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Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater
STOTEN-24418; No of Pages 7 Science of the Total Environment xxx (2017) xxx–xxx
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Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater Yue Yin a, Junfeng Wan a,⁎, Shaozhen Li a, Hongli Li a, Christophe Dagot b,c, Yan Wang a a b c
School of Chemical Engineering and Energy, Zhengzhou University, 100 Science Avenue, 450001, PR China GRESE EA 4330, Université de Limoges, 123 Avenue Albert Thomas, F-87060 Limoges Cedex, France INSERM, U1092, Limoges, France
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
• A-O process was used to treat synthetic livestock wastewater containing ROX. • ROX and its metabolites could affect the bacterial communities in the process. • Different forms of As could be detected in the effluent, sludge and biogas.
a r t i c l e
i n f o
Article history: Received 1 August 2017 Received in revised form 15 October 2017 Accepted 19 October 2017 Available online xxxx Editor: Kevin V. Thomas Keywords: Anoxic-oxic process As speciation As transformation Roxarsone
a b s t r a c t In order to evaluate the influence of roxarsone (ROX) on the livestock wastewater treatment, a lab-scale pilot employing an anoxic–oxic (A-O) process was investigated by adding different concentrations of ROX at different periods. The mass balance of arsenic (As) in the A-O system was established through the analysis of As speciation and As migration in the gas, liquid and solid phases. The results showed that around 80% of total ROX (initial concentration was 50 mg ROX L−1) was eliminated in the anoxic reactor (R1) in which at least about 11% of total ROX was transformed to inorganic Asv (iAsv) due to the direct breaking of the C-As bond of ROX. Inorganic AsIII (iAsIII) and arsine (AsH3) were produced in R1, while the generated iAsIII in the effluent of R1 was almost completely oxidized to iAsV in the aerobic reactor (R2). However, the concentration of ROX in the effluent of R2 was almost the same as that in the effluent of R1. After 85 days operation, iAsV and residual ROX as the main forms of As were observed after the A-O process. Furthermore, the mass balance of As at steady state revealed that around 0.08%, 3.91% and 96.01% of total As was transformed into gas (biogas), solid (excess sludge) and liquid (effluent). Additionally, the 16S rRNA analysis demonstrated that the existence of ROX in livestock wastewater may play a crucial role in the diversity of bacterial community in the A-O system. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail address: [email protected] (J. Wan).
Roxarsone (3-nitro-4-hydroxy benzene arsenic acid, ROX) is widely used as a feed additive in livestock and poultry breeding to control coccidial intestinal parasites, improve feed efficiency and promote
https://doi.org/10.1016/j.scitotenv.2017.10.194 0048-9697/© 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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animal growth (Garbarino et al., 2003; Makris et al., 2008; Mandal and Suzuki, 2002; Yao et al., 2016). Up until 2011, ROX was fed to about 88% of the 9 billion broiler chickens produced each year in America. It is also legal that ROX is applied as feed additives for agriculture in China (Walrod et al., 2016). The Ministry of Agriculture of the People's Republic of China regulated that the addition of ROX in the feed is limited to 50 mg kg−1 (Wang and Liao, 2004). Nevertheless, as a typical organoarsenic chemical, ROX has a potential hazard to the environment. It was reported that after the animal digestion, N 90% of ROX in feeding was released with manure to environment (Fu et al., 2016). Hence, most of residual ROX would finally be transported to the natural environment and it should be degraded or accumulated in the relative environment such as water or soils (Brown et al., 2005; Nachman et al., 2013). Recently, the studies on the migration and transformation of ROX in environment have received enormous attention (Fisher et al., 2015; Huang et al., 2014). Normally, the treatment of livestock wastewater is mainly focused on the removal of high concentrated organic matter (COD), nitrogen and phosphorus (Harrington and Mcinnes, 2009; Shi et al., 2011). The combined process such as anoxic-oxic process (A-O process) was usually applied to treat livestock wastewater after the anaerobic treatment which could remove about 60% of concentrated organic matter (Harwood et al., 1998; Karim and Gupta, 2003). Then the A-O process could remove the majority of the residual COD and nutrients (N and P) to meet the discharge standard (Ren et al., 2010). In fact, the effect of the residual ROX existed in livestock wastewater on the real treatment engineering was usually neglected due to its relatively low concentration (lower than 50 mg L−1). At the same time, the degradation of ROX in anaerobic process was reported in the lab-scale experiment by previous studies (Cortinas et al., 2006; Guzmán-Fierro et al., 2015; Shui et al., 2016; Zhang et al., 2014). Moreover, the biological degradation of COD could be obviously inhibited in anaerobic unit once the concentration of ROX was over 40 mg L−1 (Guo et al., 2013). Similarly, ROX was proved to disturb the efficiency of biological nitrogen removal (BNR) and enhanced biological phosphorus removal (EBPR) processes (Guo et al., 2013; Liu et al., 2014). Additionally, the existence of ROX and its toxic intermediate metabolites probably affected the activity of microorganism. For example, the activity of acetic acid-utilizing methanogenic bacteria was significantly inhibited by ROX at 263 mg L−1 (Sierra-Alvarez et al., 2010). Furthermore, ROX could influence the microbial community structure in wastewater treatment system and thus disturbed the performance of the bioreactors (Jiang et al., 2013; Stolz et al., 2007). At present, most of the relative studies are focus on the degradation of ROX and its metabolites in anaerobic reactor (Cortinas et al., 2006; Shi et al., 2014; Stolz et al., 2007). However, it was rarely reported about the interaction between ROX and the combined process such as the A-O process during livestock wastewater treatment. Therefore, the objectives of this study are: 1) to test the influence of ROX at various concentration on the performance of the A-O process; 2) to analyze the fate of ROX and establish the mass balance of As in the A-O process; 3) to investigate the long-term effect of ROX on the biodiversity and community stucture of microorganism in the A-O process. 2. Material and methods 2.1. Experimental set-up The two bioreactors (working volume = 4.0 L) were installed and connected to function as the A-O process. The activated sludge extracted from local wastewater treatment plant was inoculated in the anoxic reactor (R1) and the aerobic reactor (R2). Both reactors with the volume exchange rate of 50% functioned as sequencing batch reactor (SBR) model. One cycle of 12 h includes four steps: feeding (20 min), reaction (630 min), settling (60 min) and discharging (1 min). During the whole 85 days operation, four phases were established according to the
Table 1 Strategy of ROX addition at different phases. Phases
ROX (mg L−1)
Time (d)
I II III IV
10 50 0 50
1–15 16–55 56–65 66–85
addition of ROX (C6H6AsNO6, Purity: 98%, Rongyao Biotech. Co., Ltd., China) at different concentrations as Table 1. The composition of synthetic wastewater was shown in Table 2. 2.2. Sampling and analysis Mixed liquid suspended solids (MLSS), Mixed liquid volatile suspended solids (MLVSS), Chemical oxygen demand (COD), nitrogen − − forms (NH+ 4 , NO2 , and NO3 ), phosphate were analyzed according to 2− 2− standard methods (APHA, 2002). The sulfur forms (SO2− ) 4 , SO3 , S were measured by Ion Chromatography (Thermo Scientific ICS900). O-aminophenol in the effluent was determined by the high performance liquid chromatograph (HPLC) (ELITE P230II) with a quantification limit of 10 μg L−1 (Wakamatsu et al., 2014). The produced biogas in anoxic reactor R1 were gathered through an on-line gas collector filled with sodium alginate-silver particles in order to analyze the quantity of arsine (AsH3) in gaseous phase (Mestrot et al., 2009). At the end of the reaction, 50 mL of mixed samples periodically extracted from R1 and R2. All the mixed samples were firstly centrifuged at 4500g for 15 min. The precipitants were utilized as sludge samples for analysis of As speciation in solid phase, while the supernatant was filtered by a filter (Ø = 0.45 μm) and stored at 4 °C before the analysis of As speciation in liquid phase. Additionally, the sludge samples and the supernatant were digested for 2 h with the addition of H2O2HNO3 before the total As measurement (Chu et al., 2009). The total As was determined by a hydride generation atomic fluorescence spectrometry (HG-AFS), while As speciation was analyzed by HPLC-HG-AFS. The quantification limits were 0.5, 0.1 and 0.3 μg L−1 for total As, AsIII and AsV, respectively. The detailed parameters of As detection are given in Table 3. 2.3. Microbial structure analysis 2.3.1. DNA extraction and PCR amplification Sludge samples were extracted from R1 and R2 on Day-15, Day-55, Day-65 and Day-85. Microbial DNA was extracted using the E.Z.N.A.® water DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer's protocols. The V4–V5 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 5 min) using primers 338F 5′-barcode-ACTCCT ACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′,
Table 2 Composition of synthetic wastewater. Synthetic wastewater
mg L−1
Trace elements
mg L−1
Sodium propionate NH4Cl K2HPO4 KH2PO4 Na2SO4 KHCO3 FeSO4 · 7H2O MgSO4 · 7H2O CaCl2
670 150 28 14 253 100 50 100 20
H3BO3 MnSO4 CoCl2 CuSO4 · 5H2O NiCl2 · 6H2O ZnSO4 · 7H2O
1 1 0.2 2 0.4 2
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
Y. Yin et al. / Science of the Total Environment xxx (2017) xxx–xxx Table 3 Analytical parameters of total As and As speciation measurement. Settings
HPLC-HG-AFS
HG-AFS
Carrier gas flow (mL min−1) Auxiliary gas flow (mL min−1) Lamp current (mA) Negative high voltage (V) Furnace temperature (°C) Current-carrying Reductant (m/m) Oxygenant (m/m) Injection volume (μL) Flow rate (mL min−1) Mobile phase A Mobile phase B
300 600 50 280 200 10% HCl 3% KBH4, 0.5% KOH 2% K2S2O8, 0.5% KOH 100 1 15 mM (NH4)2HPO4 (pH = 6.0)b 10 mM KHPa (pH = 6.0)b
300 600 50 280 200 3% HCl 1% KBH4, 0.5% KOH – – – – –
a b
KHP is Potassium Hydrogen Phthalate. pH is adjusted with addition of NH3·H2O.
where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 ∗ FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. 2.3.2. Illumina MiSeq sequencing Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer's instructions and quantified using QuantiFluor™-ST (Promega, U.S.). Purified amplicons were pooled in equimolar and paired-end sequenced (2 ∗ 250) on an Illumina MiSeq platform according to the standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP064834) (Quast et al., 2013). 2.3.3. Processing of sequencing data Raw fastq files were demultiplexed, quality-filtered using QIIME (Quast et al., 2013) with the following criteria: (i) The 250 bp reads were truncated at any site receiving an average quality score b 20 over a 10 bp sliding window, discarding the truncated reads that were shorter than 50 bp. (ii) exact barcode matching, 2 nucleotide mismatch in primer matching, reads containing ambiguous characters were removed. (iii) only sequences that overlap longer than 10 bp were assembled according to their overlap sequence. Reads which could not be assembled were discarded. Operational Units (OTUs) were clustered with 97% similarity cutoff using UPARSE (http://drive5.com/uparse/) and chimeric sequences were identified and removed by using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was
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analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the silva (SSU115) 16S rRNA database using confidence threshold of 70% (Amato et al., 2013). 3. Results 3.1. Performance of A-O process 3.1.1. Anoxic reactor R1 The effluent of R1 was periodically analyzed during the whole experiment as shown in Fig. 1. Once ROX in the feeding increased from 10 to 50 mg L−1 (from Phases I to II), the effluent COD and sulfate (SO2− 4 ) rapidly rised, which was probably due to the acute inhibition on microbial activity by ROX. After 7 days continuous operation in Phase II, COD and sulfate gradually decreased and stabilized at 305.3 ± 5.8 mg O2 L−1 and 6.7 ± 1.0 mg S L−1. The similar results were observed when influent ROX increased from 0 to 50 mg L−1 (from Phases III to IV). A peak at around 40 mg N L−1 for the ammonium appeared at the start-up of Phase II and then gradually decreased and stabilized at 31.1 ± 1.0 mg N L−1, which accounted for around 80% of influent ammonium. A small amount of nitrite was observed in the presence of ROX (Phases I, II and IV), while phosphate was fluctuated at around 6 mg L−1 during the whole experiment. 3.1.2. Aerobic reactor R2 After each cycle, the effluent of R1 was pumped to R2. With the increase of ROX in the feeding of R1 (from 10 to 50 mg L−1 on Day-16 and from 0 to 50 mg L−1 on Day-66), a similar trends was observed as shown in Fig. 2 for COD, nitrate and phosphate in the effluent of R2: the concentrations of these substrates immediately reached at a high level, then gradually decreased and stabilized after a few days of continuous operation. These results revealed that ROX and its possible metabolites after anoxic treatment had an acute inhibition effect on the removal of COD and nutrients (N, P) in aerobic conditions. Moreover, the acute inhibition effect at the beginning of Phase II was much more remarkable compared with Phase IV when ROX was increased to 50 mg L−1. Additionally, no ammonium was accumulated during the whole experiment. 3.2. ROX degradation and As speciation during A-O process The concentration of ROX in the feeding was controlled as the unique variable parameter in this experiment. As shown in Fig. 3, As speciation in the effluent including iAsIII, iAsV, ROX and total As were measured. When additive ROX in the feeding increased to 50 mg L−1 on Day-16 and Day-66, the effluent ROX of R1 rapidly rose to a high level and then decreased and stabilized at 2.41 ± 0.09 mg As L−1.
Fig. 1. Evaluation of substrates in the effluent of R1; (a) COD and SO2− (b); N-forms and Phosphate. 4
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Fig. 2. Evaluation of substrates in the effluent of R2; (a) COD and SO2− (b); N-forms and Phosphate. 4
Correspondingly, generated inorganic As (i.e. iAsIII and iAsV) in the effluent of R1 slowly increased and finally remained at a stable value. These results documented that ROX could be biologically degraded under anoxic conditions. Moreover, some unknown organoarsenic compounds were detected by HPLC-HG-AFS measurement. On the contrary, residual ROX in the effluent of R1 was very close to which in the effluent of R2 no matter how the initial concentration of additive ROX in the feeding changed, which proved that ROX was hardly degraded under aerobic conditions. However, the quantity of iAsV in the effluent of R2 was much more than that in the effluent of R1, which revealed that other organoarsenic compounds were oxidized to iAsV under aerobic conditions. The similar results were reported by Markley and Herbert (2009). 3.3. Bacterial community in A-O process At the end of each phase, the diversity and the abundance of the microbial communities at various ROX concentrations were analyzed by high-throughput sequencing. As shown in Fig. 4, a community abundance analysis was obtained on genus level. After a long-term operation, functional bacteria such as Desulfobulbus, Geobacter and Paracoccus for sulfate reduction, aromatic hydrocarbon degradation and denitrification were present in R1 (Blaszczyk, 1993; Coates et al., 1997; Shinoda et al., 2004; Taylor and Parkes, 1983), which means that these bacteria could resist to ROX and relative inorganic As (Anderson et al., 2003). At the end of Phase III, the proportion of Hydrogenophaga was increased in R1, while Bacteroidetes-vadinHA17 was decreased significantly without addition of ROX. At the same time, the increased proportion of some bacteria like Alphaproteobacteria was observed in R2, while Thauera was decreased obviously without addition of ROX. When ROX was increased to 50 mg L−1 again in Phase IV, the proportion of the bacteria increased in Phase III such as Hydrogenophaga decreased, and was even lower than its proportion at the same concentration of ROX during
the Phase II. Thus, the influence of ROX on the bacterial diversity and abundance cannot be ignored in the presence of ROX in the A-O process. 4. Discussion 4.1. Possible degradation mechanism of ROX ROX could be possibly degraded by the fracture of C\\As bond on the benzene ring in the anaerobic conditions (Shi et al., 2014). As a result, oaminophenol could be produced once C\\As bond on the benzene ring was broken and the nitro group of ROX was reduced together. At the same time, the released inorganic As that was derived from the direct breakage of C\\As bond could be estimated based on the oaminophenol production if regardless of the degradation of oaminophenol and other metabolic pathways. From Fig. 5a, when initial ROX concentration was 10 mg L− 1 (38.02 μM), the o-aminophenol and the sum of inorganic As (iAsIII + iAsV) in the effluent of R1 maintained at 0.09 mg L−1 (=0.83 μM) and 0.49 mg As L−1 (=6.20 μM) at the end of the reaction. When ROX concentration was increased to 50 mg L− 1 (190.11 μM), the concentration of o-aminophenol maintained at about 0.23 mg L− 1 (= 2.11 μM) in steady state (at the end of the Phase II and Phase IV), while the sum of inorganic As (iAsIII + iAsV) was 1.46 mg As L− 1 (= 18.48 μM) in R1. Considering ROX was the only source of benzene homologues in the whole process, we concluded that at least about 11% of the total amount of inorganic As was produced by the C\\As bond breaking of ROX. Furthermore, the kinetic study of R1 (Fig. 5b) showed that o-aminophenol gradually increased from 0.11 (=1.01 μM) to 0.24 mg L−1 (=2.20 μM) during 300 min of reaction. Additionally, almost no o-aminophenol was detected in the effluent of R2, which means that o-aminophenol could be readily degraded under aerobic conditions. The measured quantity of o-aminophenol was the dynamic results of its formation and degradation in R1, so it
Fig. 3. Evaluation of As speciation in the effluent of R1 (a) and R2 (b).
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Fig. 4. Percentage of community abundance on genus level for the both reactors on different phases; A15, A55, A65 and A85 were sludge samples which were extracted from R1 on Day-15, 55, 65 and 85; O15 O55 O65 and O85 were sludge samples which were extracted from R2 on Day-15, 55, 65 and 85.
was difficult to evaluate the total amount of o-aminophenol produced in the whole reaction process (Bednar et al., 2003; Stolz et al., 2007; Yang et al., 2016). Therefore, the method for judging the exact value of inorganic As generated by the direct cleavage of C\\As bond still needs to be further improved, although the present data showed only a small fraction of inorganic As in R1 caused by the direct cleavage of C\\As bond of ROX. At the same time, with the consideration that a large quantity of iAsV were obviously accumulated in R2 with addition of 50 mg L−1 of ROX after long-term operation (Fig. 3), the biologically induced oxidation of the reducible form of As (e.g. iAsIII) possibly occured in the oxic system as our previous studies (Wang et al., 2017a; Wang et al., 2017b).
4.2. Mass balance and distribution of As speciation in A-O process At the end of Phase IV from Day-80 to Day-85, the quantity of arsine (AsH3) in gaseous phase was periodically measured through the collection and analysis of the gaz. The measured arsine released to air from R1 was about 25.36 ± 2.85 μg (0.31 ± 0.03 μmol) in 1 cycle (12 h), which indicated that about 0.08% of total As in the influent was reduced to AsH3 in R1 every day. Considering that the arsine was as one of the final products of ROX degradation, the percentage of arsine on the total As transformation in A-O process cannot be ignored (Chen et al., 2011; Huang et al., 2016; Qin et al., 2006). At the same time, the contents of total arsenic in anoxic and aerobic sludge were 1.00 ±
Fig. 5. Variation of o-aminophenol: a) in the effluent of R1 during the whole experiment; b) in R1 for 1 cycle on Day-90.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Fig. 6. The distribution of As species in R1 (a) and R2 (b) on Day-85. Liquid represents different forms of As in water sample, Sludge represents different forms of As in solid sample, ND represents the undetected amount of As according to the difference between total As and the sum of various As forms in liquid and sludge samples.
0.05 mg As g−1 VSS and 2.82 ± 0.08 mg As g−1 VSS, respectively. If assumed that the anoxic and aerobic yield coefficients under pH = 7.0 and 25 °C were about 0.20 and 0.42 g VSS g−1 COD (Pipyn and Verstraete, 2010; Tawfik et al., 2008), around 0.09 and 0.18 g VSS L−1 increased in R1 and R2 because about 90 and 210 mg L−1 of COD were removed every day in R1 and R2. As a result, the amounts of As absorbed by anoxic and aerobic sludge was about 0.36 and 1.99 mg As in R1 and R2 every day. According to this hypothesis, we can conclude that around 0.08%, 3.91% and 96.01% of total As were transformed into gas (biogas), solid (excess sludge) and liquid (effluent). Furthermore, in order to evaluate the As speciation in the A-O process, the identical volume of suspended samples including sludge and water samples were directly exacted at the end of reaction from R1 and R2 on Day-85. The various As forms such as iAsIII, iAsV, MMA and ROX in sludge and liquid were measured. Moreover, the total As was measured after digestion of suspended samples. The percentage of various As forms in liquid and sludge samples were calculated as shown in Fig. 6. From Fig. 6a in R1, a small amount of As content (about 24.2% of the total As in suspended samples) existed in the anoxic sludge, in which iAsIII and ROX were the mains forms. 72.59% of the total As existed in liquid of R1, in which the unknown form and ROX were the main forms of As in liquid followed by the iAsV and iAsIII. The unknown As form was about 3946.8 ± 184.4 μg L−1 in R1 and decreased to 1552.6 ± 185.4 μg L−1 in R2 after a cycle reaction. Additionally, much more quantity of As existed as iAsV (about 46.2% of the total As in suspended samples) was detected in the aerobic sludge of R2 compared with R1. In contrast to the various forms of As in the effluent of R1 and R2 (Fig. 3), it can be seen that a small amount of organic As in the unknown form in R1 was mainly converted to iAsV in R2. In fact, ROX and the possible generated HAPA in R1 were difficult to open loop to release iAsV under the aerobic conditions (Donlon et al., 1996; Karim and Gupta, 2003; Lenke and Knackmuss, 1992; Lenke et al., 1992). Additionally, the biological open-loop ability is relatively weak under the aerobic conditions. Thus, the extra quantity of iAsV in R2 was probably produced from the unknown arsenate-containing aliphatic acids which were detected in R1. It should be pointed out that the unknown arsenate-containing aliphatic acids need to be further confirmed. 5. Conclusions ROX, as one of popular organoarsenic additives, should be given adequate attention in livestock wastewater treatment. The results suggested that the residual ROX existed in the A-O process could not only influence the removal efficiency of COD and nutrients (N and P), but also played a crucial role in the microbial community. After livestock
wastewater treatment by the A-O process, most of ROX and its metabolites were still remained in the discharged effluent, while a small amount of As (mainly presented as the forms of iAsIII and iAsV) was accumulated in the activated sludge. Therefore, it is better to utilize seperation methods such as adsorption in order to maximally capture ROX and its metabolites from the livestock wastewater. At the same time, when the excess sludge is treated and recovered as resource (e.g. organic fertilizer), the control of the content of As in the recovered product should be considered.
Acknowledgments The authors thank the National Natural Science Foundation of China (No. 21107100) and the Outstanding Young Talent Research Fund of Zhengzhou University (No. 1421324067) for the financial support of this study. References Amato, K.R., Yeoman, C.J., Kent, A., Righini, N., Carbonero, F., Estrada, A., Gaskins, H.R., Stumpf, R.M., Yildirim, S., Torralba, M., Gillis, M., Wilson, B.A., Nelson, K.E., White, B.A., Leigh, S.R., 2013. Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J. 7 (7), 1344–1353. Anderson, R.T., Vrionis, H.A., Ortizbernad, I., Resch, C.T., Long, P.E., Dayvault, R., Karp, K., Marutzky, S., Metzler, D.R., Peacock, A., 2003. Stimulating the in situ activity of geobacter species to remove uranium from the groundwater of a uraniumcontaminated aquifer. Appl. Environ. Microbiol. 69 (10), 5884–5891. APHA, 2002. Standard Methods for the Examination of Water and Wastewater. 20th edition. American Public Health Association, Washington, DC. Bednar, A.J., Garbarino, J.R., Ferrer, I., Rutherford, D.W., Wershaw, R.L., Ranville, J.F., Wildeman, T.R., 2003. Photodegradation of roxarsone in poultry litter leachates. Sci. Total Environ. 302 (1–3), 237–245. Blaszczyk, M., 1993. Effect of medium composition on the denitrification of nitrate by Paracoccus denitrificans. Appl. Environ. Microbiol. 59 (11), 3951–3953. Brown, B.L., Slaughter, A.D., Schreiber, M.E., 2005. Controls on roxarsone transport in agricultural watersheds. Appl. Geochem. 20 (1), 123–133. Chen, J., Sun, G.X., Wang, X.X., Lorenzo, V.D., Rosen, B.P., Zhu, Y.G., 2011. Volatilization of arsenic from polluted soil by pseudomonas putida engineered for expression of the arsM arsenic(III)S-adenosine methyltransferase gene. Environ. Sci. Technol. 48 (48), 10337–10344. Chu, Z., Xiao, Y., Liu, W., Zheng, W., 2009. Comparison of different acid systems in the procedure of digestion and decomposition of soil arsenic, mercury, lead and cadmium using high pressure method. Environ. Monit. China 25 (5), 57–61. Coates, J.D., Woodward, J., Allen, J., Philp, P., Lovley, D.R., 1997. Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments. Appl. Environ. Microbiol. 63 (9), 3589–3593. Cortinas, I., Field, J.A., Kopplin, M., Garbarino, J.R., Gandolfi, A.J., Sierra-Alvarez, R., 2006. Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids. Environ. Sci. Technol. 40 (9), 2951–2957. Donlon, B.A., Razoflores, E., Lettinga, G., Field, J.A., 1996. Continuous detoxification, transformation, and degradation of nitrophenols in upflow anaerobic sludge blanket (UASB) reactors. Biotechnol. Bioeng. 51 (4), 439–449. Fisher, D.J., Yonkos, L.T., Staver, K.W., 2015. Environmental concerns of roxarsone in broiler poultry feed and litter in Maryland, USA. Environ. Sci. Technol. 49 (4), 1999–2012.
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Y. Yin et al. / Science of the Total Environment xxx (2017) xxx–xxx Fu, Q.L., He, J.Z., Gong, H., Blaney, L., Zhou, D.M., 2016. Extraction and speciation analysis of roxarsone and its metabolites from soils with different physicochemical properties. J. Soils Sediments 16 (5), 1–12. Garbarino, J.R., Bednar, A.J., Rutherford, D.W., Beyer, R.S., Wershaw, R.L., 2003. Environmental fate of roxarsone in poultry litter. I. Degradation of roxarsone during composting. Environ. Sci. Technol. 37 (8), 1509–1514. Guo, Q., Liu, L., Hu, Z., Chen, G., 2013. Biological phosphorus removal inhibition by roxarsone in batch culture systems. Chemosphere 92 (1), 138–142. Guzmán-Fierro, V.G., Moraga, R., León, C.G., Campos, V.L., Smith, C., Mondaca, M.A., 2015. Isolation and characterization of an aerobic bacterial consortium able to degrade roxarsone. Int. J. Environ. Sci. Technol. 12 (4), 1353–1362. Harrington, R., Mcinnes, R., 2009. Integrated constructed wetlands (ICW) for livestock wastewater management. Bioresour. Technol. 100 (22), 5498–5505. Harwood, C.S., Burchhardt, G., Herrmann, H., Fuchs, G., 1998. Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway. FEMS Microbiol. Rev. 22 (5), 439–458. Huang, L., Yao, L., He, Z., Zhou, C., Li, G., Yang, B., Deng, X., 2014. Roxarsone and its metabolites in chicken manure significantly enhance the uptake of As species by vegetables. Chemosphere 100 (3), 57–62. Huang, K., Chen, C., Zhang, J., Tang, Z., Shen, Q., Rosen, B.P., Zhao, F.J., 2016. Efficient arsenic methylation and volatilization mediated by a novel bacterium from an arseniccontaminated paddy soil. Environ. Sci. Technol. 50 (12), 6389–6396. Jiang, Z., Li, P., Wang, Y.H., Li, B., Wang, Y.X., 2013. Effects of roxarsone on the functional diversity of soil microbial community. Int. Biodeterior. Biodegrad. 76 (1), 32–35. Karim, K., Gupta, S.K., 2003. Continuous biotransformation and removal of nitrophenols under denitrifying conditions. Water Res. 37 (12), 2953–2959. Lenke, H., Knackmuss, H.J., 1992. Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2. Appl. Environ. Microbiol. 58 (9), 2933–2937. Lenke, H., Pieper, D.H., Bruhn, C., Knackmuss, H.J., 1992. Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2. Appl. Environ. Microbiol. 58 (9), 2928–2932. Liu, H., Wang, G., Ge, J., Li, L., Chen, G., 2014. Fate of roxarsone during biological nitrogen removal process in wastewater treatment systems. Chem. Eng. J. 255 (255), 500–505. Makris, K.C., Salazar, J., Quazi, S., Andra, S.S., Sarkar, D., Bach, S.B., Datta, R., 2008. Controlling the fate of roxarsone and inorganic arsenic in poultry litter. J. Environ. Qual. 37 (3), 963–971. Mandal, B.K., Suzuki, K.T., 2002. Arsenic round the world: a review. Talanta 58 (1), 201–235. Markley, C.T., Herbert, B.E., 2009. Arsenic risk assessment: the importance of speciation in different hydrologic systems. Water Air Soil Pollut. 204 (1–4), 385–398. Mestrot, A., Uroic, M.K., Plantevin, T., Islam, M.R., Krupp, E.M., Feldmann, J., Meharg, A.A., 2009. Quantitative and qualitative trapping of arsines deployed to assess loss of volatile arsenic from paddy soil. Environ. Sci. Technol. 43 (21), 8270–8275. Nachman, K.E., Baron, P.A., Raber, G., Francesconi, K.A., Navasacien, A., Love, D.C., 2013. Roxarsone, inorganic arsenic, and other arsenic species in chicken: a U.S.-based market basket sample. Environ. Health Perspect. 121 (7), 818–824. Pipyn, P., Verstraete, W., 2010. Comparison of maximum cell yield and maintenance coefficients in axenic cultures and activated sludge communities. Biotechnol. Bioeng. 20 (12), 1883–1893. Qin, J., Rosen, B.P., Zhang, Y., Wang, G., Franke, S., Rensing, C., 2006. Arsenic detoxification and evolution of trimethylarsine gas by a microbial arsenite S-adenosylmethionine methyltransferase. Proc. Natl. Acad. Sci. 103 (7), 2075–2080. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41 (Database issue), 590–596.
7
Ren, L., Wu, Y., Ren, N., Zhang, K., Xing, D., 2010. Microbial community structure in an integrated A/O reactor treating diluted livestock wastewater during start-up period. J. Environ. Sci. 22 (5), 656–662. Shi, L.J., Ban, L.T., Liu, H.F., Hao, J.C., Zhang, W.Y., 2011. Study on livestock and poultry farming wastewater treatment using a hybrid baffled reactor. Adv. Mater. Res. 201–203, 2797–2802. Shi, L., Wang, W., Yuan, S.J., Hu, Z.H., 2014. Electrochemical stimulation of microbial roxarsone degradation under anaerobic conditions. Environ. Sci. Technol. 48 (14), 7951–7958. Shinoda, Y., Sakai, Y., Uenishi, H., Uchihashi, Y., Hiraishi, A., Yukawa, H., Yurimoto, H., Kato, N., 2004. Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1. Appl. Environ. Microbiol. 70 (3), 1385–1392. Shui, M., Ji, F., Tang, R., Yuan, S., Zhan, X., Wang, W., Hu, Z., 2016. Impact of roxarsone on the UASB reactor performance and its degradation. Front. Environ. Sci. Eng. 10 (6), 1–9. Sierra-Alvarez, R., Cortinas, I., Field, J.A., 2010. Methanogenic inhibition by roxarsone (4hydroxy-3-nitrophenylarsonic acid) and related aromatic arsenic compounds. J. Hazard. Mater. 175 (1–3), 352–358. Stolz, J.F., Perera, E., Kilonzo, B., Kail, B., Crable, B., Fisher, E., Ranganathan, M., Wormer, L., Basu, P., 2007. Biotransformation of 3-nitro-4-hydroxybenzene arsonic acid (roxarsone) and release of inorganic arsenic by Clostridium species. Environ. Sci. Technol. 41 (3), 818–823. Tawfik, A., Sobhey, M., Badawy, M., 2008. Treatment of a combined dairy and domestic wastewater in an up-flow anaerobic sludge blanket (UASB) reactor followed by activated sludge (AS system). Desalination 227 (1), 167–177. Taylor, J., Parkes, R.J., 1983. The cellular fatty acids of the sulphate-reducing bacteria, Desulfobacter sp., Desulfobulbus sp. and desulfovibrio desulfuricans. Microbiology 129 (11), 3303–3309. Wakamatsu, K., Tanaka, H., Tabuchi, K., Ojika, M., Zucca, F.A., Zecca, L., Ito, S., 2014. Reduction of the nitro group to amine by hydroiodic acid to synthesize o-aminophenol derivatives as putative degradative markers of neuromelanin. Molecules 19 (6), 8039–8050. Walrod, J.H., Burriss, D., Blue, L.Y., Beck, G.E., Atwood, D.A., 2016. Arsenic mobility in Karnak soils after multi-year application of poultry litter containing roxarsone. Main Group Chem. 15 (4), 365–373. Wang, K., Liao, X., 2004. The function in animal production and influence on environment of arsenic preparation. Ecol. Domest. Anim. 25 (2), 41–43. Wang, J., Wan, J., Li, H., Li, H., Dagot, C., Wang, Y., 2017a. Biological arsenite oxidation with nitrate as sole electron acceptor. Environ. Technol. 38 (16), 2070–2076. Wang, J., Wan, J., Wu, Z., Li, H., Li, H., Dagot, C., Wang, Y., 2017b. Flexible biological arsenite oxidation utilizing NOx and O2 as alternative electron acceptors. Chemosphere 178, 136–142. Yang, Z., Peng, H., Lu, X., Liu, Q., Huang, R., Hu, B., Kachanoski, G., Zuidhof, M.J., Le, X.C., 2016. Arsenic metabolites, including N-acetyl-4-hydroxy-m-arsanilic acid, in chicken litter from a roxarsone-feeding study involving 1600 chickens. Environ. Sci. Technol. 50 (13), 6737–6743. Yao, L., Huang, L., He, Z., Zhou, C., Lu, W., Bai, C., 2016. Delivery of roxarsone via chicken diet → chicken → chicken manure → soil → rice plant. Sci. Total Environ. 566-567, 1152–1158. Zhang, F.F., Wang, W., Yuan, S.J., Hu, Z.H., 2014. Biodegradation and speciation of roxarsone in an anaerobic granular sludge system and its impacts. J. Hazard. Mater. 279 (5), 562–568.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater Yue Yin a, Junfeng Wan a,⁎, Shaozhen Li a, Hongli Li a, Christophe Dagot b,c, Yan Wang a a b c
School of Chemical Engineering and Energy, Zhengzhou University, 100 Science Avenue, 450001, PR China GRESE EA 4330, Université de Limoges, 123 Avenue Albert Thomas, F-87060 Limoges Cedex, France INSERM, U1092, Limoges, France
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
• A-O process was used to treat synthetic livestock wastewater containing ROX. • ROX and its metabolites could affect the bacterial communities in the process. • Different forms of As could be detected in the effluent, sludge and biogas.
a r t i c l e
i n f o
Article history: Received 1 August 2017 Received in revised form 15 October 2017 Accepted 19 October 2017 Available online xxxx Editor: Kevin V. Thomas Keywords: Anoxic-oxic process As speciation As transformation Roxarsone
a b s t r a c t In order to evaluate the influence of roxarsone (ROX) on the livestock wastewater treatment, a lab-scale pilot employing an anoxic–oxic (A-O) process was investigated by adding different concentrations of ROX at different periods. The mass balance of arsenic (As) in the A-O system was established through the analysis of As speciation and As migration in the gas, liquid and solid phases. The results showed that around 80% of total ROX (initial concentration was 50 mg ROX L−1) was eliminated in the anoxic reactor (R1) in which at least about 11% of total ROX was transformed to inorganic Asv (iAsv) due to the direct breaking of the C-As bond of ROX. Inorganic AsIII (iAsIII) and arsine (AsH3) were produced in R1, while the generated iAsIII in the effluent of R1 was almost completely oxidized to iAsV in the aerobic reactor (R2). However, the concentration of ROX in the effluent of R2 was almost the same as that in the effluent of R1. After 85 days operation, iAsV and residual ROX as the main forms of As were observed after the A-O process. Furthermore, the mass balance of As at steady state revealed that around 0.08%, 3.91% and 96.01% of total As was transformed into gas (biogas), solid (excess sludge) and liquid (effluent). Additionally, the 16S rRNA analysis demonstrated that the existence of ROX in livestock wastewater may play a crucial role in the diversity of bacterial community in the A-O system. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail address: [email protected] (J. Wan).
Roxarsone (3-nitro-4-hydroxy benzene arsenic acid, ROX) is widely used as a feed additive in livestock and poultry breeding to control coccidial intestinal parasites, improve feed efficiency and promote
https://doi.org/10.1016/j.scitotenv.2017.10.194 0048-9697/© 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Y. Yin et al. / Science of the Total Environment xxx (2017) xxx–xxx
animal growth (Garbarino et al., 2003; Makris et al., 2008; Mandal and Suzuki, 2002; Yao et al., 2016). Up until 2011, ROX was fed to about 88% of the 9 billion broiler chickens produced each year in America. It is also legal that ROX is applied as feed additives for agriculture in China (Walrod et al., 2016). The Ministry of Agriculture of the People's Republic of China regulated that the addition of ROX in the feed is limited to 50 mg kg−1 (Wang and Liao, 2004). Nevertheless, as a typical organoarsenic chemical, ROX has a potential hazard to the environment. It was reported that after the animal digestion, N 90% of ROX in feeding was released with manure to environment (Fu et al., 2016). Hence, most of residual ROX would finally be transported to the natural environment and it should be degraded or accumulated in the relative environment such as water or soils (Brown et al., 2005; Nachman et al., 2013). Recently, the studies on the migration and transformation of ROX in environment have received enormous attention (Fisher et al., 2015; Huang et al., 2014). Normally, the treatment of livestock wastewater is mainly focused on the removal of high concentrated organic matter (COD), nitrogen and phosphorus (Harrington and Mcinnes, 2009; Shi et al., 2011). The combined process such as anoxic-oxic process (A-O process) was usually applied to treat livestock wastewater after the anaerobic treatment which could remove about 60% of concentrated organic matter (Harwood et al., 1998; Karim and Gupta, 2003). Then the A-O process could remove the majority of the residual COD and nutrients (N and P) to meet the discharge standard (Ren et al., 2010). In fact, the effect of the residual ROX existed in livestock wastewater on the real treatment engineering was usually neglected due to its relatively low concentration (lower than 50 mg L−1). At the same time, the degradation of ROX in anaerobic process was reported in the lab-scale experiment by previous studies (Cortinas et al., 2006; Guzmán-Fierro et al., 2015; Shui et al., 2016; Zhang et al., 2014). Moreover, the biological degradation of COD could be obviously inhibited in anaerobic unit once the concentration of ROX was over 40 mg L−1 (Guo et al., 2013). Similarly, ROX was proved to disturb the efficiency of biological nitrogen removal (BNR) and enhanced biological phosphorus removal (EBPR) processes (Guo et al., 2013; Liu et al., 2014). Additionally, the existence of ROX and its toxic intermediate metabolites probably affected the activity of microorganism. For example, the activity of acetic acid-utilizing methanogenic bacteria was significantly inhibited by ROX at 263 mg L−1 (Sierra-Alvarez et al., 2010). Furthermore, ROX could influence the microbial community structure in wastewater treatment system and thus disturbed the performance of the bioreactors (Jiang et al., 2013; Stolz et al., 2007). At present, most of the relative studies are focus on the degradation of ROX and its metabolites in anaerobic reactor (Cortinas et al., 2006; Shi et al., 2014; Stolz et al., 2007). However, it was rarely reported about the interaction between ROX and the combined process such as the A-O process during livestock wastewater treatment. Therefore, the objectives of this study are: 1) to test the influence of ROX at various concentration on the performance of the A-O process; 2) to analyze the fate of ROX and establish the mass balance of As in the A-O process; 3) to investigate the long-term effect of ROX on the biodiversity and community stucture of microorganism in the A-O process. 2. Material and methods 2.1. Experimental set-up The two bioreactors (working volume = 4.0 L) were installed and connected to function as the A-O process. The activated sludge extracted from local wastewater treatment plant was inoculated in the anoxic reactor (R1) and the aerobic reactor (R2). Both reactors with the volume exchange rate of 50% functioned as sequencing batch reactor (SBR) model. One cycle of 12 h includes four steps: feeding (20 min), reaction (630 min), settling (60 min) and discharging (1 min). During the whole 85 days operation, four phases were established according to the
Table 1 Strategy of ROX addition at different phases. Phases
ROX (mg L−1)
Time (d)
I II III IV
10 50 0 50
1–15 16–55 56–65 66–85
addition of ROX (C6H6AsNO6, Purity: 98%, Rongyao Biotech. Co., Ltd., China) at different concentrations as Table 1. The composition of synthetic wastewater was shown in Table 2. 2.2. Sampling and analysis Mixed liquid suspended solids (MLSS), Mixed liquid volatile suspended solids (MLVSS), Chemical oxygen demand (COD), nitrogen − − forms (NH+ 4 , NO2 , and NO3 ), phosphate were analyzed according to 2− 2− standard methods (APHA, 2002). The sulfur forms (SO2− ) 4 , SO3 , S were measured by Ion Chromatography (Thermo Scientific ICS900). O-aminophenol in the effluent was determined by the high performance liquid chromatograph (HPLC) (ELITE P230II) with a quantification limit of 10 μg L−1 (Wakamatsu et al., 2014). The produced biogas in anoxic reactor R1 were gathered through an on-line gas collector filled with sodium alginate-silver particles in order to analyze the quantity of arsine (AsH3) in gaseous phase (Mestrot et al., 2009). At the end of the reaction, 50 mL of mixed samples periodically extracted from R1 and R2. All the mixed samples were firstly centrifuged at 4500g for 15 min. The precipitants were utilized as sludge samples for analysis of As speciation in solid phase, while the supernatant was filtered by a filter (Ø = 0.45 μm) and stored at 4 °C before the analysis of As speciation in liquid phase. Additionally, the sludge samples and the supernatant were digested for 2 h with the addition of H2O2HNO3 before the total As measurement (Chu et al., 2009). The total As was determined by a hydride generation atomic fluorescence spectrometry (HG-AFS), while As speciation was analyzed by HPLC-HG-AFS. The quantification limits were 0.5, 0.1 and 0.3 μg L−1 for total As, AsIII and AsV, respectively. The detailed parameters of As detection are given in Table 3. 2.3. Microbial structure analysis 2.3.1. DNA extraction and PCR amplification Sludge samples were extracted from R1 and R2 on Day-15, Day-55, Day-65 and Day-85. Microbial DNA was extracted using the E.Z.N.A.® water DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer's protocols. The V4–V5 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 5 min) using primers 338F 5′-barcode-ACTCCT ACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′,
Table 2 Composition of synthetic wastewater. Synthetic wastewater
mg L−1
Trace elements
mg L−1
Sodium propionate NH4Cl K2HPO4 KH2PO4 Na2SO4 KHCO3 FeSO4 · 7H2O MgSO4 · 7H2O CaCl2
670 150 28 14 253 100 50 100 20
H3BO3 MnSO4 CoCl2 CuSO4 · 5H2O NiCl2 · 6H2O ZnSO4 · 7H2O
1 1 0.2 2 0.4 2
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
Y. Yin et al. / Science of the Total Environment xxx (2017) xxx–xxx Table 3 Analytical parameters of total As and As speciation measurement. Settings
HPLC-HG-AFS
HG-AFS
Carrier gas flow (mL min−1) Auxiliary gas flow (mL min−1) Lamp current (mA) Negative high voltage (V) Furnace temperature (°C) Current-carrying Reductant (m/m) Oxygenant (m/m) Injection volume (μL) Flow rate (mL min−1) Mobile phase A Mobile phase B
300 600 50 280 200 10% HCl 3% KBH4, 0.5% KOH 2% K2S2O8, 0.5% KOH 100 1 15 mM (NH4)2HPO4 (pH = 6.0)b 10 mM KHPa (pH = 6.0)b
300 600 50 280 200 3% HCl 1% KBH4, 0.5% KOH – – – – –
a b
KHP is Potassium Hydrogen Phthalate. pH is adjusted with addition of NH3·H2O.
where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 ∗ FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. 2.3.2. Illumina MiSeq sequencing Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer's instructions and quantified using QuantiFluor™-ST (Promega, U.S.). Purified amplicons were pooled in equimolar and paired-end sequenced (2 ∗ 250) on an Illumina MiSeq platform according to the standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP064834) (Quast et al., 2013). 2.3.3. Processing of sequencing data Raw fastq files were demultiplexed, quality-filtered using QIIME (Quast et al., 2013) with the following criteria: (i) The 250 bp reads were truncated at any site receiving an average quality score b 20 over a 10 bp sliding window, discarding the truncated reads that were shorter than 50 bp. (ii) exact barcode matching, 2 nucleotide mismatch in primer matching, reads containing ambiguous characters were removed. (iii) only sequences that overlap longer than 10 bp were assembled according to their overlap sequence. Reads which could not be assembled were discarded. Operational Units (OTUs) were clustered with 97% similarity cutoff using UPARSE (http://drive5.com/uparse/) and chimeric sequences were identified and removed by using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was
3
analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the silva (SSU115) 16S rRNA database using confidence threshold of 70% (Amato et al., 2013). 3. Results 3.1. Performance of A-O process 3.1.1. Anoxic reactor R1 The effluent of R1 was periodically analyzed during the whole experiment as shown in Fig. 1. Once ROX in the feeding increased from 10 to 50 mg L−1 (from Phases I to II), the effluent COD and sulfate (SO2− 4 ) rapidly rised, which was probably due to the acute inhibition on microbial activity by ROX. After 7 days continuous operation in Phase II, COD and sulfate gradually decreased and stabilized at 305.3 ± 5.8 mg O2 L−1 and 6.7 ± 1.0 mg S L−1. The similar results were observed when influent ROX increased from 0 to 50 mg L−1 (from Phases III to IV). A peak at around 40 mg N L−1 for the ammonium appeared at the start-up of Phase II and then gradually decreased and stabilized at 31.1 ± 1.0 mg N L−1, which accounted for around 80% of influent ammonium. A small amount of nitrite was observed in the presence of ROX (Phases I, II and IV), while phosphate was fluctuated at around 6 mg L−1 during the whole experiment. 3.1.2. Aerobic reactor R2 After each cycle, the effluent of R1 was pumped to R2. With the increase of ROX in the feeding of R1 (from 10 to 50 mg L−1 on Day-16 and from 0 to 50 mg L−1 on Day-66), a similar trends was observed as shown in Fig. 2 for COD, nitrate and phosphate in the effluent of R2: the concentrations of these substrates immediately reached at a high level, then gradually decreased and stabilized after a few days of continuous operation. These results revealed that ROX and its possible metabolites after anoxic treatment had an acute inhibition effect on the removal of COD and nutrients (N, P) in aerobic conditions. Moreover, the acute inhibition effect at the beginning of Phase II was much more remarkable compared with Phase IV when ROX was increased to 50 mg L−1. Additionally, no ammonium was accumulated during the whole experiment. 3.2. ROX degradation and As speciation during A-O process The concentration of ROX in the feeding was controlled as the unique variable parameter in this experiment. As shown in Fig. 3, As speciation in the effluent including iAsIII, iAsV, ROX and total As were measured. When additive ROX in the feeding increased to 50 mg L−1 on Day-16 and Day-66, the effluent ROX of R1 rapidly rose to a high level and then decreased and stabilized at 2.41 ± 0.09 mg As L−1.
Fig. 1. Evaluation of substrates in the effluent of R1; (a) COD and SO2− (b); N-forms and Phosphate. 4
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Fig. 2. Evaluation of substrates in the effluent of R2; (a) COD and SO2− (b); N-forms and Phosphate. 4
Correspondingly, generated inorganic As (i.e. iAsIII and iAsV) in the effluent of R1 slowly increased and finally remained at a stable value. These results documented that ROX could be biologically degraded under anoxic conditions. Moreover, some unknown organoarsenic compounds were detected by HPLC-HG-AFS measurement. On the contrary, residual ROX in the effluent of R1 was very close to which in the effluent of R2 no matter how the initial concentration of additive ROX in the feeding changed, which proved that ROX was hardly degraded under aerobic conditions. However, the quantity of iAsV in the effluent of R2 was much more than that in the effluent of R1, which revealed that other organoarsenic compounds were oxidized to iAsV under aerobic conditions. The similar results were reported by Markley and Herbert (2009). 3.3. Bacterial community in A-O process At the end of each phase, the diversity and the abundance of the microbial communities at various ROX concentrations were analyzed by high-throughput sequencing. As shown in Fig. 4, a community abundance analysis was obtained on genus level. After a long-term operation, functional bacteria such as Desulfobulbus, Geobacter and Paracoccus for sulfate reduction, aromatic hydrocarbon degradation and denitrification were present in R1 (Blaszczyk, 1993; Coates et al., 1997; Shinoda et al., 2004; Taylor and Parkes, 1983), which means that these bacteria could resist to ROX and relative inorganic As (Anderson et al., 2003). At the end of Phase III, the proportion of Hydrogenophaga was increased in R1, while Bacteroidetes-vadinHA17 was decreased significantly without addition of ROX. At the same time, the increased proportion of some bacteria like Alphaproteobacteria was observed in R2, while Thauera was decreased obviously without addition of ROX. When ROX was increased to 50 mg L−1 again in Phase IV, the proportion of the bacteria increased in Phase III such as Hydrogenophaga decreased, and was even lower than its proportion at the same concentration of ROX during
the Phase II. Thus, the influence of ROX on the bacterial diversity and abundance cannot be ignored in the presence of ROX in the A-O process. 4. Discussion 4.1. Possible degradation mechanism of ROX ROX could be possibly degraded by the fracture of C\\As bond on the benzene ring in the anaerobic conditions (Shi et al., 2014). As a result, oaminophenol could be produced once C\\As bond on the benzene ring was broken and the nitro group of ROX was reduced together. At the same time, the released inorganic As that was derived from the direct breakage of C\\As bond could be estimated based on the oaminophenol production if regardless of the degradation of oaminophenol and other metabolic pathways. From Fig. 5a, when initial ROX concentration was 10 mg L− 1 (38.02 μM), the o-aminophenol and the sum of inorganic As (iAsIII + iAsV) in the effluent of R1 maintained at 0.09 mg L−1 (=0.83 μM) and 0.49 mg As L−1 (=6.20 μM) at the end of the reaction. When ROX concentration was increased to 50 mg L− 1 (190.11 μM), the concentration of o-aminophenol maintained at about 0.23 mg L− 1 (= 2.11 μM) in steady state (at the end of the Phase II and Phase IV), while the sum of inorganic As (iAsIII + iAsV) was 1.46 mg As L− 1 (= 18.48 μM) in R1. Considering ROX was the only source of benzene homologues in the whole process, we concluded that at least about 11% of the total amount of inorganic As was produced by the C\\As bond breaking of ROX. Furthermore, the kinetic study of R1 (Fig. 5b) showed that o-aminophenol gradually increased from 0.11 (=1.01 μM) to 0.24 mg L−1 (=2.20 μM) during 300 min of reaction. Additionally, almost no o-aminophenol was detected in the effluent of R2, which means that o-aminophenol could be readily degraded under aerobic conditions. The measured quantity of o-aminophenol was the dynamic results of its formation and degradation in R1, so it
Fig. 3. Evaluation of As speciation in the effluent of R1 (a) and R2 (b).
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Fig. 4. Percentage of community abundance on genus level for the both reactors on different phases; A15, A55, A65 and A85 were sludge samples which were extracted from R1 on Day-15, 55, 65 and 85; O15 O55 O65 and O85 were sludge samples which were extracted from R2 on Day-15, 55, 65 and 85.
was difficult to evaluate the total amount of o-aminophenol produced in the whole reaction process (Bednar et al., 2003; Stolz et al., 2007; Yang et al., 2016). Therefore, the method for judging the exact value of inorganic As generated by the direct cleavage of C\\As bond still needs to be further improved, although the present data showed only a small fraction of inorganic As in R1 caused by the direct cleavage of C\\As bond of ROX. At the same time, with the consideration that a large quantity of iAsV were obviously accumulated in R2 with addition of 50 mg L−1 of ROX after long-term operation (Fig. 3), the biologically induced oxidation of the reducible form of As (e.g. iAsIII) possibly occured in the oxic system as our previous studies (Wang et al., 2017a; Wang et al., 2017b).
4.2. Mass balance and distribution of As speciation in A-O process At the end of Phase IV from Day-80 to Day-85, the quantity of arsine (AsH3) in gaseous phase was periodically measured through the collection and analysis of the gaz. The measured arsine released to air from R1 was about 25.36 ± 2.85 μg (0.31 ± 0.03 μmol) in 1 cycle (12 h), which indicated that about 0.08% of total As in the influent was reduced to AsH3 in R1 every day. Considering that the arsine was as one of the final products of ROX degradation, the percentage of arsine on the total As transformation in A-O process cannot be ignored (Chen et al., 2011; Huang et al., 2016; Qin et al., 2006). At the same time, the contents of total arsenic in anoxic and aerobic sludge were 1.00 ±
Fig. 5. Variation of o-aminophenol: a) in the effluent of R1 during the whole experiment; b) in R1 for 1 cycle on Day-90.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
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Fig. 6. The distribution of As species in R1 (a) and R2 (b) on Day-85. Liquid represents different forms of As in water sample, Sludge represents different forms of As in solid sample, ND represents the undetected amount of As according to the difference between total As and the sum of various As forms in liquid and sludge samples.
0.05 mg As g−1 VSS and 2.82 ± 0.08 mg As g−1 VSS, respectively. If assumed that the anoxic and aerobic yield coefficients under pH = 7.0 and 25 °C were about 0.20 and 0.42 g VSS g−1 COD (Pipyn and Verstraete, 2010; Tawfik et al., 2008), around 0.09 and 0.18 g VSS L−1 increased in R1 and R2 because about 90 and 210 mg L−1 of COD were removed every day in R1 and R2. As a result, the amounts of As absorbed by anoxic and aerobic sludge was about 0.36 and 1.99 mg As in R1 and R2 every day. According to this hypothesis, we can conclude that around 0.08%, 3.91% and 96.01% of total As were transformed into gas (biogas), solid (excess sludge) and liquid (effluent). Furthermore, in order to evaluate the As speciation in the A-O process, the identical volume of suspended samples including sludge and water samples were directly exacted at the end of reaction from R1 and R2 on Day-85. The various As forms such as iAsIII, iAsV, MMA and ROX in sludge and liquid were measured. Moreover, the total As was measured after digestion of suspended samples. The percentage of various As forms in liquid and sludge samples were calculated as shown in Fig. 6. From Fig. 6a in R1, a small amount of As content (about 24.2% of the total As in suspended samples) existed in the anoxic sludge, in which iAsIII and ROX were the mains forms. 72.59% of the total As existed in liquid of R1, in which the unknown form and ROX were the main forms of As in liquid followed by the iAsV and iAsIII. The unknown As form was about 3946.8 ± 184.4 μg L−1 in R1 and decreased to 1552.6 ± 185.4 μg L−1 in R2 after a cycle reaction. Additionally, much more quantity of As existed as iAsV (about 46.2% of the total As in suspended samples) was detected in the aerobic sludge of R2 compared with R1. In contrast to the various forms of As in the effluent of R1 and R2 (Fig. 3), it can be seen that a small amount of organic As in the unknown form in R1 was mainly converted to iAsV in R2. In fact, ROX and the possible generated HAPA in R1 were difficult to open loop to release iAsV under the aerobic conditions (Donlon et al., 1996; Karim and Gupta, 2003; Lenke and Knackmuss, 1992; Lenke et al., 1992). Additionally, the biological open-loop ability is relatively weak under the aerobic conditions. Thus, the extra quantity of iAsV in R2 was probably produced from the unknown arsenate-containing aliphatic acids which were detected in R1. It should be pointed out that the unknown arsenate-containing aliphatic acids need to be further confirmed. 5. Conclusions ROX, as one of popular organoarsenic additives, should be given adequate attention in livestock wastewater treatment. The results suggested that the residual ROX existed in the A-O process could not only influence the removal efficiency of COD and nutrients (N and P), but also played a crucial role in the microbial community. After livestock
wastewater treatment by the A-O process, most of ROX and its metabolites were still remained in the discharged effluent, while a small amount of As (mainly presented as the forms of iAsIII and iAsV) was accumulated in the activated sludge. Therefore, it is better to utilize seperation methods such as adsorption in order to maximally capture ROX and its metabolites from the livestock wastewater. At the same time, when the excess sludge is treated and recovered as resource (e.g. organic fertilizer), the control of the content of As in the recovered product should be considered.
Acknowledgments The authors thank the National Natural Science Foundation of China (No. 21107100) and the Outstanding Young Talent Research Fund of Zhengzhou University (No. 1421324067) for the financial support of this study. References Amato, K.R., Yeoman, C.J., Kent, A., Righini, N., Carbonero, F., Estrada, A., Gaskins, H.R., Stumpf, R.M., Yildirim, S., Torralba, M., Gillis, M., Wilson, B.A., Nelson, K.E., White, B.A., Leigh, S.R., 2013. Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J. 7 (7), 1344–1353. Anderson, R.T., Vrionis, H.A., Ortizbernad, I., Resch, C.T., Long, P.E., Dayvault, R., Karp, K., Marutzky, S., Metzler, D.R., Peacock, A., 2003. Stimulating the in situ activity of geobacter species to remove uranium from the groundwater of a uraniumcontaminated aquifer. Appl. Environ. Microbiol. 69 (10), 5884–5891. APHA, 2002. Standard Methods for the Examination of Water and Wastewater. 20th edition. American Public Health Association, Washington, DC. Bednar, A.J., Garbarino, J.R., Ferrer, I., Rutherford, D.W., Wershaw, R.L., Ranville, J.F., Wildeman, T.R., 2003. Photodegradation of roxarsone in poultry litter leachates. Sci. Total Environ. 302 (1–3), 237–245. Blaszczyk, M., 1993. Effect of medium composition on the denitrification of nitrate by Paracoccus denitrificans. Appl. Environ. Microbiol. 59 (11), 3951–3953. Brown, B.L., Slaughter, A.D., Schreiber, M.E., 2005. Controls on roxarsone transport in agricultural watersheds. Appl. Geochem. 20 (1), 123–133. Chen, J., Sun, G.X., Wang, X.X., Lorenzo, V.D., Rosen, B.P., Zhu, Y.G., 2011. Volatilization of arsenic from polluted soil by pseudomonas putida engineered for expression of the arsM arsenic(III)S-adenosine methyltransferase gene. Environ. Sci. Technol. 48 (48), 10337–10344. Chu, Z., Xiao, Y., Liu, W., Zheng, W., 2009. Comparison of different acid systems in the procedure of digestion and decomposition of soil arsenic, mercury, lead and cadmium using high pressure method. Environ. Monit. China 25 (5), 57–61. Coates, J.D., Woodward, J., Allen, J., Philp, P., Lovley, D.R., 1997. Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments. Appl. Environ. Microbiol. 63 (9), 3589–3593. Cortinas, I., Field, J.A., Kopplin, M., Garbarino, J.R., Gandolfi, A.J., Sierra-Alvarez, R., 2006. Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids. Environ. Sci. Technol. 40 (9), 2951–2957. Donlon, B.A., Razoflores, E., Lettinga, G., Field, J.A., 1996. Continuous detoxification, transformation, and degradation of nitrophenols in upflow anaerobic sludge blanket (UASB) reactors. Biotechnol. Bioeng. 51 (4), 439–449. Fisher, D.J., Yonkos, L.T., Staver, K.W., 2015. Environmental concerns of roxarsone in broiler poultry feed and litter in Maryland, USA. Environ. Sci. Technol. 49 (4), 1999–2012.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194
Y. Yin et al. / Science of the Total Environment xxx (2017) xxx–xxx Fu, Q.L., He, J.Z., Gong, H., Blaney, L., Zhou, D.M., 2016. Extraction and speciation analysis of roxarsone and its metabolites from soils with different physicochemical properties. J. Soils Sediments 16 (5), 1–12. Garbarino, J.R., Bednar, A.J., Rutherford, D.W., Beyer, R.S., Wershaw, R.L., 2003. Environmental fate of roxarsone in poultry litter. I. Degradation of roxarsone during composting. Environ. Sci. Technol. 37 (8), 1509–1514. Guo, Q., Liu, L., Hu, Z., Chen, G., 2013. Biological phosphorus removal inhibition by roxarsone in batch culture systems. Chemosphere 92 (1), 138–142. Guzmán-Fierro, V.G., Moraga, R., León, C.G., Campos, V.L., Smith, C., Mondaca, M.A., 2015. Isolation and characterization of an aerobic bacterial consortium able to degrade roxarsone. Int. J. Environ. Sci. Technol. 12 (4), 1353–1362. Harrington, R., Mcinnes, R., 2009. Integrated constructed wetlands (ICW) for livestock wastewater management. Bioresour. Technol. 100 (22), 5498–5505. Harwood, C.S., Burchhardt, G., Herrmann, H., Fuchs, G., 1998. Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway. FEMS Microbiol. Rev. 22 (5), 439–458. Huang, L., Yao, L., He, Z., Zhou, C., Li, G., Yang, B., Deng, X., 2014. Roxarsone and its metabolites in chicken manure significantly enhance the uptake of As species by vegetables. Chemosphere 100 (3), 57–62. Huang, K., Chen, C., Zhang, J., Tang, Z., Shen, Q., Rosen, B.P., Zhao, F.J., 2016. Efficient arsenic methylation and volatilization mediated by a novel bacterium from an arseniccontaminated paddy soil. Environ. Sci. Technol. 50 (12), 6389–6396. Jiang, Z., Li, P., Wang, Y.H., Li, B., Wang, Y.X., 2013. Effects of roxarsone on the functional diversity of soil microbial community. Int. Biodeterior. Biodegrad. 76 (1), 32–35. Karim, K., Gupta, S.K., 2003. Continuous biotransformation and removal of nitrophenols under denitrifying conditions. Water Res. 37 (12), 2953–2959. Lenke, H., Knackmuss, H.J., 1992. Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2. Appl. Environ. Microbiol. 58 (9), 2933–2937. Lenke, H., Pieper, D.H., Bruhn, C., Knackmuss, H.J., 1992. Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2. Appl. Environ. Microbiol. 58 (9), 2928–2932. Liu, H., Wang, G., Ge, J., Li, L., Chen, G., 2014. Fate of roxarsone during biological nitrogen removal process in wastewater treatment systems. Chem. Eng. J. 255 (255), 500–505. Makris, K.C., Salazar, J., Quazi, S., Andra, S.S., Sarkar, D., Bach, S.B., Datta, R., 2008. Controlling the fate of roxarsone and inorganic arsenic in poultry litter. J. Environ. Qual. 37 (3), 963–971. Mandal, B.K., Suzuki, K.T., 2002. Arsenic round the world: a review. Talanta 58 (1), 201–235. Markley, C.T., Herbert, B.E., 2009. Arsenic risk assessment: the importance of speciation in different hydrologic systems. Water Air Soil Pollut. 204 (1–4), 385–398. Mestrot, A., Uroic, M.K., Plantevin, T., Islam, M.R., Krupp, E.M., Feldmann, J., Meharg, A.A., 2009. Quantitative and qualitative trapping of arsines deployed to assess loss of volatile arsenic from paddy soil. Environ. Sci. Technol. 43 (21), 8270–8275. Nachman, K.E., Baron, P.A., Raber, G., Francesconi, K.A., Navasacien, A., Love, D.C., 2013. Roxarsone, inorganic arsenic, and other arsenic species in chicken: a U.S.-based market basket sample. Environ. Health Perspect. 121 (7), 818–824. Pipyn, P., Verstraete, W., 2010. Comparison of maximum cell yield and maintenance coefficients in axenic cultures and activated sludge communities. Biotechnol. Bioeng. 20 (12), 1883–1893. Qin, J., Rosen, B.P., Zhang, Y., Wang, G., Franke, S., Rensing, C., 2006. Arsenic detoxification and evolution of trimethylarsine gas by a microbial arsenite S-adenosylmethionine methyltransferase. Proc. Natl. Acad. Sci. 103 (7), 2075–2080. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41 (Database issue), 590–596.
7
Ren, L., Wu, Y., Ren, N., Zhang, K., Xing, D., 2010. Microbial community structure in an integrated A/O reactor treating diluted livestock wastewater during start-up period. J. Environ. Sci. 22 (5), 656–662. Shi, L.J., Ban, L.T., Liu, H.F., Hao, J.C., Zhang, W.Y., 2011. Study on livestock and poultry farming wastewater treatment using a hybrid baffled reactor. Adv. Mater. Res. 201–203, 2797–2802. Shi, L., Wang, W., Yuan, S.J., Hu, Z.H., 2014. Electrochemical stimulation of microbial roxarsone degradation under anaerobic conditions. Environ. Sci. Technol. 48 (14), 7951–7958. Shinoda, Y., Sakai, Y., Uenishi, H., Uchihashi, Y., Hiraishi, A., Yukawa, H., Yurimoto, H., Kato, N., 2004. Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1. Appl. Environ. Microbiol. 70 (3), 1385–1392. Shui, M., Ji, F., Tang, R., Yuan, S., Zhan, X., Wang, W., Hu, Z., 2016. Impact of roxarsone on the UASB reactor performance and its degradation. Front. Environ. Sci. Eng. 10 (6), 1–9. Sierra-Alvarez, R., Cortinas, I., Field, J.A., 2010. Methanogenic inhibition by roxarsone (4hydroxy-3-nitrophenylarsonic acid) and related aromatic arsenic compounds. J. Hazard. Mater. 175 (1–3), 352–358. Stolz, J.F., Perera, E., Kilonzo, B., Kail, B., Crable, B., Fisher, E., Ranganathan, M., Wormer, L., Basu, P., 2007. Biotransformation of 3-nitro-4-hydroxybenzene arsonic acid (roxarsone) and release of inorganic arsenic by Clostridium species. Environ. Sci. Technol. 41 (3), 818–823. Tawfik, A., Sobhey, M., Badawy, M., 2008. Treatment of a combined dairy and domestic wastewater in an up-flow anaerobic sludge blanket (UASB) reactor followed by activated sludge (AS system). Desalination 227 (1), 167–177. Taylor, J., Parkes, R.J., 1983. The cellular fatty acids of the sulphate-reducing bacteria, Desulfobacter sp., Desulfobulbus sp. and desulfovibrio desulfuricans. Microbiology 129 (11), 3303–3309. Wakamatsu, K., Tanaka, H., Tabuchi, K., Ojika, M., Zucca, F.A., Zecca, L., Ito, S., 2014. Reduction of the nitro group to amine by hydroiodic acid to synthesize o-aminophenol derivatives as putative degradative markers of neuromelanin. Molecules 19 (6), 8039–8050. Walrod, J.H., Burriss, D., Blue, L.Y., Beck, G.E., Atwood, D.A., 2016. Arsenic mobility in Karnak soils after multi-year application of poultry litter containing roxarsone. Main Group Chem. 15 (4), 365–373. Wang, K., Liao, X., 2004. The function in animal production and influence on environment of arsenic preparation. Ecol. Domest. Anim. 25 (2), 41–43. Wang, J., Wan, J., Li, H., Li, H., Dagot, C., Wang, Y., 2017a. Biological arsenite oxidation with nitrate as sole electron acceptor. Environ. Technol. 38 (16), 2070–2076. Wang, J., Wan, J., Wu, Z., Li, H., Li, H., Dagot, C., Wang, Y., 2017b. Flexible biological arsenite oxidation utilizing NOx and O2 as alternative electron acceptors. Chemosphere 178, 136–142. Yang, Z., Peng, H., Lu, X., Liu, Q., Huang, R., Hu, B., Kachanoski, G., Zuidhof, M.J., Le, X.C., 2016. Arsenic metabolites, including N-acetyl-4-hydroxy-m-arsanilic acid, in chicken litter from a roxarsone-feeding study involving 1600 chickens. Environ. Sci. Technol. 50 (13), 6737–6743. Yao, L., Huang, L., He, Z., Zhou, C., Lu, W., Bai, C., 2016. Delivery of roxarsone via chicken diet → chicken → chicken manure → soil → rice plant. Sci. Total Environ. 566-567, 1152–1158. Zhang, F.F., Wang, W., Yuan, S.J., Hu, Z.H., 2014. Biodegradation and speciation of roxarsone in an anaerobic granular sludge system and its impacts. J. Hazard. Mater. 279 (5), 562–568.
Please cite this article as: Yin, Y., et al., Transformation of roxarsone in the anoxic–oxic process when treating the livestock wastewater, Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.194