Bioaccumulation of titanium dioxide nanoparticles in green (Ulva sp.) and red (Palmaria palmata) seaweed.

A bioaccumulation study in red (Palmaria palmata) and green (Ulva sp.) seaweed has been carried out after exposure to different concentrations of citrate-coated titanium dioxide nanoparticles (5 and 25 nm) for 28 days. The concentration of total titanium and the number and size of accumulated nanoparticles in the seaweeds has been determined throughout the study by inductively coupled plasma mass spectrometry (ICP-MS) and single particle-ICP-MS (SP-ICP-MS), respectively. Ammonia was used as a reaction gas to minimize the effect of the interferences in the 48Ti determination by ICP-MS. Titanium concentrations measured in Ulva sp. were higher than those found in Palmaria palmata for the same exposure conditions. The maximum concentration of titanium (61.96 ± 15.49 µg g-1) was found in Ulva sp. after 28 days of exposure to 1.0 mg L-1 of 5 nm TiO2NPs. The concentration and sizes of TiO2NPs determined by SP-ICP-MS in alkaline seaweed extracts were similar for both seaweeds exposed to 5 and 25 nm TiO2NPs, which indicates that probably the element is accumulated in Ulva sp. mainly as ionic titanium or nanoparticles smaller than the limit of detection in size (27 nm). The implementation of TiO2NPs in Ulva sp. was confirmed by electron microscopy (TEM/STEM) in combination with energy dispersive X-Ray analysis (EDX).

Proteomics reveals multiple effects of titanium dioxide and silver nanoparticles in the metabolism of turbot, Scophthalmus maximus.

Titanium dioxide (TiO2) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO2 or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO2 NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/long-term exposures and the need for additional studies to validate specific biological applications of these NPs.

Ultrasonication followed by enzymatic hydrolysis as a sample pre-treatment for the determination of Ag nanoparticles in edible seaweed by SP-ICP-MS.

Seaweed can bioaccumulate nanomaterials that would be transferred to the trophic chain. This work describes the optimization of a method for the separation of silver nanoparticles (AgNPs) from seaweed using an ultrasound-assisted enzymatic hydrolysis method and ulterior determination by single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). The following parameters affecting the isolation of AgNPs were optimized using a Palmaria palmata (red seaweed) sample previously exposed to AgNPs: type of sonication (bath vs. ultrasonic probe), ultrasound amplitude, sonication time, sonication mode (pulsed vs. continuous sonication), concentration of the enzymes mixture (Macerozyme R-10®), and enzymatic hydrolysis time. The stability of AgNPs during extraction was tested by transmission electron microscopy (TEM) and using a standard of 15 nm of polyvinylpyrrolidone (PVP)-coated AgNPs analyzed by SP-ICP-MS. The analytical performance was evaluated with good results. For total Ag determination, the limits of detection and quantification were 2.2 and 7.7 ng g-1, respectively; and for AgNPs determination, the limits of detection in size and number were 14 nm and 4.34 × 107 part g-1, respectively. Besides, the matrix effect, the repeatability and the analytical recovery were also studied. Finally, the method was applied to the analysis of several red (Palmaria palmata) and green (Ulva sp.) seaweed samples.

Metal Content in Textile and (Nano)Textile Products.

Metals, metallic compounds, and, recently, metallic nanoparticles appear in textiles due to impurities from raw materials, contamination during the manufacturing process, and/or their deliberate addition. However, the presence of lead, cadmium, chromium (VI), arsenic, mercury, and dioctyltin in textile products is regulated in Europe (Regulation 1907/2006). Metal determination in fabrics was performed by inductively coupled plasma-mass spectrometry (ICP-MS) after microwave-assisted acid digestion. The ICP-MS procedure has been successfully validated; relative standard deviations were up to 3% and analytical recoveries were within the 90-107% range. The developed method was applied to several commercial textiles, and special attention has been focused on textiles with nanofinishing (fabrics prepared with metallic nanoparticles for providing certain functionalities). Arsenic content (in textile T4) and lead content (in subsamples T1-1, T1-2, and T3-3) were found to exceed the maximum limits established by the European Regulation 1907/2006. Although impregnation of yarns with mercury compounds is not allowed, mercury was quantified in fabrics T1-2, T5, and T6. Further speciation studies for determining hexavalent chromium species in sample T9 are necessary (hexavalent chromium is the only species of chromium regulated). Some textile products commercialised in Europe included in this study do not comply with European regulation 1907/2006.

Titanium dioxide nanoparticles assessment in seaweeds by single particle inductively coupled plasma - Mass spectrometry.

In this study, a first attempt for isolating and determining (characterising) background levels of titanium dioxide nanoparticles (TiO2 NPs) in seaweed has been developed by using single particle inductively coupled plasma - mass spectrometry (SP-ICP-MS). Seaweeds were processed using an optimised ultrasound assisted extraction (UAE) procedure based on tetramethylammonium hydroxide (TMAH) before dilution and SP-ICP-MS analysis. The effect of the TMAH percentage in the extracting solution, as well as the volume of extracting solution and sonication (extraction) time, has been fully assessed. Additional experiments also showed that TiO2 NPs were quantitatively released from the seaweed matrix in one UAE step since the analysis of residues gave TiO2 NPs concentrations lower than the limit of quantification (LOQ) of the method. Validation of the method with 50 and 100 nm TiO2 NPs (10 µg L-1 as Ti) showed good analytical recovery (115% and 112% for 50 and 100 nm TiO2 NPs, respectively), and good reproducibility (2% for size and 16% for number of TiO2 NPs). Experiments regarding TiO2 NPs stability showed that the extracted NPs are stable since there were not changes on the number of TiO2 NPs and TiO2 NPs size distributions when exposing TiO2 NPs standards to the optimised extractive conditions.

In vitro assessment of major and trace element bioaccessibility in tea samples.

Bioaccessibility of trace elements (Li, Be, Ti, Ga, Cu, Ag, Hg, Cd, Cs, Pt, Tl, Pb, As, Cr, Co, Ni, V, Se, Sn and Sb) and major elements (Rb, Ba, Al, Fe, Zn, Si, Ca, Mg, Mn, Mo, Sr, P and K) in tea infusions has been assessed using an in vitro dialyzability protocol. Gastric simulation (using pepsin solution) and intestinal simulation (using pancreatin and bile salts) were used to perform the in vitro digestion. ICP-MS, ICP-OES and FAES were used for elements determination in digested tea leaves, their infusions and the dialyzate fractions from tea infusions. Microwaves assisted acid digestion was used for the total element determination in tea leaves, while tea infusions were prepared by brewing tea leaves for 5 min in boiling water. The LODs for elements determined in tea leaves were in the range of 0.11-656 ng g-1 and 0.02-145.6 µg g-1 for trace and major elements, respectively. For elements' determination in tea infusions, the LODs were ranged between 0.23 and 399.9 ng L-1 for trace elements and 0.2-1248 µg L-1 for major elements. The LODs for the elements in the dialyzable fraction varied from 0.018 to 142 µg L-1. The accuracy of the total element determination was evaluated using certified reference materials (Tea Leaves INCT-TL-1 and Rye Grass). The analytical recoveries were also assessed for analyzed elements in digested tea leaves (95-114%) and their infusions (92-115%), showing good recoveries. Among the studied elements, K was the most abundant element in tea leaves and tea infusions in almost all samples, followed by Ca, Mg, and P. Zn, Cs, and K showed the highest dialyzability percentages up to 84%, 76%, and 54%, respectively, followed by Si and Ca and K that show moderate to high dialyzability percentages. The accuracy of the dialysis process was evaluated using a mass-balance study.

Development of a sensitive method for the analysis of four phthalates in tea samples: Tea bag contribution to the total amount in tea infusion.

A sensitive, precise and selective method for the analysis of butyl benzyl phthalate (BBP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and dimethyl phthalate (DMP) in tea samples has been applied. Molecularly Imprinted Polymer-Solid Phase Extraction (MIP-SPE) has been used for the separation and preconcentration of these compounds. Phthalates extracted by SPE were analysed by high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS). The method was sensitive (LOD < 2 µg L-1), precise (RSD <10%) and accurate with recovery percentages ranging from 84% to 97%. Finally, the developed method was applied for the analysis of these phthalates in several tea samples marketed in bags. Migration studies were also performed to evaluate the concentration of phthalates released from the bags into the infusions, and teabag filters were analysed by Fourier-transform infrared spectroscopy. The migration study shows that tea filter bags contribute to the total phthalates concentration in tea infusion, and this contribution varies between 1.8 to 93.5 % of the total phthalates' concentrations. Tea filter bags release higher DBP than BBP, DMP, and DEP.

The bioavailability of arsenic species in rice.

Rice is the principal food in many countries for billions of people and one of the most consumed cereals in the world. The rice plant has the ability to bioaccumulate essential and toxic trace elements such as arsenic. The toxicity of the elements depends not only on their concentration but also on their chemical form and their bioavailability. The inorganic forms of arsenic are more toxic than the organic forms and the toxicity increases with decreasing oxidation states. The consumers of rice in Europe who are the most exposed to inorganic arsenic are children under three, thorough diet (rice-based food). Recently, the European Commission established the maximum levels of inorganic arsenic in foodstuffs. This regulation establishes a maximum level of inorganic arsenic of 100 µg kg-1 in rice destined for the production of food for infants and young children. In order to know the relation between the As ingested and the arsenic absorbed, studies of bioavailability are necessary. We proposed an in vitro digestion method with dialysis to estimate this relation. Furthermore, a bioavailability study of As species in rice was performed in order to know if a change in As species occurred during the gastrointestinal digestion process. Arsenic species were determined in rice and in the dialysate fraction by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The proposed method has been applied to different rice samples acquired in the local Spanish market. Graphical abstract.

Simultaneous determination and speciation analysis of arsenic and chromium in iron supplements used for iron-deficiency anemia treatment by HPLC-ICP-MS.

This work proposes the use of high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for simultaneous speciation of arsenic and chromium in iron supplements used for the treatment of anemia. The sample preparation procedure recommended for the total determination of arsenic and chromium was established using acid digestion in a microwave assisted oven. For speciation analysis, however, the microwave-assisted extraction procedure involved the use of water as extraction solvent at 90°C for 30min. The chromatographic separation was performed using a mobile phase containing 1.0mM tetrabutylammonium hydroxide (TBAH), 0.7mM ethylenediaminetetraacetic acid (EDTA) and 5% methanol at pH 7.2. Helium was used in the collision cell for elimination of the interferences. Under optimized conditions, the separation and detection of the As(III), As(V), Cr(III) and Cr(VI) species can be performed in 5min, permitting their quantification with the external calibration technique with standards prepared in the mobile phase. The limits of quantification obtained were 0.008, 0.010, 0.5 and 0.14µgg-1, for As(III), As(V), Cr(III) and Cr(VI), respectively. The accuracy of the method was evaluated and confirmed by addition/recovery tests. The recoveries obtained varied from 81% to 110%. The proposed method was applied to the speciation analysis of arsenic and chromium in commercially available iron supplements acquired in several cities in Brazil and Spain. The content of the species ranged from 0.01 to 1.3µgg-1 for arsenic, and from 0.4 to 61.2µgg-1 for chromium.

Assessment of metals bound to marine plankton proteins and to dissolved proteins in seawater.

Studies based on laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) have been performed to assess metal bound to dissolved proteins and proteins from marine plankton after two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Dissolved proteins were pre-concentrated from surface seawater (60 L) by tangential ultrafiltration with 10 kDa molecular weight cut-off (MWCO) membranes and further centrifugal ultrafiltration (10 kDa) before proteins isolation by methanol/chloroform/water precipitation. Proteins isolation from plankton was assessed after different trichloroacetic acid (TCA)/acetone and methanol washing stages, and further proteins extraction with a phenol solution. LA-ICP-MS analysis of the electrophoretic profiles obtained for dissolved proteins shows the presence of Cd, Cr, Cu, and Zn in five spots analyzed. These proteins exhibit quite similar molecular weights (within the 10-14 kDa range) and pIs (from 5.8 to 7.3). Cd, Cr, Cu, and Zn have also been found to be associated to proteins isolated from plankton samples. In this case, Cd has been found to be bound to proteins of quite different molecular weight (9, 13 and 22 kDa) and pIs (4.5, 5.2, 5.5, and 10). However, trace elements such as Cr, Cu and Zn appear to be mainly bound to plankton proteins of low molecular weight and variable pI.

Two-dimensional isoelectric focusing OFFGEL and microfluidic lab-on-chip electrophoresis for assessing dissolved proteins in seawater.

Dissolved proteins were assessed in surface and deep seawater by two-dimensional isoelectric focusing (IEF) OFFGEL-lab-on-chip (LOC) electrophoresis after tangential flow ultrafiltration followed by centrifugal ultrafiltration (preconcentration factor of 3000). Dissolved protein isolation was performed by treating the ultrafiltrated retentate with cold acetone and also with chloroform as precipitating reagents. The best electrophoretic behavior of the isolated proteins was obtained after protein precipitation with chloroform before different rinsing stages for removing methanol and water interferences. Metals bound to proteins in the different OFFGEL fractions were assessed by inductively coupled plasma-optical emission spectrometry and electrothermal atomic absorption spectrometry, under optimized operating conditions. Experiments regarding stability of the metal-binding proteins [superoxide dismutase (SOD) and alcohol dehydrogenase (ADH) as protein models] showed the integrity of the Zn-binding SOD/ADH under the OFFGEL electrophoretic conditions. However, stability of Cu bound to SOD is not guaranteed. The first electrophoretic dimension (IEF OFFGEL) showed that dissolved proteins in surface seawater exhibit alkaline isoelectric points (pIs of 8.10 and 8.37) and also acid Ips (4.82, 5.13, 5.43, and 5.73), while LOC showed that the isolated proteins exhibit a spread molecular weight range (within 15 - 63 kDa); although, high molecular weights were the most commonly found. Regarding deep seawater, isolated proteins were of acid Ips (from 3.30 to 4.22) and low molecular weight (within the 21-24 kDa range). Elements such as Cd, Cu, Mn, and Ni were mainly associated with dissolved proteins of alkaline pIs in surface seawater, while Zn was mainly associated to proteins of acid pIs. However, only Cu and Mn were found to be bound to dissolved proteins of higher Ips in deep seawater, and the amount of Mn (from 68 to 84 µg L(-1)) was higher than that found in dissolved proteins in surface seawater (22.4 µg L(-1)).

Matrix solid phase dispersion-assisted BCR sequential extraction method for metal partitioning in surface estuarine sediments.

The BCR (the Community Bureau of Reference) of the European Union sequential extraction scheme for metal partitioning in estuarine sediments has been accelerated by using a matrix solid phase dispersion (MSPD) approach. The MSPD assisted BCR procedure consists of passing the extractants proposed by conventional BCR protocol (0.11 M acetic acid, 0.1M hydroxylammonium chloride and 8.8M hydrogen peroxide plus 1M ammonium acetate) through the dispersed sample packaged inside a disposable syringe. Different silica-, magnesium- and aluminium-based materials were tested as dispersing agents and sea sand was found to offer the best performances. Variables for assisting the three stages of the BCR protocol were optimized, and accurate results were obtained when assisting the first and the third stages (exchangeable and oxidizable fractions, respectively). However, lack of accuracy was observed when assisting the second step (reducible fraction) and this result agrees with most of the assisted BCR procedures for which extracting the reducible fraction is the most troublesome stage. The organic matter oxidation (third stage) was successfully assisted by passing hydrogen peroxide at 50°C through the dispersed sample inside de syringe just before passing ammonium acetate. Therefore, the time-consuming and unsafe conventional organic matter oxidation processes, commonly performed even for microwave/ultrasounds assisted BCR procedures, are totally avoided. Inductively coupled plasma-mass spectrometry (ICP-MS) was used as a selective detector. The target elements were Cd, Co, Cr, Mn, Ni, Sr and Zn (first stage), Cd, Co and Ni (second stage), and Co, Cr, Mn, Ni, Sr and Zn (third stage). Repeatability of the method (n=7) was good, and RSDs values of 9, 10, 10, 8, 8, 3 and 8% was obtained for Cd, Co, Cr, Mn, Ni, Sr and Zn, respectively (first stage); 10, 9 and 9% for Cd, Co and Ni, respectively (second stage); and 6, 2, 3, 4, 7 and 9% Co, Cr, Mn, Ni, Sr and Zn, respectively (third stage). The procedure was also validated by analysing two certified reference materials (CRM 601 and CRM 701). Good accuracy was obtained for the target elements extracted at the first stage: Cd (4.0 ± 0.1 and 7.3 ± 0.09 µg g(-1) in CRM 601 and CRM 701, respectively), Cr (0.36 ± 0.008 and 2.21 ± 0.08 µg g(-1) in CRM 601 and CRM 701, respectively), Ni (8.0 ± 0.3 and 15.4 ± 0.3 µg g(-1) in CRM 601 and CRM 701, respectively) and Zn (262 ± 3 and 203 ± 3 µg g(-1) in CRM 601 and CRM 701, respectively). Also, good accuracy was observed for elements extracted at the third step: Cd (1.8 ± 0.09 and 0.29 ± 0.03 µg g(-1) in CRM 601 and CRM 701, respectively), Cr (145 ± 4 µg g(-1) in CRM 701), Ni (8.2 ± 0.7 and 15.1 ± 0.5 µg g(-1) in CRM 601 and CRM 701, respectively) and Zn (45 ± 0.7 µg g(-1) in CRM 701).

Characterization of edible seaweed harvested on the Galician coast (northwestern Spain) using pattern recognition techniques and major and trace element data.

Major and trace elements in North Atlantic seaweed originating from Galicia (northwestern Spain) were determined by using inductively coupled plasma-optical emission spectrometry (ICP-OES) (Ba, Ca, Cu, K, Mg, Mn, Na, Sr, and Zn), inductively coupled plasma-mass spectrometry (ICP-MS) (Br and I) and hydride generation-atomic fluorescence spectrometry (HG-AFS) (As). Pattern recognition techniques were then used to classify the edible seaweed according to their type (red, brown, and green seaweed) and also their variety (Wakame, Fucus, Sea Spaghetti, Kombu, Dulse, Nori, and Sea Lettuce). Principal component analysis (PCA) and cluster analysis (CA) were used as exploratory techniques, and linear discriminant analysis (LDA) and soft independent modeling of class analogy (SIMCA) were used as classification procedures. In total, t12 elements were determined in a range of 35 edible seaweed samples (20 brown seaweed, 10 red seaweed, 4 green seaweed, and 1 canned seaweed). Natural groupings of the samples (brown, red, and green types) were observed using PCA and CA (squared Euclidean distance between objects and Ward method as clustering procedure). The application of LDA gave correct assignation percentages of 100% for brown, red, and green types at a significance level of 5%. However, a satisfactory classification (recognition and prediction) using SIMCA was obtained only for red seaweed (100% of cases correctly classified), whereas percentages of 89 and 80% were obtained for brown seaweed for recognition (training set) and prediction (testing set), respectively.

Phthalates determination in pharmaceutical formulae used in parenteral nutrition by LC-ES-MS: importance in public health.

A method for determining a group of phthalate esters in pharmaceutical formulae used in parenteral nutrition samples (with and without vitamins) has been developed. The phthalic acid esters (PAEs) studied were dimethyl phthalate, diethyl phthalate, butyl benzyl phthalate, dibutyl phthalate, di-(2-ethylhexyl) phthalate, and dioctyl phthalate. This group of phthalates was determined by high performance liquid chromatography (HPLC)-electrospray ionization-mass spectrometry, working in positive ion mode. The phthalates analyzed were extracted from the sample using hexane and sodium hydroxide. The hexane was then evaporated, and the compounds were redissolved in acetonitrile. The compounds were separated by HPLC working in gradient mode with acetonitrile-ultrapure water starting from 5% to 75% acetonitrile in 5 min, followed by isocratic elution for 27 min. Standard calibration curves were linear for all the analytes over the concentration range 10-250 microg L(-1). The method was precise (with RSD from 3.3% to 12.9%) and sensitive. The proposed analytical method has been applied to the analysis of these compounds in different pharmaceutical formulae (with different compositions) for parenteral nutrition samples in order to check the presence of phthalates and determine their concentration.

Determinación de plomo y cadmio en sangre y su relación con fuentes de exposición. Estudio PESA, 2008 / Blood lead and cadmium levels and sources of exposure. PESA study, 2008

Introducción. La exposición al plomo y al cadmio es un problema de salud pública debido a la amplia exposición a estos tóxicos en la población general. El objetivo del estudio es determinar la concentración de plomo y cadmio en sangre en una población laboral procedente de 4 centros hospitalarios universitarios de Madrid, Getafe, Cartagena y Santiago de Compostela e identificar los factores asociados. Material y métodos. En el estudio participaron 252 sujetos a los que se les administró el cuestionario estandarizado PESA® de exposición al plomo y al cadmio. La concentración de plomo y cadmio en sangre se midió por espectrometría de absorción atómica con atomización electrotérmica y corrección de fondo por efecto Zeeman en espectrómetros Perkin-Elmer; se garantizó la transferibilidad de los resultados. Resultados. La mediana de la concentración global de plomo en sangre fue de 2,0μg/dl (rango intercuartílico [RIC]: 1,2–3,1) y la de cadmio fue de 0,2μg/l (RIC: 0,1–0,4). La mediana de plomo en las mujeres menopáusicas fue superior (2,9μg/dl) a la de las mujeres premenopáusicas (1,3μg/dl) (p<0,001). La mediana de cadmio de los sujetos fumadores (0,54μg/l) fue mayor que la de los sujetos no fumadores (0,21μg/l) (p<0,001). Conclusiones. Se observa un descenso en las concentraciones de plomo y cadmio en sangre respecto a estudios previos realizados en España. Sin embargo, los resultados sugieren que existen factores de mayor riesgo, como la edad, la menopausia, la antigüedad de la vivienda y la exposición al humo de tabaco (AU)

Introduction. Exposure to lead and cadmium is a public health problem owing to the wide exposure to these toxic substances among the general population. The objective of this study is to determine blood lead and cadmium concentrations in a working population drawn from four university hospitals in Madrid, Getafe, Cartagena and Santiago de Compostela, and to identify associated factors. Materials and Methods. A total of 252 subjects took part in the study and were given the standardised PESA® questionnaire on exposure to lead and cadmium. The blood lead and cadmium concentrations were measured by electrothermal atomization atomic absorption spectrometry with Zeeman background correction in Perkin-Elmer spectrometers, guaranteeing the transferability of the results. Results. The median overall blood lead concentration was: 2.0μg/dL ( Interquartile Range (IQR):1.2–3.1) and that of cadmium was: 0.2μg/L (IQR: 0,1–0,4). The median of lead in post-menopausal women was higher (2.9μg/dL) than that of pre-menopausal women (1.3μg/dL) P<0.001. The median of cadmium in subjects who smoked (0.54μg/L) was higher than that in non-smokers (0.21μg/L) P<0.001. Conclusions. A reduction in blood lead and cadmium levels was observed with respect to previous studies carried out in Spain. Nevertheless, the results suggest there are certain factors which increase the risk, such as age, menopause, age of housing and exposure to cigarette smoke (AU)