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Journal of Environmental Science and Health, PartA: Toxic/Hazardous Substances and EnvironmentalEngineeringPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/lesa20

Changes of solid phase toxicity during sewage sludgecomposting in relation to bioavailability of polycyclicaromatic hydrocarbonsPatryk Oleszczuk aa Institute of Soil Science and Environmental Management, University of Agriculture , Lublin,PolandPublished online: 02 Jan 2009.

To cite this article: Patryk Oleszczuk (2009) Changes of solid phase toxicity during sewage sludge composting in relation tobioavailability of polycyclic aromatic hydrocarbons, Journal of Environmental Science and Health, Part A: Toxic/HazardousSubstances and Environmental Engineering, 44:2, 137-145, DOI: 10.1080/10934520802539681

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Journal of Environmental Science and Health Part A (2008) 44, 137–145Copyright C© Taylor & Francis Group, LLCISSN: 1093-4529 (Print); 1532-4117 (Online)DOI: 10.1080/10934520802539681

Changes of solid phase toxicity during sewage sludgecomposting in relation to bioavailability of polycyclicaromatic hydrocarbons

PATRYK OLESZCZUK

Institute of Soil Science and Environmental Management, University of Agriculture, Lublin, Poland

The aim of the present study was to determine the content of the bioaccessible fraction of polycyclic aromatic hydrocarbons (PAHs) insewage sludges and composts by means of three techniques (solid phase extraction with Tenax-TA and non-exhaustive extraction withhydroxypropyl[β]cyclodextrin and n-butanol) and at the same time to establish their influence on toxicity for Heterocypris incongruens(OstracodtoxkitTM test) and Lepidium sativum (PhytotoxkitTM test). In the majority of cases sewage sludges negatively influencedon organisms. Generally, sewage sludge composting exerted a positive influence on phytotoxicity, whereas in the case of ecotoxicitya negative effect was noted. The content of the potentially bioaccessible PAHs fraction varied depending on the method applied.Composting usually lowered the content of the potentially bioaccessible fraction. Significant positive relationships were observedmainly between Tenax-TA extracted fraction of individual PAHs and growth inhibition of H. incongruens. Only negative correlationswere noted in the case of phytotoxicity.

Keywords: Toxicity, sewage sludge, compost, polycyclic aromatic hydrocarbons, bioavailability, tenax, HPCD, n-butanol.

Introduction

Bioaccessibility of contaminants is the main factor deter-mining their fate in the environment and their influenceon living organisms.[1,2] As a result of processes such assequestration or bound residue formation, their influencecan become limited.[2] On the other hand, the presence ofthe other substances, such as surfactants or dissolved or-ganic carbon, can increase the bioaccumulation or toxicityof contaminants.[3,4] Currently, there is considerable scien-tific interest in finding chemical techniques capable of reli-ably predicting the bioavailability of organic compounds tobiota in soils and sediments.[2] In most cases known from lit-erature, research work has concentrated mainly on soils andsediments neglecting other environmental matrices. How-ever, an in-depth investigation of this problem in relationto sewage sludges and composts is of utmost importance assewage sludges or composts are applied to soils. Since thislast method of biosolid utilisation is becoming more com-mon, it may bring about irreversible environmental conse-

Address correspondence to Dr. Patryk Oleszczuk, Institute of SoilScience and Environmental Managemenet, University of Agri-culture, ul. Leszczynskiego 7, 20-069 Lublin, Poland; E-mail: pa-tryk.oleszczuk@ar.lublin.plReceived July 30, 2008.

quences with an indirect influence on elements of the en-vironment (soil, water, plants and animals). Contaminantspresent in sewage sludges may accumulate in soils, migratedeeper into the soil profile or be uptaken by plants.[5−7]

Hence, sewage sludges and their utilisation have become aglobal problem for all elements of the environment and alsofor humans.

Sewage sludge toxicity varies, mainly as a result oftheir origin, and hence the content of various contami-nant groups also varies. In sewage sludges there are manycontaminants that can have a potentially negative influ-ence on living organisms.[8,9] The latest proposals fromthe European Union[10] on sludge utilisation underlinethe necessity of evaluating not only the heavy metal con-tent in sludges but also other contaminants such as: or-ganic halogen compounds (AOX), nonylphenol ethoxy-lates (NPE), di-(2-ethylhexyl) phthalate (DEHP), linearalkylbenzene sulphonates (LAS), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F), polychlorinatedbiphenyl (PCBs) and polycyclic aromatic hydrocarbons(PAHs). However, there is little data in the literature toconfirm decisively the influence of a given substance onsewage sludge toxicity. Polycyclic aromatic hydrocarbonsare a common group among the above contaminants, andit has been assumed that they are one of the potential toxic-ity elements in sewage sludges. PAHs often occur in sewage

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sludges[8] at levels that suggest a negative influence on liv-ing organisms. A high content of organic matter can signifi-cantly limit this influence; hence, evaluation of the bioacces-sible fraction and its influence on the sewage sludge toxicityis important.

One of the proposed methods of decreasing the load ofPAHs and other organic pollutants in sludges is compost-ing. This method is relatively cheap and effective; however,there is little data in the literature on the fate of the poten-tially bioaccessible fractions and their influence on sewagesludge toxicity. Recent research studies showed that sewagesludge composting can result in an increase of their eco- andphytotoxicity,[11] despite a visible lowering of the total PAHcontent.[12] A suggested reason behind this phenomenonis an increase in the content of the potentially bioacces-sible fraction of contaminants[13] resulting from their re-mobilisation which, in turn, poses a danger of their sec-ondary release into the natural environment. Hence, it isof utmost importance to investigate the fate of the releasedcontaminants, and to determine their potential influenceon living organisms.

The aim of the present study was to attempt to answerthe following questions: (i) Is it possible to determine thePAH fraction responsible for the toxicity of sewage sludgesand composts using some selected chemical techniques ap-plied for the determination of the potentially bioaccessiblePAH fraction? and (ii) Is the increase in the content of thepotentially bioaccessible PAH fraction during compostingrelated to the increase in the sewage sludge toxicity?

Materials and methods

Toxicity assessment

Four sewage sludges (named KR, ZM, LB and BJ) collectedfrom municipal treatment sewage plants were compostedin two parallel bins for 76 days. A detailed description ofsewage sludges and the course of composting process werepresented in another works.[11,12] A standard OECD soil[14]

was used for all tests, and it was mixed with sewage sludge orcompost in an amount which represented sludges or com-post concentrations in the soil at the level of 6% and 24%,respectively.

Two tests were used for the evaluation of the solid phasetoxicity, i.e., Ostracodtoxkit FTM with Heterocypris incon-gruens and Phytotoxkit FTM with Lepidium sativum in thepresent study. Ostracodtoxkit FTM is a six days ‘direct’contact test using the crustacean Heterocypris incongru-ens. Mortality of the test organisms and growth inhibitionof the surviving ones were determined.[15] The phytotoxkitmicrobiotest measures the decrease (or the absence) of seedgermination and of the growth of the young roots after afew days of exposure of seeds of selected higher plants tocontaminated matrix in comparison to the controls in areference soil. At the end of the incubation period a digital

picture was taken of the test plates with the germinatedplants. The analyses and the length measurements were per-formed using the Image Tool 3.0 for Windows (UTHSCSA,San Antonio, TX, USA). The bioassays were carried out inthree replicates.[16]

The soil (with sewage sludge or compost addition −6and 24%) was placed in plastic containers. Then, 10 seedsof Lepidium sativum were sown into each of the pots. After14 days, the plant material obtained was collected. In thisstage biomass increase/decrease in the plant material wereevaluated.

Growth inhibition of H. incongruens was calculated as:

GI = 100 −(

AB

· 100)

(1)

where:

A – length increment of the ostracods in the sewage sludgeB – increment of the ostracods in the reference soil

The percent inhibition of seed germination (SG), rootgrowth inhibition (RI) and biomass inhibition (BI) for plantwere calculated with the formula:

SG/RI/BI = A − BA

· 100 (2)

where:

A – mean seed germinaton, root length and biomass in thecontrol soil

B – mean seed germination, root length and biomass in thetest soil

Chemical analysis

For PAHs analysis samples (15 g) were extracted inan ultrasonic bath (Sonic-3, Polsonic, Poland). Theextracts were centrifuged, decanted and evaporated todryness. The residues were then dissolved in acetoni-trile:water mixture (1:1 v/v) and purified by solid phaseextraction.[17] A qualitative and quantitative analysis ofPAHs was carried out on the liquid chromatograph withUV detection (TermoSeparation Products). For PAHsseparation an analytical Spherisorb S5 PAH (250 × 4.6mm I.D., 5 µg by Schambeck SFD GmbH, Germany)column was used. Data acquisition and analysis wasperformed using the Clarity Lite ChromatographicStation (DataApex, Czech Republic). Ten PAHs fromEPA list were determined: phenanthrene, anthracene,fluoranthene, pyrene, benzo[a]anthracene, chrysene,benzo[b]fluoranthene, benzo[a]pyrene, benzo[ghi]peryleneand indeno[1,2,3-cd]pyrene.

The method using hydroxypropyl[β]cyclodextrin(HPCD) was adopted from the works of Stokes et al.[18]

The sewage sludges or composts were extracted usingan aqueous HPCD shake extraction. Samples (5 g) were

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PAH bioavailability 139

weighed into 200-mL Teflon centrifuge tubes and 100 mLof a 50-mM aqueous solution of HPCD was added. Thetubes were shaken for 20 h and centrifuged for 30 min.The supernatant was discarded, and the residue sewagesludge was shaken with deionized water and centrifugedagain; the supernatant then was discarded. The PAHscontent was determined in the residue after extraction withdichloromethane in accordance with the method describedabove. The differences between the total PAH content (asdetermined in the dichloromethane) and the residue (afterextraction with HPCD) was determined as a HPCD orpotentially bioavailable (bioaccessible) fraction.

The method using mild solvent extraction with n-butanol(BtOH) was adopted from the works of Kelsey et al.[19]

Mild-solvent extraction was carried out by the extractionof samples of sewage sludge or compost (15 g) with 80 mL n-butanol after which, the extracts were centrifuged and thePAH content was determined in the residue after extrac-tion in accordance with the method described above. Thedifferences between the total PAH content (as determinedin the dichloromethane) and the residue (after extractionwith n-butanol) was determined as a n-butanol or poten-tially bioavailable (bioaccessible) fraction.

The method using Tenax-TA was adopted from the worksof Cornelissen et al.[20] Desorption was determined at 20◦Cby means of a single Tenax-TA solid phase extractionmethod. During desorption a mixture of Tenax-TA (3 g),sewage sludge or compost (2 g dry wt), HgCl2 (1 mg) as abiocide and deionised water (70 mL) was constantly shakenin a 100 mL separation funnel for 6h. After desorption,both the Tenax-TA beads and the sewage sludge or com-post samples were separated. The collected Tenax-TA beadswere extracted with 2 × 30 mL of acetone for a few seconds.The extracts were evaporated to dryness and then residueswere dissolved in 1 mL of acetonitrile:water mixture. PAHcontent was determined in accordance with the method de-scribed above.

Quality control and data presentation

The procedural blank was determined by going through thesame extraction and cleanup procedures for each series ofsamples. None of the analytical blanks were found to havedetectable contamination of the monitoring PAHs, and thusthe results were not blank corrected. All reported concen-tration of PAHs in samples on a dry-weight basis of sewagesludge or compost (determined by drying the samples for24 h at 105◦C) and are the average of triplicate extraction.

Statistical analysis

ANOVA tests were performed for differences between con-centrations of each compound and toxicity parametersbefore and after composting. Correlation analysis (Pear-son’s) was performed between the concentration of indi-vidual PAHs and mortality and growth inhibition of Het-erocypris incongruens and seed germination, inhibition ofroot growth and inhibition of biomass synthesis of Lepid-ium sativum. Significance was set at *P<0.05. All statisticalanalyses were performed using SPSS 14.0 for Windows.

Results

Toxicity of sewage sludges and compost and its changesduring composting

A detailed description of toxicity in relation to Lepidiumsativum and Heterocypris incongruens in the sewage sludgesand composts together with the influence of compostingon the evaluated parameters was presented in the otherwork.[11] In these studies, attention was drawn only to themost significant tendencies and changes observed.

Depending on the dose applied, a different phytotoxicityof the sewage sludges was observed (Table 1). The stimulat-ing effect of sludges on seed germination was only noted at

Table 1. Phytotoxicity of sewage sludges and compost samples to Lepidium sativum dependence on sewage sludge or compost dose(6/24%).

Inhibition of seed germination Root growth inhibition Biomass inhibition

Biosolid 6% 24% 6% 24% 6% 24%

Sewage sludgeKR −12.5 ± 0.8 0 20.0 ± 1.0 32.4 ± 2.3 6.8 ± 0.3 15.4 ± 1.4ZM −12.5 ± 1.1 100 ± 0.0 70.6 ± 3.8 100.0 ± 0.0 18.9 ± 1.1 37.2 ± 2.6LB −12.5 ± 1.0 100 ± 0.0 70.5 ± 4.2 100.0 ± 0.0 12.8 ± 0.9 69.6 ± 5.6BJ −25.0 ± 1.9 25 ± 2.7 −10.3 ± 0.3 64.2 ± 5.9 −8.7 ± 0.4 33.0 ± 2.0

CompostKR −12.5 ± 0.9 −12.5 ± 1.0 46.8 ± 10.0 −2.5 ± 0.3 −3.0 ± 0.2 −14.7 ± 0.9ZM −12.5 ± 1.0 12.5 ± 1.2 68.1 ± 5.2 89.0 ± 7.4 6.6 ± 0.6 13.8 ± 1.2LB −25.0 ± 1.9 100 ± 0.0 74.0 ± 2.3 100.0 ± 0.0 46.5 ± 3.7 28.5 ± 2.3BJ −12.5 ± 1.1 −25 ± 1.9 −16.8 ± 0.9 30.8 ± 1.1 32.3 ± 2.0 −18.4 ± 0.9

Values with ‘−’ exerted a positive influence on phytotoxicity.

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their lowest dose. An increase of sludge dose to 24% nega-tively influenced this last parameter. In the case of sludgesKR, ZM and LB, root growth and biomass synthesis wasinhibited already at the lowest doses. An increase in thedose caused a further negative influence by sludges on rootgrowth and biomass synthesis, similarly as for seed germi-nation (Table 1).

The influence of composting on the phytotoxic proper-ties was varied and depended both on compost and its dose.Generally, the lowest compost dose, as in the case of sludges,stimulated seed germination (Table 1). The negative influ-ence of composts on seed germination (with an exceptionof sludge LB) with increases in the dose was not as signifi-cant as those observed for sewage sludges. Composting didnot exert a positive influence on root growth for sludgesZM and LB. In compost BJ, composting significantly de-creased root growth inhibition, especially visible at a sludgedose of 24%. In compost KR, only at the lowest dose wasa negative influence found for composting on the inhibi-tion of root growth. In the remaining cases, compostingexerted a significant positive influence on this parameter.In sludges KR and ZM, composting positively influencedbiomass synthesis, whereas in sludges LB and BJ, a negativeeffect was observed. At the highest compost concentration,the influence on the biomass synthesis was positive for allsludges.

Depending on the sewage sludge type and dose, the mor-tality of Heterocypris incongruens ranged from 0 for sludgesBJ to 90% for LB. An increase of dose always resulted ina significant increase in the mortality of the test organismsfor all sludges tested (Table 2). In the case of two sludges(KR and ZM), the lowest dose caused more than a 50%growth inhibition. Growth inhibition of H. incongruens inrelation to sludges LB and ZM was similar to that obtainedfrom the control sample. With the exception of sludge LB,

Table 2. Ecotoxicity of sewage sludges and compost samples toHeterocypris incongruens dependence on sewage sludge or com-post dose (6/24%).

Mortality Growth inhibition

Biosolid 6% 24% 6% 24%

Sewage sludgeKR 17 ± 3.5 30 ± 5.0 54.5 ± 1.3 −7.3 ± 0.5ZM 17 ± 3.3 43 ± 2.5 53.0 ± 2.4 27.8 ± 0.8LB 13 ± 2.5 90 ± 2.9 7.8 ± 0.4 44.8 ± 2.8BJ 0 10 ± 2.9 −7.9 ± 1.0 −21.1 ± 1.5

CompostKR 13 ± 3.8 0 46.8 ± 10.0 −10.8 ± 0.7ZM 17 ± 4.8 67 ± 1.9 74.0 ± 2.3 76.5 ± 21.1LB 37 ± 1.0 70 ± 1.7 −16.8 ± 0.9 −11.8 ± 0.7BJ 7 ± 1.9 0 68.1 ± 5.2 0.9 ± 0.1

Values with ‘−’ exerted a positive influence on ecotoxicity.

an increase of does of sludges KR, ZM and BJ positivelyinfluenced the growth of H. incongruens.

The influence of composting on mortality and growthinhibition of H. incongruens showed a greater differenti-ation than for the phytotoxicity evaluation (Table 2). Atthe lowest sewage sludge dose (6%), composting decreasedmortality for sludge KR, did not change for sludge ZMand increased for sludges LB and BJ. Growth inhibitiondecreased for sludges KR and LB and increased for sludgesZM and BJ.

In composts KR and BJ, an increase in their dose resultedin a decrease in their toxicity, both in terms of mortalityand growth inhibition. In sludges ZM and LB, a reversedtendency was noted. At the highest dose for sludges KR,LB and BJ, mortality was lower than prior to composting.This last tendency was repeated also by sludges KR and LBfor growth inhibition. In the remaining cases, compostingnegatively influenced the tested parameters (Table 2).

Potentially bioaccessible PAHs content

Figure 1 presents the content of potentially bioac-cessible PAHs determined with Tenax-TA and mild(non-exhaustive) extraction with n-butanol (BtOH) andhydroxypropyl[β]cyclodextrin (HPCD) methods togetherwith the change resulting from composting. The content ofthe PAH sum was similar for BtOH and HPCD methods insludges KR, ZM and LB. For the method using the Tenax-TA adsorbent, 50% less PAHs was usually noted than forthe BtOH and HPCD methods. Sludge BJ was the only ex-ception, with content measured using BtOH and Tenax-TA50% lower than from the HPCD method. A similar relationwas noted for individual compounds (data not presented).

Composting influenced the decrease in the content andcontribution of bioaccessible fraction in the case of almostall methods and sludges tested (Fig. 1). The content of thebioaccessible fraction of the PAH sum in individual sludgesdecreased after composting by 42.7–83.8% (KR), 45.2–50.7(LB), 20.1–62.2% (ZM) and 40.8–43.0% (BJ). The decreasedepended on the method applied. In sludge BJ with theBtOH method, an increase in the content of the bioacces-sible fraction by 43.9% (Fig. 1) was observed.

In the case of individual PAHs, an increase in their con-tent after composting was noted (Fig. 2, Tables 3 and 4).In the case of the BtOH method, composting resulted ina increase of phenanthrene and anthracene content lev-els in sludge KR and for most individual PAHs in sludgeBJ (with the exception of benzo[a]pyrene) (Table 3). Usingthe HPCD method, only sludge ZM showed an increasein the content of individual PAHs (Table 4). In sludgesKR, BJ and LB, an increase was observed for phenan-threne (KR, BJ), anthracene (KR), benzo[a]anthracene(LB), benzo[ghi]perylene (LB) and indeno[1,2,3-cd]pyrene(LB). In the case of the Tenax-TA method, an increase wasnoted for benzo[a]anthracene content in all sludges (Fig. 2).In sludges LB, ZM and BJ, an additional increase was also

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PAH bioavailability 141

Fig. 1. The content and contribution of potentially bioavailable PAHs (sum of 10 compounds) in sewage sludges and composts.Tenax-TA – Tenax-TA fraction, BtOH - n-butanol fraction and HPCD - hydroxypropyl[β]cyclodextrin fraction. Error bars representstandard deviation error (n = 3).

noted in the content of anthracene (LB, ZM, BJ), fluoran-thene (LB), benzo[a]pyrene (LB, ZM), benzo[ghi]perylene(ZM, BJ) and indeno[1,2,3-cd]pyrene (LB).

Relationships between potentially bioaccessible PAHs insewage sludge and toxicity parameters

Positive significant relationships that may suggest a nega-tive PAH influence on H. incongruens were found mainly fora sewage sludge dose of 6% and for growth inhibition andbenzo[a]anthracene (Tenax-TA), chrysene (DCM, HPCD)and benzo[b]fluoranthene (Tenax-TA), as well as for adose of 24% between growth inhibition and indeno[1,2,3-cd]pyrene (Tenax-TA) (Fig. 3). Whereas in composts, neg-ative correlations were noted only for mortality and forthe following PAHs: benoz[a]anthracene (DCM), chrysene(DCM, HPCD) and benzo[a]pyrene (HPCD) (data notpresented).

Table 3. The content of BtOH and HPCD sewage sludge and compost fraction in biosolids KR and ZM (mg·kg−1, dry weight basis).

KR ZM

BtOH HPCD BtOH HPCD

PAHs SL C SL C SL C SL C

Phen 0.054 ± 0.003 0.103 ± 0.007 0.009 ± 0.001 0.029 ± 0.003 0.046 ± 0.003 0.024 ± 0.001 0.076 ± 0.008 0.031 ± 0.002Ant 0.222 ± 0.017 0.420 ± 0.058 0.200 ± 0.014 0.643 ± 0.051 0.031 ± 0.002 0.025 ± 0.001 0.040 ± 0.004 0.024 ± 0.002Fluo 0.559 ± 0.048 0.283 ± 0.017 0.503 ± 0.063 0.169 ± 0.013 0.304 ± 0.017 0.250 ± 0.026 0.344 ± 0.028 0.306 ± 0.021Pyr 4.641 ± 0.311 2.970 ± 0.202 4.426 ± 0.433 2.881 ± 0.248 0.628 ± 0.028 0.363 ± 0.023 0.626 ± 0.078 0.389 ± 0.012BaA 0.313 ± 0.010 0.080 ± 0.004 0.360 ± 0.028 0.182 ± 0.012 0.248 ± 0.016 0.029 ± 0.002 0.277 ± 0.028 0.082 ± 0.005Ch 0.182 ± 0.008 0.126 ± 0.011 0.205 ± 0.021 0.192 ± 0.008 0.107 ± 0.009 0.033 ± 0.003 0.136 ± 0.013 0.072 ± 0.006BbF 0.300 ± 0.018 0.271 ± 0.034 0.308 ± 0.028 0.297 ± 0.030 0.123 ± 0.007 0.059 ± 0.003 0.197 ± 0.016 0.053 ± 0.002BaP 1.045 ± 0.055 0.018 ± 0.001 1.103 ± 0.115 0.046 ± 0.003 0.372 ± 0.017 0.059 ± 0.003 0.409 ± 0.037 0.029 ± 0.002BghiP 0.558 ± 0.017 n.d. 0.631 ± 0.041 0.103 ± 0.011 0.228 ± 0.014 n.d. 0.246 ± 0.021 0.011 ± 0.001Ind 0.174 ± 0.008 0.032 ± 0.004 0.207 ± 0.017 0.014 ± 0.001 0.296 ± 0.016 0.062 ± 0.005 0.284 ± 0.020 0.064 ± 0.005

SL – sewage sludge, C – compost. BtOH – n-butanol; HPCD – hydroxypropyl[β]cyclodextrin; Phen – phenantherene; Ant – anthracene; Fluo –fluoranthene; Pyr – pyrene, BaA – benzo[a]anthracene; Ch – chryzene; BbF – benzo[b]fluoranthene; BaP – benzo[a]pyrene, BghiP – benzo[ghi]perylene;Ind – indeno[1,2,3-cd]pyrene; ± - standard deviation (n = 3).

The only negative correlations were observed in the eval-uation of various PAH forms and phytotoxicity parameterswhen tested both with the sewage sludges and the compostsobtained from them (data not presented).

Discussion

Little is known about the dose–response relationship be-tween PAHs and ostracod mortality or about the typeof interactions among PAHs in contaminated soils. Somerelationships were noted in soils contaminated by PAHsand in H. incongruens mortality.[21,22] However, studies intothese issues are very rare. Phytotoxicity tests predominateamong studies evaluating the toxicity of sewage sludgesand compost, and among them tests using leachates aremost common.[23,24] Studies on other groups of organismsare relatively rare.[25−27] In the present study, significant

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Fig. 2. The changes of Tenax-TA fraction of individual PAHs during sewage sludge composting. Error bars represent standarddeviation error (n = 3).

-20 0 20 40 60

Growth inhibition [%]

0.02

0.04

0.06

0.08

0.10

BaA

(T

enax

-TA

) [m

g⋅kg

-1]

-20 0 20 40 60

Growth inhibition [%]

0.12

0.14

0.16

0.18

0.20

0.22

Chr

ysen

e (H

PC

D)

[mg⋅

kg-1

]

-20 0 20 40 60

Growth inhibition [%]

0.01

0.02

0.03

0.04

0.05

0.06

BbF

(Te

nax-

TA)

[mg⋅

kg-1

]

-40 -20 0 20 40 60

Growth inhibition [%]

0.00

0.05

0.10

0.15

0.20

0.25

Ind

(Ten

ax-T

A)

[mg⋅

kg-1

]

Fig. 3. The relationships between H. incongruens growth inhibition and content of individual PAHs in different Tenax-TA (BaA, BbFand Ind) and HPCD (chrysene) fraction. BaA – benzo[a]anthracene, BbF – benzo[b]fluoranthene and Ind – indeno[1,2,3-cd]pyrene.

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PAH bioavailability 143

Table 4. The content of BtOH and HPCD sewage sludge and compost fraction in biosolids LB and BJ (mg·kg−1, dry weight basis).

LB BJ

BtOH HPCD BtOH HPCD

AHs SL C SL C SL C SL C

Phen 0.084 ± 0.006 0.078 ± 0.005 0.139 ± 0.012 0.055 ± 0.004 0.080 ± 0.005 0.092 ± 0.007 0.099 ± 0.008 0.112 ± 0.009Ant 0.045 ± 0.002 0.044 ± 0.004 0.061 ± 0.005 0.041 ± 0.002 0.026 ± 0.002 0.030 ± 0.002 0.084 ± 0.008 0.056 ± 0.005Fluo 0.446 ± 0.035 0.369 ± 0.031 0.489 ± 0.031 0.383 ± 0.026 0.248 ± 0.014 0.338 ± 0.030 0.742 ± 0.054 0.526 ± 0.060Pyr 0.728 ± 0.060 0.304 ± 0.017 0.792 ± 0.042 0.331 ± 0.025 0.140 ± 0.007 0.192 ± 0.015 0.967 ± 0.044 0.531 ± 0.050BaA 0.226 ± 0.012 0.057 ± 0.003 0.002 ± 0.0001 0.110 ± 0.010 0.255 ± 0.017 0.320 ± 0.027 0.150 ± 0.009 0.0004 ± 0.00003Ch 0.127 ± 0.006 0.067 ± 0.006 0.131 ± 0.009 0.054 ± 0.004 0.056 ± 0.003 0.258 ± 0.017 0.200 ± 0.017 0.201 ± 0.013BbF 0.201 ± 0.012 0.078 ± 0.008 0.215 ± 0.013 0.032 ± 0.002 0.100 ± 0.006 0.140 ± 0.016 0.239 ± 0.017 0.096 ± 0.009BaP 0.194 ± 0.012 0.060 ± 0.003 0.116 ± 0.006 n.d. 0.213 ± 0.018 0.210 ± 0.019 0.395 ± 0.023 0.065 ± 0.005BghiP 0.043 ± 0.003 0.001 ± 0.0001 0.062 ± 0.005 0.081 ± 0.006 0.079 ± 0.007 0.118 ± 0.013 0.001 ± 0.0001 0.045 ± 0.003Ind 0.050 ± 0.005 0.001 ± 0.0001 0.019 ± 0.002 0.024 ± 0.001 0.081 ± 0.007 0.139 ± 0.013 0.112 ± 0.007 0.072 ± 0.005

SL – sewage sludge, C – compost. BtOH – n-butanol; HPCD – hydroxypropyl[β]cyclodextrin; Phen – phenantherene; Ant – anthracene; Fluo –fluoranthene; Pyr – pyrene, BaA – benzo[a]anthracene; Ch – chryzene; BbF – benzo[b]fluoranthene; BaP – benzo[a]pyrene, BghiP – benzo[ghi]perylene;Ind – indeno[1,2,3-cd]pyrene; ± - standard deviation (n = 3).

relationships between total PAHs content in sewage sludgesand growth inhibition of H. incongruens was noted only forchrysene. The above relations remained few in number aftercomposting and took on negative values, mainly for mor-tality (supporting information).

Most of the research studies carried out so far on soils,sediments and sewage sludges showed that the total con-tent of organic contaminants is a poor predictor of theirtoxicity.[27] In the studies quoted, correlations between con-taminant content and waste toxicity are relatively rare[26] ornonexistent.[27] This situation may be related to the com-plicated matrix compositions as well as the complex in-teractions that occur between the individual components.The cause for this lack of correlations may be that thebioaccessibility of the contaminants determines their influ-ence and bioavailability rather than the total contaminantcontent (usually determined in such studies). The methodused for the evaluation of bioaccessibility in the presentstudy made it possible to determine significant relation-ships for a larger number of PAHs than achieved usingthe DCM method (total PAH content). The best resultswere obtained with the application of the Tenax-TA adsor-bent, which showed a potentially negative influence of threePAHs, i.e. benzo[a]anthracene, benzo[b]fluoranthene andindeno[1,2,3-cd]pyrene on growth inhibition (Fig. 3). Otherresearchers[28] found significant relationships between PAHtoxicity for nematode and earthworms when using theTenax-TA method.

Judging from a model proposed by Sverdrup et al.[29]

benzo[a]anthracene is expected to be toxic while heavierPAHs are not. The negative influence of benzo[a]anthraceneon Oniscus asellus was also shown by van Brummelen etal.[30] In some earlier studies[31] on sewage sludges, a poten-tially negative influence of benzo[a]anthracene on mortal-ity and growth inhibition of Heterocypris incongruens was

noted. However, there is too little data available to makeany conclusions on the validity of the results for organismsother than those tested. In soil, exposure of compounds toliving organisms mainly occur through porewater, and tox-icity therefore depends on the solubility and bioavailabilityof the compound in question.[32] Tenax-TA adsorbent takesup pollutants from the aqueous phase,[20] i.e., the fractionwhich can potentially exert a negative influence on organ-isms.

When analysing the bioaccessible fraction content ofcontaminants by means of the Tenax-TA method (Fig. 3),benzo[a]anthracene was found to be the only compoundswhere the content increased after composting in all thesludges examined. In sewage sludges LB and ZM, wherethe negative influence of composting on H. incongruens wasmost frequently determined (Table 2), an increase was alsonoted in the content of other PAHs such as anthracene(LB, ZM), fluoranthene (LB), benzo[a]pyrene (LB, ZM),benzo[ghi]perylene (ZM) and indeno[1,2,3-cd]pyrene (LB)(Fig. 3). The above confirmed the assumption suggested inthe beginning that an increase in the sewage sludge tox-icity after composting may be related to an increase inthe potentially bioaccessible fraction. The possibility of atoxic influence by some PAHs such as fluoranthene andbenzo[a]pyrene has been described in the literature manytimes.[29,30,33] Moreover, PAH toxicity can also increase asa result of synergistic effects between them.[34]

The studies conducted so far showed that Lepidiumsativum can be a sensitive indicator of soil contaminationby polycyclic aromatic hydrocarbons.[34] In presented stud-ies, no satisfactory results were achieved as the correlationsobserved only took negative values, perhaps indicating apositive influence by the PAHs present in sewage sludges onseed germination, growth inhibition and biomass synthe-sis. A simulating effect by xenobiotics at the relatively low

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144 Oleszczuk

content levels they maintained was also observed in soilsby other authors.[35] The above phenomenon is sometimesknown as hormesis, and may be explained by the possibilitythat PAHs act as plant growth activator.[36]

Conclusion

The studies presented here, as far as the present authoris aware, are the first attempt at the evaluation of thebioaccessible fraction of polycyclic aromatic hydrocarbonsin sewage sludges and composts against the backgroundof their toxicity for living organisms. Two direct con-tact tests and three methods (Tenax-TA, n-butanol andhydroxypropyl[β]cyclodextrin) were used in the presentstudy for the evaluation of the potentially bioaccessiblefraction. The best results were obtained for the method us-ing the Tenax-TA adsorbent, this last method allowed thedetermination of significant relationships between the tox-icity and contaminant content for a larger PAH group thanduring the total content evaluation for these compounds. Itwas found that in those sewage sludges showing an increasein their toxicity, an increase in the content of the bioaccessi-ble fraction of some PAHs could also be observed. This maymean they have a potentially negative influence on organ-isms. Attention should also be drawn to a possible syner-gistic influence by the contaminants. Hence, when studyingsewage sludge usability, research could be extended by in-cluding studies on the bioaccessible contaminant fractionwhen evaluating composting and assessing risks.

Acknowledgment

The work was founded in the frame of grant No. 2 P06S005 29 financed in 2005–2008 from the budget of Ministryof Science and Information Society Technologies.

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