A new selenized yeast reference material (SELM-1) produced by the Institute for National Measurement Standards, National Research Council of Canada (INMS, NRC) certified for total selenium (2,059+/-64 mg kg(-1)), methionine (Met, 5,758+/-277 mg kg(-1)) and selenomethionine (SeMet, 3,431+/-157 mg kg(-1)) content is described. The +/-value represents an expanded uncertainty with a coverage factor of 2. SeMet and Met amount contents were established following a methanesulfonic acid digestion of the yeast using GC-MS and LC-MS quantitation. Isotope dilution (ID) calibration was used for both compounds, using 13C-labelled SeMet and Met. Total Se was determined after complete microwave acid digestion based on ID ICP-MS using a 82Se spike or ICP-OES spectrometry using external calibration. An international intercomparison exercise was piloted by NRC to assess the state-of-the-art of measurement of selenomethione in SELM-1. Determination of total Se and methionine was also attempted. Seven laboratories submitted results (2 National Metrology Institutes (NMIs) and 5 university/government laboratories). For SeMet, ten independent mean values were generated. Various acid digestion and enzymatic procedures followed by LC ICP-MS, LC AFS or GC-MS quantitation were used. Four values were based on species-specific ID calibration, one on non-species-specific ID with the remainder using standard addition (SA) or external calibration (EC). For total selenium, laboratories employed various acid digestion procedures followed by ICP-MS, AFS or GC-MS quantitation. Four laboratories employed ID calibration, the remaining used SA or EC. A total of seven independent results were submitted. Results for methionine were reported by only three laboratories, all of which used various acid digestion protocols combined with determination by GC-MS and LC UV. The majority of participants submitted values within the certified range for SeMet and total Se, whereas the intercomparison was judged unsuccessful for Met because only two external laboratories provided values, both of which were outside the certified range.

The effect of metal ions (Co, Cu, Ni, Pb, Zn) on the molecular weight distribution of humic substances (HSs) obtained from compost is studied. We believe this is the first of this type of study applied in this way to humic substances. Size exclusion chromatography is coupled with two on-line detection systems (spectrophotometric and ICPMS) to study the binding of metal ions by humic substances leached from compost. ICPMS provided highly specific, sensitive, and multielement analytical information that enabled obtaining direct experimental evidence for the participation of metal ions in molecular size distributions of humic compounds. The compost extract or its high molecular weight fraction (>5,000) was put in contact with EDTA or citrate ions, thereby competing with HSs for binding metals. The experiments were carried out by varying the pH maintained by Tris-HCl or CAPS buffer (pH 8.0 and 10.3) and keeping the ionic strength constant. The elution profile of humic substances using UV/ visible detection was compared with those from ICPMS detection of Co, Cu, Ni, Pb, and Zn in the same chromatographic runs. The results obtained suggested that both bridging between small molecules and complexation/ chelation by individual molecules are involved in metal ion binding to humic substances. The use of ICPMS to study the role of metal ions in aggregation/disassociation of humic substances proposed in this work is promising. Coupling element-specific detection with SEC or other separation systems allows better understanding of the mobility and bioaccessibility of elemental species in the environment and further elucidation of the dissolved humic structure.

A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium-phytochelatin complexes (Cd-PC 2 , Cd-PC 3 , and Cd-PC 4 ) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversedphase separation. The detection limits were found to be 91.8 ng l −1 , 77.2 ng l −1 and 49.2 ng l −1 for Cd-PC 2 , Cd-PC 3 , and Cd-PC 4 respectively. The regression coefficients (r 2 ) for Cd-PC 2 to Cd-PC 4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium-phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC 2 . In both lines, higher levels of Cd-PC 2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC 2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC 2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC 2 than the wild type. The enhanced role of PC 2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.

In this work, the speciation of elements in compost was studied with emphasis on their binding to humic substances. In order to assess the distribution of As, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, U, Th and Zn among molecular weight fractions of humic substances, the compost extract (extracted by 0.1 mol l(-1) sodium pyrophosphate) was analyzed by size exclusion chromatography coupled on-line with UV-Vis spectrophotometric and ICP-MS detection. Similar chromatograms were obtained for standard humic acid (Fluka) and for compost extract (254 nm, 400 nm) and three size fractions were operationally defined that corresponded to the apparent molecular weight ranges > 15 kDa, 1-15 kDa and < 1 kDa. The percentage of total element content in compost that was leached to the extract ranged from 30% up to 100% for different elements. The elution profiles of Co, Cr, Cu, Ni and Pb (ICP-MS) followed that of humic substances, while for other elements the bulk elution peak matched the retention time observed for the element in the absence of compost extract. Spiking experiments were carried out to confirm elements' binding and to estimate the affinity of individual elements for humic substances derived from compost. The results obtained indicated the following order of decreasing affinity: Cu > Ni > Co > Pb > Cd > (Cr, U, Th) >> (As, Mn, Mo, Zn). After standard addition, further binding of Cu, Ni and Co with the two molecular weight fractions of humic substances was observed, indicating that humic substances derived from compost were not saturated with these elements.

A preconcentration and determination method for humic and fulvic acids at trace levels in natural water samples was developed. Cloud point extraction was successfully employed for the preconcentration of humic acid (HA) and fulvic acid (FA) prior to the determination by using a flow injection (FI) system coupled to a spectrophotometric UV-Vis detector. The quantitative extraction of HA and FA within the pH range 1-12 was obtained by neutralization of the anionic charge on the humic substances with a cationic surfactant, hexadecyltrimethylammonium bromide (CTAB). This generated a hydrophobic species that was subsequently incorporated (solubilized) into the micelles of a non-ionic surfactant polyethylene glycol, tert-octylphenyl ether (Triton X-114). The FI method for HA and FA determination was developed by injection of 100 microl of the extracted surfactant-rich phase using an HPLC pump with spectrophotometric detection at 350 nm. A 50 ml sample solution preconcentration allowed an enrichment factor of 167. The limit of detection (LOD) obtained under the optimal conditions was 5 microg l(-1). The precision for ten replicate determinations at 0.2 mg l(-1) HA was 3.1% relative standard deviation (RSD), calculated from the peak heights. The calibration using the preconcentration system for HA and FA was linear with a correlation coefficient (r2) of 0.9997 at levels near the detection limits up to at least 1 mg l(-1). The method was successfully applied to the determination of HA and FA in natural water samples (river water).