Chemosphere 122 (2015) 168–175

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Chemosphere

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Dioxins and dioxin-like compounds in composts and digestates fromEuropean countries as determined by the in vitro bioassay and chemicalanalysis

http://dx.doi.org/10.1016/j.chemosphere.2014.11.0390045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.

Martin Beníšek a, Petr Kukucka a, Giulio Mariani b, Gert Suurkuusk b, Bernd M. Gawlik b, Giovanni Locoro b,John P. Giesy c,d,e,f, Ludek Bláha a,⇑a Masaryk University, Faculty of Science, RECETOX, Brno, Czech Republicb European Commission, DG Joint Research Centre (JRC), Institute for Environment and Sustainability, Unit H.01-Water Resources Unit, Ispra, Italyc Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, SK, Canadad Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Regione School of Biological Sciences, University of Hong Kong, Hong Kong Special Administrative Regionf State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China

h i g h l i g h t s

� Pan-European study of dioxins and dioxin-like compounds in composts and digestates.� PCDD/Fs, PCBs, OCPs, PAHs and dioxin effects compared in various types of samples.� Compliance with conservative limits confirmed for most of the samples.� High added value of the biodetection tools and effect-based monitoring demonstrated.

a r t i c l e i n f o

Article history:Received 18 July 2014Received in revised form 11 November 2014Accepted 15 November 2014Available online 15 December 2014

Handling Editor: Gang Yu

Keywords:Biodetection toolsEffect-based monitoringDioxinPAHsCompostDigestate

a b s t r a c t

Aerobic composting and anaerobic digestion plays an important role in reduction of organic waste bytransforming the waste into humus, which is an excellent soil conditioner. However, applications of chem-ical-contaminated composts on soils may have unwanted consequences such as accumulation of persistentcompounds and their transfer into food chains. The present study investigated burden of composts and dig-estates collected in 16 European countries (88 samples) by the compounds causing dioxin-like effects asdetermined by use of an in vitro transactivation assay to quantify total concentrations of aryl hydrocarbonreceptor-(AhR) mediated potency. Measured concentrations of 2,3,7,8-Tetrachlorodibeno-p-dioxin(2,3,7,8-TCDD) equivalents (TEQbio) were compared to concentrations of polycyclic aromatic hydrocarbons(PAHs) and selected chlorinated compounds, including polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), co-planar polychlorinated biphenyls (PCBs), indicator PCB congeners and organochlorine pesticides(OCPs). Median concentrations of TEQbio (dioxin-like compounds) determined by the in vitro assay in crudeextracts of various types of composts ranged from 0.05 to 1.2 with a maximum 8.22 lg (TEQbio) kg�1 drymass. Potencies were mostly associated with less persistent compounds such as PAHs because treatmentwith sulfuric acid removed bioactivity from most samples. The pan-European investigation of contamina-tion by organic contaminants showed generally good quality of the composts, the majority of which werein compliance with conservative limits applied in some countries. Results demonstrate performance andadded value of rapid, inexpensive, effect-based monitoring, and points out the need to derive correspond-ing effect-based trigger values for the risk assessment of complex contaminated matrices such ascomposts.

� 2014 Elsevier Ltd. All rights reserved.

M. Beníšek et al. / Chemosphere 122 (2015) 168–175 169

1. Introduction

Composting (aerobic process) and digestion (anaerobic process)can be useful for reduction of various wastes, because these pro-cesses can transform organic waste to humus, which is an excellentsoil conditioner (Grossi et al., 1998). On the other hand, the pres-ence of or formation of persistent toxicants in composts or dige-states is an issue that might limit their widespread use. Use ofcompost and digestate as amendments to improve fertility increaseorganic matter, reduce erosion, and improve physical chemicalproperties of soils such as retention of water (Semple, 2001;Pedra et al., 2007). There are several requirements placed on qualityof compost that need to be met to protect the environment fromadverse effects of both inorganic and organic contaminants. Themost important parameters include minimum content of metalsand organic chemicals at toxic concentrations and the absence ofpathogens that pose risks to health of humans (Lasaridi et al.,2006). Composts and digestates can be contaminated by variouschemicals that can cause adverse effects on humans and wildlife.While heavy metals in composts are relatively well studied andcontrolled, there is still limited knowledge about the content, fateand effects of organic pollutants. The focus of this study waspersistent organic pollutants (POPs), including organochlorineinsecticides (OCs), constituents of personal care products, industrialchemicals, such as polychlorinated biphenyls (PCBs), polycyclicaromatic hydrocarbons (PAHs), and polychlorinateddibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF)(Brändli et al., 2007a,b; Grossi et al., 1998; Hseu, 2004). Somenon-persistent organic pollutants, such as lesser molecular massPAHs, can be degraded during composting (Brändli et al., 2007b),but other contaminants such as PCBs or PCDDs/Fs could accumulatein soil when contaminated compost or digestate is applied repeat-edly (Umlauf et al., 2011). In several European countries some limitvalues for the content of organic pollutants such as PCDDs/Fs, PCBs,PAHs and others have been established (Saveyn and Eder, 2014;WRAP, 2002).

Various toxic effects have been associated with the above men-tioned organic pollutants. Probably the most important and mostwidely studied is activation of the arylhydrocarbon receptor(AhR) which results in dioxin-like toxicity (Sorg, 2013). Toxicpotential of dioxin-active compounds is often calculated from theresults of chemical analyses using the Toxic Equivalency, TEQ,approach (I-TEQ or WHO-TEQ) (Lee et al., 2013). TEQs, to calculateequivalents of 2,3,7,8-tetrachlorodibenzo-p-dioxin, (TCDD) (usu-ally expressed in ng of TCDD per kg material) are calculated asthe sum of the product of concentrations of individual AhR-activecompounds multiplied by their corresponding toxic equivalencyfactors (TEFs), or relative response factors (Lee et al., 2013;Machala et al., 2001). In addition, TEQbio can be estimated by useof bioanalytical tools such as ethoxyresorufin O-deethylase (EROD)assay (Joung et al., 2007) or transactivation, reporter gene bioas-says, such as H4IIE-luc cells (Giesy et al., 2002), DR-CALUX (Murket al., 1996) and others where expression of the reporter gene,luciferase, is up-regulated by exposure to agonists of the AhR.

Both chemical and biological methods used for the analyses ofdioxin-active compounds have their specific advantages and disad-vantages. Chemical analysis allows assessment of only a limitednumber of compounds such as the 17 US EPA PCDDs/Fs or 16 USEPA PAHs, while bioanalytical methods measure the integratedpotency of all AhR-active compounds present in the sample includ-ing also e.g. brominated derivatives (Samara et al., 2009) or variousPAH metabolites and analogs (Sovadinova et al., 2006). Chemicalanalysis can also be costly, especially when considering analysesof PCDD/Fs and co-planar PCBs (Joung et al., 2007). The conceptof chemical TEQs is based on additive effects of toxic compounds

but these compounds present in complex samples might elicitinfra- or supra-additive interactions (Suzuki et al., 2006), whichcan be detected by the use of biodetection tools. Bioanalyticaldetection systems can also be more sensitive because they areresponding to the complex of chemicals in aggregate instead ofeach chemical individually. Alternatively, limitations of bioanalyt-ical tools include greater variability which is natural to biologicaltesting systems, a lesser degree of standardization and generallylesser acceptance by regulatory authorities. Nevertheless, theeffect-based monitoring is becoming widely applied (Escheret al., 2013; Hecker and Giesy, 2011) and certain biodetection toolshave already been standardized and suggested for practical use,including the assessment of endocrine disruptive (estrogenic)(ISO, 2014) or dioxin-like compounds (Hecker and Giesy, 2011).

Concentrations of persistent organic pollutants and PAHs incomposts determined by chemical analyses have been reportedpreviously (Grossi et al., 1998; Brändli et al., 2007a,b) and somestudies also investigated the dioxin-like activity using thebiodetection tools such as H4IIE-luc cells (Takigami et al., 2010;Suzuki et al., 2006). However, detailed comparisons of the chemi-cal- and effect-based monitoring of AhR active compounds usingthe broader set of compost samples are few. In previous studiessome PAHs such as benzo[k]fluoranthene, dibenz[a,h]anthraceneor benzo[a]pyrene detected in composts (Brändli et al., 2007a)were shown to be potent inducers of AhR in vitro (Machala et al.,2001). Also some other PAHs, which may have lesser dioxin-likepotentials (IEF – Induction Equivalency Factor) but great abun-dance in composts (such as fluoranthene and others) could signif-icantly contribute to the dioxin-like effects of the whole sample(Lee et al., 2013). Available data on concentrations of persistentorganic pollutants indicate that composts contain congeners withlesser TEFs or IEFs such as octachlorodibenzo-p-dioxin orheptachlorodibenzo-p-dioxin (OCDD or HpCDD) (Takigami et al.,2010; Muñoz et al., 2013). In addition to PCDDs/Fs and dioxin-likePCBs, other chlorinated compounds could be found in compostsincluding organochlorinated pesticides (OCPs) such as dichlorodi-phenyltrichloroethane (DDT), hexachlorocyclohexane (HCH), hexa-chlorobenzene (HCB), pentachlorobenzene (PeCB) and others(Brändli et al., 2004). Although toxicity of these compounds isnot primarily mediated via the AhR, some have been shown toact as weak AhR agonists and could also contribute to the effectof the whole mixture (Mrema et al., 2013). To our knowledge, con-centrations of other potential AhR-acting dioxin-like compounds(such as polybrominated diphenyl ethers, PBDEs) have only rarelybeen investigated in compost or digestates. In summary, the avail-able data indicate that PAHs may be the dominant compounds con-tributing to the dioxin-like effects of the complex compost samplesbut the role of different AhR-active compounds has not been stud-ied in detail.

The main objective of the present study was to investigate con-centrations of a range of compounds causing dioxin-like toxicity incomposts and digestates collected throughout 16 countries inEurope. The present study compared the concentrations of PAHsand diverse chlorinated compounds (indicator PCBs, OCPs, PCDD/Fs and co-planar PCBs) with dioxin-like effects observed in vitro.

2. Materials and methods

2.1. Design of the study

The present study investigated various categories of compostssuch as organic waste from households, green compost from gar-dens and parks, sewage sludge compost and also some compostsand digestates after Mechanical Biological Treatment. Screening

170 M. Beníšek et al. / Chemosphere 122 (2015) 168–175

of crude organic extracts of 88 samples of composts was performedby use of the H4IIE-luc bioassay while PAHs were also quantified.Based on results of the screening, seventeen samples were selectedfor detailed chemical analysis of PCDDs/Fs, PCBs and other organo-chlorine compounds. In addition, dioxin-like activity was tested inthis subset of samples also after the treatment of the sulfuric acid(H2SO4) which is known to remove labile compounds (such asPAHs) retaining only highly persistent organochlorine compounds.

2.2. Compost samples

Composts and digestates were collected and provided by theowners of various compost plants all over the Europe. The sam-pling according to EN 12579 was recommended but with respectto the pan-European character of the study, complete standardiza-tion was not possible and each plant was allowed to collect thesamples using their own protocol. Overall, 96 composts or dige-states were collected from which 88 samples (from 16 Europeancountries) were selected, divided into categories, and analyzed.The respective categories were:

(A) Compost produced from separately collected organic wastefrom households and similar commercial institutions, includinggarden and park waste; (B) compost produced from garden andpark waste only (green compost); (C) sewage sludge compostproduced from good quality sewage sludge and other separatelycollected organic waste (e.g. garden and park waste, straw, etc.);(D) Municipal Solid Waste compost generated by Mechanical Bio-logical Treatment (MBT) aimed at producing compost (derivedfrom non-hazardous household waste and similar commercialwaste where no separate collection of household waste is in place);(E) biowaste digestates from source separated biowaste fromhouseholds and similar commercial institutions (not investigatedin the present study for technical reasons); (F) digestates frommanure and source separated biowastes from households and sim-ilar commercial institutions; (G) digestates from manure andenergy crops; (H) digestate derived from Mechanical BiologicalTreatment of Municipal Solid Waste, aimed at producing digestatefor use in agriculture (derived from nonhazardous householdwaste and similar commercial waste); (I) other, minor categories.These include bark compost or Municipal Solid Waste compost likeoutput generated by Mechanical Biological Treatment aimed atstabilizing a rest fraction sent to landfill.

2.3. Processing of samples for biological analyses

Lyophilized samples (2 g, dry mass; dm) were extracted (auto-mated warm Soxhlet extractor, BüchiB-811, Switzerland) bydichloromethane (DCM) (150 mL, 2 h). Extracts were evaporatedto approximately 5 mL and transferred to vials. Samples were con-centrated by nitrogen stream to the last drop and then dissolved inmethanol (0.5 mL) and stored frozen until testing. For 17 selectedsamples, aliquots (200 lL) were treated with sulfuric acid silicacolumn to remove less persistent pollutants like PAHs. Persistentorganic compounds were then eluted from the column by a mix-ture of dichloromethane/hexane (40 mL), concentrated by nitrogenstream. One aliquot of the sample was used for analyses of persis-tent compounds and the second aliquot was evaporated, dissolvedin methanol and tested for their dioxin-like effects in vitro usingthe H4IIE-luc bioassay.

2.4. Quantification of PAHs

All native and labeled mixture standards of PAHs werepurchased from Chiron AS (Norway). Custom PAH Neat StandardMixture (Product No. S-4582-25-IO, batch 9184) containing: Phen-anthrene, Anthracene, Fluoranthene, Pyrene, Benzo(a)anthracene,

Chrysene, Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(e)-pyrene, Benzo(a)pyrene, Perilene, Indeno(1,2,3-cd)pyrene,Dibenzo(a,h)anthracene, Benzo(g,h,i)perilene, Dibenzo(a,l)pyrene,Dibenzo(a,h)pyrene, Dibenzo(a,i)pyrene, Dibenzo(a,e)pyrene andCoronene. Custom PAH Surrogate Standard Mixture (Product No.S-4582-50-IO, batch 92010) containing: Phenanthrene-d10,Anthracene-d10, Fluoranthene-d10, Pyrene-d10, Chrysene-d12,Benzo(b)fluoranthene-d12, Benzo(e)pyrene-d12, Benzo(a)pyrene-d12, Perilene-d12, Indeno(1,2,3-cd)pyrene-d12, Dibenzo(a,h)anthracene-d14, Benzo(g,h,i)perilene-12, Dibenzo(a,i)pyrene-d14,and Coronene-d12. Custom PAH Syringe Standard Mixture (Prod-uct No. S-4581-50-IO, batch 9171) containing: p-Terphenyl-d14,Benzo(a)anthracene-d12, Benzo(k)fluoranthene-d12. All solvents(Acetone, n-hexane) were dioxin grade and purchased by Riedel-de Haen (Germany).

Lyophilized samples (0.1 g) were spiked with 25 ng of SurrogateStandard Mixture and extracted twice for 30 min. by ultrasonicbath with 0.5 mL of a mixture of n-hexane/acetone (80:20, %V/V).After 25 ng of Syringe Standard Mixture spike the extracts withadded samples were submitted to gas chromatography–massspectrometry (GC–MS) analysis. PAHs were analyzed on a high res-olution gas chromatography (HRGC) (Agilent 6890N) coupled witha Mass Selective Detector (Agilent 5973N). For all analytes themolecular ions were recorded for both native and labeled congen-ers. Quantified isomers were identified by comparison of retentiontimes of the corresponding standard. Quantification was per-formed by isotopic dilution. PAHs were separated on a BPX-5060 m long with 0.25 mm i.d. (inner diameter) and 0.25 lm films(SGE, Victoria, Australia). Gas chromatographic conditions were:PTV injector from 100 to 300 �C at 12 �C s�1, constant flow at1.0 mL min�1 of He, GC–MS interface at 280 �C. The GC programrate was: 100 �C with a 3 min hold, then 15 �C min�1 to 220 �Cfor 0 min, then 2 �C min�1 to 300 �C for 20 min finally 3 �C min�1

to 340 �C and a final hold of 30 min. The selectivity, linearity,detection and quantification limits, trueness, repeatability, recov-ery, and stability of the extracts were determined and the uncer-tainty estimated. Detailed recoveries and limits of quantificationsfor measured PAHs are reported in Supplementary materialsTable S1-part F. The detailed method description was reported byTavazzi et al. (2013)

2.5. Analyses of indicator PCBs, OCPs, PCDDs/Fs and dl-PCBs

All standards (PCDDs/Fs, dl-PCBs) were purchased from Wel-lington Laboratories (Canada) or LGC Europe (PCBs and OCPs).For indicator PCBs and for organochlorine pesticides, instrumentalanalysis used GC Agilent 68890N-: Micromass Qauttro Micro GC(tandem quadrupole system), splitless injections, columns:60 m � 0.25 mm � 0.25 lm DB5-MS (J&W, Agilent, USA). Heliumwas used as a carrier gas at constant flow 1.5 mL min�1. quantifica-tion of PCDD/Fs and coplanar (dioxin-like, dl) PCBs, aliquots ofextracts prepared in dichloromethane were spiked with internalstandards of 13C PCDDs/Fs (according to EN-1948) and 13C dl-PCBs(77, 81, 126, 169, 105, 114, 118, 123, 156, 157, 167 and 189). Theconcentrated extracts were cleaned-up on a sulfuric acid-modified(44% w/w) silica column, eluted with 40 mL DCM/n-hexane mix-ture (1:1). Fractionation was achieved in a micro column (6 mmi.d.) containing from the bottom to top: 50 mg silica, 70 mg char-coal (Darco G60, Sigma–Aldrich)/silica (1:40) and 50 mg of silica.The column was pre-washed with 5 mL of toluene, followed by5 mL of DCM/cyclohexane mixture (30%), then the sample wasapplied and eluted with 9 mL DCM/cyclohexane mixture (30%) infraction 1 (mono–ortho dl-PCBs) and 40 mL of toluene in fraction2 (PCDDs/Fs, non-ortho dl-PCBs). Each fraction was concentratedusing the stream of nitrogen in a TurboVap II concentrator unit(Caliper LifeSciences, USA) and transferred into an insert in a vial.

M. Beníšek et al. / Chemosphere 122 (2015) 168–175 171

The sensitivity (syringe) standards (13C 1,2,3,4-TCDD and1,2,3,7,8,9-HxCDD, 13C PCBs 70, 111, 138 and 170) were added toall samples. The final volume prepared for analyses was 50 lL.High resolution gas chromatography/high resolution mass spec-trometry (HRGC/HRMS) instrumental analysis of PCDDs/Fs anddl-PCBs was performed on an 7890A GC (Agilent, USA) equippedwith a 60 m � 0.25 mm � 0.25 lm DB5-MS column (Agilent J&W,USA) coupled to an AutoSpec Premier MS (Waters, Micromass,UK). The MS was operated in EI + mode at the resolution of>10000. Detailed recoveries and limits of quantifications forPCDDs/Fs and dioxin-like PCBs are reported in Supplementarymaterials Table S2-B, S3-B.

Concentrations of co-planar PCBs and PCDD/Fs obtained fromHRGC/HRMS were multiplied by respective toxic equivalencyfactors (TEFs) and expressed as upper bound TEQ values (non-detected compounds are substituted by its limit of detection forcalculation) and lower bound TEQ values (non-detected com-pounds are substituted by 0 for calculation). To compare theresults from the bioassay with analytical results, concentrationsof co-planar PCBs and PCDDs/Fs were multiplied with the assay-specific relative potencies for chlorinated AhR active compounds(RePs reported by Lee et al., 2013; Behnisch, 2003) to calculateassay-specific TEQH4IIE. Concentrations of PAHs were multipliedby the induction equivalency factors for PAHs (IEFs reported byMachala et al., 2001), to calculate assay-specific TEQPAH.

2.6. H4IIE-luc bioassay

H4IIE-luc, rat hepato-carcinoma cells stably transfected withthe luciferase gene under control of the arylhydrocarbon receptor(AhR) were used for analysis of dioxin-like activity of the samples(Giesy et al., 2002). H4IIE-luc cells were cultured in Dulbecco’smodified Eagle medium – DMEM (PAA, Austria) with 10% fetal calfserum in incubator with 5% CO2 at 37 �C. H4IIE-luc cells wereseeded into 96-well plates (15000 cells per well). After 24 h, dilu-tion series of tested samples, calibration (0.4–500 pM TCDD-dose-response curve is shown in Supplementary materials-Fig. S1) andnegative (solvent) control were added (final concentration of thesolvent was 0.5%). Exposures were conducted in three replicatesfor 24 h. After exposure to extracts of samples or standards, micro-scopic evaluation of each well was performed to check cytotoxiceffects and luminescence intensity was measured by use of thePromega Steady Glo kit (Promega, Mannheim, Germany). Dioxin-like potencies were determined by use of the equi-effectiveapproach and the results were expressed as dioxin-like equivalents(TEQbio) with respect to standard 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TEQbio was calculated from the dose-response curvemodel fitted to the Hill function, based on the comparison of EC50

Table 1Results of PAHs, TEQPAHs and TEQbio for respective groups of compounds.

Group N PAHs (lg kg�1) TEQPAH (lg

Median 90% int Max Median

A 28 1.6E+03 3.2E+03 5.5E+03 1.7E�01B 23 1.2E+03 2.9E+03 8.5E+03 1.4E�01C 16 1.3E+03 3.0E+03 1.1E+04 1.6E�01D 8 6.9E+02 1.6E+03 2.2E+03 8.8E�02F 5 9.3E+02 1.2E+03 1.2E+03 1.0E�01G 1 – – 7.4E+02 –H 2 1.5E+03 1.6E+03 1.6E+03 2.4E�01I 5 1.2E+03 1.7E+04 2.6E+04 1.4E�01

A – biobin + green waste compost, B – green waste compost, C – sewage sludge compodigestate, H – MBT digestate, I – Other; PAH – polycyclic aromatic hydrocarbons, TEQPAH

induction equivalency factors for PAHs, TEQbio – toxic equivalent of 2,3,7,8 TCDD calcul

of standard TCDD to EC50 of samples (Villeneuve et al., 2000). Allcalculations were performed in GraphPad Prism 5.0.

2.7. Statistical analysis

Correlations among the parameters were tested using the Pear-son’s correlation and controlled by the non-parametric Spearman’sR. P-values less than 0.05 were considered statistically significant.

3. Results and discussion

The chemical contamination of composts repeatedly applied onthe arable soil can have adverse effects on the quality of soils andfood chains (Déportes et al., 1995). To our knowledge, only a fewisolated studies investigated the concentrations of organic contam-inants and their potential to induce dioxin-like effects in compostsamples but broader representative investigation and assessmentof the contribution of individual chemical classes to the dioxin-likeeffects has been missing.

In the present study concentrations of 19 PAHs in 88 sampleswere quantified, 12 of them were US EPA priority PAHs (Supple-mentary Table S1) and exhibited a broad range of contaminationfrom 1.2 � 102 to 2.6 � 104 lg kg�1, dry mass (dm) (Table 1). Themost abundant of analyzed PAHs in most of the samples were fluo-ranthene and pyrene (Supplementary materials Table S1). Greatermedian concentrations were found in compost categories A, B, C,H and I, while lesser concentrations were observed in other com-post categories (Table 1). Several studies investigated concentra-tions of PAHs in composts (Table 3), and the range of PAHspreviously reported (2.7 � 101–2.1 � 104 lg kg�1, dm) well corre-spond to the findings of the present study. Only a single sample(category ‘‘Others‘‘) had greater concentrations of 19 PAHs(2.6 � 104 lg kg�1, dm; see Supplemental Table S1-E). The samesample contained greater concentrations of 11 US EPA PAHs(2.2 � 104 lg kg�1, dm; i.e. sum of 12 US EPA PAHs withoutdibenz[a,h]anthracene), which is directly comparable to the maxi-mum concentration of 2.1 � 104 lg kg�1 reported in the literature(McGowin et al., 2001). Some limits have been suggested in theEuropean countries ranging from 4 to 10 mg kg�1, dm for US EPA16 priority PAHs (Brändli et al., 2004). With the exception of themost contaminated composts, most concentrations were less thanthe limit of 10 mg kg�1, dm when 12 analyzed US EPA priority PAHswere considered (Supplementary Table S1). Since a significantcorrelation between the sums of PAH16 and PAH12 has beenpreviously demonstrated with an average ratio of 1.073 (Brändliet al., 2007a), it can be concluded that most of the composts com-plied with the given limit value of 10 mg kg�1, dm. When comparedwith the lesser suggested limit (4 mg kg�1, dm), few samples from

kg�1) TEQbio (lg kg�1)

90% int Max Median 90% int Max

4.7E�01 8.3E�01 5.7E�01 1.6E+00 3.3E+005.4E�01 1.9E+00 6.4E�01 1.8E+00 3.8E+004.5E�01 2.0E+00 5.1E�01 1.7E+00 4.4E+001.8E�01 2.9E�01 5.9E�01 1.1E+00 1.3E+001.3E�01 1.3E�01 5.0E�02 4.1E�01 4.7E�01– 1.2E�01 – – 9.0E�022.9E�01 3.0E�01 1.2E+00 1.2E+00 1.3E+001.7E+00 2.8E+00 5.6E�01 7.4E+00 8.2E+00

st, D – MBT compost, F – manure + biowaste digestate, G – manure + energy crops– toxic equivalent of 2,3,7,8 TCDD for PAHs calculated from analytical results and

ated from bioassay results.

Tabl

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Resu

lts

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lm

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chem

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biol

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alda

tafo

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sele

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com

post

sam

ples

.

Sam

ple

no.

P12

US-

EPA

PAH

s( l

gkg�

1)

P19

PAH

s(l

gkg�

1)

TEQ

PA

Hs

(lg

kg�

1)

PPC

DD

s/Fs

(lg

kg�

1)

TEQ

H4

IIE

PCD

Ds/

Fs-

UB

( lg

kg�

1)

PD

L-PC

Bs

(lg

kg�

1)

TEQ

H4

IIE

DL-

PCB

s-U

B( l

gkg�

1)

TEQ

bio

(lg

kg�

1)

P6P

CB

ind.

(lg

kg�

1)

PD

DT

(lg

kg�

1)

PH

CH

(lg

kg�

1)

HC

B(l

gkg�

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PeC

B(l

gkg�

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1A1.

6E+0

31.

7E+0

32.

1E�

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1E�

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0E�

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3E+0

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1.0E

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172 M. Beníšek et al. / Chemosphere 122 (2015) 168–175

categories A (N = 2), B (N = 2), C (N = 1) and I (N = 1) were greaterthan this more conservative criterion. In some countries limits forPAHs consider also the sums of fewer PAHs, such as 6 PAHs withthe limit of 6 mg kg�1, dm in Austria or 11 PAHs with the limit of3 mg kg�1, dm in Denmark (Brändli et al., 2004).

A subset of seventeen samples was further analyzed for organo-chlorine compounds (Table 2). Among PCDDs/Fs and dioxin-likePCBs, the most abundant congener was OCDD, followed by1,2,3,4,6,7,8 HpCDD (Supplementary materials – Table S2-A).Concentrations of TEQ, based on concentrations of the PCDDs/Fs,ranged from 2.9 to 15.1 ng WHO-TEQ kg�1, dm. Compared to con-centrations in the literature (Table 3), similar concentrations werereported by Brändli et al. (2007b), lesser values were measured byMuñoz et al. (2013), and greater TEQs were found in an older studyfrom Brazil (Grossi et al., 1998). Various values for quality limitshave been accepted in different countries ranging from 20 ng Inter-national TEQ kg�1, dm (I-TEQ) in Switzerland (Brändli et al., 2004)to 100 ng I-TEQ kg�1, dm in Belgium (Tavazzi et al., 2013; Saveynand Eder, 2014). In all cases concentrations of TEQ were less thanthe conservative limits, with a maximum measured concentrationof 15 ng WHO-TEQ kg�1, dm (or 14 ng I-TEQ kg�1, dm; Supplemen-tary Table S2-A) confirming thus again generally good quality ofEuropean composts.

The contribution of dioxin-like PCBs to TEQs was less than thatof PCDDs/Fs (Table 2), with the exception of a sample 1C, whichhad relatively great concentrations of PCB 126 (70 ng dl-PCBTEQ kg�1, dm; Table 2 and Supplementary Table S3-A). Upperbound concentrations in other samples ranged from 0.1 to2.5 ng dl-PCB TEQ kg�1, dm. These are comparable to values avail-able in the literature, which ranged from 0.4 to 6.8 ng TEQ kg�1, dm(Brändli et al., 2007b) (Table 3).

Concentrations of six indicator PCBs ranged from 1 to44 lg kg�1, dm (Table 2, Supplementary Table S4) with PCB 153,138 and 180 being the most abundant congeners. Concentrationsin the present study were less than those in another Europeanstudy (Lazzari et al., 1999), which ranged from 1.4 � 101 to1.3 � 102 lg kg�1, dm, and less than the most conservative limitof 80 lg kg�1, dm used in Denmark (Tavazzi et al., 2013). Also forcongener PCBs limits vary among countries from 80 lg kg�1 to800 lg kg�1, dm for PCB7 and from 1 � 102 to 1 � 103 lg kg�1,dm for PCB6, respectively (Tavazzi et al., 2013). Concentrations oforganochlorine pesticides are given in Table 2. Sum of DDTs rangedfrom 0.49 to 56 lg kg�1, dm, while maximum concentrations ofHCH (sum of congeners), HCB and PeCB were 11, 5.9 and0.3 lg kg�1, dm, respectively. Only limited comparable data couldbe found in the literature but similar mean concentrations of sumsof DDTs and HCHs (about 18 and 3.5 lg kg�1, dm, respectively)were reported by Brändli et al. (2004). The same study foundslightly greater mean concentrations of PeCB (mean 1.2 lg kg�1,dm). In summary, analyses of several classes of chemical contam-inants revealed good compliance of the studied composts withthe limits available for POPs or PAHs.

The in vitro H4IIE-luc bioassay revealed significant biologicalactivities with the effective concentrations ranging from 0.5 to1.2 lg TEQbio kg�1, dm for most of the categories (Table 1). Theobserved effects were comparable available literature (Table 3,Takigami et al., 2010).

Lesser concentrations were found in category F (digestates frommanure and source separated biowaste) with a median concentra-tion of 0.05 lg TEQbio kg�1, dm, while greater concentrations weredetected in category I (‘‘Others’’) (maximum 8.22 lg TEQbio kg�1,dm, which included some bark and municipal waste composts –Supplementary materials-Table S5). A subset of 17 samples wasfurther tested after a treatment with sulfuric acid, which substan-tially reduced AhR-effects, and only a single sample (No. 3C)induced an AhR-mediated response near to the limit of detection

Table 3Comparison of the results with literature.

This study Literature values References

PAHs 1.2 � 102–2.6 � 104 lg kg�1 (P

19 PAHs) 2.1 � 102–1.1 � 104 lg kg�1 (P

15 PAHs) Grossi et al. (1998)4 � 102–2.1 � 104 lg kg�1 (

P11 PAHs) McGowin et al. (2001)

27–2.1 � 102 lg kg�1 (P

16 PAHs) Takigami et al. (2010)

PCDDs/Fs 3–15 ng WHO–TEQ kg�1 upper bound 3–1.6 � 102 ng I-TEQ kg�1 Grossi et al. (1998))0.52–21 ng I-TEQ kg�1 Brändli et al. (2007b)0.25–2.5 ng WHO TEQ kg�1 Muñoz et al. (2013)

DL-PCBs 0.8–70 ng WHO TEQ kg�1 upper bound 0.4–6.8 ng WHO–TEQ kg�1 Brändli et al. (2007b)

TEQ bio <10 ng kg�1–8.2 � 103 ng kg�1 22–3.9 � 103 ng kg�1 Takigami et al. (2010)360 ng kg�1 Suzuki et al. (2006)

Ind. PCBs 1–44 lg kg�1 14–1.3 � 102 lg kg�1 Lazzari et al. (1999)

PAHs – polycyclic aromatic hydrocarbons; PCDDs/Fs – polychlorinated dibenzodioxins/furans; DL-PCB – dioxin-like polychlorinated biphenyls; TEQbio – toxic equivalent of2,3,7,8 tetrachlorodibenzo-p-dioxin calculated from bioassay; Ind. PCBs – indicator polychlorinated biphenyls.

Fig. 1. Correlation between TEQPAH and TEQbio. (A) biobin + green waste compost, (B) green waste compost, (C) sewage sludge compost, (D) MBT compost, (E) all parallel data(TEQPAH and TEQbio); TEQPAH – toxic equivalent of 2,3,7,8 TCDD for PAHs calculated from analytical results and induction equivalency factors for PAHs, TEQbio-toxic equivalentof 2,3,7,8 TCDD calculated from bioassay results; RP – Pearson’s correlation, RS – Spearman’s correlation, ns – not significant.

M. Beníšek et al. / Chemosphere 122 (2015) 168–175 173

174 M. Beníšek et al. / Chemosphere 122 (2015) 168–175

(LOD) TEQbio – 10 ng TEQbio kg�1, dm. Dose-response curves of rawextract and sulfuric acid treated extract of the sample 3C are pre-sented in Supplementary materials-Fig. S1.

Comparison of TEQbio and TEQPAH indicated that in some sam-ples PAHs were likely responsible for the majority of the dioxin-like effects since concentrations of TEQPAH were even greater thanTEQbio (N = 4 samples from category A, N = 2 from category F, N = 1from categories C and G, see Supplementary materials Table S5). Inmost of the samples (N = 80) concentrations of TEQPAH calculatedfrom RePs and concentrations of measured PAHs could accountfor 10–90% of TEQbio. Statistically significant correlations betweenTEQbio and

PTEQPAH were observed (Spearman’s R, p < 0.05, Fig. 1),

and the correlations were also significant for the subset of 17 sam-ples (Pearson P < 0.0001; Spearman P = 0.007). Contrary, correla-tions between TEQbio in crude extracts and calculated TEQs basedon PCDDs/Fs and dioxin-like PCBs TEQs were not significant(Spearman’s R, p > 0.05) confirming again the importance of PAHsfor dioxin-like effects. Comparison with the chemical analysesshowed that benzo(k)fluoranthene and dibenz(a,h)anthracenewere the greatest contributors to the observed dioxin-like activity.

Although PAHs seem to be important chemicals causing dioxin-like effects not only in composts but also in sediments (Hilscherovaet al., 2001), results of some other studies have indicated they mayhave a relatively minor role. For example Takigami et al. (2010)estimated the proportion of TEQ contributed by PAHs compostbeing only 0.2–1.8%, and the same study also confirmed minorcontributions of persistent compounds that accounted for maxi-mum 0.3% of the total TEQbio in crude extracts These findings, alsoin agreement with other studies focusing e.g. on sediments(Hilscherova et al., 2001), indicate the toxicological importance ofother non-analyzed chemicals, which could be detected only bythe biological assay such as heterocyclic PAHs (Sovadinova et al.,2006) or diverse metabolites of PAHs such as hydroxides and epox-ides (Jeuken et al., 2003).

4. Conclusions

Analyses of a unique set of European compost samples (88samples from 16 European countries, 8 different types/categories)indicate that in the regulatory controlled parameters, such as con-centrations of PAHs, PCDD/Fs, PCBs or TEQs, few of the samplesexceeded the most conservative limits applied within differentcountries. The values found in the composts were in general agree-ment with other previously published studies. The results of theeffect-based analyses, using the in vitro test for dioxin-like effects,were correlated with concentrations of PAHs. However, persistentdioxin-active compounds such as PCDDs/Fs and dioxin-like PCBswere detected at generally small concentrations and did not signif-icantly contribute to the observed dioxin-like effects. The presentstudy provides one of few comprehensive investigations of thecompost contamination by organic contaminants and shows gen-erally good quality of the composts at the European level (withrespect to chemical contamination by traditionally analyzed PAHsand POPs). The study also demonstrates the need to introduce theeffect-based monitoring of the complex contaminated samples,and derive corresponding effect-based trigger values as also sug-gested for other environmental matrices (Tang et al., 2013).

Acknowledgements

The research was supported by the European Social Fund, thestate budget of the Czech Republic, and by the grants from theMinistry of Education of the Czech Republic (LM2011028 andLO1214). It was performed in the framework of the end-of-wasteproject of the EU (http://susproc.jrc.ec.europa.eu/activities/waste/

). Prof. Giesy was supported by the Canada Research Chair pro-gram, a Visiting Distinguished Professorship in the Department ofBiology and Chemistry and State Key Laboratory in Marine Pollu-tion, City University of Hong Kong, the 2012 ‘‘Great ConcentrationForeign Experts’’ (#GDM20123200120) program, funded by theState Administration of Foreign Experts Affairs, the P.R. China toNanjing University and the Einstein Professor Program of the Chi-nese Academy of Sciences.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2014.11.039.

References

Behnisch, P., 2003. Brominated dioxin-like compounds: in vitro assessment incomparison to classical dioxin-like compounds and other polyaromaticcompounds. Environ. Int. 29, 861–877.

Brändli, R., Kupper, T., Bucheli, T., Mayer, J., Stadelmann, F.X., Tarradellas, J., 2004.Occurrence and relevance of organic pollutants in compost, digestate andorganic residues – literature review. Agroscope FAL/Reckenholz/EPF Lausanne/ENAC/ISTE/CECOTOX. p. 193. (<http://www.bafu.admin.ch/abfall/01472/01480/01742/index.html?lang=en>).

Brändli, R.C., Bucheli, T.D., Kupper, T., Furrer, R., Stahel, W.A., Stadelmann, F.X.,Tarradellas, J., 2007a. Organic pollutants in compost and digestate. Part 1.Polychlorinated biphenyls, polycyclic aromatic hydrocarbons and molecularmarkers. J. Environ. Monit. 9, 456–464.

Brändli, R.C., Kupper, T., Bucheli, T.D., Zennegg, M., Huber, S., Ortelli, D., Müller, J.,Schaffner, C., Iozza, S., Schmid, P., Berger, U., Edder, P., Oehme, M., Stadelmann,F.X., Tarradellas, J., 2007b. Organic pollutants in compost and digestate. Part 2.Polychlorinated dibenzo-p-dioxins, and -furans, dioxin-like polychlorinatedbiphenyls, brominated flame retardants, perfluorinated alkyl substances,pesticides, and other compounds. J. Environ. Monit. 9, 465–472.

Déportes, I., Benoit-Guyod, J.-L., Zmirou, D., 1995. Hazard to man and theenvironment posed by the use of urban waste compost: a review. Sci. TotalEnviron., 197–222.

Escher, B.I., van Daele, C., Dutt, M., Tang, J.Y.M., Altenburger, R., 2013. Mostoxidative stress response in water samples comes from unknown chemicals:the need for effect-based water quality trigger values. Environ. Sci. Technol. 47,7002–7011.

Giesy, J.P., Hilscherova, K., Jones, P.D., Kannan, K., Machala, M., 2002. Cell bioassaysfor detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediatedactivity in environmental samples. Mar. Pollut. Bull. 45, 3–16.

Grossi, G., Lichtig, J., Kraub, P., 1998. PCDD/F, PCB and PAH content of Braziliancompost. Chemosphere 37, 2153–2160.

Hecker, M., Giesy, J.P., 2011. Effect-directed analysis of Ah-receptor mediatedtoxicants, mutagens and endocrine disruptors in sediments and biota. In: Brac,W. (Ed.), Eff. Anal. Complex Environ. Contam., vol. 15. Springer, BerlinHeidelberg, pp. 285–313.

Hilscherova, K., Kannan, K., Kang, Y.S., Holoubek, I., Machala, M., Masunaga, S.,Nakanishi, J., Giesy, J.P., 2001. Characterization of dioxin-like activity ofsediments from a Czech river basin. Environ. Toxicol. Chem. 20, 2768–2777.

Hseu, Z.-Y., 2004. Evaluating heavy metal contents in nine composts using fourdigestion methods. Bioresour. Technol. 95, 53–59.

ISO, 2014. ISO (2014) NWIP ISO/NP 19040–3 – Water quality – determination of theestrogenic potential of water and waste water – Part 3: in vitro human cell-based reporter gene assay [draft norm in the validation process].

Jeuken, A., Keser, B.J.G., Khan, E., Brouwer, A., Koeman, J., Denison, M.S., 2003.Activation of the Ah receptor by extracts of dietary herbal supplements,vegetables, and fruits. J. Agric. Food Chem. 51, 5478–5487.

Joung, K.E., Chung, Y.H., Sheen, Y.Y., 2007. DRE-CALUX bioassay in comparison withHRGC/MS for measurement of toxic equivalence in environmental samples. Sci.Total Environ. 372, 657–667.

Lasaridi, K., Protopapa, I., Kotsou, M., Pilidis, G., Manios, T., Kyriacou, A., 2006.Quality assessment of composts in the Greek market: the need for standardsand quality assurance. J. Environ. Manage. 80, 58–65.

Lazzari, L., Sperni, L., Salizzato, M., Pavoni, B., 1999. Gas chromatographicdetermination of organic micropollutants in samples of sewage sludge andcompost: behaviour of PCB and PAH during composting. Chemosphere 38,1925–1935.

Lee, K.T., Hong, S., Lee, J.S., Chung, K.H., Hilscherová, K., Giesy, J.P., Khim, J.S., 2013.Revised relative potency values for PCDDs, PCDFs, and non-ortho-substitutedPCBs for the optimized H4IIE-luc in vitro bioassay. Environ. Sci. Pollut. Res. Int.20, 8590–8599.

Machala, M., Vondrácek, J., Bláha, L., Ciganek, M., Neca, J., 2001. Aryl hydrocarbonreceptor-mediated activity of mutagenic polycyclic aromatic hydrocarbonsdetermined using in vitro reporter gene assay. Mutat. Res. Toxicol. Environ.Mutagen. 497, 49–62.

M. Beníšek et al. / Chemosphere 122 (2015) 168–175 175

McGowin, A.E., Adom, K.K., Obubuafo, A.K., 2001. Screening of compost for PAHsand pesticides using static subcritical water extraction. Chemosphere 45, 857–864.

Mrema, E.J., Rubino, F.M., Brambilla, G., Moretto, A., Tsatsakis, A.M., Colosio, C.,2013. Persistent organochlorinated pesticides and mechanisms of their toxicity.Toxicology 307, 74–88.

Muñoz, M., Gomez-Rico, M.F., Font, R., 2013. Dioxin-like PCB concentrations duringmunicipal solid waste biomethanation and subsequent composting.Chemosphere.

Murk, A.J., Legler, J., Denison, M.S., Giesy, J.P., van de Guchte, C., Brouwer, A., 1996.Chemical-activated luciferase gene expression (CALUX): a novel in vitrobioassay for Ah receptor active compounds in sediments and pore water.Fundam. Appl. Toxicol. 33, 149–160.

Pedra, F., Polo, A., Ribeiro, A., Domingues, H., 2007. Effects of municipal solid wastecompost and sewage sludge on mineralization of soil organic matter. Soil Biol.Biochem. 39, 1375–1382.

Samara, F., Gullett, B.K., Harrison, R.O., Chu, A., Clark, G.C., 2009. Determination ofrelative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activatedluciferase gene expression cell bioassay (CALUX). Environ. Int. 35, 588–593.

Saveyn, H., Eder, P., 2014. End-of-waste criteria for biodegradable waste subjectedto biological treatment (compost & digestate): Technical proposal. EC JRCScientific and Policy Reports (EUR 26425 EN).

Semple, K., 2001. Impact of composting strategies on the treatment of soilscontaminated with organic pollutants. Environ. Pollut. 112, 269–283.

Sorg, O., 2013. AhR signalling and dioxin toxicity. Toxicol. Lett.. http://dx.doi.org/10.1016/j.toxlet.2013.10.039.

Sovadinova, I., Blaha, L., Janosek, J., Hilscherova, K., Giesy, J.P., Jones, P.D., Holoubek,I., 2006. Cytotoxicity and aryl hydrocarbon receptor-mediated activity of

N-heterocyclic polycyclic aromatic hydrocarbons: structure-activityrelationships. Environ. Toxicol. Chem. 25, 1291–1297.

Suzuki, G., Takigami, H., Kushi, Y., Sakai, S., 2006. Time-course changes of mixtureeffects on AhR binding-dependent luciferase activity in a crude extract from acompost sample. Toxicol. Lett. 161, 174–187.

Takigami, H., Suzuki, G., Sakai, S., 2010. Screening of dioxin-like compounds in bio-composts and their materials: chemical analysis and fractionation-directedevaluation of AhR ligand activities using an in vitro bioassay. J. Environ. Monit.12, 2080–2087.

Tang, J.Y.M., McCarty, S., Glenn, E., Neale, P.A., Warne, M.S.J., Escher, B.I., 2013.Mixture effects of organic micropollutants present in water: towards thedevelopment of effect-based water quality trigger values for baseline toxicity.Water Res. 47, 3300–3314.

Tavazzi, S., Locoro, G., Comero, S., Sobiecka, E., Loos, R., Eder, P., Saveyn, H., Blaha, L.,Benisek, M., Gans, O., Hartl, W., Voorspoels, S., Ghiani, M., Umlauf, G., Mariani,G., Suurkuusk, G., Paracchini, B., Cristache, C., Fissiaux, I., Alonzo-Ruiz, A.,Gawlik, B., 2013. Occurrence and levels of selected compounds in Europeancompost and digestate samples. European Commission – Joint Research Centre,JRC Scientific and Policy Reports (EUR 26164 EN/ISBN 978-92-79-33195-4).

Umlauf, G., Christoph, E.H., Lanzini, L., Savolainen, R., Skejo, H., Bidoglio, G.,Clemens, J., Goldbach, H., Scherer, H., 2011. PCDD/F and dioxin-like PCB profilesin soils amended with sewage sludge, compost, farmyard manure, and mineralfertilizer since 1962. Environ. Sci. Pollut. Res. Int. 18, 461–470.

Villeneuve, D., Blankenship, A., Giesy, J., 2000. Derivation and application of relativepotency estimates based on in vitro bioassay results. Environ. Toxicol. Chem. 19,2835–2843.

WRAP, 2002. The Waste and Resources Action Program (WRAP): Comparison ofCompost Standards within the EU, North America and Australasia. Main report-section 1, WRAP, Banbury.

SUPPLEMENTARY MATERIALS:

Dioxins and dioxin-like compounds in composts and digestates from European countries as determined by the in vitro bioassay and

chemical analysis

Martin Beníšek1, Petr Kukučka1, Giulio Mariani2, Gert Suurkuusk2, Bernd M. Gawlik2, Giovanni Locoro2, John P. Giesy3,4,5,6, Luděk Bláha1*

1- Masaryk University, Faculty of Science, RECETOX

2-European Commission, DG Joint Research Centre (JRC), Institute for Environment and Sustainability,Unit H.01-Water Resources Unit, Ispra,

Italy

3- Department of Veterinary Biomedical sciences and Toxicology Centre, University of Saskatchewan, SK, Canada

4- Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong,

SAR, China

5- School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China

6- State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic

of China

* corresponding author

Table S1-A – Results of PAHs measurement for biobin+greenwaste compost (type A)-samples 1-14

Location Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Type A A A A A A A A A A A A A A

Country of origin Belgium France France Finland Italy Luxembourg Belgium Netherland Spain Sweden Netherland Denmark Germany Germany

PAH (µg/kg dm) Phenantrene* 5.2E+02 4.3E+01 6.0E+02 5.0E+01 4.1E+02 1.2E+01 2.2E+01 4.1E+02 8.3E+01 2.9E+01 1.5E+02 1.4E+02 3.3E+02 1.2E+01

Antracene* 9.4E+00 4.2E+01 4.2E+01 7.3E+00 3.1E+00 2.7E+01 1.6E+01 8.3E+01 4.6E+00 2.6E+01 4.8E+01 1.2E+01 1.4E+01 1.1E+01

Fluoranthene* 1.0E+03 7.9E+01 6.5E+02 1.0E+02 2.5E+02 1.4E+02 4.3E+02 7.8E+02 8.9E+01 1.4E+02 5.9E+02 1.7E+02 4.0E+02 2.1E+02

Pyrene* 5.9E+02 4.4E+01 3.7E+02 5.7E+01 1.3E+02 1.2E+02 2.9E+02 4.4E+02 7.8E+01 8.5E+01 4.5E+02 1.3E+02 2.4E+02 1.5E+02

Benzo(a)antracene* 8.5E+01 1.7E+01 1.3E+02 1.0E+01 2.7E+01 2.0E+02 1.1E+02 1.4E+02 1.9E+01 4.3E+01 1.3E+02 3.0E+01 7.1E+01 7.9E+01

Chrysene* 4.5E+02 2.5E+01 2.1E+02 1.3E+01 4.5E+03 3.6E+02 2.3E+02 2.0E+02 2.4E+01 7.2E+01 2.0E+02 4.8E+01 1.2E+02 1.3E+02

Benzo(b)fluoranthene* 4.1E+02 2.3E+01 1.2E+02 2.2E+01 3.7E+01 2.8E+02 1.5E+02 1.9E+02 2.3E+01 4.0E+01 1.1E+02 4.2E+01 7.9E+01 1.3E+02

Benzo(k)fluoranthene* 1.9E+02 1.4E+01 6.1E+01 1.6E+01 2.2E+01 1.4E+02 6.4E+01 8.1E+01 2.2E+01 2.1E+01 5.3E+01 2.6E+01 3.9E+01 6.5E+01

Benzo(e)pyrene 2.7E+02 1.9E+01 8.6E+01 1.6E+01 2.5E+01 2.2E+02 1.1E+02 1.3E+02 2.7E+00 2.3E+01 8.3E+01 4.0E+01 5.5E+01 8.9E+01

Benzo(a)pyrene* 2.4E+02 1.3E+01 3.9E+01 9.7E+00 2.8E+00 1.5E+02 5.3E+01 1.2E+02 2.8E+00 1.2E+01 6.3E+01 2.5E+01 3.6E+01 6.0E+01

Perylene 4.0E+01 1.4E+01 2.9E+01 1.7E+01 3.1E+00 5.8E+01 2.6E+01 5.0E+01 3.1E+00 1.0E+01 3.1E+01 1.8E+01 2.6E+01 4.7E+01

Indeno(1,2,3-cd)pyrene* 2.9E+02 4.9E+01 6.1E+01 1.9E+01 2.8E+01 1.6E+02 6.7E+01 1.3E+02 3.0E+00 1.9E+01 5.9E+01 2.7E+01 6.0E+01 7.0E+01

Dibenz(a,h)antracene* 9.7E+01 3.2E+00 4.4E+01 3.2E+00 3.2E+00 9.7E+01 4.2E+01 3.2E+00 3.2E+00 1.4E+01 3.8E+01 1.5E+01 3.0E+01 4.4E+01

Benzo(g,h,i)perylene* 2.8E+02 3.4E+00 7.3E+01 1.4E+01 3.4E+00 2.0E+02 8.7E+01 9.9E+01 2.4E+01 2.5E+01 7.2E+01 2.8E+01 7.0E+01 7.9E+01

Dibenzo(a,l)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,h)pyrene 4.6E+02 BLOQ BLOQ BLOQ BLOQ 1.1E+02 BLOQ BLOQ BLOQ BLOQ 1.8E+01 BLOQ BLOQ BLOQ

Dibenzo(a,i)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Coronene 1.1E+02 5.7E+00 4.7E+01 5.7E+00 5.7E+00 7.1E+01 5.7E+00 5.7E+00 5.7E+00 5.7E+00 1.7E+01 5.7E+00 5.7E+00 5.7E+00

Summaries ∑ all 19 PAHs (µg/kg dm) 5.1E+03 3.9E+02 2.6E+03 3.6E+02 5.5E+03 2.4E+03 1.7E+03 2.9E+03 3.9E+02 5.7E+02 2.1E+03 7.5E+02 1.6E+03 1.2E+03

∑12 EPA PAHs (µg/kg dm) 4.2E+03 3.5E+02 2.4E+03 3.2E+02 5.4E+03 1.9E+03 1.6E+03 2.7E+03 3.7E+02 5.3E+02 2.0E+03 6.9E+02 1.5E+03 1.0E+03

TEQPAH (µg/kg dm) 6.0E-01 4.5E-02 2.0E-01 3.9E-02 5.0E-01 4.5E-01 2.1E-01 2.1E-01 4.4E-02 6.7E-02 1.8E-01 7.7E-02 1.4E-01 2.0E-01

Table S1 –A - Results of PAHs measurement for biobin+greenwaste compost (type A)-samples 15-28

Location Code 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Type A A A A A A A A A A A A A A

Country of origin Germany Germany France Germany Italy Italy Italy Portugal Belgium UK Suisse Suisse Germany Belgium

PAH (µg/kg dm) Phenantrene* 8.6E+01 2.6E+01 5.0E+02 1.2E+02 4.5E+01 1.3E+02 1.6E+02 6.6E+01 3.1E+02 8.7E+01 1.1E+02 2.5E+02 2.4E+02 5.8E+01

Antracene* 2.6E+01 1.2E+01 1.0E+02 3.3E+01 4.9E+00 1.2E+01 3.9E+00 3.6E+00 3.5E+01 1.5E+01 2.6E+01 2.9E+01 2.9E+01 2.8E+01

Fluoranthene* 3.9E+02 6.9E+01 6.7E+02 2.7E+02 1.2E+02 2.4E+02 1.6E+02 1.2E+02 4.2E+02 1.8E+02 1.8E+02 2.8E+02 4.5E+02 7.0E+02

Pyrene* 2.6E+02 3.4E+01 4.4E+02 1.9E+02 1.0E+02 2.0E+02 1.7E+02 9.5E+01 2.5E+02 1.9E+02 1.3E+02 2.4E+02 2.6E+02 4.8E+02

Benzo(a)antracene* 9.6E+01 2.0E+02 2.0E+02 1.1E+02 2.5E+01 5.4E+01 2.9E+01 1.7E+01 8.1E+01 6.4E+02 7.4E+01 7.6E+01 9.2E+01 2.0E+02

Chrysene* 1.4E+02 3.6E+02 2.7E+02 1.6E+02 5.2E+01 9.0E+01 7.4E+01 4.6E+01 1.4E+02 1.2E+02 1.0E+02 1.1E+02 1.4E+02 3.5E+02

Benzo(b)fluoranthene* 9.1E+01 5.5E+02 2.0E+02 1.3E+02 4.2E+01 6.4E+01 3.1E+01 2.9E+01 9.9E+01 1.1E+02 1.0E+02 1.0E+02 9.8E+01 2.5E+02

Benzo(k)fluoranthene* 4.3E+01 2.5E+02 9.1E+01 7.1E+01 2.2E+01 3.6E+01 1.2E+01 1.5E+01 4.8E+01 5.0E+01 5.4E+01 5.9E+01 4.6E+01 1.2E+02

Benzo(e)pyrene 6.6E+01 3.7E+02 1.4E+02 9.5E+01 3.6E+01 5.2E+01 2.5E+01 2.4E+01 7.1E+01 9.0E+01 8.9E+01 8.2E+01 7.0E+01 2.0E+02

Benzo(a)pyrene* 4.6E+01 4.0E+02 1.2E+02 8.9E+01 2.6E+01 4.1E+01 2.8E+00 1.6E+01 4.7E+01 6.4E+01 8.2E+01 6.7E+01 5.3E+01 1.3E+02

Perylene 2.7E+01 1.6E+02 3.5E+01 4.8E+01 1.8E+01 2.3E+01 3.1E+00 1.2E+01 2.6E+01 2.5E+01 3.5E+01 3.6E+01 2.2E+01 4.4E+01

Indeno(1,2,3-cd)pyrene* 5.2E+01 3.8E+02 9.9E+01 1.0E+02 3.4E+01 7.1E+01 1.9E+01 5.4E+01 5.4E+01 7.7E+01 7.8E+01 6.1E+02 4.7E+01 1.4E+02

Dibenz(a,h)antracene* 3.2E+00 1.8E+02 6.0E+01 3.2E+00 1.7E+01 1.5E+01 8.0E+00 1.3E+01 3.4E+01 3.4E+01 3.8E+01 4.0E+01 3.1E+01 5.7E+01

Benzo(g,h,i)perylene* 6.1E+01 3.7E+02 1.2E+02 1.0E+02 4.0E+01 4.2E+01 3.5E+01 3.7E+01 4.9E+01 1.1E+02 9.7E+01 8.4E+01 6.6E+01 1.3E+02

Dibenzo(a,l)pyrene BLOQ BLOQ 9.8E+00 BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,h)pyrene BLOQ BLOQ 5.5E+01 BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 3.2E+01 4.5E+01 BLOQ BLOQ 7.2E+01

Dibenzo(a,i)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Coronene 5.7E+00 5.7E+00 5.2E+01 5.7E+00 1.0E+01 2.0E+01 1.8E+01 2.1E+01 5.7E+00 6.2E+01 4.1E+01 2.4E+01 3.8E+01 6.1E+01

Summaries ∑ all 19 PAHs (µg/kg dm) 1.4E+03 3.4E+03 3.2E+03 1.5E+03 6.0E+02 1.1E+03 7.5E+02 5.7E+02 1.7E+03 1.9E+03 1.3E+03 2.1E+03 1.7E+03 3.0E+03

∑12 EPA PAHs (µg/kg dm) 1.3E+03 2.8E+03 2.9E+03 1.4E+03 5.3E+02 9.9E+02 7.1E+02 5.1E+02 1.6E+03 1.7E+03 1.1E+03 2.0E+03 1.6E+03 2.6E+03

TEQPAH (µg/kg dm) 1.1E-01 8.3E-01 3.0E-01 1.8E-01 7.5E-02 1.1E-01 4.3E-02 6.3E-02 1.6E-01 1.7E-01 1.8E-01 3.5E-01 1.5E-01 3.6E-01

Table S1-B Results of PAHs measurement for greenwaste compost (Type B)-samples 1-12

Location Code 1 2 3 4 5 6 7 8 9 10 11 12

Type B B B B B B B B B B B B

Country of origin Belgium France Luxembourg Belgium Netherland Netherland Spain Sweden Denmark Germany Germany France

PAH (µg/kg dm) Phenantrene* 3.5E+02 1.1E+01 2.1E+01 1.6E+02 4.6E+00 1.5E+02 6.8E+01 5.9E+01 9.7E+00 4.5E+01 8.4E+00 8.8E+01

Antracene* 8.8E+01 1.3E+01 1.7E+02 3.1E+01 4.6E+00 3.2E+01 1.3E+01 2.6E+01 3.8E+00 1.5E+01 2.6E+00 2.3E+01

Fluoranthene* 6.6E+02 1.7E+02 3.4E+02 2.9E+02 2.6E+01 2.5E+02 1.2E+02 1.1E+02 7.1E+01 1.0E+02 4.1E+01 1.9E+02

Pyrene* 3.2E+02 7.7E+01 2.6E+02 2.0E+02 1.9E+01 1.7E+02 9.1E+01 8.2E+01 4.4E+01 2.9E+01 2.4E+01 1.5E+02

Benzo(a)antracene* 1.4E+02 4.8E+01 5.4E+02 9.0E+01 3.5E+01 7.5E+01 2.3E+01 5.0E+01 1.9E+01 3.8E+01 2.1E+01 7.3E+01

Chrysene* 2.4E+02 8.6E+01 7.1E+02 1.7E+02 7.8E+01 1.4E+02 3.8E+01 9.2E+01 5.1E+01 9.6E+01 7.9E+01 1.3E+02

Benzo(b)fluoranthene* 1.5E+02 7.2E+01 7.3E+02 1.5E+02 6.1E+01 1.3E+02 2.0E+01 8.9E+01 4.4E+01 1.0E+02 7.8E+01 1.1E+02

Benzo(k)fluoranthene* 7.2E+01 4.2E+01 3.5E+02 7.3E+01 2.9E+01 5.6E+01 1.0E+01 4.1E+01 2.1E+01 4.2E+01 2.5E+01 5.1E+01

Benzo(e)pyrene 9.6E+01 5.6E+01 5.1E+02 1.0E+02 3.8E+01 9.9E+01 1.5E+01 7.4E+01 3.5E+01 7.3E+01 5.4E+01 7.9E+01

Benzo(a)pyrene* 7.6E+01 4.4E+01 5.3E+02 8.6E+01 2.8E+00 7.5E+01 1.7E+01 6.4E+01 2.4E+01 4.7E+01 3.4E+01 5.4E+01

Perylene 3.7E+01 2.1E+01 2.0E+02 8.1E+00 3.1E+00 3.5E+01 5.4E+00 2.8E+01 1.2E+01 2.3E+01 1.8E+01 2.3E+01

Indeno(1,2,3-cd)pyrene* 7.5E+01 5.7E+01 4.2E+02 9.2E+01 3.4E+01 7.4E+01 1.2E+01 6.6E+01 2.8E+01 7.1E+01 3.9E+01 6.6E+01

Dibenz(a,h)antracene* 3.0E+01 3.2E+00 2.7E+02 5.9E+01 3.2E+00 4.1E+01 4.5E+00 3.5E+01 9.3E+00 3.2E+00 1.6E+01 4.2E+01

Benzo(g,h,i)perylene* 8.6E+01 4.9E+01 4.7E+02 1.2E+02 3.6E+01 9.4E+01 2.5E+01 8.2E+02 2.7E+01 7.4E+01 4.9E+01 9.0E+01

Dibenzo(a,l)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,h)pyrene BLOQ BLOQ 7.9E+02 1.1E+02 BLOQ BLOQ BLOQ 3.1E+01 BLOQ BLOQ BLOQ 3.2E+01

Dibenzo(a,i)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Coronene 5.7E+00 5.7E+00 1.4E+02 3.3E+01 5.7E+00 2.3E+01 1.2E+01 3.9E+01 5.7E+00 5.7E+00 5.7E+00 3.9E+01

Summaries ∑ all 19 PAHs (µg/kg dm) 2.4E+03 7.5E+02 6.4E+03 1.8E+03 3.8E+02 1.5E+03 4.8E+02 1.7E+03 4.0E+02 7.7E+02 5.0E+02 1.2E+03

∑12 EPA PAHs (µg/kg dm) 2.3E+03 6.7E+02 4.8E+03 1.5E+03 3.3E+02 1.3E+03 4.5E+02 1.5E+03 3.5E+02 6.6E+02 4.2E+02 1.1E+03

TEQPAH (µg/kg dm) 2.1E-01 1.1E-01 1.2E+00 2.5E-01 7.1E-02 1.9E-01 3.2E-02 1.5E-01 6.2E-02 1.1E-01 8.5E-02 1.7E-01

Table S1-B Results of PAHs measurement for greenwaste kompost (Type B)-Samples 13-23

Location Code 13 14 15 16 17 18 19 20 21 22 23

Type B B B B B B B B B B B

Country of origin France France France France France France Belgium Belgium Suisse Italy France

PAH (µg/kg dm) Phenantrene* 1.8E+01 6.6E+01 4.5E+02 7.8E+01 1.0E+01 1.9E+01 3.4E+01 2.1E+02 7.6E+01 3.0E+01 5.0E+01

Antracene* 2.5E+01 4.4E+01 2.3E+01 1.6E+01 1.1E+01 4.6E+01 2.0E+01 9.0E+01 1.4E+01 2.7E+00 4.1E+00

Fluoranthene* 1.1E+02 1.8E+02 3.4E+02 3.6E+02 3.8E+01 4.5E+01 5.5E+02 5.0E+02 1.4E+02 3.7E+01 9.1E+01

Pyrene* 9.5E+01 1.1E+02 1.9E+02 2.2E+02 2.4E+01 2.7E+01 2.6E+02 3.4E+02 9.5E+01 3.2E+01 4.4E+01

Benzo(a)antracene* 5.5E+01 1.2E+02 6.5E+01 7.4E+01 2.8E+01 3.7E+01 1.1E+02 5.7E+02 4.4E+01 1.9E+01 2.2E+01

Chrysene* 1.4E+02 2.5E+02 1.0E+02 1.3E+02 8.5E+01 1.5E+02 1.9E+02 8.4E+02 7.6E+01 2.4E+01 5.3E+01

Benzo(b)fluoranthene* 9.0E+01 3.8E+02 7.6E+01 9.3E+01 9.1E+01 1.8E+02 1.1E+02 1.1E+03 7.4E+01 2.4E+01 4.1E+01

Benzo(k)fluoranthene* 3.6E+01 1.7E+02 4.5E+01 5.1E+01 3.1E+01 5.9E+01 5.4E+01 6.0E+02 3.7E+01 1.2E+01 2.1E+01

Benzo(e)pyrene 7.2E+01 2.8E+02 5.5E+01 6.9E+01 7.0E+01 1.4E+02 8.2E+01 8.5E+02 6.0E+01 1.8E+01 2.9E+01

Benzo(a)pyrene* 3.0E+01 2.3E+02 4.6E+01 5.3E+01 4.2E+01 6.1E+01 5.2E+01 9.0E+02 4.7E+01 2.2E+01 1.7E+01

Perylene 1.7E+01 7.9E+01 3.0E+01 2.8E+01 1.8E+01 2.9E+01 3.0E+01 2.1E+02 2.2E+01 4.5E+00 1.1E+01

Indeno(1.2.3-cd)pyrene* 6.2E+01 3.0E+02 2.5E+01 6.7E+01 5.4E+01 1.6E+02 6.2E+01 8.2E+02 5.3E+01 2.0E+01 2.8E+01

Dibenz(a,h)antracene* 2.5E+01 1.6E+02 3.6E+01 4.0E+01 2.6E+01 6.5E+01 4.0E+01 4.0E+02 2.6E+01 7.2E+00 1.2E+01

Benzo(g,h,i)perylene* 7.7E+01 3.1E+02 4.9E+01 6.4E+01 6.4E+01 1.6E+02 6.7E+01 5.0E+02 7.2E+01 2.2E+01 3.0E+01

Dibenzo(a,l)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,h)pyrene BLOQ 2.6E+02 BLOQ BLOQ BLOQ 9.2E+01 BLOQ 3.7E+02 BLOQ BLOQ BLOQ

Dibenzo(a,i)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Coronene 5.7E+00 1.0E+02 5.7E+00 5.7E+00 3.4E+01 5.7E+01 5.7E+00 1.9E+02 3.1E+01 5.7E+00 5.7E+00

Summaries ∑ all 19 PAHs (µg/kg dm) 8.6E+02 3.0E+03 1.5E+03 1.3E+03 6.3E+02 1.3E+03 1.7E+03 8.5E+03 8.7E+02 2.8E+02 4.6E+02

∑12 EPA PAHs (µg/kg dm) 7.7E+02 2.3E+03 1.4E+03 1.2E+03 5.0E+02 1.0E+03 1.5E+03 6.9E+03 7.5E+02 2.5E+02 4.1E+02

TEQPAH (µg/kg dm) 1.3E-01 6.1E-01 1.4E-01 1.7E-01 1.1E-01 2.5E-01 1.8E-01 1.9E+00 1.2E-01 3.9E-02 6.6E-02

Table S1 – C Results of PAHs measurment for sewage sludge compost (Type C)

Location Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Type C C C C C C C C C C C C C C C C

Country of origin France Finland Luxembourg Spain Germany Austria Austria Austria France France France France France France Czech Rep. United Kingdom

PAH (µg/kg dm) Phenantrene* 2.3E+02 1.0E+02 2.8E+01 3.4E+01 1.4E+02 1.4E+02 5.5E+02 1.5E+02 4.6E+01 2.2E+01 3.2E+01 6.8E+01 6.9E+01 3.1E+02 5.4E+01 1.8E+01

Antracene* 4.9E+01 1.5E+01 8.5E+01 4.6E+00 4.2E+01 2.0E+01 3.9E+01 2.2E+01 9.0E+00 8.2E+00 3.1E+01 1.3E+01 1.4E+01 4.6E+01 2.0E+01 1.8E+01

Fluoranthene* 2.6E+02 2.0E+02 1.0E+03 2.9E+01 3.3E+02 2.4E+02 5.9E+02 4.1E+02 1.1E+02 1.8E+02 3.6E+02 2.2E+02 8.1E+01 3.1E+02 1.8E+02 4.5E+01

Pyrene* 1.6E+02 1.9E+02 8.7E+02 5.6E+01 2.2E+02 1.7E+02 4.2E+02 2.9E+02 8.5E+01 1.2E+02 2.7E+02 1.7E+02 6.7E+01 2.3E+02 1.7E+02 4.5E+01

Benzo(a)antracene* 8.2E+01 5.9E+01 9.3E+02 2.0E+01 8.3E+01 6.3E+01 9.0E+01 8.6E+01 3.0E+01 7.6E+01 9.2E+01 7.2E+01 2.5E+01 9.2E+01 2.0E+02 7.2E+01

Chrysene* 6.5E+01 1.3E+02 1.4E+03 3.7E+01 1.5E+02 9.3E+01 1.3E+02 1.1E+02 4.7E+01 1.2E+02 1.3E+02 1.1E+02 3.4E+01 1.2E+02 3.2E+02 1.8E+02

Benzo(b)fluoranthene* 9.4E+01 1.2E+02 1.3E+03 5.3E+01 1.1E+02 8.0E+01 7.1E+01 1.0E+02 4.4E+01 8.7E+01 1.3E+02 1.0E+02 3.6E+01 9.1E+01 4.1E+02 1.6E+02

Benzo(k)fluoranthene* 2.9E+01 6.6E+01 6.4E+02 2.3E+01 5.3E+01 3.9E+01 3.3E+01 5.6E+01 2.7E+01 4.8E+01 6.7E+01 5.5E+01 2.2E+01 5.4E+01 1.9E+02 7.0E+01

Benzo(e)pyrene 3.2E+01 8.0E+01 9.6E+02 5.7E+01 7.9E+01 5.5E+01 4.4E+01 7.2E+01 3.6E+01 6.7E+01 1.0E+02 7.1E+01 2.5E+01 5.5E+01 3.4E+02 1.4E+02

Benzo(a)pyrene* 7.3E+01 7.0E+01 7.7E+02 4.3E+01 4.9E+01 5.5E+01 3.9E+01 7.0E+01 3.0E+01 5.1E+01 9.4E+01 4.8E+01 2.0E+01 6.3E+01 3.6E+02 8.1E+01

Perylene 9.3E+00 3.5E+01 2.5E+02 3.1E+00 3.3E+01 2.4E+01 9.7E+00 3.5E+01 2.6E+01 2.0E+01 2.8E+01 2.6E+01 3.1E+00 1.2E+01 1.3E+02 3.0E+01

Indeno(1,2,3-cd)pyrene* 3.4E+01 9.5E+01 7.2E+02 2.5E+01 7.3E+01 7.5E+01 3.5E+01 6.6E+01 4.3E+01 5.0E+01 8.7E+01 1.0E+02 4.1E+01 8.7E+01 3.4E+02 9.0E+01

Dibenz(a,h)antracene* 2.0E+01 3.3E+01 4.1E+02 3.2E+00 4.4E+01 3.1E+01 1.7E+01 2.9E+01 2.3E+01 2.8E+01 3.7E+01 3.2E+00 3.2E+00 4.5E+01 1.5E+02 4.9E+01

Benzo(g,h,i)perylene* 4.3E+01 9.0E+01 8.7E+02 6.7E+01 8.6E+01 8.6E+01 5.5E+01 9.1E+01 5.4E+01 6.0E+01 8.2E+01 9.6E+01 2.7E+01 8.1E+01 4.1E+02 1.2E+02

Dibenzo(a,l)pyrene BLOQ BLOQ 7.1E+01 BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 8.6E+00 BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,h)pyrene BLOQ 3.4E+01 4.3E+02 BLOQ BLOQ BLOQ BLOQ 3.7E+01 BLOQ BLOQ 4.8E+01 BLOQ BLOQ BLOQ 3.5E+02 3.5E+01

Dibenzo(a,i)pyrene BLOQ BLOQ 2.8E+01 BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ 7.6E+01 BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Coronene 5.7E+00 4.3E+01 3.6E+02 5.7E+00 5.7E+00 5.7E+00 4.7E+01 5.0E+01 5.7E+00 5.7E+00 4.4E+01 5.7E+00 5.7E+00 1.5E+01 1.8E+02 5.0E+01

Summaries ∑ all 19 PAHs (µg/kg dm) 1.2E+03 1.4E+03 1.1E+04 4.6E+02 1.5E+03 1.2E+03 2.2E+03 1.7E+03 6.1E+02 9.4E+02 1.6E+03 1.2E+03 4.7E+02 1.6E+03 3.8E+03 1.2E+03

∑12 EPA PAHs (µg/kg dm) 1.1E+03 1.2E+03 9.1E+03 3.9E+02 1.4E+03 1.1E+03 2.1E+03 1.5E+03 5.4E+02 8.5E+02 1.4E+03 1.1E+03 4.4E+02 1.5E+03 2.8E+03 9.4E+02

TEQPAH (µg/kg dm) 9.9E-02 2.0E-01 2.0E+00 5.8E-02 1.8E-01 1.4E-01 1.0E-01 1.7E-01 9.2E-02 1.5E-01 2.1E-01 1.4E-01 5.9E-02 1.9E-01 6.7E-01 2.3E-01

Table S1-D Results of PAHs measurement for mechanical biological treatment compost (Type D)

Location Code 1 2 3 4 5 6 7 8

Type D D D D D D D D

Country of origin France France France France France Portugal Portugal UK

PAH (µg/kg dm) Phenantrene* 1.2E+02 1.7E+02 2.2E+02 9.6E+01 9.1E+01 9.8E+01 4.9E+01 2.2E+02

Antracene* 5.7E+00 4.6E+00 2.4E+01 1.0E+01 9.6E+00 5.2E+00 3.5E+00 3.3E+01

Fluoranthene* 9.1E+01 8.5E+01 3.0E+02 1.1E+02 9.6E+01 6.5E+01 3.4E+01 3.2E+02

Pyrene* 6.4E+01 8.7E+01 1.9E+02 9.6E+01 7.7E+01 6.6E+01 4.2E+01 2.3E+02

Benzo(a)antracene* 3.0E+01 2.4E+01 1.0E+01 4.1E+01 4.7E+01 2.0E+01 2.5E+01 1.7E+02

Chrysene* 4.0E+01 4.9E+01 1.5E+02 6.1E+01 6.1E+01 3.8E+01 3.3E+01 2.1E+02

Benzo(b)fluoranthene* 4.6E+01 3.4E+01 1.1E+02 5.0E+01 5.2E+01 2.7E+01 2.3E+01 1.7E+02

Benzo(k)fluoranthene* 3.0E+01 1.4E+01 5.6E+01 2.9E+01 3.3E+01 1.5E+01 1.3E+01 8.9E+01

Benzo(e)pyrene 3.6E+01 8.4E+01 8.5E+01 4.3E+01 4.7E+01 2.5E+01 2.0E+01 1.4E+02

Benzo(a)pyrene* 3.3E+01 2.5E+01 6.2E+01 3.5E+01 4.6E+01 1.8E+01 2.3E+01 1.4E+02

Perylene 1.9E+01 2.0E+01 3.2E+01 2.2E+01 2.8E+01 1.4E+01 5.8E+00 5.6E+01

Indeno(1,2,3-cd)pyrene* 6.4E+01 2.3E+01 6.2E+01 3.9E+01 4.0E+01 2.4E+01 1.9E+01 1.3E+02

Dibenz(a,h)antracene* 3.2E+00 2.0E+01 3.2E+00 2.2E+01 2.3E+01 1.2E+01 1.6E+01 5.9E+01

Benzo(g,h,i)perylene* 4.6E+01 3.3E+01 7.1E+01 5.1E+01 4.2E+01 3.0E+01 4.0E+01 1.8E+02

Dibenzo(a,l)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,h)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,i)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Coronene 5.7E+00 5.7E+00 5.7E+00 2.2E+01 5.7E+00 5.7E+00 3.2E+01 5.7E+00

Summaries ∑ all 19 PAHs (µg/kg dm) 6.3E+02 6.8E+02 1.4E+03 7.2E+02 7.0E+02 4.6E+02 3.8E+02 2.2E+03

∑12 EPA PAHs (µg/kg dm) 5.7E+02 5.7E+02 1.3E+03 6.4E+02 6.2E+02 4.2E+02 3.2E+02 2.0E+03

TEQPAH (µg/kg dm) 8.1E-02 6.2E-02 1.4E-01 9.7E-02 1.1E-01 5.2E-02 5.2E-02 3.0E-01

Table S1 – part E – Results of PAHs measurement for manure and biowaste digestate (Type F), manure and energy crop digestate (Type G), MBT digestate (Type H) and other compost samples (Type i)

Location Code 1 2 3 4 5 1 1 2 1 2 3 4 5

Type F F F F F G H H i i i i i

Country of origin Belgium Luxembourg Austria Belgium Belgium Austria Netherland France France Netherland Austria Austria France

PAH (µg/kg dm) Phenantrene* 5.5E+01 4.9E+01 2.7E+01 2.4E+02 2.7E+02 6.9E+01 2.2E+01 9.3E+01 3.3E+01 3.9E+03 5.3E+01 7.0E+01 9.6E+01

Antracene* 4.6E+00 4.6E+00 4.6E+00 3.8E+01 3.3E+01 1.1E+01 1.7E+01 1.9E+01 9.5E+00 3.5E+02 2.5E+01 2.0E+01 1.0E+01

Fluoranthene* 2.5E+01 1.4E+02 1.7E+01 1.7E+02 2.0E+02 1.2E+02 1.3E+02 2.8E+02 5.0E+01 4.8E+03 6.3E+01 4.5E+02 1.5E+02

Pyrene* 3.1E+01 1.1E+02 1.9E+01 1.1E+02 1.5E+02 1.0E+02 1.1E+02 1.7E+02 2.2E+01 3.2E+03 5.4E+01 4.7E+02 1.1E+02

Benzo(a)antracene* 2.5E+01 6.2E+01 5.7E+00 6.4E+01 5.8E+01 4.5E+01 9.0E+01 9.5E+01 2.0E+01 1.9E+03 3.4E+01 1.3E+02 6.8E+01

Chrysene* 2.7E+01 9.0E+01 1.0E+01 6.6E+01 7.2E+01 6.8E+01 1.5E+02 1.4E+02 6.6E+01 2.1E+03 4.5E+01 1.7E+02 9.5E+01

Benzo(b)fluoranthene* 6.3E+01 8.6E+01 9.9E+00 1.5E+02 7.5E+01 6.1E+01 1.9E+02 1.1E+02 9.2E+01 1.6E+03 6.6E+01 8.7E+01 9.1E+01

Benzo(k)fluoranthene* 2.1E+01 4.7E+01 2.4E+00 6.1E+01 4.6E+01 4.3E+01 8.8E+01 5.3E+01 3.5E+01 8.5E+02 3.3E+01 4.1E+01 5.0E+01

Benzo(e)pyrene 2.4E+01 6.4E+01 3.4E+00 7.6E+01 1.1E+02 3.9E+01 1.4E+02 8.3E+01 6.8E+01 1.5E+03 6.5E+01 7.1E+01 8.6E+01

Benzo(a)pyrene* 2.6E+01 8.3E+01 2.8E+00 1.8E+02 1.4E+02 4.5E+01 1.5E+02 7.3E+01 5.2E+01 1.4E+03 6.4E+01 5.3E+01 8.9E+01

Perylene 3.1E+00 3.0E+01 3.1E+00 4.1E+01 8.7E+00 1.8E+01 5.7E+01 2.9E+01 2.8E+01 3.9E+02 2.4E+01 3.1E+01 3.1E+01

Indeno(1,2,3-cd)pyrene* 2.7E+01 8.0E+01 3.0E+00 3.0E+00 3.0E+00 3.3E+01 1.5E+02 6.9E+01 7.0E+01 9.1E+02 8.3E+01 4.5E+01 7.2E+01

Dibenz(a,h)antracene* 1.4E+01 BLOQ 3.2E+00 3.2E+00 3.2E+00 2.7E+01 6.8E+01 3.9E+01 2.4E+01 5.8E+02 3.0E+01 3.2E+01 4.0E+01

Benzo(g,h,i)perylene* 4.0E+01 8.6E+01 3.4E+00 3.4E+00 3.4E+00 6.0E+01 1.7E+02 7.5E+01 7.4E+01 1.3E+03 8.2E+01 8.1E+01 8.5E+01

Dibenzo(a,l)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 1.2E+02 1.2E+01 BLOQ 1.2E+01

Dibenzo(a,h)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 7.7E+01 BLOQ BLOQ 6.5E+02 5.1E+01 1.8E+01 4.9E+01

Dibenzo(a,i)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

Dibenzo(a,e)pyrene BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 2.1E+02 BLOQ BLOQ BLOQ

Coronene 5.7E+00 5.7E+00 5.7E+00 5.7E+00 5.7E+00 5.7E+00 4.0E+01 4.0E+01 2.8E+01 5.0E+02 8.7E+01 7.9E+01 4.3E+01

Summaries ∑ all 19 PAHs (µg/kg dm) 3.9E+02 9.3E+02 1.2E+02 1.2E+03 1.2E+03 7.4E+02 1.6E+03 1.4E+03 6.7E+02 2.6E+04 8.7E+02 1.8E+03 1.2E+03

∑12 EPA PAHs (µg/kg dm) 3.6E+02 8.3E+02 1.1E+02 1.1E+03 1.0E+03 6.8E+02 1.3E+03 1.2E+03 5.5E+02 2.3E+04 6.3E+02 1.6E+03 9.6E+02

TEQPAH (µg/kg dm) 6.6E-02 1.2E-01 1.0E-02 1.3E-01 1.0E-01 1.2E-01 3.0E-01 1.8E-01 1.2E-01 2.8E+00 1.3E-01 1.5E-01 1.7E-01

In italic bold are indicated values as LOD/2, BLOQ-below limit of quantification, PAHs – polycyclic aromatic hydrocarbons, *-12 EPA PAHs, TEQPAH – toxic equivalent of 2,3,7,8 TCDD for PAHs calculated from analytical results and induction equivalency factors for PAHs

Table S1-part F – Recoveries and limits of quantification of studied PAHs

PAH LOQ µg/kg Recovery (%)

Phenanthrene 10.7 62

Anthracene 7.8 64

Fluoranthene 5.3 67

Pyrene 6.0 68

Benzo(a)anthracene 5.6 74

Chrysene 6.8 74

Benzo(b)fluoranthene 10.7 75

Benzo(k)fluoranthene 11.6 75

Benzo(e)pyrene 11.5 70

Benzo(a)pyrene 6.4 76

Perilene 7.4 69

Indeno(1,2,3-cd)pyrene 13.6 67

Dibenzo(a,h)anthracene 7.9 74

Benzo(g,h,i)perilene 11.6 56

Dibenzo(a,l)pyrene 92.9 63

Dibenzo(a,h)pyrene 97.0 63

Dibenzo(a,i)pyrene 848.0 63

Dibenzo(a,e)pyrene 961.9 63

Coronene 88.1 31

Table S2-A : 17 EPA PCDDs/Fs values for 17 selected samples

Sample number 1 2 3 4 1 2 1 2 3 1 2 1 2 1 1 2 1

Sample type A A A A B B C C C D D F F G H H i

Country of origin Belgium Germany Belgium Belgium Belgium Germany Luxembourg Germany France France United Kingdom Luxembourg Belgium Austria Netherland France Netherland

PCDDs/Fs 2378-TCDF 2.1E+00 <1.1E+00 1.6E+00 9.1E-01 1.8E+00 1.4E+00 <5.9E+00 2.3E+00 <1.7E+00 7.9E-01 1.6E+00 <7.7E-01 <8.4E-01 <6.1E-01 1.1E+00 1.1E+00 3.2E+00

ng/kg dm 12378-PeCDF 1.0E+00 <7.4E-01 <1.1E+00 <1.3E+00 <9.9E-01 1.6E+00 <2.6E+00 <1.8E+00 <1.6E+00 <7.0E-01 <9.0E-01 <4.5E-01 <7.3E-01 <8.0E-01 7.6E-01 <8.6E-01 1.9E+00

23478-PeCDF 1.6E+00 1.0E+00 1.2E+00 <1.3E+00 1.1E+00 1.9E+00 <2.3E+00 <1.8E+00 <1.6E+00 <8.2E-01 <1.8E+00 <5.5E-01 <7.2E-01 <8.3E-01 9.0E-01 <8.1E-01 <2.6E+00

123478-HxCDF 1.1E+00 1.1E+00 <1.2E+00 <1.4E+00 8.3E-01 5.3E+00 <5.0E+00 <3.7E+00 <1.9E+00 <1.3E+00 1.4E+00 <5.1E+00 <1.8E+01 <1.8E+01 <7.1E-01 <2.4E+00 <7.3E+00

123678-HxCDF 1.0E+00 1.7E+00 <1.2E+00 <1.4E+00 8.1E-01 5.8E+00 <4.9E+00 <3.6E+00 <1.8E+00 <1.3E+00 1.9E+00 <8.0E+00 <1.8E+01 <1.7E+01 7.7E-01 <2.3E+00 <6.1E+00

234678-HxCDF <9.7E-01 <9.6E-01 <1.2E+00 <1.5E+00 <8.5E-01 5.2E+00 <4.9E+00 <3.8E+00 <1.9E+00 <1.3E+00 <1.5E+00 <5.9E+00 <2.3E+01 <2.5E+01 <7.6E-01 <2.5E+00 <8.1E+00

123789-HxCDF <1.2E+00 <1.2E+00 <1.6E+00 <1.7E+00 <1.0E+00 1.1E+00 <5.1E+00 <4.6E+00 <2.3E+00 <1.7E+00 <2.4E+00 <8.1E+00 <2.3E+01 <3.6E+01 <9.5E-01 <2.9E+00 <1.2E+01

1234678-HpCDF 7.0E+00 3.7E+00 5.3E+00 2.2E+00 3.5E+00 4.0E+01 <3.3E+00 4.6E+00 2.5E+00 3.6E+00 9.7E+00 <3.4E+00 <4.5E+00 <1.1E+01 3.7E+00 <2.6E+00 3.6E+01

1234789-HpCDF <1.9E+00 <1.2E+00 <1.8E+00 <1.8E+00 <1.9E+00 3.9E+00 <3.7E+00 <3.1E+00 <2.6E+00 <2.2E+00 <3.7E+00 <2.9E+00 <3.3E+00 <4.8E+00 <1.9E+00 <3.4E+00 1.2E+01

OCDF 4.9E+00 5.3E+00 5.7E+00 2.9E+00 6.1E+00 3.9E+01 2.5E+00 6.6E+00 5.6E+00 7.5E+00 1.5E+01 <1.6E+00 <2.3E+00 <1.7E+00 4.9E+00 2.9E+00 1.5E+01

2378-TCDD <9.9E-01 <9.9E-01 <9.3E-01 <1.3E+00 <1.1E+00 <8.2E-01 <5.3E+00 <1.7E+00 <1.4E+00 <9.8E-01 <1.6E+00 <9.5E+00 <9.0E-01 <7.0E-01 <7.1E-01 <1.2E+00 <1.9E+00

12378-PeCDD <9.7E-01 <1.0E+00 <1.3E+00 <1.5E+00 <1.3E+00 <1.1E+00 <3.3E+00 <2.1E+00 <2.2E+00 <1.2E+00 <1.2E+00 <1.9E+00 <1.7E+00 <1.5E+00 <1.1E+00 <1.3E+00 <3.8E+00

123478-HxCDD <7.4E-01 <9.1E-01 <1.5E+00 <1.8E+00 <1.2E+00 <6.6E-01 <3.9E+00 <2.8E+00 <2.4E+00 2.3E+00 <1.6E+00 <2.2E+00 <4.9E+00 <1.1E+01 <1.0E+00 <1.5E+00 <4.9E+00

123678-HxCDD 1.6E+00 <9.3E-01 <1.5E+00 <1.8E+00 <1.3E+00 <6.6E-01 <3.8E+00 <2.8E+00 4.1E+00 <1.2E+00 <1.9E+00 <2.2E+00 <4.8E+00 <9.1E+00 <9.8E-01 <1.5E+00 <5.4E+00

123789-HxCDD <1.3E-02 <1.3E-02 <1.4E+00 <1.0E-02 <1.0E-02 <6.3E-01 <3.7E+00 <2.6E+00 <1.3E-02 2.1E+00 <1.0E-02 <2.1E+00 <4.6E+00 <9.9E+00 <9.4E-01 <1.0E-02 <4.6E+00

1234678-HpCDD 7.1E+01 1.7E+01 3.2E+01 4.3E+01 1.6E+01 1.4E+01 8.7E+00 2.2E+01 9.9E+01 2.3E+01 4.7E+01 1.8E+00 3.8E+00 <4.2E+00 1.0E+01 5.9E+01 1.1E+02

OCDD 3.2E+02 6.7E+01 1.8E+02 1.7E+02 9.7E+01 5.3E+01 2.8E+01 8.6E+01 2.3E+02 1.9E+02 3.9E+02 7.3E+00 1.1E+01 7.9E+00 5.5E+01 2.1E+02 3.6E+02

Total 17 EPA PCDDs/Fs 4.1E+02 9.7E+01 2.3E+02 2.2E+02 1.3E+02 1.7E+02 4.0E+01 1.2E+02 3.4E+02 2.3E+02 4.7E+02 9.2E+00 1.5E+01 7.9E+00 7.7E+01 2.7E+02 5.4E+02

Total-TEQ LB WHO98 2.3E+00 1.0E+00 1.1E+00 5.6E-01 1.1E+00 3.4E+00 9.0E-02 5.1E-01 2.2E+00 8.1E-01 1.1E+00 3.0E-02 4.0E-02 0.0E+00 8.1E-01 1.3E+00 2.0E+00

Total-TEQ UB WHO98 4.5E+00 3.6E+00 4.4E+00 5.1E+00 3.9E+00 5.6E+00 1.4E+01 7.8E+00 7.9E+00 4.2E+00 5.7E+00 1.5E+01 1.3E+01 1.5E+01 3.2E+00 5.6E+00 1.4E+01

Total-TEQ LB WHO2006 2.0E+00 8.3E-01 9.4E-01 5.9E-01 8.9E-01 3.0E+00 1.0E-01 5.3E-01 2.2E+00 8.5E-01 1.2E+00 3.0E-02 4.0E-02 0.0E+00 6.3E-01 1.4E+00 2.1E+00

Total-TEQ UB WHO2006 4.3E+00 3.4E+00 4.2E+00 4.8E+00 3.7E+00 5.2E+00 1.3E+01 7.4E+00 7.6E+00 4.0E+00 5.4E+00 1.5E+01 1.3E+01 1.5E+01 3.0E+00 5.5E+00 1.3E+01

Total I-TEQ LB 2.6E+00 1.1E+00 1.3E+00 7.1E-01 1.2E+00 3.5E+00 1.2E-01 5.9E-01 1.7E+00 9.9E-01 1.5E+00 3.0E-02 5.0E-02 1.0E-02 8.6E-01 9.1E-01 2.4E+00

Total I-TEQ UB 4.3E+00 3.1E+00 3.9E+00 4.5E+00 3.4E+00 5.1E+00 1.2E+01 6.8E+00 6.3E+00 3.7E+00 5.4E+00 1.4E+01 1.2E+01 1.5E+01 2.7E+00 4.5E+00 1.2E+01

Total TEQH4IIELB 5.8E+00 1.7E+00 2.8E+00 2.4E+00 2.1E+00 5.8E+00 4.3E-01 2.0E+00 5.3E+00 2.4E+00 3.5E+00 8.8E-02 1.8E-01 3.9E-03 1.6E+00 3.2E+00 8.3E+00

Total TEQH4IIE UB 7.9E+00 4.5E+00 6.1E+00 7.0E+00 5.0E+00 7.7E+00 1.6E+01 9.4E+00 1.1E+01 5.5E+00 8.3E+00 1.5E+01 1.5E+01 1.9E+01 3.8E+00 7.7E+00 2.0E+01

Table S2-B – Recoveries and limits of quantification of studied PCDDs/Fs

PCDDs/Fs % ng/kg

recovery min recovery max recovery average LOQ min LOQ max LOQ average 2378-TCDF 57 74 68 0.6 5.9 1.2

12378-PeCDF 61 146 78 0.5 2.6 1.1

23478-PeCDF 44 118 76 0.6 2.6 1.1

123478-HxCDF 30 102 79 0.7 17.9 3.7

123678-HxCDF 32 93 69 0.7 18.1 3.7

234678-HxCDF 27 102 77 0.8 24.6 4.4

123789-HxCDF 23 109 77 1.0 36.1 5.6

1234678-HpCDF 17 117 72 0.9 12.4 3.0

1234789-HpCDF 25 136 73 1.2 10.0 2.9

OCDF 65 185 87 1.2 3.7 2.0

2378-TCDD 7 66 58 0.7 9.5 1.8

12378-PeCDD 63 107 76 1.0 3.8 1.7

123478-HxCDD 20 109 80 0.7 10.5 2.4

123678-HxCDD 30 106 72 0.7 9.1 2.4

123789-HxCDD 25 121 75 0.6 9.9 2.9

1234678-HpCDD 20 136 78 1.1 4.2 1.8

OCDD 65 203 93 1.2 3.7 2.2

Table S3-A– Dioxin-like PCBs values for 17 selected samples

Number of sample 1 2 3 4 1 2 1 2 3 1 2 1 2 1 1 2 1

Sample code A A A A B B C C C D D F F G H H i

Country of origin Belgium Germany Belgium Belgium Belgium Germany Luxembourg Germany France France UK Luxembourg Belgium Austria Netherland France Netherland

Dl PCBs PCB77 5.7E+01 3.4E+01 9.7E+01 6.3E+01 5.3E+01 3.8E+01 1.0E+02 7.9E+01 1.4E+02 5.9E+01 1.5E+02 6.0E+00 3.4E+01 1.3E+01 2.3E+01 2.1E+02 3.4E+02

ng/kg, dm PCB81 3.8E+00 3.0E+00 8.6E+00 7.0E+00 3.9E+00 4.2E+00 2.3E+01 4.7E+00 1.2E+01 3.9E+00 8.1E+00 <6.0E-01 2.2E+00 <2.1E+00 1.4E+00 1.4E+01 2.2E+01

PCB126 1.1E+01 7.9E+00 1.4E+01 8.7E+00 9.5E+00 1.0E+01 <7.0E+02 2.3E+01 1.6E+01 6.5E+00 3.4E+00 <6.0E-01 4.5E+00 <2.0E+00 2.7E+00 9.9E+00 1.2E+01

PCB169 1.4E+00 <1.2E+00 <1.6E+00 <1.9E+00 <1.5E+00 2.3E+00 <3.2E+00 3.2E+00 <2.2E+00 <1.3E+00 <1.9E+00 <1.3E+00 <2.2E+00 <2.7E+00 <8.0E-01 <2.0E+00 <5.1E+00

PCB105 5.0E+02 1.9E+02 6.1E+02 5.3E+02 4.0E+02 1.7E+02 3.1E+02 4.0E+02 1.0E+03 1.0E+03 3.5E+02 2.1E+01 2.1E+02 8.0E+01 1.5E+02 1.9E+03 1.4E+03

PCB114 2.7E+01 1.2E+01 3.3E+01 3.1E+01 2.0E+01 1.1E+01 8.2E+02 2.2E+01 4.8E+01 5.6E+01 2.3E+01 1.7E+00 1.2E+01 5.0E+00 8.8E+00 9.5E+01 1.0E+02

PCB118 1.6E+03 8.4E+02 2.1E+03 2.1E+03 1.3E+03 7.5E+02 1.4E+02 1.7E+03 3.4E+03 2.8E+03 1.1E+03 6.0E+01 8.8E+02 8.1E+01 6.8E+02 4.4E+03 4.0E+02

PCB123 2.5E+02 1.8E+02 3.5E+02 5.0E+02 1.9E+02 1.6E+02 1.6E+02 4.0E+02 4.1E+02 3.2E+02 1.5E+02 8.3E+00 1.4E+02 8.1E+01 1.2E+02 4.9E+02 5.5E+02

PCB156 4.9E+02 3.8E+02 5.4E+02 1.0E+03 4.2E+02 3.3E+02 2.5E+02 7.3E+02 6.7E+02 4.4E+02 2.6E+02 8.9E+00 2.3E+02 1.6E+02 2.6E+02 7.0E+02 7.5E+02

PCB157 6.0E+01 4.0E+01 6.8E+01 9.9E+01 5.2E+01 3.7E+01 3.9E+01 8.0E+01 1.2E+02 7.7E+01 3.0E+01 1.6E+00 2.6E+01 1.3E+01 2.6E+01 1.2E+02 1.1E+02

PCB167 2.3E+02 1.8E+02 2.6E+02 4.3E+02 1.9E+02 1.7E+02 1.0E+02 3.7E+02 2.8E+02 1.9E+02 1.2E+02 3.9E+00 1.1E+02 8.4E+01 1.3E+02 2.8E+02 2.7E+02

PCB189 5.1E+01 5.0E+01 5.2E+01 1.8E+02 5.1E+01 5.7E+01 3.4E+01 1.0E+02 5.0E+01 2.2E+01 2.0E+01 1.1E+00 2.2E+01 1.1E+01 2.7E+01 5.0E+01 6.7E+01

Total dl-PCBs 3.3E+03 1.9E+03 4.2E+03 5.0E+03 2.7E+03 1.7E+03 2.0E+03 4.0E+03 6.1E+03 5.0E+03 2.2E+03 1.1E+02 1.7E+03 5.3E+02 1.4E+03 8.3E+03 4.1E+03

Total-TEQ LB WHO98 1.7E+00 1.1E+00 2.1E+00 1.8E+00 1.4E+00 1.4E+00 6.4E-01 3.0E+00 2.5E+00 1.4E+00 6.8E-01 1.0E-02 7.1E-01 1.1E-01 5.3E-01 2.2E+00 1.9E+00

Total-TEQ UB WHO98 1.7E+00 1.2E+00 2.1E+00 1.8E+00 1.4E+00 1.4E+00 7.0E+01 3.0E+00 2.5E+00 1.4E+00 7.0E-01 9.0E-02 7.4E-01 3.5E-01 5.3E-01 2.2E+00 2.0E+00

Total-TEQ LB WHO2006 1.3E+00 8.5E-01 1.6E+00 1.0E+00 1.0E+00 1.2E+00 8.0E-02 2.5E+00 1.8E+00 8.0E-01 4.3E-01 0.0E+00 5.0E-01 1.0E-02 3.3E-01 1.3E+00 1.3E+00

Total-TEQ UB WHO2006 1.3E+00 8.9E-01 1.6E+00 1.1E+00 1.1E+00 1.2E+00 7.0E+01 2.5E+00 1.8E+00 8.4E-01 4.8E-01 1.0E-01 5.8E-01 3.0E-01 3.5E-01 1.3E+00 1.5E+00

Total TEQH4IIE LB 1.1E+00 8.1E-01 1.5E+00 9.3E-01 9.8E-01 1.1E+00 9.2E-02 2.4E+00 1.7E+00 6.8E-01 3.9E-01 9.4E-04 4.7E-01 5.9E-03 2.9E-01 1.1E+00 1.3E+00

Total TEQH4IIE UB 1.1E+00 8.1E-01 1.5E+00 9.3E-01 9.8E-01 1.1E+00 7.0E+01 2.4E+00 1.7E+00 6.8E-01 3.9E-01 6.3E-02 4.7E-01 2.1E-01 2.9E-01 1.1E+00 1.3E+00

Legend: PCB-polychlorinated biphenyl, TEQ – toxic equivalent of 2,3,7,8-tetrachlorodibenzo-p-dioxin, LB-lower bound value, UB-upper bound value, TEQH4IIE-toxic equivalent of 2,3,7,8 TCDD calculated from analytical results and relative potencies (REPs) of PCDDs/Fs or DL-PCBs

Table S3-B Recoveries and limits of quantification (LOQ) for dioxin-like PCBs

)

dlPCBs % ng/kg

recovery min recovery max recovery average LOQ min

LOQ max

LOQ average

PCB77 2 81 67 0.5 22.0 3.3

PCB81 3 82 69 0.6 20.2 3.1

PCB126 60 77 69 0.6 697.2 36.7

PCB169 47 81 67 0.8 5.1 2.0

PCB105 62 136 78 0.5 14.3 1.9

PCB114 58 135 73 0.5 20.7 2.2

PCB118 59 137 72 0.4 21.9 2.2

PCB123 59 143 73 0.4 24.0 2.3

PCB156 65 148 79 0.7 7.5 2.4

PCB157 62 137 77 0.6 6.2 2.4

PCB167 23 149 77 0.7 12.8 3.0

PCB189 37 63 54 0.7 3.6 1.7

Table S4: Results of 6 indicator PCBs and OCPs for 17 selected samples

Nr of sample 1 2 3 4 1 2 1 2 3 1 2 1 2 1 1 2 1

Sample code A A A A B B C C C D D F F G H H i

Country of origin Belgium Germany Belgium Belgium Belgium Germany Luxembourg Germany France France United Kingdom Luxembourg Belgium Austria Netherland France Netherland

Cl-mix PCB 28 6.1E-01 4.1E-01 1.1E+00 8.3E-01 6.6E-01 3.2E-01 1.0E+00 8.9E-01 9.8E-01 5.6E-01 2.1E+00 3.0E-01 8.5E-01 6.2E-01 2.1E-01 2.9E+00 5.3E+00

µg/kg PCB 52 8.2E-01 5.1E-01 1.0E+00 1.2E+00 6.1E-01 4.5E-01 7.6E-01 9.5E-01 1.9E+00 1.3E+00 1.2E+00 1.6E-01 6.4E-01 4.2E-01 2.9E-01 3.4E+00 4.2E+00

dm PCB 101 3.1E+00 2.4E+00 4.1E+00 5.6E+00 2.2E+00 1.9E+00 1.1E+00 4.1E+00 4.6E+00 4.5E+00 1.9E+00 1.6E-01 1.5E+00 1.2E+00 1.3E+00 5.2E+00 4.2E+00

PCB 153 6.7E+00 5.7E+00 8.8E+00 1.4E+01 5.3E+00 4.6E+00 2.7E+00 1.1E+01 8.6E+00 6.3E+00 3.6E+00 1.4E-01 3.6E+00 2.7E+00 3.4E+00 7.1E+00 6.2E+00

PCB 138 6.2E+00 5.2E+00 7.4E+00 1.1E+01 5.0E+00 4.1E+00 2.4E+00 9.3E+00 7.6E+00 5.9E+00 3.2E+00 1.3E-01 2.8E+00 2.3E+00 2.9E+00 7.2E+00 6.0E+00

PCB 180 4.2E+00 3.9E+00 4.8E+00 1.2E+01 4.2E+00 3.5E+00 2.4E+00 7.7E+00 4.8E+00 2.3E+00 2.3E+00 9.9E-02 2.0E+00 1.2E+00 2.0E+00 4.0E+00 4.1E+00

PeCB 9.1E-02 6.2E-02 3.3E-01 3.9E-02 1.8E-01 8.1E-02 8.8E-02 8.1E-02 9.2E-02 5.9E-02 6.7E-02 4.9E-02 2.8E-01 1.0E-01 1.9E-01 7.6E-02 3.4E-01

HCB 3.2E-01 6.4E-01 1.1E+00 1.6E-01 4.0E-01 3.6E-01 3.9E-01 4.6E-01 2.5E-01 1.5E-01 4.7E-01 6.9E-02 3.6E-01 1.5E-01 1.5E+00 1.9E-01 5.9E+00

a-HCH 1.6E-01 1.9E-01 1.3E-01 1.6E-01 1.7E-01 1.6E-01 4.2E+00 1.6E-01 1.5E-01 5.6E-01 2.2E-01 1.1E+00 1.3E-01 1.5E-01 1.2E-01 1.5E-01 8.9E-01

b-HCH 4.5E-02 1.2E-01 2.5E-02 3.9E-02 1.5E-01 3.2E-02 1.8E-01 2.7E-02 1.2E-01 5.9E-01 1.2E-01 4.9E-02 - 2.5E-02 2.6E-02 7.6E-02 5.5E-01

Lindane 3.0E-01 3.5E-01 3.0E-01 2.4E-01 5.0E-01 2.6E-01 5.2E+00 3.0E-01 2.8E-01 4.3E+00 3.6E+00 1.6E+00 2.0E-01 2.8E-01 2.0E-01 3.0E-01 4.9E-01

d-HCH 9.1E-02 1.2E-01 1.0E-01 1.2E-01 1.1E-01 1.1E-01 1.3E+00 1.1E-01 9.2E-02 3.6E-01 1.5E-01 6.9E-01 8.7E-02 1.4E-01 9.2E-02 7.6E-02 3.4E-01

o.p'-DDE 1.2E+00 4.1E-02 2.5E-01 1.2E-01 1.8E-01 4.9E-02 1.1E-01 1.1E-01 1.8E-01 4.2E-01 1.0E-01 3.0E-02 1.0E-01 3.8E-02 2.6E-02 2.3E-01 1.2E-01

p.p'-DDE 4.4E+01 1.3E+00 1.4E+01 3.5E+00 9.5E+00 1.2E+00 5.6E+00 3.6E+00 8.5E+00 2.8E+00 2.7E+00 1.0E+00 1.3E+00 3.1E-01 1.0E+00 5.9E+00 1.4E+00

o.p'-DDD 3.2E+00 1.4E-01 3.3E-01 3.6E-01 1.8E-01 1.9E-01 6.7E+00 1.9E+00 3.1E-01 5.6E-01 5.4E-01 3.0E-02 1.5E-02 1.3E-02 1.3E-01 5.7E-01 9.2E-01

p.p'-DDD 5.7E+00 4.9E-01 1.5E+00 7.5E-01 8.8E-01 3.6E-01 5.9E-01 4.9E-01 3.7E-01 3.0E+00 2.6E+00 6.9E-02 7.3E-02 2.5E-02 4.9E-01 1.9E+00 3.6E+00

o.p'-DDT 3.0E-01 6.2E-02 7.6E-02 1.2E-01 2.8E-01 6.5E-02 1.6E-01 2.7E-02 9.2E-02 3.3E-01 1.5E-01 4.9E-02 2.9E-02 3.8E-02 6.6E-02 1.9E-01 1.8E-01

p.p'DDT 1.1E+00 1.9E-01 1.3E-01 3.6E-01 1.4E+00 1.9E-01 4.6E-01 1.6E-01 2.5E-01 1.8E+00 6.7E-01 1.1E-01 2.9E-02 6.3E-02 1.7E-01 6.8E-01 5.8E-01

e-HCH 4.5E-02 4.1E-02 - 3.9E-02 - - 2.5E-01 5.4E-02 3.1E-02 8.9E-02 6.7E-02 8.9E-02 4.4E-02 1.3E-02 - 3.8E-02 9.2E-02

SUMA PCBs 2.2E+01 1.8E+01 2.7E+01 4.4E+01 1.8E+01 1.5E+01 1.0E+01 3.4E+01 2.9E+01 2.1E+01 1.4E+01 9.8E-01 1.1E+01 8.5E+00 1.0E+01 3.0E+01 3.0E+01

SUMA HCHs 5.9E-01 7.8E-01 5.6E-01 5.5E-01 9.2E-01 5.7E-01 1.1E+01 6.0E-01 6.4E-01 5.8E+00 4.1E+00 3.4E+00 4.2E-01 5.9E-01 4.4E-01 6.1E-01 2.3E+00

SUMA DDTs 5.6E+01 2.2E+00 1.7E+01 5.2E+00 1.2E+01 2.1E+00 1.4E+01 6.3E+00 9.7E+00 8.9E+00 6.8E+00 1.3E+00 1.6E+00 4.9E-01 1.9E+00 9.4E+00 6.8E+00

Legend: OCP-organochlorine pesticides, PCB- polychlorinated biphenyl, PeCB – pentachlorobenzene, HCH-hexachlorocyclohexane, DDE-dichlorodiphenyldichloroethylene, DDT-dichlorodiphenyltrichloroethane, HCB-hexachlorobenzene

Table S5: Results of H4IIE-luc bioassay (TEQbio) and TEQPAHs for 88 tested samples

Code Type Country of origin TEQbio (µg/kg,dm) TEQPAHs (µg/kg,dm) 1 B Belgium 6.4E-01 2.1E-01

2 B France 2.9E-01 1.0E-01 3 B Luxembourg 2.0E+00 1.2E+00 4 B Belgium 3.8E+00 2.5E-01 5 B The Netherlands 3.8E-01 7.1E-02 6 B The Netherlands 5.7E-01 1.9E-01 7 B Spain 1.6E-01 3.2E-02 8 B Sweden 1.1E+00 1.5E-01 9 B Denmark 8.8E-01 6.2E-02

10 B Germany 5.1E-01 1.1E-01 11 B Germany 6.5E-01 8.5E-02 12 B France 1.4E+00 1.7E-01 13 B France 1.3E-01 1.3E-01 14 B France 1.5E+00 6.1E-01 15 B France 2.7E-01 1.4E-01 16 B France 1.4E+00 1.7E-01 17 B France 7.3E-01 1.1E-01 18 B France 1.2E+00 2.5E-01 19 B Belgium 6.0E-01 1.8E-01 20 B Belgium 2.7E+00 1.9E+00 21 B Suisse 2.4E-01 1.2E-01 22 B Italy 2.1E-01 3.9E-02 23 B France 2.5E-01 6.6E-02

Code Type Country of origin TEQbio (µg/kg,dm) TEQPAHs (µg/kg ,dm)

1 A Belgium 3.6E-01 6.0E-01

2 A France 1.4E-01 4.5E-02 3 A France 5.7E-01 2.0E-01 4 A Finland 1.2E-01 3.9E-02 5 A Italy 3.3E-01 5.0E-01 6 A Luxembourg 6.5E-01 4.5E-01 7 A Belgium 2.7E+00 2.1E-01 8 A The Netherlands 4.4E-01 2.1E-01 9 A Spain <1.0E-02 4.4E-02

10 A Sweden 4.1E-01 6.7E-02 11 A The Netherlands 1.4E+00 1.8E-01 12 A Denmark 1.8E-01 7.7E-02 13 A Germany 8.6E-01 1.4E-01 14 A Germany 5.2E-01 2.0E-01 15 A Germany 1.1E+00 1.1E-01 16 A Germany 5.3E-01 8.3E-01 17 A France 4.8E-01 2.9E-01 18 A Germany 1.1E+00 1.8E-01 19 A Italy 4.7E-01 7.5E-02 20 A Italy 1.2E+00 1.1E-01 21 A Italy 1.2E-01 4.3E-02 22 A Portugal 5.6E-01 6.3E-02 23 A Belgium 3.3E+00 1.6E-01 24 A United Kingdom 1.2E+00 1.7E-01 25 A Suisse 6.4E-01 1.8E-01 26 A Suisse 1.4E+00 3.5E-01 27 A Germany 1.2E+00 1.5E-01 28 A Belgium 2.0E+00 3.6E-01

Code Type Country of origin TEQbio (µg/kg,dm) TEQPAHs (µg/kg,dm)

1 C France <1.0E-02 9.9E-02 2 C Finland 4.8E-01 2.0E-01 3 C Luxembourg 4.4E+00 2.0E+00 4 C Spain 3.3E-01 5.8E-02 5 C Germany 1.1E+00 1.8E-01 6 C Austria 1.1E+00 1.4E-01 7 C Austria 4.5E-01 1.0E-01 8 C Austria 1.2E+00 1.7E-01 9 C France 2.8E-01 9.2E-02

10 C France 1.8E+00 1.5E-01 11 C France 4.4E-01 2.1E-01 12 C France 5.4E-01 1.4E-01 13 C France 2.9E-01 5.9E-02 14 C France 2.7E-01 1.9E-01 15 C Czech Rep. 1.6E+00 6.7E-01 16 C United Kingdom 9.5E-01 2.3E-01 1 D France 5.6E-01 8.1E-02 2 D France 2.3E-01 6.2E-02 3 D France 6.4E-01 1.4E-01 4 D France 6.2E-01 9.7E-02 5 D France 1.3E+00 1.1E-01 6 D Portugal 4.7E-01 5.2E-02 7 D Portugal 1.6E-01 5.2E-02 8 D United Kingdom 9.6E-01 2.9E-01

Legend: A-Biobin + green waste compost, B – green waste compost, C- sewage sludge compost, D – MBT compost, F – manure + biowaste digestate, G – manure + energy crops digestate, H – MBT digestate, i – Other; TEQbio- toxic equivalent of 2,3,7,8 tetrachlorodibenzo-p-dioxin calculated from bioassay; TEQPAH – toxic equivalent of 2,3,7,8 TCDD for PAHs calculated from analytical results and induction equivalency factors for PAHs, dm-dry mass Values of TEQbio are average of three replicates, CV<30%, in italic are signed TEQsPAH higher than TEQbio

Code Type Country of origin TEQbio (µg/kg,dm) TEQPAHs (µg/kg,dm) 1 F Belgium <1.0E-02 6.6E-02 2 F Luxembourg 3.4E-01 1.2E-01 3 F Austria 5.0E-02 1.0E-02 4 F Belgium 4.6E-01 1.3E-01 5 F Belgium <1.0E-02 1.0E-01 1 G Austria 9.0E-02 1.2E-01 1 H Netherlands 1.3E+00 3.0E-01 2 H France 1.2E+00 1.8E-01 1 i France 5.6E-01 1.2E-01 2 i Netherlands 8.2E+00 2.8E+00 3 i Austria 6.3E+00 1.3E-01 4 i Austria 3.7E-01 1.4E-01 5 i France 2.6E-01 1.7E-01

Fig. S1 Dose-response curve of standard compound – 2,3,7,8-TCDD and one of selected sample (C3) before and after sulphur acid treatment

Sample C3

-6 -5 -4 -3 -2 -10

50

100 raw extractH2SO4 treated

log c (g/ml)

% T

CD

D 5

00pM

TCDD calibration

-13 -12 -11 -10 -90

50

100

log c (M)

% T

CD

D 5

00pM