Trimethylselenonium ion determination in human urine by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry optimization of the hydride generation step.

This work describes the intricacies of the determination of the trimethylselenonium ion (TMSe) in human urine via high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). By definition, this technique requires that the separated TMSe can be online converted into a volatile compound. Literature data for the determination of TMSe via the hydride generation technique are contradictory; i.e., some authors claim that direct formation of volatile compounds is possible under reduction with NaBH4, whereas others reported that a digestion step is mandatory prior to conversion. We studied and optimized the conditions for online conversion by varying the mobile phase composition (pyridine, phosphate, and acetate), testing different reaction coils, and optimizing the hydride generation conditions, although technically no hydride (H2Se) is formed but a dimethylselenide (DMSe). The optimized conditions were used for the analysis of 64 urine samples of 16 (unexposed) volunteers and the determination of low amounts of TMSe (LOD = 0.2 ng mL-1). Total (specific gravity-corrected) selenium concentrations in the urine samples ranged from 7.9 ± 0.7 to 29.7 ± 5.0 ng mL-1 for individual volunteers. Four volunteers were characterized as TMSe producers (hINMT genotype GA) and 12 were non-producers (hINMT genotype GG). Urine of TMSe producers contained 2.5 ± 1.7 ng mL-1 of TMSe, compared to 0.2 ± 0.2 ng mL-1 for non-producers.

Arsenic metabolites; selenium; and AS3MT, MTHFR, AQP4, AQP9, SELENOP, INMT, and MT2A polymorphisms in Croatian-Slovenian population from PHIME-CROME study.

The relationships between inorganic arsenic (iAs) metabolism, selenium (Se) status, and genetic polymorphisms of various genes, commonly studied in populations exposed to high levels of iAs from drinking water, were studied in a Croatian-Slovenian population from the wider PHIME-CROME project. Population consisted of 136 pregnant women in the 3rd trimester and 176 non-pregnant women with their children (n = 176, 8-9 years old). Their exposure to iAs, defined by As (speciation) analyses of biological samples, was low. The sums of biologically active metabolites (arsenite + arsenate + methylated As forms) for pregnant women, non-pregnant women, and children, respectively were: 3.23 (2.84-3.68), 1.83 (1.54-2.16) and 2.18 (1.86-2.54) ng/mLSG; GM (95 CI). Corresponding plasma Se levels were: 54.8 (52.8-56.9), 82.3 (80.4-84.0) and 65.8 (64.3-67.3) ng/mL; GM (95 CI). As methylation efficiency indexes confirmed the relationship between pregnancy/childhood and better methylation efficiency. Archived blood and/or saliva samples were used for single nucleotide polymorphism (SNP) genotyping of arsenic(3+) methyltransferase - AS3MT (rs7085104, rs3740400, rs3740393, rs3740390, rs11191439, rs10748835, rs1046778 and the corresponding AS3MT haplotype); methylene tetrahydrofolate reductase - MTHFR (rs1801131, rs1801133); aquaporin - AQP 4 and 9 (rs9951307 and rs2414539); selenoprotein P1 - SELENOP (rs7579, rs3877899); indolethylamine N-methyltransferase - INMT (rs6970396); and metallothionein 2A - MT2A (rs28366003). Associations of SNPs with As parameters and urine Se were determined through multiple regression analyses adjusted using appropriate confounders (blood As, plasma Se, ever smoking, etc.). SNPs' influence on As methylation, defined particularly by the secondary methylation index (SMI), confirmed the 'protective' role of minor alleles of six AS3MT SNPs and their haplotype only among non-pregnant women. Among the other investigated genes, the carriers of AQP9 (rs2414539) were associated with more efficient As methylation and higher urine concentration of As and Se among non-pregnant women; poorer methylation was observed for carriers of AQP4 (rs9951307) among pregnant women and SELENOP (rs7579) among non-pregnant women; MT2A (rs28366003) was associated with higher urine concentration of AsIII regardless of the pregnancy status; and INMT (rs6970396) was associated with higher As and Se concentration in non-pregnant women. Among confounders, the strongest influence was observed for plasma Se; it reduced urine AsIII concentration during pregnancy and increased secondary methylation index among non-pregnant women. In the present study of populations with low As exposure, we observed a few new As-gene associations (particularly with AQPs). More reliable interpretations will be possible after their confirmation in larger populations with higher As exposure levels.

Exceptions in patterns of arsenic compounds in urine of acute promyelocytic leukaemia patients treated with As2O3.

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As2O3/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15-25% of As(III), 35-55% of DMA, 25-30% of MMA and 1-5% of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2% As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4% of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40%) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.

Bioaccumulation of arsenic species in rays from the northern Adriatic Sea.

The difference in arsenic concentration and speciation between benthic (Pteromylaeus bovinus, Myliobatis aquila) and pelagic rays (Pteroplatytrygon violacea) from the northern Adriatic Sea (Gulf of Trieste) in relation to their size (age) was investigated. High arsenic concentrations were found in both groups with tendency of more efficient arsenic accumulation in benthic species, particularly in muscle (32.4 to 362 µg·g-1 of total arsenic). This was attributed to species differences in arsenic access, uptake and retention. In liver most arsenic was present in a form of arsenobetaine, dimethylarsinic acid and arsenoipids, whereas in muscle mainly arsenobetaine was found. The good correlations between total arsenic/arsenobetaine and size reflect the importance of accumulation of arsenobetaine with age. Arsenobetaine is an analogue of glycine betaine, a known osmoregulator in marine animals and both are very abundant in mussels, representing an important source of food for benthic species P. bovinus and M. aquila.

Risk of arsenic exposure from drinking water and dietary components: implications for risk management in rural Bengal.

This study investigates the risk of arsenic (As) exposure to the communities in rural Bengal, even when they have been supplied with As safe drinking water. The estimates of exposure via dietary and drinking water routes show that, when people are consuming water with an As concentration of less than 10 µg L(-1), the total daily intake of inorganic As (TDI-iAs) exceeds the previous provisional tolerable daily intake (PTDI) value of 2.1 µg day(-1) kg(-1) BW, recommended by the World Health Organization (WHO) in 35% of the cases due to consumption of rice. When the level of As concentration in drinking water is above 10 µg L(-1), the TDI-iAs exceeds the previous PTDI for all the participants. These results imply that, when rice consumption is a significant contributor to the TDI-iAs, supplying water with an As concentration at the current national drinking water standard for India and Bangladesh would place many people above the safety threshold of PTDI. We also found that the consumption of vegetables in rural Bengal does not pose a significant health threat to the population independently. This study suggests that any effort to mitigate the As exposure of the villagers in Bengal must consider the risk of As exposure from rice consumption together with drinking water.

Chemical composition of natural sea salt from the Secovlje salina (Gulf of Trieste, northern Adriatic).

In our research the concentrations of major and minor elements were determined in natural sea salts from the Se~ovlje salina (Piran salts, Slovenia) and compared to those of selected samples of commercially available unrefined salts with different geographical origins (Croatia, Austria, Italy, Portugal, India, and Pakistan). In the case of major element contents such as sodium (Na), iron (Fe), aluminum (Al), manganese (Mn), and titanium (Ti) many similarities were observed among the analysed salt samples. On the other hand, Piran salts are characterized by lower silicon (Si) values. Among the salts from the Secovlje salina, the salt with the trade name Piran salt has a higher Mg content while Flower of salt has a lower concentration of calcium (Ca). In Slovenian samples the majority of trace element values were lower than 0.5 µg g-1, which was comparable to the results from commercially available unrefined salts. The salt composition differences observed indicate area-specific signatures related to geographic origin and diverse salt production processes. The quality of the studied salt samples is in accordance with standards established by the Codex Alimentarius Commission and the Piran salts are also suitable regarding issues of national food control.

Human urinary arsenic species, associated exposure determinants and potential health risks assessed in the HBM4EU Aligned Studies.

The European Joint Programme HBM4EU coordinated and advanced human biomonitoring (HBM) in Europe in order to provide science-based evidence for chemical policy development and improve chemical management. Arsenic (As) was selected as a priority substance under the HBM4EU initiative for which open, policy relevant questions like the status of exposure had to be answered. Internal exposure to inorganic arsenic (iAs), measured as Toxic Relevant Arsenic (TRA) (the sum of As(III), As(V), MMA, DMA) in urine samples of teenagers differed among the sampling sites (BEA (Spain) > Riksmaten adolescents (Sweden), ESTEBAN (France) > FLEHS IV (Belgium), SLO CRP (Slovenia)) with geometric means between 3.84 and 8.47 µg/L. The ratio TRA to TRA + arsenobetaine or the ratio TRA to total arsenic varied between 0.22 and 0.49. Main exposure determinants for TRA were the consumption of rice and seafood. When all studies were combined, Pearson correlation analysis showed significant associations between all considered As species. Higher concentrations of DMA, quantitatively a major constituent of TRA, were found with increasing arsenobetaine concentrations, a marker for organic As intake, e.g. through seafood, indicating that other sources of DMA than metabolism of inorganic As exist, e.g. direct intake of DMA or via the intake of arsenosugars or -lipids. Given the lower toxicity of DMA(V) versus iAs, estimating the amount of DMA not originating from iAs, or normalizing TRA for arsenobetaine intake could be useful for estimating iAs exposure and risk. Comparing urinary TRA concentrations with formerly derived biomonitoring equivalent (BE) for non-carcinogenic effects (6.4 µg/L) clearly shows that all 95th percentile exposure values in the different studies exceeded this BE. This together with the fact that cancer risk may not be excluded even at lower iAs levels, suggests a possible health concern for the general population of Europe.

How to use human biomonitoring in chemical risk assessment: Methodological aspects, recommendations, and lessons learned from HBM4EU.

One of the aims of the European Human Biomonitoring Initiative, HBM4EU, was to provide examples of and good practices for the effective use of human biomonitoring (HBM) data in human health risk assessment (RA). The need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/poly-fluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.

Arsenic trioxide versus tetraarsenic oxide in biomedical research: misunderstandings and misinterpretations.

This work presents an analytical chemist's view on the sometimes unconscious use of arsenic trioxide in (bio)medical research. Arsenic trioxide is a frequently used chemical in cancer treatment research and its action to various malignant cells has been extensively studied and published. Unfortunately some research articles show trivial errors with regards to background knowledge of the chemical, handling the chemical, experimental design and interpretation of results like e.g. in a range of articles comparing advantages of tetraarsenic oxide over arsenic trioxide (dimeric/monomeric) although the dissolution of both yields the same active compound (HAsO(2)). To fully understand the implications of these errors we will highlight some of them with the intent to harmonize future work in this field.

As(2)O (3) oxidation by vitamin C: cell culture studies.

The ability of As(2)O(3) to induce apoptosis in various malignant cell lines has made it a potential treatment agent for several malignancies. In this study the chemical stability of As(2)O(3) (As(III)) in cell-free growth media with various compositions was studied (MEM with different amount of amino acids and DMEM). Special attention was given to evaluate the influence of serum (FBS; fetal bovine serum) absence and vitamin C addition on the oxidation of As(III) to As(V) in cell-free growth media. FBS is an important source of antioxidants and vitamin C (ascorbic acid) is acting as a prooxidant in millimolar concentrations. Media were incubated with As(III) (0.6, 2 and 7 µmol l(-1)) up to 72 h. Experiments were performed at 37°C in light or/and in the dark, with or without added serum (10%) or vitamin C (1.4, 0.14 mM). Metabolites were followed with high-performance liquid chromatography directly coupled to a hydride generation-atomic fluorescence spectrometry system. After 72 h up to 30% of As(III) was transformed into As(V) in MEMs and up to 35% in DMEM when exposed in dark. Light had no influence on transformations in MEMs, but changed the situation dramatically in DMEM where almost all As(III) was oxidized to As(V) after 72 h when exposed to light. Except for some faster oxidation rate the absence of FBS had little effect on the transformation rate in all media. The most visible impact on As(III) oxidation was observed by addition of vitamin C. Addition of vitamin C (1.4 mM) transformed almost all As(III) to As(V) within 72 h. In lower concentrations (0.14 mM) a pro-oxidative effect was still observed reaching approximately 60% oxidation of As(III) during 72 h. All oxidation processes could be explained by pseudo first order reaction kinetics, yielding reaction rates increasing with initial As(III) concentration and vitamin C concentration whereas the FBS content additionally increased the As(III) oxidation rate in the DMEM (light). The temporal oxidation of As(III) to As(V) in various cell-free growth media necessitates routine checking of the valence state of arsenic during cell culture experiments and the results of biological effects attributed to As(III) should be interpreted with caution. Special attention is needed particularly in cases with vitamin C which was acting pro-oxidatively in all conditions examined.

Multiple kinetic Langmuir modeling to predict the environmental behaviour of As(v) in soils.

A soil with a relatively high Fe content (2.82% [w/w]) was loaded for up to one year with As(v) by equilibrating it with a solution containing 1000 mg l(-1) As(v) at a soil mass-to-solution ratio of 0.1 kg l(-1). The incorporation of As(v) into the soil and its distribution over the soil phases were monitored by sampling at strategic time intervals using an operationally defined five-step sequential extraction procedure (Wenzel et al., Anal. Chim. Acta, 2001, 436, 309) and subsequent As measurement. A multiple kinetic Langmuir model was developed to retrieve the dynamic parameters (adsorption and desorption rate constants, capacities and Langmuir equilibrium constants) for each of the soil phases by numerical fitting of the experimental adsorption data to the model. Under the equilibration conditions used the adsorption rate constants for all five operationally defined soil phases were very similar but the desorption rate constants decreased by a factor of ca. 150 from soil phase 1 (non-specifically sorbed As) to 5 (residual phases). This implies that As(v) incorporation "deeper" into the soil leads to stronger binding which is associated with the Langmuir equilibrium constants (adsorption rate constants/desorption rate constants). Equilibration of the soil with As(v) was complete in ca. 10 days with As(v) predominantly bound to soil phase 2 (specifically sorbed As) and soil phase 3 (amorphous and poorly crystalline hydrous oxides). X-Ray absorption spectroscopy techniques revealed that these binding characteristics may be related to adsorption of As(v) on Si- and/or Al-containing structures and natural hydrous iron oxide (HFO) surface sites, respectively. Since the model is independent of the initial As(v) concentration in the solution and the soil mass-to-solution ratio, the behaviour of the thus characterized soil-As(v) system can be predicted for a range of conditions. Simulations showed that in an accidental As(v) spill the soil studied would actively scavenge As(v) by instantaneous adsorption onto all soil phases followed by redistribution of As(v) from weaker binding sites to stronger ones over time.

Arsenic speciation and elemental composition of rice samples from the Slovenian market.

A survey of highly toxic arsenic compounds, together with some other elements was carried out on 40 polished rice samples (white, basmati and parboiled) and 10 brown rice samples from the Slovenian market. The average total As concentration was 157 ± 60 µg kg-1; highest levels were found in parboiled and brown rice and lowest in basmati. The average inorganic As concentration was 90 ± 35 µg kg-1. Dimethylarsinic acid and monomethylarsonic acid, which also exhibit high toxicity levels in some cases constitute >50% of total arsenic and might deserve more attention. Contrary to other foods, the total arsenic concentration in rice may even be a better health hazard indicator than the inorganic arsenic concentration. Elemental analysis of rice revealed large differences between polished and brown rice, especially for Mg, Mn, P, Fe and K, which were 2-4 times higher in brown rice than in polished rice.

Spatial and temporal distribution of trace elements in Padina pavonica from the northern Adriatic Sea.

Chemical pollution is a major environmental concern especially in coastal areas, having adverse impacts on marine organisms and ecosystem services. Macroalgae can accumulate trace elements, but available studies are restricted to a limited number of elements and species. The goal of this research was to assess, seasonally, the concentrations of 22 elements in the brown alga Padina pavonica from monitoring sampling sites in Slovenian waters. The concentration of most elements in thalli differed significantly between spring and autumn, with generally higher levels in autumn samples. However, it was not possible to correlate these concentrations with the ecological status of macroalgae. The maximum values set by European regulations for the potentially hazardous As, Cd and Hg in food and feed were never exceeded, while Pb concentrations were slightly higher. The results show that P. pavonica can act as an effective bioindicator of chemical pollution.

Differential role of cathepsins B and L in autophagy-associated cell death induced by arsenic trioxide in U87 human glioblastoma cells.

Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

Assessment of physical leaching processes of some elements in soil upon ingestion by continuous leaching and modeling.

The physical processes controlling the desorption of some elements (B, Cd, Co, Mn, Ni, and Sr) from soils in a continuous leaching system representing the human stomach are investigated here by fitting experimental leaching data to a mathematical particle diffusion model. Soil samples (50 mg) from Cornwall, UK, contained in a flow-through extraction chamber (ca. 6.5 mL) were intimately contacted with artificial gastric solution at various flow rates (0.42-1.42 mL min(-1)) for up to ca. 4 h, followed by analysis of the fractions collected with inductively coupled plasma mass spectrometry (ICP-MS). The leaching profiles of the various elements were fitted to a mathematical model incorporating two mass transfer processes (liquid film diffusion and apparent solid phase diffusion) to determine the effective external mass transfer coefficient (beta) and the apparent intraparticle soil diffusion coefficient (D(a)). A system of partial differential equations was solved numerically with a finite difference discretization of the computational domain allowing the rate limiting physical desorption process(es) for each element to be determined. The (thermodynamic) driving force of the leaching process is defined by the distribution coefficient (K(d0)) between soil and leachant. Although the K(d0) values investigated are very similar (ca. 6-15 L kg(-1)) for the elements studied with the exception of B (ca. 2.7 L kg(-1)), the leaching profiles are very different due to diffusion-limited processes. The elements may be classified as limited by beta (B, Sr, and Cd), by D(a) (Co, and Mn) or by beta and D(a) (Ni). This results in quantifiable parameters for the liability of elements in soil upon ingestion which may be implemented in future risk assessment protocols.

Trace elements and ALAD gene polymorphisms in general population from three uranium legacy sites - A case study in Kyrgyzstan.

At three uranium (U) legacy sites in Kyrgyzstan, namely, Kadji Sai, Mailuu-Suu and Sumsar, an initial human bio-monitoring programme was introduced as a complementary activity to environmental impact studies in these areas. The aim was to assess trace element (TE) contents in blood and genetic susceptibility for Pb as one of the contaminants. The programme included the determination of 9 TE in blood samples from 123 residents living permanently in this environment. The analyses included U and the potentially toxic TE, lead (Pb), cadmium, mercury (Hg), and arsenic (As), together with essential elements iron (Fe), copper, selenium (Se) and manganese (Mn). TE were analysed by inductively coupled plasma mass spectrometry (ICPMS) and genetic background effect by three single nucleotide polymorphisms (SNPs) of delta-aminolevulinic acid dehydratase (ALAD; rs1805313, rs818708, rs1800435) genotyped by quantitative polymerase chain reaction (qPCR). The obtained results were generally similar to literature reference values obtained from the U non-exposed environments. However, some significant findings indicated elevated levels of certain contaminants typical of the studied environment (U, Pb). Several essential (Se, Mn) and toxic TE (Pb, Hg, As, U) in blood showed statistically significant differences among the studied areas. All areas showed diminished Fe blood levels. Altogether, this indicated specific and different environmental conditions at three industrial legacy sites for U milling and processing along with the accompanying chemical (pollutant) elements. Blood U concentrations were slightly higher at Mailuu-Suu, known for elevated technogenic and naturally occurring U. At Sumsar, the distribution of elevated blood Pb concentrations indicated an airborne source of pollution that was different from the anticipated aqueous exposure pathway. Pb blood variability was found associated with ALAD polymorphisms (SNPs rs1805313, rs1800435). Results are confirming that human data will be a useful and scientifically important additional tool for environmental impact assessment studies at industrial legacy sites in Kyrgyzstan.

Results of the first national human biomonitoring in Slovenia: Trace elements in men and lactating women, predictors of exposure and reference values.

The first national human biomonitoring in Slovenia surveyed cadmium (Cd), lead (Pb), mercury (Hg), arsenic (As), manganese (Mn), selenium (Se), copper (Cu) and zinc (Zn) in a childbearing population (18-49 years) selected from lactating primiparous women and men (N = 1084). The overall aim was to estimate trace elements' levels and geographical variations in order to identify sources of possible exposures and set the national reference values. The study population was selected evenly from 12 study areas across Slovenia, including rural, urban and known or potentially contaminated environments. Within 6-8 weeks after delivery, venous blood, spot urine, scalp hair and breast milk samples were collected to determine the selected elements. The data analysis included descriptive statistics and multiple linear regression using elemental concentrations in biological matrices, questionnaire data and environmental datasets. Essential elements showed no significant deficiencies or excessive levels in the study population and were largely determined by sex and/or the participating women's physiological status (postpartum, lactation), as well as by certain dietary sources. Toxic elements' levels were mainly below the levels considered to present increased health risk. Lifestyle and nutritional habits appeared as significant determinants of exposure to Cd (smoking and game meat consumption), Hg (seafood and amalgam fillings), As (seafood) and Pb (alcohol consumption, smoking, game meat consumption and type of water supply). A distinctive geographical pattern was confirmed, due to past mining activities combined with naturally elevated background levels in the cases of Pb (Mezica Valley), Hg (Idrija and Posocje) and As exposure (Zasavje). Increased seafood consumption in the coastal study area contributed to higher Hg and As (arsenobetaine) levels. Extensive sample size database accompanied with life-style and environmental data improved the prediction of exposure patterns, set the reference values for the childbearing population living in Slovenia, and provided a strong basis for evaluating spatial and temporal trends in exposure. To our best knowledge, this is the first study to establish reference values for lactating primiparous women.

Migration of arsenic from old tailings ponds--a case study on the King Edward Mine, Cornwall, UK.

A methodology is presented to study the physico-chemical processes in old tailings ponds using an array of analytical-physical chemistry approaches. A case study was conducted on the sorption/desorption behaviour of arsenic in tailings pond 2406, at the King Edward Mine (KEM) in Cornwall, UK. The tailings pond was in operation from approximately 1907 to 1921. The methodology involves two principal stages: (1) sequential extraction followed by subsequent arsenic species determination to characterise the material with regards to the association of arsenic with soil phases and identification of As (III/V) in the easily accessible soil phase; (2) batch contacting/equilibrating the tailings pond material with As(III/V), followed by a similar procedure as in stage 1 to establish the material's As(III/V) phase distribution kinetics/thermodynamics. By extrapolating the data from present day samples we infer past and future elemental mobility. From this study it is concluded that adsorption and desorption from tailings material is a rapid process for the most unstable soil phases (non-specific and specific) and a slow process for the more stable phases (poorly crystalline and well crystalline). The hypothetical application of this conclusion to the tailings from dam 2406 is that, during the initial phases of the dam's creation (ca. 100 years ago), when arsenic was both in solution and bound to mineralogical components, arsenic must have dispersed into the environment as a result of slow As(V) adsorption/phase distribution processes. Aging of the tailings material sees the movement of the arsenic to the more stable soil phases, producing a situation that is seen at present day.

Uptake and biotransformation of arsenate in the lichen Hypogymnia physodes (L.) Nyl.

The uptake and metabolism of arsenate, As(V), as a function of time and concentration were examined in the lichen Hypogymnia physodes (L.) Nyl. Lichen thalli were exposed to As(V) in the form of a solution. Exponential uptake of As(V) from 4 microg mL(-1) As(V) solution was accompanied by constant arsenite, As(III), excretion back into the solution. Arsenate taken up into the lichens from 0, 0.1, 1, 10 microg mL(-1) As(V) solutions was partially transformed into As(III), dimethylarsinic acid (DMA) and (mono)methylarsonic acid (MA). 48 h after exposure, the main arsenic compound in the lichens was DMA in 0.1, As(III) in 1 and As(V) in 10 microg mL(-1) treatment. The proportion of methylated arsenic compounds decreased with increasing arsenate concentration in the exposure solution. These results suggest that at least two types of As(V) detoxification exist in lichens; arsenite excretion and methylation.

Comparative effects of arsenite (As(III)) and arsenate (As(V)) on whole plants and cell lines of the arsenic-resistant halophyte plant species Atriplex atacamensis.

Whole plants and hypocotyl-derived calli of the halophyte plant species Atriplex atacamensis were exposed to 50 µM arsenate (As(V)) or 50 µM arsenite (As(III)). At the whole plant level, As(III) was more toxic than As(V): it reduced plant growth, stomatal conductance, photosystem II efficiency while As(V) did not. In roots, As accumulated to higher level in response to As(III) than in response to As(V). Within root tissues, both arsenate and arsenite were identified in response to each treatment suggesting that oxidation of As(III) may occur. More than 40% of As was bound to the cell wall in the roots of As(V)-treated plants while this proportion strongly decreased in As(III)-treated ones. In leaves, total As and the proportion of As bound to the cell wall were similar in response to As(V) and As(III). Non-protein thiol increased to higher extent in response to As(V) than in response to As(III) while ethylene synthesis was increased in As(III)-treated plants only. Polyamine profile was modified in a contrasting way in response to As(V) and As(III). At the callus level, As(V) and As(III) 50 µM did not reduce growth despite an important As accumulation within tissues. Calli exposed to 50 µM As did not increase the endogenous non-protein thiol. In contrast to the whole plants, arsenite was not more toxic than arsenate at the cell line level and As(V)-treated calli produced higher amounts of ethylene and malondialdehyde. A very high dose of As(V) (1000 µM) strongly reduced callus growth and lead to non-protein thiols accumulation. It is concluded that As(III) was more toxic than As(V) at the plant level but not at the cellular level and that differential toxicity was not fully explained by speciation of accumulated As. Arsenic resistance in A. atacamensis exhibited a cellular component which however did not reflect the behavior of whole plant when exposed to As(V) or As(III).