Chemosphere 118 (2015) 315–321
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Aerobic biodegradation of selected polybrominated diphenyl ethers (PBDEs) in wastewater sewage sludge Hana Stiborova a,⇑, Jana Vrkoslavova a, Petra Lovecka a, Jana Pulkrabova b, Petra Hradkova b, Jana Hajslova b, Katerina Demnerova a a b
ICT Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technická 3, 16628 Prague 6, Czech Republic ICT Prague, Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, Technická 3, 16628 Prague 6, Czech Republic
h i g h l i g h t s PBDEs were degraded in sewage sludge under aerobic condition by indigenous microflora. Their elimination was reaching 62–78% of their initial amounts. Degradation was significantly enhanced by addition of yeast extract and 4-bromobiphenyl. The half-lives of the most abundant congener BDE 209 ranged between 6.0 and 8.2 months.
a r t i c l e
i n f o
Article history: Received 14 May 2014 Received in revised form 17 September 2014 Accepted 18 September 2014 Available online 28 October 2014 Handling Editor: Myrto Petreas Keywords: Aerobic degradation Polybrominated diphenyl ethers Persistent organic pollutants Sewage sludge
a b s t r a c t Due to widespread accumulation of polybrominated diphenyl ethers (PBDEs) in our surroundings, it is important to clarify their fate in the environment and the options of their elimination. The aim of this study was to monitor the biodegradation of the most frequent congeners (BDE 28, 47, 49, 66, 85, 99, 100, 153, 154, 183 and 209) under aerobic condition by indigenous microflora in 2 industrially contaminated sewage sludge samples. BDE 209 was detected as the predominating congener in concentrations 685 ng/g and 1403 ng/g dry weight in sewage sludge from WWTPs (waste water treatment plants) Hradec Kralove and Brno, respectively. The total amount of 10 lower PBDEs was 605 and 205 ng/g dry weight, respectively. The aerobic degradation was significantly enhanced by the addition of yeast extract and 4-bromobiphenyl. The total concentrations of all 11 PBDE congeners were lowered and their elimination was detected reaching 62–78% of their initial amounts after 11 months of cultivation. The degradation of most abundant congener BDE 209 followed the first-order kinetics with constant detected between 2.77 103 d1 and 3.79 103 d1 and the half-lives of BDE 209 degradation ranged between 6.0 and 8.2 months. This work clearly demonstrates that both lower brominated PBDEs as well as the major representative BDE 209 could be successfully removed from municipally contaminated sludge under aerobic conditions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Polybrominated diphenyl ethers (PBDEs) are anthropogenic chemicals that are added to a wide variety of consumer/commercial products in order to improve their fire resistance for more than 40 years. Due to their wide-spread use and their properties, namely chemical persistence and ability to bioaccumulate, considerable levels of these compounds have been found world-wide and their levels in the environment have been increasing since the 1970s (Law et al., 2006; Vonderheide et al., 2008). Despite their ⇑ Corresponding author. Tel.: +420 220 44 5204. E-mail address: [email protected] (H. Stiborova). http://dx.doi.org/10.1016/j.chemosphere.2014.09.048 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
ecotoxicological concerns, the use of PBDEs has been restricted. By 2004 and 2006, pentaBDE and octaBDE technical mixtures were phased-out from the production and the usage in Europe and USA, respectively (EU, 2003; EPA, 2006). DecaBDE mixture was considered to be a recalcitrant chemical and remained in worldwide commercial use as a flame retardant (BSEF, 2012). Therefore, the commercial production of decaBDE mixture was rising. The total volume of decaBDE sold in 2008 increased from 7500 metric tonnes to 10 000 metric tonnes in 2011 (VECAP, 2011). However, nowadays usage, manufacturing, import, or processing of decaBDE is restricted. Recent studies showed that BDE 209 could be debrominated down to lower PBDEs which may be even more toxic than parent compounds (Gerecke et al., 2005; Deng et al., 2011).
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H. Stiborova et al. / Chemosphere 118 (2015) 315–321
Current scientific opinion suggests that these chemicals will accumulate in the environment and their fate is attributed to the structural stability and subsequently limited degradation (Shin et al., 2010). The ability of soil microorganisms to degrade these persistent organic pollutants (POPs) is an important feature of determining the fate of PBDEs in the environment. Microbial degradation is usually considered as an effective and safe way to remove contaminants from the polluted ecosystem. Until now, there are only several studies showing the aerobic degradation of these compounds. Kim et al. (2007) described the aerobic transformation of mono-, di- and tri-BDEs by Sphingomonas sp. PH-07 growing on diphenyl ether. Deng et al. (2011) identified bacterium Lysinibacillus fusiformis DB-1 which is capable to debrominate BDE 209 using lactate as the carbon source. Other studies describing the ability of bacteria to transform BDE 209 were reported by Shi et al. (2013) who studied debromination under the influence of co-metabolic substrates and Lu et al. (2013) who demonstrated that Bacillus cereus JP12 metabolize BDE 209 through debromination. To be able to successfully remediate PBDE-contaminated soil it is important to plot the degradation of mixture PBDE congeners. Wastewater treatment plants (WWTPs) may play an important role in the environmental PBDE debromination and the original xenobiotics or their metabolic products could be transferred throughout the urban system. PBDEs were found in sludge samples in high concentrations (Cincinelli et al., 2012) and the application of enormous volumes of contaminated sewage sludge in agricultures areas could provide an avenue for the input of PBDEs into the food chain (Vrkoslavova et al., 2010). Only few studies were addressed to the degradation of the mixture of most abundant congeners present in contaminated soil/sludge and their removal by indigenous microflora (Nyholm et al., 2010; Shin et al., 2010). There is some information about PBDE fate in soil/sludge under anaerobic conditions but the evidence of microbial degradation under aerobic conditions by indigenous microflora is still lacking. To extend the knowledge about the fate of PBDEs in sewage sludge we intended to track the dynamics of removal of 11 PBDE congeners to find out the potential of autochthonous microflora to degrade PBDEs. Due to their hydrophobic character, these chemicals are strongly bound to solid particles in soil, sediments and sewage sludge (Law et al., 2006) and in fact, before reaching the anaerobic stratum, they are probably exposed to aerobic microorganisms. To the best of our knowledge the degradation of mixture of lower brominated congeners and the most abundant BDE 209 under aerobic conditions has not been studied yet. Further studies are therefore necessary to investigate the possible ways of PBDEs removal from contaminated sewage sludge to be able to achieve safe re-using of sludge in the environment. Results presented in this paper will therefore help us elucidate the possible strategies of PBDEs elimination in the contaminated environment. 2. Materials and Methods 2.1. Chemicals 4-bromobiphenyl (purity 98%) was obtained from Sigma Aldrich. An analytical set of standard solutions containing PBDE congeners (concentration 50 lg ml1 in nonane) was as follows: 2,4,40 -triBDE (BDE 28); 3,4,40 -BDE (BDE 37); 2,20 ,4,40 -tetraBDE(BDE 47); 2,20 ,4,50 -tetraBDE (BDE 49); 2,30 ,4,40 -tetraBDE (BDE 66); 2,20 ,3,4,40 -pentaBDE (BDE 85); 2,20 ,4,40 ,5-pentaBDE (BDE 99); 2,20 ,4,40 ,6-pentaBDE (BDE 100); 2,20 ,4,40 ,5,50 -hexaBDE (BDE 153); 2,2,40 ,4,50 ,60 -hexaBDE (BDE 154); 2,20 ,4,40 ,50 ,6-BDE (BDE 183) and deca-BDE (BDE 209), all with declared purity 98% were obtained from Cambridge Isotope Laboratories (CIL, Andover, USA). Standard solution of PCB-112 (10 lg ml1 in isooctane) was purchased from Gr. Ehrenstorfer GmBH (Augsburg, Germany).
The organic solvents (cyclohexane, dichloromethane, ethylacetate and isooctane), all of analytical grade were all supplied by Merck (Darmstadt, Germany). Anhydrous sodium sulphate supplied by Penta Chrudim (Chrudim, Czech Republic) was heated at 600 °C for 5 h and then stored in dessicator prior to use. Styrene– divinylbenzene gel (Bio Beads S-X3, 200–400 mesh) was purchased from Biorad Laboratories (Hercules, CA, USA). Sulphuric acid (98%) was obtained from Merck (Darmstadt, Germany). All other chemicals and reagents were of analytical grade. 2.2. Sewage sludge collection and slurry preparation Two sewage sludge samples collected in WWTPs Hradec Kralove and Brno were selected for the monitoring of PBDE degradation. (WWTP Hradec Kralove – the amount of cleaned wastewater: 16 million m3; the total length of sewerage net: 496 km; the number of sewage connection: 16 775; WWTP Brno – the amount of cleaned wastewater: 31 million m3; the total length of sewerage net: 1350 km; the number of sewage connection: 49 930). Samples were collected in May, 2007. The samples were pooled in jars and stored on ice during the transport and then stored at 4 °C for no longer than 2 weeks. Slurries were prepared by mixing 15 g of wet sewage sludge and 35 ml of mineral medium (Hickey and Focht, 1990). Three different experimental patterns were set up: (A) sewage sludge mixed with mineral medium; (B) sewage sludge mixed with mineral medium with yeast extract (YE) added to the final concentration of 50 lg ml1; (C) sewage sludge mixed with mineral medium with YE – 50 lg ml1 and 4-bromobiphenyl (4-BB) – 0.6 lg ml1. Flasks were incubated at 28 °C in the incubator (150 RPM) for 11 months in the dark. Due to the evaporation the mineral medium was regularly filled in. PBDEs removal was monitored after 3, 7 and 11 months of incubation under defined conditions. Three flasks were harvested at each time for the determination of the PBDE content. 2.3. Analysis A dried sample was mixed with anhydrous sodium sulphate to form flowing powder which was transferred into Soxhlet extraction thimbles (PCB 112 used as a recovery standard). Extraction was performed by dichloromethane in a Soxhlet apparatus. The crude extract was carefully evaporated and then the sample was dissolved in solvent mixture cyclohexane–ethylacetate (1:1, v/v) which was used as a mobile phase in gel permeation chromatography (GPC) employing a Bio Beads S-X3 column for the separation of interfering co-extracts. This fraction was after evaporation re-dissolved in isooctane with syringe standard (BDE 37). The isooctane phase was transferred to a glass vial and analysed by an Agilent 6890 (Agilent, USA) gas chromatograph equipped with a single quadrupole mass analyser Agilent 5975 XL operated in negative chemical ionization mode (GC/MS–NCI). The GC conditions were as follows: capillary column DB-XLB column (30 m 0.25 mm i.d. 0.1 lm film thickness, J & W Scientific, Folsom, USA), a column temperature program: from 105 °C (hold 2 min) to 300 °C at 20 °C min1 (hold 5 min); carrier gas: helium (Linde, Prague, Czech Republic) with constant flow 1.5 ml min1; injection temperature: 275 °C; injection volume: 1 ll using pulsed splitless injection mode (splitless time: 2 min). Monitored ions (m/z) were 79, 81, 159 and 161. Ion m/z 79 was used for quantification of all target analytes. Methane, which was used as a reagent gas (purity 99.995%, Linde, Prague, Czech Republic), was set at a pressure 2 104 mbar. Ion source and quadrupole temperatures were 150 °C. The presence of BDE 209 was monitored using the same GC/MSNCI employing a shorter capillary column BD-XLB (15 m 0.25 mm i.d. 0.1 lm film thickness J & W Scientific, USA). The temperature
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H. Stiborova et al. / Chemosphere 118 (2015) 315–321
program was as follows: from 80 °C (hold 2 min) to 280 °C at 20 °C min1 and to 320 °C at 5 °C min1 (hold 5 min); carrier gas: helium with constant flow 3 ml min1; injection temperature: 285 °C; injection volume: 1 ll using pulsed splitless injection mode (splitless time: 2 min). Monitored ions (m/z) were 485 and 487; ion at m/z 487 was used for quantification.
MQLs were defined as the lowest concentration of each analyte in the matrix at which the quantification and identity confirmation provided signal-to-noise ratio (S/N) higher than 10. MQLs were as follows 0.1–1.2 ng g1 dry weight. 2.5. Statistic
2.4. QA/ QC
Data were analysed using the statistical and graphical functions of SigmaPlot 8.0 and SigmaStat 3.0 (SPSS Inc., Chicago, IL, USA). If in a one-way analysis of variance a significant F-value of P < 0.05 was obtained, a Dunnett’s multiple comparison test between the treated and control group was conducted. For all statistical tests the significance level was established at P < 0.05.
To ensure the homogeneity of the samples the sewage sludge was thoroughly mixed and triplicate analyses were performed to confirm the uniform distribution of PBDEs within the samples. To distinguish the adsorption from microbial degradation the same experiments were performed with heat-sterilized sewage sludge. 8 flasks of slurries prepared by mixing 15 g of wet sewage sludge and 35 ml of mineral medium were used to elucidate abiotic degradation of PBDEs. The slurries were heat sterilized for 60 min twice apart 24 h at 120 °C. The effect of autoclaving on PBDEs was verified and 3 flasks were harvested. There were no significant changes in PBDE content and the concentration after heat sterilization. The concentrations of PBDEs in sewage sludge Hradec Kralove were before and after autoclaving 685 ± 26 and 642 ± 27 ng g1 dry weight of BDE 209 and 605 ± 6 and 595 ± 13 ng g1 dry weight of P 10BDEs, respectively. The concentrations of PBDEs in sewage sludge sampled in Brno were before and after autoclaving 1403 ± 45 and 1363 ± 29 ng g1 dry weight of BDE 209 and 205 ± 3 P and 216 ± 2 ng g1 dry weight of 10BDEs, respectively. Two flasks were used for testing the biological activity by growing the microorganisms on agar plates. No CFU were detected. The sterile controls were processed under identical conditions as experimental samples. To prevent photodegradation the flasks were incubated in the dark for 11 months. No significant changes in PBDE content and concentration after 11 months incubation period were observed. Analysis was carried out in an accredited testing laboratory (No. 1316.2) in the Czech Republic. The quality control was performed by the regular analysis of procedural blanks (one in each batch of nine samples), i.e. samples processed as mentioned above, but without the use of the test matrix. No contamination during the overall method by any of the target compounds was observed. Recoveries (%) and repeatabilities (expressed as relative standard deviation – RSD, %) were calculated as a mean value from the data obtained by the analysis of six replicates of respective fortified blank sewage sludge sample using the above mentioned procedures. Recoveries (%) and repeatabilities (expressed as relative standard deviation – RSD, %) were calculated from the data obtained by the analysis of six replicates of blank sewage sludge sample materials fortified 15 min prior to extraction with all PBDEs at 10 lg kg1 dry weight. Method recoveries ranged from 88% to 106% and repeatabilities from 4% to 11%. The method quantification limits (MQLs) for PBDEs were estimated based on preliminary measurements using matrix samples contaminated at low levels.
3. Results and discussion 3.1. Sewage sludge PBDEs contamination The fate of PBDEs has gained attention with increasing contamination in the environment. The main source of PBDEs in soil is application of enormous volumes of contaminated sewage sludge to agricultures areas (US EPA, 2009; Cincinelly et al., 2012), therefore the present study focuses on the removal of 11 major representatives of the PBDE group (from tri-BDEs to deca-BDE) from sewage sludge. Based on the results from our previous study (Stiborova et al., 2007) when 15 WWTPs sewage sludge samples were analysed for PBDEs content, we have chosen 2 sewage sludge samples (WWTPs Hradec Kralove and Brno) with the highest concentration and different PBDEs profile. The major congener in both sludge samples was BDE 209, followed by BDE 47 and BDE 99. In Table 1 we have compared the current PBDE contamination of the sewage sludge reported in recent studies from other European countries with our 2 sewage sludge samples. Even the relative abundance of each congener varies among the sewage sludge; BDE 209 dominated in the congener profile in all samples. The contribution of major contaminating congener BDE 209 usually ranges between 75% and 99.8% of the total PBDEs (Table 1), which clearly shows the extent of decaBDE technical mixture usage (Cincinelli et al., 2012). In our samples BDE 209 represents 53% and 87% of the total PBDE content in sludge samples in Hradec Kralove and Brno, respectively. The concentration of major lower brominated congeners BDE 47 and BDE 99 were higher in sludge from Hradec Kralove in comparison with other samples collected from the European sewage sludge. With the respect to the regulation of lower PBDEs usage in Europe from 2004 (EU, 2003), this contamination has to represent the old burden of the environment. The total concentration of 10 monitored lower brominated congeners (No. 28, 47, 49, 66, 85, 99, 100, 153, 154, 183) were 605 and 205 ng g1 dry weight in samples from WWTPs Hradec Kralove and Brno, respectively. BDE 209 ranged from 685 ng g1 dry weight in WWTP Hradec
Table 1 Comparison of PBDE concentration levels in sewage sludge samples from different locations in Europe.
Location
BDE concentration [ng g1 d.w] 47 99 100
153
154
183
209
Reference
Sweden (2000) Spain (2005) Spain (2010) Spain (2009) Switzerland (2003–2005) Italy (2009–2010) Germany (2002–2003) Brno (2007) Hradec Kralove (2007)
7.0–100 17.0–40.9 4.3–17.2
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Aerobic biodegradation of selected polybrominated diphenyl ethers (PBDEs) in wastewater sewage sludge Hana Stiborova a,⇑, Jana Vrkoslavova a, Petra Lovecka a, Jana Pulkrabova b, Petra Hradkova b, Jana Hajslova b, Katerina Demnerova a a b
ICT Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technická 3, 16628 Prague 6, Czech Republic ICT Prague, Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, Technická 3, 16628 Prague 6, Czech Republic
h i g h l i g h t s PBDEs were degraded in sewage sludge under aerobic condition by indigenous microflora. Their elimination was reaching 62–78% of their initial amounts. Degradation was significantly enhanced by addition of yeast extract and 4-bromobiphenyl. The half-lives of the most abundant congener BDE 209 ranged between 6.0 and 8.2 months.
a r t i c l e
i n f o
Article history: Received 14 May 2014 Received in revised form 17 September 2014 Accepted 18 September 2014 Available online 28 October 2014 Handling Editor: Myrto Petreas Keywords: Aerobic degradation Polybrominated diphenyl ethers Persistent organic pollutants Sewage sludge
a b s t r a c t Due to widespread accumulation of polybrominated diphenyl ethers (PBDEs) in our surroundings, it is important to clarify their fate in the environment and the options of their elimination. The aim of this study was to monitor the biodegradation of the most frequent congeners (BDE 28, 47, 49, 66, 85, 99, 100, 153, 154, 183 and 209) under aerobic condition by indigenous microflora in 2 industrially contaminated sewage sludge samples. BDE 209 was detected as the predominating congener in concentrations 685 ng/g and 1403 ng/g dry weight in sewage sludge from WWTPs (waste water treatment plants) Hradec Kralove and Brno, respectively. The total amount of 10 lower PBDEs was 605 and 205 ng/g dry weight, respectively. The aerobic degradation was significantly enhanced by the addition of yeast extract and 4-bromobiphenyl. The total concentrations of all 11 PBDE congeners were lowered and their elimination was detected reaching 62–78% of their initial amounts after 11 months of cultivation. The degradation of most abundant congener BDE 209 followed the first-order kinetics with constant detected between 2.77 103 d1 and 3.79 103 d1 and the half-lives of BDE 209 degradation ranged between 6.0 and 8.2 months. This work clearly demonstrates that both lower brominated PBDEs as well as the major representative BDE 209 could be successfully removed from municipally contaminated sludge under aerobic conditions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Polybrominated diphenyl ethers (PBDEs) are anthropogenic chemicals that are added to a wide variety of consumer/commercial products in order to improve their fire resistance for more than 40 years. Due to their wide-spread use and their properties, namely chemical persistence and ability to bioaccumulate, considerable levels of these compounds have been found world-wide and their levels in the environment have been increasing since the 1970s (Law et al., 2006; Vonderheide et al., 2008). Despite their ⇑ Corresponding author. Tel.: +420 220 44 5204. E-mail address: [email protected] (H. Stiborova). http://dx.doi.org/10.1016/j.chemosphere.2014.09.048 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
ecotoxicological concerns, the use of PBDEs has been restricted. By 2004 and 2006, pentaBDE and octaBDE technical mixtures were phased-out from the production and the usage in Europe and USA, respectively (EU, 2003; EPA, 2006). DecaBDE mixture was considered to be a recalcitrant chemical and remained in worldwide commercial use as a flame retardant (BSEF, 2012). Therefore, the commercial production of decaBDE mixture was rising. The total volume of decaBDE sold in 2008 increased from 7500 metric tonnes to 10 000 metric tonnes in 2011 (VECAP, 2011). However, nowadays usage, manufacturing, import, or processing of decaBDE is restricted. Recent studies showed that BDE 209 could be debrominated down to lower PBDEs which may be even more toxic than parent compounds (Gerecke et al., 2005; Deng et al., 2011).
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H. Stiborova et al. / Chemosphere 118 (2015) 315–321
Current scientific opinion suggests that these chemicals will accumulate in the environment and their fate is attributed to the structural stability and subsequently limited degradation (Shin et al., 2010). The ability of soil microorganisms to degrade these persistent organic pollutants (POPs) is an important feature of determining the fate of PBDEs in the environment. Microbial degradation is usually considered as an effective and safe way to remove contaminants from the polluted ecosystem. Until now, there are only several studies showing the aerobic degradation of these compounds. Kim et al. (2007) described the aerobic transformation of mono-, di- and tri-BDEs by Sphingomonas sp. PH-07 growing on diphenyl ether. Deng et al. (2011) identified bacterium Lysinibacillus fusiformis DB-1 which is capable to debrominate BDE 209 using lactate as the carbon source. Other studies describing the ability of bacteria to transform BDE 209 were reported by Shi et al. (2013) who studied debromination under the influence of co-metabolic substrates and Lu et al. (2013) who demonstrated that Bacillus cereus JP12 metabolize BDE 209 through debromination. To be able to successfully remediate PBDE-contaminated soil it is important to plot the degradation of mixture PBDE congeners. Wastewater treatment plants (WWTPs) may play an important role in the environmental PBDE debromination and the original xenobiotics or their metabolic products could be transferred throughout the urban system. PBDEs were found in sludge samples in high concentrations (Cincinelli et al., 2012) and the application of enormous volumes of contaminated sewage sludge in agricultures areas could provide an avenue for the input of PBDEs into the food chain (Vrkoslavova et al., 2010). Only few studies were addressed to the degradation of the mixture of most abundant congeners present in contaminated soil/sludge and their removal by indigenous microflora (Nyholm et al., 2010; Shin et al., 2010). There is some information about PBDE fate in soil/sludge under anaerobic conditions but the evidence of microbial degradation under aerobic conditions by indigenous microflora is still lacking. To extend the knowledge about the fate of PBDEs in sewage sludge we intended to track the dynamics of removal of 11 PBDE congeners to find out the potential of autochthonous microflora to degrade PBDEs. Due to their hydrophobic character, these chemicals are strongly bound to solid particles in soil, sediments and sewage sludge (Law et al., 2006) and in fact, before reaching the anaerobic stratum, they are probably exposed to aerobic microorganisms. To the best of our knowledge the degradation of mixture of lower brominated congeners and the most abundant BDE 209 under aerobic conditions has not been studied yet. Further studies are therefore necessary to investigate the possible ways of PBDEs removal from contaminated sewage sludge to be able to achieve safe re-using of sludge in the environment. Results presented in this paper will therefore help us elucidate the possible strategies of PBDEs elimination in the contaminated environment. 2. Materials and Methods 2.1. Chemicals 4-bromobiphenyl (purity 98%) was obtained from Sigma Aldrich. An analytical set of standard solutions containing PBDE congeners (concentration 50 lg ml1 in nonane) was as follows: 2,4,40 -triBDE (BDE 28); 3,4,40 -BDE (BDE 37); 2,20 ,4,40 -tetraBDE(BDE 47); 2,20 ,4,50 -tetraBDE (BDE 49); 2,30 ,4,40 -tetraBDE (BDE 66); 2,20 ,3,4,40 -pentaBDE (BDE 85); 2,20 ,4,40 ,5-pentaBDE (BDE 99); 2,20 ,4,40 ,6-pentaBDE (BDE 100); 2,20 ,4,40 ,5,50 -hexaBDE (BDE 153); 2,2,40 ,4,50 ,60 -hexaBDE (BDE 154); 2,20 ,4,40 ,50 ,6-BDE (BDE 183) and deca-BDE (BDE 209), all with declared purity 98% were obtained from Cambridge Isotope Laboratories (CIL, Andover, USA). Standard solution of PCB-112 (10 lg ml1 in isooctane) was purchased from Gr. Ehrenstorfer GmBH (Augsburg, Germany).
The organic solvents (cyclohexane, dichloromethane, ethylacetate and isooctane), all of analytical grade were all supplied by Merck (Darmstadt, Germany). Anhydrous sodium sulphate supplied by Penta Chrudim (Chrudim, Czech Republic) was heated at 600 °C for 5 h and then stored in dessicator prior to use. Styrene– divinylbenzene gel (Bio Beads S-X3, 200–400 mesh) was purchased from Biorad Laboratories (Hercules, CA, USA). Sulphuric acid (98%) was obtained from Merck (Darmstadt, Germany). All other chemicals and reagents were of analytical grade. 2.2. Sewage sludge collection and slurry preparation Two sewage sludge samples collected in WWTPs Hradec Kralove and Brno were selected for the monitoring of PBDE degradation. (WWTP Hradec Kralove – the amount of cleaned wastewater: 16 million m3; the total length of sewerage net: 496 km; the number of sewage connection: 16 775; WWTP Brno – the amount of cleaned wastewater: 31 million m3; the total length of sewerage net: 1350 km; the number of sewage connection: 49 930). Samples were collected in May, 2007. The samples were pooled in jars and stored on ice during the transport and then stored at 4 °C for no longer than 2 weeks. Slurries were prepared by mixing 15 g of wet sewage sludge and 35 ml of mineral medium (Hickey and Focht, 1990). Three different experimental patterns were set up: (A) sewage sludge mixed with mineral medium; (B) sewage sludge mixed with mineral medium with yeast extract (YE) added to the final concentration of 50 lg ml1; (C) sewage sludge mixed with mineral medium with YE – 50 lg ml1 and 4-bromobiphenyl (4-BB) – 0.6 lg ml1. Flasks were incubated at 28 °C in the incubator (150 RPM) for 11 months in the dark. Due to the evaporation the mineral medium was regularly filled in. PBDEs removal was monitored after 3, 7 and 11 months of incubation under defined conditions. Three flasks were harvested at each time for the determination of the PBDE content. 2.3. Analysis A dried sample was mixed with anhydrous sodium sulphate to form flowing powder which was transferred into Soxhlet extraction thimbles (PCB 112 used as a recovery standard). Extraction was performed by dichloromethane in a Soxhlet apparatus. The crude extract was carefully evaporated and then the sample was dissolved in solvent mixture cyclohexane–ethylacetate (1:1, v/v) which was used as a mobile phase in gel permeation chromatography (GPC) employing a Bio Beads S-X3 column for the separation of interfering co-extracts. This fraction was after evaporation re-dissolved in isooctane with syringe standard (BDE 37). The isooctane phase was transferred to a glass vial and analysed by an Agilent 6890 (Agilent, USA) gas chromatograph equipped with a single quadrupole mass analyser Agilent 5975 XL operated in negative chemical ionization mode (GC/MS–NCI). The GC conditions were as follows: capillary column DB-XLB column (30 m 0.25 mm i.d. 0.1 lm film thickness, J & W Scientific, Folsom, USA), a column temperature program: from 105 °C (hold 2 min) to 300 °C at 20 °C min1 (hold 5 min); carrier gas: helium (Linde, Prague, Czech Republic) with constant flow 1.5 ml min1; injection temperature: 275 °C; injection volume: 1 ll using pulsed splitless injection mode (splitless time: 2 min). Monitored ions (m/z) were 79, 81, 159 and 161. Ion m/z 79 was used for quantification of all target analytes. Methane, which was used as a reagent gas (purity 99.995%, Linde, Prague, Czech Republic), was set at a pressure 2 104 mbar. Ion source and quadrupole temperatures were 150 °C. The presence of BDE 209 was monitored using the same GC/MSNCI employing a shorter capillary column BD-XLB (15 m 0.25 mm i.d. 0.1 lm film thickness J & W Scientific, USA). The temperature
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H. Stiborova et al. / Chemosphere 118 (2015) 315–321
program was as follows: from 80 °C (hold 2 min) to 280 °C at 20 °C min1 and to 320 °C at 5 °C min1 (hold 5 min); carrier gas: helium with constant flow 3 ml min1; injection temperature: 285 °C; injection volume: 1 ll using pulsed splitless injection mode (splitless time: 2 min). Monitored ions (m/z) were 485 and 487; ion at m/z 487 was used for quantification.
MQLs were defined as the lowest concentration of each analyte in the matrix at which the quantification and identity confirmation provided signal-to-noise ratio (S/N) higher than 10. MQLs were as follows 0.1–1.2 ng g1 dry weight. 2.5. Statistic
2.4. QA/ QC
Data were analysed using the statistical and graphical functions of SigmaPlot 8.0 and SigmaStat 3.0 (SPSS Inc., Chicago, IL, USA). If in a one-way analysis of variance a significant F-value of P < 0.05 was obtained, a Dunnett’s multiple comparison test between the treated and control group was conducted. For all statistical tests the significance level was established at P < 0.05.
To ensure the homogeneity of the samples the sewage sludge was thoroughly mixed and triplicate analyses were performed to confirm the uniform distribution of PBDEs within the samples. To distinguish the adsorption from microbial degradation the same experiments were performed with heat-sterilized sewage sludge. 8 flasks of slurries prepared by mixing 15 g of wet sewage sludge and 35 ml of mineral medium were used to elucidate abiotic degradation of PBDEs. The slurries were heat sterilized for 60 min twice apart 24 h at 120 °C. The effect of autoclaving on PBDEs was verified and 3 flasks were harvested. There were no significant changes in PBDE content and the concentration after heat sterilization. The concentrations of PBDEs in sewage sludge Hradec Kralove were before and after autoclaving 685 ± 26 and 642 ± 27 ng g1 dry weight of BDE 209 and 605 ± 6 and 595 ± 13 ng g1 dry weight of P 10BDEs, respectively. The concentrations of PBDEs in sewage sludge sampled in Brno were before and after autoclaving 1403 ± 45 and 1363 ± 29 ng g1 dry weight of BDE 209 and 205 ± 3 P and 216 ± 2 ng g1 dry weight of 10BDEs, respectively. Two flasks were used for testing the biological activity by growing the microorganisms on agar plates. No CFU were detected. The sterile controls were processed under identical conditions as experimental samples. To prevent photodegradation the flasks were incubated in the dark for 11 months. No significant changes in PBDE content and concentration after 11 months incubation period were observed. Analysis was carried out in an accredited testing laboratory (No. 1316.2) in the Czech Republic. The quality control was performed by the regular analysis of procedural blanks (one in each batch of nine samples), i.e. samples processed as mentioned above, but without the use of the test matrix. No contamination during the overall method by any of the target compounds was observed. Recoveries (%) and repeatabilities (expressed as relative standard deviation – RSD, %) were calculated as a mean value from the data obtained by the analysis of six replicates of respective fortified blank sewage sludge sample using the above mentioned procedures. Recoveries (%) and repeatabilities (expressed as relative standard deviation – RSD, %) were calculated from the data obtained by the analysis of six replicates of blank sewage sludge sample materials fortified 15 min prior to extraction with all PBDEs at 10 lg kg1 dry weight. Method recoveries ranged from 88% to 106% and repeatabilities from 4% to 11%. The method quantification limits (MQLs) for PBDEs were estimated based on preliminary measurements using matrix samples contaminated at low levels.
3. Results and discussion 3.1. Sewage sludge PBDEs contamination The fate of PBDEs has gained attention with increasing contamination in the environment. The main source of PBDEs in soil is application of enormous volumes of contaminated sewage sludge to agricultures areas (US EPA, 2009; Cincinelly et al., 2012), therefore the present study focuses on the removal of 11 major representatives of the PBDE group (from tri-BDEs to deca-BDE) from sewage sludge. Based on the results from our previous study (Stiborova et al., 2007) when 15 WWTPs sewage sludge samples were analysed for PBDEs content, we have chosen 2 sewage sludge samples (WWTPs Hradec Kralove and Brno) with the highest concentration and different PBDEs profile. The major congener in both sludge samples was BDE 209, followed by BDE 47 and BDE 99. In Table 1 we have compared the current PBDE contamination of the sewage sludge reported in recent studies from other European countries with our 2 sewage sludge samples. Even the relative abundance of each congener varies among the sewage sludge; BDE 209 dominated in the congener profile in all samples. The contribution of major contaminating congener BDE 209 usually ranges between 75% and 99.8% of the total PBDEs (Table 1), which clearly shows the extent of decaBDE technical mixture usage (Cincinelli et al., 2012). In our samples BDE 209 represents 53% and 87% of the total PBDE content in sludge samples in Hradec Kralove and Brno, respectively. The concentration of major lower brominated congeners BDE 47 and BDE 99 were higher in sludge from Hradec Kralove in comparison with other samples collected from the European sewage sludge. With the respect to the regulation of lower PBDEs usage in Europe from 2004 (EU, 2003), this contamination has to represent the old burden of the environment. The total concentration of 10 monitored lower brominated congeners (No. 28, 47, 49, 66, 85, 99, 100, 153, 154, 183) were 605 and 205 ng g1 dry weight in samples from WWTPs Hradec Kralove and Brno, respectively. BDE 209 ranged from 685 ng g1 dry weight in WWTP Hradec
Table 1 Comparison of PBDE concentration levels in sewage sludge samples from different locations in Europe.
Location
BDE concentration [ng g1 d.w] 47 99 100
153
154
183
209
Reference
Sweden (2000) Spain (2005) Spain (2010) Spain (2009) Switzerland (2003–2005) Italy (2009–2010) Germany (2002–2003) Brno (2007) Hradec Kralove (2007)
7.0–100 17.0–40.9 4.3–17.2
