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Sequential extraction of metals from mixed and digested sludge

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Waste Management 29 (2009) 418–424 www.elsevier.com/locate/wasman

Sequential extraction of metals from mixed and digested sludge from aerobic WWTPs sited in the south of Spain
E. Alonso *, I. Aparicio, J.L. Santos, P. Villar, A. Santos
? Department of Analytical Chemistry, Industrial Engineering School, University of Seville, c/Virgen de Africa 7, 41011, Sevilla, Spain Accepted 17 January 2008 Available online 5 March 2008

Abstract The content of heavy metals is the major limitation to the application of sewage sludge in soil. However, assessment of the pollution by total metal determination does not reveal the true environmental impact. It is necessary to apply sequential extraction techniques to obtain suitable information about their bioavailability or toxicity. In this paper, sequential extraction of metals from sludge before and after aerobic digestion was applied to sludge from ?ve WWTPs in southern Spain to obtain information about the in?uence of the digestion treatment in the concentration of the metals. The percentage of each metal as residual, oxidizable, reducible and exchangeable form was calculated. For this purpose, sludge samples were collected from two di?erent points of the plants, namely, sludge from the mixture (primary and secondary sludge) tank (mixed sludge, MS) and the digested-dewatered sludge (?nal sludge, FS). Heavy metals, Al, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti and Zn, were extracted following the sequential extraction scheme proposed by the Standards, Measurements and Testing Programme of the European Commission and determined by inductively-coupled plasma atomic emission spectrometry. The total concentration of heavy metals in the measured sludge samples did not exceed the limits set out by European legislation and were mainly associated with the two less-available fractions (27–28% as oxidizable metal and 44–50% as residual metal). However, metals as Co (64% in MS and 52% in FS samples), Mn (82% in MS and 79% in FS), Ni (32% in MS and 26% in FS) and Zn (79% in MS and 62% in FS) were present at important percentages as available forms. In addition, results showed a clear increase of the concentration of metals after sludge treatment in the proportion of two less-available fractions (oxidizable and residual metal). ? 2008 Elsevier Ltd. All rights reserved.

1. Introduction In view of the increasing production of sludge in Europe and the simultaneous tightening of waste disposal practices, the preferred choices for sludge disposal in Europe used to be between land spreading (agricultural or other) and incineration. The practical choice of sludge disposal will be made locally according to many cultural, economic and scienti?c parameters (Bontoux et al., 1998). So far, there is general agreement that agricultural use can be a safe and viable option. It is certainly one of the most likely ways forward, although in some countries, such as France, Germany, Sweden, and The Netherlands, fears about the e?ects on soils and crops are disabling this outlet. In south*

Corresponding author. Tel.: +34 9 5455 2858; fax: +34 9 5428 2777. E-mail address: ealonso@us.es (E. Alonso).

ern Spain the use of such sludge (also sludge from aerobic treatment processes) as fertilizers or as organic soil regenerators is the main sludge disposal option. The European Directive 86/278/EEC (and also the third draft of the updated directive) concerning the use of wastewater sludge for agricultural purposes refers to the control of a set of parameters that guarantee the suitability of this sludge for agricultural application. These parameters include heavy metal levels that should not exceed speci?ed limits. In accordance with current legislation (and overall trends), total metal levels are determined in sludge samples. However, sequential extraction procedures for fractionation of metals have also been shown to be a useful tool for the environmental control of samples (Alonso et al., ? ? 2004; Lopez-Sanchez et al., 1996; Mossop and Davidson, 2003; Ure and Davidson, 2002) and also, therefore, for sewage sludge (Angelidis and Gibbs, 1989; Carlson and

0956-053X/$ - see front matter ? 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.wasman.2008.01.009

E. Alonso et al. / Waste Management 29 (2009) 418–424


? Morrison, 1992; Fuentes et al., 2004a; Legret, 1992; Sol?s et al., 2002; Zu?aurre et al., 1998). To evaluate the environmental impact of these metals in sludge and sludgeamended soil is not su?cient to determine their total content since their behavior in a given medium and their capacity for mobilization are equally important. In contrast, the metal extractable forms cannot only provide information about the general degree of contamination but can also provide an assessment of the mobility of these elements into sludge and sludge-amended soil, and may help to predict the release of metals in soil solution. In this context, the distribution of metals in sludge and their possible synergic and antagonist e?ects depend not only on the amount of these elements in sewage sludge but also on the stabilization process applied to the sludge (Fuentes et al., 2008). The main aim of this work was to study the distribution of metal forms and to establish whether aerobically digested sludge can be reused for agricultural purposes. The di?erent metal fractions of aluminum, cadmium, cobalt, chromium, copper, iron, mercury, manganese, molybdenum, nickel, lead, titanium and zinc (Al, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti and Zn) were determined by the sequential extraction scheme proposed by the Standards, Measurements and Testing Program of the European Commission, formerly Bureau Community of Reference (Ure et al., 1993). This paper is a follow on from a study carried out on the sequential metal extraction in sludge samples from ten anaerobic wastewater stabilization ponds published in Waste Management (Alonso et al., 2006). Owing to the scarcity of data associated with the characterisation and fractionation of heavy metals in sludge from aerobic sludge treatment plants, this work produces a useful database for an area lacking comprehensive information.

2. Methods 2.1. Sampling and sample pre-treatment Sludge samples were collected from ?ve wastewater treatment plants (WWTPs) based on aerobic sludge digestion. Aerobic digestion is an extension of the activated sludge aeration process where primary and secondary sludge are continually aerated for long periods of time. In aerobic stabilization processes, organic matter from the sludge is oxidized by microorganisms to carbon dioxide, water and ammonia. The ammonia is further converted to nitrates during the digestion process. All the treatment plants are in the Andalusian region in south Spain (their precise geographical location is indicated in Fig. 1). Sludge samples were collected from two di?erent points of the plants, namely, sludge from the mixture (primary and secondary sludge) tank (mixed sludge, MS) and the digesteddewatered sludge (?nal sludge, FS). Before analysis, samples were air-dried and the particle size was reduced by grinding in an agate mortar. Samples were then sieved into two particle sizes: 2 mm for the generic characterisation of parameters and afterwards to 63 lm to determine metal levels and for metal fractionation. Samples were stored in polyethylene containers at 4 °C until analysis. 2.2. Sludge analysis A set of parameters (water content, organic and mineral matter, pH, conductivity, ammonium (NH4+) and total Kjeldahl nitrogen (TKN), phosphate (PO43?) and total phosphorus (P), boron (B), calcium (Ca2+), potassium (K+), magnesium (Mg2+), sodium (Na+) and sulphur (S)) were determined for the generic characterisation of the sludge in an attempt to assess their agricultural value.

Fig. 1. Wastewater treatment plants sampled.


E. Alonso et al. / Waste Management 29 (2009) 418–424

The physico-chemical parameters were measured in triplicate by standard methods approved by the Spanish legislation for soil analysis (MAPA, 1986) and standard techniques compiled by APHA-AWWA-WPCF (1992). 2.3. Total concentration of metals Total metal levels were determined according to a previously reported and validated method (Alonso et al., 2000). The metals analysed were Al, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti and Zn. The analytical method was based on sample treatment by microwave digestion of 0.500 g of sludge sample and determination by inductively-coupled plasma atomic emission spectrometry (ICP-AES). 2.4. Multi-step sequential extraction

The microwave extraction system for total metals determination was a Microwave Ethos 900 apparatus (Milestone, Sorisole, Italy) with a programmable power and irradiation time. The Microwave Ethos 900 apparatus is equipped with a carousel that is able to hold six extraction vessels. Crison 501 pH meter, a Nitro-Kjeldhal apparatus, a Crison 522 conduct-meter and a Selecta heater were also used. All reagents were of analytical-reagent grade. Deionized water, further puri?ed using a Millipore (Bedford, MA, USA) Milli-Q system, was used throughout. Aqueous stock solutions of Al, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti, Zn, B, Ca, K, Mg, Na and S were prepared by dilution of the respective standard 1000 mg L?1 solutions (Merck, Germany). All standard and reagent solutions were stored in polyethylene bottles. 3. Results and discussion

Multi-step sequential extraction was carried out according to Alonso et al. (2006). The extraction was successively carried out over an initial mass of 1.00 g of sample. The extracts were grouped into four di?erent fractions, and arranged in relation to the mobility and bioavailability of the metals. Determined fractions were exchangeable, associated with water and acid-soluble species; reducible, associated with iron and manganese oxides; oxidizable, containing species bound to organic matter; and residual, strongly associated with mineral matter. 2.5. Instrumentation and reagents An ICP Applied Research Laboratories (Fisons Instruments) model 3410+ was used for the determination of the total amounts, and fractions of Al, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti and Zn. B, Ca, K, Mg, Na and S were also analysed by ICP-AES.

3.1. Characterisation of the sludge Table 1 compiles the data (mean, median, maximum and minimum values and standard deviation) of the parameters of the generic characterisation for the MS and FS samples analysed (from ?ve WWTPs). The mean organic matter content was 62.1% and 63.2% in mixed and ?nal sludge, respectively. These similar values could be explained by the poor removal e?ciencies of organic matter observed during aerobic stabilization processes involved in the studied WWTPs and the loss of weight, mainly due to the loss of volatile organic matter. The organic matter contents observed in aerobically digested sludge samples were higher than the contents, from 34% to 61%, detected in anaerobic sludge conventional treatment plants (Alonso et al., 2002). The mean value of pH, close to neutral, and the moderate electrical

Table 1 Descriptive statistics of generic parameters in mixed and ?nal sludge samples from ?ve WWTPs Mixed sludge Mean Water content (%) Dry matter (%) Mineral matter (%) Organic matter (%) pH Conductivity (lS cm?1) NH4+–N (mg Kg?1) PO43—P (mg Kg?1) Total Kjeldahl N (%) Total P (%) B (mg Kg?1) S (%) Ca (%) K (%) Mg (%) Na (%) 97.3 2.7 37.9 62.1 6.6 3878 1584 765 4.0 1.5 97.6 1.78 6.48 0.71 0.76 2.12 Median 97.9 2.1 38.5 61.5 6.7 4110 1570 687 4.0 1.4 82.3 1.72 6.89 0.58 0.83 1.78 Minimum 95.1 1.6 33.2 59.4 6.2 1618 664 87 3.7 1.1 57.5 1.58 4.82 0.41 0.47 0.94 Maximum 98.4 4.9 40.6 66.8 7.1 6940 2996 1212 4.7 1.8 153 2.19 7.39 1.32 1.08 4.70 Standard deviation 1.3 1.3 2.9 2.9 0.3 20 18 9 0.1 0.1 12.1 0.24 0.12 0.16 0.20 0.08 Final sludge Mean 81.9 18.1 36.8 63.2 6.8 1346 1482 546 5.1 1.8 53.8 1.89 6.90 0.83 0.61 0.57 Median 81.9 18.1 35.7 64.3 6.7 1017 1496 575 5.6 1.9 52.4 1.50 7.11 0.95 0.55 0.38 Minimum 79.8 16.5 33.3 58.5 6.6 876 1248 90 4.4 1.3 45.5 1.31 5.13 0.32 0.40 0.14 Maximum 83.5 20.2 41.5 66.7 6.9 2620 1683 1200 5.7 1.9 71.1 3.32 8.08 1.27 0.88 1.56 Standard deviation 1.6 1.6 3.4 3.4 0.1 725 175 465 0.7 0.3 1.04 0.11 0.08 0.14 0.06 0.10

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conductivity could suggest an absence of industrial waste contamination during the monitoring period and re?ect the main urban origin of the waters from the sludge. With regard to the nutrients, sludge samples presented nitrogenous and phosphorus values lower than those present in commercial fertilizers. About inorganic components, calcium was present in the highest concentrations associated to the mineral matter of the sludge. The highest levels of the remaining elements, except sulphur and potassium were present in the mixed sludge. This fact could be explained through the chemical changes of these species, mainly oxidation processes, which occurs during the aerobic stabilization process. The products of these reactions are usually soluble salts which are removed during the dewatering process of the sludge. These values were in the following order: sodium > sulphur > magnesium > potassium > boron for MS samples and sulphur > potassium > magnesium > sodium > boron for FS samples. These characterisation parameters make these sludges useful as plant fertilizer. The results agree with data in literature (Petersen et al., 2003). Table 2a and b show, for MS and FS samples, respectively, the mean values of the total amounts and fractions of metals in sludge from ?ve WWTPs versus the legal limit

concentrations (mg kg?1, dry weight) in the European Union. As previously was reported for sludge from anaerobic ponds (Alonso et al., 2006), attending total metal concentrations, four major groups can be distinguished. The metals Cd, Co, Hg and Mo, present in concentrations below 10 mg kg?1 for MS and FS samples, constituted the ?rst group. The metals Ni, Cr and Pb made up a second group with concentrations below 100 mg kg?1 for both kinds of sludge. Mn and Cu presented mean levels below 300 mg kg?1 and Zn below 1100 mg kg?1. The metals Al, Fe and Ti presented, in decreasing order, the highest concentrations. Similar relative abundances and metal concentration levels have also been reported by other researchers for sewage sludge (Karvelas et al., 2003; Scancar et al., 2000). For all metals, except Cu, the maximum values were recorded for the ?nal sludge samples. This fact is related to the loss of weight of the fresh sludge during digestion processes mainly due to the loss of inorganic matter (Alonso et al., 2002). It is noteworthy that none of the metals studied exceeded the limits established by Spanish or European legislation (Directive 86/278/CEE) or the maximum permitted levels predicted to appear in the updated European

Table 2 Mean values of total and fraction concentrations (mg kg?1, dry weight) of metals in mixed sludges (a) and ?nal sludges (b) from ?ve WWTPs versus legal limit concentrations (mg kg?1, dry weight) in European Union Metal Exchangeable Mean (a) Al Cd Co Cr Cu Fe Hg Mn Mo Ni Pb Ti Zn (b) Al Cd Co Cr Cu Fe Hg Mn Mo Ni Pb Ti Zn 26.3 0.04 <0.01 0.17 8.52 36.6 <0.05 70.4 <0.01 3.87 <0.01 <0.01 372 55.1 0.03 0.17 0.07 4.74 30.5 <0.05 88.9 <0.01 4.37 <0.01 <0.01 147 SD 12.1 0.10 0.03 0.31 6.0 24.2 – 42.8 – 2.51 – – 68.1 9.4 0.10 0.26 0.16 2.5 28.7 – 82.6 – 1.71 – – 47 Reducible Mean 719 0.17 <0.01 0.13 7.76 1060 <0.05 31.0 0.12 2.15 1.15 <0.01 451 518 0.26 0.03 0.07 2.68 802 <0.05 47.4 <0.01 2.6 0.14 <0.01 223 SD 667 0.10 0.19 0.33 15.0 1109 0.08 14.9 0.18 1.61 4.65 – 614 844 0.12 0.17 0.60 11.6 789 – 24.5 – 1.62 1.51 – 84 Oxidizable Mean 2761 0.02 <0.01 15.5 175 585 <0.05 8.61 3.83 3.29 17.6 15.1 126 3360 0.17 0.19 22.7 167 741 <0.05 14.4 3.48 5.7 14.9 11.1 99.4 SD 1545 0.43 0.04 2.8 76 176 – 3.6 1.98 1.52 8.34 8.44 184 1352 0.28 0.13 9.8 64 586 – 2.1 1.09 2.66 28.1 14.0 34.9 Residual Mean 5258 0.13 <0.01 27.7 22.2 5629 <0.05 13.0 2.84 9.21 39.0 1490 84.8 7084 0.41 <0.01 43.9 31.9 8582 <0.05 20.3 3.94 13.6 54.3 1842 128 SD 3602 0.27 – 6.3 12.3 2333 – 2.4 0.91 1.10 36.5 502 21.4 5190 0.42 – 6.6 11.1 4132 – 15.4 1.51 1.39 38.2 654 175 Total concentration Mean 8764 0.36 <0.01 43.5 213 7311 <0.05 123 6.79 18.5 57.7 1505 1034 11017 0.87 0.39 66.7 206 10156 <0.05 171 7.43 26.5 69.4 1853 597 SD 4790 0.65 0.28 19.0 95 3381 0.08 60.3 1.85 4.91 40.5 504 1594 6873 0.76 0.02 18.2 232 5826 – 127.3 1.52 1.98 64.7 660 204 – 20–40 – 1000–1750 1000–1750 – 16–25 – – 300–400 750–1200 – 2500–4000 – 20–40 – 1000–1750 1000–1750 – 16–25 – – 300–400 750–1200 – 2500–4000 – 10 – 1000 1000 – 16 – – 300 750 – 2500 – 10 – 1000 1000 – 16 – – 300 750 – 2500 Legal limit (86/278/EEC) Legal limit (3rd draft EU)


E. Alonso et al. / Waste Management 29 (2009) 418–424

directive on sludge from treated wastewaters destined for agricultural use (3rd draft). 3.2. Fractionation of heavy metals In the sequential extraction scheme used in this study, the mobility and hence possible bioavailability of metals decrease from readily exchangeable to residual. Fig. 2a and b show, for MS and FS, respectively, the mean percentages, in relation to the total concentration, of each of the metal fractions obtained after fractionation of the ?ve samples studied (n = 3). The values for mercury were not included since these levels are below the detection level of the technique (ICP-AES) used for this element. Fig. 3a and b give, for MS and FS, respectively, the overall proportion of each of the fractions for all the metals studied and show that the metals are predominantly present in forms retained by the sludge. The following results were obtained for the metal fractions. 3.2.1. Exchangeable Overall, a very small amount of metal was extracted in this form. Ti, Mo and Pb presented levels below the detection limit for this technique. Exceptions to this were shown

by Co (45% for FS samples), Mn (57% for MS and 52% for FS), Ni (21% for MS and 16% for FS) and Zn (36% for MS samples and 25% for FS samples). Similar percentages were ? also found by Sol?s et al. (2002) and Alonso et al. (2002, 2006) in sludge from anaerobic digestion systems and anaerobic stabilization ponds. The metals Co and Ni were present in low total concentrations and would not be expected to have a high toxicity potential. However, Mn, although not usually with a high toxicity potential, and especially Zn, should be carefully monitored if these sludges are to be reused for agricultural purposes. The remaining metals were present in percentages lower than 11% and 3% in MS and FS samples, respectively. Overall, these results were in concordance with several previously reported works in the recent bibliography for the same extraction scheme in related samples (Alonso et al., 2006, ? 2002; Fuentes et al., 2004a,b; Sol?s et al., 2002). 3.2.2. Reducible Cd, Mn and Zn were the predominant metals in this fraction (47%, 25% and 43%, respectively in MS samples, and 30%, 28% and 37% in FS samples). Similar results were found by Alonso et al. (2002) in heavy metals fractionation of sludge with di?erent stabilization degrees. These could

Fig. 2. Distribution of fractions for each metal in mixed sludges (a) and ?nal sludges (b).

E. Alonso et al. / Waste Management 29 (2009) 418–424


Fig. 3. Global distribution of metal fractions in mixed sludges (a) and ?nal sludges (b).

be released into the medium in reducing conditions. For all analysed metals, the relative percentage of reducible metals in MS was higher than in FS. The same observations can be made for this fraction as for the exchangeable fraction. It could be explained assuming that aerobic stabilization processes reduce the metals to more available forms. The rest of the metals were present in low percentages, e.g., below 14.5% for the metal Fe in MS and below 7.8% for the same metal in FS. For Mo and Ti, levels did not exceed the detection limits in FS. 3.2.3. Oxidizable For most of the metals, this fraction comprised a much larger proportion of the whole sample than the previous two fractions. Cu was mainly associated with the oxidizable fraction. This metal is predominantly associated with the organic matter fraction. Results obtained in this study con?rmed this with 82% for MS and 81% for FS, in accordance with the high stability constant of the copper complexes with organic matter (Ashworth and Alloway, 2004; Fjallborg and Dave, 2003). Other metals with high percent¨ ages in this fraction were Co (37% for MS and 48% for FS) and Mo (56% for MS and 47% for FS). In contrast, the metal Ti only corresponds to a low percentage (1%). This fraction corresponded to percentages between 20% and 40% of the remaining metals. 3.2.4. Total residual This was the predominant fraction for most of the metals. The metals with the highest concentrations (Al, Fe and Ti) mainly formed species bound to the mineral matter of the sludge. Other metals, with an important toxicity such as Cd, Cr, Ni or Pb, were also mainly present in this fraction, a result that re?ects the ?ndings of Alonso et al. (2002, 2006) and Fuentes et al. (2004b). 4. Conclusions The organic matter content (around 60%), higher than in sludge from anaerobic treatment plants; the presence of nitrogen, phosphorus and other inorganic elements,

especially potassium; and the low heavy metal content which is below the legal limits in all of the analysed samples, make this type of sludge a suitable product for agricultural applications. The determination of the concentrations of total metals does not re?ect whether the metals are present in mobile or available forms, explaining the need for fractionation analysis. The results of the sequential extraction study showed that most of the metals were mainly available in forms strongly associated to the sludge in spite of important percentages of metals as Co, Mn, Ni and Zn present in available forms. Whereas the low levels of Co and Ni seem to indicate negligible toxic e?ects of these metals, the higher levels of Mn and Zn in bioavailable forms mean that these metals should be monitored after application of the sludge to soil. Majority metals such as Al, Fe and Ti, and others such as Cd, Cr, Ni or Pb were mainly present in the residual fraction, thus reducing their toxicity potential. Overall, according to the results reported here, only 14% for MS and 12% for FS of the metal was present in forms easily available whereas 80% is retained by the sludge. It can be deduced, therefore, that, with the exception of Zn, very low amounts of metal are available in mobile or bioavailable forms and then these sludges could be safely applied to soils. Thus, data derived from fractionation analysis should not be used instead of total metal concentrations but as complementary information in order to guarantee a safe use and disposal of sludge from aerobic digestion systems. Acknowledgments The authors wish to thank European Regional Development Fund and DGICYT (1FD97-1533 project) for the ?nancial support provided for the conduct of this study. References
? ? Alonso, E., Callejon, M., Jimenez, J.C., Ternero, M., 2000. Determination of heavy metal in sewage sludge by microwave acid digestion and inductively-coupled plasma atomic emission spectrometry. Toxicological Environmental Chemistry 75, 207–214. ? ? Alonso, E., Callejon, M., Jimenez, J.C., Ternero, M., 2002. Heavy metal extractable forms in sludge from wastewater treatment plants. Chemosphere 47, 765–775. ? ? Alonso, E., Santos, A., Callejon, M., Jimenez, J.C., 2004. Speciation as a screening tool for the determination of heavy metal surface water pollution in the Guadiamar river basin. Chemosphere 56, 561– 570. Alonso, E., Villar, P., Santos, A., Aparicio, I., 2006. Fractionation of heavy metals in sludge from anaerobic wastewater stabilization ponds in southern Spain. Waste Management 26, 1270–1276. Angelidis, M., Gibbs, R., 1989. Chemistry of metals in an treated sludge. Water Resources 23, 29–38. APHA-AWWA-WPCF, 1992. Standard Methods for the Examination of Water and Wastewater. Washington, DC. Ashworth, D.J., Alloway, B.J., 2004. Soil mobility of sewage sludgederived dissolved organic matter, copper, nickel and zinc. Environmental Pollution 127, 137–144.


E. Alonso et al. / Waste Management 29 (2009) 418–424 ? ? MAPA, 1986. Metodos O?ciales Para el Analisis de Suelos y Fertilizantes. Madrid. Mossop, K.F., Davidson, C.M., 2003. Comparison of original and modi?ed BCR sequential extraction procedures for the fractionation of copper, iron, lead, manganese and zinc in soils and sediments. Analytica Chimica Acta 478, 111–118. Petersen, S.O., Petersen, J., Rubaek, G.H., 2003. Dynamics and plant uptake of nitrogen and phosphorus in soil amended with sewage sludge. Applied Soil Ecology 24, 187–195. Scancar, J., Milacic, M., Strazar, M., Burica, O., 2000. Total metal concentrations and partitioning of Cd, Cu, Fe, Ni and Zn in sewage sludge. Science of the Total Environment 250, 9–19. ? Sol?s, G., Alonso, E., Riesco, P., 2002. Distribution of metal extractable fractions during anaerobic sludge treatment in southern Spain WWTPs. Water, Air and Soil Pollution 140 (2002), 139–156. Ure, A.M., Davidson, C.M., 2002. In: Ure, A.M., Davidson, C.M. (Eds.), Chemical Speciation in the Environment, second ed. Blackwell Scienti?c Publications, Oxford, p. 451. Ure, A.M., Quevauviller, Ph., Muntau, H., Griepink, B., 1993. Improvements in the determination of extractable contents of trace metals in soil and sediment prior to certi?cation. EUR Report 14763 EN. CEC, Brussels. ? Zu?aurre, R., Olivar, A., Chamorro, P., Ner?n, C., Callizo, A., 1998. Speciation of metals in sewage sludge for agricultural uses. Analyst 123, 255–259.

Bontoux, L., Vega, M., Papameletiou, D., 1998. Urban wastewater treatment in Europe: what about the sludge? IPTS Report, 1–5. Seville, Spain. Carlson, C.E., Morrison, G.M., 1992. Fractionation and toxicity of metals in sewage sludge. Environmental Technology 13, 751–759. Fjallborg, B., Dave, G., 2003. Toxicity of copper in sewage sludge. ¨ Environmental International 28, 761–769. ? ? Fuentes, A., Llorens, M., Saez, J., Soler, A., Aguilar, M.I., Ortuno, J.F., ? Meseguer, V.F., 2004a. Simple and sequential extractions of heavy metals from di?erent sewage sludges. Chemosphere 54, 1039–1047. ? ? Fuentes, A., Llorens, M., Saez, J., Aguilar, M.I., Ortuno, J.F., Meseguer, ? V.F., 2004b. Phytotoxicity and heavy metals speciation of stabilised sewage sludges. Journal of Hazardous Materials 108, 161–169. ? ? Fuentes, A., Llorens, M., Saez, J., Soler, A., Aguilar, M.I., Ortuno, J.F., ? Meseguer, V.F., 2008. Comparative study of six di?erent sludges by sequential speciation of heavy metals. Bioresource Technology 99, 517–525. Karvelas, M., Katsoyiannis, A., Samara, C., 2003. Occurrence and fate of heavy metals in wastewater treatment process. Chemosphere 53, 1201– 1210. Legret, M., 1992. Speciation of heavy metals in sewage sludge and sludgeamended soil. International Journal of Environmental Analytical Chemistry 51, 161–165. ? ? Lopez-Sanchez, J., Rubio, R., Samitier, C., Rauret, G., 1996. Trace metal partitioning in marine sediments and sludges deposited o? the coast of Barcelona (Spain). Water Resources 30, 153–159.

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