Characterization of the Plasmatic and Erythroid Multielemental Biodistribution in Childhood Obesity Using a High-Throughput Method for Size Fractionation of Metal Species.

In this chapter, we describe a metallomics method based on protein precipitation under non-denaturing conditions and further analysis by inductively coupled plasma mass spectrometry for high-throughput metal speciation in plasma and erythrocyte samples. This methodology enables to study the total multielemental profile of these biological matrices, as well as to quantify the metal fractions conforming the metallometabolome and the metalloproteome. Furthermore, the analytical coverage comprises several essential and toxic metal elements, namely aluminum, arsenic, cadmium, cobalt, chromium, copper, iron, lithium, manganese, molybdenum, nickel, lead, selenium, vanadium, and zinc. Altogether, the metallomics method here proposed represents an excellent approach to comprehensively characterize the metal biodistribution in human peripheral blood, which would enable to decipher the role of metal homeostasis in health and disease, and particularly in childhood obesity.

Impact of the SARS-CoV-2 lockdown measures in Southern Spain on PM10 trace element and gaseous pollutant concentrations.

Trace element concentrations within PM10, gaseous pollutants (NO2 and SO2), and PM10 levels were studied during the Covid-19 lockdown at a regional level in Southern Spain (Andalusia). Pollutant concentrations were compared considering different mobility periods (pre-lockdown, lockdown, and relaxation) in 2020 and previous years (2013-2016). An acute decrease in NO2 levels (<50%) was observed as a consequence of traffic diminution during the confinement period. Moreover, a lower reduction in PM10 levels and a non-clear pattern for SO2 levels were observed. During the lockdown period, PM10 elements released from traffic emissions (Sn and Sb) showed the highest concentration diminution in the study area. Regarding the primary industrial sites, there were no significant differences in V, Ni, La, and Cr concentration reduction during 2020 associated with industrial activity (stainless steel and oil refinery) in Algeciras Bay. Similarly, concentrations of Zn showed the same behaviour at Cordoba, indicating that the Zn-smelter activity was not affected by the lockdown. Nevertheless, stronger reductions of Cu, Zn, and As in Huelva during the confinement period indicated a decrease in the nearby Cu-smelter emissions. Brick factories in Bailen were also influenced by the confinement measures, as corroborated by the marked decrease in concentrations of Ni, V, Cu, and Zn during the lockdown compared to that from previous years. This work has shown the baseline concentrations of trace elements of PM10, which is of great value to air quality managers in order to minimise pollution levels by applying the confinement of the population, affecting both traffic and industrial anthropogenic activities.

Contribution of anthropogenic and natural sources in PM10 during North African dust events in Southern Europe.

The influence of North African (NAF) dust events on the air quality at the regional level (12 representative monitoring stations) in Southern Europe during a long time series (2007-2014) was studied. PM10 levels and chemical composition were separated by Atlantic (ATL) and NAF air masses. An increase in the average PM10 concentrations was observed on sampling days with NAF dust influence (42 µg m-3) when compared to ATL air masses (29 µg m-3). Major compounds such as crustal components and secondary inorganic compounds (SIC), as well as toxic trace elements derived from industrial emissions, also showed higher concentrations of NAF events. A source contribution analysis using positive matrix factorisation (PMF) 5.0 of the PM10 chemical data, discriminating ATL and NAF air mass origins, allowed the identification of five sources: crustal, sea salt, traffic, regional, and industrial. A higher contribution (74%) of the natural sources to PM10 concentrations was confirmed under NAF episodes compared with ATL. Furthermore, there was an increase in anthropogenic sources during these events (51%), indicating the important influence of the NAF air masses on these sources. The results of this study highlight that environmental managers should take appropriate actions to reduce local emissions during NAF events to ensure good air quality.

Source contribution and origin of PM10 and arsenic in a complex industrial region (Huelva, SW Spain).

Air pollution coming from industrial activities is a matter of interest since their emissions can seriously affect to the human health of nearby populations. A more detailed study about industrial emissions is required in order to discriminate different activities contributing to pollutant sources. In this sense, gaseous pollutants (NO2, SO2 and O3) and PM10 levels has been studied in a complex industrial area in the southwest of Spain (La Rabida and the nearby city of Huelva) during the period 1996-2017. Hourly, daily, monthly and annual variations of PM10 and gaseous pollutants concentrations point to the industrial activity as the main SO2 source. Furthermore, traffic and resuspension emissions contribute to the NO2 and PM10 levels, respectively. Results from chemical composition of PM10 at both sites during the period 2015-2017 are characterized by high concentrations of the crustal components derived from natural and local resuspension. Arsenic is found to be the main geochemical anomaly at La Rabida (annual mean of 7 ng m-3), exceeding the European annual target of 6 ng m-3, which supposes a risk for the nearby population. An emission source from Cu-smelter has been identified in La Rabida and Huelva. A second source corresponding to emissions from polymetallic sulfides handling in a port area has been described for the first time in La Rabida. In addition, arsenic speciation results have identified three different As impacts scenarios as a function of the dominant wind direction, the SO2 episodes and the As extraction efficiency: impact of the Cu-smelter, impact of the bulk polymetallic sulfides and a mixed impact of both sources.

As3MT and GST Polymorphisms Influencing Arsenic Metabolism in Human Exposure to Drinking Groundwater.

The urinary arsenic metabolites may vary among individuals and the genetic factors have been reported to explain part of the variation. We assessed the influence of polymorphic variants of Arsenic-3-methyl-transferase and Glutathione-S-transferase on urinary arsenic metabolites. Twenty-two groundwater wells for human consumption from municipalities of Colombia were analyzed for assessed the exposure by lifetime average daily dose (LADD) (µg/kg bw/day). Surveys on 151 participants aged between 18 and 81 years old were applied to collect demographic information and other factors. In addition, genetic polymorphisms (GSTO2-rs156697, GSTP1-rs1695, As3MT-rs3740400, GSTT1 and GSTM1) were evaluated by real time and/or conventional PCR. Arsenic metabolites: AsIII, AsV, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were measured using HPLC-HG-AFS. The influence of polymorphic variants, LADD and other factors were tested using multivariate analyses. The median of total arsenic concentration in groundwater was of 33.3 µg/L and the median of LADD for the high exposure dose was 0.33 µg/kg bw/day. Univariate analyses among arsenic metabolites and genetic polymorphisms showed MMA concentrations higher in heterozygous and/or homozygous genotypes of As3MT compared to the wild-type genotype. Besides, DMA concentrations were lower in heterozygous and/or homozygous genotypes of GSTP1 compared to the wild-type genotype. Both DMA and MMA concentrations were higher in GSTM1-null genotypes compared to the active genotype. Multivariate analyses showed statistically significant association among interactions gene-gene and gene-covariates to modify the MMA and DMA excretion. Interactions between polymorphic variants As3MT*GSTM1 and GSTO2*GSTP1 could be potential modifiers of urinary excretion of arsenic and covariates as age, LADD, and alcohol consumption contribute to largely vary the arsenic individual metabolic capacity in exposed people.

Arsenic accumulation and speciation in strawberry plants exposed to inorganic arsenic enriched irrigation.

The accumulation and transformation of arsenic species have been studied in the context of hydroponic cultivation of strawberry plants. Cultivation experiments have been performed by adding inorganic arsenic at concentrations of 10, 100 and 1000 µg L-1 via root irrigation. The total arsenic content was determined by Hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS). The accumulation was dependent on the concentration of arsenic added to the irrigation and the arsenic species. Arsenic (III) accumulated at higher rates than arsenic (V). A greater accumulation of arsenic was found in roots (0.44-4.10 mg kg-1) than in stems (0.43-1.27 mg kg-1) and fruits (0.22-0.30 mg kg-1). The speciation results obtained by HPLC-HG-AFS analysis indicated that the addition of As(III) resulted in a partial methylation producing monomethyl arsenic (MMA) and dimethyl arsenic (DMA). After As(V) addition, only MMA was observed and this was accompanied with a notable reduction in the ratio of As(V) to As(III).

Hazardous trace elements in thoracic fraction of airborne particulate matter: Assessment of temporal variations, sources, and health risks in a megacity.

The deleterious health effects of thoracic fractions seem to be more related to the chemical composition of the particles than to their mass concentration. The presence of hazardous materials in PM10 (e.g., heavy metals and metalloids) causes risks to human health. In this study, twelve trace elements (Cd, Cr, Pb, Zn, Cu, Ni, Sn, Ba, Co, As, V, and Sb) in 315 samples of ambient PM10 were analyzed. The samples were collected at an urban background site in a Latin American megacity (Bogota, Colombia) for one year. The concentrations and temporal variabilities of these elements were examined. According to the results, Cu (52 ng/m3), Zn (44 ng/m3), Pb (25 ng/m3), and Ba (20 ng/m3) were the traces with the highest concentrations, particularly during the dry season (January to March), which was characterized by barbecue (BBQ) charcoal combustion and forest fires. In addition, the differences between the results of weekdays and weekends were identified. The determined enrichment factor (EF) indicated that Zn, Pb, Sn, Cu, Cd, and Sb mainly originated from anthropogenic sources. Moreover, a speciation analysis of inorganic Sb (EF > 300) was conducted, which revealed that Sb(V) was the main Sb species in the PM10 samples (>80%). Six causes for the hazardous elements were identified based on the positive matrix factorization (PMF) model: fossil fuel combustion and forest fires (60%), road dust (19%), traffic-related emissions (9%), copper smelting (8%), the iron and steel industry (2%), and an unidentified industrial sector (2%). Furthermore, a health risk assessment of the carcinogenic elements was performed. Accordingly, the cancer risk of inhalation exposure to Co, Ni, As, Cd, Sb(III), and Pb was negligible for children and adults at the sampling site. For adults, the adjusted Cr(VI) level was slightly higher than the minimal acceptable risk level during the study period (1.4 × 10-6).

The geochemical evolution of brines from phosphogypsum deposits in Huelva (SW Spain) and its environmental implications.

The present study focuses on the geochemistry of large phosphogypsum deposits in Huelva (SW Spain). Phosphogypsum slurry waste from fertiliser production was disposed in large ponds containing aqueous waste (i.e. brines) and exposed to weathering. These evaporation ponds were found to be dynamic environments far from attaining steady state conditions where a number of trace pollutants are subjected to temporal variations in response to changing environmental conditions. Chemical, mineralogical and morphological data were used to improve our understanding on the dynamics of a large number of elements in the phosphogypsum-brine-evaporation deposits system. Weekly sampling of brines over the course of 1 yr indicated a substantial enrichment in potentially harmful elements (e.g. As, Cr, Cu, F, Ni, U, V, Zn) present in time-dependent concentrations. The evaporation deposits formed multi-layered precipitates of chlorides, sulphates, phosphates and fluorides containing a large number of pollutants in readily soluble forms. The precipitation sequence revealed a time-dependent composition reflecting alternating precipitation and re-dissolution processes associated with seasonal changes in the local weather conditions. Concatenation of precipitation/re-dissolution stages was found to progressively enrich the brines in pollutants. These findings were supported by the observations from a tank experiment simulating the phosphogypsum-brine-evaporation deposits system under laboratory conditions. Given the substantially high concentrations of pollutants present in mobile forms in the brine-salt system, actions to abate these compounds should be implemented.

Arsenic exposure, profiles of urinary arsenic species, and polymorphism effects of glutathione-s-transferase and metallothioneins.

This study assessed the effects of polymorphic variants of gutathione-S-transferase and metallothioneins on profiles of urinary arsenic species. Drinking groundwater from Margarita and San Fernando, Colombia were analyzed and the lifetime average daily dose (LADD) of arsenic was determined. Specific surveys were applied to collect demographic information and other exposure factors. In addition, GSTT1-null, GSTM1-null, GSTP1-rs1695 and MT-2A-rs28366003 genetic polymorphisms were evaluated, either by direct PCR or PCR-RFLP. Urinary speciated arsenic concentrations were determined by HPLC-HG-AFS for species such as AsIII, AsV, monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and total urinary As (TuAs). Primary methylation index (PMI) and secondary methylation index (SMI) were also calculated as indicators of the metabolic capacity. Polymorphisms effects were tested using multivariate analysis, adjusted by potential confounders. The As concentrations in groundwater were on average 34.6 ± 24.7 µg/L greater than the WHO guideline for As (10 µg/L). There was a correlation between As concentrations in groundwater and TuAs (r = 0.59; p = 0.000). Urinary inorganic arsenic (%InAs) was associated with GSTP1, LADD, GSTP1*Age, GSTP1*alcohol consumption (r2 = 0.43; likelihood-ratio test, p = 0.000). PMI was associated with sex (r2 = 0.20; likelihood-ratio test, p = 0.007). GSTP1 (AG + GG) homozygotes/heterozygotes could increase urinary %InAs and decrease the PMI ratio in people exposed to low and high As from drinking groundwater. Therefore, the explanatory models showed the participation of some covariates that could influence the effects of the polymorphisms on these exposure biomarkers to As.

Geochemical anomalies of household dust in an industrialized city (Huelva, SW Spain).

Geochemical anomalies of sulphide like elements (Cu, As and Cd) derived from the industrial activity have been identified in household dust of Huelva (SW Spain) using geochemical maps. Major and trace elements were analysed by ICP-OES and ICP-MS, respectively. Electron images of single particles were analysed by SEM-EDS in order to know their size, shape and composition. The geochemistry of the household dust has been compared to anomalies in deposition particles, PM10 and soils. A zonation has been observed: the eastern part of the city displays higher concentrations of sulphide like elements than the western part, supporting the origin of these elements related to the vicinity of industrial estates (Cu-smelter processes). Other domestic sources (e.g. wall painting) did not contain any geochemical anomalies related to sulphide like elements in household dust. Principal Component Analysis (PCA) was applied for grouping elements with similar sources, and reinforced the identification of a major industrial source in the eastern part. In this context, geochemical composition of household dust is considered as a fingerprint in order to identify industrial sources in the indoor air quality of Huelva.

Antimony speciation as geochemical tracer for anthropogenic emissions of atmospheric particulate matter.

The chemical composition of atmospheric particulate matter (PM) has been studied at the cities of Cordoba and Granada (South of Spain) between 2007 and 2013, considering urban background, traffic and industrial monitoring stations. The results of Principal Component Analysis (PCA) indicated that geochemical anomalies observed in the ambient air of Cordoba (mainly Cu, Zn, Pb and Cd) are closely related to the geochemical profile obtained from fugitive metallurgy emissions of brass industries. These findings have been confirmed performing an Sb speciation analysis of PM10 samples, which allowed to distinguish between Sb(III) and Sb(V). The percentage of Sb(V) in PM10 found in the traffic station of Granada was 64-69%. At Cordoba, the percentage of Sb(V) was found to be higher (73-77%) at both urban background and traffic stations, indicating a possible second source of Sb in the PM of this city. The PM10 samples from the industrial station of Cordoba showed a 85-86% of Sb(V). A similar percentage (84-88%) of Sb(V) was found for the fugitive emissions of the brass industries, confirming this industrial source of Sb. These results show that Sb speciation can be a useful geochemical tracer to identify anthropogenic sources (traffic and industrial) emissions of PM.

Analytical approaches for arsenic determination in air: A critical review.

This review describes the different steps involved in the determination of arsenic in air, considering the particulate matter (PM) and the gaseous phase. The review focuses on sampling, sample preparation and instrumental analytical techniques for both total arsenic determination and speciation analysis. The origin, concentration and legislation concerning arsenic in ambient air are also considered. The review intends to describe the procedures for sample collection of total suspended particles (TSP) or particles with a certain diameter expressed in microns (e.g. PM10 and PM2.5), or the collection of the gaseous phase containing gaseous arsenic species. Sample digestion of the collecting media for PM is described, indicating proposed and established procedures that use acids or mixtures of acids aided with different heating procedures. The detection techniques are summarized and compared (ICP-MS, ICP-OES and ET-AAS), as well those techniques capable of direct analysis of the solid sample (PIXE, INAA and XRF). The studies about speciation in PM are also discussed, considering the initial works that employed a cold trap in combination with atomic spectroscopy detectors, or the more recent studies based on chromatography (GC or HPLC) combined with atomic or mass detectors (AFS, ICP-MS and MS). Further trends and challenges about determination of As in air are also addressed.

Geochemical anomalies of toxic elements and arsenic speciation in airborne particles from Cu mining and smelting activities: influence on air quality.

A characterization of chemical composition and source contribution of PM10 in three representative environments of southwest Spain related to mining activities (mineral extraction, mining waste and Cu-smelting) has been performed. A study of geochemical anomalies was conducted in the samples collected at the three stations between July 2012 and October 2013. The influence of Cu-smelting processes was compared to other mining activities, where common tracers were identified. The Cu and As concentrations in the study area are higher than in other rural and urban stations of Spain, in which geochemical anomalies of As, Se, Bi, Cd, and Pb have been reported. The results of source contribution showed similar geochemical signatures in the industrial and mining factors. However, the contribution to PM10 is different according to the type of industrial activity. These results have been confirmed performing an arsenic speciation analysis of the PM10 samples, in which the mean extraction efficiency of arsenic depended on the origin of the samples. These finding indicate that the atmospheric particulate matter emitted from Cu-smelting has a high residence time in the atmosphere. This indicates that the Cu-smelter can impact areas of high ecological interest and considered as clean air.

A simplified method for inorganic selenium and selenoaminoacids speciation based on HPLC-TR-HG-AFS.

A simplified speciation method for the determination of selenite, selenate and three selenoaminoacids (selenocystine, selenomethylselenocysteine and selenomethionine) has being developed, based on the coupling of high performance liquid chromatography (HPLC), thermoreduction (TR), hydride generation (HG) and atomic fluorescence spectrometry (AFS). Most of the existing methods based on AFS detection employ a two step procedure to reduce selenate to selenite before HG: (i) Ultraviolet radiation followed by (ii) heating, to produce volatile hydrides of the selenium compounds. The proposed simplified method HPLC-TR-HG-AFS does not require ultraviolet radiation. Instead, KBr dissolved in a HCl solution is added during the heating step (thermoreduction), resulting in an effective hydride generation of the selenium species. Different variables (temperature, HCl and NaBH4 concentrations) have been optimized, using both univariant and multivariant experimental designs. The proposed method is therefore less complex and allows limits of detection, reproducibility and repeatability values similar or better than the existing AFS detection methods described in the literature. A Certified Reference Material (SELM-1 with certified selenomethionine content) and a Se-enriched algae sample have been successfully analyzed with the proposed method. The results were also compared to an alternative technique (GC-MS) that provided similar results.

Evaluation of heavy metals and arsenic speciation discharged by the industrial activity on the Tinto-Odiel estuary, SW Spain.

This study reports the annual amount of heavy metals discharged by industrial activity into the estuary of the Ría of Huelva (SW Spain). The findings showed that the discharged metals found in highest amounts were Fe (11 t y⁻¹), Zn (3.4 t y⁻¹) and Mo (0.88 t y⁻¹). There were other metals with high pollutant charge, such as Ti (232 kg y⁻¹), As (228 kg y⁻¹), Ni (195 kg y⁻¹), Pb (100 kg y⁻¹), Cr (39 kg y⁻¹) and Cd (33 kg y⁻¹). These results were compared with pollutants transported via the Tinto and Odiel rivers from abandoned mining activities in the Iberian Pyrite Belt (IPB), and it was deduced that the amounts spilled exclusively by industries were less than 1% in relation to the total discharge. Hence, the treatment of residues from the IPB should be the priority goal to improve water quality in the estuary.

Diel cycles of arsenic speciation due to photooxidation in acid mine drainage from the Iberian Pyrite Belt (Sw Spain).

Twenty four hours diel cycles of arsenic speciation in Acid Mine Drainage (AMD) due to photooxidation have been reported for the first time. AMD samples were taken during 48 h (31st March and 1st April, 2005) at 6 h intervals from the effluent of a massive abandoned polymetallic sulphide mine of the Iberian Pyrite Belt (Sw Spain). Samples were preserved in situ using cationic exchange prior to analysis by coupled high performance liquid chromatography, hydride generation and atomic fluorescence spectrometry (HPLC-HG-AFS) for arsenic speciation. The results indicated the presence of inorganic arsenic species with daily means of 262mugl(-1) for As(V) and 107 microg l(-1) for As(III). No marked diel trend was observed for As(V). However, a marked diel trend was observed for As(III) in the two studied days, with maximum concentrations during nighttime (141-143 microg l(-1)) and minimum concentrations at daytime (72-77 microg l(-1)). This difference in concentration during daytime and nighttime is ca. 100%. A similar diel cycle was observed for iron. An explanation for the arsenic diel cycles observed is the light induced photooxidation of As(III) and the elimination of As(V) due to its adsorption onto Fe precipitates during the daytime. Furthermore, the diel changes in arsenic speciation emphasize the importance of designing suitable sampling strategies in AMD systems.

Arsenic speciation of atmospheric particulate matter (PM10) in an industrialised urban site in southwestern Spain.

An arsenic speciation study has been performed in PM10 samples collected on a fortnight basis in the city of Huelva (SW Spain) during 2001 and 2002. The arsenic species were extracted from the PM10 filters using a NH2OH x HCl solution and sonication, and determined by HPLC-HG-AFS. The mean bulk As concentration of the samples analyzed during 2001 and 2002 slightly exceed the mean annual 6 ng m(-3) target value proposed by the European Commission for 2013, arsenate [As(V)] being responsible for the high level of arsenic. The speciation analyses showed that As(V) was the main arsenic species found, followed by arsenite [As(III)] (mean 6.5 and 7.8 ng m(-3) for As(V), mean 1.2 and 2.1 ng m(-3) for As(III), in 2001 and 2002, respectively). The high levels of arsenic species found in PM10 in Huelva have a predominant industrial origin, such as the one from a nearby copper smelter, and do not present a seasonal pattern. The highest daily levels of arsenic species correspond to synoptic conditions in which the winds with S and SW components transport the contaminants from the main emission source. The frequent African dust outbreaks over Huelva may result in an increment of mass levels of PM10, but do not represent a significant input of arsenic in comparison to the anthropogenic source. The rural background levels of arsenic around Huelva are rather high, in comparison to other rural or urban areas in Spain, showing a relatively high atmosphere residence time of arsenic. This work shows the importance of arsenic speciation in studies of aerosol chemistry, due to the presence of arsenic species [As(III) and As(V)] with distinct toxicity.

Development of a rapid extraction procedure for speciation of arsenic in chicken meat.

A rapid extraction procedure has been developed for speciation of arsenic in chicken tissue. Water, methanol-water (1:1), and methanol-chloroform (1:1) were tested as extraction media. Individual use of an ultrasonic bath, a microwave oven, or an ultrasonic probe was not sufficient for quantitative recovery of As(III), dimethylarsinate, monomethylarsonate, As(V), and arsenobetaine in spiked samples of chicken tissue. A new extraction procedure using a methanol-water mixture and a microwave oven then an ultrasonic probe enabled extraction of the arsenic species in 7 min with efficiencies ranging from 80 to 100%. HPLC-UV-HG-AFS was used for the determinations. The extraction procedure was 100% efficient when applied to real samples of chicken tissue. AsB (48+/-5 microg As kg (-1)) and one containing-arsenic feed additive, Nitarsone (227+/-5 microg As kg (-1)) were detected.

Preservation procedures for arsenic speciation in a stream affected by acid mine drainage in southwestern Spain.

A preservation study has been performed for arsenic speciation in surface freshwaters affected by acid mine drainage (AMD), a pollution source characterized by low pH and high metallic content. Two sample preservation procedures described in the literature were attempted using opaque glass containers and refrigeration: i) addition of 0.25 mol L(-1) EDTA to the samples, which maintained the stability of the arsenic species for 3 h; and ii) in situ sample clean-up with a cationic exchange resin, in order to reduce the metallic load, which resulted in a partial co-adsorption of arsenic onto Fe precipitates. A new proposed method was also tried: sample acidification with 6 mol L(-1) HCl followed by in situ clean-up with a cationic exchange resin, which allowed a longer preservation time of at least 48 h. The proposed method was successfully applied to water samples with high arsenic content, taken from the Aguas Agrias Stream (Odiel River Basin, SW Spain), which is severely affected by AMD that originates at the nearby polymetallic sulfide mine of Tharsis. The speciation results obtained by liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) indicated that during the summer the main arsenic species was As(V) at the hundred microg L(-1) level, followed by DMA (dimethyl arsenic) and As(III) below the ten microg L(-1) level. In winter, As(V) and As(III) increased at least fivefold, whereas the DMA was not detected.

Arsenic speciation in river and estuarine waters from southwest Spain.

An arsenic speciation survey was carried out in water samples from the Tinto and Odiel Rivers (southwest of Spain), as well as their common estuary. Both rivers are affected by acid mine drainage (AMD) and represent an input of heavy metals into the estuary, which also suffers from industrial water discharges. Samples were taken in December 2000 and July 2001. The arsenic species considered were arsenite (As(III)), arsenate (As(V)), monomethylarsonic (MMA) and dimethylarsinic (DMA) ions using coupled high-performance liquid chromatography-hydride generation-inductively coupled plasma-mass spectrometry (HPLC-HG-ICP-MS) for their determination. Parameters such as pH, salinity, redox potential and dissolved O2 were also measured. The results revealed that the acid mine drainage originating mainly during winter along the upper part of the Tinto River course causes high inorganic concentrations of dissolved arsenic, up to 600 microg l(-1) of As(III) and 200 microg l(-1) of As(V). In summer, As(III) levels decreased due to the diminution of the input from acid mine drainage and also because of oxidation, with a corresponding increase of As(V) level. Furthermore, the extreme acidic conditions of this river (pH 2.3-2-6) do not allow biological activity sufficient to produce significant concentrations of methylated arsenic species. The arsenic concentrations in the nearby Odiel River were always 5-10 times lower than in the Tinto River, with arsenic levels usually below 100 microg l(-1), dominated by As(V), indicating that it is less affected by acid mine drainage. The highest inorganic arsenic species concentrations were found where the river crosses a mining site, which corresponds to the highest As(III) values. Significant biological activity in this river produced methylated species that were detected along the water-course, with the highest concentrations at the lower course of the river, accounting for up to 53-61% of the total dissolved arsenic. At the common estuary formed by both rivers, only arsenate was detected in most samples at lower concentrations than in the riverine water samples. The tidal cycle showed a similar pattern of dilution of the arsenate when seawater comes into the estuary. Methylated species were not found either in summer or winter, at least at the 0.1 microg l(-1) level, possibly because of the high turbidity of the waters, producing an inhibition of the phytoplankton activity. In addition to the riverine inputs into the common estuary, industrial activity also represents an important source of arsenic as the discharge from a Cu smelter produced the highest arsenate level of all samples in estuary and also the only sample with significant arsenite concentration. Furthermore, the underlying iron-oxide-rich sediments represent an importance source of arsenic into the water column. In three nearby estuaries not affected by industrial activity or acid mine drainage, arsenic levels remained below detection limits.