Recent Advances in the Study of the Remediation of Polycyclic Aromatic Compound (PAC)-Contaminated Soils: Transformation Products, Toxicity, and Bioavailability Analyses

Abstract

Polycyclic aromatic compounds (PACs) encompass a diverse group of compounds, often found in historically contaminated sites. Different experimental techniques have been used to remediate PACs-contaminated soils. This brief review surveyed over 270 studies concerning remediation of PACs-contaminated soils and found that, while these studies often measured the concentration of 16 parent polycyclic aromatic hydrocarbons (PAHs) pre- and post-remediation, only a fraction of the studies included the measurement of PAC-transformation products (PAC-TPs) and other PACs (n = 33). Only a few studies also incorporated genotoxicity/toxicity/mutagenicity analysis pre- and post-remediation (n = 5). Another aspect that these studies often neglected to include was bioavailability, as none of the studies that included measurement of PAH-TPs and PACs included bioavailability investigation. Based on the literature analysis, future remediation studies need to consider chemical analysis of PAH-TPs and PACs, genotoxicity/toxicity/mutagenicity, and bioavailability analyses pre- and post-remediation. These assessments will help address numerous concerns including, among others, the presence, properties, and toxicity of PACs and PAH-TPs, risk assessment of soil post-remediation, and the bioavailability of PAH-TPs. Other supplementary techniques that help assist these analyses and recommendations for future analyses are also discussed.

Graphical Abstract

INTRODUCTION

Polycyclic aromatic compounds (PACs) are a ubiquitous group of contaminants found in soil.^1,2 Due to their potential human and environmental health impacts,^3,4 PACs-contaminated sites must be assessed for potential risks and remediated before the sites are deemed safe for public use.^5–7 PACs are one of the most common groups of chemicals remediated in the U.S. and soil represents a major portion of remediated sites under the Superfund program in the U.S.⁵ For these reasons, various remediation techniques have been proposed and selected in order to degrade PACs in soil,^5,8 some of which may include bioremediation,^9,10 chemical oxidation,^11,12 and phytoremediation.¹³

The success of a given remediation technique is typically measured based on the post-remediation concentration of the 16 polycyclic aromatic hydrocarbons (PAHs) that are included in the U.S. Environmental Protection Agency’s list of priority pollutants (PAH 16). Consequently, risk assessment of remediated soil sites are measured based on PAH 16 concentrations pre- and post-remediation.⁷ However, some studies have shown that PAC-contaminated soils contain a diverse group of compounds beyond the PAH 16, such as oxygenated-PAHs (OPAHs), azaarenes (PANHs) and alkylated-PAHs,^1,14,15 of which their concentrations have typically not been measured and not included in the risk assessment pre- and post-remediation. Other groups of compounds that have often not been studied are the transformation products (TPs) of parent-PAHs (PAH-TPs), such as hydroxylated-PAHs (OHPAHs), and other PACs, such as sulfur heterocyclic PAHs (PASHs) and oxygen heterocyclic PAHs (PAOHs).^6,16,17 Screening PAH-TPs and other PACs is important not only because such an effort will result in better representation of the actual profile of PAC-contaminated soil sites, but also because some of these compounds are known to be toxic and/or mutagenic.^18–20 Recent studies on the remediation of PAC-contaminated soils showed the association between an increase in toxicity or genotoxicity with the increase in concentrations of PAH-TPs and PAC.^6,14,16 Another important aspect in remediation of PAC-contaminated soil sites that was also typically not assessed was the bioavailability of fractions of the soil post-remediation. Some studies have shown that bioavailability of PAC increased post-remediation,^21–23 which is relevant to measure because of the potential environmental health implications for organisms in the terrestrial ecosystem. The chemical, toxicity, and bioavailability assessments are needed to address the knowledge gap regarding the presence, properties, and toxicity of PAH-TPs and other PACs. There is also a need to determine if remediation is complete, particularly to ensure that toxic compounds are not formed post-remediation and that the soil fraction that contains toxic, bioavailable compounds does not increase post-remediation.

The purpose of this brief review is to highlight studies that go beyond the screening of PAH 16 in any remediation studies, particularly those studies that measure the concentrations of PAH-TPs and PACs post-remediation. Studies that evaluate toxicity/genotoxicity/mutagenicity are also included in this brief review. Example studies that include the bioavailability of PACs in soil extracts pre- and post-remediation are also included, although these studies only measured PAH 16.

This brief review does not intend to expand on the specific mechanism and theories behind the different remediation techniques. Readers who would like to learn more about the different remediation techniques are directed to other reviews that have carefully examined these individual techniques, such as reviews on biodegradation,^24,25 bioremediation,^{7,9,10,26–29} chemical oxidation,^11,12 electrokinetics,³⁰ and phytoremedation.¹³ These reviews were only focused on each individual technique and did not provide a comparison with other remediation techniques. The discussion regarding the formation of TPs post-remediation, toxicity, and bioavailability was also often not included in these reviews and these reviews typically only included a single PAH compound, rather than a mixture of compounds. Therefore, this brief review is intended to fill these knowledge gaps, specifically the formation of TPs from different remediation techniques and to provide a discussion on the toxicity and bioavailability of the soil extracts post-remediation. The brief review also intends to highlight example studies that incorporate chemical analysis of PAH-TPs and PACs, genotoxicity/toxicity/mutagenicity analysis, and bioavailability analysis pre- and post-remediation and propose the inclusion of all three analyses in future remediation studies. This brief review also collates a list of PAH-TPs and other PACs whose concentrations increase post-remediation in order to provide other researchers, stakeholders, and policymakers with a list of compounds that may be included in future research. Such a comprehensive list is currently not available and is yet to be covered by previous review articles on the topic of remediation of PACs in soil. Other techniques that may complement the analysis of soil remediation, which include effects-directed analysis, suspect screening, and non-targeted analysis (EDA, SSA, and NTA, respectively) are briefly discussed as effective techniques that can be used in future soil remediation studies.

CHEMICAL ANALYSIS OF PAH-TPs AND PACs PRE- AND POST-REMEDIATION

Over 270 studies on remediation of PAC-contaminated sites, from 1990 to 2019, was reviewed from Scopus by searching for “PAH” or “polycyclic aromatic hydrocarbon” or “PAC” or “polycyclic aromatic compounds” and “soil” and “remediate” as keywords. The scope of the search was limited to soil samples and did not include sediment samples. Both historically- and artificially-contaminated (spiked) soil samples were included in this search. Studies that measured the formation PAH-TPs and increased concentrations of other PACs post-remediation are listed in the Supporting Information (Table S1). With regard to specific compounds, 9,10-anthracenedione, 9,10-phenanthrenedione, and 9-fluorenone are three of the most commonly measured PAH-TPs post-remediation (Table 1), which is likely because of the availability of commercial standards that could be purchased to confirm compound identity. In a recent bioremediation study, 2H-naphtho[2,1,8-def]chromen-2-one has been identified as a pyrene-TP that is responsible for the increase in toxicity post-remediation.^31,32 Some chemicals are identified to be genotoxic/toxic/mutagenic in the whole sample extracts, but they are not confirmed to be the reason for increase in genotoxicity/toxicity/mutagenicity post-remediation. These chemicals include diaminotriazole, acridine, methylazaphenanthrene (no information provided on the specific isomers), 4-oxapyrene-5-one, and 7H-benzo[de]anthracene-7-one (Table 1). The remaining chemicals in Table 1 are included because their bioactivity was >70%. The >70% threshold is gathered from PubChem search,³³ and it is arbitrarily chosen to represent chemicals that are potentially genotoxic. This list may be used as chemicals for suspect screening in future remediation studies.

Table 1.

List of PAH-TPs and PACs to be included in future remediation studies based on previous remediation studies.

No.	Chemical	Molecular Formula	Molecular Weight (g/mol)	CAS Number	aBioassay Activity Frequency (%)	bLog Kow Predicted Range	References
1.	9-fluorenone	C13H8O	180.2	486–25-9	0.8	2.33 – 3.58	6,36,38,43,45,47,48,51,58
2.	9,10-phenanthrenedione	C14H8O2	208.2	84–11-7	27	1.70 – 3.56	6,36,38,49,54
3.	9,10-anthracenedione	C14H8O2	208.2	84–65-1	4	3.39	2,6,38,41–43,45–47,49–51,53,54,57,60,61
4.	2H-naphtho[2,1,8-def]chromen-2-one	C15H8O2	220.2	NA	NA	NA	32
5.	Diaminotriazole	C2H5N5	99.1	1455–77-2	NA	−1.61	2
6.	Acridine	C13H9N	179.2	260–94-6	15	1.91 – 3.43	2
7.	Methylazaphenanthrene	C14H11N	193.2	-	-	-	2
8.	4-oxapyrene-5-one	C15H8O2	220.2	23702–49-0	0	2.25 – 4.20	31
9.	7H-benzo[de]anthracene-7-one	C17H10O	230.3	82–05-3	11	3.10 – 4.81	2
10.	Phenalenone	C13H8O	180.2	548–39-0	94	1.75 – 3.45	6,17,47,48
11.	2-hydroxyphenanthrene	C14H10O	194.2	605–55-0	100	2.45 – 4.19	6
12.	3-hydroxyphenanthrene	C14H10O	194.2	605–87-8	100	2.54 – 4.19	6
13.	1-azapyrene	C15H9N	203.2	313–80-4	75	2.34 – 4.32	2
14.	1-chloropyrene	C16H9Cl	236.7	34244–14-9	100	4.49 – 5.77	54
15.	7-(hydroxymethyl)benzo[a] anthracene	C19H14O	258.3	16110–13-7	100	2.40 – 4.80	52

The list is divided into those that are most commonly studied in any remediation studies (1–3), a PAH-TP that has been identified to be the chemical that cause the increase in soil extract genotoxicity post-bioremediation (4), those that have been identified in mutagenic soil extracts post-bioremediation (5–9),

^aand those that have bioactivity frequency >70% based on PubChem search (10–15).³³

^bLog K_ow values are obtained from the U.S. EPA CompTox Chemicals Dashboard.¹¹² A comprehensive list of all chemicals can be found in Table S1. “NA” represents not available data, “-“ indicates no information available because the specific isomer is not identified.

Of the studies that have measured the concentrations of PAH-TPs and PACs pre- and post-remediation (Table S1), bioremediation^{2,16,17,31,32,34–43} and chemical oxidation^44–54 are the two most prominent types of remediation that researchers prefer to use and investigate. Most of the bioremediation studies relied on bioslurry or bioreactor treatment, although studies by Steinhart and co-workers showed the applicability of composting to reduce PAC concentrations.^38,42 Bioremediation studies also covered other compounds beyond PAH 16. For example, formation of alkylated-PAHs and PASHs TPs are measured in several of the bioremediation studies,^35,38,42 while measurements of PANHs, PAOHs, and PASHs pre-bioremediation^35,39 and carboxylic acids and other smaller TPs post-remediation have also been performed.^35,38 Lemieux et al. measured an increase in PASH concentration post-remediation, though the exact biological mechanism leading to the increase of PASHs was not elucidated.³¹ The inclusion of a diverse group of compounds provides a fuller picture of the presence and biodegradation of PACs in contaminated soil samples. Although the breadth of PAH-TPs and PAC that are measured in the various bioremediation studies suggests that bioremediation of PACs contaminated soil is a mature area of study, there are still research gaps that need to be explored, such as the toxicity analysis of PAH-TPs and the prediction of which TPs might be formed post-remediation.⁵⁵ The field of bioremediation should also examine the TPs that are formed from the remaining six PAH 16 that have not been previously studied yet, namely chrysene, benzo[b]- and [k]-fluoranthene, benzo[g,h,i]perylene, dibenzo[a,h]anthracene, and indeno[1,2,3-cd]pyrene, as well as TPs from other PACs.

With regard to chemical oxidation, the three most common techniques were Fenton-,^{44,46,47,49,53} persulfate-^45,48,50,52 and permanganate-based.^45,47,51 Some of the chemical oxidation experiments use a mixture of PAH 16,^45,47,48 rather than individual compounds. Several of the chemical oxidation studies have also measured the formation of alkylated-PAHs,^47,52,53 PASHs,⁵² PANHs,⁵² PAOHs,^47,52 and chlorinated-TPs⁵⁴ following chemical oxidation remediation. Some of the TPs observed following chemical oxidation studies are also observed in bioremediation studies, including phthalic acid, 1-acenaphthenone, 9-fluorenone, phenalenone, 4H-cyclopenta[def]phenanthrenone, and 9,10-phenanthrenedione (Table S1). However, small molecular weight TPs (90 – 150 a.m.u.), such as propanal-2,3-dihydroxy, phenol, 2-hydroxybenzaldehyde, quinolone, 1-indanone, and 1,4-benzopyrone to name a few, are only measured in bioremediation studies and not in chemical oxidation studies (Table S1). Hydroxylated-PAHs are also primarily observed in bioremediation, although 9-anthraceneethanol, 1-hydroxy-9,10-anthracenedione, 2-hydroxy-9,10-anthracenedione, and 7-(hydroxymethyl)benz[a]anthracene are measured in two chemical oxidation studies.^49,52 These observations combined to indicate that the degradation pathways of parent-PAHs is likely to be different with and without the presence of organisms.

Other remediation techniques, where formation of PAH-TPs and PACs were measured, included remediation by pulsed corona discharge,⁵⁶ remediation by fungi,^57,58 phytoremediation,^59,60 chemical reduction,⁶¹ and thermal remediation⁶ (Table S1). In the pulsed corona discharge study, a pulsed corona discharge plasma system is used to degrade fluorene-contaminated soil.⁵⁶ While limited in scope, the pulsed corona discharge study offers a promising technique to degrade fluorene into carboxylic acids and smaller TPs. In the field of remediation by fungi, works by Andersson and co-workers have shown that the ability of white-rot fungi to degrade soil samples artificially contaminated by fluorene, phenanthrene, anthracene, pyrene, benzo[a]anthracene, and dibenzo[a,h]anthracene.^57,58 Depending on the specific strain of the white-rot fungi, different PAH-TPs are formed post -remediation. In the field of thermal remediation, steam enhanced extraction thermal degradation has been used to degrade PAC in creosote-contaminated soil in a laboratory setting.⁶ PAH-TPs and some PACs that are measured post-remediation include hydroxylated-fluoranthene, -chrysene, -benzo[b]fluoranthene, -benzo[k]fluoranthene, -benzo[a]pyrene, and -indeno[1,2,3-cd]pyrene, indicating that thermal remediation is able to transform higher molecular weight-PAHs. More studies in the above mentioned remediation techniques are needed to fully understand whether these techniques are applicable in field scale. Furthermore, the toxicity of the TPs and PACs formed post-remediation should also be analyzed.

TOXICITY TESTING OF SOIL POST-REMEDIATION

The success of a remediation technique is often only measured in the lab based on chemical analysis, even though toxicity analysis post-remediation provides complementary, and often insightful, results that may help researchers, stakeholders, and policy makers assess whether or not a given remediation technique should be tested in the field. However, the majority of the studies that included the analysis of PAH-TPs and PACs post-remediation do not include toxicity testing post-remediation. Only five studies included any genotoxicity/toxicity/mutagenicity testing in their assessments (Table 2). Contaminated soil samples from former manufactured gas plant site in North Carolina was bioremediated and the genotoxicity of the soil extracts was analyzed.^16,32,62 Increases in the genotoxicity of the soil extract post-bioremediation was identified with the chicken DT40 B-lymphocyte isogenic cell line and its DNA-repair-deficient mutants.^16,32 Furthermore, the toxicity of the soil extracts was confirmed with the embryonic zebrafish (Danio rerio) developmental toxicity testing.¹⁶ In another study, creosote contaminated soil from a Superfund site in Washington was thermally remediated with steam enhanced extraction.⁶ The extract was also analyzed with the zebrafish developmental toxicity testing and an increase in developmental toxicity was associated with the formation of PAH-TPs post-remediation.⁶ With regard to mutagenicity analysis, an earlier study by Brooks et al. applied bioremediation to remediate creosote contaminated soils from Minnesota.² They used the Salmonella typhimurium strain YG1041, with and without liver metabolic activation, post-bioremediation and identified the specific compounds that were found to be mutagenic: benzanthrone, 1-azapyrene, and anthracenedione (specific isomer not indicated).² The study was one of the first publications that successfully identified PAH-TPs that were responsible for the increase in bioactivity post-bioremediation. In another study, contaminated soil samples from a former gasworks site in Stockholm, Sweden was bioremediated. The mutagenicity of contaminated soil extracts pre- and post-bioremediation was monitored using Salmonella typhimurium, but with four different strains (Table 2), with and without S9 activation, and the mutagenicity was correlated with the increase in OPAHs and PASHs.³¹ However, the study did not identify the specific compounds that resulted in the increase in mutagenicity post-bioremediation. We recommend any of these analyses to be replicated in future remediation studies to help further understand the genotoxicity/toxicity/mutagenicity post-remediation.

Table 2.

List of different toxicity/genotoxicity/mutagenicity tests used in the analysis of soil extracts from various PAC-remediation studies.

Type of Test	Name	Cell Line/Test Organism/Strain	References
Genotoxicity	Chicken cell line assay	DT40 B-lymphocyte	16,32
Toxicity	Developmental toxicity	Zebrafish (Danio rerio)	6,16
Mutagenicity	Ames	Salmonella typhimurium
		TA98	31
		TA100	31
		YG1041	2,31
		YG1042	31

Only tests where PAH-TPs are formed or PACs concentration increase post-remediation are included in this list. Examples of other tests such as the umu test,¹¹³ green fluorescent protein,⁶⁸ Microtox^®,^{65–67,71,72} Esenia worms,^69–73,73 and seed germination assay^70,71,73,114 have previously been used in PAC-remediation studies, but are not included in the list because these studies do not include the measurement of PAH-TPs and/or PACs post-remediation.

Other studies have included genotoxicity/toxicity/mutagenicity analysis pre- and post-remediation, but only focused on measurement of PAH 16. The Salmonella/microsome assay has been used to analyze the mutagenicity of soil extracts pre- and post-bioremediation, which further highlights the application of mutagenicity analysis as an effective tool to measure the efficacy of bioremediation.⁶³ A combined bioanalytical approach with chemical analysis of remediated soil samples suggests that the inclusion of battery of bioassay reporters may prove to be helpful to minimize any potential human health risks that may occur post-remediation.⁶⁴ Analysis of genotoxicity/toxicity/mutagenicity would also ensure that the soil organisms would not be adversely affected post-remediation, thus sustaining the terrestrial ecosystem.

Even when identification of specific compound(s) that result in the increase of genotoxicity/toxicity/mutagenicity proved to be difficult, some studies still include the assessment of the genotoxicity/toxicity/mutagenicity of the whole sample extracts pre- and post-remediation—although most of these studies were performed specifically in bioremediation studies. Microtox^® acute toxicity test using Aliivibrio fischeri is the most common test to measure toxicity post-bioremediation.^65–67 Another study used toxicity testing to assess the efficacy of bioremediation, such as the CALUX aryl-hydrocarbon receptor (AhR) agonist bioassay and the Comet genotoxicity bioassay with RTL-W1 cells.²³ In yet another study, Escherichia coli DH5α cells that expressed green fluorescent has been used to show the reduction in toxicity post-bioremediation.⁶⁸ Researchers have also used ecotoxicity tests in bioremediation with the Eisenia worms as the most commonly used organism.^69,70 These tests may be combined with plant test, such as the germination assays,⁷¹ to assist with the characterization of soil extracts post-remediation. These studies have shown that there are other options that may be applied to assess the toxicity of the soil extracts post-remediation. However, the effectiveness of these tests will be greatly enhanced if it is combined with the chemical analysis post-remediation.

There are scarce examples of genotoxicity/toxicity/mutagenicity analysis in other types of remediation other than bioremediation. Joner et al. use both Aliivibrio fischeri and earthworm (Eisenia fetida) to assess the toxicity of contaminated soil extracts pre- and post-phytoremediation,⁷² while Sivaram et al. used earthworm (Eisenia fetida) together with garden pea (Pisum sativum) to assess the efficacy of phytoremediation.⁷³ The lack of genotoxicity/toxicity/mutagenicity analysis in other types of remediation other than bioremediation represents a research gap that needs to be filled. Future studies that aim to link toxicity with chemical analysis should also consider the bioavailability analysis of the soil extracts to further understand the effect of a given remediation technique.

BIOAVAILABILITY ANALYSIS OF SOIL PRE- AND POST-REMEDIATION

None of the remediation studies that have measured PAH-TPs and PACs post-remediation include any measurements of bioavailability, although work by Hu et al. found that OPAHs are more desorbable and biodegradable than the parent compound, as exemplified by higher desorption and biodegradation of 9,10-anthracenedione relative to anthracene.⁷⁴ The same study also found that genotoxicity increased post-bioremediation. There are also other studies that combined bioavailability and toxicity, such as the work by Andersson et al and Cipullo et al.,^23,71 but both studies are focused on PAH 16. Therefore, while the discussion in this section would include remediation studies that have included bioavailability component in the studies, the studies are limited to only those that measure PAH 16 concentrations pre- and post-remediation.

The application of bioavailability in the framework of PAC-contaminated soil remediation may be considered from two perspectives. The first perspective is through the study of bioavailability of compounds in soil pre- and post-remediation. Similar to chemical and genotoxicity/toxicity/mutagenicity analyses, bioavailability analysis of compounds in soil pre- and post-remediation is still uncommon and studies that have included bioavailability analysis in soil are related to bioremediation.^23,71,75 There are currently limited bioavailability studies in other types of remediations than bioremediation. Researchers who are interested in including bioavailability analysis in the future may consider multiple bioavailability techniques. Cui et al. categorizes bioavailability techniques into two categories: partial extraction techniques, which include mild solvent, cyclodextrin, and Tenax extractions, and passive sampling methods to measure freely dissolved concentrations, which use of semi-permeable membrane device (SPMD), polyethylene device (PED), or solid phase microextraction (SPME).⁷⁶ In addition to the partial extraction and passive sampling methods, biological methods can also be considered to assess bioavailability of compounds in soil.⁷⁷ A particular technique that is of interest to other researchers is the use of polyoxymethylene (POM) passive sampling device to measure the freely dissolved concentrations.^1,78 The POM method has been proven to not only account for the bioavailability of PAHs in soil, but also OPAHs and PANHs,¹ which makes the POM method an advantageous technique for researchers in the area of remediation study.

The second perspective of (bio)availability, in relation to remediation, is the use of surfactants to enhance availability of compounds in soil and increase the efficacy of a remediation technique. Due to the sorption to soil and low bioavailability of some PACs, surfactants have been used, in conjunction with remediation, to increase the removal of PACs from contaminated soil samples.⁷⁹ Aitken and colleagues have explored the use of different surfactants, in conjunction with bioremediation, to degrade a range of PAHs,^80–84 including alkylated-PAHs.⁸² In Adrion et al., they tested five nonionic surfactants to assist the biodegradation of PAHs and found that polyoxyethylene sorbitol hexaoleate (POESH) had the greatest removal effect.⁸² Furthermore, while addition of POESH did not result in the increase of soil cytotoxicity and ecotoxity,⁸³ the genotoxicity post-bioremediation increased following the addition of POESH.⁸² Brij 30 was found to be the only surfactant that decreased soil genotoxicity post-bioremediation.⁸² Works by Aitken and colleagues on surfactant and biodegradation also extend into the analysis on bacterial community pre- and post-bioremediation, with and without the addition of surfactants, and found different effects of surfactants on the bacteria population.^81,84 Yang et al. compare the efficiency of different surfactants to remove anthracene and pyrene from contaminated soil and found that mixed surfactants performed better than individual surfactant.⁸⁵ Surfactants may also be used in conjunction with other remediation techniques, such as electroremediation and chemical oxidation.^52,86 Further discussions on the variety of surfactants available for researchers to use are not the focus of this brief review and are available elsewhere.⁷⁹

CONSIDERATIONS FOR FUTURE REMEDIATIONs OF PAC-CONTAMINATED SOILS

Other remediation or treatment techniques are not included in this brief review because these studies do not analyze PAH-TPs and PACs post-remediation. Some examples of these techniques include biochar remediation,⁸⁷ vermiremediation,⁸⁸ and in situ remediation with activated carbon.⁸⁹ The most recent U.S. Superfund Remedy Report also lists other techniques that are yet to be explored in terms of PAH-TPs and PACs post remediation, including nanoremediation, flushing, and monitored natural attenuation.⁵ With regard to remediation studies that have been discussed in this brief review, the area of fungal remediation may benefit from new studies that explore the use of different types of fungi, studies that analyze a more diverse group of compounds to degrade, and studies that examine the degradation of PACs from historically contaminated soils, rather than artificially contaminated samples. Future studies may also consider the combination of different remediation techniques to degrade PACs. Regardless of the remediation technique that is chosen, a complete assessment for any remediation effort should consider incorporating all three aspects that have been discussed throughout this brief review: chemical analysis of PAH-TPs and PACs of soil extracts, genotoxicity/toxicity/mutagenicity analysis of soil extracts, and bioavailability analysis of compounds in soil samples pre- and post-remediation.

Within the framework of chemical analysis, researchers should expand the list of PACs in their studies. The TPs of chrysene and indeno [1,2,3-cd]pyrene have only been identified in one study,⁶ while the TPs of benzo[b]- and [k]fluoranthene, benzo[g,h,i]perylene, and dibenzo[a,h]anthracene, the remaining high molecular weight PAH 16, have not been identified yet. The concentrations of the TPs of these PAHs may be low relative to the TPs of the other PAH 16 because of the strong sorption of the parent compounds to soil. The concentrations of PAH 16 are often used to characterize the sources of PAHs in the environment, yet they are not representative of the actual profile of PACs in the environment. Thus, more PACs need to be screened in order to better profile the PAC-contaminated sites.⁹⁰ The list of chemicals on Table 1 can also serve as a starting point for researchers who are interested in expanding the list of target chemicals to monitor pre- and post-remediation. Alkylated-PAHs are often neglected in the analysis of soil extracts pre- and post-remediation, even though they are known to be toxic.^18,91 OHPAHs are another group of compounds that should be analyzed because of their potential for developmental toxicity.^20,92 PAHs with molecular weight of 302 a.m.u. are yet another group of compounds that have received little attention. Only a few studies have examined the presence of these compounds pre- and post-remediation,^6,17,52 though a particular concern regarding their low bioavailability has also been raised previously.¹⁷ Bioremediation studies also often tracked the formation of carboxylic acids,^35,38,41 but such analysis has not been replicated in other types of remediation and, thus, are needed to better understand if the remediation is complete. There is also a research gap regarding the mechanisms that lead to the formation or presence of PASHs and PANHs post-remediation. These compounds have either been detected post-remediation or found to have increased post-remediation,^{31,34,37–39,52} but the exact mechanism was still unknown and would need to be elucidated. Only Oberoi et al. and Vila et al. have discussed possible mechanisms on PANHs formation,^35,39 but the mechanism was related to the formation of oxygenated-PANHs and not PANHs themselves.

Chemical analysis in PAC-remediation studies often relies on gas chromatography coupled with mass spectrometry (GC-MS). However, future studies that are interested in analyzing alkylated-PAHs may consider the use of two dimensional GC system, which is capable of separating isomers^93–95—a relevant factor in the separation of alkylated-PAHs. Furthermore, as researchers expand their analysis to PAH-TPs and other PACs, some of which are more polar than PAH 16 and parent-PAHs, the use of liquid chromatography (LC) system may be considered.^32,39,96 Although derivatization of OHPAHs followed by GC-MS has been used in a previous remediation study,⁶ new methods on the analysis OHPAHs using LC-MS will greatly benefit researchers who are interested in quantifying OHPAHs post-remediation. The availability of standards is another area that requires further attention as researchers identify more and more compounds in their remediation studies. For example, the identification of 2H-naphtho[2,1,8-def]chromen-2-one as the compound that was responsible for the increase in genotoxicity post-bioremediation was only possible due to the synthesis of this compound.³²

Given the scientific advancement in the field of chemical analysis, future studies may benefit from the application of non-target analysis (NTA) to determine the formation of any TPs post-remediation. NTA was previously used to identify PAH-TPs that were formed post-bioremediation.^17,32 When combined with various fragmentation prediction programs, such as CFM-ID^97,98 or MetFrag,⁹⁹ NTA can provide researchers with a list of new PAH-TPs and/or PACs that have not been previously studied. Full scan GC-MS analysis, combined with access to the NIST MS library, can provide researchers with a comprehensive list of PAH-TPs and PACs that are formed following remediation, as demonstrated by Li et al.⁵² Other approaches for NTA include the combination of GC and LC, high resolution MS, and computational calculation to predict the formation of PAH-TPs.^55,96,100 The use of Kendrick Mass Defects to analyze for the presence of homologous series of PACs can also be useful as demonstrated by Vila and colleagues in the case of PANHs in soil extracts.^14,39 In cases where NTA is not viable, researchers can apply suspect screening analysis, such as by using the comprehensive list of analytes listed in this brief review (Table S1).

Example studies on whole sample analysis for genotoxicity/toxicity/mutagenicity are already discussed in the earlier section. Most of these studies are primarily limited to bioremediation, therefore other remediation techniques will benefit from the inclusion of genotoxicity/toxicity/mutagenicity analysis in their studies. As an example, the developmental toxicity test can be of interest to future researchers due to genetic similarities between zebrafish (Danio rerio) and human¹⁰¹ and its use in high throughput screening.¹⁰² When combined with chemical analysis, the results from genotoxicity/toxicity/mutagenicity analysis provide a better picture of the success of a given remediation technique, particularly when the genotoxicity/toxicity/mutagenicity decrease post-remediation. These toxicity tests should be performed not only immediately post-remediation, but also at several time points post-remediation to ensure that degradation of the parent compounds are complete and to ensure real risk abatement of the remediation.

The identification process of the specific compounds that are responsible for any genotoxicity/toxicity/mutagenicity may prove to be cumbersome. To alleviate this problem, and streamline the identification process, future studies may benefit from the use of effects-directed analysis (EDA). EDA refers to the technique where toxic extract of a sample is fractionated and tested for genotoxicity/toxicity/mutagenicity, followed by a cycle of identification and verification of the compound(s) responsible for the genotoxicity/toxicity/mutagenicity of the fraction.^103–105 EDA has been applied to plenty of matrices, but the specific application on soil samples pre- and post-remediation is still rare. Through EDA, researchers can focus their efforts on specific fractions that are deemed to be genotoxic/toxic/mutagenic and, as such, identification of the compounds that are responsible for increase in genotoxicity/toxicity/mutagenicity pre- and post-remediation is limited to select sub-sample(s) rather than the whole sample. An application of EDA, in combination with NTA, has been demonstrated in the works of Chibwe et al. and Tian et al. to identify genotoxic compound that is formed post-bioremediation.^16,17,32 There are plenty of options to fractionate a sample extract, but a promising tool in EDA is a GC-Fractionation system coupled to MS,^106,107 which allows researchers to overlay bioassay and chemical chromatograms. In turn, researchers can isolate the specific fraction of interest and simultaneously identify compounds that correspond with the observed bioactivity. A similar system is also available for LC,^108,109 which benefits researchers who are interested in studying polar compounds.

The application of bioavailability analysis of compounds in soil pre- and post-remediation is still limited, thus providing a research gap that needs to be filled. Most of the bioavailability extraction techniques are also optimized for parent-PAHs. Therefore, as researchers analyze a more diverse group of compounds, there may be a need to develop bioavailability extraction techniques that can encompass PACs with a wide range of polarity and in combination with toxicity tests.

Post-remediation risk assessment needs to consider cost and carbon footprint. Using the U.S. EPA’s remediation of manufacturing gas plant sites as a model, remedial action represents almost 75% of total remediation cost.⁸ Future remediation studies can also take into account the carbon footprint of a given technique, specifically when the technique is applied in field scale. A recent risk assessment framework proposed by Volchko et al. incorporated consideration for greenhouse gas emissions for any remediation actions and assessed ways to minimize socio-economic losses.¹¹⁰ Considerations for both cost and carbon footprint are also particularly relevant for projects where researchers are obligated to provide accountability reports to pertinent stakeholders and policymakers.

One of the United Nations Sustainable Development Goals concerns the Life on Land (Goal no. 15)¹¹¹ and the proposed approach that incorporates chemical analysis of PAH-TPs and PACs of soil extracts, genotoxicity/toxicity/mutagenicity analysis of soil extracts, and bioavailability analysis of soil samples pre- and post-remediation directly addresses this goal. By identifying PAH-TPs and/or PACs that are formed and/or increase in concentration post-remediation and by using toxicity tests, researchers will be able to help protect and restore terrestrial ecosystems. At the same time, identification of potentially genotoxic/toxic/mutagenic PAH-TPs and/or PACs, complemented by bioavailability analysis of compounds in soil samples pre- and post-remediation, will help researchers prevent any negative effects to the ecosystems and promote sustainable use of soil.