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Effects of combined composting and vermicomposting of waste sludge on arsenic fate and bioavailability
Journal of Hazardous Materials 280 (2014) 544–551
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Effects of combined composting and vermicomposting of waste sludge on arsenic fate and bioavailability ˇ Blanka Manáková, Jan Kuta, Markéta Svobodová, Jakub Hofman ∗ Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 753/5, CZ-62500, Czech Republic
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Industrial sludge with high As con• • • •
tent was treated by composting and vermicomposting. The volume of compost decreased, which led to an increase in total As content. The labile arsenic fraction was significantly decreased. AsV was the predominant arsenic species formed. The mobile fraction was not directly related to bioavailability to earthworms.
a r t i c l e
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Article history: Received 20 March 2014 Received in revised form 4 August 2014 Accepted 7 August 2014 Available online 25 August 2014 Keywords: Sludge Arsenic Composting Vermicomposting Bioavailability
a b s t r a c t Composting and vermicomposting are traditional processes for the treatment of sludge. During these processes, the humification of organic matter has a significant effect on the physicochemical form and distribution of heavy metals. In this study, industrial sludge (groundwater treatment waste) contaminated by arsenic (396 ± 1 mg kg−1 ) was used. Such sludge poses a significant challenge with respect to effective treatment. Composting, vermicomposting (with Eisenia fetida), and the combined approach of composting and vermicomposting were performed to determine the evolution of arsenic speciation, mobility and bioavailability. The composting/vermicomposting was done with sludge, horse manure, and grass in the ratios of 3:6:1. A solution of 0.1 M NH4 COOCH3 was used as a single extraction solvent for determination of the mobile arsenic pool and targeted arsenic species (AsIII , AsV , monomethylarsenic acid – MMAV , dimethylarsenic acid – DMAV ). The analysis of arsenic in the extracts was carried out by means of HPLC–ICP-MS spectrometry. In addition, the earthworm species E. fetida was used for bioaccumulation tests that followed the compost and vermicompost processes. The obtained results indicate a reduction in arsenic mobility and bioavailability in all matured composts and vermicomposts. The combined process exhibited a greater effect than compost or vermicompost alone. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The overproduction of anthropogenic waste sludge has led to the use of inappropriate disposal practices and caused the introduction of metals into the environment. Nowadays, composting
∗ Corresponding author. Tel.: +420 549 494 267; fax: +420 549 492 840. E-mail address: [email protected] (J. Hofman). http://dx.doi.org/10.1016/j.jhazmat.2014.08.024 0304-3894/© 2014 Elsevier B.V. All rights reserved.
and vermicomposting are two of the best-known processes for the biological stabilization of sludge [1]. During these processes, heavy metals are redistributed to a newly formed matrix and the level of metal contamination generally grows [2–4]. From another point of view, the role of composting could be considered as an important environmental sink for the elimination of metals. Arsenic bioavailability and toxicity is strongly dependent on arsenic species. The mobility of sludge-born arsenic entering into composting/vermicomposting processes is strongly related to
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Table 1 Selected properties of sludge used in the study. As (mg kg−1 )
AsIII (mg kg−1 )
DMA (mg kg−1 )
MMA (mg kg−1 )
AsV (mg kg−1 )
396 ± 1
<0.004
<0.003
<0.003
1.26 ± 0.04
pH
TC (%)
TOC (%)
TIC (%)
Dry mass (%)
8.5
1.03 ± 0.01
0.23 ± 0.03
0.80 ± 0.03
63 ± 1
redox processes, microbial activity, and the degradation of organic matter [5]. Arsenic interaction with organic matter may include redox reaction, complexation, colloid formation, and sorption competition; however, the direct chemical effect of organic matter on the redox speciation of arsenic has not yet been proven. Beside AsIII oxidation and various methylation reactions [6], microbial processes are a potential cause of high variability in AsV reduction, during which microorganisms use AsV as an electron acceptor and mediate transformation to the more toxic and mobile AsIII species [7]; however, chemical reactions may also contribute to this process. AsV usually remains bound to iron, alumina and manganese oxides, which limit its mobility and bioavailability [8]. The toxicity and behavior of arsenic and its compounds in the environment is summarized in many reviews [6,9]. The aim of this work was to understand the influence of composting, vermicomposting, and both processes combined (following vermicomposting of composted material) on the concentration, mobility, and chemical speciation of arsenic. The main emphasis included assessment of the mobile As fraction, targeted arsenic species, and arsenic bioavailability. Chemical studies were undertaken using bioaccumulation tests with earthworms performed before and after composting and vermicomposting.
2.2. Composting A container for home compost production was used and filled with approximately 290 l of the following materials: sewage sludge, horse manure with sawdust, and grass clippings. The components were mixed in a dry state (except for the fresh grass) in the volumetric ratio of 3:6:1 (sludge/manure/grass). After that, water was added equivalent to 50% of the dry material weight. Composting was allowed to run for 90 days outside the laboratory at ambient temperature. The container was placed under the roof to avoid contamination by rain. Turning was done weekly to ensure aeration. The moisture content was maintained at approximately 50% of water holding capacity (WHC) by the squeeze-test and periodic addition of water. Changes in temperature were automatically monitored daily in the middle of the compost with a digital temperature probe. In addition, the pH value and bulk density of the compost was monitored monthly in collected representative samples. 5 kg sample (composites of five sub-samples taken randomly) was collected before composting (C-0) and similar samples taken after 30, 60 and 90 days of composting (C-30, C-60 and C-90). Each sample was air-dried, thoroughly homogenized by hand (the sludge particle size was not maintained), and stored at laboratory temperature before analysis. The pH value (of a suspension of 1 g of sample in 10 ml of water) and dry bulk density were determined for each sample.
2. Materials and methods 2.3. Vermicomposting 2.1. Materials Dewatered sludge with a high arsenic concentration (396 ± 1 mg kg−1 dry weight) was used as the initial material for the study. The sludge was produced by a groundwater treatment plant, which cleans groundwater seriously contaminated by arsenic from phosphogypsum (PG) deposits in Fosfa Postorna (Breclav, Czech Republic). PG is a by-product from processing fluorapatite by the “wet acid” method for phosphoric acid production in fertilizer plants [10]. The cleaning process of contaminated water consisted of the addition of slaked lime (Ca(OH)2 ) to adjust pH and also precipitation by means of the addition of ferric sulfate (Fe2 (SO4 )3 ). Metal precipitates were removed from the water by filtration and a yellow sludge was the final waste product of the remediation. Immediately after sampling, the sludge sample was air-dried for 25 days on a plastic tarp. The dried sludge was pulverized to a grain size <8 mm and ground. The basic properties of the sludge and metal content are shown in Tables 1 and 2. On the basis of the total arsenic content, this sludge is considered to be a highly hazardous waste material, according to EPA [11]. Horse manure mixed with sawdust was taken from a small horse farm which does not extensively use chemistry, pesticides or pharmaceuticals. The horse manure was spread for 20 days on a plastic tarp for air-drying. Grass was acquired from a meadow covered mostly by Trifolium pratense, Festuca pratensis, Lolium perenne, Poa pratensis, and Festuca rubra. The grass was cut into 25–30 cm long pieces. The basic chemical characteristics of the horse manure and grass clippings are shown in Table 2.
An earthworm culture (Eisenia fetida) was cultured at the laboratories of the Research Centre for Toxic Compounds in the Environment (Brno, Czech Republic) in a mixture of garden substrate (50%), granulated cattle manure (40%), and Sphagnum peat (10%). The water content of the substrate was approximately 80% WHC (water holding capacity) and the pH was adjusted to 6–7 with CaCO3 . The earthworms were fed with granulated cattle manure and the culture was maintained at 20 ± 1 ◦ C in darkness. The vermicomposting experiments were carried out in the laboratory using plastic vermicomposting boxes of 10 l capacity. Two vermicomposts were carried out with different substrates: (a) for vermicompost VC1 , the material C-0 was used (C-0 is hereinafter called VC1 -0), (b) for vermicompost VC2 , the material C-90 was used (C-90 is hereinafter called VC2 -0). Eight liters of dry substrate were placed in vermicomposting box and water was added to reach 50% of the dry material weight. Then, 200 earthworms E. fetida per 1 l of dry matter (i.e. 1600 individuals per box) were added. Each vermicompost was established in duplicate to ensure a sufficient amount of material for analysis. The mean weight of adult earthworms was approximately 200 mg at the beginning of the experiment. All vermicomposts were kept in the dark at a laboratory temperature of 22 ◦ C for a period of 90 days. The moisture content was maintained at approximately 50% WHC by the squeeze-test and by the periodic sprinkling of water on filter paper, which covered the substrates in the box. The mixtures were turned over manually every 30 days. During the process of vermicomposting no extra feed was added. Samples were collected at the beginning of the experiment (VC1 0 and VC2 -0) and after 30, 60 and 90 days of vermicomposting
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Table 2 Total contents of elements in initial materials used in the experiments. Elements (mg kg−1 )
Sludge
Grass
Horse manure
Soil
As Ca Cd Co Cr Fe Ni P Pb V Zn
396 ± 1 117,700 ± 300 0.83 ± 0.01 19.5 ± 0.07 8.2 ± 0.1 15,270 ± 40 51.8 ± 0.1 84,400 ± 500 5.23 ± 0.06 14.67 ± 0.05 31.5 ± 0.2
0.45 ± 0.04 Not measured 0.060 ± 0.006 0.33 ± 0.04 2.1 ± 0.3 Not measured 1.4 ± 0.1 Not measured 1.1 ± 0.1 2.0 ± 0.3 42 ± 2
0.7 ± 0.1 Not measured 0.18 ± 0.01 0.44 ± 0.04 1.6 ± 0.1 Not measured 1.62 ± 0.09 Not measured 1.2 ± 0.2 1.7 ± 0.1 25 ± 3
7.7 ± 0.8 Not measured 0.20 ± 0.01 8.8 ± 0.1 41.1 ± 0.7 Not measured 21 ± 1 Not measured 18.1 ± 0.1 43 ± 1 57.6 ± 0.1
(VC1 -30, VC1 -60, VC1 -90 and VC2 -30, VC2 -60 and VC2 -90) as five representative sub-samples taken randomly from various parts of the vermicompost and mixed to give a composite sample of approximately 2 kg. This composite sample was pooled with the sample from the respective vermicompost duplicate. All earthworms were separated from these samples by hand sorting and put back into the boxes. The samples of vermicomposts were air-dried at laboratory temperature, thoroughly homogenized by hand and stored at lab temperature before analyses. The pH value and dry bulk density were determined for each sample.
mobile arsenic concentration was determined by shaking a 5.00 (±0.01) g sample with 50 ml 0.1 M CH3 COONH4 for 5 h (150 rpm) in glass flasks. The suspensions were centrifuged at 6000 rpm for 10 min and filtered through a 0.45 m membrane filter. Each extraction was performed in three replicates. 4 ml sample solutions were stored for analysis in plastic test tubes and acidified with 100 l of nitric acid. For the analysis of arsenic species, 1 ml sample solutions were kept in GC micro vials and deep-frozen (−20 ◦ C). The determination of arsenic content in the extracts was carried out using ICP-MS spectrometry.
2.4. Bioaccumulation tests with earthworms Four materials were tested with respect to the bioavailability of arsenic to earthworms: C-0, C-90, VC1 -90 and VC2 -90, i.e. materials before and after composting and/or vermicomposting. Due to the high acute toxicity, especially of the vermicomposted substrates (>20% mortality of exposed earthworms), a ratio of 1:9 (substrate:soil) was used. The bioaccumulation test was performed according to OECD guidelines [12]. Non-contaminated surface soil (0–20 cm) was collected from Mokrá (Brno, Czech Republic). Subsamples of soils were air-dried for 25 days on a plastic tarp, then sieved through a 2 mm sieve to remove large soil fractions, such as stones and gravel. After air-drying, the soils were sealed in plastic packets at room temperature. The physicochemical properties of the soil are shown in Table 2. The collections of 10 earthworms from these tests were lyophilized, weighed, ground in a mortar, and digested using the microwave extraction procedure (Section 2.7). Earthworm bioaccumulation factors (BAFs) were calculated as total earthworm arsenic contents (mg kg−1 ) divided by total arsenic (mg kg−1 ) in the substrate. 2.5. Total As content determination Total arsenic content in compost and vermicompost samples was estimated from aqua regia extraction. 10 g samples were derived as a composite of five sub-samples randomly collected from compost and vermicompost samples. These samples were deep-frozen in liquid nitrogen and homogenized to a fine powder (particles <10 m) in a laboratory ball mill (Mixer Mill MM 301, Germany), following the procedure recommended by the International Organization for Standardization [13]. Each extraction was performed in three replicates. The digested samples were then analyzed for total amounts of heavy metals using an inductively coupled plasma mass spectrometer (Agilent 7500ce ICP-MS, Japan). All glassware and plastic were cleaned by soaking in 5% nitric acid for at least 24 h and then rinsed three times with distilled water. 2.6. Mobile As content 25 g samples (composites of five sub-samples taken randomly) were collected from compost and vermicompost substrates. The
2.7. Arsenic content in earthworms Earthworm tissue samples were digested using a Speedwave MWS-3+ laboratory microwave system (Berghof, Germany) with automatic temperature control. For this purpose, about 300 mg of representative and homogenous sample was weighed into a Teflon vessel and 4 ml of sub-boiled HNO3 (65%) and 2 ml of hydrogen peroxide (30%) were added. Heating of the Teflon vessels in a microwave oven was achieved with a temperature program in five steps (5 min at 180 ◦ C, 10 min at 190 ◦ C, and 3 × 5 min at 100 ◦ C) without evaporation. After digestion, the content of Teflon vessels were allowed to cool down, transferred quantitatively to a volumetric flask, and filled to 50 ml with Milli-Q water. The determination of arsenic content in the extracts was carried out using ICP-MS spectrometry.
2.8. Arsenic speciation analysis Stock solutions (1000 ppm As) of arsenite (AsIII ), arsenate (AsV ), dimethylarsenic acid (DMAV ) and monomethylarsenic acid (MMAV ) were prepared separately by dissolving 0.0434 g of NaAsO2 (p.a., Penta), 0.1041 g of Na2 HAsO4 ·7H2 O (p.a., Supelco), 0.0714 g of CH3 Na2 AsO3 ·6H2 O (p.a., Fluka) and 0.094 g of (CH3 )2 Na2 AsO2 ·3H2 O (p.a., Fluka) in a volumetric flasks and filling the flasks to 25 ml with Milli-Q water (Millipore Simplicity 185). All stock solutions were stored in a refrigerator at 4 ◦ C. Working calibration solutions of arsenic were prepared daily by appropriate dilution from the stock solutions with Milli-Q water. A buffer solution of 0.04 mol NH4 COOCH3 was prepared for HPLC–ICP-MS analysis. 2.28 ml of acetic acid was dissolved into 1 l of Milli-Q water. The pH of the buffer solution was adjusted to 6.0 by the drop wise addition of a diluted ammoniac solution. Under chromatographic conditions on a Hailton PRP-X100 column (Hamilton Company, USA), the elution order (according to retention times) was AsIII , DMA, MMA and AsV . To check the accuracy of the results obtained, one calibration sample was analyzed periodically during the measurement.
B. Manáková et al. / Journal of Hazardous Materials 280 (2014) 544–551 ˇ Table 3 Characteristics of compost and final products of composting and vermicomposting. All parameters were validated by duplicated analysis of the sample and the extended combined uncertainty was below 10%.
TOC (%) Ntot (%) Q4/6 HA (%) FA (%) HS (%) pH CEC (mmol kg−1 ) Ca (mg kg−1 ) K (mg kg−1 ) Mg (mg kg−1 )
C-0
C-90
VC1 -90
VC2 -90
20.0 0.63 4.9 0.91 1.20 2.11 8.4 n.a. 23.6 6.0 5.8
15.4 0.76 6.1 1.31 1.02 2.39 6.8 1.93 23.9 7.3 6.6
18.3 1.07 5.8 1.43 1.18 2.2 6.6 2.11 24.9 9.9 6.8
14.4 0.67 6.7 1.04 0.96 1.98 7.9 2.17 27.1 8.1 7.0
2.9. Statistical analysis All measurements were made in triplicate and results are presented as their mean value. The differences between concentrations over time and between composted and vermicomposted samples were tested by one-way analysis of variance ANOVA followed by Tukey’s test. Differences with ˛ = 0.05 were considered as statistically significant. All statistical analysis was performed in GraphPad Prism 5 for Windows version 5.01 (GraphPad Software, Inc., 2007). 3. Results 3.1. Composting 3.1.1. Basic characteristics of the composting system The main physico-chemical properties of the initial compost material (C-0) and its final product (C-90) are presented in Table 3. The temperature in the compost system increased quickly to 50 ◦ C during the first four days, remained at this temperature for the next two days and then began slowly to decrease. During the following experimental period, the temperature remained at a lower level of 24–28 ◦ C (during the 2nd–4th week) and then decreased spontaneously to ambient temperature and fluctuated at around 14 ◦ C for the next two months. As the process proceeded, the initial pH value decreased slightly from an alkaline value of 8.4 to a final value of 6.8. The initial C/N ratio in the compost was 32:1 and, over time, slowly decreased to 20:1. The proceeding mineralization of organic matter led to mass changes in compost weight (C-0: 311 ± 56 kg m−3 ). After 30 days of composting, the compost volume increases to 122% of total dry mass weight. A similar rate of organic matter loss was observed during the second month and the degradation was very slow over the third month. At the end of the composting period, the bulk density had increased to 477 ± 47 kg m−3 (154%). 3.1.2. Total content The results show that the total As concentration followed a significant (ANOVA, p < 0.05, Tukey, p < 0.05) increasing trend from 253 ± 11 mg kg−1 at the beginning of the process to 283 ± 3 mg kg−1 at the end of the third month of composting (Fig. 1a), with the main increase occurring during the second month of the composting cycle. Over the composting process, arsenic became more concentrated (equivalent to 111% of the initial material value). 3.1.3. Mobile fraction The easily mobile arsenic pool was measured by extraction with 0.1 M NH4 COOCH3 . Over the 90-day period, the extractable arsenic fraction tended to decrease from 1.9% of total content
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(4.9 ± 0.2 mg kg−1 ) at the beginning of the process to 1.3% of total content (3.66 ± 0.04 mg kg−1 ) at the end, as shown in Fig. 2a. The mobile fraction was therefore significantly (ANOVA, p < 0.05, Tukey, p < 0.05) reduced to 2/3 of its initial value by the process of composting. 3.1.4. Speciation analysis Speciation analysis was focused on four arsenic species: AsIII , DMA, MMA and AsV in the ammonium acetate extract, corresponding to the sum of metal species in each extract and determining the changes in As speciation over the composting process. The relative contributions of the four species are shown in Fig. 3a. Inorganic AsV was the main species extracted in the compost (88–96%), followed by AsIII (4–8%). There was no significant change in AsV content, but over the experimental period the AsIII content was significantly reduced from 8% (0.412 ± 0.007 mg kg−1 ) to 4% (0.152 ± 0.001 mg kg−1 ) (ANOVA, p < 0.05, Tukey, p < 0.05). At the beginning of the process, the content of the organic forms DMA and MMA were 0.3% and 0.2%, respectively. After the third month, DMA content slightly decreased to 0.2% and MMA content decreased below LOD (<0.003 mg kg−1 ). 3.2. Vermicomposting 3.2.1. Basic characteristics of the vermicomposting system In contrast to composting, vermicomposting does not involve exothermic reactions; the daily measured ambient temperature remained stable at 22 ± 1 ◦ C during the whole experimental period. Moisture content was maintained in the range 70–80% for both vermicomposts. Vermicompost VC1 was represented by samples C-0, VC1 -30, VC1 -60 and VC1 -90; vermicompost VC2 was represented by samples C-90, VC2 -30, VC2 -60 and VC2 -90. The main physicochemical properties of the initial material of the vermicomposts (C-0, C-90) and its final products (VC1 -90, VC2 -90) are presented in Table 3. There were only slight changes in pH values in both treatments. The pH value in VC1 decreased from 8.4 (C-0) to a slightly acidic pH of 6.6 (VC1 -90), whereas the pH value of VC2 increased slightly and moved from 6.8 (C-90) toward the neutrality of 7.1 (VC2 -90). The differences in pH were significant over the whole experimental period. In vermicompost VC1 , the C/N ratio decreased progressively from 32:1 (C-0) to 20:1 (VC1 -90). For vermicompost VC2 , the ratio was stable (21:1 for C-90; 22:1 for VC2 -90) and no significant changes were obtained. The bulk density of vermicompost VC1 had increased by 40% at the end of the vermicomposting process while VC2 showed a 20% increase. Bulk density in all vermicomposts showed a significant increase compared to its initial value (C-0; C-90) and was 439 ± 55 kg m−3 and 547 ± 72 kg m−3 respectively in VC1 -90 and VC2 -90. 3.2.2. Total content The analysis of total As content showed that the total As concentration (Astot ) for VC1 decreased significantly from 253 ± 11 mg kg−1 at the beginning to 226 ± 4 mg kg−1 at the end of the third month of vermicomposting (Fig. 1b) (ANOVA, p < 0.05, Tukey test, p < 0.05). The As content decreased significantly, falling to 88% of the original total As value at the end of the experiment. There were no significant differences in the total As concentration in vermicompost VC2 (Fig. 1c). At the beginning of the process, the As content was 283 ± 3 mg kg−1 and remained stable over the experimental period. 3.2.3. Mobile fraction Fig. 2b and c shows the changes in the mobile arsenic pool during the vermicomposting processes. In vermicompost VC1 , the mobile As fractions decreased significantly from 1.9% of total content (4.9 ± 0.2 mg kg−1 ) to 0.7% of total content (1.56 ± 0.02 mg kg−1 ).
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Fig. 1. Total arsenic concentrations (column: [mg kg−1 ]; curve: [%]) during sludge composting and vermicomposting: (a) compost C-(0–90); (b) vermicompost VC1 -(0–90); and (c) vermicompost VC2 -(0–90).
The mobile arsenic pool in vermicompost VC2 decreased significantly from 1.3% of total content (3.66 ± 0.04 mg kg−1 ) to 0.8% of total content (2.25 ± 0.02 mg kg−1 ). All changes found were highly statistically significant (ANOVA, p < 0.05, Tukey test, p < 0.05).
3.2.4. Arsenic speciation The results showed that AsV was the predominant species in VC1 and VC2 over the whole three month period of vermicomposting, and the trends in AsIII and AsV contents were consistent (Fig. 3b and c). For VC1 the content of AsV significantly decreased from 92% (VC1 -0) to 85% (VC1 -90) of arsenic in the mobile fraction. At the beginning of the process, the amount of AsIII was 8% of the mobile content (0.412 ± 0.007 mg kg−1 ) and then increased slightly to 14% the mobile content (0.21 ± 0.03 mg kg−1 ) after the third month. For vermicompost VC2 (Fig. 3c), the concentration of inorganic AsV slightly increased from 96% (VC2 -0) to 99% (VC2 -90) of the mobile arsenic fraction and represented the predominant species during the whole three months. AsIII amounts ranged from 4% (VC2 -0) to 0.5% (VC2 -90) of the mobile arsenic fraction. The results showed a significant increase in the AsIII amount after the first month, but over the whole process, the content of AsIII significantly decreased to 0.5%. All these changes were statistically significant (ANOVA, p < 0.05, Tukey, p < 0.05). In both vermicomposts the sum of the amounts of MMA and DMA forms were just above limit of detection and did not change significantly during the vermicomposting processes.
3.3. Bioaccumulation in earthworms Earthworm bioaccumulation factors (BAFs) were observed for soils amended with compost/vermicompost in the ratio of 9:1. All materials were checked for their toxicity to earthworms without dilution; the mortality of earthworms achieved 100% in all tested samples. During the composting, arsenic absorption by earthworms decreased significantly from 35 ± 5 mg kg−1 (C-0) to 14 ± 1 mg kg−1 (C-90), 14 ± 1 mg kg−1 (VC1 -90) and 17 ± 1 mg kg−1 (VC2 -90). The mean BAF values (n = 3) were reduced from 1.4 (C-0) to 0.5 (C-90), 0.5 (VC1 -90) and 0.5 (VC2 -90) for compost. 4. Discussion 4.1. Composting process The composting process involves the accelerated degradation of organic matter by microbial activity. Intensive decomposition produces heat which is reflected by a rapid increase in temperature. The compost material in our study was not energy-rich, so the temperature level observed was lower. The alkaline components of the sludge could not buffer the increased acidity of the compost. This was probably because of the presence of nitrous and nitric acid formations [14]. The C/N ratio gradually decreased to 20/1 in the finished product, corresponding to good quality compost [15]. The mineralization process resulted in a significant increase in compost bulk density and the non-biodegradable part (e.g. heavy metals)
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Fig. 2. Fraction of 0.1 M CH3 COONH4 extractable arsenic (column: [mg kg−1 ]; curve: [%]) during sludge composting and vermicomposting: (a) compost C-(0–90); (b) vermicompost VC1 -(0–90); and (c) vermicompost VC2 -(0–90).
tended to increased during the degradation process. Arsenic, like other heavy metals, is not destroyed during the composting process but, on the contrary, tends to accumulate (Fig. 1). The total heavy metals concentration is considered to be an overall pollution indicator. The obtained total arsenic fraction easily exceeded the limit of 20 mg kg−1 for As in compost [16], which may be appropriate for the estimation of long-term risks. Arsenic is one of those metals (such as Cr, Mo, Sb) which form oxyanions in solution at all pH values [17]. The results showed the gradual release of arsenic from sludge, reflected in the changes of total As content mainly during the second and third months of the composting. The arsenic became more concentrated, a phenomenon corresponding to studies describing similar trends for metals (e.g. As, Mn, Cu, Zn) in compost [18–20]. It should be noted that toxic arsenic concentrations in compost are potentially high and that the leachability of arsenic associated with compost is of particular concern. However, in our study, the transformation of compost matter influenced the bond strength of arsenic in this matrix and increased residual arsenic fraction in mature compost. In the present study, 0.1 mol l−1 NH4 COOCH3 was used as a mild extractant to evaluate the labile arsenic pool in compost. This labile fraction is positively the most biologically active and has the highest potential for environmental contamination. The results
showed that arsenic became more available in the matrix of compost (1.2–1.9%) compared to the available As fraction in sludge (0.3%). Arsenic is mobilized partly due to the chemical or microbial reduction of AsV to AsIII [21] and the dissolution of As contained in minerals and organic phases under changing redox conditions [22]. At the end of the composting process, a very large arsenic pool (98.7%) was not extractable and was strongly sorbed on the compost matrix. This indicated the high stability of the As pool present in the compost under experimental conditions. It was confirmed that arsenic can be included in the group of metals containing Zn, Cu, Cd and Pb, whose easily-mobile fractions are, in general, low and do not change very much during the composting process [23]. The predominant arsenic species in sludge was AsV (99.9%). The presence of organic matter changes the redox speciation of arsenic in compost both chemically and microbially. At the beginning of the composting process, AsIII constituted a small proportion of the total amount of arsenic (8%). Probably, therefore, a direct chemical effect of organic matter on the redox speciation of arsenic can be suggested. During the composting process, AsIII was slowly decreased and transformed to AsV . The methylation of arsenic by compost microorganisms must have been brought about by dimethylarsinic acid (DMA) and monomethylarsinic acid (MMA). Smaller concentrations of MMA (<0.2%) and DMA (<0.3%) were present and their contents did not change significantly.
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Fig. 3. Percentage contribution of arsenic species in 0.1 M CH3 COONH4 during sludge composting and vermicomposting: (a) compost C-(0–90); (b) vermicompost VC1 -(0–90); and (c) vermicompost VC2 -(0–90).
4.2. Vermicomposting Similarly to the composting process, vermicomposting showed the potential of organic matter to influence the retention and mobilization behavior of arsenic by altering As speciation by means of interaction with the digestive systems of earthworms. The pH decrease in vermicompost VC1 was probably caused by the mineralization of nitrogen and phosphorus and by the bioconversion of organic material into intermediate species of organic acids [24]. The pH of this substrate slowly decreased to a value of 6.8; E. fetida earthworms are relatively tolerant with regard to pH, enjoying an optimal pH range of between 5 and 9 [25]. The extent of the volume reduction of vermicompost substrates was not significant and did not lead to an increase in total metal content, although both vermicomposts were highly As contaminated when considering the current CSN Guideline Values for As of 10 mg kg−1 (I. class) and 20 mg kg−1 (II. class) [16]. The effects of earthworms on the mobility of heavy metals have been extensively studied by many authors, and both epigeic and anecic species of earthworms appear to increase the availability and mobility of metals [26–28]. In contrast to these studies, we demonstrated the ability of earthworms to decrease As mobility over the whole vermicomposting process. For both vermicompost end products, the extractable arsenic fraction was reduced to less
than half of the NH4 COOCH3 extract and less than 2.5% of the total As content. Vermicomposting reduced arsenic availability by 1/3. With regards to the total arsenic concentration, it has been suggested that the reason for the decrease in arsenic availability is the accumulation within the tissue of the earthworm (VC1 : 286 ± 18 mg kg−1 As) itself, which shows their ability to eliminate an excess of arsenic. In any case, the results indicate the obvious impact of earthworms on arsenic availability. Arsenic speciation was similar in both vermicomposts, with AsV and AsIII present as the dominant inorganic species. In the case of vermicompost VC1 , all arsenic species were present at the same time. This is with agreement with the observations of Langdon et al., who found the effect of biotransformation by both earthworms and microorganisms to arsenic content and bioaccumulation [29]. The stabilization of substrates (VC1 ) through composting made them more resistant to arsenic methylation and even though MMA was just above the limit of detection, DMA was still present and slowly decreased over time. Regarding the content of AsIII in vermicompost VC1 , the chemical or microbial reduction of AsV to AsIII was significant. In contrast to vermicompost VC1 , AsV was not reduced during the VC2 vermicomposting period; instead, AsIII oxidation was observed. As speciation analysis was not performed on earthworm tissues. Recent physiological studies on the metabolism of As
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in earthworm species suggests that earthworms simply accumulate arsenic through passive diffusion and metabolize it into less toxic organic derivatives or inorganic forms (AsIII and AsV ) [30–32]. 4.3. Bioaccumulation in earthworms In our study, the dilution of compost/vermicompost samples with soil could be associated with lower compost/vermicompost application rates, and the BAF values could reflect the arsenic fraction directly accessible to earthworms. The results of arsenic bioaccumulation confirmed the significant influence of composting and vermicomposting processes on the stability of arsenic interactions, although the end products showed a similar potential with respect to the bioavailability of arsenic to earthworms. 5. Conclusion The study concludes that combined composting and vermicomposting proved to be an effective method for the stabilization of the arsenic mobile fraction from the matured products. The combination of the actions of microorganisms and earthworms promoted significant changes related to the chemistry of arsenic present in the processed material. Total arsenic concentrations were enhanced and persisted during the composting and vermicomposting processes. By contrast, these processes significantly reduced the labile arsenic pool and changed the contents of arsenic species to AsIII and AsV in end products. Mobile arsenic contents were reduced to 2/3 by composting and to 1/3 by vermicomposting. Further compost vermicomposting then reduced the mobile As pool to 4/9 of its initial value (the most effective). The largest proportion of arsenic was found in the fraction more resistant to extraction, indicating that arsenic was in more stable forms and consequently less available for plant uptake. In end products, the amount of potentially bioavailable arsenic was in the range 0.7–1.3%. Composting and vermicomposting processes affected arsenic speciation; AsV was found to be the dominant species in all easily extractable fractions. The investigation of arsenic BAFs showed that composting and vermicomposting processes decreased the arsenic fraction easily taken up by earthworms. Despite the fact that the labile arsenic content was greatly reduced, the produced composts/vermicomposts cannot be considered suitable for agronomic application due to the fact that the arsenic contents do not comply with the limits set for compost. Acknowledgment This research was supported by the Ministry of Education of the Czech Republic (projects LM2011028 and LO1214). References [1] J. Dominguez, C.A. Edwards, Relationships between composting and vermicomposting: relative values of the products, in: C.A. Edwards, N.Q. Arancon, R.L. Sherman (Eds.), Vermiculture Technology: Earthworms, Organic Waste and Environmental Management, CRC Press, Boca Raton, FL, 2010, pp. 11–26. [2] S. Hait, V. Tare, Transformation and availability of nutrients and heavy metals during integrated composting–vermicomposting of sewage sludges, Ecotoxicol. Environ. Saf. 79 (2012) 214–224. [3] R. Gupta, V.K. Garg, Stabilization of primary sewage sludge during vermicomposting, J. Hazard. Mater. 153 (2008) 1023–1030.
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[11] EPA Standards for the Use or Disposal of Sewage Sludge, Final Rules, 40 CFR Part 257, 1993, http://water.epa.gov/scitech/wastetech/biosolids/ upload/fr2-19-93.pdf (accessed 27.02.14). [12] OECD Guidelines for the Testing of Chemicals, Bioaccumulation in Oligochaetes, 2009, http://www.oecd.org/chemicalsafety/ Terrestrial testing/44098118.pdf (accessed 27.02.14). [13] ISO 11466, Soil Quality, Extraction of Trace Elements Soluble in Aqua Regia, ISO, Geneva, Switzerland, 1995. [14] J.H. Hsu, S.L. Lo, Chemical and spectroscopic analysis of organic matter formations during composting of pig manure, Environ. Pollut. 104 (1998) 189–196. [15] V.K. Sharma, M. Canditelli, F. Fortuna, G. Cornacchia, Processing of urban and agro-industrial residues by aerobic composting: review, Energy Convers. Manage. 38 (1997) 453–478. [16] CSN 46 5735, Prumyslove komposty, technicka norma, CSN, Prague, Czech Republic, 1991 (in Czech). [17] C. Vandecasteele, G. Cornelis, Oxyanions in waste: occurrence, leaching, stabilisation, relation to wastewater treatment, in: M. Vaclavikova, K. Vitale, G.P. Gallios, L. Ivanicova (Eds.), Water Treatment Technologies for the Removal of High-toxicity Pollutants, Springer, 2010, pp. 149–159. [18] J.H. Hsu, S.L. Lo, Effect of composting on characterization and leaching of copper, manganese, and zinc from swine manure, Environ. Pollut. 114 (2001) 119–127. [19] A. Mahdi, I. Azni, O.S.R. Syed, Physico-chemical characterization of compost of the industrial tannery sludge, J. Eng. Sci. Technol. 2 (2007) 81–94. [20] G.M. Greenway, Q.J. Song, Heavy metal speciation in the composting process, J. Environ. Monit. 4 (2002) 300–305. [21] A.C. Heimann, C. Blodau, D. Postma, F. Larsen, P.H. Viet, P.Q. Nhan, S. Jessen, M.T. Duc, N.T.M. Hue, R. Jakobsen, Hydrogen thresholds and steady-state concentrations associated with microbial arsenate respiration, Environ. Sci. Technol. 41 (2007) 2311–2317. [22] M. Bissen, F.H. Frimmel, Arsenic–- a review: Part I. Occurrences, toxicity, speciation, mobility, Acta Hydrochim. Hydrobiol. 31 (2003) 9–18. [23] J. Singh, A.S. Kalamdhad, Bioavailability and leachability of heavy metals during composting – a review, Int. Res. J. Environ. Sci. 2 (2013) 59–64. [24] V.K. Garg, Y.K. Yadav, A. Sheoran, S. Chand, P. Kaushik, Livestock excreta management through vermicomposting using an epigeic earthworm Eisenia foetida, Environmentalist 26 (2006) 269–276. [25] C.A. Edwards, Earthworm Ecology, 2nd ed., CRC Press, Boca Raton, FL, 2004. [26] T. Sizmur, E.L. Tilston, J. Charnock, B. Palumbo-Roe, M.J. Watts, M.E. Hodson, Impacts of epigeic, anecic and endogeic earthworms on metal and metalloid mobility and availability, J. Environ. Monit. 13 (2011) 266–273. [27] M.I. Zorn, C.A.M. Van Gestel, H. Eijsackers, The effect of Lumbricus rubellus and Lumbricus terrestris on zinc distribution and availability in artificial soil columns, Biol. Fertil. Soils 41 (2005) 212–215. [28] L. Bernard, L. Chapuis-Lardy, T. Razafimbelo, M. Razafindrakoto, A.L. Pablo, E. Legname, J. Poulain, T. Brüls, M. O’Donohue, A. Brauman, J.L. Chotte, E. Blanchart, Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil, ISME J. 6 (2012) 213–222. [29] C.J. Langdon, A.A. Meharg, J. Feldmann, T. Balgarm, J. Charnock, M. Farquhar, T.G. Piearce, K.T. Semple, J. Cotter-Howells, Arsenic-speciation in arsenate-resistant and non-resistant populations of the earthworm, Lubricus rubellus, J. Environ. Monit. 4 (2002) 608–630. [30] M. Button, G.R. Jenkin, C.F. Harrington, M.J. Watts, Arsenic biotransformation in earthworms from contaminated soils, J. Environ. Monit. 11 (2009) 1484–1491. [31] T. Sizmur, M.J. Watts, G.D. Brown, B. Palumbo-Roe, M.E. Hodson, Impact of gut passage and mucus secretion by the earthworm Lumbricus terrestris on mobility and speciation of arsenic in contaminated soil, J. Hazard. Mater. 197 (2011) 169–175. 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Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Effects of combined composting and vermicomposting of waste sludge on arsenic fate and bioavailability ˇ Blanka Manáková, Jan Kuta, Markéta Svobodová, Jakub Hofman ∗ Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 753/5, CZ-62500, Czech Republic
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Industrial sludge with high As con• • • •
tent was treated by composting and vermicomposting. The volume of compost decreased, which led to an increase in total As content. The labile arsenic fraction was significantly decreased. AsV was the predominant arsenic species formed. The mobile fraction was not directly related to bioavailability to earthworms.
a r t i c l e
i n f o
Article history: Received 20 March 2014 Received in revised form 4 August 2014 Accepted 7 August 2014 Available online 25 August 2014 Keywords: Sludge Arsenic Composting Vermicomposting Bioavailability
a b s t r a c t Composting and vermicomposting are traditional processes for the treatment of sludge. During these processes, the humification of organic matter has a significant effect on the physicochemical form and distribution of heavy metals. In this study, industrial sludge (groundwater treatment waste) contaminated by arsenic (396 ± 1 mg kg−1 ) was used. Such sludge poses a significant challenge with respect to effective treatment. Composting, vermicomposting (with Eisenia fetida), and the combined approach of composting and vermicomposting were performed to determine the evolution of arsenic speciation, mobility and bioavailability. The composting/vermicomposting was done with sludge, horse manure, and grass in the ratios of 3:6:1. A solution of 0.1 M NH4 COOCH3 was used as a single extraction solvent for determination of the mobile arsenic pool and targeted arsenic species (AsIII , AsV , monomethylarsenic acid – MMAV , dimethylarsenic acid – DMAV ). The analysis of arsenic in the extracts was carried out by means of HPLC–ICP-MS spectrometry. In addition, the earthworm species E. fetida was used for bioaccumulation tests that followed the compost and vermicompost processes. The obtained results indicate a reduction in arsenic mobility and bioavailability in all matured composts and vermicomposts. The combined process exhibited a greater effect than compost or vermicompost alone. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The overproduction of anthropogenic waste sludge has led to the use of inappropriate disposal practices and caused the introduction of metals into the environment. Nowadays, composting
∗ Corresponding author. Tel.: +420 549 494 267; fax: +420 549 492 840. E-mail address: [email protected] (J. Hofman). http://dx.doi.org/10.1016/j.jhazmat.2014.08.024 0304-3894/© 2014 Elsevier B.V. All rights reserved.
and vermicomposting are two of the best-known processes for the biological stabilization of sludge [1]. During these processes, heavy metals are redistributed to a newly formed matrix and the level of metal contamination generally grows [2–4]. From another point of view, the role of composting could be considered as an important environmental sink for the elimination of metals. Arsenic bioavailability and toxicity is strongly dependent on arsenic species. The mobility of sludge-born arsenic entering into composting/vermicomposting processes is strongly related to
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Table 1 Selected properties of sludge used in the study. As (mg kg−1 )
AsIII (mg kg−1 )
DMA (mg kg−1 )
MMA (mg kg−1 )
AsV (mg kg−1 )
396 ± 1
<0.004
<0.003
<0.003
1.26 ± 0.04
pH
TC (%)
TOC (%)
TIC (%)
Dry mass (%)
8.5
1.03 ± 0.01
0.23 ± 0.03
0.80 ± 0.03
63 ± 1
redox processes, microbial activity, and the degradation of organic matter [5]. Arsenic interaction with organic matter may include redox reaction, complexation, colloid formation, and sorption competition; however, the direct chemical effect of organic matter on the redox speciation of arsenic has not yet been proven. Beside AsIII oxidation and various methylation reactions [6], microbial processes are a potential cause of high variability in AsV reduction, during which microorganisms use AsV as an electron acceptor and mediate transformation to the more toxic and mobile AsIII species [7]; however, chemical reactions may also contribute to this process. AsV usually remains bound to iron, alumina and manganese oxides, which limit its mobility and bioavailability [8]. The toxicity and behavior of arsenic and its compounds in the environment is summarized in many reviews [6,9]. The aim of this work was to understand the influence of composting, vermicomposting, and both processes combined (following vermicomposting of composted material) on the concentration, mobility, and chemical speciation of arsenic. The main emphasis included assessment of the mobile As fraction, targeted arsenic species, and arsenic bioavailability. Chemical studies were undertaken using bioaccumulation tests with earthworms performed before and after composting and vermicomposting.
2.2. Composting A container for home compost production was used and filled with approximately 290 l of the following materials: sewage sludge, horse manure with sawdust, and grass clippings. The components were mixed in a dry state (except for the fresh grass) in the volumetric ratio of 3:6:1 (sludge/manure/grass). After that, water was added equivalent to 50% of the dry material weight. Composting was allowed to run for 90 days outside the laboratory at ambient temperature. The container was placed under the roof to avoid contamination by rain. Turning was done weekly to ensure aeration. The moisture content was maintained at approximately 50% of water holding capacity (WHC) by the squeeze-test and periodic addition of water. Changes in temperature were automatically monitored daily in the middle of the compost with a digital temperature probe. In addition, the pH value and bulk density of the compost was monitored monthly in collected representative samples. 5 kg sample (composites of five sub-samples taken randomly) was collected before composting (C-0) and similar samples taken after 30, 60 and 90 days of composting (C-30, C-60 and C-90). Each sample was air-dried, thoroughly homogenized by hand (the sludge particle size was not maintained), and stored at laboratory temperature before analysis. The pH value (of a suspension of 1 g of sample in 10 ml of water) and dry bulk density were determined for each sample.
2. Materials and methods 2.3. Vermicomposting 2.1. Materials Dewatered sludge with a high arsenic concentration (396 ± 1 mg kg−1 dry weight) was used as the initial material for the study. The sludge was produced by a groundwater treatment plant, which cleans groundwater seriously contaminated by arsenic from phosphogypsum (PG) deposits in Fosfa Postorna (Breclav, Czech Republic). PG is a by-product from processing fluorapatite by the “wet acid” method for phosphoric acid production in fertilizer plants [10]. The cleaning process of contaminated water consisted of the addition of slaked lime (Ca(OH)2 ) to adjust pH and also precipitation by means of the addition of ferric sulfate (Fe2 (SO4 )3 ). Metal precipitates were removed from the water by filtration and a yellow sludge was the final waste product of the remediation. Immediately after sampling, the sludge sample was air-dried for 25 days on a plastic tarp. The dried sludge was pulverized to a grain size <8 mm and ground. The basic properties of the sludge and metal content are shown in Tables 1 and 2. On the basis of the total arsenic content, this sludge is considered to be a highly hazardous waste material, according to EPA [11]. Horse manure mixed with sawdust was taken from a small horse farm which does not extensively use chemistry, pesticides or pharmaceuticals. The horse manure was spread for 20 days on a plastic tarp for air-drying. Grass was acquired from a meadow covered mostly by Trifolium pratense, Festuca pratensis, Lolium perenne, Poa pratensis, and Festuca rubra. The grass was cut into 25–30 cm long pieces. The basic chemical characteristics of the horse manure and grass clippings are shown in Table 2.
An earthworm culture (Eisenia fetida) was cultured at the laboratories of the Research Centre for Toxic Compounds in the Environment (Brno, Czech Republic) in a mixture of garden substrate (50%), granulated cattle manure (40%), and Sphagnum peat (10%). The water content of the substrate was approximately 80% WHC (water holding capacity) and the pH was adjusted to 6–7 with CaCO3 . The earthworms were fed with granulated cattle manure and the culture was maintained at 20 ± 1 ◦ C in darkness. The vermicomposting experiments were carried out in the laboratory using plastic vermicomposting boxes of 10 l capacity. Two vermicomposts were carried out with different substrates: (a) for vermicompost VC1 , the material C-0 was used (C-0 is hereinafter called VC1 -0), (b) for vermicompost VC2 , the material C-90 was used (C-90 is hereinafter called VC2 -0). Eight liters of dry substrate were placed in vermicomposting box and water was added to reach 50% of the dry material weight. Then, 200 earthworms E. fetida per 1 l of dry matter (i.e. 1600 individuals per box) were added. Each vermicompost was established in duplicate to ensure a sufficient amount of material for analysis. The mean weight of adult earthworms was approximately 200 mg at the beginning of the experiment. All vermicomposts were kept in the dark at a laboratory temperature of 22 ◦ C for a period of 90 days. The moisture content was maintained at approximately 50% WHC by the squeeze-test and by the periodic sprinkling of water on filter paper, which covered the substrates in the box. The mixtures were turned over manually every 30 days. During the process of vermicomposting no extra feed was added. Samples were collected at the beginning of the experiment (VC1 0 and VC2 -0) and after 30, 60 and 90 days of vermicomposting
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Table 2 Total contents of elements in initial materials used in the experiments. Elements (mg kg−1 )
Sludge
Grass
Horse manure
Soil
As Ca Cd Co Cr Fe Ni P Pb V Zn
396 ± 1 117,700 ± 300 0.83 ± 0.01 19.5 ± 0.07 8.2 ± 0.1 15,270 ± 40 51.8 ± 0.1 84,400 ± 500 5.23 ± 0.06 14.67 ± 0.05 31.5 ± 0.2
0.45 ± 0.04 Not measured 0.060 ± 0.006 0.33 ± 0.04 2.1 ± 0.3 Not measured 1.4 ± 0.1 Not measured 1.1 ± 0.1 2.0 ± 0.3 42 ± 2
0.7 ± 0.1 Not measured 0.18 ± 0.01 0.44 ± 0.04 1.6 ± 0.1 Not measured 1.62 ± 0.09 Not measured 1.2 ± 0.2 1.7 ± 0.1 25 ± 3
7.7 ± 0.8 Not measured 0.20 ± 0.01 8.8 ± 0.1 41.1 ± 0.7 Not measured 21 ± 1 Not measured 18.1 ± 0.1 43 ± 1 57.6 ± 0.1
(VC1 -30, VC1 -60, VC1 -90 and VC2 -30, VC2 -60 and VC2 -90) as five representative sub-samples taken randomly from various parts of the vermicompost and mixed to give a composite sample of approximately 2 kg. This composite sample was pooled with the sample from the respective vermicompost duplicate. All earthworms were separated from these samples by hand sorting and put back into the boxes. The samples of vermicomposts were air-dried at laboratory temperature, thoroughly homogenized by hand and stored at lab temperature before analyses. The pH value and dry bulk density were determined for each sample.
mobile arsenic concentration was determined by shaking a 5.00 (±0.01) g sample with 50 ml 0.1 M CH3 COONH4 for 5 h (150 rpm) in glass flasks. The suspensions were centrifuged at 6000 rpm for 10 min and filtered through a 0.45 m membrane filter. Each extraction was performed in three replicates. 4 ml sample solutions were stored for analysis in plastic test tubes and acidified with 100 l of nitric acid. For the analysis of arsenic species, 1 ml sample solutions were kept in GC micro vials and deep-frozen (−20 ◦ C). The determination of arsenic content in the extracts was carried out using ICP-MS spectrometry.
2.4. Bioaccumulation tests with earthworms Four materials were tested with respect to the bioavailability of arsenic to earthworms: C-0, C-90, VC1 -90 and VC2 -90, i.e. materials before and after composting and/or vermicomposting. Due to the high acute toxicity, especially of the vermicomposted substrates (>20% mortality of exposed earthworms), a ratio of 1:9 (substrate:soil) was used. The bioaccumulation test was performed according to OECD guidelines [12]. Non-contaminated surface soil (0–20 cm) was collected from Mokrá (Brno, Czech Republic). Subsamples of soils were air-dried for 25 days on a plastic tarp, then sieved through a 2 mm sieve to remove large soil fractions, such as stones and gravel. After air-drying, the soils were sealed in plastic packets at room temperature. The physicochemical properties of the soil are shown in Table 2. The collections of 10 earthworms from these tests were lyophilized, weighed, ground in a mortar, and digested using the microwave extraction procedure (Section 2.7). Earthworm bioaccumulation factors (BAFs) were calculated as total earthworm arsenic contents (mg kg−1 ) divided by total arsenic (mg kg−1 ) in the substrate. 2.5. Total As content determination Total arsenic content in compost and vermicompost samples was estimated from aqua regia extraction. 10 g samples were derived as a composite of five sub-samples randomly collected from compost and vermicompost samples. These samples were deep-frozen in liquid nitrogen and homogenized to a fine powder (particles <10 m) in a laboratory ball mill (Mixer Mill MM 301, Germany), following the procedure recommended by the International Organization for Standardization [13]. Each extraction was performed in three replicates. The digested samples were then analyzed for total amounts of heavy metals using an inductively coupled plasma mass spectrometer (Agilent 7500ce ICP-MS, Japan). All glassware and plastic were cleaned by soaking in 5% nitric acid for at least 24 h and then rinsed three times with distilled water. 2.6. Mobile As content 25 g samples (composites of five sub-samples taken randomly) were collected from compost and vermicompost substrates. The
2.7. Arsenic content in earthworms Earthworm tissue samples were digested using a Speedwave MWS-3+ laboratory microwave system (Berghof, Germany) with automatic temperature control. For this purpose, about 300 mg of representative and homogenous sample was weighed into a Teflon vessel and 4 ml of sub-boiled HNO3 (65%) and 2 ml of hydrogen peroxide (30%) were added. Heating of the Teflon vessels in a microwave oven was achieved with a temperature program in five steps (5 min at 180 ◦ C, 10 min at 190 ◦ C, and 3 × 5 min at 100 ◦ C) without evaporation. After digestion, the content of Teflon vessels were allowed to cool down, transferred quantitatively to a volumetric flask, and filled to 50 ml with Milli-Q water. The determination of arsenic content in the extracts was carried out using ICP-MS spectrometry.
2.8. Arsenic speciation analysis Stock solutions (1000 ppm As) of arsenite (AsIII ), arsenate (AsV ), dimethylarsenic acid (DMAV ) and monomethylarsenic acid (MMAV ) were prepared separately by dissolving 0.0434 g of NaAsO2 (p.a., Penta), 0.1041 g of Na2 HAsO4 ·7H2 O (p.a., Supelco), 0.0714 g of CH3 Na2 AsO3 ·6H2 O (p.a., Fluka) and 0.094 g of (CH3 )2 Na2 AsO2 ·3H2 O (p.a., Fluka) in a volumetric flasks and filling the flasks to 25 ml with Milli-Q water (Millipore Simplicity 185). All stock solutions were stored in a refrigerator at 4 ◦ C. Working calibration solutions of arsenic were prepared daily by appropriate dilution from the stock solutions with Milli-Q water. A buffer solution of 0.04 mol NH4 COOCH3 was prepared for HPLC–ICP-MS analysis. 2.28 ml of acetic acid was dissolved into 1 l of Milli-Q water. The pH of the buffer solution was adjusted to 6.0 by the drop wise addition of a diluted ammoniac solution. Under chromatographic conditions on a Hailton PRP-X100 column (Hamilton Company, USA), the elution order (according to retention times) was AsIII , DMA, MMA and AsV . To check the accuracy of the results obtained, one calibration sample was analyzed periodically during the measurement.
B. Manáková et al. / Journal of Hazardous Materials 280 (2014) 544–551 ˇ Table 3 Characteristics of compost and final products of composting and vermicomposting. All parameters were validated by duplicated analysis of the sample and the extended combined uncertainty was below 10%.
TOC (%) Ntot (%) Q4/6 HA (%) FA (%) HS (%) pH CEC (mmol kg−1 ) Ca (mg kg−1 ) K (mg kg−1 ) Mg (mg kg−1 )
C-0
C-90
VC1 -90
VC2 -90
20.0 0.63 4.9 0.91 1.20 2.11 8.4 n.a. 23.6 6.0 5.8
15.4 0.76 6.1 1.31 1.02 2.39 6.8 1.93 23.9 7.3 6.6
18.3 1.07 5.8 1.43 1.18 2.2 6.6 2.11 24.9 9.9 6.8
14.4 0.67 6.7 1.04 0.96 1.98 7.9 2.17 27.1 8.1 7.0
2.9. Statistical analysis All measurements were made in triplicate and results are presented as their mean value. The differences between concentrations over time and between composted and vermicomposted samples were tested by one-way analysis of variance ANOVA followed by Tukey’s test. Differences with ˛ = 0.05 were considered as statistically significant. All statistical analysis was performed in GraphPad Prism 5 for Windows version 5.01 (GraphPad Software, Inc., 2007). 3. Results 3.1. Composting 3.1.1. Basic characteristics of the composting system The main physico-chemical properties of the initial compost material (C-0) and its final product (C-90) are presented in Table 3. The temperature in the compost system increased quickly to 50 ◦ C during the first four days, remained at this temperature for the next two days and then began slowly to decrease. During the following experimental period, the temperature remained at a lower level of 24–28 ◦ C (during the 2nd–4th week) and then decreased spontaneously to ambient temperature and fluctuated at around 14 ◦ C for the next two months. As the process proceeded, the initial pH value decreased slightly from an alkaline value of 8.4 to a final value of 6.8. The initial C/N ratio in the compost was 32:1 and, over time, slowly decreased to 20:1. The proceeding mineralization of organic matter led to mass changes in compost weight (C-0: 311 ± 56 kg m−3 ). After 30 days of composting, the compost volume increases to 122% of total dry mass weight. A similar rate of organic matter loss was observed during the second month and the degradation was very slow over the third month. At the end of the composting period, the bulk density had increased to 477 ± 47 kg m−3 (154%). 3.1.2. Total content The results show that the total As concentration followed a significant (ANOVA, p < 0.05, Tukey, p < 0.05) increasing trend from 253 ± 11 mg kg−1 at the beginning of the process to 283 ± 3 mg kg−1 at the end of the third month of composting (Fig. 1a), with the main increase occurring during the second month of the composting cycle. Over the composting process, arsenic became more concentrated (equivalent to 111% of the initial material value). 3.1.3. Mobile fraction The easily mobile arsenic pool was measured by extraction with 0.1 M NH4 COOCH3 . Over the 90-day period, the extractable arsenic fraction tended to decrease from 1.9% of total content
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(4.9 ± 0.2 mg kg−1 ) at the beginning of the process to 1.3% of total content (3.66 ± 0.04 mg kg−1 ) at the end, as shown in Fig. 2a. The mobile fraction was therefore significantly (ANOVA, p < 0.05, Tukey, p < 0.05) reduced to 2/3 of its initial value by the process of composting. 3.1.4. Speciation analysis Speciation analysis was focused on four arsenic species: AsIII , DMA, MMA and AsV in the ammonium acetate extract, corresponding to the sum of metal species in each extract and determining the changes in As speciation over the composting process. The relative contributions of the four species are shown in Fig. 3a. Inorganic AsV was the main species extracted in the compost (88–96%), followed by AsIII (4–8%). There was no significant change in AsV content, but over the experimental period the AsIII content was significantly reduced from 8% (0.412 ± 0.007 mg kg−1 ) to 4% (0.152 ± 0.001 mg kg−1 ) (ANOVA, p < 0.05, Tukey, p < 0.05). At the beginning of the process, the content of the organic forms DMA and MMA were 0.3% and 0.2%, respectively. After the third month, DMA content slightly decreased to 0.2% and MMA content decreased below LOD (<0.003 mg kg−1 ). 3.2. Vermicomposting 3.2.1. Basic characteristics of the vermicomposting system In contrast to composting, vermicomposting does not involve exothermic reactions; the daily measured ambient temperature remained stable at 22 ± 1 ◦ C during the whole experimental period. Moisture content was maintained in the range 70–80% for both vermicomposts. Vermicompost VC1 was represented by samples C-0, VC1 -30, VC1 -60 and VC1 -90; vermicompost VC2 was represented by samples C-90, VC2 -30, VC2 -60 and VC2 -90. The main physicochemical properties of the initial material of the vermicomposts (C-0, C-90) and its final products (VC1 -90, VC2 -90) are presented in Table 3. There were only slight changes in pH values in both treatments. The pH value in VC1 decreased from 8.4 (C-0) to a slightly acidic pH of 6.6 (VC1 -90), whereas the pH value of VC2 increased slightly and moved from 6.8 (C-90) toward the neutrality of 7.1 (VC2 -90). The differences in pH were significant over the whole experimental period. In vermicompost VC1 , the C/N ratio decreased progressively from 32:1 (C-0) to 20:1 (VC1 -90). For vermicompost VC2 , the ratio was stable (21:1 for C-90; 22:1 for VC2 -90) and no significant changes were obtained. The bulk density of vermicompost VC1 had increased by 40% at the end of the vermicomposting process while VC2 showed a 20% increase. Bulk density in all vermicomposts showed a significant increase compared to its initial value (C-0; C-90) and was 439 ± 55 kg m−3 and 547 ± 72 kg m−3 respectively in VC1 -90 and VC2 -90. 3.2.2. Total content The analysis of total As content showed that the total As concentration (Astot ) for VC1 decreased significantly from 253 ± 11 mg kg−1 at the beginning to 226 ± 4 mg kg−1 at the end of the third month of vermicomposting (Fig. 1b) (ANOVA, p < 0.05, Tukey test, p < 0.05). The As content decreased significantly, falling to 88% of the original total As value at the end of the experiment. There were no significant differences in the total As concentration in vermicompost VC2 (Fig. 1c). At the beginning of the process, the As content was 283 ± 3 mg kg−1 and remained stable over the experimental period. 3.2.3. Mobile fraction Fig. 2b and c shows the changes in the mobile arsenic pool during the vermicomposting processes. In vermicompost VC1 , the mobile As fractions decreased significantly from 1.9% of total content (4.9 ± 0.2 mg kg−1 ) to 0.7% of total content (1.56 ± 0.02 mg kg−1 ).
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Fig. 1. Total arsenic concentrations (column: [mg kg−1 ]; curve: [%]) during sludge composting and vermicomposting: (a) compost C-(0–90); (b) vermicompost VC1 -(0–90); and (c) vermicompost VC2 -(0–90).
The mobile arsenic pool in vermicompost VC2 decreased significantly from 1.3% of total content (3.66 ± 0.04 mg kg−1 ) to 0.8% of total content (2.25 ± 0.02 mg kg−1 ). All changes found were highly statistically significant (ANOVA, p < 0.05, Tukey test, p < 0.05).
3.2.4. Arsenic speciation The results showed that AsV was the predominant species in VC1 and VC2 over the whole three month period of vermicomposting, and the trends in AsIII and AsV contents were consistent (Fig. 3b and c). For VC1 the content of AsV significantly decreased from 92% (VC1 -0) to 85% (VC1 -90) of arsenic in the mobile fraction. At the beginning of the process, the amount of AsIII was 8% of the mobile content (0.412 ± 0.007 mg kg−1 ) and then increased slightly to 14% the mobile content (0.21 ± 0.03 mg kg−1 ) after the third month. For vermicompost VC2 (Fig. 3c), the concentration of inorganic AsV slightly increased from 96% (VC2 -0) to 99% (VC2 -90) of the mobile arsenic fraction and represented the predominant species during the whole three months. AsIII amounts ranged from 4% (VC2 -0) to 0.5% (VC2 -90) of the mobile arsenic fraction. The results showed a significant increase in the AsIII amount after the first month, but over the whole process, the content of AsIII significantly decreased to 0.5%. All these changes were statistically significant (ANOVA, p < 0.05, Tukey, p < 0.05). In both vermicomposts the sum of the amounts of MMA and DMA forms were just above limit of detection and did not change significantly during the vermicomposting processes.
3.3. Bioaccumulation in earthworms Earthworm bioaccumulation factors (BAFs) were observed for soils amended with compost/vermicompost in the ratio of 9:1. All materials were checked for their toxicity to earthworms without dilution; the mortality of earthworms achieved 100% in all tested samples. During the composting, arsenic absorption by earthworms decreased significantly from 35 ± 5 mg kg−1 (C-0) to 14 ± 1 mg kg−1 (C-90), 14 ± 1 mg kg−1 (VC1 -90) and 17 ± 1 mg kg−1 (VC2 -90). The mean BAF values (n = 3) were reduced from 1.4 (C-0) to 0.5 (C-90), 0.5 (VC1 -90) and 0.5 (VC2 -90) for compost. 4. Discussion 4.1. Composting process The composting process involves the accelerated degradation of organic matter by microbial activity. Intensive decomposition produces heat which is reflected by a rapid increase in temperature. The compost material in our study was not energy-rich, so the temperature level observed was lower. The alkaline components of the sludge could not buffer the increased acidity of the compost. This was probably because of the presence of nitrous and nitric acid formations [14]. The C/N ratio gradually decreased to 20/1 in the finished product, corresponding to good quality compost [15]. The mineralization process resulted in a significant increase in compost bulk density and the non-biodegradable part (e.g. heavy metals)
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Fig. 2. Fraction of 0.1 M CH3 COONH4 extractable arsenic (column: [mg kg−1 ]; curve: [%]) during sludge composting and vermicomposting: (a) compost C-(0–90); (b) vermicompost VC1 -(0–90); and (c) vermicompost VC2 -(0–90).
tended to increased during the degradation process. Arsenic, like other heavy metals, is not destroyed during the composting process but, on the contrary, tends to accumulate (Fig. 1). The total heavy metals concentration is considered to be an overall pollution indicator. The obtained total arsenic fraction easily exceeded the limit of 20 mg kg−1 for As in compost [16], which may be appropriate for the estimation of long-term risks. Arsenic is one of those metals (such as Cr, Mo, Sb) which form oxyanions in solution at all pH values [17]. The results showed the gradual release of arsenic from sludge, reflected in the changes of total As content mainly during the second and third months of the composting. The arsenic became more concentrated, a phenomenon corresponding to studies describing similar trends for metals (e.g. As, Mn, Cu, Zn) in compost [18–20]. It should be noted that toxic arsenic concentrations in compost are potentially high and that the leachability of arsenic associated with compost is of particular concern. However, in our study, the transformation of compost matter influenced the bond strength of arsenic in this matrix and increased residual arsenic fraction in mature compost. In the present study, 0.1 mol l−1 NH4 COOCH3 was used as a mild extractant to evaluate the labile arsenic pool in compost. This labile fraction is positively the most biologically active and has the highest potential for environmental contamination. The results
showed that arsenic became more available in the matrix of compost (1.2–1.9%) compared to the available As fraction in sludge (0.3%). Arsenic is mobilized partly due to the chemical or microbial reduction of AsV to AsIII [21] and the dissolution of As contained in minerals and organic phases under changing redox conditions [22]. At the end of the composting process, a very large arsenic pool (98.7%) was not extractable and was strongly sorbed on the compost matrix. This indicated the high stability of the As pool present in the compost under experimental conditions. It was confirmed that arsenic can be included in the group of metals containing Zn, Cu, Cd and Pb, whose easily-mobile fractions are, in general, low and do not change very much during the composting process [23]. The predominant arsenic species in sludge was AsV (99.9%). The presence of organic matter changes the redox speciation of arsenic in compost both chemically and microbially. At the beginning of the composting process, AsIII constituted a small proportion of the total amount of arsenic (8%). Probably, therefore, a direct chemical effect of organic matter on the redox speciation of arsenic can be suggested. During the composting process, AsIII was slowly decreased and transformed to AsV . The methylation of arsenic by compost microorganisms must have been brought about by dimethylarsinic acid (DMA) and monomethylarsinic acid (MMA). Smaller concentrations of MMA (<0.2%) and DMA (<0.3%) were present and their contents did not change significantly.
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Fig. 3. Percentage contribution of arsenic species in 0.1 M CH3 COONH4 during sludge composting and vermicomposting: (a) compost C-(0–90); (b) vermicompost VC1 -(0–90); and (c) vermicompost VC2 -(0–90).
4.2. Vermicomposting Similarly to the composting process, vermicomposting showed the potential of organic matter to influence the retention and mobilization behavior of arsenic by altering As speciation by means of interaction with the digestive systems of earthworms. The pH decrease in vermicompost VC1 was probably caused by the mineralization of nitrogen and phosphorus and by the bioconversion of organic material into intermediate species of organic acids [24]. The pH of this substrate slowly decreased to a value of 6.8; E. fetida earthworms are relatively tolerant with regard to pH, enjoying an optimal pH range of between 5 and 9 [25]. The extent of the volume reduction of vermicompost substrates was not significant and did not lead to an increase in total metal content, although both vermicomposts were highly As contaminated when considering the current CSN Guideline Values for As of 10 mg kg−1 (I. class) and 20 mg kg−1 (II. class) [16]. The effects of earthworms on the mobility of heavy metals have been extensively studied by many authors, and both epigeic and anecic species of earthworms appear to increase the availability and mobility of metals [26–28]. In contrast to these studies, we demonstrated the ability of earthworms to decrease As mobility over the whole vermicomposting process. For both vermicompost end products, the extractable arsenic fraction was reduced to less
than half of the NH4 COOCH3 extract and less than 2.5% of the total As content. Vermicomposting reduced arsenic availability by 1/3. With regards to the total arsenic concentration, it has been suggested that the reason for the decrease in arsenic availability is the accumulation within the tissue of the earthworm (VC1 : 286 ± 18 mg kg−1 As) itself, which shows their ability to eliminate an excess of arsenic. In any case, the results indicate the obvious impact of earthworms on arsenic availability. Arsenic speciation was similar in both vermicomposts, with AsV and AsIII present as the dominant inorganic species. In the case of vermicompost VC1 , all arsenic species were present at the same time. This is with agreement with the observations of Langdon et al., who found the effect of biotransformation by both earthworms and microorganisms to arsenic content and bioaccumulation [29]. The stabilization of substrates (VC1 ) through composting made them more resistant to arsenic methylation and even though MMA was just above the limit of detection, DMA was still present and slowly decreased over time. Regarding the content of AsIII in vermicompost VC1 , the chemical or microbial reduction of AsV to AsIII was significant. In contrast to vermicompost VC1 , AsV was not reduced during the VC2 vermicomposting period; instead, AsIII oxidation was observed. As speciation analysis was not performed on earthworm tissues. Recent physiological studies on the metabolism of As
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in earthworm species suggests that earthworms simply accumulate arsenic through passive diffusion and metabolize it into less toxic organic derivatives or inorganic forms (AsIII and AsV ) [30–32]. 4.3. Bioaccumulation in earthworms In our study, the dilution of compost/vermicompost samples with soil could be associated with lower compost/vermicompost application rates, and the BAF values could reflect the arsenic fraction directly accessible to earthworms. The results of arsenic bioaccumulation confirmed the significant influence of composting and vermicomposting processes on the stability of arsenic interactions, although the end products showed a similar potential with respect to the bioavailability of arsenic to earthworms. 5. Conclusion The study concludes that combined composting and vermicomposting proved to be an effective method for the stabilization of the arsenic mobile fraction from the matured products. The combination of the actions of microorganisms and earthworms promoted significant changes related to the chemistry of arsenic present in the processed material. Total arsenic concentrations were enhanced and persisted during the composting and vermicomposting processes. By contrast, these processes significantly reduced the labile arsenic pool and changed the contents of arsenic species to AsIII and AsV in end products. Mobile arsenic contents were reduced to 2/3 by composting and to 1/3 by vermicomposting. Further compost vermicomposting then reduced the mobile As pool to 4/9 of its initial value (the most effective). The largest proportion of arsenic was found in the fraction more resistant to extraction, indicating that arsenic was in more stable forms and consequently less available for plant uptake. In end products, the amount of potentially bioavailable arsenic was in the range 0.7–1.3%. Composting and vermicomposting processes affected arsenic speciation; AsV was found to be the dominant species in all easily extractable fractions. The investigation of arsenic BAFs showed that composting and vermicomposting processes decreased the arsenic fraction easily taken up by earthworms. Despite the fact that the labile arsenic content was greatly reduced, the produced composts/vermicomposts cannot be considered suitable for agronomic application due to the fact that the arsenic contents do not comply with the limits set for compost. Acknowledgment This research was supported by the Ministry of Education of the Czech Republic (projects LM2011028 and LO1214). References [1] J. Dominguez, C.A. Edwards, Relationships between composting and vermicomposting: relative values of the products, in: C.A. Edwards, N.Q. Arancon, R.L. Sherman (Eds.), Vermiculture Technology: Earthworms, Organic Waste and Environmental Management, CRC Press, Boca Raton, FL, 2010, pp. 11–26. [2] S. Hait, V. Tare, Transformation and availability of nutrients and heavy metals during integrated composting–vermicomposting of sewage sludges, Ecotoxicol. Environ. Saf. 79 (2012) 214–224. [3] R. Gupta, V.K. Garg, Stabilization of primary sewage sludge during vermicomposting, J. Hazard. Mater. 153 (2008) 1023–1030.
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