Jörg Feldmann

Uptake kinetics of arsenic species in rice plants.

Arsenic (As) finds its way into soils used for rice (Oryza sativa) cultivation through polluted irrigation water, and through historic contamination with As-based pesticides. As is known to be present as a number of chemical species in such soils, so we wished to investigate how these species were accumulated by rice. As species found in soil solution from a greenhouse experiment where rice was irrigated with arsenate contaminated water were arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The short-term uptake kinetics for these four As species were determined in 7-d-old excised rice roots. High-affinity uptake (0–0.0532 mm) for arsenite and arsenate with eight rice varieties, covering two growing seasons, rice var. Boro (dry season) and rice var. Aman (wet season), showed that uptake of both arsenite and arsenate by Boro varieties was less than that of Aman varieties. Arsenite uptake was active, and was taken up at approximately the same rate as arsenate. Greater uptake of arsenite, compared with arsenate, was found at higher substrate concentration (low-affinity uptake system). Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly suppressed in the presence of phosphate, whereas arsenite transport was not affected by phosphate. At a slow rate, there was a hyperbolic uptake of monomethylarsonic acid, and limited uptake of dimethylarsinic acid.

The Nature of Arsenic-Phytochelatin Complexes in Holcus lanatus and Pteris cretica

We have developed a method to extract and separate phytochelatins (PCs)—metal(loid) complexes using parallel metal(loid)-specific (inductively coupled plasma-mass spectrometry) and organic-specific (electrospray ionization-mass spectrometry) detection systems—and use it here to ascertain the nature of arsenic (As)-PC complexes in plant extracts. This study is the first unequivocal report, to our knowledge, of PC complex coordination chemistry in plant extracts for any metal or metalloid ion. The As-tolerant grass Holcus lanatus and the As hyperaccumulator Pteris cretica were used as model plants. In an in vitro experiment using a mixture of reduced glutathione (GS), PC2, and PC3, As preferred the formation of the arsenite [As(III)]-PC3 complex over GS-As(III)-PC2, As(III)-(GS)3, As(III)-PC2, or As(III)-(PC2)2 (GS: glutathione bound to arsenic via sulphur of cysteine). In H. lanatus, the As(III)-PC3 complex was the dominant complex, although reduced glutathione, PC2, and PC3 were found in the extract. P. cretica only synthesizes PC2 and forms dominantly the GS-As(III)-PC2 complex. This is the first evidence, to our knowledge, for the existence of mixed glutathione-PC-metal(loid) complexes in plant tissues or in vitro. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica.

The Rice Aquaporin Lsi1 Mediates Uptake of Methylated Arsenic Species

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher Vmax than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.

Uptake and translocation of inorganic and methylated arsenic species by plants

Environmental context. The molecular occurrence of arsenic in soils can vary as a result of soil conditions and/or application of arsenic-containing herbicides or fertiliser. Although large amounts of As-containing herbicides are used for different crops, there is still a lack of understanding as to how the molecular form of As determines the uptake of arsenic into plants and, in particular, the translocation into shoot and grain. Abstract. The uptake and translocation into shoots of arsenate, methylarsonate (MA), and dimethylarsinate (DMA) by 46 different plant species were studied. The plants (n = 3 per As species) were exposed for 24 h to 1 mg of As per litre under identical conditions. Total arsenic was measured in the roots and the shoots by acid digestion and inductively coupled plasma mass spectrometry from which, besides total As values, root absorption factors and shoot-to-root transfer factors were calculated. As uptake into the root for the different plant species ranged from 1.2 to 95 (μg of As per g of dry weight) for AsV, from 0.9 to 44 for MAV and from 0.8 to 13 for DMAV, whereas in shoots the As concentration ranged from 0.10 to 17 for AsV, 0.1 to 13 for MAV, and 0.2 to 17 for DMAV. The mean root absorption factor for AsV (1.2 to 95%) was five times higher than for DMAV (0.8 to 13%) and 2.5 times higher than for MAV (0.9 to 44%). Although the uptake of arsenic in the form of AsV was significantly higher than that of MAV and DMAV, the translocation of the methylated species was more efficient in most plant species studied. Thus, an exposure of plants to DMAV or MAV can result in higher arsenic concentrations in the shoots than when exposed to AsV. Shoot-to-root transfer factors (TFs) for all plants varied with plant and arsenic species. While AsV had a median TF of 0.09, the TF of DMAV was nearly a factor of 10 higher (0.81). The median TF for MAV was in between (0.30). Although the TF for MAV correlates well with the TF for DMAV, the plants can be separated into two groups according to their TF of DMAV in relation to their TF of AsV. One group can immobilise DMAV in the roots, while the other group translocates DMAV very efficiently into the shoot. The reason for this is as yet unknown.

Complexation of Arsenite with Phytochelatins Reduces Arsenite Efflux and Translocation from Roots to Shoots in Arabidopsis

Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

Arsenic in the Meager Creek hot springs environment, British Columbia, Canada

Levels of arsenic in water from Meager Creek hot springs, British Columbia, Canada, were found to be naturally elevated. Biota including microbial mats, green algae, sedge, cedar, fleabane, monkey flower, moss, mushrooms and lichens, that were expected to be impacted by the water, were analyzed for total levels of arsenic and for arsenic species. The major arsenic species extracted from all samples were arsenate and arsenite, which are toxic forms of arsenic. Additionally, small amounts of arsenosugars X and XI were detected in microbial mats and green algae, implying that cyanobacteria/bacteria, and possibly green algae are capable of synthesizing arsenosugars from arsenate. Low to trace amounts of arsenosugars X and XI were detected in lichens and the fungus Tarzetta cupularis. A large fraction (on average, greater than 50%) of arsenic was not extracted by using methanol/water (1:1) and the chemical and toxicological significance of this arsenic remains unknown.

Occurrence of Volatile Metal and Metalloid Species in Landfill and Sewage Gases

Speciation of volatile metals and metalloids in the environment is extensively described in literature. To investigate unstable volatile organometallics, online coupling of gas chromatog. with inductively coupled plasma-mass spectrometry was used. Preliminary results for gases of sewage sludge fermn. under thermophilic and mesophilic conditions are compared with metal and metalloid speciation in landfill gases. In each case, 20 L of gas were sampled by cryogenic trapping. Species were identified by element-specific detection either by retention time of stds. or by calcn. of the b.p. correlation. Characteristic of the sepn. is the linear correlation of b.p. (bp/ Deg) vs. retention time (rt/min) (bp = 6.39*rt-109.2, r2 = 0.9926). The amts. of total volatile elements were estd. by semi-quantification. Cd, Sn, Hg, and Pb (sewage gas) and Se, Te, Hg, and Pb (landfill gas) were detd. in ng/m3 levels; As, Sb, Te, and Bi (sewage gas) and As, Sn, Sb, and Bi (landfill gas) in mg/m3 levels.

Arsenic–glutathione complexes—their stability in solution and during separation by different HPLC modes

Complexes of arsenic compounds and glutathione are believed to play an essential part in the metabolism and transport of inorganic arsenic and its methylated species. Up to now, the evidence of their presence is mostly indirect. We studied the stability and chromatographic behaviour of glutathione complexes with trivalent arsenic: i.e. AsIII(GS)3, MAIII(GS)2 and DMAIII(GS) under different conditions. Standard ion chromatography using PRP X-100 and carbonate or formic acid buffer disintegrated the complexes, while all three complexes are stable and separable by reversed phase chromatography (0.1% formic acid/acetonitrile gradient). AsIII(GS)3 and MAIII(GS)2 were more stable than DMAIII(GS), which even under optimal conditions tended to degrade on the column at 25 °C. Chromatography at 6 °C can retain the integrity of the samples. These results shed more light on the interpretation of a vast number of previously published arsenic speciation studies, which have used chromatographic separation techniques with the assumption that the integrity of the arsenic species is guaranteed.

Laser ablation of soft tissue using a cryogenically cooled ablation cell

A cryogenically cooled ablation cell enables the direct analysis of thin sections from fresh soft tissue samples, such as liver or kidney, for trace elements using laser ablation ICP-MS. We show here, for the first time, that reproducibilities of about 2–6% can be achieved if the tissue sample can be ablated at a temperature below −60 °C. All ablation and detection parameters, such as energy, spot size, focus, fire frequency and integration time at the ICP-TOF-MS, were optimised. A calibration method using three different tissue samples (sheep kidney, pig liver and sheep liver), from which the bulk element concentrations were determined, was validated with CRM Pig Liver (LGC 7112), which was pressed and frozen in the form of a thin slice. Good recoveries (86–124% for the certified values) were achieved for Cd (0.31 mg kg−1; 0.25 ± 0.04 certified), Cu (101 mg kg−1; 117 ± 8 certified), Zn (43.0 mg kg−1; 43.0 ± 2.7 certified) and Mo (1.8 mg kg−1; ∼1 indicative value). Therefore this CRM can be used for the quantification of other tissues with similar C-content using a one-point calibration. Detection limits in the lower µg kg−1 range (Cd: 15 µg kg−1, Cu: 50 µg kg−1, Zn: 20 µg kg−1, Mo: 10 µg kg−1 and Pb: 2 µg kg−1) were determined based on 3σ of the blank signal with a spatial resolution of less than 200 μm. Using the CRM Pig Liver, it was shown that the use of an internal standard (13C) can account for fluctuations in the ablated material during a line scan. Instead of 12% RSD without internal standard, the stability of the signal was improved using the normalized signal (5.2%) compared to 2.5–3.5% precision when a NIST 610 Glass standard was ablated. Hence, LA coupled to ICP-MS with a cryogenically cooled ablation chamber is the ideal technique for 2D mapping of trace elements in soft tissues. Depending on the concentration of element present, it may be possible to determine trace elements directly in tissue samples at a spatial resolution of <20 µm.

Critical review or scientific opinion paper: Arsenosugars—a class of benign arsenic species or justification for developing partly speciated arsenic fractionation in foodstuffs?

In this opinion paper the toxicokinetic behaviour of arsenosugars is reviewed and compared with that of inorganic arsenic and arsenobetaine. It is concluded that the arsenosugars are similar to inorganic arsenic in terms of metabolite formation and tissue accumulation. As a pragmatic means of generating uniform data sets which adequately represent the toxicity of arsenic in food we recommend reporting partly speciated arsenic concentrations in food commodities in three fractions: i) toxic inorganic arsenic as arsenate (after oxidation); ii) arsenobetaine as established non-toxic arsenic; and iii) potentially toxic arsenic, which includes arsenosugars and other organoarsenicals.