RESEARCH ARTICLE

Hydroxypropyl-β-cyclodextrin extractability and bioavailabilityof phenanthrene in humin and humic acid fractions from differentsoils and sediments

Huipeng Gao & Jing Ma & Li Xu & Lingyun Jia

Received: 13 December 2013 /Accepted: 24 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Organic matter (OM) plays a vital role in control-ling polycyclic aromatic hydrocarbon (PAH) bioavailability insoils and sediments. In this study, both a hydroxypropyl-β-cyclodextrin (HPCD) extraction test and a biodegradation testwere performed to evaluate the bioavailability of phenan-threne in seven different bulk soil/sediment samples and twoOM components (humin fractions and humic acid (HA) frac-tions) separated from these soils/sediments. Results showedthat both the extent of HPCD-extractable phenanthrene andthe extent of biodegradable phenanthrene in humin fractionwere lower than those in the respective HA fraction and sourcesoil/sediment, demonstrating the limited bioavailability ofphenanthrene in the humin fraction. For the source soils/sediments and the humin fractions, significant inverse rela-tionships were observed between the sorption capacities forphenanthrene and the amounts of HPCD-extractable or bio-degradable phenanthrene (p<0.05), suggesting the impor-tance of the sorption capacity in affecting desorption andbiodegradation of phenanthrene. Strong linear relationshipswere observed between the amount of HPCD-extractablephenanthrene and the amount degraded in both the bulksoils/sediments and the humin fractions, with both slopesclose to 1. On the other hand, in the case of phenanthrenecontained in HA, a poor relationship was observed betweenthe amount of phenanthrene extracted by HPCD and theamount degraded, with the former being much less than thelatter. The results revealed the importance of humin fraction inaffecting the bioavailability of phenanthrene in the bulk soils/sediments, which would deepen our understanding of the

organic matter fractions in affecting desorption and biodegra-dation of organic pollutants and provide theoretical supportfor remediation and risk assessment of contaminated soils andsediments.

Keywords HPCD . Biodegradation . Phenanthrene .

Bioavailability . Humin . Humic acid

Introduction

Polycyclic aromatic hydrocarbons (PAHs) in soils and sedi-ments can pose a substantial risk to microorganism, plants,animals, and humans. An accurate estimation of the bioavail-ability of PAHs would be important for establishing the reme-diation strategy and assessment of their risks (Kördel et al.2013). Due to the strong interactions between PAHs andsoil/sediment matrix, the bioavailability was often decreasedwith the aging time increased (George and Andreas 2006). Asbiodegradation of PAHs is an important process in the biore-mediation of PAHs in soils and sediments, the limited bio-availability of PAHs to microorganisms is also one of thecritical factors affecting their degradation, particularly atlong-term contaminated sites (George and Andreas 2006;Mahmoudi et al. 2013). In general, desorption of PAHs fromsoils or sediments into the aqueous phase is biphasic ortriphasic, whereby a short period of rapid desorption is follow-ed by a longer period of slow desorption or a very slowdesorption (Gomez-Lahoz and Ortega-Calvo 2005). The bio-available PAHs are mainly attributed to the rapidly desorbingfraction from soils or sediments (Rhodes et al. 2010), and theslowly desorbing fraction was less accessible to the degradingmicroorganisms (Li et al. 2009; Scelza et al. 2010).

Organic matter is regarded as the most important compo-nent that controls the behavior of PAHs in soils and sediments(Luthy et al. 1997; Nam et al. 1998). Humic substances (HSs)

Responsible editor: Ester Heath

H. Gao : J. Ma : L. Xu : L. Jia (*)School of Life Science and Biotechnology, Dalian University ofTechnology, No. 2 Linggong Road, Dalian 116024, People’sRepublic of Chinae-mail: lyj81@dlut.edu.cn

Environ Sci Pollut ResDOI 10.1007/s11356-014-2701-6

are the major organic matter content in soils, and they domi-nate the sorption of hydrophobic organic compounds (HOCs)(Perminova et al. 1999). Depending on the extractability indilute base solutions and the solubility in dilute acid solutions,HS can be separated into humic acid (HA), fulvic acid (FA),and humin (Stevenson 1994). The sorption capacity of FA hasseldom been studied as FA is soluble in both acid and basesolutions. The sorption capacity of humin and HA has beeninvestigated by many researchers. Different trends of HA andhumin have been reported with respect to their sorption ca-pacities for PAHs. It was found that the humin fraction exhib-ited a higher sorption capacity for phenanthrene than HAfraction (Bonin and Simpson 2007). Wen et al. (2007) sug-gested that the humin fraction was highly heterogeneous,which was considered to be responsible for the nonlinearsorption of phenanthrene. Pan et al. (2006) found that huminfraction was the main region for slow and nonlinear sorptionof phenanthrene and pyrene after sorption experiment. Thedifferent sorption capacities of organic matter factions wereattributed to their different molecular characteristics such aspolarity (Watanabe et al. 2005), the contents of aromaticcarbon and aliphatic carbon (Xing 2001), as well as thecontent of carbonaceous materials (Cornelissen et al. 2005).However, less study was concentrated on the bioavailability ofPAHs in different organic matter fractions. As the humin andHA fractions exhibited different sorption capacities for PAHs,we assumed that PAHs aged in humin and HA might exhibitdifferent bioavailabilities.

Measuring the bioavailability of PAHs is an important stepfor risk assessment and bioremediation in soils and sediments.Biological assays using earthworms or biodegradability ofPAHs by microorganisms could reflect the bioavailability,but these methods are usually time-consuming (Kördel et al.2013). Thus, a range of techniques have been developed topredict the bioavailability of PAHs, such as extraction ofPAHs by solid phase, chemical solvent, or supercritical fluid(George and Andreas 2006). Hydroxypropyl-β-cyclodextrin(HPCD) had been shown to enhance the solubility of a widerange of HOCs by forming inclusion complexes between itshydrophobic cavity and HOCs (Wang and Brusseau 1993;Wong and Bidleman 2010). It was reported that HPCD couldenhance the mass transfer of the pollutant from soil solidphase into soil solution (Brusseau et al. 1994; Wong andBidleman 2010; Villaverde et al. 2013). The method that usesHPCD extraction to estimate bioavailability of PAH wasfirstly developed by Reid et al. (2000). The study showed a1:1 relationship between the amount of HPCD-extractablephenanthrene and the amount of microbially mineralizedphenanthrene in the soils. It was supposed that the excessamount of added HPCD could extract the rapidly desorbingfraction of phenanthrene from soils and sediments, but not theslowly desorbing fraction (Reid et al. 2000, 2003; Pattersonet al. 2004; Schulze et al. 2012). Even though the extraction of

phenanthrene by HPCD solution has already been tested insoils and sediments, it has been never applied to the humin andHA fractions. We assume that the HPCD extraction methodcould be a useful tool in determining the amounts of rapidlydesorbing phenanthrene in the humin and HA fractions.

In this study, both a HPCD extraction assay and a biodeg-radation assay were performed in the bulk soils/sediments andhumin and HA fractions spiked with phenanthrene, with theaim of determining the rapidly desorbing fraction and biode-gradable fraction of phenanthrene in these samples. The ef-fects exerted by the sorption capacity of the sorbent (the bulksoils/sediments and humin and HA fractions) on HPCD ex-tractability and biodegradability of phenanthrene were alsoexamined.

Materials and methods

Materials

Phenanthrene (>96 %, high-performance liquid chromatogra-phy (HPLC) grade) was purchased from Alfa Aesar. HPCD(purity >99 %) were purchased from Aladdin (China).Methanol, n-hexane, and acetone were of HPLC grade, andall other chemicals were of analytical grade. Milli-Q® water(Millipore, France) was used for the preparation of test solu-tions and reagents.

Soil preparation and fractionation of HS

A total of seven samples which contained varying levels oforganic carbon (1.12–8.80 %) were collected and used for thisstudy. Soil samples 1 and 2 were collected from the farmlandin Jilin (China), soil 3 was collected from the farmland inHeilongjiang (China), and soil 4 was collected from the grass-land in the campus of Dalian University of Technology.Sediment samples 1–3 were collected from Qing River, LiuRiver, and Chai River in Liaoning (China), respectively. Thesoil/sediment samples used in the study have not been pollut-ed by PAHs. Each of the samples was passed through a 10-mm sieve and air-dried. These were then passed through a 2-mm sieve to remove roots and other vascular materials. Thephysical and chemical properties of the samples are listed inTable 1.

The samples were then fractionated into FA, HA, andhumin fractions using the classical base extraction procedure(Salloum et al. 2001). First, each sample was mixed with0.1 M NaOH solution at a sample to NaOH ratio of 1:10under nitrogen. The mixture was allowed to react at 25±2 °C with shaking at 180 rpm for approximately 20 h. Next,the mixture was centrifuged at 4,000×g for 30 min to separatethe solid material from the liquid. The solid material was againmixed with 0.1 M NaOH and extracted as described. This

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extraction was repeated until no more organic material couldbe removed from the solid material. The precipitated solid wasconsidered as humin fraction. All the supernatants obtainedfrom the extraction with NaOH extraction were pooled andacidified with 6 M HCl to a pH<1 and then allowed to standfor 24 h to precipitate the HA fraction. The solid HA wasseparated by centrifugation at 9,000×g for 30 min. The super-natant contained FA fraction. Excess chlorides in humin andHA fractions were removed by repeated rinsing with deion-ized water until chlorides were no longer detected by theaddition of silver nitrate. The humin and HA were freeze-dried and stored at room temperature or further use.

Batch sorption experiment

The bulk soils/sediments and the humin and HA fractionswere each separately weighed into 11-ml vials with Teflon-lined screw caps. Phenanthrene solutions were prepared inmethanol and then diluted with a solution containing 0.01 MCaCl2 and 100 mg l−1 NaN3. The concentration of methanolwas maintained at below 0.1 % (v/v) to avoid the effect ofmethanol on the sorption of phenanthrene (Bonin andSimpson 2007). Ten milliliters of the sample was added intothe vial containing the solid samples. The pH value wasadjusted to 7.0 for soil/sediment and humin samples and 5.0for HA samples. The low pH value (pH 5.0) was used for HAsamples to ensure that the HAwould remain in the solid phase.Preliminary experiments were conducted to estimate the timeneeded to reach apparent equilibrium between the solid phaseand the aqueous phase. It has been reported that if the con-centration of phenanthrene remaining in the aqueous phase istoo high or too low (after equilibrium), the error associatedwith the determination of sorption isotherm would increase(Bonin and Simpson 2007). In order to minimize the error, theexperiments were conducted to ensure that the final concen-tration of phenanthrene in the aqueous phase remained in the

range of 20–80% of the total phenanthrene concentration afterattainment of the apparent equilibrium. Sorption isotherm wasdetermined at seven different concentrations of phenanthrene(ranging from 0.05 to 1.0 mg l−1), each in triplicate. The vialswere rotated vertically at 30 rpm for 48 h at room temperaturesince preliminary tests indicated that the apparent equilibriumbetween the solid and aqueous phases at each phenanthreneconcentration was reached before 36 h. After equilibrium, thevials were centrifuged at 1,000×g for 20 min, and then, 2-mlaliquot was removed from the supernatant of each vial andtransferred into separate 2-ml amber vials. The concentrationsof phenanthrene in the vials were analyzed using HPLC.Controls without addition of solid samples were run in tripli-cate. No phenanthrene was sorbed by the wall of the test tubeor HPLC vial.

The data obtained from the batch sorption experiment wasfitted with Freundlich model

logCs ¼ logKd þ N logCe ð1Þ

where Cs is the concentration of solute sorbed on solid sorbent(mg kg−1), Ce is the concentration of solute in the aqueousphase, Kd is the sorption coefficient, and N is a factor thatdescribes the site energy heterogeneity and the nonlinearity ofthe isotherm. Organic carbon-normalized distribution coeffi-cient (Koc) could be calculated as

Koc ¼ 100Kd= f oc% ð2Þ

where foc is the total organic carbon content of the solidsorbent.

Soil spiking and aging

The bulk soils/sediments and the humin and HA fractions(each 5 g) were separately transfer into a 20-ml brown bottle.Each sample was added with phenanthrene that had beendissolved in hexane, and the sample was allowed to evaporate

Table 1 Physicochemical properties of soils/sediments

Soil/sediment Organic carbon analyses pH Particle size (%)

foc (%)a FHA (%)b Fhumin (%)c Sand Silt Clay

Soil 1 2.57 25.08 30.56 5.37 14.6 52.0 33.4

Soil 2 8.80 17.99 15.32 5.23 18.6 48.9 32.6

Soil 3 3.66 25.07 49.72 5.86 21.5 58.7 19.7

Soil 4 2.71 27.16 34.55 6.39 35.3 44.4 20.3

Sediment 1 1.12 19.04 43.40 6.19 12.4 78.7 8.9

Sediment 2 1.35 8.87 48.18 7.33 10.7 80.0 9.3

Sediment 3 4.02 27.08 60.31 4.65 36.0 60.0 4.0

a The total organic carbon content of soil/sedimentb Percentage of organic carbon content of HA to the total organic carbonc Percentage of organic carbon content of humin to the total organic carbon

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by leaving the vial open for 24 h. Deionized water was thenadded to each sample to give 30 % water content. To comparethe differences in the amounts of biodegradable and HPCD-extractable phenanthrene among different samples, theamount of phenanthrene spiked in each sample waspredetermined to ensure that they all have the same concen-tration of phenanthrene per unit organic carbon (OC). Thefinal concentration of phenanthrene in each sample (the bulksoil/sediment, humin fraction, or HA fraction) was 50mg kg−1

(100foc)−1. The samples were stored at 25±2 °C in the dark

and aged for 20 days. After 20 days of aging, analysis of theconcentrations of phenanthrene in each sample was also con-ducted. The result showed no significant loss of phenanthrene(less than 2 %). All the samples prepared were subjected toextraction by HPCD and biodegradation test.

Extraction of phenanthrene by HPCD solution

All the bulk soils/sediments and the humin and HA fractionsaged for 20 days were subjected to extraction by HPCDsolution. The HPCD concentration must be kept high enoughto enable desorption of all readily bioavailable phenanthrene.The successively use of the HPCD extraction method toestimate bioavailability of phenanthrene has been reportedby many researchers (Reid et al. 2000, 2003). According tothese studies, a mass ratio of HPCD to OC at 20:1 was enoughto estimate all the readily desorbed fractions of phenanthrene.A single extraction by HPCD was used as it has been reportedthat one single extraction by HPCD was enough to get a goodestimation of bioavailability of phenanthrene in soils (Reidet al. 2000, 2002). The mass of sample in each bottle waspredetermined to ensure that all samples have the same levelof organic carbon (0.025 g OC). Briefly, the bulk soils/sediments and the humin and HA fractions containing0.025 g OC were each weighed into separate 11-ml amberbottles (three bottles for each), and 10 ml of 50 mM HPCDsolution was subsequently added to each bottle. Each bottlewas sealed and placed on an orbital shaker and shaken at 25±2 °C and 30 rpm for 24 h. After extraction, the bottles werecentrifuged at 3,000×g for 1 h, and 2ml of the supernatant wastaken from each vial and subjected to HPLC to determine theconcentration of phenanthrene.

Biodegradation

Pseudomonas pseudoalcaligenes strain JM2 was used as thephenanthrene degrader. It was isolated from a sludge samplecollected in a wastewater treatment factory in the northeasternregion of China. It can utilize phenanthrene as a sole carbonsource, and it also has the capacity to degrade low molecularweight PAHs in soil (Ma et al. 2012). Prior to each experi-ment, P. pseudoalcaligenes strain JM2 was grown in 250-mlErlenmeyer flasks, each containing 100 ml of mineral salt

medium (MSM) (pH 7.0) supplemented with 200 mg phen-anthrene as the carbon source. The composition of MSMmedium contained per liter the following: 4.4 g K2HPO4,1.7 g KH2PO4, 2.1 g NH4Cl, 3.0 g NaCl, 0.05 g yeast extract,0.195 g MgSO4, 0.05 g MnSO4·H2O, 0.01 g FeSO4·H2O, and0.003 g CaCl2·H2O. The cultures were incubated at 30 °Cwith shaking at 180 rpm. Cultures in the late exponentialphase of growth were collected and used in the degradationexperiment. The cultures were first passed through a glass fritto remove the phenanthrene crystals remaining in the solution.The cells were separated by centrifugation at 4,000×g for5 min and washed with MSM. This process was repeatedtwo times, and the cells were finally resuspended in MSM.The cell density of the sample was adjusted to 1.0×107 cfu ml−1 before it was used in the biodegradationexperiment.

Biodegradation experiments were carried out bymeasuringphenanthrene disappearance in closed test systems. All thebulk soils/sediments and the humin and HA fractions aged for20 days were subjected to the biodegradation test. Briefly, thehumin and HA fractions and the bulk soils/sediments contain-ing 0.025 g OC were each weighed into separate 11-ml amberbottles (three bottles for each). The mass of sample in eachbottle was the same as the mass used in the HPCD extractionassay in “Extraction of phenanthrene by HPCD solution”. Thesamples were each inoculated with 10 ml of MSM culture thatwas prepared by incubating the microbes in MSM with phen-anthrene as the sole carbon source. The pH value was adjustedto 7.0 for the soils/sediments and humin fractions and 5.0 forHA fractions. The preliminary test showed that the biodegra-dation of phenanthrene was not affected when the low pHvalue (pH 5.0) was in use. MSM plus phenanthrene incubatedwithout the microbes was used as a control. It was demon-strated that less than 5 % of the total phenanthrene was lostduring the whole biodegradation period for each of the solidmatrixes. All the bottles were incubated on a rotary tumbler at30 rpm and 25±2 °C. To retain the activity of the aerobicbacteria, during the course of biodegradation, the headspaceoxygen content was maintained by flushing with air for 5 minin laminar flow cabinet at 48-h intervals. In this study, a periodof 14 days was sufficient to remove the rapidly desorbing(readily bioavailable) PAHs. After 14 days, triplicate sampleswere sacrificed to determine the amount of phenanthreneremaining in the samples.

Chemical extraction and analysis

The residual phenanthrene concentrations after biodegrada-tion test were analyzed. After biodegradation, the bottles werefirst centrifuged at 4,000×g for 30 min, and the supernatantswere removed. Two milliliters of the supernatant was extract-ed with 4 ml hexane in an 11-ml amber bottle by vortexing for5 min. After standing for 30 min, 1.0 ml of the hexane phase

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was transferred into a sample vial, and the residual concentra-tion of phenanthrene in the aqueous phase was then deter-mined by HPLC. The solid residues in the bottles were ex-tracted three times with 8 ml of a hexane-acetone mixture (1:1v/v). Each extraction was conducted by incubating the bottleson a rotary tumbler at 30 rpm for 24 h. After the finalextraction, the extracts were combined, filtered, and concen-trated using a rotary evaporator. The concentrate was trans-ferred into a 2-ml sample vial and further concentrated underN2 flow and then re-dissolved in 1 ml of hexane and subjectedto HPLC analysis to determine the concentration ofphenanthrene.

The concentration of phenanthrene was analyzed using anAgilent 1200 HPLC (Agilent, CA, USA) fitted with anautosampler and a UV detector. The wavelength used fordetecting phenanthrene was 254 nm. HPLC was operated ata flow rate of 1.0 ml min−1 and a mobile phase of 15 % waterand 85 %methanol. A reversed phase C18 column ZORBAXEclipse Plus C18, containing 5-μm particles, was used. Thetemperature of the column was maintained at 25 °C.Quantification of the desired peak of phenanthrene wasachieved using standard solutions. The detection limit ofphenanthrene using these parameters was found to be10 μg l−1.

Statistical analysis

Statistical analysis of the results was performed using SPSSStatistics 18. Comparisons between HPCD extractability andbiodegradation were performed using Student’s t test (p=0.05) and linear regression modeling.

Results and discussion

Phenanthrene sorption isotherm and coefficients

The organic carbon contents of the seven soil/sediment sam-ples ranged from 1.12 to 8.80 % (Table 1). As shown inTable 2, the HA fractions had higher organic contents (11.46to 33.95 %) than the corresponding soil/sediment samples,whereas the humin fractions had the lowest organic contents(0.564 to 2.691). Percentage of organic carbon content ofhumin or HA to the total organic carbon content ofsoil/sediment (Fhumin or FHA) was also calculated by evaluat-ing the dry weight of HA and humin after NaOH extraction(Table 1). While soil sample 2 exhibited no differences be-tween Fhumin and FHA, Fhumin of the other six soil/sedimentsamples was higher than the corresponding FHA.

The sorption isotherms of phenanthrene to bulk soils/sediments, humin fractions, and HA fractions wereestablished, and the calculated parameters (N and Koc) arelisted in Table 2. Data obtained from the batch sorption

experiment fitted well to Freundlich equation with high r2

values (r2>0.9138). The sorption isotherms of phenanthreneto the bulk soils/sediments and the humin and HA fractionswere nonlinear (N<1). The N values of HA ranged from0.8775 to 0.9920, which were significantly (p<0.05) higherthan the respective values of the bulk soils/sediments, whilethe N values of humin fractions were lower than the respectivevalues of the bulk soils/sediments. As N values can representthe heterogeneous sorption sites of the sorbent (Weber et al.1992;Wang et al. 2011), the relatively lowerN value of huminfraction suggested that the composition of humin was moreheterogeneous than that of HA and the whole soil/sediment.The result was consistent with previous reports, which sug-gested that humin fraction generally produces higher nonlin-earity (higher N values) than HA fraction during the sorptionof PAHs (Bonin and Simpson 2007; Wen et al. 2007; Wanget al. 2011).

TheKoc value, representing the sorption coefficient per unitorganic carbon, is often used to describe the sorption capaci-ties of different sorbents (Weber et al. 1992). While the Koc

values of source soil/sediment samples varied with a rangefrom 10,063 to 15,986 (Table 2), the Koc values of humin orHA fractions from different soils/sediments also varied(13,661–28,814 for humin fractions and 9,037–19,794 for

Table 2 Freundlich isotherm parameters of the bulk soils/sediments andthe humin and HA fractions for phenanthrene

Sample foc Kd (ml g−1) Koc (ml g−1)a N r2b

Soil 1 2.570 363 14,124 0.9559 0.9834

Humin 0.849 140 16,443 0.6785 0.9912

HA 22.95 2,074 9,037 0.9525 0.9982

Soil 2 8.800 886 10,063 0.8925 0.9950

Humin 2.119 289 13,661 0.8272 0.9724

HA 32.03 2,430 7,587 0.9693 0.8949

Soil 3 3.664 547 14,527 0.7198 0.9922

Humin 1.767 221 28,814 0.7154 0.9884

HA 11.46 1,881 15,517 0.8775 0.9907

Soil 4 2.710 433 15,986 0.8431 0.9911

Humin 1.073 216 20,156 0.7971 0.9138

HA 33.95 3,805 11,206 0.9789 0.9810

Sediment 1 1.115 144 12,520 0.8619 0.9878

Humin 0.564 113 20,059 0.7817 0.9773

HA 19.11 1,910 9,997 0.9379 0.9957

Sediment 2 1.447 198 13,713 0.8090 0.9715

Humin 0.830 178 21,463 0.7710 0.9962

HA 31.99 4,728 14,783 0.9563 0.9995

Sediment 3 4.017 543 13,524 0.7988 0.9961

Humin 2.619 392 24,212 0.8547 0.9980

HA 30.66 6,069 19,794 0.9920 0.9982

aOrganic carbon-normalized distribution coefficients Koc=100Kd/foc%bCorrelation coefficient of Freundlich model fitting for phenanthrene

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HA fractions). Differences inKoc values exhibited by differentsamples suggested that the sorption capacities for phenan-threne were different among the soils/sediments collectedfrom different areas, which contained different chemical com-positions and physical conformations of sorbent organic mat-ter (SOM) (Watanabe et al. 2005). Meanwhile, for each typeof soil/sediment sample, the Koc value of humin was generallyhigher than the corresponding values of source soil/sedimentsample and HA. Except for the humin from soil samples 1 and2, the Koc values of the other five humin samples were higherthan 20,000. The high sorption capacity of humin has alsobeen reported by earlier studies (Pan et al. 2006; Bonin andSimpson 2007;Wang et al. 2011). They proposed that the highsorption coefficients for humin were due to the high aroma-ticity or aliphaticity of the organic carbon (Ran et al. 2007;Sun et al. 2008;Wang et al. 2011). Furthermore, black carbon,which has been suggested to be a sorbent with high sorptioncapacity for PAHs in soils and sediments, might also presentin the humin fraction (George and Andreas 2006). Since alarge percentage of the total organic carbon is taken by humin(Table 1), humin would play a major role in affecting thesorption capacity of the bulk soil/sediment samples.

Extraction of phenanthrene from the bulk soils/sedimentsand the humin and HA fractions by HPCD solution

In this study, the amounts of phenanthrene that were extract-able by HPCD solution in the bulk soils/sediments and thehumin and HA fractions after 20 days of aging were deter-mined. As shown in Table 3 and Fig. 1(a–c), the amounts ofHPCD-extractable phenanthrene in humin or HA fractionsfrom different source soil/sediment samples varied, and therewas a significant (p<0.05) positive relationship between theamounts of HPCD-extractable phenanthrene in humin or HAfractions and the amounts in their respective source soils/sediments. The similar trend in HPCD extractability betweenthe HA or humin fraction and their respective source

soil/sediment demonstrated that both the humin fraction andthe HA fraction affect the rapidly desorbing fraction of phen-anthrene in the source soils/sediments. In comparing theamount of HPCD-extractable phenanthrene in humin fractionwith that in the respective source soil/sediment, the amount ofHPCD-extractable phenanthrene in humin fraction from soilsample 2 was not significantly different from the amount ofHPCD-extractable phenanthrene in the respective sourcesoil/sediment (p>0.05), but for humin fraction from each ofthe other six soil/sediment samples, HPCD extractability wassignificantly (p<0.05) lower than that in the correspondingsource of soils/sediments. In comparing the amount of HPCD-extractable phenanthrene in humin fraction with that in therespective HA fraction, it can be seen that for soil sample 1and sediment samples 1–3, the amount of HPCD-extractablephenanthrene in humin fraction was significantly (p<0.05)lower than that in the respective HA fraction, and for soilsamples 2–4, the difference in extractability was not signifi-cant (p>0.05).

The relatively lower amounts of HPCD-extractable phen-anthrene in the humin fractions demonstrated that desorptionof phenanthrene in humin was more limited. It was reportedthat the HPCD-extractable phenanthrene was associated withthe OC contents of soils (Reid et al. 2000; Rhodes et al. 2008a,b). However, the masses of both the bulk soils/sediments andthe humin and HA fractions used in this assay have the samelevel of organic carbon. Thus, differences in the amount ofHPCD-extractable phenanthrene might be connected with theproperties of unit organic matter from different organic matterfractions. As indicated in the phenanthrene sorption experi-ment, humin fractions exhibited higher Koc values and non-linearity (lower N values) than the respective values of HAand the bulk soils/sediments. It was indicated that the highersorption capacity of humin could be an important factor thatresults in the lower rapidly desorbing fraction and, therefore,the lower amount of HPCD-extractable phenanthrene inhumin, compared to that in HA.

Table 3 Extent of biodegradable phenanthrene and HPCD-extractable phenanthrene in the bulk soils/sediments and the humin and HA fractions

Soil/sediment Biodegradation (%) HPCD extraction (%)

Soil/sediment Humin HA Soil/sediment Humin HA

Soil 1 80.02±2.21Aaab 74.79±0.89Ab 98.81±1.53Ac 79.43±1.72Aa 75.71±0.55Ab 89.71±1.15Ad

Soil 2 94.29±1.25Ba 90.73±1.92Ba 98.10±2.50Aa 95.43±1.69Ba 92.27±1.58Ba 94.29±0.86Ba

Soil 3 75.03±1.65Ca 65.99±2.88Cb 92.87±2.90Bc 74.69±1.33Ca 67.43±0.85Cb 69.43±2.13Cb

Soil 4 75.98±1.73Ca 73.13±1.68Aa 97.38±1.33Ab 77.86±2.25Aa 70.29±2.35Cc 72.86±1.52Ca

Sediment 1 85.25±2.53Aa 69.80±2.55Ab 98.10±1.68Ac 87.59±1.60 Da 70.54±2.20Cb 88.11±0.91Ad

Sediment 2 83.35±2.19Aa 64.57±3.15Cb 98.81±1.18Ac 85.39±0.65 Da 68.38±1.25Cb 96.76±1.49Bc

Sediment 3 78.60±2.95Aa 67.18±2.46Cb 99.29±0.98Ac 79.51±0.85Aa 68.92±1.67Cb 75.41±1.19Cd

a Comparison between values within the same column; the same letter represents no significant difference (p>0.05)b Comparison between values within the same row; the same letter represents no significant difference (p>0.05)

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The correlation analysis was conducted between phenan-threne Koc value and amount of HPCD-extractable phenan-threne in the bulk soils/sediments, humin fractions, and HAfractions (Fig. 1(a–c)). There was a significant inverse rela-tionship between Koc value and HPCD-extractable phenan-threne in the bulk soils/sediments and the humin fractions(p<0.05; Fig. 1(a, b)). That demonstrated sorption capacityof the humin fractions, and the bulk soils/sediments for phen-anthrene had a significant impact on the HPCD extractability.While the regression model for the bulk soils/sediments waslinear, the regression for the humin fractions were more fittedwith the exponential decay model. There was a steep increasein the amount of HPCD-extractable phenanthrene for Koc

values of humin fractions lower than 16,000. In contrast, thisrelationship was not significant for HA fractions (p>0.05;Fig. 1(c)), which demonstrated that the ability of HPCD toextract phenanthrene from HA fractions was not significantlyinfluenced by the sorption capacity.

Biodegradation of phenanthrene in the bulk soils/sedimentsand the humin and HA fractions

The biodegradation of phenanthrene in the bulk soils/sediments and the humin and HA fractions was examinedt h r o ug h a 1 4 - d a y b i o d e g r a d a t i o n t e s t u s i n gP. pseudoalcaligenes strain JM2 as the phenanthrene degrader.

Fig. 1 Comparison of organic carbon-normalized sorption coefficient(Koc) of phenanthrene with extent of HPCD-extractable phenanthrene inbulk soils/sediments (a), humin fractions (b), and HA fractions (c) at50 mg kg−1 (100foc)

−1 aged for 20 days. Comparison ofKoc with extent ofbiodegradable phenanthrene in bulk soils/sediments (d), humin fractions(e), and HA fractions (f). Comparison of extent of HPCD-extractable

phenanthrene with the total extent of biodegradable phenanthrene in bulksoils/sediments (g), humin fractions (h), HA fractions (i). Solid linesrepresent the regression line for the raw data. Dashed lines in g, h, and irepresent the reference lines y=x. Error bars represent the deviation fromthe mean of triplicate samples

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With respect to the quantitative method, the recoveries of thephenanthrene extraction method obtained from all the bulksoils/sediments and humin fractions were ≥85 %, and therecoveries from the HA fractions were ≥90 %. For the huminfractions, the extent of phenanthrene degraded by JM2 rangedfrom 64.57 to 90.73 % (Table 3). The amount of biodegrad-able phenanthrene in the humin fraction was significantly(p<0.05) lower than that in the respective sourcesoil/sediment and HA fraction. As for HA, more than 95 %phenanthrene was degraded by JM2 except for the HA frac-tion from soil sample 3, in which only 92.87 % of phenan-threne was degraded. The extent of phenanthrene biodegradedin the HA fraction was significantly (p<0.05) higher than thatin the respective humin fraction, especially for sediment sam-ples 1–3, in which the extent of phenanthrene biodegraded inHA fraction was at least 20 % more than that in the respectivehumin fraction. The results were consistent with higher sorp-tion capacity (Koc) and nonlinearity (N) for the humin fractionthan those for the respective source soil/sediment and HAfraction, and this demonstrated that the sorption capacitymight also be an important factor that cause the differentamounts of biodegradable phenanthrene among the sourcesoil/sediment and the humin and HA fraction.

The correlation analysis was conducted between phenan-threneKoc value and the extent of biodegradable phenanthrenein the bulk soils/sediments and the humin and HA fractions(Fig. 1(d–f)). The results showed that there was a significant(p<0.05) inverse relationship between the Koc value and theamount of biodegradable phenanthrene in the bulk soils/sediments and the humin fractions (Fig. 1(a, b)). The resultsdemonstrated that the sorption capacity of the humin fractionsor the bulk soils/sediments play an important role on thebioavailability of phenanthrene. That was consistent with theprevious studies that found that samples with high sorptioncapacity for organic contaminants could result in the lowbioavailability (Bogan and Sullivan 2003; Yang et al. 2009).Like the relationship between HPCD extractability and Koc

values, the extent of biodegradation of phenanthrene forhumin fractions also exhibited an exponential decay withincreasing Koc values. There was a steep increase of the extentof biodegradation when Koc values were lower than 16,000.That may indicate that, for humin fractions with sorptioncapacity reduced to a certain degree, the aging effect whichcauses the hysteresis between sorption and desorption weak-ened dramatically. A threshold value of Koc may exist forhumin fractions to fully gain ability in limiting the bioavail-ability of phenanthrene. Meanwhile, no significant relation-ship was observed in the case of the HA fractions (p>0.05;Fig. 1(f)), and although the Koc value for phenanthrene variedamong different HA samples, the degradation of phenanthrenein these HA samples was all above 90 %. This suggested thatphenanthrene aged in the HA fractions was more easily bio-degradable than phenanthrene in the humin fractions and the

bulk soils/sediments, and the Koc value could not have asignificant effect on the bioavailability of phenanthrene inthe HA fractions.

Comparison of amount of biodegradable phenanthrenewith amount of HPCD-extractable phenanthrene in the bulksoils/sediments and the humin and HA fractions

The correlation between the HPCD-extractable and biode-gradable phenanthrene was examined in the bulk soils/sediments and the humin and HA fractions (Fig. 1(g–i)).There was a significant linear relationship between the amountof HPCD-extractable phenanthrene and the amount of biode-gradable phenanthrene in the bulk soils/sediments (r2=0.984;slope of best fit line=0.95; intercept=2.97; p<0.01; Fig. 1(g)).The slope of the best fit line was also close to 1. The nearly 1:1relationship demonstrates that HPCD extraction techniquecould accurately predict the phenanthrene biodegradation indifferent types of soils/sediments in this study. This result wasconsistent with the previous results which showed that HPCDextraction techniques could accurately evaluate the bioavail-ability of phenanthrene in both laboratory-spiked and field-contaminated soils (Reid et al. 2000; Doick et al. 2005, 2006;Rhodes et al. 2008a, b). Meanwhile, a significant linear rela-tionship between the amount of HPCD-extractable phenan-threne and the amount of biodegradable phenanthrene in thehumin fractions was obtained (r2=0.935; slope of best fit line=0.94; intercept=3.68; p<0.01; Fig. 1(h)). The ratio of HPCD-extractable phenanthrene to biodegradable phenanthrene in thehumin fractions was approximately 1:1. The nearly 1:1 ratiodemonstrated that the rapidly desorbing fraction of phenan-threne determined by HPCD extraction was directly related tothe biodegradable fraction in the humin fractions.

In contrast, an insignificant relationship was found betweenthe amount of HPCD-extractable phenanthrene and theamount of biodegradable phenanthrene in HA (r2=0.101;slope of best fit line=0.037; intercept=95.35; p>0.05;Fig. 1(i)). The result demonstrated that the rapidly desorbedfraction determined by HPCD extraction could not accuratelypredict the biodegradable fraction of phenanthrene in HA. Asseen from Fig. 1(b), in the cases of HAs from soil sample 2and sediment sample 2, the amount of phenanthrene extractedby HPCD (94.29 and 96.76 %) was not significantly differentfrom the extent of its biodegradation (98.10 and 98.81 %).However, as for the HA fractions from the other five soil orsediment samples, the amount of biodegradable phenanthrenewas significantly higher (p<0.05) than the amount of HPCD-extractable phenanthrene. This suggested that JM2 degradedthe phenanthrene in the HA fractions to a much greater extentthan estimated by the HPCD extraction, and a portion of thebiodegradable phenanthrene in HA could not be extracted byHPCD. Even though many investigations have demonstratedthat only phenanthrene that exists in the free and dissolved

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form in the aqueous phase could be degraded by microorgan-isms, there have been a few investigations, which showed thatphenanthrene that exists in the sorbed form was also biode-gradable (Laor et al. 1999; Vacca et al. 2005; Smith et al.2009). Laor et al. (1999) reported that phenanthrene mineral-ization by Pseudomonas sp. enriched from the coal tar-contaminated soil was enhanced upon sorption to mineral-HA complexes, and the degree of enhancement was positivelycorrelated with the fraction of sorbed phenanthrene. Vaccaet al. (2005) has shown that some competent strains maydirectly access phenanthrene sorbed by the HA, such thatphenanthrene sorbed by HA is bioavailable. Smith et al.(2009) had also reported that the presence of HA could in-creased the rates of phenanthrene degradation bySphingomonas sp. LH162. On the other hand, HPCD couldonly extract phenanthrene that desorbed from the solid phaseinto the aqueous phase (Reid et al. 2000). Therefore, asdescribed above, a possible explanation of the result in thepresent study was that sorbed phenanthrene in HA might bedirectly degraded by JM2, and this could cause a portion ofsorbed phenanthrene to be still bioavailable apart from theHPCD-extractable portion.

As described above, the 1:1 relationship between amountsof HPCD-extractable phenanthrene and amounts of biode-gradable phenanthrene in both the bulk soils/sediments andthe humin fractions indicated the importance of humin frac-tion in affecting the bioavailability of phenanthrene in the bulksoils/sediments. Despite of the existence of HA fraction thatcould lead to a higher amount of biodegradable phenanthrenethan the amount of rapidly desorbing phenanthrene, consider-ing that phenanthrene present in soils/sediments has under-gone 20 days of aging, the phenanthrene weakly sorbed byHA could gradually transfer to the stronger sorption sites inhumin. Compared with long-term contaminated sites, 20 daysof aging time used in the present study was undoubtedly ashort aging time. Even so, the importance of humin fractionsin affecting the bioavailability of the bulk soils/sediments hasbeen demonstrated for samples with the 20 days of aging, andwe think that, as the aging time increased, more amounts ofphenanthrene would be transferred into the strong sorptionsites in humin fractions. Nam and Kim (2002) reported thatcontaminants were initially partitioned into the HA and FApolymer layers at the surface of soil particles, followed bydiffusion into micropores in the humin core of the particles.On the other hand, the percentage of organic carbon content ofHA to the total organic carbon content of soil/sediment (FHA)was significantly lower than that of the respective humin(Fhumin) among all the soil/sediment samples in this experi-ment, which also supported the finding that the humin frac-tions played a bigger role in controlling the bioavailability ofphenanthrene in the bulk soils/sediments. This work is limitedin scope to only phenanthrene with a relatively short agingtime. As the HPCD extraction method has been suggested to

successfully estimate the bioavailability of other PAHs andeven a mixture of PAHs and aliphatic hydrocarbon in a rangeof soils/sediments (Allan et al. 2006; Papadopoulos et al.2007a, b; Stroud et al. 2009; Hickman et al. 2008; Bernhardtet al. 2013), further works could be performed to providemuch broader application of the method used in this study todifferent PAHs and a range of soils/sediments with differentaging times. By evaluating the sorption capacity and contentof humin fraction in different soils, we could possibly assessthe difficulty of remediation and the risk of the contaminants.In addition, for contaminated soils/sediments which have ahigh HA content or sites with a heavy pollution, the effect ofHA on the bioavailability of phenanthrene might not be ig-nored. Given this, further research should be undertaken tosoils with high HA content and the highly contaminatedindustrial sites to further conform the relative contribution ofhumin and HA fractions on the bioavailability of the bulksoils/sediments.

Conclusion

In this study, the amounts of HPCD-extractable and biode-gradable phenanthrene in both the bulk soils/sediments andthe humin and HA fractions were examined. The extent ofHPCD-extractable or biodegradable phenanthrene in huminfraction was lower than that in the respective HA fraction andbulk soil/sediment, demonstrating the relatively lower rapidlydesorbing fraction and bioavailability of phenanthrene in thehumin fraction. For the bulk soils/sediments and the huminfractions, a significant inverse relationship was observed be-tween the Koc values and the amounts of HPCD-extractable orbiodegradable phenanthrene, demonstrating the importance ofthe sorption capacity in affecting the rapidly desorbing frac-tion and biodegradation of phenanthrene. Finally, the biodeg-radation of phenanthrene in the bulk soils/sediments and thehumin and HA fractions was compared with its extraction byHPCD. For the bulk soils/sediments and the humin fractions, astrong linear relationship was observed between the amount ofphenanthrene degraded and the amount extracted by HPCD,with the slope close to 1. On the other hand, in the case ofphenanthrene contained in HA, a poor relationship was ob-served between the amount of phenanthrene extracted byHPCD and the amount degraded, with the former being muchless than the latter. It was assumed that phenanthrene existedin some sorbed forms in HA might be directly degraded. The1:1 relationship between amounts of HPCD-extractable phen-anthrene and amounts of biodegradable phenanthrene in boththe bulk soils/sediments and the humin fractions may indicatethe importance of humin fraction in affecting the bioavailabil-ity of phenanthrene in the bulk soils/sediments. The resultswould deepen our understanding of the organic matter frac-tions in affecting desorption and biodegradation of organic

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pollutants and provide theoretical support for remediation andrisk assessment of contaminated soils and sediments.

Acknowledgments This work was supported by the National NaturalScience Foundation of China (Grant No. 21277021).

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