Environmental Technology & Innovation 4 (2015) 98–109

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Risk based land management requires focus beyond thetarget contaminants—A case study involving weatheredhydrocarbon contaminated soilsPalanisami Thavamani a,b,c,∗, Euan Smith a, Ramadass Kavitha a,Grant Mathieson a, Mallavarapu Megharaj a,b,c, Prashant Srivastava b,Ravi Naidu a,b,c

a Centre for Environmental Risk Assessment and Remediation (CERAR), University of South Australia, Mawson Lakes, SA 5095, Australiab Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), University ofNewcastle, Callaghan, NSW, Australiac Global Center for Environmental Remediation(GCER), University of Newcastle, Callaghan, New South Wales, Australia

h i g h l i g h t s

• Weathered hydrocarbons may not pose risk to environmental receptors.• Multispecies ecotoxicity assessment should include site based local receptors.• Field level application of Risk based land management is complicated using current methods.• Risk based land management should focus beyond target contaminants.

a r t i c l e i n f o

Article history:Received 15 December 2014Received in revised form 27 April 2015Accepted 28 April 2015Available online 28 May 2015

Keywords:Weathered hydrocarbonsBioavailabilityRisk-based land management (RBLM)Residual riskMulti-species toxicity assay

a b s t r a c t

Irrespective of the nature of contamination, the use of total contaminant loading as ameasure of risk together with conservative policy guidance is proving major stumblingblock towards remediation of contaminated sites. The objective of this study was toinvestigate the use of risk based approach to manage contaminated sites at field scale. Thisstudy recognizes the presence of weathered hydrocarbon compounds in long-term totalpetroleum hydrocarbon (TPH) contaminated soils and that such compounds may not poserisk to local receptors. A multispecies ecotoxicological assessment was used to determinethe potential risk from weathered hydrocarbons to the surrounding environment. Theecotoxicity of soil residual TPHswas evaluatedusing earthworm,water-flea, twonative andtwo non-native Australian plants, and soil microbial activity. Plant germination was 100%in all soils but post germination, seedlings except Ryegrass failed to establish. Earthwormtoxicity studies found that there was a negative impact on earthworm reproductionand mortality. Further investigation of the poor plant growth and earthworm mortalityrevealed that it was due to the elevated salinity that developed due to surface evaporationof the saturated calcium sulphate and not residual soil TPHs. Toxicity assessment of thesoil leachate on the aquatic environment showed no effect on the survival of water-fleaeven though the TPH concentrations in the first year leachate were as high as 1.6 mg TPHL−1. The study concluded that the residual TPHs in soils had little impact on a range ofenvironmental receptors. Assessment of the residual TPH ecotoxicity was complicated by

∗ Correspondence to: Global Center for Environmental Remediation(GCER), University of Newcastle, Callaghan, New South Wales, Australia. Tel.: +61 240339411.

E-mail address: Thava.Palanisami@newcastle.edu.au (P. Thavamani).

http://dx.doi.org/10.1016/j.eti.2015.04.0052352-1864/© 2015 Elsevier B.V. All rights reserved.

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the elevated salinity of stockpile soils which impacted on the earthworm and phytotoxicityassessments. Therefore results of this study suggest that it is paramount to focus beyondtarget contaminants while implementing risk-basedmanagement approach. Indicators forrisk based assessment are considered critical for regulatory decisionmaking. The results ofthis study provide a valuable input in to the risk basedmanagement of contaminated sites.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Currently, there are more than 3,000,000 potentially contaminated sites around the world which represent a losteconomic opportunity and threat to the health and wellbeing of human as well as the environment (Singh and Naidu,2012; Thavamani et al., 2011). Of the 160,000 contaminated sites in Australia (CEI, 2005), 60% comprise hydrocarbon-contaminated sites (NEPC, 1999). Ageing of hydrocarbon contaminated sites results in the loss of volatile hydrocarbonswith residual weathered compounds remaining in soils (Tang et al., 2012). Land-farming has commonly been used for manyyears in the management and disposal of hydrocarbon contaminated soils (Theodore and Reynolds, 1987). It is one of thesimplest and cheapest bioremediation treatment options for large volumes of hydrocarbon contaminated soils. Properlymanaged land-farming treatments often lead to a significant decline in total hydrocarbon concentration via volatilization oflowmolecular weight TPHs (e.g. gasoline range fractions). Biodegradation is more significant than evaporation in removingmediummolecular weight range hydrocarbon products (e.g. diesel fuel, kerosene) (Atlas and Hazen, 2011). However, land-farming often leaves behind heavier complex and less bioavailable hydrocarbon fractions (e.g. lubricating oils) due to thefact that they are not volatile and take an extended time period to biodegrade (US EPA, 1994; Alexander, 2000; Thavamaniet al., 2012a). The presence of residual petroleum hydrocarbons including weathered products has been of much concernto regulators and as a consequence, industry is left with stockpiles of land-farmed soils and stockpile management costs inexcess of ∼AUD 5M per annum.

Risk-based land management (RBLM) is increasingly being adopted by regulators as a means of managing contaminatedsites. RBLM works on the underlying basis of demonstrating no or negligible risk from exposure to contaminants to bothhumans and environmental receptors (Naidu et al., 2008). RBLM involves diverse approaches including an assessment ofthe concentration and nature of contamination, potential risk that these contaminants pose to environmental and humanhealth, the planned use of the soil and the impact on surrounding ecosystems. RBLM can be applied at various stages asappropriate to manage contaminated soils (Kwame, 2006; Luo et al., 2009; Naidu et al., 2008). It represents a mature andsustainable approach in management of increasing numbers of contaminated sites and associated costs in remediation(Naidu et al., 2013; Nathanail, 2009). RBLM, in particular, can be applied in setting an overall sustainable developmentstrategy for management of contaminated land in a particular region (Naidu et al., 2008; Kwame, 2006). However, there arelimited field based studies demonstrating the application of RBLM tomanagement of long-term hydrocarbon contaminatedsoils. The presence of approximately 10,000 tonnes of post-land farm soils stockpiled at an industrial site at Newman,Western Australia provides an excellent opportunity to demonstrate application of such a strategy. Analysis of compositestockpile samples indicated that the total petroleum hydrocarbon (TPH) concentrations in the stockpile soils are lowerthan the locally applicable Soil Ecological Investigation Levels (EILs) (WA DEC, 2010) but did not achieve the target TPHconcentrations stated in the remediation licence. The TPH residual fraction comprises mainly the C29–C36 fractions andthe presence of these fractions limits further bioremediation of the stockpile soils. The presence of large volumes of land-farmed soils therefore presents an opportunity to test risk-based approach and especially its acceptance by regulators.Clearly from regulatory perspectives, risk based frameworks are not always supported by suitable and robust indicatorsfor risk assessment, especially in the case of matrices contaminated with weathered hydrocarbons. Such indicators for riskbased assessment are considered critical for regulatory decision making.

There are a number of approaches that may be adopted when considering environmental ecotoxicity testing but there isincreasing interest to incorporate a range of tests (with a battery of different assays) that integrate contaminant exposure,and on a range of bioassays (O’Halloran, 2006; Chapman et al., 2012). Standardized bioassays can be used for path-related,toxicological characterization of soils and soil materials, taking into account possible transfer of pollutants to groundwaterand potential effects on soil micro-organisms, earthworms and plants (Hatcher, 2002). In recent years, ecotoxicological testshave also been used as supplementary tools tomonitor bioremediation of hydrocarbons, both in laboratory and field studies(Wang and Bartha, 1990; Salanitro et al., 1997; Saterbak et al., 1999;Megharaj et al., 2000, 2011; Sinnett et al., 2011). Variousbioassays representing different trophic levels have been used for screening soil toxicity (Juvonen et al., 2000; Spurgeonet al., 2005). Soil quality tests using bacteria, plants and invertebrates are all promising tools for risk-based corrective action(Płaza et al., 2005). Utilization of standard ecological receptors facilitates the standardization of ecotoxicological endpointsto assess the environmental impact of the contaminant when undertaking risk-based ecotoxicity assessments (Alexander,2000).

The objective of this study was to develop the underlying scientific basis for the risk based assessment of long-termTPH contaminated sites. In particular, the study focuses on an evaluation of the residual toxicity of stockpiled long-term

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Table 1Relevant eco-toxicological endpoints used in this RBLM study.

hydrocarbon contaminated soils using a combination of chemical and bioassays. Along with this an assessment of thepotential release of TPHs from the stockpile soils by using column leaching studies is also considered. Data gathered aspart of this study was then used to formulate a strategy for the risk based management of the contaminated soils.

2. Materials and methods

2.1. Stockpile soil characterization

Fifty composite samples from stockpile soils which had been remediated by land-farming were collected from theNewman, WA, Australia. All soils were screened for total TPH soil concentrations against the Tier 1 regulatory soil guidelinevalues (WA DEC, 2010). Total TPHs were extracted using an ultrasonic solvent extraction method (USEPA 3550) andquantification by GC FID was performed according to the protocol described in USEPA, 8015C. Based upon total TPHs, sixsoils: SP1-7 (240 mg TPH kg−1); SP4-7 (1020 mg TPH kg−1); SP4-19 (980 mg TPH kg−1); SP4-20 (770 mg TPH kg−1); SP1-8(560 mg TPH kg−1); SP4-8 (830 mg TPH kg−1) were selected for further ecological risk assessment. The selection of the sixsoils was based solely on the residual TPH concentration in the soils.

2.2. Ecotoxicological assessment of residual TPHs

Bioassays are general indicators of the presence of stressors in the environment. They can be divided into acute or chronictests by looking at the selected endpoints (Hansen et al., 2007). Table 1 presents the battery of bioassays used to assessthe toxicity end points of weathered TPHs in the soils. The test species were representative of two major groups of soilorganisms—plants and soil invertebrates. The species used for the tests were consistent with earlier work completed insupport of the development of the petroleum hydrocarbon toxicity standards (Cermak et al., 2010). Micro-organisms are themost sensitive organism for the effect of the pollutants and themicrobial toxicity tests are needed for the risk assessment ofpolluted soils and sediments because soil and sediment quality guidelines derived from animal toxicity data are not alwayslow enough to protect micro-organisms (van Beelen, 2003). Hence the microbial toxicity tests were included among thetoxicity assays by studying the changes in the soil enzyme activity (Oleszczuk et al., 2014). Daphnia sp. as an ecologicalindicator used for common ecotoxicological tests to assess water quality and this organisms was used in our study to testthe soil leachate toxicity. Daphnia carinata is the most abundant and widely distributed daphnid species in Australia andSouth East Asia (Zalizniak and Nugegoda, 2004).

2.2.1. Leachate experimentSelected soils were dried and sieved prior to re-packing into stainless steel columns (20 × 10 cm) to a soil density of

1.8 g cm−3. Simulated annual rainfall (average for the Pilbara region) was applied for the equivalent period of 10 years toinvestigate thepotential long term residual TPH risks to the environment. Rainwaterwas collected froma14,000 Lhouseholdrainwater tank fitted with a ‘‘first-first flush’’ leaf litter collection systems. Fifty litres of rainwater was collected in 10× 5 L

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plastic containers which had been washed 3 times with deionized water. Prior to collection each container was rinsed 3times with rain water. The water was full at the time of collection. The characteristics of the rainwater were; pH 6.5, EC51 µS cm−1. The major metals identified in the rainwater were; Ca 200 µg L−1, Fe 52 µg L−1, Zn 650 µg L−1, Al 32 µg L−1,Cu 60 µg L−1, Mn 10 µg L−1. The characteristics of the rainwater are similar to those reported in rainwater tank surveysChapman et al. (2006). Simulated rainfall was applied for a 4–5 day period and then the soil columns were allowed to drynaturally for three days, before the cycle was repeated. Each column cycle represented the equivalent of one year in thePilbara region. Column leachates were collected at the end of each cycle and analysed for TPH concentrations. The acutetoxicity of the collected leachates to aquatic organisms was examined according OECD Method 202 using Daphnia carinataexposed to leachate solution for 48 h to determine the EC50 (OECD, 2004; Liu et al., 2009).

2.2.2. Earthworm ecotoxicity assayThe earthworm studies were conducted following the standard OECD Method 207 (OECD, 1984) & Method 222 (OECD,

2004) using Eisenia fetida as the model organism. Earthworm survival, growth and reproduction were recorded over an 8-week incubation period. A 500 g of soil was weighed in to 1 L glass jars and the soil moisture content adjusted to 40%–60%water holding capacity. Ten adult worms per treatment were depurated for 24–48 h, cleaned and weighed before beingadded to individual jars. The treatments were incubated at 20 °C in a 16 h day/night cycle, and the worms were fed withground oatmeal once a week. After 4 weeks, earthworms were separated, depurated, and weighed before freezing prior toTPH analysis. The incubated jars were maintained for another 4 weeks to study earthworm reproduction. At the end of 8weeks, the number of cocoons and juveniles were counted and validated against the results from the OECD control soil.

2.2.3. Microbial enzyme activityEnzyme activities have often been used as indicators of soil microbial activity and can be useful to interpret the

effect of contaminants on microbial activity in soil (Thavamani et al., 2012b). Soils were mixed with natural carbon andnitrogen sources (OECD, 2000) and incubated at 25 °C for 28 days to revitalize the native microbial communities. Followingincubation, dehydrogenase (Casida et al., 1964) and urease activities (Tabatabai and Bremner, 1972), nitrification (Sauvéet al., 1999) were assayed.

2.2.4. Phytotoxicity assessmentFour plant species were utilized to assess the potential residual TPH phytotoxicity; Golden Beard (Chrysopogon fallax)

grass, Salt Rush (Juncus krausii), Ryegrass (Lolium perenne), and Sunflower (Helianthus annuus). Both Golden Beard and SaltRush are native species endemic to the Pilbara study area in Western Australia. Ryegrass was selected for the study as it isan OECD model plant which is tolerant of base-rich soil conditions and Sunflower as a representative of a dicotyledonousplant species often used in phytotoxicity tests (OECD, 2006). Seedling germination (%) was estimated as a key criterion inthis toxicity assay. All the plant species were grown for 2 months and harvested for the determination of plant biomass(Dry matter production) and the elemental concentration accumulated in the plant tissue. Four selected plant species werealso grown in the agricultural soil collected from South Australian wheat growing district and the same parameters weremeasured. To compare the effect of residual hydrocarbons, growth parameters obtained from the plants experiments in theagricultural soil (sandy loam soils) were treated as control values.

2.2.5. Statistical analysisStatistical analysis were performed using established statistical software tool SPSS version 18.0.0, SPSS, Inc. (SPSS Inc.,

Chicago, USA) to find out whether the residual TPHs have significant (P < 0.001) effect in the stockpile soils in the toxico-logical assays through comparison with the background control soils.

3. Results and discussion

3.1. Characterization of contaminated soils

Total TPH concentrations in the fifty soils analysed ranged from <100–1000 mg kg−1, with a median TPH concentrationof 350mg kg−1. The highly volatile fractions such as the C6–C9 and C10–14were less than the analytically quantifiable limit(10 and 50 ng ul−1 respectively) indicating that these TPH fractions were removed during the land-farming treatment. TheC15–C36 hydrocarbon fractions were the predominant TPH fractions present in the stockpile soils and among these, theC15–C28 fraction ranged from <100 to 420 mg kg−1. These hydrocarbon concentrations are below the regulatory guidelinevalue (data not given).

Themajority of the hydrocarbons present in the stockpile soilswere in the C29–C36 fractionswhich represented between51%–100% of the total TPHs. Currently there is no listed Environmental Screening Level guideline value for this hydrocarbonfraction in the NEPM documents as it is generally accepted that this hydrocarbon fraction poses little toxicity to theenvironment due its poor solubility and mobility (Brassington et al., 2007).

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Table 2Physico-chemical characteristics of six stockpile soils selected for multi species ecotoxicity assessment.

Soils Physical and chemical characteristicsTPH (mg kg−1) pH EC (µS cm−1) Total N (%) Total C (%) H2O extractable elements

(1 : 5 mg L−1)Particledistribution (%)

Ca K Mg Na S Sand Silt Clay

SP1-7 240 7.51 295 0.007 0.273 75 18 58 119 100 46 45 9SP4-7 1020 8.34 619 0.013 0.662 273 26 109 217 410 48 43 9SP1-8 560 8.33 462 0.023 0.618 373 28 142 238 442 46 49 5SP4-8 860 8.12 804 0.030 0.741 265 26 114 232 379 47 48 5SP4-19 980 8.32 628 0.015 0.583 341 24 96 215 452 50 42 8SP4-20 770 8.25 675 0.016 0.513 203 31 65 157 297 45 46 9

3.2. Characterization of the soils selected for toxicity assays

Of the 50 soils screened above, we selected 6 soils varying widely in TPHs concentration for leachability and toxicitystudies. The residual TPH concentrations in the selected soils ranged from 240 to 1020 mg kg−1 which is markedly lowerthan the WA DEC guideline values. In addition to residual TPH concentration, the six soils were also assessed for variousphysico-chemical characteristics (Table 2). All soils were alkaline range with the pH ranging from 7.51 to 8.34. The electricalconductivity (EC) of the 1:5 soilwater extract ranged from295 to 700µS cm−1, indicating that all the soils are highly saline aswell. The EC of the soil SP1-7was considerably lower than that of the other soils. Elevatedwater-extractable (1:5 soil:water)concentrations of calcium (Ca), potassium (K), magnesium (Mg) and sulphate (measured as sulphur (S)) were higher in thefive soil leachates compared to SP1-7. Natural soil fertility is poor with low levels of nitrogen (N) and carbon (C) present(Table 2). Based on particle analysis all soils can be classified as sandy loam.

3.3. Leachate ecotoxicity assessment

3.3.1. TPH concentration in column leachatesThe fate and transport of TPH constituents varies as a function of their individual chemical and physical properties.

The primary route of subsurface movement of TPHs is through leaching and is directly proportional to the solubility andhydrophobicity of the hydrocarbon fractions (Stroo et al., 2000). The TPHs which are more mobile are likely to enter thegroundwater and also exert more toxicity on soil organisms (Cvancarova et al., 2013). To monitor the leachable fractions ofresidual TPHs, we used the site specific rainfall simulated leachability test. Column leaching studies found that leachate TPHconcentrations were highest in first year, ranging from 0.4 mg L−1 (SP1-7) to 1.6 mg L−1 (SP1-8), after which, they rapidlydeclined by approximately 50% in third year as compared to first year (Fig. 1). The elevated TPH leachate concentration infirst year is not unexpected as colloidal transport in repacked soil columns is initiallymuch greater in the initial experimentalperiod compared to those actually observed under field conditions. The elevated TPH concentrations detected in the firstfew years of leaching are associated with colloidal transport of TPH materials. Colloid transport of TPH was not verifiedby direct measurement but a visual inspection of the collection vessels identified a deposition of fine colloidal materialon the bottom of the container. Many studies have observed similar colloidal transport behaviour using repacked soilcolumns (de Jonge et al., 2004; Sharma et al., 2011) and the colloid bound transport of nutrients and contaminants is awell-recognized phenomenon (de Jonge et al., 2004). At the end of the 10 years simulated rainfall, TPH concentrations werereduced to <0.6 mg L−1 for all the stockpile soils studied. Further analysis of the simulated rainfall identified that theC15–C28 comprised >98% of the TPHs present in the leachate. The C15–C28 petroleum hydrocarbons are relatively morewater-soluble than the >C29 TPH fraction (Brassington et al., 2007).

3.3.2. Leachate ecotoxicity study using Daphnia carinataLeachate ecotoxicity test indicated the toxicity ofwater soluble/leachable fractions of hydrocarbons to aquatic organisms.

Daphnia carinata has been widely used and considered to be very sensitive to even lowest concentrations of chemicals(Cáceres et al., 2007). The stockpile soil columns leachates from the second year onwards showed no toxicity on Daphniacarinata survival (Table 3). Daphnia survival was >80% in the first year, rising to >90% in the leachate collected in thefollowing years. As reported above the first year leachate comprised of C15–28 fractions, which are more mobile, but theconcentration was less than 1.6 mg L−1 (SP1-8). The residual fractions above C28 did not leach. Abernethy et al. (1986)reported similar results by studying the acute toxicities of 38 hydrocarbons and chlorinated hydrocarbons on Daphniacarinata, the median lethal concentrations (LC50 values) were strongly correlated with aqueous solubility. Also, Daphniasurvival was less than in the control solution only in first year, which is probably attributed to the much higher salinity ofthe first year simulated rainfall leachate compared to other years. The EC of the simulated rainfall leachate in the first yearranged from 1000–2500µS cm−1 but by second year these leachate EC values had dropped to between 85 and 310µS cm−1.Although values in the range 1000–2500 µS cm−1 are not considered elevated, they are considerably higher than the EC ofthe control solution (<100 µS cm−1) and may have had a minor impact on Daphnia survival in the first year leachate.

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Fig. 1. Concentration of TPHs in simulated rainfall leachate.

Table 3Daphnia carinata survival (%) in simulated rainfall leachate solutions collected from repacked stockpile soil columns.

Control SP1-7 SP4-7 SP1-8 SP4-8 SP4-19 SP4-20

First year 95 80 80 80 90 85 90Second year 95 85 90 90 90 85 90Third year 100 90 95 90 95 95 95Fourth year 95 90 90 95 90 90 95Fifth year 100 95 95 95 90 95 95Tenth year 95 95 95 90 95 95 95

Note: Five animals were used in each vial and mortality calculated against control vials. Control—(MilliQ water:Spring water) 70:30.

3.4. Earthworm ecotoxicity assessment

Earthworm, being a common soil invertebrate, plays an important role in maintaining soil health via decomposition,recycling of nutrients; their sensitivity to pollutants and their survival and reproduction indicates the restoration ofsoil health while tissue analysis indicate the bioavailability of contaminants (Lamb et al., 2013; Duan et al., 2013a).Physico chemical soil characteristics are not sufficient to prove whether soil functions have been re-established after theremediation. Therefore it is important to include reproduction or other sub-lethal tests for earthworms when estimatingthe efficiency of soil restoration/remediation (Shin and Kim, 2001; Geissen et al., 2008). The toxic effects of chemicalson earthworms can be used to determine the limits and the extent of the remediation process, and to construct a risk-based corrective action framework. Toxicity of hydrocarbons on earthworms is not only by ingestion but also throughdermal uptake. So far there is no published study about earthworm toxicity endpoint within the context of risk based framework.

In this study, earthworm survival was markedly impacted by 28 days in all soils except soil SP4-20 (Figs. 2–3). InSP1-7, mortality was 100% but there was marked reduction in numbers in the other soils which are outside the acceptableassessment range (≤10%). The reduced earthworm survival is reflected in the small number of cocoons and juvenilesidentified in the soils compared to the OECD standard soil. The elevated earthwormmortality in all soils except SP4-20 is ofconcern. However, the TPH soil concentrations are low in comparison to other ecotoxicity studies reported elsewhere (Tanget al., 2011). In our study, earthwormmortality and poor reproductionmay be due to soil parameters other than TPH contentsuch as salinity, as the presence of salts causes desiccation of earthworms and eventually their death. To assess the potentialeffects of salinity on earthworm survival and reproduction, soils which had been leached with rainwater were air-dried andutilized in a replicate earthworm study. Earthworm survival, growth and reproduction were all similar to the values forthe OECD model soil (Figs. 2–3). Analysis of leachate water collected during column studies showed a decline in EC fromapproximately 1665 µS cm−1 across all columns to 166 µS cm1. The initial elevated EC of leachate was due to the presenceof calcium, magnesium; sodium and sulphate present in the soils (Fig. 5). Soil TPH concentrations appear to have littleinfluence on earthworm survival and reproduction but EC appears to be responsible for high earthwormmortality and poorreproduction in the initial study. So far there is no report similar to this on salinity interference on worm toxicity. However,some of the published materials available on toxicity of diesel and oil fractions on different species of earthworm indicatethat the concentrations present in our soil are unlikely to pose toxicity (Cermak et al., 2013). For example, Salanitro et al.(1997) reported the LC50 for diesel contaminated soil wasmore than 1% after bioremediation, Similarly, (Hanna andWeaver,2002) reported that 1.2% of highmolecular weight hydrocarbons did not affect the survival and reproduction in earthworms

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Fig. 2. Comparison of earthworm survival at 14 and 28 days in soils before and after leaching.

Fig. 3. Comparison of earthworm reproduction rates in terms of number of (A) cocoons, and (B) juveniles produced in soils at 28 days before and afterleaching.

and in fact some of the bulking agents (Vermiculite and gay grass) used in the bioremediation study interfered with wormsurvival and affected their growth. Even though salinity affected the worm growth, it is not linked to any industrial activityin this situation. Thus, the salinity observed in stockpile soils of this study has been attributed to nature of these soils.

3.5. Microbial enzyme activity

The microbial population is a very sensitive indicator of soil contamination due to the intimate relationship betweensoil micro-organisms and the soil solution. Soil enzymes are the catalyst of importance metabolic processes including thedegradation and detoxification of organic pollutants (Tejada and Masciandaro, 2011). The use of soil enzymes activitiesto determine the extent of soil pollution has proved a successful approach in soil ecological risk assessment as they aresensitive to the impact of soil pollutants. Several of the soil enzymes have been proposed for evaluating and monitoringremediation of hydrocarbon contaminated soils. In this enzyme activity of dehydrogenase and ureases was consideredas indicators of assessing recovery of hydrocarbon impacted soils (Mikkonen, 2012). Enzyme activities in all soils variedmarkedly depending on the enzyme system studied (Fig. 4). Urease and DHA activity were similar to, or higher than in thesoils compared to the SP-1 7 soil which contained the lowest TPH concentration. By contrast, NIF activity was less than thatof SP1-7 in the SP1-8, SP4-19 and SP4-20 soils. In general, nitrifying microbial populations are very sensitive to even smallenvironmental perturbations and also a direct reflection of the poor fertility (low N content) of the soils (Thavamani et al.,2012b). The lowest NIF activity was recorded in SP4-19, where 100% mortality of the earthworms was also observed. Of thethree enzymes activities assessed in the soils, DHA is themost important as it is an indicator of overall soil microbial activityand occur intracellular in all micro-organisms. The marked similarity in DHA activity in the soils tested implies that there islittle impact of TPH concentrations on the soil microbial activity.

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Fig. 4. Soil enzyme activities in stockpile soils (expressed as % of control soil).

Fig. 5. X-RAY diffraction (XRD) analysis of salt crust appeared during phytotoxicity studies in stockpile soils; Note: The salt crusts appeared in top of thepots(inside picture) are shown to be saturated gypsum particles consisting of elevated calcium and sulphur peaks in the diffraction spectrum.

3.6. Plant ecotoxicity assessment

3.6.1. Seed germination assessmentSeed germination of the four test plants is summarized in Table 4. Percentage germinations for Salt Rush and Sunflower

were approximately 100% in all soils but Golden Beardwas considerably less successful andmore variable between the soils,ranging from 28%–80%. It should be noted however, that the germination percentages reported are for soils which have beenleached with rainwater. Slightly lower seed germination rates for Ryegrass, Salt Rush and Golden Beard (Sunflower was nottrialled) were obtained for soils which were not leached.

3.6.2. Plant growth assessmentPlant growth (biomass) was assessed for Ryegrass, Salt Rush and Sunflower plant species grown in the TPH contaminated

soils. Initially, itwas observed that seeds planted in these soilswould germinate successfully but once the germination coverswere removed, the small seedlings failed to establish furtherwhichmay be an indicator of soil phytotoxicity. In all soils, poorseedling establishmentwas associatedwith the development of awhitemineral crust on the soil surface of all pots. Removaland analysis of the crust by X-ray Diffraction spectroscopy (Fig. 5) identified it to be hydrated gypsum (CaSO4 ·2H2O) whichwas probably a result of excessive capillary rise followedby surface evaporation from the pots in the glasshouse environment

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Table 4Germination percentages in the plant ecotoxicity assessment.

Soils Germination (%)Ryegrass Golden Beard Salt Rush Sunflower

SP1-7 90 80 100 100SP1-8 83 60 100 100SP4-7 85 58 100 100SP4-8 70 40 100 100SP4-19 63 45 100 100SP4-20 75 28 100 100

Table 5Dry matter production (DMP) for Ryegrass, Sunflower and Salt Rush.

Soils DMP (%) Percentage change in DMPRyegrass Sunflower Salt Rush Ryegrass Sunflower Salt Rush

SP1-7 19 ± 2b 26 ± 2a 34 ± 3b −44 −27 26SP4-8 27 ± 2b 24 ± 6b 36 ± 3b −21 −31 33SP4-7 45 ± 20a 27 ± 2a 36 ± 2b 34 −23 33SP4-20 12 ± 1b 21 ± 2b 37 ± 3b −64 −40 37SP4-19 13 ± 4b 21 ± 3b 38 ± 3b −61 −40 41SP1-8 24 ± 2b 23 ± 3b 28 ± 5a −30 −34 −33Agricultural soil (control) 34 ± 9a 35 ± 2a 27 ± 2a

Means with different letters within the same column for the same plant are significantly different at P < 0.05.

once the seed germination covers had been removed. Prior to the formation of gypsum, the soil salinity near the surface ofthe plant pot would have increased substantially and it is likely that this was responsible for seedling death. To assess thehypothesis, soils were leached with rainwater for the equivalent rainfall period of 10 years. Leaching of the excess ionsreduced the average stockpile soil EC from 1665 to 165 µS cm−1. Whereas leaching the soils made a marked difference instockpile soil EC, the TPH concentrations were only reduced by between 8% and 12% of the original concentrations in thesoil.

Subsequent plant growth studies were conducted with leached soils and the plant dry matter produced compared toagricultural soil (sandy loam) collected from the northern South Australian wheat growing district. The study identifiedthat plant dry matter production (DMP) varied markedly between the test soils and the sandy loam soil (control soil) aswell as between the different test plant species (Table 5). DMP observed in the sandy loam soil were considered as controlvalues (100%) and compared against the DMP values obtained from the leached soils. Ryegrass and Sunflower DMP wereconsiderably lower than the DMP production in the agricultural soil whereas Salt Rush production was markedly higher.The greater DMP in the more fertile agricultural soil for Ryegrass and Sunflower plants is not unexpected but the SaltRush DMP in the contaminated soils was investigated further and compared to Sunflower DMP. Analysis of the elementalaccumulation in the plant dry matter (DM) identified marked differences in several key elements between the agriculturalsoil for Sunflower and Salt Rush. For example, Na and SO4 are markedly higher in the plants grown for Sunflower in thecontaminated soils compared to the agricultural soil (Table 6). In contrast, Salt Rush contained elevated Na concentrationbut the SO4 concentrations were similar in all the soils studied. Salt Rush, a salt-marsh species, is known to bemore tolerantto soil salinity than Sunflower accounting for the similar DMP in both the contaminated and the agricultural soils.

3.7. Multi-species ecotoxicity assessment as a tool for risk based land management

The primary goal in managing contaminated sites is to render the soil environmentally acceptable throughmanagementor remediation so the site can be used for specified purpose. This goal applies equally to situations where there have beenfresh chemical releases, where the releases have occurred over some time, or where the chemicals have been ‘‘weathered’’for many decades (Duan et al., 2013a). The latter is the situation in our case study; the residual hydrocarbon contaminatedsoils have been stockpiled for several years due to the requirement by regulators to remediate up to the background levels.It has long been recognized that land-farming TPH contaminated soils can remove low molecular weight hydrocarbonsvia volatilization or bioremediation while high molecular weight long-chain hydrocarbon compounds remain in theresidual fraction for long periods. The presence of residual compounds has been attributed to their low bioavailability andinaccessibility of microbes for biodegradation. Bioavailability is the underlying basis for risk-based landmanagement as it isthe bioavailable fraction that induces toxic effects (Naidu et al., 2008, 2013). For many chemical substances, bioavailabilitydecreases with ageing (Vig et al., 2002; Duan et al., 2013a,b). A risk based approach identifies land as contaminated on thebasis of the risk that the contaminants pose to environmental receptors and not merely on the presence of contaminantsof concern (Naidu et al., 2008). Thus, for land to be designated as contaminated a ‘‘significant pollutant linkage’’ must existby the presence of a receptor exposed to a contaminant source by means of a pathway (Catney et al., 2006; Naidu et al.,2008). For successful implementation of risk based approach, a selection of reliable indicators which are directly linked

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Table 6Elemental concentration in Sunflower DM, and Salt Rush DM in contaminated and agricultural soils.

Soil Elemental concentration in (mg kg−1)Na Mg Al P S K Ca

Sunflower DM

SP1-7 8446 ± 1099 7606 ± 1132 378 ± 72 1263 ± 517 2991 ± 941 21738 ± 491 12000 ± 2002SP1-8 6622 ± 1259 8325 ± 856 128 ± 11 908 ± 219 5204 ± 1323 9768 ± 2059 10647 ± 517SP4-7 7621 ± 695 8008 ± 361 394 ± 94 960 ± 147 6197 ± 605 22865 ± 852 16423 ± 501SP4-8 13926 ± 732 10933 ± 39 284 ± 72 1005 ± 117 9453 ± 1241 27237 ± 4269 18597 ± 464SP4-19 5783 ± 481 8265 ± 730 229 ± 13 821 ± 41 4940 ± 136 25722 ± 1190 18630 ± 1035SP4-20 8451 ± 1177 8593 ± 465 341 ± 48 1010 ± 94 7745 ± 406 38195 ± 4135 18792 ± 1094Agricultural soil-control 3869 ± 1909 5430 ± 722 1199 ± 496 1104 ± 74 2896 ± 932 42467 ± 5151 21060 ± 1953

Salt Rush DM

SP1-7 10005 ± 336 3382 ± 185 972 ± 312 724 ± 42 1739 ± 50 8427 ± 197 3403 ± 236SP1-8 8122 ± 1337 2948 ± 350 393 ± 45 609 ± 152 2172 ± 105 13347 ± 1783 3232 ± 198SP4-7 7196 ± 172 2235 ± 95 866 ± 30 800 ± 97 1802 ± 89 12191 ± 1051 2075 ± 115SP4-8 7621 ± 569 2291 ± 56 1315 ± 794 1101 ± 42 2002 ± 240 18136 ± 1992 1986 ± 158SP4-19 9048 ± 106 2142 ± 136 577 ± 205 947 ± 117 1826 ± 78 16603 ± 3601 2464 ± 45SP4-20 7791 ± 533 2357 ± 141 1076 ± 145 1039 ± 88 2336 ± 163 1864 ± 2098 3341 ± 245Agricultural soil-control 2318 ± 234 1272 ± 134 2106 ± 1105 568 ± 93 1385 ± 307 12763 ± 1400 3799 ± 433

Data are mean +SE of triplicate values.

to the particular contaminated site is necessary. Soil is fundamental to the diverse communities of microbes, plants, andinvertebrate and vertebrate animals that comprise the terrestrial ecosystem, and it is important to understand the effectsand risks of contaminated sites in relation to these ecological receptors (Shin and Kim, 2001). The ecotoxicological assaysshow a clear linkwith risks posed by contaminants in soils (Stroo et al., 2000; Cvancarova et al., 2013) therefore, these assaysare invaluable as indicators of risk in risk-based remediation approach.

Using multispecies ecotoxicity assessment we demonstrate no significant risk to environment or human health fromresidual TPH present in stockpile soils. Tests reveal that in the stock pile soils, the low molecular weight TPH’s hadweathered during land farming with the residual TPH being long-chain high molecular weight compounds that are notbioavailable. Hence, a battery of toxicity tests representing different trophic levels such as primary producers (algae, plants)and consumers (daphnia, earthworm, etc.) in addition to leachability tests is proposed as part of the indicators of risk-basedapproach (Duan et al., 2013a). In this study we applied systematic RBLM approach for the stockpile soils using differentindicators (both standard species as well as endemic species) of toxicity as discussed above. This study however, provides avaluable input in to the RBLM concept cautioning remediators to ensure a more comprehensive test of contaminated soilsas seen in the present study that the risk was not from residual TPH rather from salinity that was the result of using salinewater for moisture additions to enhance remediation. Increased understanding on the availability of contaminants in soilenvironment will have considerable benefits for their risk assessment and therefore be very instructive for designing RBLMstrategies for contaminated sites.

4. Conclusion and implications

The presence of large quantities of residual hydrocarbon-contaminated soils presents a valuable opportunity to test theRBLMhypothesis. The field level implementation of risk based landmanagement is not a straight forward lab to fieldmethodand requires careful consideration in case by case scenario. Our case study using the real contaminated soil revealed thechallenges due to the interfering soil chemical propertieswhich can lead to over estimation of toxicity. The results of differentstandard toxicity test employed in this study can be translated into risk based land management decisions. This case studysuggests that cautionmust be exercised when using ecotoxicity tests for risk-basedmanagement of contaminated soils. Theoutcomes from this study which at first glance may not support risk based approach given the general toxicity shown by allindicators. However following recognition of salinity and further tests conducted after leaching the soil demonstrated thatresidual weathered hydrocarbons do not pose risk to the sensitive environmental receptors.

Acknowledgements

This researchwas funded and supported by BHP Billiton Iron Ore (Project 8.1.1) through the Cooperative Research Centrefor Contamination Assessment and Remediation of the Environment (CRC CARE).

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