Chemosphere 100 (2014) 57–62
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Roxarsone and its metabolites in chicken manure significantly enhance the uptake of As species by vegetables Lianxi Huang a,b,c, Lixian Yao d,⇑, Zhaohuan He a,b,c, Changmin Zhou a,b,c, Guoliang Li a,b,c, Baomei Yang a,b,c, Xiancai Deng d a
Institute of Agricultural Resources and Environment, Guangdong Academy of Agricultural Sciences, China Key Laboratory of Plant Nutrition and Fertilizer in South Region, Ministry of Agriculture, China Guangdong Key Laboratory of Nutrient Cycling and Farmland Conservation, China d South China Agricultural University, Guangzhou 510640, China b c
h i g h l i g h t s Roxarsone was degraded in animal manure and converted to higher toxic As forms. Roxarsone and it metabolites in animal manure increased As uptake of vegetables. As(III) was the predominant As species in vegetables. As can enter food chain via the way: roxarsone ? animal ? manure ? soil ? crop. Roxarsone and it metabolites in organic fertilizer lead to As exposure for human.
a r t i c l e
i n f o
Article history: Received 9 June 2013 Received in revised form 16 December 2013 Accepted 31 December 2013 Available online 22 January 2014 Keywords: Roxarsone As species Chicken manure Vegetable
a b s t r a c t Roxarsone is an organoarsenic feed additive which can be finally degraded to other higher toxic metabolites after excreted by animal. In this work, the uptake of As species by vegetables treated with chicken manure bearing roxarsone and its metabolites was investigated. It was showed that more than 96% of roxarsone added in chicken feed was degraded and converted to arsenite, monomethylarsonic acid, dimethylarsinic acid, arsenate, 4-hydroxyphenylarsonic acid and other unknown As species. Arsenite and arsenate could be found in roots of vegetables but only arsenite transported up to shoots. Chicken manure bearing roxarsone and its metabolites increased 33–175% of arsenite and 28%seven times of arsenate in vegetable roots, 68–175% of arsenite in edible vegetable shoots. Arsenite, the most toxic As form, was the major extractable As species in vegetables accounted for 79–98%. The results reflected that toxic element As could be absorbed by vegetables via the way: roxarsone in feed ? animal ? animal manure ? soil ? crop and the uptake of As species would be enhanced by using chicken manure bearing roxarsone and its metabolites as organic fertilizer. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction As is one of the most serious global environmental toxicants which can be taken up by crops from soil via root absorption, and eventually transferred into human beings via the food chain. As in environment, plants and organisms usually presents in various chemical forms such as arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), 4-hydroxyphenylarsonic acid (4-HPA), 3-amino-4-hydroxyphenylarsonic acid (3-A-HPA), trimethylarsine oxide (TMAO), tetramethylarsonium cation (TETRA), arsenobetaine (AsB), arsenocholine (AsC) and arsenosugars. Generally, the toxicity and mobility of As ⇑ Corresponding author. Tel./fax: +86 20 38604958. E-mail address: [email protected] (L. Yao). 0045-6535/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.12.074
species in organisms is considered to be: As(III) > As(V) > organoarsenic (Vahter et al., 1988; Cullen and Reimer, 1989). Roxarsone (3-nitro-4-hydroxarsoneyphenylarsonic acid) is an organoarsenic feed additive to promote growth, control intestinal parasites and improve feed efficiency in animal production (Moore et al., 1998; Chapman and Johnson, 2002). The use of roxarsone as a feed additive had been banned by the European Union in 1999 and the sales of roxarsone also had been suspended by the United States in 2011, but roxarsone was still a widely used feed additive in China (Yao et al., 2013). The toxicity of roxarsone is low but it can be finally excreted by animal and degraded into higher toxic metabolites soon after the animal manure enters environment or when the animal manure is composted (Bednar et al., 2003; Garbarino et al., 2003). The metabolites of roxarsone in animal manure primarily occur as As(III), DMA, MMA, As(V), 3-A-HPA, As(V),
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4-HPA and other unknown As species (Jackson and Bertsch, 2001; Rosal et al., 2005; Makris et al., 2008). It was reported that As content in animal manure is from undetectable level to 315.1 mg kg1 (Cang et al., 2004; Yao et al., 2006), and 70–75% of the As in poultry manure is water soluble (Jackson and Bertsch, 2001; Jackson et al., 2003). Moreover, animal manure is commonly applied as organic fertilizer. Hence, the bioavailability of roxarsone and its metabolites in animal manure might be high and should be of great concern. It was reported that As concentration in plant growing in contaminated sites ranged from 1.14 mg kg1 to 98.5 mg kg1 (dry mass), but in a non-contaminated area with similar geological characteristics only 0.06–0.58 mg kg1 (Ruiz-Chancho et al., 2008). The research (Yao et al., 2009) showed that As(V), As(III) and DMA were found in turnip tissues applied with chicken manure containing roxarsone and its metabolites, and the content of As species increased with the increasing used rate of chicken manure. It was also demonstrated that As concentrations in different parts of rice plants increased with the elevating concentrations of roxarsone and arsanilic acid added in soils and varied with the growth stage (Wang et al., 2006). Moreover, the capability of As uptake and accumulation by different varieties of crop was different. The sequence of As concentration in the edible part of various crops was as follow: celery > mustard > spinach > lettuce > taro > bokchoi > cowpea > caulifl ower > eggplant (Huang et al., 2006). The following pattern of As concentration in several crops: red cabbage curly endive > barley wheat sugar beet leek > cabbage green pepper was also reported (De La Fuente et al., 2010). In this work, pot experiment was carried out to investigate the uptake of As species in vegetables treated with chicken manure bearing roxarsone and its metabolites. The objective was to reveal the pathway of As species entering human food chain from organoarsenic additives in animal feed. The potential risk of using roxarsone as feed additive and using chicken manure bearing roxarsone and its metabolites as fertilizer were also evaluated. 2. Materials and methods 2.1. Soil, chicken manure and vegetables Soil used in this study was a typical lateritic red soil collected at a depth of 0–25 cm from the Crop Experiment Station of the Guangdong Academy of Agricultural Sciences (23°80 4300 N, 113°200 5000 E) located in Guangzhou, South China. Chicken manure was taken from an intensive chicken farm located in Huizhou city, Guangdong province in South China. The first chicken manure (CKCM) was excreted by chickens fed with non-roxarsone feedstuff and the second chicken manure (As-CM) was excreted by the same species of chickens fed with feedstuff containing 55 mg kg1 roxarsone. The basic properties such as total content of As, N, P, K, organic matter and pH value of soil and chicken manures used in this experiment were listed in Table 1. Seeds of coriander herb (Coriandrum sativum L.), crowndaisy (Chrysanthemum coronarium), flowering cabbage (Brassica campestris L.), leaf mustard (Brassica juncea) and spinach (Spinacia oleracea) were purchased from the seed market of Guangdong Academy of Agricultural Sciences. 2.2. Pot experiment The pot experiment was carried out with two chicken manures (CK-CM and As-CM) and four replications of each vegetable. The amount of chicken manure amended in soil was 2% (w/w, manure/soil) and CK-CM was considered as the control. After completely mixed with 2% chicken manure, the soil was kept at 70% field water holding capacity for 10 d by adding deionized water. In order to obtain sufficient biomass, five seedlings of crowndaisy,
flowering cabbage, leaf mustard, spinach and six seedlings of coriander herb were kept in corresponding pot. Approximately 100 mL of deionized water was dispersedly sprinkled on the topsoil of every pot when the soil was lack of sufficient water. All the vegetables were grown at natural temperature and photoperiod of the green house, no additional thermal source and light were supplied. 2.3. Sample collection and preparation Before sowing, one soil sample was collected in each pot. According to the individual characteristic of vegetables, the samples of coriander herb, crowndaisy, flowering cabbage, leaf mustard, and spinach was harvested after 48, 54, 33, 50 and 68 days’ growing respectively. Shoots and roots of vegetable plants were harvested separately with stainless knife, washed with tap water and then rinsed with deionized water, then immediately lyophilized (Alpha 1–4/LD-plus, Christ German). Fresh weight and dry weight of the vegetable tissues were recorded. Rhizosphere soil samples were synchronously collected and then lyophilized. The plant samples were quickly pulverized to fine powder using retsch grinder (ZM200, Germany), and the soil samples were grinded using a agate mortar then passed a 150 lm sieve for total As and As species analysis. Plant and soil samples were kept in 80 °C refrigerator to assure the stabilization of As species before extraction. 2.4. Chemical reagents Roxarsone with a purity of 97.5% was purchased from Dr. Ehrenstorfer Gmbh in Germany, 3-A-HPA (99%) from Sigma–Aldrich (USA) and 4-HPA (98%) from TCI Tokyo Kasei (Japan). The standard stock solutions of As(V) (17.5 ± 0.4 mg L1), As(III) (75.7 ± 1.2 mg L1), MMA (25.1 ± 0.8 mg L1), and DMA (52.9 ± 1.8 mg L1) presented as the concentration of As were provided by the Chinese National Standard Materials Center and stored in the dark at 4 °C. The HPLC grade methanol was used and all other reagents were analytical grade. The water used in chemical analysis was prepared by Millipore Milli-Q Academic. 2.5. Chemical analysis The methods of total As extraction and detection were NY/ T1121.11-2006 (chicken manure and soil samples) and GB/ T5009.11-2003 (vegetable samples) issued by China. Firstly, chicken manure and soil samples were digested with 50% aqua regia, then the extracted total As was pre-deoxidized to As(III) using a mixture of 5.0% H2NCSNH2 and 5.0% C8H8O6. Vegetable samples were digested with a mixture of concentrated HNO3, H2SO4 and HClO4, then the extracted total As was pre-deoxidized to As(III) using 0.5% H2NCSNH2. The As(III) in chicken manure, soil and vegetables was finally deoxidized to AsH3 by 2.0% KBH4 and 5% HCl and determined by hydride generation-atomic fluorescence spectrometry (AFS8130, Jitian Beijing, China). The running speed of carrier gas (argon) was 300 mL min1 and shield gas 800 mL min1. Photomultiplier tube voltage was 270 V and the hollow cathode lamp current 60 mA (primary)/30 mA (boosted). Two reference materials GBW07408 (soil) and GBW07602 (bush plant), purchased from the Chinese National Reference Materials Center, were used to control the analytical quality of total As in soil, chicken manure and plant samples with detected error 3.2 ± 1.2%. The extraction and detection of As species are the same with the methods mentioned in our previous studies (Huang et al., 2010, 2013; Yao et al., 2013) and presented as follow. As species in chicken manure and soil samples were extracted with a mixture of 0.1 mol L1 H3PO4 and 0.1 mol L1 NaH2PO42H2O (1:9, V/V) (Jackson and Miller, 2000), and those in vegetables by ultrapure water (Tomohiro Narukawa et al., 2008). Approximately 0.25 g of chicken
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L. Huang et al. / Chemosphere 100 (2014) 57–62 Table 1 Basic properties of soil and chicken manures used in the experiment. Item c
pH Organic matter (g kg1) Total N (g kg1) Total P (g kg1) Total K (g kg1) Total As (mg kg1) As(III) (mg kg1) Dimethylarsinic acid (As, mg kg1) Monomethylarsonic acid (As, mg kg1) 3-amino-4-hydroxyphenylarsonic acid(As, mg kg1) As(V) (mg kg1) 4-hydroxyphenylarsonic acid (As, mg kg1) Unknown As species Roxarsone (As, mg kg1) a b c d e
Soil
CK-CMa
As-CMb
6.88 ± 0.11 20.6 ± 0.00 1.27 ± 0.01 1.12 ± 0.01 13.1 ± 0.11 7.35 ± 0.13 0.12 ± 0.00 NDd ND ND 1.53 ± 0.01 ND ND ND
5.99 ± 0.02 426 ± 0.00 22.6 ± 1.44 20.5 ± 0.46 32.9 ± 0.14 8.85 ± 0.09 0.33 ± 0.04 1.07 ± 0.04 ND ND 1.27 ± 0.11 ND ND ND
5.82 ± 0.06 512 ± 0.00 33.2 ± 0.00 17.7 ± 0.23 38.8 ± 0.14 58.3 ± 2.10 2.96 ± 0.05 3.00 ± 0.15 1.73 ± 0.23 ND 20.5 ± 0.38 0.36 ± 0.14 Detectablee 1.58 ± 0.31
Represents chicken manure without roxarsone and its metabolites. Represents chicken manure containing roxarsone and its metabolites. Measured in a 1:2.5 soil or chicken manure to water (w/v) suspension. Not detectable. Detectable but could not be quantitatively calculated owing to absence of internal standard materials.
manure, 0.50 g of vegetable or 1.0 g of soil samples were placed into Teflon vessels with 10 mL extractant, and kept in water bath at 55 °C for 10 h, then sonicated for 10 min. The supernatant was collected after centrifugation at 4000 rpm for 5 min. This extraction procedure was repeated three times. The three supernatants were mixed and passed through a solid-phase extraction cartridge of C18 to remove pigments or hydrophobic organic compounds. The filtrate was lyophilized and dissolved using 3 mL of Milli-Q water then filtered through a 0.22 lm Millipore membrane before Liquid Chromatography (20AT, SHIMADZU, Japan)-Hydrogen GenerationAtomic fluorescence Spectrometry analysis. Reversed C18 chromatographic column (250 mm 4.6 mm i.d.,5 lm, Phenomenex, USA) filled with ODS3 was used to separate the As species. Mobile phase A and mobile phase B for As species separation were 10 mmol L1 NaH2PO4 and 50 mmol L1 NaH2PO4 both containing 0.5 mmol L1 TBAB and CH3OH (3% for mobile phase A and 5% for mobile phase B). The pH value of mobile phase A and B was accommodated to 6.22 using 50% ammonia. Gradient elution procedure was 100% A (0–11 min), 100% B (11–20 min) and 100% A (20– 30 min). Flow rate was 1.0 mL min1 and injection volume 100 lL. After complete separation, As species were firstly mixed with 20 g L1 K2S2O8 (some complex As forms such as 3-A-HPA, 4-HPA and roxarsone were broken down at this step), then reduced to As(III) by 20 g L1 KBH4 and 10% (V/V) HCl, and finally detected by AFS. The running speed of carrier gas (argon) was 400 mL min1 and shield gas 600 mL min1. Photomultiplier tube voltage was 295 V and the hollow cathode lamp current 90 mA (primary)/ 40 mA (boosted). Detection limits of As(III), MMA, DMA, 3-AHPA, As(V), 4-HPA and roxarsone were 1.8, 1.9, 3.6, 12.1, 4.7, 3.8 and 9.5 lg L1, respectively. Recoveries of standard addition of As(III), MMA, DMA, 3-A-HPA, As(V), 4-HPA and roxarsone were 82.4 ± 1.3%, 91.8 ± 2.1%, 90.6 ± 2.1%, 45.8 ± 1.9%, 98.8 ± 2.0%, 103.3 ± 6.0% and 93.0 ± 2.3% in chicken manures, 87.8 ± 2.9%, 103.2 ± 3.3%, 105.6 ± 3.9%, 60.3 ± 2.7%, 93.1 ± 3.1%, 94.6 ± 2.9% and 104.1 ± 3.7% in soils, 95.2 ± 2.4%, 86.5 ± 2.2%, 86.4 ± 1.7%, 58.6 ± 5.4%, 99.1 ± 2.9%, 90.3 ± 2.7% and 85.3 ± 1.3% in shoots of vegetables, 96.0 ± 2.1%, 83.3 ± 3.2%, 81.4 ± 2.4%, 60.1 ± 6.9%, 98.4 ± 2.8%, 96.5 ± 3.8% and 87.4 ± 2.3% in roots of vegetables, respectively. Unless specially pointed out, the contents of As species in all samples were presented as elemental As. 2.6. Data and statistics Owing to the low biomass, the four replications of flowering cabbage roots treated with CK-CM were combined into one sample
to obtain sufficient amount for analysis. All the other As contents were the means of four replications. Uptake amount of As was the product of biomass multiplied by As content in plant tissues. Extraction efficiency of As species were calculated as the ratio of total As species uptake amount in shoot or root of vegetable/total As uptake amount in shoot or root of vegetable. Transfer factors (TFs) of As species were calculated as the ratio of As content in shoot/As content in the root of vegetable. ANOVA with LSD was performed using SAS/STAT software (SAS V9 by SAS Institute Inc., Cary, NC, USA). Figures in this paper were made using Origin 8.0 software. 3. Results and discussion 3.1. The metabolism of roxarsone in chicken manure Besides roxarsone, As(III), DMA, MMA, As(V), 4-HPA and two unknown As species were detected in As-CM (Fig. 1B). Total As content in CK-CM and As-CM was 8.85 ± 0.09 mg kg1 and 58.3 ± 2.1 mg kg1 respectively (Table 1), indicating that about 49.4 mg kg1 of As in the As-CM originated from the roxarsone added in chicken feed. The residual roxarsone in the As-CM was 1.58 ± 0.31 mg kg1, implying that more than 96% of roxarsone was degraded and converted to As(III), DMA, MMA, As(V), 4-HPA and other unknown As species. additionally, the total concentration of knowable As species including As(V), As(III), MMA, DMA, 4-HPA and roxarsone in the As-CM was 30.1 mg kg1, meaning that about 48% of As was the forms unknown and/or not be extracted efficiently. The separation of seven chemical As standard compounds and As species in As-CM was shown in Fig. 1. 3.2. The biomass in tissues of vegetables The biomass of both shoots and roots of five vegetables was presented in Table 2. ANOVA statistics analysis indicated that there was significant difference in the biomass of vegetable varieties (p = 0.0057 for vegetable shoots and P < 0.0001 for vegetable roots). As-CM could enhance the shoot biomass and root biomass of vegetables as compared to the CK-CM treatment. There was significant difference (p < 0.0001) in shoot biomass of vegetables between As-CM and CK-CM treatment but no difference in root biomass (p = 0.4999). The reason why As-CM in soil had positive effect on the growth of vegetables was probably due to different characteristic and nutrient components between As-CM and CK-CM showed in Table 1.
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Intensity (mV)
1000
As(III)
800
MMA
600
A
DMA 3-A-HPA 4-HPA
400
Rox
As(V)
200 0 0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30
Intensity (mV)
200
Unknown compound
As(V)
160
B
120 80
As(III) MMA DMA
40 0
0
2
4
6
4-HPA
Rox
8 10 12 14 16 18 20 22 24 26 28 30
Time (min) Fig. 1. Separation of As species. (A) seven As standard compounds, As(III) 160 lg L1, As(V) 160 lg L1, DMA 160 lg L1, MMA 160 lg L1, 3-A-HPA 160 lg L1, 4-HPA 156.8 lg L1, ROX 160 lg L1; B: Roxarsone and its metabolites in chicken manure excreted by chickens fed with feedstuff containing roxarsone.
Table 2 Biomass in tissues of five vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites (g pot1, dry weight). Vegetable
Treatment
Shoot
Root
Coriander herb
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
5.69 ± 1.66 9.59 ± 0.75 10.24 ± 3.02 13.88 ± 1.25 4.59 ± 1.07 7.43 ± 2.16 7.49 ± 2.80 14.19 ± 5.38 7.70 ± 3.65 11.35 ± 6.42
0.92 ± 0.24 1.09 ± 0.14 2.36 ± 0.32 2.46 ± 0.25 0.33 ± 0.03 0.51 ± 0.12 0.98 ± 0.44 1.26 ± 0.50 2.52 ± 1.34 2.56 ± 1.63
Crowndaisy Flowering cabbage Leaf mustard Spinach
3.3. As species in tissues of vegetables Two As species (As(III) and As(V)) were detected in tissues of five vegetables (Table 3) which indicated that As(V) and As(III) could be taken up by vegetables via root absorption from soils. It was reported DMA could be determined in turnip plants fertilized with chicken manure containing high content of DMA (66.8 ± 4.2 mg kg1) in the previous study (Yao et al., 2009), however, DMA was not detected in any tissues of vegetables in this experiment which indicated DMA was not taken up by roots of the five vegetables or the DMA content in tissues of the five vegetables was under detection limit. Roxarsone, MMA and 4-HPA were all detected in As-CM but also not found in any tissues of five vegetables. This result was in accordance with the viewpoints of several reports that organic forms of As were absorbed less effectively than inorganic forms in some plants (Marin et al., 1992; Raab et al.,
2007; Zhao et al., 2009). Moreover, it had been pointed out that aquaporins is in relation to the absorption of As(III) (Isayenkov and Maathuis, 2008) and phosphate transporters play an important role in the absorption of As(V) (Esteban et al., 2003). Results showed the contents of As(III) and As(V) in shoots and roots of vegetables treated with As-CM were all higher than those treated with CK-CM. Significant difference (p < 0.0001) of As(III) and As(V) content between CK-CM and As-CM treated vegetables were obtained from the ANOVA analysis. Compared to CK-CM treatment, the As-CM increased 12%, 122%, 111%, 95% and 62% of As(III), 32%, 46%, 272%, 101% and 29% of As(V) in roots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach respectively. While the enhanced proportion of As(III) in edible shoots of the five vegetables treated with As-CM were 26%, 29%, 16%, 44% and 20% respectively. It was confirmed that the uptake and accumulation of As species such as As(III) and As(V) in vegetables was increased by amended As-CM in farmland and the risk of As exposure by consuming vegetables applied with As-CM was significantly enhanced.
3.4. The uptake amount of As species in vegetables Uptake amount of As species (Table 4) was used to estimate the effect of As-CM on the uptake of As species by vegetables because of the different biomass due to the different species of vegetables and different chicken manure amended treatments. The uptake amount of As species in shoots and roots of five vegetables treated with As-CM were all higher than those treated with CK-CM which was in agreement with the results of As species content in Table 3. There was significant difference of As species uptake amount (p < 0.0001 for shoot As(III), p < 0.0001 for root As(III) and p = 0.0218 for root As(V)) between CK-CM and As-CM amended treatments. Compared to CK-CM treatment, As-CM increased 33%, 114%, 175%, 144% and 61% of As(III) uptake amount; 59%, 39%, seven times, 143% and 28% of As(V) uptake amount in roots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach, respectively. While the enhanced proportion of As(III) uptake amount in edible shoots of the five vegetables treated with As-CM were 114%, 74%, 91%, 175% and 68% as compared to CK-CM treatment respectively. Consequently, nearly two folds or more than two folds of As(III) will be taken into human body when the same amount of As-CM amended vegetables is consumed compared to CK-CM amended vegetables. The uptake amount of total As in shoots and roots of vegetables was also presented in Table 4. It was showed that the average extraction efficiency of As species in shoots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach were 82%, 78%, 83%, 84% and 91%, in roots were 102%, 57%, 97%, 68% and 59% respectively. This result indicating that most of the As species in vegetable shoots were extracted, but there were still some unknown complex As species combined by other components in
Table 3 Concentrations of As species in five vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites (lg pot1, dry weight). Vegetable
Treatment
Shoot As(III)
Root As(III)
Root As(V)
Coriander Herb
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
87.10 ± 5.20 109.39 ± 3.04 169.84 ± 8.91 219.15 ± 10.24 97.40 ± 6.87 112.59 ± 6.87 64.98 ± 4.54 93.80 ± 2.76 72.55 ± 7.06 86.95 ± 8.50
3882.58 ± 255.73 4343.28 ± 194.04 678.19 ± 19.44 1504.06 ± 88.13 616.89 1302.96 ± 18.34 638.25 ± 11.40 1247.01 ± 151.24 624.99 ± 65.43 1013.07 ± 21.04
245.62 ± 17.26 324.12 ± 31.03 280.80 ± 31.42 410.63 ± 63.10 47.34 176.31 ± 13.14 140.45 ± 18.78 282.81 ± 27.43 222.00 ± 27.50 285.52 ± 35.90
Crowndaisy Flowering cabbage Leaf mustard Spinach
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L. Huang et al. / Chemosphere 100 (2014) 57–62 Table 4 Uptake amount of As species and total As in tissues of vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites (lg pot1). Vegetable
Treatme-nt
Shoot As(III)
Root As(III)
Root As(V)
Total As(III)
Root total As species
Shoot total As
Root total As
Coriander -Herb Crownda -isy Flowering -cabbage Leaf -mustard Spinach
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
0.49 ± 0.13 1.05 ± 0.08 1.75 ± 0.56 3.04 ± 0.25 0.44 ± 0.06 0.84 ± 0.27 0.48 ± 0.17 1.32 ± 0.48 0.57 ± 0.28 0.96 ± 0.46
3.55 ± 1.98 4.73 ± 0.78 1.74 ± 0.37 3.72 ± 0.59 0.20 0.55 ± 0.18 0.63 ± 0.28 1.54 ± 0.68 1.60 ± 0.89 2.57 ± 1.28
0.22 ± 0.05 0.35 ± 0.02 0.72 ± 0.18 1.00 ± 0.08 0.01 0.08 ± 0.03 0.14 ± 0.07 0.34 ± 0.12 0.58 ± 0.34 0.74 ± 0.52
4.04 ± 1.06 5.78 ± 0.85 3.49 ± 0.91 6.76 ± 0.73 0.64 1.40 ± 0.34 1.11 ± 0.45 2.86 ± 1.10 2.17 ± 1.17 3.53 ± 2.02
3.77 ± 1.00 5.08 ± 0.79 2.46 ± 0.54 4.72 ± 0.57 0.21 0.63 ± 0.21 0.77 ± 0.35 1.88 ± 0.80 2.18 ± 1.22 3.31 ± 2.08
0.66 ± 0.21 1.14 ± 0.29 2.32 ± 1.08 3.76 ± 0.26 0.52 ± 0.18 1.03 ± 0.36 0.68 ± 0.24 1.36 ± 0.42 0.62 ± 0.33 1.07 ± 0.68
3.77 ± 0.96 4.90 ± 0.65 4.57 ± 0.96 7.37 ± 0.78 0.19 0.76 ± 0.19 1.19 ± 0.15 2.66 ± 1.28 3.67 ± 1.88 5.60 ± 3.27
roots of leaf mustard, crowndaisy and spinach not totally extracted or detected. 3.5. Transport and distribution of As species in vegetables As(V) and As(III) were detected in roots but only As(III) was found in shoots of all vegetables which is coincided with previous research (Duan et al., 2005) that As transported in some plants is primarily in the form of As(III). As TFs value in Table 5 were used to evaluate the upward transport capacity of As(III) in five vegetables. Results showed that the TFs of As(III) in crowndaisy, flowering cabbage, leaf mustard and spinach treated with As-CM were all lower than those treated with CK-CM but in coriander herb there was no distinction between the two treatments. Significant difference (P < 0.0001) of As(III) TFs was obtained from vegetables between CK-CM and As-CM amended treatments, and significant difference (P < 0.0001) of As(III) TFs also existed among different vegetables. So As transport from root to frond in vegetables was depressed by roxarsone and its metabolites in chicken manure and the translocation of As was also affected by the internal characteristic of vegetable species. The transport of As compounds in tissues of vegetables resulted in various distribution of As species. The distribution of As species in Table 5 showed that As(III) was the major As species which taken up 79–98% in five vegetables. There was significant difference (p < 0.0001) of As(III) distribution among the same tissues of five vegetables. However, the allocation of As(III) in vegetables treated with two chicken manures only had slight difference. 3.6. As species in soils Before sowing, the major As forms in soils amended with two chicken manures were As(V) and As(III), As-CM addition increased the concentrations of both inorganic As compounds and introduced DMA into the soil compared to the CK-CM application as presented in Fig. 2. As(V) was the dominant As compound in soils amended with two chicken manures but the majority of As in five
vegetable tissues was As(III) as presented in Table 5. It was possible that As(V) in soil was first absorbed by roots of vegetables then converted into As(III) in vegetable tissues. It had been described that once inside the cell, As(V) is reduced to As(III) which is catalyzed by arsenate reductase and consumes reduced glutathione (Verbruggen et al., 2009). After growing vegetables, the content of As(V) and As(III) in CK-CM amended soil decreased. The content of As(V) in following soils ranked the order: soil growing flowering cabbage > soil growing leaf mustard > soil growing coriander herb > soil growing spinach > soil growing crowndaisy and the content of As(III) in soils ranked the following order: soil growing flowering cabbage > soil growing leaf mustard > soil growing spinach > soil growing crowndaisy > soil growing coriander herb. In addition, the uptake amount of As(V) in flowering cabbage < leaf mustard < coriander herb < spinach < crowndaisy and total As(III) uptake amount in flowering cabbage < leaf mustard < spinach < crowndaisy < coriander herb was presented in Table 4. The results showed that the decrease of As species in CK-CM amended soil grown vegetables was corresponding to the uptake of As species by vegetable plants. The changed pattern of As species remained in As-CM amended soil after vegetable harvested and accumulated in vegetable plants was the same as that treated with CK-CM. It should be noted that in As-CM amended soils there were still 20–49% of As(III) and 12–18% of As(V) more than CK-CM amended treatment after vegetable harvested. If As-CM was successively used in the next crop in the same soil, higher content of As could be remained in the soil and the extra inorganic As would continuously affect the uptake of As species of the successive second or third crop planted in the soil. Hence, the potential risk of As contamination by fertilizing of chicken manure bearing roxarsone and its metabolites should be of great concern. 4. Conclusion After excreted, more than 96% of roxarsone in animal manure is degraded into higher toxic metabolites such as As(V), As(III), MMA,
Table 5 As transfer and distribution of As species in five vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites. Vegetable
Coriander Herb Crowndaisy Flowering cabbage Leaf mustard Crowndaisy Spinach
Treatment
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
TFs
0.02 ± 0.00 0.02 ± 0.00 0.25 ± 0.01 0.15 ± 0.01 0.16 0.09 ± 0.00 0.10 ± 0.01 0.08 ± 0.01 0.25 ± 0.01 0.15 ± 0.01 0.12 ± 0.02 0.09 ± 0.01
Percentage of As species (%) Shoot As(III)
Root As(III)
Root As(V)
11.5 ± 1.3 17.2 ± 1.3 40.9 ± 4.9 39.2 ± 2.4 67.0 ± 3.4 56.3 ± 6.4 40.0 ± 4.4 41.7 ± 5.0 40.9 ± 4.9 39.2 ± 2.4 22.4 ± 4.4 23.5 ± 3.7
83.2 ± 1.6 77.0 ± 1.9 41.8 ± 2.7 47.8 ± 4.0 30.6 ± 2.9 38.5 ± 5.7 49.2 ± 3.5 47.7 ± 5.1 41.8 ± 2.7 47.8 ± 4.0 57.2 ± 2.3 59.7 ± 3.7
5.3 ± 0.4 5.8 ± 0.6 17.3 ± 2.5 13.0 ± 1.5 2.3 ± 0.2 5.2 ± 0.7 10.8 ± 1.5 10.6 ± 0.3 17.3 ± 2.5 13.0 ± 1.5 20.4 ± 2.4 16.8 ± 1.7
L. Huang et al. / Chemosphere 100 (2014) 57–62
2200
As(III) As(V) DMA
1650
CK-CM
As-CM
1100
550
0
Be
fo r Co e so w ria in nd g er H Fl Cr e ow ow rb n er in dai g ca sy Le bb af ag m e us t Sp ard in Be ac fo h Co re ria sow nd in er g Fl Cr He ow ow rb er in nda i g c sy Le abb af ag m e us t Sp ard in ac h
As speciesin soils before vegetable sowing and -1 after vegetable harvested ( µg.kg )
62
Fig. 2. Concentrations of As species in soils of two treatments before vegetable sowing and after vegetable harvested.
DMA, 4-HPA and other unknown As species. As(III) and As(V) were detected in vegetables and the most toxic As(III) was the major As species accounted for 79–98% in five vegetables. Application of chicken manure bearing roxarsone and its metabolites in farmland increased the biomass and As content of five vegetables. Compared to the control treatments, the chicken manure bearing roxarsone and its metabolites increased 33%, 114%, 175%, 144% and 61% of As(III) uptake amount, 59%, 39%, seven times, 143% and 28% of As(V) uptake amount in roots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach, respectively. While the enhanced proportion of As(III) uptake amount in edible shoots of the five vegetables treated with As-CM were 114%, 74%, 91%, 175% and 68% as compared to CK-CM treatment respectively. Consequently, the uptake and accumulation of As species such as As(III) and As(V) in vegetables was increased by amended As-CM in cultivated soil and nearly two folds or more than two folds of As(III) would be taken into human body when the same amount of As-CM amended vegetables is consumed compared to CK-CM amended vegetables. The results reflected that the toxic element As could come into human body via the way: roxarsone in feed ? animal ? animal manure ? soil ? crop and the edible security of vegetables applied with animal manure containing roxarsone and its metabolites need to be pay great attention. Moreover, the residual As would be increased when animal manure containing roxarsone and its metabolites was sequentially applied in the farmland and would continuously affect the uptake of As species of the successive second or third crop planted. Acknowledgment This work was supported by National Natural Science Foundation of China (Nos. 41071316 and 40871226) and Natural Science Foundation of Guangdong province of China (No. 10151064001000010). References 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, 237–245.
Cang, L., Wang, Y.J., Zhou, D.M., Dong, Y.H., 2004. Heavy metals pollution in poultry and livestock feeds and manures under intensive farming in Jiangsu Province, China. J. Environ. Sci. (China) 16, 371–374. Chapman, H.D., Johnson, Z.B., 2002. Use of antibiotics and roxarsone in broiler chickens in the USA: analysis for the years 1995–2000. Poultry. Sci. 81, 356– 364. Cullen, W.R., Reimer, K.J., 1989. Arsenic speciation in the environment. Chem. Rev. 89, 713–764. De La Fuente, C., Clemente, R., Alburquerque, J.A., Vélez, D., Bernal, M.P., 2010. Implications of the use of As-rich groundwater for agricultural purposes and the effects of soil amendments on As solubility. Environ. Sci. Technol. 44, 9463– 9469. Duan, G.L., Zhu, Y.G., Tong, Y.P., Cai, C., Kneer, R., 2005. Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator. Plant Physiol. 138, 461–469. Esteban, E., Carpena, R.O., Meharg, A.A., 2003. High-affinity phosphate/arsenate transport in white lupin (Lupinus albus) is relatively insensitive to phosphate status. New Phytol. 158, 165–173. 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, 1509–1514. Huang, R.Q., Gao, S.F., Wang, W.L., Staunton, S., Wang, G., 2006. Soil arsenic availability and the transfer of soil arsenic to crops in suburban areas in Fujian Province, southeast China. Sci. Total Environ. 368, 531–541. Huang, L.X., He, Z.H., Zeng, F., Yao, L.X., Zhou, C.M., Guo, B., 2010. Simultaneous analysis of roxarsone and its metabolites by liquid chromatography-hydrodide generation-atomic fluorescence spectrometry. Chin. J. Anal. Chem. 38, 1321– 1324. Huang, L.X., Yao, L.X., He, Z.H., Zhou, C.M., Li, G.L., Yang, B.M., Li, Y.F., 2013. Uptake of arsenic species by turnip (Brassica rapa L.) and lettuce (Lactuca sativa L.) treated with roxarsone and its metabolites in chicken manure. Food. Addit. Contam. A 30, 1546–1555. Isayenkov, S.V., Maathuis, F.J.M., 2008. The Arabidopsis thaliana aquaglyceroporin AtNIP7; 1 is a pathway for arsenite uptake. FEBS Lett. 582, 1625–1628. Jackson, B.P., Bertsch, P.M., 2001. Determination of arsenic speciation in poultry wastes by IC-ICP-MS. Environ. Sci. Technol. 35, 4868–4873. Jackson, B.P., Miller, W.P., 2000. Effectiveness of phosphate and hydroxide for desorption of arsenic and selenium species from iron oxides. Soil Sci. Soc. Am. J. 64, 1616–1622. Jackson, B.P., Bertsch, P.M., Cabrera, M.L., Camberato, J.J., Seaman, J.C., Wood, C.W., 2003. Trace element speciation in poultry litter. J. Environ. Qual. 32, 535–540. Makris, K.C., Quazi, S., Punamiya, P., Sarkar, D., Datta, R., 2008. Fate of arsenic in swine waste from concentrated animal feeding operations. J. Environ. 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Ruiz-Chancho, M.J., López-Sánchez, J.F., Schmeisser, E., Goessler, W., Francesconi, K.A., Rubio, R., 2008. Arsenic speciation in plants growing in arseniccontaminated sites. Chemosphere 71, 1522–1530. Vahter, M., Marafante, E., 1988. In vivo methylation and detoxification of arsenics. In: Craig, P.J., Glockling, F. (Eds.), The Biological Alkylation of Heavy Elements. Royal Society of Chemistry Special Publications. Special Publications, London, pp. 105–119. Verbruggen, N., Hermans, C., Schat, H., 2009. Mechanisms to cope with arsenic or cadmium excess in plants. Curr. Opin. Plant Biol. 12, 364–372. Wang, F., Chen, Z., Zhang, L., Gao, Y., Sun, Y., 2006. Arsenic uptake and accumulation in rice (Oryza sativa L.) at different growth stages following soil incorporation of roxarsone and arsanilic acid. Plant Soil 285, 359–367. Yao, L.X., Li, G.L., Dang, Z., 2006. Major chemical components of poultry and livestock manures under intensive breeding. Chin. J. Appl. Ecol. 17, 1989–1992. 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Roxarsone and its metabolites in chicken manure significantly enhance the uptake of As species by vegetables Lianxi Huang a,b,c, Lixian Yao d,⇑, Zhaohuan He a,b,c, Changmin Zhou a,b,c, Guoliang Li a,b,c, Baomei Yang a,b,c, Xiancai Deng d a
Institute of Agricultural Resources and Environment, Guangdong Academy of Agricultural Sciences, China Key Laboratory of Plant Nutrition and Fertilizer in South Region, Ministry of Agriculture, China Guangdong Key Laboratory of Nutrient Cycling and Farmland Conservation, China d South China Agricultural University, Guangzhou 510640, China b c
h i g h l i g h t s Roxarsone was degraded in animal manure and converted to higher toxic As forms. Roxarsone and it metabolites in animal manure increased As uptake of vegetables. As(III) was the predominant As species in vegetables. As can enter food chain via the way: roxarsone ? animal ? manure ? soil ? crop. Roxarsone and it metabolites in organic fertilizer lead to As exposure for human.
a r t i c l e
i n f o
Article history: Received 9 June 2013 Received in revised form 16 December 2013 Accepted 31 December 2013 Available online 22 January 2014 Keywords: Roxarsone As species Chicken manure Vegetable
a b s t r a c t Roxarsone is an organoarsenic feed additive which can be finally degraded to other higher toxic metabolites after excreted by animal. In this work, the uptake of As species by vegetables treated with chicken manure bearing roxarsone and its metabolites was investigated. It was showed that more than 96% of roxarsone added in chicken feed was degraded and converted to arsenite, monomethylarsonic acid, dimethylarsinic acid, arsenate, 4-hydroxyphenylarsonic acid and other unknown As species. Arsenite and arsenate could be found in roots of vegetables but only arsenite transported up to shoots. Chicken manure bearing roxarsone and its metabolites increased 33–175% of arsenite and 28%seven times of arsenate in vegetable roots, 68–175% of arsenite in edible vegetable shoots. Arsenite, the most toxic As form, was the major extractable As species in vegetables accounted for 79–98%. The results reflected that toxic element As could be absorbed by vegetables via the way: roxarsone in feed ? animal ? animal manure ? soil ? crop and the uptake of As species would be enhanced by using chicken manure bearing roxarsone and its metabolites as organic fertilizer. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction As is one of the most serious global environmental toxicants which can be taken up by crops from soil via root absorption, and eventually transferred into human beings via the food chain. As in environment, plants and organisms usually presents in various chemical forms such as arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), 4-hydroxyphenylarsonic acid (4-HPA), 3-amino-4-hydroxyphenylarsonic acid (3-A-HPA), trimethylarsine oxide (TMAO), tetramethylarsonium cation (TETRA), arsenobetaine (AsB), arsenocholine (AsC) and arsenosugars. Generally, the toxicity and mobility of As ⇑ Corresponding author. Tel./fax: +86 20 38604958. E-mail address: [email protected] (L. Yao). 0045-6535/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.12.074
species in organisms is considered to be: As(III) > As(V) > organoarsenic (Vahter et al., 1988; Cullen and Reimer, 1989). Roxarsone (3-nitro-4-hydroxarsoneyphenylarsonic acid) is an organoarsenic feed additive to promote growth, control intestinal parasites and improve feed efficiency in animal production (Moore et al., 1998; Chapman and Johnson, 2002). The use of roxarsone as a feed additive had been banned by the European Union in 1999 and the sales of roxarsone also had been suspended by the United States in 2011, but roxarsone was still a widely used feed additive in China (Yao et al., 2013). The toxicity of roxarsone is low but it can be finally excreted by animal and degraded into higher toxic metabolites soon after the animal manure enters environment or when the animal manure is composted (Bednar et al., 2003; Garbarino et al., 2003). The metabolites of roxarsone in animal manure primarily occur as As(III), DMA, MMA, As(V), 3-A-HPA, As(V),
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L. Huang et al. / Chemosphere 100 (2014) 57–62
4-HPA and other unknown As species (Jackson and Bertsch, 2001; Rosal et al., 2005; Makris et al., 2008). It was reported that As content in animal manure is from undetectable level to 315.1 mg kg1 (Cang et al., 2004; Yao et al., 2006), and 70–75% of the As in poultry manure is water soluble (Jackson and Bertsch, 2001; Jackson et al., 2003). Moreover, animal manure is commonly applied as organic fertilizer. Hence, the bioavailability of roxarsone and its metabolites in animal manure might be high and should be of great concern. It was reported that As concentration in plant growing in contaminated sites ranged from 1.14 mg kg1 to 98.5 mg kg1 (dry mass), but in a non-contaminated area with similar geological characteristics only 0.06–0.58 mg kg1 (Ruiz-Chancho et al., 2008). The research (Yao et al., 2009) showed that As(V), As(III) and DMA were found in turnip tissues applied with chicken manure containing roxarsone and its metabolites, and the content of As species increased with the increasing used rate of chicken manure. It was also demonstrated that As concentrations in different parts of rice plants increased with the elevating concentrations of roxarsone and arsanilic acid added in soils and varied with the growth stage (Wang et al., 2006). Moreover, the capability of As uptake and accumulation by different varieties of crop was different. The sequence of As concentration in the edible part of various crops was as follow: celery > mustard > spinach > lettuce > taro > bokchoi > cowpea > caulifl ower > eggplant (Huang et al., 2006). The following pattern of As concentration in several crops: red cabbage curly endive > barley wheat sugar beet leek > cabbage green pepper was also reported (De La Fuente et al., 2010). In this work, pot experiment was carried out to investigate the uptake of As species in vegetables treated with chicken manure bearing roxarsone and its metabolites. The objective was to reveal the pathway of As species entering human food chain from organoarsenic additives in animal feed. The potential risk of using roxarsone as feed additive and using chicken manure bearing roxarsone and its metabolites as fertilizer were also evaluated. 2. Materials and methods 2.1. Soil, chicken manure and vegetables Soil used in this study was a typical lateritic red soil collected at a depth of 0–25 cm from the Crop Experiment Station of the Guangdong Academy of Agricultural Sciences (23°80 4300 N, 113°200 5000 E) located in Guangzhou, South China. Chicken manure was taken from an intensive chicken farm located in Huizhou city, Guangdong province in South China. The first chicken manure (CKCM) was excreted by chickens fed with non-roxarsone feedstuff and the second chicken manure (As-CM) was excreted by the same species of chickens fed with feedstuff containing 55 mg kg1 roxarsone. The basic properties such as total content of As, N, P, K, organic matter and pH value of soil and chicken manures used in this experiment were listed in Table 1. Seeds of coriander herb (Coriandrum sativum L.), crowndaisy (Chrysanthemum coronarium), flowering cabbage (Brassica campestris L.), leaf mustard (Brassica juncea) and spinach (Spinacia oleracea) were purchased from the seed market of Guangdong Academy of Agricultural Sciences. 2.2. Pot experiment The pot experiment was carried out with two chicken manures (CK-CM and As-CM) and four replications of each vegetable. The amount of chicken manure amended in soil was 2% (w/w, manure/soil) and CK-CM was considered as the control. After completely mixed with 2% chicken manure, the soil was kept at 70% field water holding capacity for 10 d by adding deionized water. In order to obtain sufficient biomass, five seedlings of crowndaisy,
flowering cabbage, leaf mustard, spinach and six seedlings of coriander herb were kept in corresponding pot. Approximately 100 mL of deionized water was dispersedly sprinkled on the topsoil of every pot when the soil was lack of sufficient water. All the vegetables were grown at natural temperature and photoperiod of the green house, no additional thermal source and light were supplied. 2.3. Sample collection and preparation Before sowing, one soil sample was collected in each pot. According to the individual characteristic of vegetables, the samples of coriander herb, crowndaisy, flowering cabbage, leaf mustard, and spinach was harvested after 48, 54, 33, 50 and 68 days’ growing respectively. Shoots and roots of vegetable plants were harvested separately with stainless knife, washed with tap water and then rinsed with deionized water, then immediately lyophilized (Alpha 1–4/LD-plus, Christ German). Fresh weight and dry weight of the vegetable tissues were recorded. Rhizosphere soil samples were synchronously collected and then lyophilized. The plant samples were quickly pulverized to fine powder using retsch grinder (ZM200, Germany), and the soil samples were grinded using a agate mortar then passed a 150 lm sieve for total As and As species analysis. Plant and soil samples were kept in 80 °C refrigerator to assure the stabilization of As species before extraction. 2.4. Chemical reagents Roxarsone with a purity of 97.5% was purchased from Dr. Ehrenstorfer Gmbh in Germany, 3-A-HPA (99%) from Sigma–Aldrich (USA) and 4-HPA (98%) from TCI Tokyo Kasei (Japan). The standard stock solutions of As(V) (17.5 ± 0.4 mg L1), As(III) (75.7 ± 1.2 mg L1), MMA (25.1 ± 0.8 mg L1), and DMA (52.9 ± 1.8 mg L1) presented as the concentration of As were provided by the Chinese National Standard Materials Center and stored in the dark at 4 °C. The HPLC grade methanol was used and all other reagents were analytical grade. The water used in chemical analysis was prepared by Millipore Milli-Q Academic. 2.5. Chemical analysis The methods of total As extraction and detection were NY/ T1121.11-2006 (chicken manure and soil samples) and GB/ T5009.11-2003 (vegetable samples) issued by China. Firstly, chicken manure and soil samples were digested with 50% aqua regia, then the extracted total As was pre-deoxidized to As(III) using a mixture of 5.0% H2NCSNH2 and 5.0% C8H8O6. Vegetable samples were digested with a mixture of concentrated HNO3, H2SO4 and HClO4, then the extracted total As was pre-deoxidized to As(III) using 0.5% H2NCSNH2. The As(III) in chicken manure, soil and vegetables was finally deoxidized to AsH3 by 2.0% KBH4 and 5% HCl and determined by hydride generation-atomic fluorescence spectrometry (AFS8130, Jitian Beijing, China). The running speed of carrier gas (argon) was 300 mL min1 and shield gas 800 mL min1. Photomultiplier tube voltage was 270 V and the hollow cathode lamp current 60 mA (primary)/30 mA (boosted). Two reference materials GBW07408 (soil) and GBW07602 (bush plant), purchased from the Chinese National Reference Materials Center, were used to control the analytical quality of total As in soil, chicken manure and plant samples with detected error 3.2 ± 1.2%. The extraction and detection of As species are the same with the methods mentioned in our previous studies (Huang et al., 2010, 2013; Yao et al., 2013) and presented as follow. As species in chicken manure and soil samples were extracted with a mixture of 0.1 mol L1 H3PO4 and 0.1 mol L1 NaH2PO42H2O (1:9, V/V) (Jackson and Miller, 2000), and those in vegetables by ultrapure water (Tomohiro Narukawa et al., 2008). Approximately 0.25 g of chicken
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L. Huang et al. / Chemosphere 100 (2014) 57–62 Table 1 Basic properties of soil and chicken manures used in the experiment. Item c
pH Organic matter (g kg1) Total N (g kg1) Total P (g kg1) Total K (g kg1) Total As (mg kg1) As(III) (mg kg1) Dimethylarsinic acid (As, mg kg1) Monomethylarsonic acid (As, mg kg1) 3-amino-4-hydroxyphenylarsonic acid(As, mg kg1) As(V) (mg kg1) 4-hydroxyphenylarsonic acid (As, mg kg1) Unknown As species Roxarsone (As, mg kg1) a b c d e
Soil
CK-CMa
As-CMb
6.88 ± 0.11 20.6 ± 0.00 1.27 ± 0.01 1.12 ± 0.01 13.1 ± 0.11 7.35 ± 0.13 0.12 ± 0.00 NDd ND ND 1.53 ± 0.01 ND ND ND
5.99 ± 0.02 426 ± 0.00 22.6 ± 1.44 20.5 ± 0.46 32.9 ± 0.14 8.85 ± 0.09 0.33 ± 0.04 1.07 ± 0.04 ND ND 1.27 ± 0.11 ND ND ND
5.82 ± 0.06 512 ± 0.00 33.2 ± 0.00 17.7 ± 0.23 38.8 ± 0.14 58.3 ± 2.10 2.96 ± 0.05 3.00 ± 0.15 1.73 ± 0.23 ND 20.5 ± 0.38 0.36 ± 0.14 Detectablee 1.58 ± 0.31
Represents chicken manure without roxarsone and its metabolites. Represents chicken manure containing roxarsone and its metabolites. Measured in a 1:2.5 soil or chicken manure to water (w/v) suspension. Not detectable. Detectable but could not be quantitatively calculated owing to absence of internal standard materials.
manure, 0.50 g of vegetable or 1.0 g of soil samples were placed into Teflon vessels with 10 mL extractant, and kept in water bath at 55 °C for 10 h, then sonicated for 10 min. The supernatant was collected after centrifugation at 4000 rpm for 5 min. This extraction procedure was repeated three times. The three supernatants were mixed and passed through a solid-phase extraction cartridge of C18 to remove pigments or hydrophobic organic compounds. The filtrate was lyophilized and dissolved using 3 mL of Milli-Q water then filtered through a 0.22 lm Millipore membrane before Liquid Chromatography (20AT, SHIMADZU, Japan)-Hydrogen GenerationAtomic fluorescence Spectrometry analysis. Reversed C18 chromatographic column (250 mm 4.6 mm i.d.,5 lm, Phenomenex, USA) filled with ODS3 was used to separate the As species. Mobile phase A and mobile phase B for As species separation were 10 mmol L1 NaH2PO4 and 50 mmol L1 NaH2PO4 both containing 0.5 mmol L1 TBAB and CH3OH (3% for mobile phase A and 5% for mobile phase B). The pH value of mobile phase A and B was accommodated to 6.22 using 50% ammonia. Gradient elution procedure was 100% A (0–11 min), 100% B (11–20 min) and 100% A (20– 30 min). Flow rate was 1.0 mL min1 and injection volume 100 lL. After complete separation, As species were firstly mixed with 20 g L1 K2S2O8 (some complex As forms such as 3-A-HPA, 4-HPA and roxarsone were broken down at this step), then reduced to As(III) by 20 g L1 KBH4 and 10% (V/V) HCl, and finally detected by AFS. The running speed of carrier gas (argon) was 400 mL min1 and shield gas 600 mL min1. Photomultiplier tube voltage was 295 V and the hollow cathode lamp current 90 mA (primary)/ 40 mA (boosted). Detection limits of As(III), MMA, DMA, 3-AHPA, As(V), 4-HPA and roxarsone were 1.8, 1.9, 3.6, 12.1, 4.7, 3.8 and 9.5 lg L1, respectively. Recoveries of standard addition of As(III), MMA, DMA, 3-A-HPA, As(V), 4-HPA and roxarsone were 82.4 ± 1.3%, 91.8 ± 2.1%, 90.6 ± 2.1%, 45.8 ± 1.9%, 98.8 ± 2.0%, 103.3 ± 6.0% and 93.0 ± 2.3% in chicken manures, 87.8 ± 2.9%, 103.2 ± 3.3%, 105.6 ± 3.9%, 60.3 ± 2.7%, 93.1 ± 3.1%, 94.6 ± 2.9% and 104.1 ± 3.7% in soils, 95.2 ± 2.4%, 86.5 ± 2.2%, 86.4 ± 1.7%, 58.6 ± 5.4%, 99.1 ± 2.9%, 90.3 ± 2.7% and 85.3 ± 1.3% in shoots of vegetables, 96.0 ± 2.1%, 83.3 ± 3.2%, 81.4 ± 2.4%, 60.1 ± 6.9%, 98.4 ± 2.8%, 96.5 ± 3.8% and 87.4 ± 2.3% in roots of vegetables, respectively. Unless specially pointed out, the contents of As species in all samples were presented as elemental As. 2.6. Data and statistics Owing to the low biomass, the four replications of flowering cabbage roots treated with CK-CM were combined into one sample
to obtain sufficient amount for analysis. All the other As contents were the means of four replications. Uptake amount of As was the product of biomass multiplied by As content in plant tissues. Extraction efficiency of As species were calculated as the ratio of total As species uptake amount in shoot or root of vegetable/total As uptake amount in shoot or root of vegetable. Transfer factors (TFs) of As species were calculated as the ratio of As content in shoot/As content in the root of vegetable. ANOVA with LSD was performed using SAS/STAT software (SAS V9 by SAS Institute Inc., Cary, NC, USA). Figures in this paper were made using Origin 8.0 software. 3. Results and discussion 3.1. The metabolism of roxarsone in chicken manure Besides roxarsone, As(III), DMA, MMA, As(V), 4-HPA and two unknown As species were detected in As-CM (Fig. 1B). Total As content in CK-CM and As-CM was 8.85 ± 0.09 mg kg1 and 58.3 ± 2.1 mg kg1 respectively (Table 1), indicating that about 49.4 mg kg1 of As in the As-CM originated from the roxarsone added in chicken feed. The residual roxarsone in the As-CM was 1.58 ± 0.31 mg kg1, implying that more than 96% of roxarsone was degraded and converted to As(III), DMA, MMA, As(V), 4-HPA and other unknown As species. additionally, the total concentration of knowable As species including As(V), As(III), MMA, DMA, 4-HPA and roxarsone in the As-CM was 30.1 mg kg1, meaning that about 48% of As was the forms unknown and/or not be extracted efficiently. The separation of seven chemical As standard compounds and As species in As-CM was shown in Fig. 1. 3.2. The biomass in tissues of vegetables The biomass of both shoots and roots of five vegetables was presented in Table 2. ANOVA statistics analysis indicated that there was significant difference in the biomass of vegetable varieties (p = 0.0057 for vegetable shoots and P < 0.0001 for vegetable roots). As-CM could enhance the shoot biomass and root biomass of vegetables as compared to the CK-CM treatment. There was significant difference (p < 0.0001) in shoot biomass of vegetables between As-CM and CK-CM treatment but no difference in root biomass (p = 0.4999). The reason why As-CM in soil had positive effect on the growth of vegetables was probably due to different characteristic and nutrient components between As-CM and CK-CM showed in Table 1.
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L. Huang et al. / Chemosphere 100 (2014) 57–62
Intensity (mV)
1000
As(III)
800
MMA
600
A
DMA 3-A-HPA 4-HPA
400
Rox
As(V)
200 0 0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30
Intensity (mV)
200
Unknown compound
As(V)
160
B
120 80
As(III) MMA DMA
40 0
0
2
4
6
4-HPA
Rox
8 10 12 14 16 18 20 22 24 26 28 30
Time (min) Fig. 1. Separation of As species. (A) seven As standard compounds, As(III) 160 lg L1, As(V) 160 lg L1, DMA 160 lg L1, MMA 160 lg L1, 3-A-HPA 160 lg L1, 4-HPA 156.8 lg L1, ROX 160 lg L1; B: Roxarsone and its metabolites in chicken manure excreted by chickens fed with feedstuff containing roxarsone.
Table 2 Biomass in tissues of five vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites (g pot1, dry weight). Vegetable
Treatment
Shoot
Root
Coriander herb
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
5.69 ± 1.66 9.59 ± 0.75 10.24 ± 3.02 13.88 ± 1.25 4.59 ± 1.07 7.43 ± 2.16 7.49 ± 2.80 14.19 ± 5.38 7.70 ± 3.65 11.35 ± 6.42
0.92 ± 0.24 1.09 ± 0.14 2.36 ± 0.32 2.46 ± 0.25 0.33 ± 0.03 0.51 ± 0.12 0.98 ± 0.44 1.26 ± 0.50 2.52 ± 1.34 2.56 ± 1.63
Crowndaisy Flowering cabbage Leaf mustard Spinach
3.3. As species in tissues of vegetables Two As species (As(III) and As(V)) were detected in tissues of five vegetables (Table 3) which indicated that As(V) and As(III) could be taken up by vegetables via root absorption from soils. It was reported DMA could be determined in turnip plants fertilized with chicken manure containing high content of DMA (66.8 ± 4.2 mg kg1) in the previous study (Yao et al., 2009), however, DMA was not detected in any tissues of vegetables in this experiment which indicated DMA was not taken up by roots of the five vegetables or the DMA content in tissues of the five vegetables was under detection limit. Roxarsone, MMA and 4-HPA were all detected in As-CM but also not found in any tissues of five vegetables. This result was in accordance with the viewpoints of several reports that organic forms of As were absorbed less effectively than inorganic forms in some plants (Marin et al., 1992; Raab et al.,
2007; Zhao et al., 2009). Moreover, it had been pointed out that aquaporins is in relation to the absorption of As(III) (Isayenkov and Maathuis, 2008) and phosphate transporters play an important role in the absorption of As(V) (Esteban et al., 2003). Results showed the contents of As(III) and As(V) in shoots and roots of vegetables treated with As-CM were all higher than those treated with CK-CM. Significant difference (p < 0.0001) of As(III) and As(V) content between CK-CM and As-CM treated vegetables were obtained from the ANOVA analysis. Compared to CK-CM treatment, the As-CM increased 12%, 122%, 111%, 95% and 62% of As(III), 32%, 46%, 272%, 101% and 29% of As(V) in roots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach respectively. While the enhanced proportion of As(III) in edible shoots of the five vegetables treated with As-CM were 26%, 29%, 16%, 44% and 20% respectively. It was confirmed that the uptake and accumulation of As species such as As(III) and As(V) in vegetables was increased by amended As-CM in farmland and the risk of As exposure by consuming vegetables applied with As-CM was significantly enhanced.
3.4. The uptake amount of As species in vegetables Uptake amount of As species (Table 4) was used to estimate the effect of As-CM on the uptake of As species by vegetables because of the different biomass due to the different species of vegetables and different chicken manure amended treatments. The uptake amount of As species in shoots and roots of five vegetables treated with As-CM were all higher than those treated with CK-CM which was in agreement with the results of As species content in Table 3. There was significant difference of As species uptake amount (p < 0.0001 for shoot As(III), p < 0.0001 for root As(III) and p = 0.0218 for root As(V)) between CK-CM and As-CM amended treatments. Compared to CK-CM treatment, As-CM increased 33%, 114%, 175%, 144% and 61% of As(III) uptake amount; 59%, 39%, seven times, 143% and 28% of As(V) uptake amount in roots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach, respectively. While the enhanced proportion of As(III) uptake amount in edible shoots of the five vegetables treated with As-CM were 114%, 74%, 91%, 175% and 68% as compared to CK-CM treatment respectively. Consequently, nearly two folds or more than two folds of As(III) will be taken into human body when the same amount of As-CM amended vegetables is consumed compared to CK-CM amended vegetables. The uptake amount of total As in shoots and roots of vegetables was also presented in Table 4. It was showed that the average extraction efficiency of As species in shoots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach were 82%, 78%, 83%, 84% and 91%, in roots were 102%, 57%, 97%, 68% and 59% respectively. This result indicating that most of the As species in vegetable shoots were extracted, but there were still some unknown complex As species combined by other components in
Table 3 Concentrations of As species in five vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites (lg pot1, dry weight). Vegetable
Treatment
Shoot As(III)
Root As(III)
Root As(V)
Coriander Herb
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
87.10 ± 5.20 109.39 ± 3.04 169.84 ± 8.91 219.15 ± 10.24 97.40 ± 6.87 112.59 ± 6.87 64.98 ± 4.54 93.80 ± 2.76 72.55 ± 7.06 86.95 ± 8.50
3882.58 ± 255.73 4343.28 ± 194.04 678.19 ± 19.44 1504.06 ± 88.13 616.89 1302.96 ± 18.34 638.25 ± 11.40 1247.01 ± 151.24 624.99 ± 65.43 1013.07 ± 21.04
245.62 ± 17.26 324.12 ± 31.03 280.80 ± 31.42 410.63 ± 63.10 47.34 176.31 ± 13.14 140.45 ± 18.78 282.81 ± 27.43 222.00 ± 27.50 285.52 ± 35.90
Crowndaisy Flowering cabbage Leaf mustard Spinach
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L. Huang et al. / Chemosphere 100 (2014) 57–62 Table 4 Uptake amount of As species and total As in tissues of vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites (lg pot1). Vegetable
Treatme-nt
Shoot As(III)
Root As(III)
Root As(V)
Total As(III)
Root total As species
Shoot total As
Root total As
Coriander -Herb Crownda -isy Flowering -cabbage Leaf -mustard Spinach
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
0.49 ± 0.13 1.05 ± 0.08 1.75 ± 0.56 3.04 ± 0.25 0.44 ± 0.06 0.84 ± 0.27 0.48 ± 0.17 1.32 ± 0.48 0.57 ± 0.28 0.96 ± 0.46
3.55 ± 1.98 4.73 ± 0.78 1.74 ± 0.37 3.72 ± 0.59 0.20 0.55 ± 0.18 0.63 ± 0.28 1.54 ± 0.68 1.60 ± 0.89 2.57 ± 1.28
0.22 ± 0.05 0.35 ± 0.02 0.72 ± 0.18 1.00 ± 0.08 0.01 0.08 ± 0.03 0.14 ± 0.07 0.34 ± 0.12 0.58 ± 0.34 0.74 ± 0.52
4.04 ± 1.06 5.78 ± 0.85 3.49 ± 0.91 6.76 ± 0.73 0.64 1.40 ± 0.34 1.11 ± 0.45 2.86 ± 1.10 2.17 ± 1.17 3.53 ± 2.02
3.77 ± 1.00 5.08 ± 0.79 2.46 ± 0.54 4.72 ± 0.57 0.21 0.63 ± 0.21 0.77 ± 0.35 1.88 ± 0.80 2.18 ± 1.22 3.31 ± 2.08
0.66 ± 0.21 1.14 ± 0.29 2.32 ± 1.08 3.76 ± 0.26 0.52 ± 0.18 1.03 ± 0.36 0.68 ± 0.24 1.36 ± 0.42 0.62 ± 0.33 1.07 ± 0.68
3.77 ± 0.96 4.90 ± 0.65 4.57 ± 0.96 7.37 ± 0.78 0.19 0.76 ± 0.19 1.19 ± 0.15 2.66 ± 1.28 3.67 ± 1.88 5.60 ± 3.27
roots of leaf mustard, crowndaisy and spinach not totally extracted or detected. 3.5. Transport and distribution of As species in vegetables As(V) and As(III) were detected in roots but only As(III) was found in shoots of all vegetables which is coincided with previous research (Duan et al., 2005) that As transported in some plants is primarily in the form of As(III). As TFs value in Table 5 were used to evaluate the upward transport capacity of As(III) in five vegetables. Results showed that the TFs of As(III) in crowndaisy, flowering cabbage, leaf mustard and spinach treated with As-CM were all lower than those treated with CK-CM but in coriander herb there was no distinction between the two treatments. Significant difference (P < 0.0001) of As(III) TFs was obtained from vegetables between CK-CM and As-CM amended treatments, and significant difference (P < 0.0001) of As(III) TFs also existed among different vegetables. So As transport from root to frond in vegetables was depressed by roxarsone and its metabolites in chicken manure and the translocation of As was also affected by the internal characteristic of vegetable species. The transport of As compounds in tissues of vegetables resulted in various distribution of As species. The distribution of As species in Table 5 showed that As(III) was the major As species which taken up 79–98% in five vegetables. There was significant difference (p < 0.0001) of As(III) distribution among the same tissues of five vegetables. However, the allocation of As(III) in vegetables treated with two chicken manures only had slight difference. 3.6. As species in soils Before sowing, the major As forms in soils amended with two chicken manures were As(V) and As(III), As-CM addition increased the concentrations of both inorganic As compounds and introduced DMA into the soil compared to the CK-CM application as presented in Fig. 2. As(V) was the dominant As compound in soils amended with two chicken manures but the majority of As in five
vegetable tissues was As(III) as presented in Table 5. It was possible that As(V) in soil was first absorbed by roots of vegetables then converted into As(III) in vegetable tissues. It had been described that once inside the cell, As(V) is reduced to As(III) which is catalyzed by arsenate reductase and consumes reduced glutathione (Verbruggen et al., 2009). After growing vegetables, the content of As(V) and As(III) in CK-CM amended soil decreased. The content of As(V) in following soils ranked the order: soil growing flowering cabbage > soil growing leaf mustard > soil growing coriander herb > soil growing spinach > soil growing crowndaisy and the content of As(III) in soils ranked the following order: soil growing flowering cabbage > soil growing leaf mustard > soil growing spinach > soil growing crowndaisy > soil growing coriander herb. In addition, the uptake amount of As(V) in flowering cabbage < leaf mustard < coriander herb < spinach < crowndaisy and total As(III) uptake amount in flowering cabbage < leaf mustard < spinach < crowndaisy < coriander herb was presented in Table 4. The results showed that the decrease of As species in CK-CM amended soil grown vegetables was corresponding to the uptake of As species by vegetable plants. The changed pattern of As species remained in As-CM amended soil after vegetable harvested and accumulated in vegetable plants was the same as that treated with CK-CM. It should be noted that in As-CM amended soils there were still 20–49% of As(III) and 12–18% of As(V) more than CK-CM amended treatment after vegetable harvested. If As-CM was successively used in the next crop in the same soil, higher content of As could be remained in the soil and the extra inorganic As would continuously affect the uptake of As species of the successive second or third crop planted in the soil. Hence, the potential risk of As contamination by fertilizing of chicken manure bearing roxarsone and its metabolites should be of great concern. 4. Conclusion After excreted, more than 96% of roxarsone in animal manure is degraded into higher toxic metabolites such as As(V), As(III), MMA,
Table 5 As transfer and distribution of As species in five vegetables fertilized with chicken manure bearing and not bearing roxarsone and its metabolites. Vegetable
Coriander Herb Crowndaisy Flowering cabbage Leaf mustard Crowndaisy Spinach
Treatment
CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM CK-CM As-CM
TFs
0.02 ± 0.00 0.02 ± 0.00 0.25 ± 0.01 0.15 ± 0.01 0.16 0.09 ± 0.00 0.10 ± 0.01 0.08 ± 0.01 0.25 ± 0.01 0.15 ± 0.01 0.12 ± 0.02 0.09 ± 0.01
Percentage of As species (%) Shoot As(III)
Root As(III)
Root As(V)
11.5 ± 1.3 17.2 ± 1.3 40.9 ± 4.9 39.2 ± 2.4 67.0 ± 3.4 56.3 ± 6.4 40.0 ± 4.4 41.7 ± 5.0 40.9 ± 4.9 39.2 ± 2.4 22.4 ± 4.4 23.5 ± 3.7
83.2 ± 1.6 77.0 ± 1.9 41.8 ± 2.7 47.8 ± 4.0 30.6 ± 2.9 38.5 ± 5.7 49.2 ± 3.5 47.7 ± 5.1 41.8 ± 2.7 47.8 ± 4.0 57.2 ± 2.3 59.7 ± 3.7
5.3 ± 0.4 5.8 ± 0.6 17.3 ± 2.5 13.0 ± 1.5 2.3 ± 0.2 5.2 ± 0.7 10.8 ± 1.5 10.6 ± 0.3 17.3 ± 2.5 13.0 ± 1.5 20.4 ± 2.4 16.8 ± 1.7
L. Huang et al. / Chemosphere 100 (2014) 57–62
2200
As(III) As(V) DMA
1650
CK-CM
As-CM
1100
550
0
Be
fo r Co e so w ria in nd g er H Fl Cr e ow ow rb n er in dai g ca sy Le bb af ag m e us t Sp ard in Be ac fo h Co re ria sow nd in er g Fl Cr He ow ow rb er in nda i g c sy Le abb af ag m e us t Sp ard in ac h
As speciesin soils before vegetable sowing and -1 after vegetable harvested ( µg.kg )
62
Fig. 2. Concentrations of As species in soils of two treatments before vegetable sowing and after vegetable harvested.
DMA, 4-HPA and other unknown As species. As(III) and As(V) were detected in vegetables and the most toxic As(III) was the major As species accounted for 79–98% in five vegetables. Application of chicken manure bearing roxarsone and its metabolites in farmland increased the biomass and As content of five vegetables. Compared to the control treatments, the chicken manure bearing roxarsone and its metabolites increased 33%, 114%, 175%, 144% and 61% of As(III) uptake amount, 59%, 39%, seven times, 143% and 28% of As(V) uptake amount in roots of coriander herb, crowndaisy, flowering cabbage, leaf mustard and spinach, respectively. While the enhanced proportion of As(III) uptake amount in edible shoots of the five vegetables treated with As-CM were 114%, 74%, 91%, 175% and 68% as compared to CK-CM treatment respectively. Consequently, the uptake and accumulation of As species such as As(III) and As(V) in vegetables was increased by amended As-CM in cultivated soil and nearly two folds or more than two folds of As(III) would be taken into human body when the same amount of As-CM amended vegetables is consumed compared to CK-CM amended vegetables. The results reflected that the toxic element As could come into human body via the way: roxarsone in feed ? animal ? animal manure ? soil ? crop and the edible security of vegetables applied with animal manure containing roxarsone and its metabolites need to be pay great attention. Moreover, the residual As would be increased when animal manure containing roxarsone and its metabolites was sequentially applied in the farmland and would continuously affect the uptake of As species of the successive second or third crop planted. Acknowledgment This work was supported by National Natural Science Foundation of China (Nos. 41071316 and 40871226) and Natural Science Foundation of Guangdong province of China (No. 10151064001000010). References 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, 237–245.
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