Environment International 37 (2011) 184–189

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Finger printing of mixed contaminants from former manufactured gas plant (MGP)site soils: Implications to bioremediation

Palanisami Thavamani a,b, Mallavarapu Megharaj a,b,⁎, G.S.R. Krishnamurti a,b,Ross McFarland c, Ravi Naidu a,b

a Centre for Environmental Risk Assessment and Remediation (CERAR), University of South Australia, Mawson Lakes, SA, Australiab Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Mawson Lakes, SA, Australiac National Contaminated Land Practice Leader, AECOM, Warabrook, NSW 2304, Australia

⁎ Corresponding author. Centre for Environmental RiskUniversity of South Australia, Mawson Lakes, South Aust8 83025044; fax: +61 8 8302 3057.

E-mail address: Megharaj.Mallavarapu@unisa.edu.au

0160-4120/$ – see front matter © 2010 Elsevier Ltd. Aldoi:10.1016/j.envint.2010.08.017

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 June 2010Accepted 31 August 2010Available online 26 September 2010

Keywords:Mixed contaminationMGP sitePAHsHeavy metalsSpeciationBioremediation

Contaminants in general do not occur as single chemicals but as mixtures at any contaminated site. Gasworkssites are the typical mixed contaminated sites. These sites are not only subjected to PAH contamination butalso varying degrees of heavy metal contamination. Bioremediation in these sites is often hindered by thepresence of heavy metals. The co-occurrence of PAHs with heavy metals has not been systematicallyinvestigated. Metals are reported to inhibit the general soil microbiological processes. The total concentrationof solublemetal in the system includes both freemetal ion and complexed forms.Within bioavailable fraction,the most toxic form is the free metal species, which was not addressed well so far in gas works sitecharacterisation. This study underpins the science and importance of metal bioavailability and speciationbased site characterisation inmixed contaminated sites. In this study a detailed elemental chemistry of the gasworks site soils are discussed using different methods. The PAH contamination was contributed by both lowand high molecular weight PAHs. The total PAHs concentration ranged from 335 to 8645 mg/kg. Among mosttoxic metals Pb was found in high concentration ranging from 88 to 671 mg/kg, Cd 8 to 112 mg/kg and Znvaried from 64 to 488 mg/kg. Thermodynamic chemical equilibrium model VMINTEQ (Ver 2.52) was used tocalculate the free metal species in gas works site soils. The percentage free metal species showed a differenttrend compared to total metal concentrations, free Zn species ranged 18–86%, free Cd was 26–87% and Pbshowed lowest free metal percentage (0–17%). The bioavailable metal species and its implications tobioremediation have also been discussed.

Assessment and Remediation,ralia, 5095, Australia. Tel.: +61

(M. Megharaj).

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© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The polycyclic aromatic hydrocarbons (PAHs) are chemicalcompounds consisting of atoms of C and H, arranged in the form oftwo or more aromatic rings.

PAHs are ubiquitous environmental pollutants that have beenfound to have toxic, mutagenic, and carcinogenic properties (Yu et al.,2006). Consequently, the U.S. Environmental Protection Agency(USEPA) as listed PAHs among the priority pollutants to be monitoredin the environment. PAH-contaminated sites are generally associatedwith industrial activities such as wood preservation, petroleumrefining, transportation; former manufactured gas plants (MGPs),lignite pyrolysis sites, military installations, and municipal andhazardous waste landfills. In many cases, PAHs at these sites arefound with other contaminants such as heavy metals. Forty percent of

the hazardous waste sites on the U. S. Environmental ProtectionAgency's National Priority List (NPL) are contaminated with bothmetal and organic pollutants (USEPA, 2000b).

Manufactured gas plants were used to produce energy for lighting,heating and cooking between 1820 and 1950. While the manufactur-ing processes have been gradually phased out (USEPA, 2000a,b), it hasleft behind a legacy of coal tar and other residues that impact soil andgroundwater (Thomas and Lester, 1993). Manufactured Gas Plant(MGP) site wastes comprises of mixtures of organic and inorganicsubstances including volatile and semi volatile organic compounds,polycyclic aromatic hydrocarbons (PAHs), phenolics, cyanides, thio-cyanates, metals (arsenic, chromium, copper, lead, nickel, and zinc),ammoniates, nitrates, and sulphate/sulphides (Lundstedt, 2003;Reddy et al., 2006; Thomas and Lester, 1993).

The presence of such a cocktail of mixed contaminants presents acomplex and challenging problem for remediating MGP sites. Hence,the most common approach to remediating MGP sites has sinceinvolved excavation and transport to prescribed land fill sites.However, with the increase in landfill costs, owners of MGP sitesare faced with increasing costs for in situ management of such

185P. Thavamani et al. / Environment International 37 (2011) 184–189

contaminated sites. Bioremediation, often a very economical optionfor soil decontamination, has often shown to be limited by thepresence of heavymetals. Co-existence of heavymetals with PAHs is aparticular challenge for bioremediation of MGP sites. Metals affect amyriad of microbial activities suggests that metals have the potentialto affect the biodegradation of PAHs in mixed contaminated soils suchas MGP sites.

It is clear that MGP sites are characterised using different ways andthe methods used to measure each vary and continue to evolve(Maier, 2000). There has been no systematic investigation of co-occurrence of PAHs andmetals in MGP sites and their potential effectson bioremediation. Most of the published studies on characterisationof former MGP sites mainly focused on concentration of PAHs due totheir carcinogenicity and high levels of occurrence. However very fewpublished studies reported about the co-occurrence of PAHs withvarious heavy metals (Lundstedt, 2003; Reddy et al., 2006; Thomasand Lester, 1993). The mobility and bioavailability of heavy metalslargely depends on its form or chemical species. In typical soilsolution, there may be 10–20 different metal cations that can reactwith as many different inorganic and organic ligands to form 300 to400 soluble complexes and up to 80 solid phases. Chemical speciationmodelling can be used to predict these soluble complexes (Adriano,2001). A lucid and comprehensive understanding of metal speciationin relation to microbial toxicity may lead to the development of noveltechnologies to remediate MGP sites.

In this study, we have characterised the mixed contaminated(MGP site) soils with special emphasis on heavy metal availability,speciation and implications to the bioremediation of mixed contam-inated soils.

2. Materials and methods

2.1. Soil samples

The contaminated soil samples used for this study were collectedfrom former MGP site in Sydney, New South Wales, Australia. Thegasworks commenced in 1902 and was used for the production oftown gas through pyrolysis of coal in retorts. The by-products of thisprocess, such as, coal tar, ash, coke, spent oxides, were in partdisposed of on site. Nine different surface soil samples were collected(1–50 cm) from various points of gasometer, retort house, gascompressor, and drainage, where the soil had a strong hydrocarbonodour, and lenses of tarry material, broken steel pipes and otherdebris dispersed throughout the site (Figure S1). The uncontaminatedsoil (Control) was collected at an uncontaminated site next to theMGP site. The soil samples were mildly crushed and passed through a2 mm sieve and homogenized and stored for further analysis.

2.2. Soil characterisation

Thegeneral physicochemical propertiesof the soilsweredeterminedusing various standard procedures. The pH and conductivity weremeasured in 1:5 (w/v) soil–water suspensions (Smart CHEM-Lab, TPSPty Ltd, Brisbane, Australia). The mechanical analysis of the soils wasdetermined following the micropipette method (Miller and Miller,1987). The soil organic carbon (SOC)was determined following thewetoxidation method (Walkley and Black, 1934). The cation exchangecapacity of the soil was determined following the method outlined byGillman and Sumpter (1986). Dissolved organic carbon (DOC) wasdetermined using TOC organic carbon analyzer 1010 (O-I-Analytical,College Station, TX, USA). Soil water extract [soil:water; 1:5 (w/w)]wasobtained after overnight shaking in an end-over-end shaker and thesupernatant was separated by centrifugation at 4000 rpm for 10 min.The concentration of anions and cations in the soil–water extract wasdetermined using ion chromatography (IC) with a Dionex ISC-2000 Ion

Analyzer fitted with an AS18 column (Dionex), and ICPMS (Agilent7500c, Agilent Technologies, Tokoyo, Japan) respectively.

2.3. Determination of total and available heavy metal concentrations ofthe soils

The total (aqua regia extractable) heavymetal concentration of thesoils was determined after extraction in a micro-wave acceleratedreaction system (CEM-MARS X®, CEM Corporation, Matthews, NC, U.S.A) according to USEPA method EPA 3051H-HP 500 (USEPA, 1997).The available heavymetal concentration was determined according tostandard DIN 19730 method (DIN, 1995). A 50 ml of 1 M ammoniumnitrate (NH4NO3) solutionwas added to 20 g soil (solution to soil ratio2.5:1), shaken for 2 h at room temperature and suspension waspassed through 0.45 μM membrane filter and preserved for analysis.The heavy metals content in all extracts were analysed using Agilent7500c (Agilent Technologies, Tokyo, Japan) ICPMS.

2.4. Extraction of PAHs

Soil samples (0.5 g) were extracted in microwave using USEPA3546 procedure for extracting water insoluble or slightly watersoluble organic compounds from soils. The Soil sample (10 g) wasweighed into a Teflon container and a 25 ml of solvent mixture(Acetone–hexane 1:1) was added to the soil. The contents weredigested in a micro-wave accelerated reaction system (CEM-MARSX®, CEM Corporation, Matthews, NC, U.S.A) following the methodoutlined in USEPA 3546 (USEPA, 2000a).

The extracted samples were cleaned using Pasteur pipette columnmethod (Guerin, 1999) with slight modification. The absorbent usedwas florisil (Sigma-Aldrich, Melbourne, Australia) with activity No. 1previously activated in a muffle furnace at 400 °C for 4 h. The dryingagent was sodium sulfate, which was treated in the same way as theflorisil. Methylene chloride was used as an eluent for the PAHs. APasteur pipette was packed into three layers (bottom, middle, top).The bottom was first plugged with sodium sulphate (10 mm) and themiddlewas packedwith activated florisil (50 mmheight). The Pasteurpipette was tapped gently to ensure even packing, followed by 10–20 mm of sodium sulfate, to form a top layer. The columns were pre-eluted with 5–10 ml of hexane or DCM prior to their single use. Thesamples were concentrated to 1 ml under gentle stream of nitrogen.

2.5. Analysis of PAHs

PAHs were analyzed using gas chromatograph with a massselection detector (Hewlett-Packard 5890 Series II supplied by AgilentTechnologies, Delaware, USA) according to the standard USEPA 8270C method (USEPA, 1996). The PAHs were separated using a 30-m highresolution glass capillary column DB-5(i.d. 0.25 mm, 0.25um film)coated with a 0.25 μm film (J&W Scientific, Agilent Technologies,Delaware, USA). The oven temperature was held initially at 40 °C for4 min, and then ramped up to 270 °C at 10 °C min−1. The calibrationwas done for each PAH by external calibration, using a certifiedmixture (TCL Polynuclear Aromatic Hydrocarbons Mix-Ref 4-8905,Supelco, Bellefonte, PA, USA). Deuterated surrogate standards (1-methylnaphthalene-d8, fluorene-d10, anthracene-d10, pyrene-d10,p-terphenyl-d14, BaP-d12, and benzo(ghi)perylene-d12) were usedtomonitor PAH losses during extraction and cleanup. The reliability ofthe calibration was checked periodically by injecting known stan-dards and solvent in to the column.

2.6. Heavy metal speciation

The solution speciation of the metals in MGP gas works site soilswas determined using a Thermodynamic chemical equilibriummodelvisual MINTEQ (Ver 2.52) (Gustafsson, 2009).

186 P. Thavamani et al. / Environment International 37 (2011) 184–189

3. Results and discussion

3.1. Soil characterisation

The general physico-chemical characteristics of the soil samples are summarised inTable 1. All the soils were classified as sandy loam and contained very low percentagesof clay (b1%) indicating that the sand and silt contents are likely to play a significantrole in the binding of the contaminants (Jonsson et al., 2007). The soil organic carbon(OC) content varied considerably between the samples ranging from 1.7 to 4.9%. TheDOC content ranged from 44.3 to 303 mg/l and is likely to play a significant role inbiogeochemical processes, such as mineralization, distribution, mobility and bioavail-ability of contaminants (O'Loughlin and Chin, 2004). The pH of the soils varied fromstrongly acidic to near neutral. Soils S1, S2, S3, S4 and S7 can be classified as stronglyacidic due the very low pH (3.7 to 5.4) and all other samples had neutral to near neutralpH. Soil pH is an important for successful bioremediation and should be amended to thepH range of optimum bacterial growth and metabolism. The electrical conductivity(EC), measured in the same extract, varied from 63 μS to 4240 μS. Cation exchangecapacity (CEC), which is a measure of the quantity of readily exchangeable cations(Stalikas et al., 1997), did not show significant variation amongst the soils.

The soil solution concentration of the anions is summarised in Table 2. Sulphate isthe most abundant anion ranging from 38 to 533 mg/kg, which may be attributed tosulphur added during the gas and liquor purification processes. However, the majorportion of the sulphur was removed during desulfurization step. The other anions werelower than sulphate, chloride concentration ranged from 5 to 21 mg/kg, nitrate from 4to 138 mg/kg and phosphate from 1.4 to 12.6 mg/kg.

The major nutrients such as nitrogen (N), phosphorus (P) and potassium (K)contents were analysed in all the soils (Table 2). The soil available nutrients areimportant for successful application of bioremediation methods. The mean concentra-tion of NH3-N, Olsen-P and K of the soils were 124 mg/kg, 7 mg/kg, and 33 mg/kg,respectively (Table 2). For bioremediation of soils, the optimum C: N: P: K ratio shouldbe 100:10:1:2.5 (Alexander, 1995). The average CNPK ratio of the soils in the presentstudy is 100:5:0.25:1.25. There is a need to adjust the NPK ratio based on carboncontent including PAHs.

3.2. Concentrations of polycyclic aromatic hydrocarbons in MGP soils

All the soil samples contain most of the EPA priority PAHs (Table 3). The meanconcentration of the total PAHs (tPAHs) ranged from 335 to 8645 mg/kg. Similar range inconcentrations was reported in other gasworks and wood impregnation sites (Jonssonet al., 2007). Soils originating from gas works and coke production sites are generallycharacterized by relatively high concentrations of high molecular weight PAHs (HMWPAHs) (Lundstedt, 2003; Yang et al., 1991). The lowmolecular weight PAHs (LMW PAHs)(12670 mg/kg) andHMWPAHs (11409 mg/kg)were contributing equally to the load ofΣtPAHs (24079 mg/kg) in the MGP site soils of the present study. The slightly higherproportion of HMW PAHs is indicative of coal tar (by-product of gasification process)contamination with long term industrial activity.

The soil samples were collected both from “hot spots” and from areas presumed tobe less contaminated. The spatial distribution among nine samples differs significantly.The samples, S8 and S9, sampled from near gas station had higher tPAHs relative toother samples suggesting that varying degrees of contamination and contaminantprofiles in the sample site (Jonsson et al., 2007).

The concentration profile of individual PAHs showed a relatively even distributionof compounds amongst the soils. Phenanthrene was most abundant amongst the lowmolecular weight (LMW) compounds as was pyrene in high molecular weight (HMW)compounds. However it was notable that BaP, the most potent carcinogenic PAH,ranged between 58 and 738 mg/kg with a mean concentration of 240 mg/kg.The presence of high concentration of HMW PAHs poses significant challengefor remediation given the lack of demonstrated technologies to metabolise thesecompounds.

Table 1Selected physicochemical characteristics of the manufactured gas plant site soils.

Soil pH EC(uS)

Sand(%)

Clay(%)

Silt(%)

CECb

(cmol(+)/kg)OC(%)

DOC(mg/L)

S1 3.7 283 61.7 b1 37.6 0.7 2.4 49.3S2 4.5 73 75.9 b1 23.7 0.7 4.7 69.7S3 5.4 80 32.4 b1 67.1 1.8 2.3 142.5S4 5.0 4240 59.9 b1 39.7 0.7 1.7 38.1S5 7.3 63 84.8 b1 15.0 1.0 3.0 44.4S6 6.8 1395 81.2 b1 18.5 1.4 4.9 107.1S7 4.5 1410 74.1 b1 25.6 0.7 4.3 243.4S8 7.3 930 28.6 b1 70.9 2.9 3.8 242.5S9 7.2 850 25.2 b1 74.1 2.2 3.6 302.8Control 6.3 14 82.6 b1 17.2 0.7 1.6 49.3

Various factors, such as diffusion in to soil organic matter, formation of residues,entrapment in soil micropores and co-occurrence of other compounds, affect thebioavailability and biodegradation in long-term contaminated soils (Wild and Jones,1995). It has been shown that degradability of organic compounds in soil decreaseswith time (Hatzinger and Alexander, 1995). This phenomenon is often referred to as“ageing” of contaminants. Although PAHs are ubiquitous pollutants, elevatedconcentrations have been found in urban soils (NEPC, 1999) and roadside soils (Yanget al., 1991), while highest concentrations have been reported for industrial sites(Haeseler et al., 1999; Wilson and Jones, 1993). The concentrations of PAHs in the MGPsoils under study have all exceeded the National Environment Protection Measure(NEPM) level of 100 mg/kg for industrial sites (NEPC, 1999).

3.3. The elemental chemistry of gasworks site soils

Gasworks sites are not only subjected to PAH contamination but also to varyingdegree of metal contamination. Although there are several papers published on gaswork sites contamination, only few have reported the presence of metals (Lundstedt,2003; Mi et al., 1998; Thomas and Lester, 1993; Yuan et al., 1999). The presence ofmetals is likely to adversely influence microbiological processes. The elementalchemistry of the MGP gas work site soils is discussed below.

3.3.1. Total metal concentrations of MGP site soilsThe concentration of aqua regia extractable metals varied considerably amongst

the soils within the gasworks site (Table 4). Among the toxic metals Pb was found inhigh concentration ranging from 88 to 671 mg/kg. Whereas Zn varied from 64 to488 mg/kg, Cd from 8 to 112 mg/kg and Cr from 33 to 379 mg/kg. Pb has been reportedto be the major co-contaminant in PAH contaminated sites (Mielke et al., 2004).The other toxic elements Cu, As, Mn and Ni, were found in varying range from 18 to57 mg/kg, 5 to 9 mg/kg, 52 to 181 mg/kg and 10 to 230 mg/kg, respectively (Table 4).The order of mean concentrations of all elements in gasworks site soils in this studywasas follows, PbNZnNCrNMnNNiNCd. The total heavy metal concentrations for Pb, Zn andCd did not exceed NEPM health investigation levels (Pb-1500 mg/kg; Zn-35000 mg/kg;Cd-100 mg/kg) for commercial/industrial soils. Based on the total concentrations ofindividual metals, the MGP site of the present study may not be classified as heavymetal contaminated soil. However, the bioavailability of the toxic elements has thepotential to affect microbial properties.

3.3.2. Available metal concentrations of the MGP site soilThe different chemical forms in which metals occur within the soil matrix

determine their mobility and bioavailability. The use of single salt extractions toascertain the potentially available pool of metals within the soil matrix has been usedsince the early 1970's. Since then, the number of extraction procedures has grownexponentially (Meers et al., 2007). The German DIN method has been shown to be themost suitable method to predict metal availability for biological uptake (Belotti, 1998;Prüeβ, 1992). In this study the available metal concentrations were estimated by a 1MNH4NO3 extraction procedure (DIN, 1995) and are presented in Table 5.

Similar to total aqua regia extractable metals, available metal concentrations variedconsiderablywith the Pb, Cd, Zn, Mn and Ni concentrations ranging from 0 to 667 μg/kg, 0to 634 μg/kg, 0 to 1119 μg/kg, 16 to 690 μg/kg, and 0 to 131 μg/kg, respectively. Theavailable Cu and Aswere not detected in any of the samples studied, as well as Cr was toolow. The extent to which heavy metals may influence bioremediation depends upon theconcentrationof available orpotentially availablemetal rather than the total concentration(Babich and Stotzky, 1985). In our study the available mean concentration of mostbiologically toxic metals were in the order ZnNPbNCd.

3.3.3. Correlation of PAHs and major toxic elements in MGP site soilsVarious studies reported correlation between PAHs and soil organic matter (SOM)

(Stalikas et al., 1997; Yu et al., 2006). However, no attempt has been made to correlatebetween PAHs and toxic metals found in gasworks site soils. In this study, significantcorrelations (pb0.01 and pb0.05) were observed between PAHs and toxic elementsparticularly with Zn. The data on correlation analysis between LMW PAHs, HMW PAHs,total PAHs andmajor toxic elements (Pb, Cd, Zn) is presented in Table S1(supplementarymaterial). All individual PAHs showed strong correlation (rN0.8) with LMW, HMW andtotal PAHs, and most individual PAHs in soils have shown strong correlation with eachother (pb0.01). The strong correlation suggests that composition of the major PAHs isfairly constant at different sampling sites and may arise from similar sources.

There was a relatively strong correlation between Cd and Pb (pb0.05). Theobserved significant correlations indicate that appreciable amounts of toxic elementsare most likely incorporated into soil from the same point source (e.g. spent oxides,additives). However, the significant but a negative correlation (pb0.05) of Zn withacenapthene, phenanthrene, LMW PAHs and total PAHs suggests that not all metal ionsare directly associated with PAHs in soil, and the source of metals may differ fromsource of PAHs. But also under acidic conditions some of the metals may have leachedfrom the soil, thus some of themetals present in these soils could be due to atmosphericdeposition and other unidentified activity rather than due to gasification process.However, control soil did not show elevated amount of metals (b15 mg/kg soil), hencemetal deposition could be due to some other unidentified activities during gasmanufacturing process. Most of the MGP sites are 50–100 years old, the buildingstructures, pipelines and other structures were completely corroded and mixed withother contaminants of the site (Thomas and Lester, 1993).

Table 2Concentrations of major anions and nutrients (mg/kg dry soil) in soil solution from MGP site soils.

Soil Chloride Nitrate Sulphate Phosphate Fluoride NH3-N Olsen-P Potassium

S1 8.0±0.2 90.0±3.3 278±7 6.6±0.3 1.46±0.03 24.6±1.4 12±1.2 16.5±0.8S2 38.0±1 3.0±0.1 64±3 1.4±0.1 1.09±0.01 3.6±0.1 32.5±1.7 29.2±1.2S3 12.0±1 116.0±5 20±0.5 3.1±0.1 0.69±0.02 5.4±0.4 32.6±2.4 56.9±4.1S4 21.0±1.4 105.0±3 175±11 2.3±0.2 0.27±0.01 4.2±0.2 10.6±1.0 379.2±12S5 5.0±0.1 72.0±2 38±1.0 1.7±0.1 0.65±0.03 1.5±0.07 11.3±1.1 29.9±1.6S6 15.0±1 4.0±0.2 533±21 4.1±0.2 0.47±0.02 9.6±1.0 13.7±1.0 82.4±3.7S7 9.0±0.5 138.0±12 196±7 5.7±0.1 0.28±0.01 22.2±2.0 36.6±2.8 31.5±1.0S8 5.0±0.3 9.0±1 316±0.5 12.6±0.1 1.06±0.02 89.4±7.0 6.7±0.2 30.1±1.0S9 6.0±0.2 26.0±0.2 144±12 4.9±0.1 0.72±0.03 124.2±10 7.0±0.6 33.0±0.7Control 32±1.7 18±0.8 100±5.7 1.4±0.1 0.72±0 0.9±0 37.9±3.2 20.5±2.4

Table 3Concentration profile of polycyclic aromatic hydrocarbons (mg/kg dry soil) in MGP site soils.

PAHs M. wt(g/mol)

No. ofrings

Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6 Soil 7 Soil 8 Soil 9 Mini Max Average Sum ofindividualPAHs

NAP 128 2 95±0.5 349±0.7 53±0.4 67±0.8 51±4.4 56±4.2 66±0.6 660±26.3 564±0.7 51 660 218 1960ANY 152 3 59±0.6 118±2.7 0 56±0.9 50±1.6 52±0.9 54±4.5 584±2.4 623±15.7 0 623 177 1596ANE 154 3 51±0.7 70±1.4 0 0 0 0 49±7.6 216±8.2 222±12.4 0 222 68 608FLE 166 3 61±1.2 82±1.4 0 52±3.5 0 25±2.2 51±1.7 726±21.2 706±17.4 0 726 189 1704PHE 178 3 173±0.8 469±13.6 55±1.5 110±3.7 52±3.4 67±3.1 132±7.1 2377±0.4 2738±0.6 52 2738 686 6171ANT 178 3 74±0.8 181±4.7 0 65±0.6 0 55±3.4 64±2.1 114±4.6 73±3.2 0 181 70 626FLA 202 4 174±0.2 419±2.5 63±0.4 187±5.7 55±0.8 108±6.8 171±2.4 1245±13.6 1137±11.5 55 1245 396 3560PYR 202 4 189±0.5 504±7.5 64±1.4 191±6.2 55±4.4 115±5.2 185±6.9 1357±41.8 1245±60.6 55 1357 434 3904CHR 228 4 110±0.4 124±1.0 38±0.6 93±3.3 33±1.8 77±5.8 83±2.3 748±12.3 476±25.8 33 748 198 1782BAP 252 5 131±7.2 236±3.2 62±0.6 117±0.8 58±2.3 101±7.3 101±9.0 619±19.3 738±12.6 58 738 240 2163LMW PAHs 513 1269 108 350 153 255 416 4677 4926 108 4926 1267 Σ LMW 12670HMW PAHs 604 1283 227 588 201 401 540 3969 3596 201 3969 1141 Σ HMW 11409Total PAHs 1117 2552 335 938 354 656 956 8645 8521 335 8645 2675 Σ PAH 24079

Note: LMW – low molecular weight PAHs (2–3-ring PAHs); HMW – high molecular weight PAHs (4–6-ring PAHs). Values are means with standard deviations.NAP – Napthelene; ANY – Acenaphthylene; ANE – Acenapthene; FLE – Fluorene; PHE – Phenanthrene; ANT – Anthracene; FLA – Fluoranthene; PYR – pyrene; CHR – chrysene;BAP – Benzo(a)pyrene.Among 16 USEPA priority pollutants Benz(a)anthracene; Benzo(b+k)fluoranthene; Indeno(1,2,3-cd)pyrene; Dibenz(a,h)anthracene; Benzo(g,h,i)perylene are not reported herebecause they were not detected in the samples.PAHs were not detected (b1 mg/kg soil) in control soils.

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3.3.4. Heavy metal speciation of MGP site soilsIn soils contaminated with PAHs and metals, metal speciation is the key process to

understand the real toxicity of metals. It has been suggested that, typically, stronglycomplexed metals are less toxic to organisms than weakly complexed forms, which inturn, are less toxic to organisms than the free ions (Adriano, 2001). Chemicalequilibrium computer programs are useful for determining the distribution of speciesin soil solutions containing defined concentrations of metals and ligands. In this study,metal speciation of the soil solutions was determined using the visual MINTEQ (Ver2.52) computer software (Gustafsson, 2009). The solution phase metal concentration(water extractable) and percentage of free metal species as calculated from visualMINTEQ is shown in Fig. 1. The water extractable Zn was the highest among three toxicmetals, the solution phase concentration ranged from 315 in S6 to 4985 μg/kg in S4. Thewater soluble Pb concentration varied from 104 (S5) to 1362 (S2) μg/kg. The watersoluble Cd concentrations varied widely among samples with no detectable Cd in soilS5 to 1311 μg/kg (S6). However, Cd is known to be toxic to biota at much lowerconcentrations than other metals such as Zn, Pb, Cu etc. (Adriano, 2001).

The ionic form of heavy metal is the most toxic to biota including contaminantdegrading microorganisms (Sandrin and Maier, 2003). The speciation calculated by

Table 4Total concentrations (mg/kg dry soil) of selected toxic elements in MGP site soils.

Soil Pb Cd Zn Cr

S1 125±10 21±1.2 64±4 120±7S2 671±22 106±7 153±10 379±1S3 88±5.1 17±1.1 179±8 54±4S4 664±26 112±8 202±14 289±1S5 178±12 30±2.1 488±21 33±2S6 324±13 62±4.3 163±6 242±1S7 255±1.9 26±1.6 147±7 161±1S8 94±3.6 45±3.1 153±9 78±4S9 99±4.2 8±0.5 100±6 83±6Control 2±0.3 0 13±1.2 2±0

vMINTEQ showed different patterns as shown in Fig. 1. Among three toxic metalsstudied, Pb2+ has shown the lowest activity from 0.44 in S3 to 16.82% in S6. It is well-known thatmetal bioavailability can be greatly affected by the dissolved organic carbon(DOC). Even though the total concentration of Pb was significantly high (94–671 mg/kg), more than 80% of Pb was complexed with DOC and the inorganic ligand, sulphate.Free metal species in solution can form complexes with a number of organic andinorganic ligands (Stumm and Morgan, 1981). Often, Cd and Pb are closely scrutinizedtogether because they originate from the same emission sources such as coalcombustion, mining and smelting operations. These two elements have been shownto act synergistically on biota (Adriano, 2001).

On the other hand, Zn and Cd have shown an interesting correlation in free metalspeciation, with the activity of Zn and Cd, both known for their high mobility, followingthe same pattern of speciation in all samples irrespective of soil pH. The activity of Znvaried from 17.92 (S3) to 85.95% (S6) and Cd activity also followed the similar trendfrom 25.5 (S3) to 87.16% (S6). Though the total concentrations of Zn were more thanCd, the percent distribution of free metal activity was identical. The close association ofCd and Zn in environmental occurrence and chemical similarity of these two elementscarry over to the biological systems (Adriano, 2001; Krishnamurti and Naidu, 2003).

Cu As Mn Ni

.1 21±1 6±0.4 52±4 50±32 52±1 5±0.1 137±7.1 230±17.2 47±3 6±0.2 105±8.3 26±20 57±2 9±0.6 148±11.7 172±14

50±2 6±0.2 54±4.3 10±0.99 49±3 5±0.1 121±9.2 132±6.21 36±1 5±0.1 79±5.4 135±7.4.7 18±1 5±0.1 73±5 45±1.6.2 21±1 9±0.6 181±9.6 28±1.1.2 2±0.1 b1 15±0.8 2±0.1

Table 5Concentrations of available toxic metals (μg/kg) in MGP soils extracted using 1 Mammonium nitrate.

Soil Pb Cd Zn Cr Mn Ni

S1 96±2.2 338±13.4 367±26 13±1 140±3 41±4S2 667±19 634±13 608±13.5 4±0.1 137±4.3 44±3.8S3 33±2 22±1.2 1054±15.8 1±0.03 311±6.8 49±4.1S4 604±23.1 317±12.7 849±35 2±0.2 690±12.4 131±6S5 1±0.1 0 11±1 0 70±7 0S6 1±0.1 0 0 1±0.07 16±1 0S7 141±4.8 141±11.5 1119±79 6±0.2 316±4 73±2.3S8 71±5 125±10.4 892±57 1±0.06 305±26 48±2.6S9 0 0 7±0.4 1±0.09 51±4 2±0.1

Note: Control soil was not considered for ammonium nitrate extraction since toxicmetals were present at very low levels.

Fig. 1. Solution phasemetal concentrations and percentage of free metal species in MGPsite soils. Note: Solution phase metals were extracted using 1:5(w/v) ratio in MQwater.Percentage of free metal activity was calculated using vMINTEQ (ver.2.52). Soilnumbers are given in parentheses along with pH on X axis.

188 P. Thavamani et al. / Environment International 37 (2011) 184–189

The effect of these two metals, having similar free metal activity distribution, onmicrobial processes and remediation has not been studied in contaminated soils so far.

3.4. Implications of metal speciation for microbial remediation of PAHs

Soil contamination due to PAHs is often associated with the presence of high levelsof potentially toxic metals (Allieri et al., 2005). Unlike organics, metals cannot bedegraded by biological process. However, degradation of organic contaminants in

Table 6Reported toxic metal concentrations that cause inhibition to PAHs biodegradation.

Toxic metal PAHs Toxic metal concentrati

Cd2+ Naphthalene b25.3–50.6 mg/LNaphthalene 1 mg/La

Naphthalene 10 mg/LPhenanthrene 1 mg/La

Phenanthrene 10 mg/LPyrene 60 mg/LPyrene 100–500 mg/kgFluoranthene 2 mmolBenzo(a)anthracene 100–500 mg/kgBenzo(a)pyrene 10 mg/kg

Pb2+ Pyrene 6 mg/LAnthracene 1000 mg/LPhenanthrene 1000 mg/L

Zn2+ Naphthalene b29.5–736 mg/LPhenanthrene 720–1440 mg/kgFluoranthene 20 mmol

a Values represents solution phase metal concentration in the system. All the remaining

mixed contaminated soils, such as gas works sites, may be affected by the co-occurrence of toxic elements. The subsurface old gas work sites consist of a mixed fillcontaminated with coal tar and its distillation products, organics, heavy metals. Thecontaminants are heterogeneously distributed, but are found throughout the area.Heavy metal contamination at former gasworks sites usually occurs at or near theground surface. Themobility of heavymetals depends heavily on various factors such aspH and the other physicochemical parameters, which affect solubility and binding tothe soil.

Wide range of metal concentrations was reported to inhibit organic biodegrada-tion. Metals including copper, zinc, cadmium, chromium (III and VI), nickel, mercury,and lead have been reported to inhibit various microbial processes. Sandrin and Maier(2003) have compiled most of the available reports on the impact of various metals onthe degradation of organic compounds. Available data on the effect of metals on PAHsbiodegradation is not extensive, but demonstrates that metals have the potential toinhibit pollutant biodegradation under both aerobic and anaerobic conditions.

Although PAHs are one of the most studied organic compounds for the past twodecades, the impact of toxic metals on their degradation has not received muchattention so far. The extent of the interferences by the heavy metals on the removal ofrecalcitrant PAHs from the soil is not very clearly documented for many heavy metals.Only very few studies reported the effect of metals on PAH degradation as shown inTable 6.

The lowest Cd concentration reported to reduce biodegradation of PAHs rangedfrom between 0.002 and 1 mg/L (Springael et al., 1993; Sandrin et al., 2000; SandrinandMaier, 2003) in terms of free Cd species. Maslin andMaier (2000) reported that a 5-day increase in lag period for phenanthrene degradation in the presence of 1 and 2 mgavailable Cd/L and complete inhibition at 3 mg available Cd/L.

Most of the previous studies did not consider the available and/or free metalconcentrations, reported just total toxic metal concentrations. For example, Cd (500 mgtotal Cd/kg) and mercury (100 mg total mercury/kg) did not affect biodegradation of avariety of polycyclic aromatic hydrocarbons (PAHs) by the fungus Pleurotus ostreatus insoil (Baldrian et al., 2000). However, the information on the concentrations of availableas well as free metal speciation is not available. Most commonly, metal inhibition ofbiodegradation has been related to the total metal concentration in a system. This maynot be the most appropriate predictor of metal toxicity, as suggested by the wide rangeof total metal concentrations reported to inhibit biodegradation.

Microbes use organic compounds either as carbon source or transform them intonon toxic products using various enzymes and extracellular products. In systemscontaining a mixture of contaminants, presence of metals interferes with microbialprocesses both physically and metabolically. The effect of heavy metals on microbialprocess is not due to its total concentration but is dependent on the available orpotentially available concentration. The availability of heavy metals in soils is affectedby various factors including pH, SOM, and DOC and concentration of anions and othercations in soil solution. For instance, Pardue et al. (1996) found that only 0.01 mgsolution phase Cd/L was required to inhibit trichloroaniline dechlorination in a mineraldominated soil, while 0.2 mg solution phase Cd/L was required for inhibition in anorganic matter dominated soil. This increase in tolerance to Cd was correlated tosaturation of metal binding sites on the organic matter. Cd and Zn appear to competefor certain organic ligands in vivo, Cd competes with Zn in forming protein complexes,an antagonistic association between two expected.

A high total metal concentration in soil may not be toxic to microbe at if it is notavailable. The total concentration of soluble metal in the system includes free metal ionand complexed forms of metals. Hughes and Poole (1989) have underlined theimportance of metal speciation in understanding its bioavailability, and toxicity, in thetest system. However, the importance of free metal species, the most toxic form of theavailable fraction of the metal, has not been addressed so far in site characterisation.

In this context, the site characterisation based on metal speciation provides betterunderstanding of the risk than traditional physico-chemical characterisation.

on Study system Reference

Tris minimal media Springael et al. (1993)Dilute MSM Sandrin et al. (2000)MMO medium Malakul et al. (1998)Soil microcosms Maslin and Maier (2000)Soil microcosms Shen et al. (2006)Soil microcosms Irha et al. (2003)Soil microcosms Baldrian et al. (2000)Houba-Remarcle media Riha et al. (1992)Soil microcosms Baldrian et al. (2000)Soil microcosms Baldrian et al. (2000)Soil microcosms Irha et al. (2003)Mineral salt media Fijałkowska et al. (1998)Mineral salt media Fijałkowska et al. (1998)Tris minimal media Springael et al. (1993)Soil microcosm Wong et al. (2005)Houba-Remarcle media Riha et al. (1992)

reported metal concentrations are total concentrations in the system.

189P. Thavamani et al. / Environment International 37 (2011) 184–189

Speciation based characterisation of mixed contaminated soils may lay the foundationfor the development of new innovative site-specific remediation methods of organiccompound degradation. Considering the lack of available information on metalspeciation based characterisation of gasworks site, it will be worth looking at thedynamics of metal speciation and its effect on the degradation of organic compounds inmixed contaminated systems such as gasworks sites soils.

4. Conclusions

Soil contamination due to PAHs is often associated with thepresence of high levels of potentially toxic metals. Most commonly,metal inhibition of biodegradation has been related to the total metalconcentration in a system. This may not be the most appropriatepredictor of metal toxicity, as suggested by the wide range of totalmetal concentrations reported to inhibit biodegradation. The concen-tration of the most available form of the metal is likely a betterindicator of the extent to which a metal will inhibit biodegradation.This study underpins the science and importance of metal bioavail-ability and speciation based site characterisation. This approach is akey step to understand, quantify the impacts of metals on biodegra-dation and develop cost-effective remediation of metal and organicmixed contaminated soils.

Acknowledgements

This research was supported by the Australian Government andUniversity of South Australia through an International PostgraduateResearch Scholarship (IPRS) in collaboration with CooperativeResearch Centre for Contamination Assessment and Remediation ofthe Environment (CRC CARE).

Appendix A. Supplementary data

Supplementarydata to this article canbe foundonline at doi:10.1016/j.envint.2010.08.017.

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