Introduction

Since roxarsone (3-nitro-4-hydroxyphenylarsonic acid, ROX) can promote growth, inhibit parasites, and improve feed efficiency, it has been world widely used as a safe and excellent feed organoarsenic additive in animal production for decades (Chapman and Johnson 2002). For example, approximately 70 % of broiler production units uses ROX in the USA (Chapman and Johnson 2002), and 23 % of chicken feed is amended with ROX in Guangdong, China (Yao et al. 2013a). While animal is fed with ROX, the major As compounds in manure include ROX and its metabolites such as 3-amino-4-hydro-phenylarsonic acid (3-AHPA), 4-hydro-phenylarsonic acid (4-HPA), As(V), As(III), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and unknown As compounds (Garbarino et al. 2003; Jackson and Bertsch 2001; Rosal et al. 2005; Yao et al. 2009).

It has been found that some of ROX metabolites (DMA, As(III), and As(V)) can be accumulated in vegetables fertilized with chicken manure (CM) bearing ROX and its metabolites (Yao et al. 2013b; Yao et al. 2009). The potential environmental and public health risks associated with organoarsenic use in animal feeds have been reviewed by Silbergeld and Nachman (2008); however, the potential risk of As contamination in crop via the way ROX → animal → animal manure → soil → plant is still in underestimation.

Animal manure is traditionally applied together with phosphorous (P) fertilizer to enhance the P bioavailability via competitive adsorption between phosphate and organic acids generated from animal manure, which has been recommended for decades in China (Zhang 1988). On the other hand, the extractable As compounds including ROX, As(V), As(III), MMA, and DMA in mineral surface increase with increased phosphate level (Jackson and Miller 2000); therefore, we suppose that the uptake of ROX and its metabolites in crops might increase when animal manure bearing ROX and its metabolites is land applied together with P fertilizer.

This work aims to identify whether combined phosphate supply affects the uptake, transport, and distribution of As species in garland chrysanthemum plants grown in soils amended with CM containing ROX and its metabolites (4-HPA, As(V), As(III), MMA, DMA, etc.). We hope this work will contribute to evaluating the potential risks of ROX and its metabolites to human by food chain.

Materials and methods

Soil, CM, and plant

The soil used was collected at the 0–25 cm depth, typical of lateritic red soil from the Crop Experiment Station of the Guangdong Academy of Agricultural Sciences located in Guangzhou, southern China. Prior to use, the soil was air-dried, ground to pass through a 2-mm sieve, and well mixed.

The fresh CM was taken from an intensive chicken farm located in Huizhou City, Guangdong Province, in southern China, where ROX was used in the chicken feed. The fresh manure was composted for a month, then air-dried with removal of impurities, and passed through a 2-mm sieve.

The soil and CM used in this study were same as those used in our previous work (Yao et al. 2013b). The detectable As species in the soil included As(V) (1.48 ± 0.03 mg/kg) and As(III) (0.08 ± 0.0 mg/kg), with the total As of 7.3 mg/kg. The composted CM contained ROX (1.58 ± 0.31 mg/kg), 3-AHPA (0.35 ± 0.14 mg/kg), As(V) (20.49 ± 0.38 mg/kg), As(III) (2.96 ± 0.05 mg/kg), MMA (1.72 ± 0.23 mg/kg), DMA (2.99 ± 0.15 mg/kg), and unknown As species, with the total As of 58.3 mg/kg. The other basic properties of soil and CM could be seen in the previous work (Yao et al. 2013b).

A popular leafy vegetable garland chrysanthemum (Chrysanthemum coronarium), purchased from Guangzhou Vegetable Institute, was used.

Experimental design

A pot experiment in garland chrysanthemum was carried out in a greenhouse. The experiment was designed with five treatments and four replications. The five fertilization treatments were 0, 0.05, 0.1, 0.2, 0.4, and 0.8 g P2O5 (NaH2PO4·2H2O, AR)/kg plus 1 % CM (w/w, manure/soil), respectively, and abbreviated as P0, P1, P2, P3, and P4. The combined use levels of phosphate and CM are based on the greatly different use rates of P fertilizer and animal manure, while these two fertilizers are applied together by different farmers in practice. Phosphate and CM were mixed with 7.5 kg soil thoroughly and then put into a PVC pot (17 cm in height and 24 cm in diameter). The soil moisture was kept at approximately 70 % field water holding capacity for 10 days by adding deionized water, then 20 seeds of garland chrysanthemum were sown, with five seedlings kept in each pot eventually.

Sample collection and preparation

Before sowing, one soil sample was gathered vertically from the surface to the bottom in each pot. After sampling, the hole was filled with the soils in each pot, and then the seeds were sown. The shoots and roots of garland chrysanthemum were harvested separately with stainless steel scissors at 47 days after seeding, washed with tap water, and then rinsed with deionized water. The fresh weight of plants was recorded, followed by immediate lyophilization (Alpha 1–4/LD-plus, Christ), and the dry weight was recorded as well. The lyophilized plant samples were pulverized to a fine powder for total P, total As, and As speciation analysis.

Soils adhered to the roots of garland chrysanthemum in each pot were collected as a soil sample when harvested. All the soil samples were lyophilized immediately, then ground and well mixed for As speciation analysis.

Chemical analysis

Soil, CM, and plant samples were digested with concentrated HNO3 + H2SO4 + HClO4, and the total As was determined by hydride generation-atomic fluorescence spectrometer (HG-AFS, AFS8130, Jitian, Beijing). Two reference materials GBW07408 and GBW07602 were used to assure the analysis quality for soil, CM, and plant samples, with the recoveries of 97∼114 %.

The As compounds in soil and manure samples were extracted as follows: Approximately 1.0 g soil or 0.2 g manure sample was placed into a Teflon vessel, and 10 mL of mixture of NaH2PO4/H3PO4 (9:1) (0.1 mol/L PO4 3−) was added, kept in 55 °C water bath for 10 h, and sonicated for 20 min and then centrifuged at 4,000 r/min for 10 min and the supernatant was collected. The residue was extracted twice with 5 mL 0.1 mol/L PO4 3− and all the extracts were combined. The extracts were transferred to a 20-mL volumetric flask and diluted to 20 mL by adding ultrapure water, then filtered through a 0.22-μm membrane for As species analysis. As species were separated by liquid chromatography (LC, 20AT, Shimadzu, Japan), then detected by HG-AFS (8× dual channel system/SAP10-8130 speciation analysis, Jitian, Beijing). The operating conditions of LC coupled with HG-AFS were presented in Table 1.

Table 1 Working conditions of LC-HG-AFS
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As species in plant samples were prepared as follows: Approximately 0.25 g sample was placed into a Teflon vessel, 10 mL ultrapure water was added, and kept in 55 °C water bath for 10 h, then sonicated for 20 min, centrifuged for 10 min at 4,000 r/min, and the supernatant was collected. The residue was extracted twice and all the extracts were combined. The extracts were lyophilized, and 5 mL ultrapure water for root or 2 mL for shoot sample was added to dissolve the residue and filtered through a 0.22-μm membrane for LC-HG-AFS analysis.

Standards and reagents

The reference materials of ROX (97.5 %, Dr. Ehrenstorfer Gmbh, Germany), 3-AHPA (99 %, Sigma-Aldrich, USA), 4-HPA (98 %, TCI Tokyo Kasei, Japan), trimethylarsine oxide (98 %, Tri Chemical Laboratories Inc., Japan), trimethylarsine (99 %, Strem Chemicals, USA), and phenylarsonic acid (99 %, Aladdin, USA) were used in this work. The standard stock solutions of As(V) (Na2HAsO4·12H2O, 17.5 ± 0.4 mg/L), As(III) (Na3AsO3, 75.7 ± 1.2 mg/L), MMA (CH4AsNaO3·1.5H2O, 25.1 ± 0.8 mg/L), and DMA (C2H6AsNaO2·2H2O, 52.9 ± 1.8 mg/L) were bought from the National Standard Materials Center of China. The stock solutions were stored in the dark at −4 °C. Prior to use, the four stock solutions were diluted to 100, 75.5, 50.2, and 105.8 μg/L, respectively. High-performance liquid chromatography (HPLC) methanol (Burdick & Jackson, USA) and analytical-grade reagents were used in all the experiments. Ultrapure water was prepared with Millipore Milli-Q Academic.

The recoveries of standard addition of ROX, 3-AHPA, 4-HPA, As(V), As(III), MMA, and DMA for plant, manure, and soil could be seen in the previous report as well (Yao et al. 2013b). The detection limits for ROX, 3-AHPA, 4-HPA, As(V), As(III), MMA, and DMA were 9.5, 3.8, 8.4, 4.7, 1.8, 1.9, and 3.6 μg/L.

Data and statistics

All data were the means of four replications and expressed as mean ± standard error. The concentrations of all As compounds were presented as the elemental As. Transfer factors (TFs) of As or P were computed as the ratios of As or P contents (fresh weight) in shoots/As or P contents (fresh weight) in roots of garland chrysanthemum. Data were subjected to ANOVA and LSD (P < 0.05) using SAS/STAT software. Pearson correlation analysis was performed by SPSS 16.0.

Results and discussion

Growth, total P, and total As concentrations of plant

Generally, the biomass of garland chrysanthemum grown in soils amended with CM was increased by external phosphate addition, with the exception of slight reduction in the shoots of the P2 treatment and in the roots of the P5 treatment (Table 2). However, the yields of both shoots and roots did not correlate to the phosphate level. The total P and total As concentrations in both shoots and roots of garland chrysanthemum increased with increasing phosphate rate (P < 0.05). Significant and positive correlation between total P and total As was also observed (r = 0.932**, P < 0.01). It indicated that increased P fertilization along with CM bearing ROX and its metabolites enhanced not only the P uptake but also the As accumulation in garland chrysanthemum plants inadvertently.

Table 2 Biomass, total As, and total P concentrations of garland chrysanthemum amended with roxarsone and its metabolites in chicken manure as affected by different phosphate levels (FW)
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As species in plant

The analysis of As speciation in garland chrysanthemum plants shows that only As(III) in shoots and As(III) and As(V) in roots could be detected (Fig. 1), while ROX, 4-HPA, MMA, DMA, and the unknown As compound could not be determined at detectable levels in tissues. ROX can be detected in Tropaeolum majus grown in soil irrigated with ROX solution (Schmidt et al. 2008), and DMA can be observed in water spinach (Yao et al. 2010) and turnip (Yao et al. 2009) grown in soils fertilized with CM containing ROX and its metabolites (primarily as As(V), DMA, etc.). Therefore, the absence of ROX or DMA in garland chrysanthemum plants might be ascribed to their very low concentrations in soils in this study.

Fig. 1
figure 1

Concentrations of As species in both shoot and root of garland chrysanthemum amended with chicken manure bearing roxarsone and its metabolites plus different phosphate levels (FW). P0, P1, P2, P3, P4, and P5 refer to the use levels of 0, 0.05, 0.1, 0.2, 0.4, and 0.8 g P2O5/kg. Bars are standard errors of means (n = 4)

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The shoot As(III) concentrations varied from 8.6 ± 0.9 to 13.2 ± 0.8 μg/kg and significantly increased with increasing phosphate rate (r = 0.961**, P < 0.01) (Fig. 1). Significant and positive correlation between phosphate and As(III) content or the sum of both inorganic As species in the roots was also observed (r = 0.944**, P < 0.01; r = 0.970**, P < 0.01). However, the root As(V) content did not correlate to phosphate dose, which was not in accordance with the well-known interaction between phosphate and As(V) (Zhao et al. 2009).

The upward transport capability of P and As can be evaluated by the TFs in plants. As(III) was prone to be accumulated in roots because the TFs were 0.08∼0.11, much lower than 1. However, the TFs of P were 1.02∼1.25, approximately tenfold higher than those of As(III), indicating a much higher affinity to P than to As(III) in the transport systems of garland chrysanthemum plants. Moreover, the translocation of As(III) was not affected by elevated phosphate level. It is known that As(V) is taken up by plant roots via phosphate transporters, and As(III) is absorbed mainly as the neutral molecule As(OH)3 via some aquaglyceroporin channels, e.g., As(III) enter the root cell by the Si pathway in rice (Zhao et al. 2009), which might explain the reason why As(III) uptake is inhibited by glycerol and antimonite (Meharg and Jardine 2003) but not by phosphate in paddy rice roots (Abedin et al. 2002). Additionally, the arsenate reductase has been found in plant, and As(V) exposure increases its activity (Duan et al. 2007). Based on the above reports, we suppose that most of the As(III) accumulated in garland chrysanthemum shoots might be taken up as As(V) by roots, followed by rapid reduction of As(V) to As(III) in roots and then transport to shoots and accumulated as As(III) as reported in reference (Pickering et al. 2000; Smith et al. 2008), rather than as As(V) sharing same transporter with phosphate in tissues. It might be the conversion of As(V) to As(III) in tissues which obfuscated the clear relation between P and As(V) in roots in this study.

Elevated phosphate increased As(III) concentrations in both shoots and roots in this study, however, decreased the shoot As(V) and As(III) concentrations in water spinach grown in soils amended with CM bearing ROX and its metabolites (Yao et al. 2010), which supports the result that interaction between P and As depends on the plant species growing in soil (Otte et al. 1990). Hence, the traditional practice that animal manure is applied with P fertilizer is not always suitable for all crops while the manure contains ROX and its metabolites.

Accumulation and distribution of As species in plants

As(III) accumulated in garland chrysanthemum shoots ranged from 0.61 ± 0.12 to 1.09 ± 0.23 μg/pot and that in roots was in the range of 0.90 ± 0.38∼1.61 ± 0.61 μg/pot (Table 3). The uptake amount of the sum of both As species in whole plants varied from 1.81 ± 0.63 to 3.04 ± 0.86 μg/pot. Elevated phosphate level significantly increased the uptake rates of As(III) in shoots, the sum of two As species in both roots and total plants (P < 0.05), with the exception of As(V) accumulation in roots.

Table 3 Uptake amount of As species in garland chrysanthemum amended with roxarsone and its metabolites in chicken manure as affected by different phosphate levels (μg/pot)
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Though As(V) was the dominant As compound in both CM and soil, As(III) was the predominant As form in garland chrysanthemum plants (Fig. 2). The shoot As(III) accounted for 29.1∼35.9 % of the total extractable As species in plants, with the root As(III) proportions as 49.9∼60.6 % and the root As(V) as 9.4∼15.0 %. Totally, phosphate addition did not affect the distribution of As species in garland chrysanthemum plants. It demonstrates that ROX can be finally accumulated in the edible part of crop primarily as As(III) through series of metabolism, degradation, and conversion via the food chain. Moreover, combined application of P fertilizer and CM inadvertently and significantly increase the potential risk of As contamination in crops fertilized with CM bearing ROX and its metabolites.

Fig. 2
figure 2

Distribution of As species in garland chrysanthemum amended with chicken manure bearing roxarsone and its metabolites plus different phosphate levels. P0, P1, P2, P3, P4, and P5 refer to the use levels of 0, 0.05, 0.1, 0.2, 0.4, and 0.8 g P2O5/kg

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As species in soils

Figure 3 shows the concentration increments of As species in rhizosphere soils between before sowing and at harvest. Before sowing, soils contained 1706.4 ± 27.6∼1868.9 ± 61.2 μg As(V)/kg, 225.7 ± 33.6∼263.6 ± 18.7 μg As(III)/kg, and 35.8 ± 12.2∼57.1 ± 6.9 μg DMA/kg. ROX, 4-HPA, MMA, and the unknown As compound could not be determined in soils amended with CM due to their low concentrations in the CM or conversions between As species in soils before sowing. We had tried to identify the unknown As form by contrasting with the references of trimethylarsine oxide, trimethylarsine, and phenylarsonic acid, but all of them could not match with the unknown As compound.

Fig. 3
figure 3

Concentration increments of As species in rhizosphere soils amended with chicken manure bearing roxarsone and its metabolites between before sowing and at harvest as affected by phosphate level. P0, P1, P2, P3, P4, and P5 refer to the use levels of 0, 0.05, 0.1, 0.2, 0.4, and 0.8 g P2O5/kg. Bars are standard errors of means (n = 4)

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At harvest, only As(V) and As(III) were determined, while DMA was not detected in both garland chrysanthemum plants and rhizosphere soil, indicating that DMA might be converted to other As compound before the garland chrysanthemum roots could take up it. As uptake by garland chrysanthemum plants was closely associated with the As supply in the rhizosphere. The change of As(V) concentrations in the rhizosphere were much greater than those of As(III) and DMA in all treatments at harvest (Fig. 3) and positively correlated to the phosphate level (r = 0.945**, P < 0.01), indicating more As(V) was released by increased phosphate supply through competitive desorption of As(V) by phosphate as documented by Jackson and Miller (2000), namely, higher As(V) supply was maintained for garland chrysanthemum roots in the rhizosphere by increased phosphate addition. However, no close relation was found between phosphate and decrements of As(III) or DMA in the rhizosphere. The above indicates that elevated phosphate level supplied not only higher P nutrition for garland chrysanthemum roots but also relatively more As(V) in the rhizosphere, leading to increased uptake of both P and As by plants.

Conclusions

While CM bearing ROX and its metabolites was applied with P fertilizer, phosphate supply significantly increased the uptake of P and As in garland chrysanthemum. The As speciation analysis shows that As(III) was the sole As compound in garland chrysanthemum shoots, and As(III) and As(V) were detectable in all roots. Elevated phosphate supplied more As(V) for garland chrysanthemum roots through competitive desorption of As(V) in the rhizosphere, leading to significantly enhanced uptake of As species in plants. Phosphate could not change the allocation pattern of As species in plants. As(III) was the predominant As form in plants (85.0∼90.6 %). Conclusively, P fertilizer increased the phytoavailability of ROX and its metabolites and greatly enhanced the potential risk of As contamination in garland chrysanthemum plants generated from the utilization of ROX in animal production.