Biogeochemical Processes Of Methylated Thioarsenates In Soil-rice Systems And The Accumulation In Rice Grains

Arsenic(As)is a toxic and carcinogenic metalloid,which is ubiquitous in the environment.Arsenic contamination in paddy fields is a serious environmental problem worldwide due to human activities such as mining,smelting,irrigation with arsenic contaminated groundwater,and the use of As-containing pesticides,threatening the production of rice.Arsenic presents as various species in rice paddies,with different As species differing substantially in their toxicity and bioavailability.Inorganic arsenic(i As)such as arsenite(As(Ⅲ)and arsenate As(Ⅴ),and methylated arsenic such as monomethyl-arsenic(MMA),dimethyl-arsenic(DMA)are generally reported arsenic species in paddy soils,among which As(Ⅲ)and DMA are also the main arsenic species reported in rice grains.The rice rhizosphere significantly affects the uptake of arsenic in rice plants,but the migration and transformation mechanism of arsenic is still unclear.Becides,a new type of thioarsenate species,including inorganic and organic thioarsenate,especially a highly toxic dimethylmonothioarsenate(DMMTA),have recently been identified in rice paddies.However,the biogeochemical processes of methylated thioarsenates in paddy soils and their subsequent uptake and accumulation in rice plants remain poorly understood.In the present study,hence,the migration and transformation of arsenic in the rhizosphere were studied by using a new rhizobox and planting rice with different radial oxygen loss.In addition,we used a high performance liquid chromatography-inductively coupled plasma-mass spectrometry(HPLC-ICP-MS)to develop methods to analyze and preserve methylated thioarsenates in the porewaters of paddy soils.Based on these methods,we investigated the biogeochemical processes of DMMTA,a highly toxic arsenic species,in paddy soils,the uptake,translocation and toxicity in rice plants.Furthermore,a new enzymatic method was also developed to extract and preserve methylated thioarsenates in rice grains.Using this method,we investigated the DMMTA concentration in rice grains produced in China main rice production areas and global markets,with the main results as following:(1)Using a new rhizobox and micro porewater samplers,we revealed that the concentration of different arsenic species,especially As(Ⅲ),were enriched in rhizosphere compared to the bulk soil(1.3-2.4 times),and the spatial distribution of arsenic and iron in the rhizosphere showed a similar regularity,namely arsenic and iron is highest near the root tip,and gradually reduced along with a single root to the mature zone.In addition,from the rhizosphere to the bulk soil,the total arsenic concentration in the porewater presents a significant reciprocal function with the distance from the root.Rice varieties had little effect on the availability of arsenic in the rhizosphere,but had a significant effect on the accumulation of arsenic in rice grains.In other words,the higher the ROL of rice varieties,the lower the accumulation of arsenic in rice grains.In particular,the iron-reducing bacteria(Fe RB)Geobacter and Clostridium are the main microorganisms that affect the migration and transformation of arsenic in the rhizosphere and dominate those process.(2)Although some studies have reports about methylated thioarsenates,no commercial standards of methylated thioarsenates available.Additionally,the preservation and analytical methods of these As species are also lacking.In the present study,we initially synthesized three methylated thioarsenates standards,including monomethymonothioarsenate(MMMTA),dimethymonothioarsenate(DMMTA),and dimethydithioarsenate(DMDTA).Next,we optimized the HPLC-ICP-MS method through using a C18 reversed phase column and the 20 m M MH4H2PO4(p H 30.).This method is capable of sepearating these methylated thioarsenates with a relatively short retention time(< 7 min).It is also shown that 10 m M DTPA(diethylenetriamine pentaacetate)plus frozen treatment is able to efficiently preserve these methylated thioarsenates in paddy soil porewaters.This preservation method not only avoids the precipitation of Fe,but also minimizes the dethiolation of these methylated thioarsenates,compared with the acidification preservation method.(3)Based on the above methods,we investigated i)the dynamics of methylated thioarsenates in seven arsenic-contaminated paddy soils under flooded conditions,ii)the dynamics of DMMTA in two soils in response to anoxic-oxic transition,and iii)the dynamics of DMMTA in field rice paddies.We found that DMMTA was the main methylated thioarsenate in all soil porewaters,with the concentrations of porewater DMMTA being positively correlated with the concentrations of porewater DMA, representing 58% of DMA concentrations.According to the structural equation model(SEM),we found that DMA and DOC concentration are the two most important factors affecting the concentrations of porewater DMMTA.Using two specific inhibitors sulfate-reducing bacteria(SRB)(molybdate,Mo)and methanogens(Sodium 2-bromoethyl sulfonate,BES),we found that that addition of Mo reduced theproduction of DMMTA by inhibiting the production of DMA,whereas the addition of BES increased porewater DMMTA by inhibiting the disappearance of DMA,indicating that Moreover,the dynamics of DMMTA depended on the concentration of DMA.Finally,in the paddy fields,we also found that the DMMTA is the dominant methylated thioarsenates in paddy fields and that additions of sulfate fertilizer or rice straw incorporation increased the concentrations of DMMTA in both soil porewaters and rice grains,and the concentration of DMMTA positively related with DMA in soil porewater(R2 = 0.51,P < 0.01).(4)We also investigated the uptake,translocation and toxicity of methylated thioarsenates in rice plants.We found that that among all arsenic species,total As concentrations in the xylem sap was the highest when rice roots exposed to DMMTA.Similarly,the translocation factor(shoot As/root As ratio)was the highest for DMMTA-exposed plants,with the order being DMMTA(0.52)> DMA(0.46)> As(Ⅲ)(0.38)> MMMTA(0.03)> MMA(0.02)= As(Ⅴ)(0.02).Root length inhibition experiments showed that DMMTA was highly toxic to rice plants,with the toxicity order being As(Ⅲ)(EC50 = 15.4 μM)> DMMTA(25.9 μM)> MMMTA(90.4 μM)> DMA(91.2 μM)> MMA(100 μM)> As(Ⅴ)(291 μM).These results indicate that DMMTA can be highly taken up by rice roots and then efficiently translocated to the shoots and also be highly toxic to rice plants.(5)Rice roots can efficiently take up DMMTA.However,whether DMMTA is present in rice grian are unclear.In the present study,we initially developed an enzymatic extraction method,which is capble of efficiently extracting arsenic species in rice grains.Next,we used this method to analyze As species in 103 samples collected from the paddy fields across the rice production areas of China and 140 samples of commercial rice samples collected from major rice producing areas globally.We found that the conventional method which uses acid to digest rice grain can result in the conversion of all DMMTAs into DMA.Additionally,the traditional analysis method(HPLC-ICP-MS with an anion exchange column)can not separate and detect the methylated thioarsenates.These two factors result in the ignorance of DMMTA in rice.Using the enzymatic method,we found that the concentrations of DMMTA ranged from 4 to 35 μg/kg in polished rice in the field survey,andfrom 1 to 30 μg/kg for commercial polished rice(market-basket suvey),accounting for 1-17% of the total arsenic in rice grain.Notably,DMMTA concentrations were found to be significantly correlated with DMA concentrations(approximately representing 30% of DMA)in polished rice samples.Importantly,grain DMMTA concentrations were unrelated with soil total As.Based on these findings in the present study and the reported DMA in literature,we assessed the DMMTA concentrations in polished rice of the major rice peoduction areas on a global scale.We found that there is a geographic pattern in DMMTA both in China and around the world.Grain DMMTA is significantly higher in Northeast China than in other regions of China.Grain DMMTA concentrations in North America and Europe are significantly higher than those in Asia,Africa and other regions.The global exposure risks of DMMTA were also assessed based on its concentrations and cytotoxicity of DMMTA.The risk associated with dietary intake of DMMTA from rice would be approximately half of that of i As for Asian diet,but double that of i As for European and American diet.In summary,the reason why DMMTA was not detected in previous reports of As species in global surveys of rice grians is attributed to the inappropriateness of conventional methods used for extraction and chromatographic separation.Our study developed a series of new methods,including the qualitative and quantitative analysis of methylated thioarsenates,the preservation of methylated thioarsenates in soil porewater samples,and the extraction of methylated thioarsenates in rice grains.Using these methods,we found that DMMTA is the dominant methylated thioarsenate species and its dynamics depends on the production of DMA in paddy soils.We also found that the highly toxic DMMTA is widely present in rice globally.The current,routinely used acid-based extraction methods incorrectly identify DMMTA as DMA is especially problematic.Our results have urgent implications for altering and improving food regulations.It is imperative that new food regulation guidelines are established that account for the accumulation of DMMTA in rice.