we found the percent degradation of the recalcitrant and strongly sorbed B2 group PAHs were significantly higher in the surfactant treatments (53 ± 1) compared with the no additions, biostimulation, and bioaugmentation treatments (28–34%) (Table S4). Previous laboratory and field-based bioremediation studies have suggested that nutrient additions alone are sufficient to enhance the growth of indigenous microbial populations and degrade the more bioavailable 2- to 3-ringed PAHs.⁷⁹ However, surfactant additions were required in the later stages of the bioremediation process to enhance the bioavailability and subsequent degradation of the more recalcitrant and strongly absorbed 4- to 6-ringed PAHs.^{79, 80} In Figure S6, we see that the percent degradation of a number of the priority PAHs were linearly correlated with bioremediation time (p < 0.05, 0.27 < r < 0.88), providing further evidence that as bioremediation proceeds, surfactants can enhance the bioavailability of recalcitrant PAHs that were previously inaccessible to soil microbial populations.

As outlined above, composting treatments were most effective at degrading the carcinogenic B2 group PAHs, which was likely due to the combined influence of added nutrients and microbiota with the introduction of organic matter. In addition, the percent degradation of all of the USEPA priority PAHs, except acenaphthene and BkF, were linearly correlated with bioremediation time (p < 0.03, 0.46 < r < 0.84) (Figure S7). As stated above, the improvement in bioremediation efficacy with time is likely due to a combination of the lag time required for the new pool of exotic microorganisms to acclimate to their new environment (and potentially outcompete indigenous microorganisms) and increased bioavailability of strongly sorbed PAHs.⁵³ Previous studies have demonstrated the utility of certain bacterial and fungal strains capable of exuding extracellular enzymes or metabolites with surfactant-like properties that enhance the bioavailability and subsequent biodegradation of recalcitrant contaminants.^80–82 Similarly, the presence of humic acids and other water-soluble biomolecules in composting materials can increase PAH mobility via solubilization^{83, 84} and slowly desorbing organic matter might allow for slower release of soil-bound PAHs.⁵³ Thus, composting provides a cost-effective, environmentally friendly, effective, and sustainable approach for the bioremediation of PAH-contaminated soils.

Influence of previous bioremediation treatments

Several soils used in our collated dataset had undergone previous bioremediation steps, such as treatment in a biopile (n = 42).^{66, 68, 85–88} These partially remediated soils were then subjected to subsequent bioremediation steps, aimed at targeting technologies for more effective removal of the persistent, carcinogenic PAHs, and these additional treatments were included in our collated dataset. Generally, the 2- to 3-ringed PAHs are easily degradable and more bioavailable and thus, the soils subjected to previous remediation steps were enriched in the 4- to 6-ringed carcinogenic PAHs (Figure 3). Similar trends have been observed for aged contaminated soils, in which the 2- to 3-ringed PAHs become depleted due to a combination of abiotic processes (e.g., volatilization) and biological degradation.^{1, 34} The use of previously remediated soils in our dataset could potentially skew our analysis towards the 4- to 6-ringed PAHs. However, upon repetition of our analysis, excluding the soil treatments that had been previously bioremediated (‘no-remediation dataset’), the results were remarkably similar (Table S7). The average percent degradation for each treatment type in the no-remediation dataset fell within the range of errors observed previously, except for the ${\Sigma }_{\text{B}2}\text{PAHs}$ treated with surfactants, which showed enhanced degradation (61 ± 1% in the no-remediation dataset compared with 53 ± 1% in the original collated dataset). This enhancement is not surprising given the ‘no-remediation’ dataset was enriched with the more degradable 2- to 4-ringed PAHs. Similarly, the correlations between percent degradation and bioremediation time were equivalent for both datasets, apart from the surfactant treatments, where the strength of the positive correlation (i.e., percent degradation increases with the length of bioremediation) was reduced for all PAHs in the no-remediation dataset, except fluoranthene, pyrene, and BaP, relative to the original collated dataset.

Figure 3.

Average pre-bioremediation profiles for the 16 priority PAHs in untreated contaminated soils and aged, contaminated soils that had previously undergone bioremediation treatment, presented as a percentage of the ${\Sigma }_{16}\text{PAH}$ concentration. The average percentages for the B2 group PAHs are displayed as horizontal lines for the untreated (solid line) and previously remediated soils (dotted line). Error bars represent standard error.

Post-bioremediation cancer risk and implications for human health:

In our collated dataset, we found that, although the reductions in ELCR values (based on the carcinogenic B2 group PAHs) achieved via bioremediation were statistically significant for many of the soil treatments investigated, considerable health risks remained. The USEPA currently recommends human health risk-based regional screening levels for contaminated soils corresponding to a 10^-6 cancer risk (i.e., one in one million cases or an ELCR=1), below which human health risks are deemed acceptable.⁶¹ In our collated dataset, none of the contaminated soils exhibited ELCR values <1 either prior to or following bioremediation (i.e., all soils exceeded the USEPA acceptable health risk level of 10^-6 even after bioremediation treatment) (Figure 4). Furthermore, 60% (108/180) of the contaminated soils had pre-bioremediation ELCR values >100 (Figure 4), which exceed the acceptable health risk level by at least two orders of magnitude. Of those 108 soils, 54% maintained ELCR values above 100 following bioremediation.

Figure 4.

Distribution of cancer risk estimates for all soil treatments pre- and post-bioremediation (n = 180). The acceptable human health risk level for individual compounds corresponds to an ELCR <1 (below one cancer case in one million people or a 10⁻⁶ incidence). For multiple compounds and exposure pathways, the acceptable cumulative lifetime risk corresponds to an ELCR <100 (or an incidence of 10⁻⁴).

Our cancer risk analysis focused on the ingestion exposure assessment guidelines for indoor adult workers at commercial/industrial sites. The USEPA regional screening levels list concentrations of individual contaminants, including PAHs, that correspond to a 10^-6 cancer risk under different soil-use types (residential vs. industrial) and exposure scenarios (ingestion, dermal, or inhalation exposure) (Table 2).⁶¹ For example, the screening level concentration for BaP in soil designated for industrial use was 0.29 mg kg^-1 (assuming multiple exposure routes), whereas a more conservative value of 0.016 mg kg^-1 was recommended for use in a residential setting.⁶⁰ In our collated data set, we found that the post-bioremediation soil concentrations of the non-carcinogenic 2- to 4-ringed PAHs did not exceed their respective screening level concentrations (Table 2). Naphthalene was an exception, with 53% of the soils in which it was measured exceeding the recommended concentration (17 mg kg^-1) for industrial soil use. Conversely, the regional screening levels for the B2 group PAHs, in residential or industrial soils, were often exceeded (Table 2). Chrysene and BkF were the only B2 group PAHs with exceedance concentrations less than 50% following bioremediation, under the industrial soils guideline. Moreover, the post-bioremediation concentrations of BaA, BaP, BbF, DBahA, and Ind exceeded the carcinogenic ingestion exposure target risk, based on a residential soil-use scenario, in 100% of cases (Table 2). The screening level concentrations above are based on a 10^-6 target cancer risk for individual compounds. However, the screening guidance recommends that the cumulative cancer risk (from summed concentrations of individual contaminants and exposure pathways) should not exceed 10^-4, which effectively corresponds to an ELCR of <100 cancer cases in an exposed population of 1 million, following bioremediation.⁶¹ Our analysis showed that 32% (58/180) of the soil treatments had post-bioremediation ELCR values >100 (Figure 3). Thus, it appears that, while bioremediation strategies ultimately lower the cancer risk, they have limited success in the removal of carcinogenic PAHs from contaminated soils to concentrations below the target cancer risk screening levels recommended by the USEPA, such that the site is suitable for reuse in residential, or even industrial, settings.

Table 2.

Regional screening levels recommended by the USEPA for total risk in industrial and residential soils (assumes soil is contaminated with a single compound) and for ingestion-only exposure of carcinogenic contaminants in residential soils. The percentages of observations in our collated dataset that exceeded those screening level concentrations in soils following bioremediation treatment are also listed (values >50% are highlighted in bold). B2 group PAHs are shaded in grey.

PAHs	#a	Industrial soil (mg kg−1)	% exceeded (industrial)	Residential soil (mg kg−1)	% exceeded (residential)	Ingestion exposure (residential soil) (mg kg−1)	% exceeded (ingestion)
Acenaphthene	96	45000	0	3600	0
Anthracene	140	230000	0	18000	0
Benzo(a)anthracene	179	2.9	85	0.16	100	0.21	100
Benzo(a)pyrene	179	0.29	100	0.016	100	0.021	100
Benzo(b)fluoranthene	166	2.9	92	0.16	100	0.21	100
Benzo(k)fluoranthene	142	29	25	1.6	97	2.1	92
Chrysene	169	290	2	16	54	21	44
Dibenz(a,h)anthracene	131	0.29	90	0.016	100	0.021	100
Fluoranthene	168	30000	0	2400	0
Fluorene	122	30000	0	2400	0
Indeno(1,2,3-cd)pyrene	77	2.9	70	0.16	100	0.21	100
Naphthalene	113	17	53	3.8	75
Pyrene	169	23000	0	1800	0

^aThe number of measurements for each PAH in our collated dataset following bioremediation.

KEY RESEARCH NEEDS

In our review, we have shown that while bioremediation treatments can significantly degrade the 16 priority PAHs (most notably, the 2- to 4-ringed PAHs), they do not suitably remove the carcinogenic PAHs and cancer risks associated with exposure to contaminated soils below the health-based risk levels deemed acceptable by the USEPA. Furthermore, current strategies for risk assessment are focused on the 16 priority PAHs, despite mounting evidence suggesting that other PAHs and their transformation products pose substantial risks to human health.^{23, 89} While the eight B2 group PAHs were once thought to be the most ‘potent’ contributors to soil carcinogenicity, this is no longer the case.²⁹ PAHs with 6–7 rings and molecular weights ≥302 a.m.u. (MW302-PAHs) are more carcinogenic²⁹ and likely to be more environmentally persistent than the 16 priority PAHs.⁷¹ For example, the MW302-PAH, dibenzo(a,l)pyrene (DalP, also known as dibenzo(def,p)chrysene), has an estimated relative potency 30 times higher than that of the reference compound, BaP (MW ~252).²⁹ Recent studies have demonstrated that MW302-PAHs, including DalP, contributed an additional 4–39% to the carcinogenic potency of coal tar-based pavement sealcoat surfaces⁹⁰ and particulate matter in air during the Beijing Olympics.⁹¹ Moreover, we recently found that while DalP comprised only a small percentage of the total soil PAH concentration (0.04–0.1%), it was not significantly degraded during biostimulation treatment in an aerobic bioreactor.⁹² When we translate the findings of Chibwe et al.⁹² into ELCR equivalents, we see that DalP alone contributed an additional 35% to the ingestion cancer risk associated with the B2 group PAHs in the same remediated soil (data not shown). Similarly, many PAHs are known to undergo transformation reactions during bioremediation treatment to form more polar metabolites, such as the oxygen-containing PAHs (hydroxylated-PAHs and quinones), which are thought to be more mobile and bioavailable than their parent compounds.²³

These findings provide additional evidence that compounds other than the 16 priority PAHs may be significantly contributing to cancer risk and highlights the need for their routine measurement in bioremediation studies and inclusion in future risk assessments. We recommend that future bioremediation studies focus on methods for enhancing the degradation and removal of the most carcinogenic PAHs (e.g., the 4- to 6-ringed PAHs and MW302-PAHs) and on identification of potential transformation products that might also contribute to cancer risk and adverse human health outcomes. It is also important to consider whether the current methods used to monitor bioremediation success at contaminated sites (i.e., targeted measurements of 16 priority PAH concentrations) reflect an actual reduction in soil toxicity and/or carcinogenicity (i.e., ELCR estimates). Finally, we recommend that future research efforts extend beyond simple concentration-based reporting to focus on the bioavailable contaminant fractions for exposure assessments and their relevance to human health outcomes.