Assessment of dietary Selenium and its role in Mercury fate in cultured fish rainbow trout with two sustainable aquafeeds.

This study enhances the current limited understanding of the interaction between mercury (Hg) and selenium (Se) species in fish. Rainbow trout (Oncorhynchus mykiss), a model aquaculture fish, was exposed to Hg and Se species through controlled dietary conditions. Over a 6-month feeding trial, the impact of dietary Se on Hg bioaccumulation in fish, including flesh, brain, and liver, was tracked. Twelve dietary conditions were tested, including plant-based diets (0.25 µgSe g-1) and tuna byproduct diets (0.25 µgHg g-1, 8.0 µgSe g-1) enriched with methylmercury and/or Se as selenite or selenomethionine. The tuna byproduct diet resulted in lower Hg levels than the plant-based diets, with muscle Hg content below the European Commission's safe threshold. This study highlights the significant impact of specific Se compounds in the diet, particularly from tuna-based aquafeed, on Hg bioaccumulation. These promising results provide a strong recommendation for future use of fisheries byproducts in sustainable aquafeeds.

Iodine distribution and volatilization in contrasting forms of forest humus during a laboratory incubation experiment.

Radionuclides 129I (t1/2 = 15.7 × 106 years) and 131I (t1/2 = 8.02 days) are both introduced into the environment as a result of nuclear human activities. Environmental transfer pathways and fluxes between and within ecosystems are essential information for risk assessment. In forest ecosystems, humus degradation over time could result in re-mobilization and then downward migration and/or volatilization of intercepted 129I. In order to estimate the scale of these processes, humus (mull and moder forms) sampled under deciduous and coniferous forests were spiked with 125I- (t1/2 = 59.4 days), as a surrogate for 129I, in order to study the evolution of its water-soluble and organic fractions as well as the volatilization rate during humus degradation at laboratory scale. To our knowledge, this is the first time that interactions between iodine and contrasting forms of forest humus have been investigated. The evolution of native stable iodine (127I) pools in unspiked humus was also studied. The nature of the humus' organic matter appears to be a factor that impacts on the proportions of water-soluble and organic fractions of iodine and on their evolution. Iodine-125 was mainly organically bound (fraction for mulls and moders: ∼54-59 and 41-49%, respectively) and no clear evolution was observed within the 4-month incubation period. A large decrease in 125I water-solubility occurred, being more marked for mull (from ∼14-32 to 3-7%) than for moder (from ∼21-37 to 7-19%) humus. By contrast, a significant fraction was not extractible (∼38-43%) and varied in inverse proportion to the water-soluble fraction, suggesting a stabilization of iodine in humus after wet deposit. The nature of the humus organic matter also impacted on 125I volatilization. Although of the same order of magnitude, the total volatilization of 125I was higher for moders (∼0.039-0.323%) than for mulls (∼0.015-0.023%) within the 4-month incubation period. Volatilization rates for mulls were correlated with the water-soluble fraction, implying that volatilization of 125I could occur from the humus solution. Our results showed that humus is thus a zone of iodine accumulation by association with organic matter and that potential losses by lixiviation are significantly more important compared to volatilization.

First Time Identification of Selenoneine in Seabirds and Its Potential Role in Mercury Detoxification.

Birds are principally exposed to selenium (Se) through their diet. In long-lived and top predator seabirds, such as the giant petrel, extremely high concentrations of Se are found. Selenium speciation in biota has aroused great interest in recent years; however, there is a lack of information about the chemical form of Se in (sea)birds. The majority of publications focus on the growth performance and antioxidant status in broilers in relation to Se dietary supplementation. The present work combines elemental and molecular mass spectrometry for the characterization of Se species in wild (sea)birds. A set of eight giant petrels (Macronectes sp.) with a broad age range from the Southern Ocean were studied. Selenoneine, a Se-analogue of ergothioneine, was identified for the first time in wild avian species. This novel Se-compound, previously reported in fish, constitutes the major Se species in the water-soluble fraction of all of the internal tissues and blood samples analyzed. The levels of selenoneine found in giant petrels are the highest reported in animal tissues until now, supporting the trophic transfer in the marine food web. The characterization of selenoneine in the brain, representing between 78 and 88% of the total Se, suggests a crucial role in the nervous system. The dramatic decrease of selenoneine (from 68 to 3%) with an increase of Hg concentrations in the liver strongly supports the hypothesis of its key role in Hg detoxification.

Atmospheric iodine, selenium and caesium depositions in France: II. Influence of forest canopies.

Estimation of the canopy influence on atmospheric inputs of iodine (I), selenium (Se) and caesium (Cs) in terrestrial ecosystems is an essential condition for appropriate biogeochemical models. However, the processes involved in rain composition modifications after its passage through forest canopy have been barely studied for these elements. We monitored I, Se and Cs concentrations in both rainfall and throughfall of fourteen French forested sites throughout one year, and estimated dry deposition and canopy exchange fluxes for these elements, as well as speciation of I and Se. Comparison of rainfall and throughfall elemental composition highlighted an important impact of forest canopy on both (i) concentrations and fluxes of I, Se and Cs, and (ii) I and Se species. For the three elements, most of their throughfall concentrations were higher than corresponding rainfall. The increase of throughfall elemental fluxes was mostly due to dry deposition for I and Se although the canopy exchange model revealed some sorption within the canopy in most cases; for Cs, foliage leaching was most influencing. Regarding speciation, iodine species in rainfall were highly modified by forest canopy with an important increase of unidentified I proportion in throughfall (on average 49 and 82% in rainfall and throughfall, respectively), possibly due to washoff of dry deposition and/or to transformation into organic forms. Similarly, while rainfall was composed of 26-54% of inorganic Se, inorganic species were undetectable in throughfall. This dataset represents key information to improve modelling of I, Se and Cs cycling within forest ecosystems.

Atmospheric iodine, selenium and caesium depositions in France: I. Spatial and seasonal variations.

The spatial distribution and seasonal variations of atmospheric iodine (I), selenium (Se) and caesium (Cs) depositions remain unclear and this precludes adequate inputs for biogeochemical models. We quantified total concentrations and fluxes of these elements in rainfalls from 27 monitoring sites in France with contrasted climatic conditions; monthly measurements were taken over one year (starting in 2016/09). Since speciation of I and Se can impact their behaviour in the environment, analysis of their inorganic compounds was also conducted. Our results showed that annual I concentrations in rainfall were much higher than those of Se and Cs (annual means = 1.56, 0.044 and 0.005 µg L-1, respectively). The annual iodine concentrations were highly positively correlated with those of marine elements (i.e. Na, Cl and Mg), involving higher I concentrations under oceanic climate than for transition, continental and mountainous ones. Furthermore, common patterns were found between Se concentrations and both marine and terrestrial components consistent with the various sources of Se in atmosphere. The association of Cs with two anthropogenic components (i.e. NH4+ and NO3-) used in agriculture supports the hypothesis of its terrestrial origin (i.e. from atmospheric dusts) in rainfall. We found higher rainfall concentrations of I during the warmest months for all climates. However, no specific seasonal trend occurred for Se and Cs. On annual average, rainfall contained mostly unidentified selenium compounds (inorganic Se proportions = 25-54%) and equal proportions of inorganic and unidentified I compounds. Concentrations of iodate were higher under oceanic climate consistent with an iodine marine-origin.

Assessment of Hg contamination by a Chlor-Alkali Plant in riverine and coastal sites combining Hg speciation and isotopic signature (Sagua la Grande River, Cuba).

Chlor-alkali plants (CAP) are recognized as major sources of mercury (Hg) in the environment. In this work, Hg concentration, speciation and isotopic signature were determined in sediments and biota (fish and oyster) from Sagua La Grande River (SG River) and the adjacent coastal zone in the vicinity of a CAP (Cuba). High Hg concentrations in surface sediments (up to 5072 ng g-1), mainly occurring as inorganic Hg, decrease with the distance from the CAP along the SG River and seaward. Meanwhile, Hg concentration and speciation in riverine catfish (Claria gariepinus) muscle (1093 ± 319 ng g-1, ˜70% as MeHg) and coastal oysters (Crassostrea rizophorae) (596 ± 233 ng g-1, ˜50% as MeHg) indicate a direct impact from CAP. Hg isotopic signature in sediments, following both mass dependent (MDF) and mass independent fractionation (MIF), exhibits a clear binary mixing between CAP pollution (+0.42‰, δ202Hg; -0.18‰, Δ201Hg) and regional background end-member (˜ -0.49‰, δ202Hg; +0.01‰, Δ201Hg). The combination of speciation and isotopic information in biota and sediments allows to trace Hg contamination pathways from contaminated sediments to the biota, establishing the importance of both methylation and demethylation extent in both river and coastal sites before Hg species bioaccumulation.

Iodine budget in forest soils: Influence of environmental conditions and soil physicochemical properties.

Due to its longevity, radioisotope 129I is a health concern following potential releases in the environment which raises questions about residence and exposure times relevant for risk assessments. We determined 127I concentrations (as a surrogate for 129I) in a series of French forest soils (i.e. litters, humus and mineral soils) under different vegetation and climate conditions in order to identify the major processes affecting its accumulation and persistence in the soil column. The input fluxes linked to rainfall, throughfall and litterfall were also characterized. Main results obtained showed that: (i) rainfall iodine concentrations probably influenced those of litterfall through absorption by leaves/needles returning to the ground; (ii) throughfall was the major iodine input to soils (mean = 83%), compared to litterfall (mean = 17%); (iii) humus represented a temporary storage of iodine from atmospheric and biomass deposits; (iv) iodine concentrations in soils depended on both the iodine inputs and the soil's ability to retain iodine due to its organic matter, total iron and aluminium concentrations; (v) these soil properties were the main factors influencing the accumulation of iodine in the soil column, resulting in residence times of 419-1756 years; and (vi) the leaching of iodine-containing organic matter dissolved in soil solution may be an important source of labile organic iodine for groundwater and streams.

Iodine distribution and cycling in a beech (Fagus sylvatica) temperate forest.

Radioiodine is of health concerns in case of nuclear events. Possible pathways and rates of flow are essential information for risk assessment. Forest ecosystems could influence the global cycle of long-lived radioiodine isotope (129I) with transfer processes similar to stable isotope (127I). Understanding iodine cycling in forest involves study of the ecosystem as a whole. In this context, we determined the 127I contents and distribution in soil, tree compartments and atmospheric inputs during a three years in situ monitoring of a temperate beech forest stand. The iodine cycle was first characterized in terms of stocks by measuring its concentrations in: tree, litterfall, humus, soil, rainfall, throughfall, stemflow and soil solutions. Main annual fluxes (requirement, uptake and internal transfers) and forest input-output budget were also estimated using conceptual model calculations. Our findings show that: (i) soil is the main I reservoir accounting for about 99.9% of ecosystem total stock; (ii) iodine uptake by tree represents a minor fraction of the available pool in soil (<0.2%); (iii) iodine allocation between tree compartments involves low immobilization in wood and restricted location in the roots; (iv) translocation of excess iodine towards senescing foliage appears as an elimination process for trees, and (v) litterfall is a major pathway in the I biological cycling. In our soil conditions, the input - output budget shows that the ecosystem behaves as a potential source of I for groundwater.

Influence of Se concentrations and species in hydroponic cultures on Se uptake, translocation and assimilation in non-accumulator ryegrass.

The success of biofortification and phytoremediation practices, addressing Se deficiency and Se pollution issues, hinges crucially on the fate of selenium in the plant media in response to uptake, translocation and assimilation processes. We investigate the fate of selenium in root and shoot compartments after 3 and 6 weeks of experiment using a total of 128 plants grown in hydroponic solution supplied with 0.2, 2, 5, 20 and 100 mg L-1 of selenium in the form of selenite, selenate and a mixture of both species. Selenate-treated plants exhibited higher root-to-shoot Se translocation and total Se uptake than selenite-treated plants. Plants took advantage of the selenate mobility and presumably of the storage capacity of leaf vacuoles to circumvent selenium toxicity within the plant. Surprisingly, 28% of selenate was found in shoots of selenite-treated plants, questioning the ability of plants to oxidize selenite into selenate. Selenomethionine and methylated organo-selenium amounted to 30% and 8% respectively in shoots and 35% and 9% in roots of the identified Se, suggesting that selenium metabolization occurred concomitantly in root and shoot plant compartments and demonstrating that non-accumulator plants can synthesize notable quantities of precursor compound for volatilization. The present study demonstrated that non-accumulator plants can develop the same strategies as hyper-accumulator plants to limit selenium toxicity. When both selenate and selenite were supplied together, plants used selenate in a storage pathway and selenite in an assimilation pathway. Plants might thereby benefit from mixed supplies of selenite and selenate by saving enzymes and energy required for selenate reduction.

Field study of time-dependent selenium partitioning in soils using isotopically enriched stable selenite tracer.

A better understanding of selenium fate in soils at both short and long time scales is mandatory to consolidate risk assessment models relevant for managing both contamination and soil fertilization issues. The purpose of this study was thus to investigate Se retention processes and their kinetics by monitoring time-dependent distribution/speciation changes of both ambient and freshly added Se, in the form of stable enriched selenite-77, over a 2-years field experiment. This study clearly illustrates the complex reactivity of selenium in soil considering three methodologically defined fractions (i.e. soluble, exchangeable, organic). Time-dependent redistribution of Se-77 within solid-phases having different reactivity could be described as a combination of chemical and diffusion controlled processes leading to its stronger retention. Experimental data and their kinetic modeling evidenced that transfer towards less labile bearing phases are controlled by slow processes limiting the overall sorption of Se in soils. These results were used to estimate time needed for (77)Se to reach the distribution of naturally present selenium which may extend up to several decades. Ambient Se speciation accounted for 60% to 100% of unidentified species as function of soil type whereas (77)Se(IV) remained the more abundant species after 2-years field experiment. Modeling Se in the long-term without taking account these slow sorption kinetics would thus result in underestimation of Se retention. When using models based on Kd distribution coefficient, they should be at least reliant on ambient Se which is supposed to be at equilibrium.

Stable isotope tracing: a powerful tool for selenium speciation and metabolic studies in non-hyperaccumulator plants (ryegrass Lolium perenne L.).

Selenium is both essential and toxic for mammals; the range between the two roles is narrow and not only dose-dependent but also related to the chemical species present in foodstuff. Unraveling the metabolism of Se in plants as a function of Se source may thus lead to ways to increase efficiency of fertilization procedures in selenium deficient regions. In this study, stable-isotope tracing was applied for the first time in plants to simultaneously monitor the bio-incorporation of two inorganic Se species commonly used as foodstuff enrichment sources. Occurrence and speciation of Se coming from different Se sources were investigated in root and leaf extracts of ryegrass (Lolium perenne L.), which had been co-exposed to two labeled Se species ((77)SeIV and (82)SeVI). Although the plant absorbed similar amounts of Se when supplied in the form of selenite or selenate, the results evidenced marked differences in speciation and tissues allocation. Selenite was converted into organic forms incorporated mostly into high molecular weight compounds with limited translocation to leaves, whereas selenate was highly mobile being little assimilated into organic forms. Double-spike isotopic tracer methodology makes it possible to compare the metabolism of two species-specific Se sources simultaneously in a single experiment and to analyze Se behavior in not-hyperaccumulator plants, the ICP-MS sensitivity being improved by the use of enriched isotopes.

A new methodology involving stable isotope tracer to compare simultaneously short- and long-term selenium mobility in soils.

A better understanding of Se fate in soils is required for different environmental issues, such as radioactive waste management or soil fertilization procedures. In these contexts, the mobility and speciation of Se have to be studied at both short and long terms after Se inputs. Here, we present a new methodology to monitor simultaneously the reactivity of added (isotopic enriched tracers) and ambient Se at trace level in soils by high-performance liquid chromatography inductively coupled plasma mass spectrometry (ICP-MS) following specific extractions. To do so, the collision/reaction cell of the ICP-MS instrument and the interference corrections were optimized to measure reliably the four major Se isotopes. To exemplify the method capabilities, the behaviors of added (77)Se(IV) and ambient Se were followed up in two soils submitted to an ageing process during 3 months. The solid/liquid distribution of added Se reached a steady state after 1 month while its speciation and distribution among soil solid phases were still changing after 3 months. The results clearly demonstrate that slow processes are involved in Se retention and transformation in soils. The usual short-term experiments (<1 month) performed after Se addition are thus not suitable for long-term risk assessment. Interestingly, the behavior of added Se tended to that of ambient Se, suggesting that ambient Se would be useful to infer the fate of Se input over long time scales.

Identification in human urine and blood of a novel selenium metabolite, Se-methylselenoneine, a potential biomarker of metabolization in mammals of the naturally occurring selenoneine, by HPLC coupled to electrospray hybrid linear ion trap-orbital ion trap MS.

Speciation analysis of selenium in human urine allowed for the first time the identification of a novel selenium metabolite, Se-methylselenoneine. Despite a concentration at low ppb level, its characterization was achieved after sample purification by solid phase extraction (SPE) followed by the parallel coupling of the bidimensional RP/HILIC chromatography with ICP-MS and ESI-LTQ Orbitrap MS detection. To confirm its biological significance with regards to selenoneine, the recently discovered analog of ergothioneine, and to discard the possibility of sample preparation artifacts, a new method was developed to monitor its actual presence, as well as the occurrence of its sulfur and/or non-methylated analogs, in non-preconcentrated urine and blood samples of non-supplemented humans. It consisted in a HILIC ESI-MS(3) method in high resolution mode (resolution 30 000 at m/z 400) with large isolation width windows for precursor ions. These two particular settings allowed respectively to keep observing the specific mass defect of selenium- and sulfur-containing molecules and to maintain the characteristic selenium pattern in product ions created through MS(n) fragmentations. As a result, all four metabolites were detected in blood and three of them in urine. Moreover, different ratios "methylated/non-methylated" were observed between urine and blood samples, which seemed to indicate their active metabolization. The analytical tool developed here will be of a great importance to further study the occurrence and the potential metabolic role in mammalian organelles, cells and fluids of these very particular and promising redox metabolites.

Selenate bioaccumulation and toxicity in Chlamydomonas reinhardtii: Influence of ambient sulphate ion concentration.

The aim of the present study was to investigate selenate toxicity in the unicellular green algae Chlamydomonas reinhardtii as a function of sulphate ion concentration and the relationship with intracellular bioaccumulation. The toxicity of selenate was evaluated by measuring the effect of different selenate concentrations on algal growth during a 96h exposure period. A non-linear regression according to the Hill model was used to describe the dose-effect relationship and estimate the effect concentrations (EC) of selenate. EC(50) values of 0.40[0.24-0.52]micromolL(-1) and of 3.10[1.65-4.86]micromolL(-1) of ambient selenate were obtained, at 8 and 80micromolL(-1) of sulphate ions in the medium, respectively. For non-toxic and low-level ambient selenate concentrations, bioaccumulation in presence of 80micromolL(-1) was one tenth that of 8micromolL(-1) of sulphate ions. When expressed as intracellular selenium burden, EC(50) values determined at 8 and 80micromolL(-1) of sulphate ions were not significantly different (126 and 67nmolSe.10(9)cells(-1), respectively). In conclusion, toxicity appeared to be correlated to selenate bioaccumulation which suggests that toxicity must be linked to intracellular selenium accumulation that is directly dependent on ambient sulphate ions that may compete with selenate for transport sites.

Quantitative analysis of volatile selenium metabolites in normal urine by headspace solid phase microextraction gas chromatography-inductively coupled plasma mass spectrometry.

The combination of headspace-solid phase microextraction (HS-SPME) and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS) was evaluated for the determination of volatile selenium metabolites in normal urine samples, i.e. without selenium supplementation. HS-SPME operating conditions were optimised and a sampling time of 10 min was found to be suitable for simultaneous extraction of dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). The amount of DMSe and DMDSe extracted onto fibre coating was calculated in clean matrix, i.e. Milli-Q water, on the basis of depletion experiments. When applied to normal urine samples, the developed method allowed the detection of four volatile selenium containing species, among which DMSe and DMDSe could be quantified by standard additions.

Advantages of hydride generation interface for selenium speciation in waters by high performance liquid chromatography-inductively coupled plasma mass spectrometry coupling.

This paper focuses on the analytical performance improvement of the coupled technique HPLC-ICPMS using on-line collision/reaction cell technology for selenium elemental and speciation analyses at the ng (Se) l(-1) level in aquatic environment. Collision/reaction cell operating parameters were optimised, resulting in selected conditions of 5.5 ml min(-1) H(2) and 0.5 ml min(-1) He mixture. The detection limits obtained were around 5 ng (Se) l(-1) for total analysis, and between 7 and 15 ng (Se) l(-1) depending on the species for speciation analysis. The capability of UV irradiation-hydride generation interfacing to increase detector sensitivity was also evaluated for speciation analysis. The detection limits obtained were in the range 2-8 ng (Se) l(-1) depending on the species. Moreover, such interface allowed to prevent bromine introduction to the ICPMS which is particularly convenient for selenium trace analysis in natural waters as (80)Se is preserved free from BrH interferences. The developed method was validated using certified water with low selenium content (TM Rain 95, NWRI, Canada) and applied to the analysis of different waters.

Speciation analysis of antimony in marine biota by HPLC-(UV)-HG-AFS: Extraction procedures and stability of antimony species.

Speciation analysis of antimony in marine biota is not well documented, and no specific extraction procedure of antimony species from algae and mollusk samples can be found in the literature. This work presents a suitable methodology for the speciation of antimony in marine biota (algae and mollusk samples). The extraction efficiency of total antimony and the stability of Sb(III), Sb(V) and trimethylantimony(V) in different extraction media (water at 25 and 90 degrees C, methanol, EDTA and citric acid) were evaluated by analyzing the algae Macrosystis integrifolia (0.55+/-0.04mugSbg(-1)) and the mollusk Mytilus edulis (0.23+/-0.01mugSbg(-1)). The speciation analysis was performed by anion exchange liquid chromatography (post-column photo-oxidation) and hydride generation atomic fluorescence spectrometry as detection system (HPLC-(UV)-HG-AFS). Results demonstrated that, based on the extraction yield and the stability, EDTA proved to be the best extracting solution for the speciation analysis of antimony in these matrices. The selected procedure was applied to antimony speciation in different algae samples collected from the Chilean coast. Only the inorganic Sb(V) and Sb(III) species were detected in the extracts. In all analyzed algae the sum of total antimony extracted (determined in the extracts after digestion) and the antimony present in the residue was in good agreement with the total antimony concentration determined by HG-AFS. However, in some extracts the sum of antimony species detected was lower than the total extracted, revealing the presence of unknown antimony species, possibly retained on the column or not detected by HPLC-(UV)-HG-AFS. Further work must be carried out to elucidate the identity of these unknown species of antimony.

Development of an on-line UV decomposition system for direct coupling of liquid chromatography to atomic-fluorescence spectrometry for selenium speciation analysis.

Speciation analysis of four selenium species (selenite, selenate, selenocystine, and selenomethionine) has been performed by on-line coupling of liquid chromatography (LC), UV decomposition, hydride generation (HG), and atomic-fluorescence spectrometry (AFS). Because only selenite (Se(IV)) can generate hydrides, on-line conversion of organic and inorganic selenium species is discussed. Preliminary study showed that the use of only UV light was not sufficient to reduce selenate, because no absorption is observed for this compound at the main wavelength of the low-pressure mercury lamp (253.7 nm). Thus, new conditions based on addition of a reducing reagent (I-) were developed. Mechanisms of action are proposed to explain selenium species conversions. Because of their compatibility with on-line treatment, phosphate buffers were used for chromatographic separation on an anion exchange column (Hamilton PRP-X100). Detection limits (19-60 pg Se) and repeatability of the technique were close to those obtained by conventional quadrupole ICPMS. Applications to real samples such as water and oysters are presented and emphasize the robustness of the system.

Simultaneous speciation analysis of Sb(III), Sb(V) and (CH3)3SbCl2 by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry detection (HPLC-HG-AFS): application to antimony speciation in sea water.

This paper presents an improvement for the simultaneous separation of Sb(V), Sb(III) and (CH3)3SbCl2 species by high performance liquid chromatography (HPLC) and its detection by hydride generation-atomic fluorescence spectrometry (HG-AFS). The separation was performed on an anion exchange column PRP-X100 using a gradient elution program between EDTA/KHP (potasium hydrogen phtalate) as first mobile phase and phosphate solutions solution as the second one. The chromatographic separation and the HG-AFS parameters were optimized by experimental design. The best results were obtained by using an elution program with 20 mmol l(-1) EDTA + 2 mmol(-01) KHP solution at pH 4.5, during 1.15 min, then change to 50 mmol l(-1) (NH4)2HPO4 solution at pH 8.3, switching back after 4.0 min to the first mobile phase, until 5 min, with a constant flow rate of 1.5 ml min(-1). Retention time of Sb(V), Sb(III) and trimethylantimony species were 1.22, 2.31 and 3.45 min and the detection limits were 0.13; 0.07 and 0.13 microg l(-1), respectively. Studies on the stability of this antimony species in sea water samples on the function of the elapsed time of storage in refrigerator at 4 degrees C was performed employing the optimized method. Results revealed that Sb(III) is easily oxidized within some hours to Sb(V) in sea water stored at 4 degrees C. However, when the sea water was immediately mixed with EDTA no oxidation of Sb(III) was observed up to 1 week of storage. The proposed methodology was then applied to the antimony speciation in sea water samples.