Helle R.
Hansen
*a,
Andrea
Raab
ab,
Adam H.
Price
a,
Guilan
Duan
c,
Yongguan
Zhu
c,
Gareth J.
Norton
a,
Jörg
Feldmann
b and
Andrew A.
Meharg
a
aInstitute of Biological and Environmental Sciences, University of Aberdeen, Cruickshank Building, St Machar Drive, Aberdeen, UK AB24 3UU. E-mail: h.r.hansen@abdn.ac.uk
bTESLA (Trace Element Speciation Laboratory) and Marine Biodiscovery Laboratory, University of Aberdeen, Aberdeen, Scotland, UK AB24 3U
cResearch Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
First published on 15th November 2010
Tetramethylarsonium has for the first time been identified in a commercially grown food product, rice, constituting up to 5.8% of the total arsenic in the rice.
Environmental impactMillions of people are exposed to arsenic through consumption of contaminated drinking water or foods, such as rice. Here we identify tetramethylarsonium in rice grain, a cationic arsenic species normally not associated with a terrestrial food source. We propose that future studies on uptake, transformation and metabolism of arsenic associated with the terrestrial environment will have to include analytical techniques also capable of unequivocally identifying cationic arsenic species, which elutes in the void volume of traditionally applied analytical methods based on anion exchange chromatography. In order to understand the mechanism of arsenic uptake in plants and the correlation between exposure and toxic effect in human, it is essential to obtain the complete picture of the arsenic species actually present at each stage. |
Fig. 1 AEC-ICP-MS chromatograms of rice extract (black) and a standard solution mixture (grey). |
Rice variety | Total As in grain µg kg−1 dry mass | Front peak by AEC as % of speciation (sum of species/total) | Tetra by CEC as % of speciation (sum of species/total) |
---|---|---|---|
Rathuwee | 599 | 6.7 (87%) | 4.8 (70%) |
Shirkati | 697 | 4.3 (102%) | 3.2 (68%) |
Rathuwee | 533 | 8.1 (70%) | 6.8 (65%) |
Al-chio-hong | 868 | 5.7 (66%) | 5.0 (62%) |
Paung malaung | 785 | 2.7 (103%) | 2.1 (88%) |
Kachilon | 1223 | 2.1 (95%) | 1.5 (87%) |
The identity of the unknown As species was investigated in six of the samples with the highest content, by using cation exchange chromatography (CEC)-ICP-MS. An already established CEC method6 using a Varian Ionosphere 5C column (150 × 4.6 mm) was applied for analysis of rice extracts (hydrogen peroxide omitted). With this method a species of As present in the rice extracts co-eluted with the tetramethylarsonium (Tetra) standard (Fig. 2A). The retention times of other cationic As species (such as AB, dimethylarsinoylethanol, trimethylarsine oxide (TMAO) and arsenocholine (AC), the latter two are shown in Fig. 2A) known to be strongly retained on the column were also checked, but these had significantly different retention times from the unknown As species in the rice extracts. When spiking the rice extracts with a standard of Tetra, the co-elution was confirmed (Fig. 2B). A different CEC system,§ with a minor mobile phase gradient change and thus insignificant influence on the As response factor, was used for quantifying Tetra in the rice extracts. Comparable amounts to those determined by AEC were observed (Table 1).
Fig. 2 CEC-ICP-MS chromatograms of (A) a rice extract and standards (all offset) of Tetra, trimethylarsine oxide (TMAO) and arsenocholine (AC) and (B) of rice extract (black) and the extract spiked with 5 µg L−1Tetra (grey). |
In order to ensure that the occurrence of Tetra in the rice extracts was not due to microbiological activity during the overnight extraction procedure, extractions were repeated without the overnight extraction step and samples were immediately frozen after the microwave assisted extraction. The occurrence of Tetra may also be potentially due to thermal decomposition of tetraalkylated As species during the microwave extraction. There have been numerous reports on As species stability and conversion during thermal treatment,7 and Tetra is a known degradation product of AB, although only in small amounts (0.1–2%) at temperatures >155 °C.8 Nevertheless, to rule out that Tetra in the rice grains was due to thermal decomposition, extraction was also performed by ultrasonication (30 min) in 1% HNO3 or in a methanol/water mixture (9 + 1) (the latter also for excluding microbiological activity), prior to overnight extraction at 4 °C followed by freezing of samples. Tetra was present in all the different extracts.
To confirm the identity of Tetra in the extracts of rice, high-resolution electro spray (ES) mass spectra were obtained of purified samples on an Orbitrap Discovery (Thermo Scientific, UK) at a resolution of 30000. For purification, fractions of the rice extracts were collected from the CEC system6 at time 28.2–30.3 min, freeze dried, and re-dissolved in water prior analysis.¶ The accurate mass obtained of a Tetra standard was 135.01472 and the unknown in the purified rice extracts was 135.01474,† (mass accuracy of 1.7 and 1.5 ppm error, respectively, theoretical mass of C4H12As+ is 135.01495).
This is the first report on the presence of Tetra in rice grains (22–35 µg kg−1 dry mass). Mandal et al.9 have previously reported the presence of AB at similar concentrations (8.2–25.8 µg kg−1 dry mass) in extracts of rice from a range of sources (Indian, Japanese, and Thai rice). In that study identification was based on the co-elution between As species in the extracts and standards on an anion exchange and a size exclusion HPLC column. This can only be rated as a tentative identification, since no molecular mass spectrum and no characteristic retention time on a CEC using spiking were attempted, so it can be speculated that Tetra was misidentified as AB. Although Tetra has been mostly associated with marine organisms (mg kg−1 level),10 it has previously been identified at trace levels (low µg kg−1 levels) in several terrestrial plant tissues1 (moss, Cocksfoot grass, Red clover, Ribwort plantain, Green spleenwort, Broad buckler fern, Wild strawberry, Cowberry, and Monkey flower). It is uncertain if the occurrence of Tetra in terrestrial plants originates in soil or is produced in the plants. The main organoarsenic species reported in soil are MA(V), DMA(V),11 and TMAO,12 and their occurrence has been attributed to microorganisms, as both fungi and bacteria are capable of methylating inorganic arsenic.13Tetra, however, seems a less commonly occurring species in the soil environment, but it has been identified at trace level in porewaters of acidic fen soil.14 The origin of Tetra in the Chinese rice analysed in this study can only be speculated at. Tetra may potentially have been present in the paddy soil that the rice grew in, in which case its presence can be attributed to bacteria, fungi (higher fungi are known to produce Tetra),11 or algae, as algae growth is associated with the fields.
Footnotes |
† Electronic supplementary information (ESI) available: Accurate mass ES-MS measurements. See DOI: 10.1039/c0em00460j |
‡ Sample of dried and milled rice grain (0.2 g) was left in 1% HNO3 (10 mL for AEC and 1 mL for CEC) overnight and subjected to microwave assisted extraction (0–55 °C in 5 min, hold for 5 min, 55–75 °C in 5 min, hold for 5 min, 75–95 °C in 5 min and hold for 30 min). |
§ Zorbax 300-SCX (150 × 4.5 mm) column, mobile phases of milliQ water and 50 mM pyridine formate at pH 2.7 with gradient elution (0–4 min; 0.5 mM, 4–16 min; 0.5–5 mM, 16–20 min; 5 mM, 20–30 min; 0.5 mM) at a flow rate of 1 mL min−1. |
¶ LC-MS analysis was performed on a Hamilton PRP-X200 pre-column with 4 mM nitric acid as carrier at a flow rate of 0.2 mL min−1. |
This journal is © The Royal Society of Chemistry 2011 |