Effects of arsenic speciation and low dissolved oxygen condition on the toxicity of arsenic to a lotic mayfly

The influence of site-specific conditions on contaminant bioavailability and toxicity to benthic invertebrates is a key consideration in the environmental risk assessment process. This is particularly relevant for contaminants with complex speciation chemistries, such as arsenic. The present study addressed uncertainties regarding arsenic toxicity to a mayfly (Baetis tricaudatus) under low dissolved oxygen (DO) conditions characteristic of many contaminated sites. Arsenic toxicity (arsenite, As(III); arsenate, As(V)) to mayfly nymphs was assessed under two DO scenarios (68 and 84% saturation). Arsenic speciation ratios were determined during testing to confirm the nature of arsenic exposure. The present study found that As(III) was more lethal and bioaccumulated to a greater degree in B. tricaudatus compared to As(V), but the sublethal toxicities of the two arsenic species were similar. Nymph growth and development were significantly inhibited after 12 d of exposure to both 1 mg/L of As(III) and As(V). Exposure to arsenic under low DO conditions (6.5 mg/L, 68% saturation) did not significantly affect As(III) or As(V) toxicity and bioaccumulation over 12 d. The DO level of 6.5 mg/L, however, appeared to be marginally lethal to B. tricaudatus. Results indicate that the Canadian arsenic criterion for the protection of aquatic life (5 microg/L) is protective of B. tricaudatus and is low enough to accommodate differences in arsenic toxicity because of the interconversions between As(III) and As(V). These findings provide insight regarding the toxicity and speciation of arsenic under DO conditions considered to be low for this lotic mayfly species and representative of existing conditions at mine sites in northern Canada.     

Assessment of occupational exposure to inorganic arsenic based on urinary concentrations and speciation of arsenic

An analytical speciation method, capable of separating inorganic arsenic (As (V), As (III] and its methylated metabolites (MMAA, DMAA) from common, inert, dietary organoarsenicals, was applied to the determination of arsenic in urine from a variety of workers occupationally exposed to inorganic arsenic compounds. Mean urinary arsenic (As (V) + As (III) + MMAA + DMAA) concentrations ranged from 4.4 micrograms/g creatinine for controls to less than 10 micrograms/g for those in the electronics industry, 47.9 micrograms/g for timber treatment workers applying arsenical wood preservatives, 79.4 micrograms/g for a group of glassworkers using arsenic trioxide, and 245 micrograms/g for chemical workers engaged in manufacturing and handling inorganic arsenicals. The maximum recorded concentration was 956 micrograms/g. For the most exposed groups, the ranges in the average urinary arsenic speciation pattern were 1-6% As (V), 11-14% As (III), 14-18% MMAA, and 63-70% DMAA. The highly raised urinary arsenic concentrations for the chemical workers, in particular, and some glassworkers are shown to correspond to possible atmospheric concentrations in the workplace and intakes in excess of, or close to, recommended and statutory limits and those associated with inorganic arsenic related diseases.     

Assessment of occupational exposure to inorganic arsenic based on urinary concentrations and speciation of arsenic.

An analytical speciation method, capable of separating inorganic arsenic (As (V), As (III] and its methylated metabolites (MMAA, DMAA) from common, inert, dietary organoarsenicals, was applied to the determination of arsenic in urine from a variety of workers occupationally exposed to inorganic arsenic compounds. Mean urinary arsenic (As (V) + As (III) + MMAA + DMAA) concentrations ranged from 4.4 micrograms/g creatinine for controls to less than 10 micrograms/g for those in the electronics industry, 47.9 micrograms/g for timber treatment workers applying arsenical wood preservatives, 79.4 micrograms/g for a group of glassworkers using arsenic trioxide, and 245 micrograms/g for chemical workers engaged in manufacturing and handling inorganic arsenicals. The maximum recorded concentration was 956 micrograms/g. For the most exposed groups, the ranges in the average urinary arsenic speciation pattern were 1-6% As (V), 11-14% As (III), 14-18% MMAA, and 63-70% DMAA. The highly raised urinary arsenic concentrations for the chemical workers, in particular, and some glassworkers are shown to correspond to possible atmospheric concentrations in the workplace and intakes in excess of, or close to, recommended and statutory limits and those associated with inorganic arsenic related diseases.     

Arsenate sorption on two Chinese red soils evaluated with macroscopic measurements and extended X-ray absorption fine-structure spectroscopy

Arsenic sorption is the primary factor that affects the bioavailability and mobility of arsenic in soils. To elucidate the characteristics and mechanisms of arsenate, As(V), sorption on soils, a combination of sorption isotherms, zeta potential measurements, and extended X-ray absorption fine-structure (EXAFS) spectroscopy was used to investigate As(V) sorption on two Chinese red soils. Arsenate sorption increased with increasing As(V) concentration and was insensitive to ionic strength changes at pH 6.0. Arsenate, mainly as H2AsO4- in soil solution at pH 6.0, was strongly sorbed mainly through ligand exchange by the two soils. The sorption capacity was affected by the iron and aluminum mineral contents in the soils. The zeta potential measurements showed that As(V) sorption lowered the zeta potential and the points of zero charge of the soils. The EXAFS data indicate that adsorbed As(V) forms inner-sphere complexes with bidentate-binuclear configurations, as evidenced by an As-Fe bond distance of 3.28 +/- 0.04 A and an As-Al bond distance of 3.17 +/- 0.03 A. The two As(V) complexes were stable at different As(V) loadings, whereas the proportions were related to the aluminum and iron mineral contents in the soils. This study illuminated the importance of inclusion of microscopic and macroscopic experiments to elucidate sorption behavior and mechanisms.     

Toxicity, biotransformation, and mode of action of arsenic in two freshwater microalgae (Chlorella sp. and Monoraphidium arcuatum)

The toxicity of As(V) and As(III) to two axenic tropical freshwater microalgae, Chlorella sp. and Monoraphidium arcuatum, was determined using 72-h growth rate-inhibition bioassays. Both organisms were tolerant to As(III) (72-h concentration to cause 50% inhibition of growth rate [IC50], of 25 and 15 mg As[III]/L, respectively). Chlorella sp. also was tolerant to As(V) with no effect on growth rate over 72 h at concentrations up to 0.8 mg/L (72-h IC50 of 25 mg As[V]/L). Monoraphidium arcuatum was more sensitive to As(V) (72-h IC50 of 0.25 mg As[V]/L). An increase in phosphate in the growth medium (0.15-1.5 mg PO4(3-)/L) decreased toxicity, i.e., the 72-h IC50 value for M. arcuatum increased from 0.25 mg As(V)/L to 4.5 mg As(V)/L, while extracellular As and intracellular As decreased, indicating competition between arsenate and phosphate for cellular uptake. Both microalgae reduced As(V) to As(III) in the cell, with further biological transformation to methylated species (monomethyl arsonic acid and dimethyl arsinic acid) and phosphate arsenoriboside. Less than 0.01% of added As(V) was incorporated into algal cells, suggesting that bioaccumulation and subsequent methylation was not the primary mode of detoxification. When exposed to As(V), both species reduced As(V) to As(III); however, only M. arcuatum excreted As(III) into solution. Intracellular arsenic reduction may be coupled to thiol oxidation in both species. Arsenic toxicity most likely was due to arsenite accumulation in the cell, when the ability to excrete and/or methylate arsenite was overwhelmed at high arsenic concentrations. Arsenite may bind to intracellular thiols, such as glutathione, potentially disrupting the ratio of reduced to oxidized glutathione and, consequently, inhibiting cell division.     

Effects of arsenic on human keratinocytes: morphological,physiological, and precursor incorporation studies

Measurement of in vitro percutaneous absorption of As(III) and As(V) by artificial human skin shows a strong affinity of arsenic for the human keratinocytes, with 1-10% of the applied arsenic dose retained by the artificial skin per hour. The inordinate retention of arsenic by the skin is a risk factor for As toxicity. The calculated permeability constant (K(p)) averaged about 4.3 x 10(-5) cm/h for As(V) and 10.1 x 10(-5) cm/h for As(III). A facile calculation suggests that dermal absorption during showering and hand washing can be an important exposure route if the water contains more than 100 microg/L As(III) or As(V). The effects of the absorbed arsenic in artificial skin were evaluated in terms of morphological characteristics, integrity of the cell membrane (by means of lactate dehydrogenase and MTS assays), and rates of DNA, RNA, and protein synthesis estimated by incorporation of radioactive precursors. We found significant morphological changes, cytotoxicity associated with disruption of the cell membrane, and inhibition of DNA and protein syntheses at As(III) exposure doses as low as 10 microg/L.     

Influence of EDTA and chemical species on arsenic accumulation in Spirodela polyrhiza L. (duckweed)

The influence of ethylenediaminetetraacetic acid (EDTA) and chemical species on arsenic accumulation in aquatic floating macrophyte Spirodela polyrhiza L. (duckweed) was investigated. The uptake of inorganic arsenic species (arsenate; As(V) and arsenite; As(III)) into the plant tissue and their adsorption on iron plaque of plant surfaces were significantly (p<0.05) higher than those of organic species (monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA)). The addition of EDTA to the culture media increased the uptake of As(V) and As(III) into the plant tissue though the MMAA and DMAA uptake were not affected. About 4–6% of the inorganic arsenic species were desorbed or mobilized from iron plaque by EDTA. Desorption of organic arsenic species was not affected by EDTA addition because the co-precipitation occurs only with inorganic species. Phosphate uptake was not affected by EDTA though its concentration in citrate-bicarbonate-EDTA (CBE)-extract was much higher than that of plant tissue. Iron uptake into the plant increased significantly (p>0.05) by EDTA addition to the culture media while its concentration in CBE-extract decreased significantly (p<0.05). The As(inorganic)/Fe ratios in plant were higher than those of CBE-extract which indicate the increased uptake of these arsenic species into the plant relative to the iron. The lower As(organic)/Fe ratios in plant and on CBE-extract suggest the reduction of accumulation of these arsenic species relative to the iron.     

Equilibrium and kinetics studies on removal of arsenite by iron oxide coated activated alumina

Arsenic in drinking water is causing much concern because of its toxicity. It occurs in water naturally as As(III) and As(V). Of these As (III) is more toxic. Adsorption on activated alumina has been one of the most commonly used methods for As (V) removal from drinking water. But it is not very effective for As (III). Activated alumina was modified by coating it with Iron oxide to make it suitable for As (III) adsorption. Iron oxide coated activated alumina was tested for its effectiveness as an adsorbent for As (III). The As (III) adsorption was strongly dependent on pH and a maximum removal of 98% was observed at a pH of 12. The adsorption process followed a first order kinetics. The equilibrium was attained after 8 hours. The kinetic study was carried out with different initial As (III) concentration. It was observed that time taken to attain equilibrium was independent of initial concentration but percentage removal decreased with increasing initial concentration. The adsorption isotherms were fitted well to both Langmuir and Freundlich equation.     

Metabolism of 74As-labeled trivalent and pentavalent inorganic arsenic in mice

Man is exposed to different forms of arsenic in the environment but differences in metabolism and toxicity are poorly understood. The differences in metabolism between pentavalent and trivalent inorganic arsenic in mice following single oral administration of ⁷⁴As-labeled arsenate or arsenite (0.4 and 4 mg As/kg body wt) was studied. Valence state of the administered arsenic was strictly controlled, which has not always been the case in previous studies. In mice given the high dose, whole body retention was two to three times higher after exposure to As(III) compared to As(V). Retention was also dose dependent. Five weeks after administration, high dose/low dose retention ratios were 6 and 11 for As(V) and As(III). Differences found in most organs studied (blood, kidney, liver, bile, brain, skeleton, and skin) were most obvious in liver and bile with concentrations 2 to 10 times higher in the As(III) groups. Red cell/plasma ratios were 2–3 for As(III) and close to unity for As(V). It is suggested that the observed differences between As(V) and As(III) are due to differences in degree of methylation and subsequent elimination primarily via the kidneys.     

氢化物发生—原子荧光光谱法(HG-AFS)测定食品中不同价态的无机砷

应用氢化物发生——原子荧光分析技术建立了食品中总无机砷、三价砷［Ａｓ（Ⅲ）］和五价砷［Ａｓ（Ⅴ）］的测定方法。用６ｍ ｏｌ／ＬＨＣｌ提取食品中无机砷,在２ｍ ｏｌ／ＬＨＣｌ条件下测定总无机砷,再利用三价砷、五价砷氢化物发生酸度条件的不同对三价砷进行选择测定。本方法标准曲线线性范围：总无机砷为０～４００μｇ／Ｌ,Ａｓ（Ⅲ）为０～３００μｇ／Ｌ;检出限：总无机砷１．６μｇ／Ｌ,Ａｓ（Ⅲ）１．１μｇ／Ｌ;相对标准偏差：总无机砷为１．９３％ ,Ａｓ（Ⅲ）为２．４９％ ;样品回收率：总无机砷为８５％ ～１０５％ ,Ａｓ（Ⅲ）为８０％ ～１１５％ ;一般食品样品的测定无干扰。     

Arsenic accumulation and toxicity in the earthworm Eisenia fetida affected by chloride and phosphate

Chloride and phosphate (PO4) were added to field soils contaminated with arsenic and heavy metals, and Eisenia fetida were inoculated in these treated soils for 70 d. After 28 d, earthworm survival, body weight, and cocoon production were measured. During the exposure period, the accumulation of As and four metals (Cu, Zn, Cd, and Pb) in E. fetida was analyzed. Bulk soils and soil solutions were characterized for the contents of As, metals, organic matter, and major cations and anions; cation exchange capacity; and pH. Although addition of Cl to the soil did not impact As toxicity, it significantly reduced As uptake. The addition of PO4 induced changes in As toxicity and reduced As accumulation even though the As concentration in the soil pore water increased because of the substitution of As by P at soil sorption sites. These results imply that the addition of Cl induced the formation of an As-Cl complex, thereby resulting in decreased As bioavailability; they also imply that monovalent phosphate (H2PO4(-)) competes with As for uptake through transporter-mediated mechanisms in cells.     

Whole-body arsenic concentrations in rainbow trout during acute exposure to arsenate.

This research explored the influence of temperature and waterborne arsenate exposure level on the whole-body arsenic (As) concentration in rainbow trout (Oncorhynchus mykiss) at loss of equilibrium (LOE), an acute toxicity endpoint. The mean median times (ET50s) to LOE for fish exposed to 120 mg arsenate liter-1 at 5 degrees C, as well as 60 and 120 mg liter-1 at 15 degrees C were 56.8, 210, and 34.8 hr, respectively. Despite the differences in ET50, the fish showed respective mean (SE) whole-body As concentrations of 8.6 (0.3), 8.1 (0.6), and 8.6 (0.4) micrograms As g-1, values which were not significantly different. Fish exposed to 240 mg arsenate liter-1 at 5 degrees C, which had a LOE ET50 of 32.2 hr, were observed to have a mean (SE) whole-body arsenic concentration of 13.5 micrograms g-1, significantly higher than the levels in fish from all other treatments. A 3-hr delay between LOE and sampling did not significantly alter whole-body As concentration. The results are discussed in terms of the utility of using whole-body As concentrations to interpret the impacts of arsenate on fish populations in the field.     

Arsenic species in solutions for parenteral nutrition

BACKGROUND: In spite of its high toxicity, arsenic is a common contaminant in pharmaceuticals. This is stated by pharmacopoeias' monographs where it is not generically included with other heavy metals, but has its own specifications. Arsenic should not exceed 0.1 mg/L in most pharmaceutical products for IV administration. This limit, however, was established without taking into consideration the specific arsenic species which contribute to this amount. In this work, the presence of arsenite and arsenate species in solutions of amino acids, salts, vitamins, and lipids commercialized for IV administration was investigated. METHODS: The measurements were done by hydride generation atomic absorption spectrometry. RESULTS: The results showed that all commercial formulations contain both arsenic species in some level; however, the total arsenic content exceeded the allowed limit in only a few samples. Calcium gluconate, sodium bicarbonate, heparin, and vitamin solutions were the most contaminated, presenting total arsenic concentration ranging from 62 to 249 microg/L. The most important finding, however, was the different ratios As(V)/As(III) among the formulations. Whereas practically only As(V) was found in ampoules containing water for injection and salt solutions (NaCl, KC1, phosphates), As(III) predominated in solutions of vitamins, gluconate, and glucose. As these are reducing substances, we investigated the possibility of their reaction with As(V) and its conversion into As(III). The heating of As(V) in the presence of gluconate, glucose, ascorbic acid, methionine, isoleucine, sodium chloride, and pure water, in autoclave for 15 minutes, showed that, whereas no As(III) was found in pure water and sodium chloride solution, approximately 50% of As(V) was converted into As(III) in the remainder of the solutions. Conclusions: The results showed that As(V), the main species in these formulations, may be converted into As(III), depending on the presence of reducing substances among the formulation constituents.     

The influence of exposure history on arsenic accumulation and toxicity in the killifish, Fundulus heteroclitus

Exposure to arsenic is known to cause adverse effects in aquatic biota and wildlife and is of major concern to human health. Although numerous studies have investigated the toxicity of arsenic, little is known about the effects of acquired tolerance on arsenic accumulation and toxicity outside of cell culture models. Accordingly, studies were conducted on the estuarine fish, Fundulus heteroclitus, that were preexposed to nontoxic concentrations of arsenic (as sodium arsenite; 0.7 and 106 micromol As/L) for 96 h or na?ve to elevated arsenic to determine the effects of acclimation on arsenic toxicity and accumulation. Tolerance to arsenic was rapidly (96 h) acquired in killifish that were preexposed. In toxicity tests with arsenic-acclimated killifish, preexposure to 106 micromol As/L resulted in a reduction in toxicity when compared to na?ve animals. Toxicity in arsenic-acclimated fish also was distinguished by a delayed onset of mortality that manifested in dose-dependent fashion and was significant even for the lower acclimation concentration (0.7 micromol As/L). The increase tolerance acquired following preexposure to 106 micromol As/L for 96 h was associated with lower concentrations of arsenic in all monitored tissues (e.g., gill, liver, kidney) and the whole body when fish were exposed to 240 micromol As/L for an additional 96 h. In accordance with these observations, expression of the multidrug resistance- associated protein (MRP)-2 gene, which is responsible for transporting arsenic conjugated to glutathione out of cells, was increased in the liver of arsenic-acclimated fish.     

Effect of arsenic on growth,oxidative stress,and antioxidant system in rice seedlings

The physiological, biochemical, and proteomic changes in germinating rice seedlings were investigated under arsenic stress. A marked decrease in germination percentage, shoot, and root elongation as well as plant biomass was observed with arsenic treatments, as compared to control, whereas accumulation of arsenic and malondialdehyde (MDA) in seedlings were increased significantly with increasing arsenic concentration (both AsIII and AsV). The up-regulation of some antioxidant enzyme activities and the isozymes of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), peroxidase (POD, EC 1.11.1.7), and glutathione reductase (GR, 1.6.4.2) substantiated that arsenic accumulation generated oxidative stress, which was more pronounced in As(III) treatment. We also studied the protective effect of reduced glutathione (GSH) and cysteine (Cys) to As(III)/As(V) stressed seedlings. Both GSH and Cys imparted enhanced tolerance to seedlings against arsenic stress. Seedlings growth improved while level of MDA declined significantly when GSH and Cys were supplemented to As(III)/As(V) treatments suggesting GSH and Cys-mediated protection against oxidative stress. The arsenic content was highest in roots of seedlings grown in As(III) in the presence of GSH/Cys. However, in case of As(V) plus GSH or Cys, the arsenic content in seedlings was highest in shoots. The results are suggestive of differential metabolism of As(III) and As(V) in rice.     

Inorganic arsenic speciation in soil and groundwater near in-service chromated copper arsenate-treated wood poles

The environmental impact of chromated copper arsenate (CCA)-treated utility poles is linked to the possible soil and groundwater contamination with arsenic. The objective of the present study was to determine the arsenic speciation in soil and groundwater near in-service CCA-treated poles. Arsenite (As[III]) and arsenate (As[V]) concentrations were determined in 29 surface and subsurface soil samples collected near eight CCA-treated wood poles. Temporal variability of total arsenic concentrations and inorganic arsenic speciation was also assessed in groundwater at two sites through four sampling events over a 19-month period. Arsenic speciation was carried out by a solvent extraction method using ammonium pyrrolidine dithiocarbamate-methyl isobutyl ketone, and total arsenic was quantified by inductively coupled plasma/atomic emission spectrometry/hydride generation. Average arsenic concentrations in surface soils immediately adjacent to utility poles ranged from 153+/-49 to 410+/-150 mg/kg but approached background levels (below 5 mg/kg) within 0.50 m from the poles. A positive correlation was found between surface soil As concentration and total Fe content. In subsurface samples (0.50 m), arsenic levels were generally high in sandy soils (up to 223+/-32 mg/kg), moderate in clayey soils (up to 126+/-26 mg/kg), and relatively lower in organic soils (up to 56+/-24 mg/ kg). Arsenic(V) was the predominant arsenic species in surface (>78%) and subsurface soils (>66%). Total arsenic concentrations in groundwater below the clayey site were high and varied widely over time (79-390 microg/L), with 30 to 68% as As(III). Below the utility pole located on the organic site with a high Fe content, lower total arsenic levels (12-33 microg/L) were found, with As(III) ranging from 0 to 100%.     

Determination of monomethylarsonous acid, a key arsenic methylation intermediate, in human urine

In this study we report on the finding of monomethylarsonous acid [MMA(III)] in human urine. This newly identified arsenic species is a key intermediate in the metabolic pathway of arsenic biomethylation, which involves stepwise reduction of pentavalent to trivalent arsenic species followed by oxidative addition of a methyl group. Arsenic speciation was carried out using ion-pair chromatographic separation of arsenic compounds with hydride generation atomic fluorescence spectrometry detection. Speciation of the inorganic arsenite [As(III)], inorganic arsenate [As(V)], monomethylarsonic acid [MMA(V)], dimethylarsinic acid [DMA(V)], and MMA(III) in a urine sample was complete in 5 min. Urine samples collected from humans before and after a single oral administration of 300 mg sodium 2,3-dimercapto-1-propane sulfonate (DMPS) were analyzed for arsenic species. MMA(III) was found in 51 out of 123 urine samples collected from 41 people in inner Mongolia 0-6 hr after the administration of DMPS. MMA(III )in urine samples did not arise from the reduction of MMA(V) by DMPS. DMPS probably assisted the release of MMA(III) that was formed in the body. Along with the presence of MMA(III), there was an increase in the relative concentration of MMA(V) and a decrease in DMA(V) in the urine samples collected after the DMPS ingestion.     

常见还原性物质还原五价无机砷的实验研究

目的研究还原型谷胱甘肽(GSH)、半胱氨酸(Cys)、维生素C(VC)和亚铁离子(Fe2+)等常见还原性物质对五价无机砷[As(Ⅴ)]的还原作用及特点。方法模拟体内环境建立还原体系[37℃、pH7.0的磷酸盐缓冲液、3μmol/L As(Ⅴ)、60mmol/L巯基化合物(GSH和Cys)、0~30mmol/L VC、0~40mmol/LFe2+],采用高效液相色谱-氢化物发生-原子荧光法(HPLC-HG-AFS)检测各种反应过程中的三价无机砷[As(Ⅲ)]和As(Ⅴ)。结果As(Ⅴ)还原率和As(Ⅲ)生成率随GSH浓度增加(0~60mmol/L)而增加;当GSH浓度为60mmol/L时,As(Ⅴ)还原率和As(Ⅲ)生成率达到峰值;随着GSH浓度继续增加(60~80mmol/L),As(Ⅴ)还原率和As(Ⅲ)生成率反而下降;巯基化合物中的巯基(-SH)在还原As(Ⅴ)过程中起关键作用,60mmol/L的-SH反应30分钟后As(Ⅴ)还原率达到60%,As(Ⅴ)还原率与-SH的具体存在形式(GSH或Cys)无关;单独VC或Fe2+不能还原As(Ⅴ),但是当VC浓度大于10mmol/L或Fe2+浓度大于20mmol/L时,可以增加60mmol/L的-SH的还原作用(P<0.01)。结论巯基化合物中的-SH可以非酶促还原As(Ⅴ),VC和Fe2+可以增强-SH的还原作用。     

Can folate intake reduce arsenic toxicity?

Arsenic-contaminated groundwater is a global environmental health concern. Inorganic arsenic is a known carcinogen, and epidemiologic studies suggest that persons with impaired arsenic metabolism are at increased risk for certain cancers, including skin and bladder carcinoma. Arsenic metabolism involves methylation to monomethylarsonic acid and dimethylarsinic acid (DMA) by a folate-dependent process. Persons possessing polymorphisms in certain genes involved in folate metabolism excrete a lower proportion of urinary arsenic as DMA, which may influence susceptibility to arsenic toxicity. A double-blind placebo-controlled trial in a population with low plasma folate observed that after 12 weeks of folic acid supplementation, the proportion of total urinary arsenic excreted as DMA increased and blood arsenic concentration decreased, suggesting an improvement in arsenic metabolism. Although no studies have directly shown that high folate intake reduces the risk of arsenic toxicity, these findings provide evidence to support an interaction between folate and arsenic metabolism.     

Numerical methods for the evaluation of individual bioequivalence criteria

The evaluation of individual bioequivalence (IBE) by bootstrap resampling using common statistical software, for example SAS, is extremely time consuming. In this article, an estimation procedure that can be implemented in a high level language with the same degree of accuracy as SAS is described. The necessary parameter estimating equations under both least square (LSE) and restricted maximum likelihood (REML) methods are given. The algorithms used to numerically compute these values are outlined and tested, in FORTRAN, on several simulated data sets and shown to reproduce SAS results with at least 10(-3) precision. More importantly, the REML bootstrap algorithm reduces the time taken in SAS by a factor of 20. Secondary results indicate that LSE and REML parameter estimates are similar for mild unbalancedness. PROC MIXED, with unstructured (UN) and compound symmetry heterogeneous (CSH) variance structures give the same results except when the subject-by-treatment interaction variance, sigma(2)(D), is 0 in which case CSH significantly overestimates sigma(2)(D) and underestimates the within-treatment variances. It is concluded that bootstrap evaluation of IBE is efficiently done using either the LSE or REML algorithm in FORTRAN.