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Sample preparation for arsenic speciation
Chapter 31
Sample preparation for arsenic speciation Walter Goessler and Doris Kuehnelt
31.1
INTRODUCTION
For many centuries arsenic was known to be the element with two faces. On the one hand, arsenic, or rather its compounds, were used in special formulations for curing various diseases, as described by Allesch [1]. As an example Salvarsan (arsphenamine) was the only medicine for curing syphilis until the discovery of penicillin [2]. On the other hand, arsenic (arsenic trioxide) was known as the king of poisons because of its lack of smell and taste. Recently, the use of arsenic as poison through the ages was reviewed by Nriagu [3]. The acute toxic properties of arsenic—or rather its compounds—are certainly the reasons for all the research efforts devoted to this element in the past. Nowadays, millions of people, especially in West Bengal and Bangladesh, are exposed long-term to high levels of inorganic arsenic via the drinking water. Such exposure causes an increased risk of skin, bladder, and kidney cancer. In addition to this elevated risk of cancer, cardiovascular and neurological effects have also been attributed to the exposure to inorganic arsenic [4]. Today, this human tragedy is the driving force for many research projects focussing on arsenic, as recently reviewed by Chakraborti et al. [5]. For simplification, in this chapter the various ions deriving from arsenous acid [As(III)] and arsenic acid [As(V)] will not be distinguished according to their degree of protonation. For example, the ions deriving from arsenic acid 22 (H3AsO4), dihydrogen arsenate (H2AsO2 4 ), hydrogen arsenate (HAsO4 ), and arsenate (AsO32 ) will always be referred to as arsenate [As(V)]. Methylarsonic 4 acid (MA), methylarsonous acid [MA(III)], dimethylarsinic acid (DMA), and dimethylarsinous acid [DMA(III)] will also not be distinguished according to their degree of protonation but will be, in contrast to As(V) and As(III), referred to as the acids. Comprehensive Analytical Chemistry XLI Mester and Sturgeon (Eds.) q 2003 Elsevier B.V. All rights reserved
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31.2
OCCURRENCE AND DISTRIBUTION OF ARSENIC IN THE ENVIRONMENT
Although arsenic is only ranked 51st in elemental abundance in the earth’s crust, it is ubiquitous in our environment, albeit at low concentrations [6]. About 30 arsenic compounds have been identified thus far. The chemical structures of the most common are listed in Figs 31.1 and 31.2. Although recent findings have revealed that there is not much difference in the arsenic speciation of the marine and the terrestrial environment, these two ecosystems are discussed separately. 31.2.1 Marine environment Besides the importance of arsenic in the forensic sciences, due to its abuse as suicidal and homicidal poison, the fact that high arsenic concentrations were detected in seafood forced researchers to look into detail which form of arsenic is present. Although the arsenic concentration in seawater is usually below 2 mg As/l, Jones [7] found high arsenic concentrations in marine algae (5 – 94 mg As/kg dry mass). He speculated that different forms of arsenic must exist in the marine environment, because no toxic effects could be observed after consumption of algae with arsenic concentrations up to , 100 mg As/kg dry mass. Four years later, in 1926, Chapman [8] completed a systematic study of arsenic concentrations in marine algae and fish and concluded the presence of a more or less complex arsenic containing organic compound of low toxicity. Today the average arsenic intake of a person living in Japan, a country with a diet typically rich in seafood, was estimated by Yamauchi et al. [9] to be 273 mg As/day. In contrast, in the United States the mean total arsenic intake is ,50 mg As/day [10]. It is obvious that, especially in countries with seafood rich diets, high pressure from the public encouraged researchers to identify the arsenic compounds present in seafood because of the bad reputation of arsenic. In 1977, Edmonds et al. [11], an Australian research team, identified for the first time the arsenic compound present in the Western rock lobster to be arsenobetaine (AB). Subsequent work revealed that AB is the dominant arsenic compound in all marine animals. About 4 years later, Edmonds and Francesconi [12] identified arsenosugars (dimethylarsinoylribosides) as the dominant arsenic compounds in brown kelp. Presently, 16 arsenosugars have been identified in marine algae, but only four (Fig. 31.2) are usually present at high concentrations. Typical total arsenic concentrations in marine animals and plants including algae, fish, and crustaceans, range from 0.5 to 50 mg As/ kg (wet mass). Data have been recently compiled by Francesconi and Kuehnelt [13]. With the identification and the subsequent synthesis of AB, toxicological studies revealed that AB is not metabolised in the human body and, therefore, does not pose any risk to humans. The acute toxicity (LD50 mouse oral) of AB
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Fig. 31.1. Chemical structures of the most common environmental arsenic compounds (abbreviations used in the text in brackets).
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Fig. 31.2. Chemical structures of the four most common arsenosugars.
was determined by Kaise et al. [14] to be .10 g/kg body mass. This means that AB is about three orders of magnitude less acute toxic than As(III) and As(V). Thus far, only limited toxicity data are available for arsenosugars. Difficulties in the synthesis of these compounds on a large scale are certainly to blame for this lack of data. Nevertheless, authorities assume that no risk for humans occurs after consumption of marine algae. In a recent study conducted by Francesconi et al. [15], it was shown that after a single oral dose of arsenosugar 1 (Fig. 31.2), up to 12 metabolites could be detected in the urine of the consumer. About half of the metabolites have thus far not been identified. Further research is necessary to identify these unknown metabolites to ensure exclusion of any risk to humans. For further, more detailed information about arsenic in the marine environment, two review articles published by Francesconi and Edmonds [16,17] are highly recommended. More recent findings about arsenic in the marine environment have been summarised by Francesconi and Kuehnelt [13].
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31.2.2 Terrestrial environment Whereas the marine environment has been studied intensively, less research effort has been devoted to the terrestrial ecosystem because of the lack of appropriate analytical techniques to handle the low arsenic concentrations. Typical arsenic concentrations in rocks range from 0.5 to 2.5 mg As/kg. In areas with high volcanic activity, such as Japan or Mexico the soil arsenic concentrations can be at least one order of magnitude higher than the average soil arsenic concentration of less than 10 mg As/kg [18]. Apart from the arsenic input into the biosphere through weathering processes and volcanic activities, the human contribution has been estimated to be 40% of the total arsenic input [19]. The use of arsenic containing insecticides and herbicides is forbidden nowadays in many countries. Arsenic compounds are still in use as wood preservatives, in glass the industry, and in farming for protection against parasitic diseases. Additionally, there is a big demand for arsenic in the electronic industries for semiconductor production. Besides the anthropogenic input of arsenic into our environment, microbiological activities are responsible for annually releasing ,2 £ 107 kg of arsenic from land surfaces into the atmosphere, as noted by Frankenberger and Arshad [20]. For a long time it was thought that arsenic compounds in the terrestrial environment were restricted to As(III), As(V), MA, DMA, and trimethylarsine [21]. The more complex arsenic compounds such as AB and arsenosugars, seemed to be a privilege of marine biota. With improvement of analytical methods, especially with the use of ICP-MS as an arsenic-specific detector, Byrne et al. [22] identified AB for the first time in terrestrial mushrooms in 1995. Two years later, arsenocholine (AC) and the tetramethylarsonium cation (TETRA) were discovered by Kuehnelt et al. [23,24] in the mushroom Amanita muscaria collected at a former arsenic roasting facility. After these reports several research groups devoted their efforts to the analysis of terrestrial samples for arsenic compounds. Arsenoriboses were detected for the first time in earthworms by Geiszinger et al. [25]. Nowadays, it is well accepted that identical arsenic compounds are present in marine and in terrestrial biota, albeit at lower concentrations and different concentration ratios. Typical arsenic concentrations in terrestrial plants from uncontaminated sites seldom exceed 20 mg As/kg (wet mass). As an exception, mushrooms have to be mentioned, which may contain several mg As/kg (wet mass) [26]. 31.2.3 Humans Humans are exposed to arsenic via air, food, and drinking water. Except from burning arsenic rich coals and workplace exposure in certain industries, exposure to humans via air is minor. Total arsenic concentrations derived from food as well as drinking water are certainly the most important sources of exposure. In the case of food, one has to keep in mind that only seafood
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(sometimes mushrooms) contributes substantially to the total arsenic burden. Fortunately, only a few percent of the arsenic present in seafood is inorganic arsenic [27]. The highest health risk for humans is certainly the consumption of arsenic contaminated water, because almost all the arsenic is present either as As(V) or As(III). Although the World Health Organisation [28] has defined a drinking water limit of 10 mg As/l to be safe, there are still some countries in the world having a limit of 50 mg As/l. The usefulness of discussing which limits are safe is questionable, especially when even the higher limits are exceeded in some countries, such as Bangladesh due to the geological situation. Tube wells with arsenic concentrations in the mg As/l range are not uncommon, especially in West Bengal and Bangladesh where millions of people suffer from chronic arsenicism which is manifested in elevated skin and internal cancer rates [5]. Usage of surface water as drinking water is not an alternative because of the bacteriological situation. High temperatures and high humidity in this part of the world are ideal for bacterial growth. Other actions, such as collection of rain water or labelling safe tube wells (arsenic concentration ,50 mg As/l) green and the ones with arsenic concentrations above 50 mg As/l, red have not been very successful until now. Many researchers are still looking for an appropriate solution that is on the one hand affordable and on the other hand acceptable for the people living in these areas. Humans excrete most ingested arsenic via the kidneys. Urinary arsenic concentrations of unexposed people are typically below 10 mg As/l [29]. The ratio of the excreted arsenic compounds is typically 60– 80% DMA, 10 –30% inorganic arsenic [sum of As(III) and As(V)], and 10–20% MA. These numbers can be significantly changed by recent (3 –4 days) seafood consumption. Recently, it was shown that there appears to be a polymorphism in arsenic methylation. Whereas Andean people from north-west of Argentina have low percentages of MA (, 2%), in their urine, people from Taiwan have an average relative MA concentration of 27% [29]. The biomethylation of inorganic arsenic is considered to occur via reduction of As(V) to As(III), addition of a methyl group from S-adenosylmethionine, reduction of the formed MA to MA(III), and further transfer of a methyl group to give DMA. Still, little is known about the enzymes involved in this process. The liver seems to be an important site for arsenic methylation, but methylation has been documented in other tissues as well. The organic forms (DMA and MA) are less reactive with tissue constituents, less toxic, and faster excreted in the urine than As(III) and As(V). As(III) was found to be the arsenic compound interacting most with tissue [29]. The methylation of arsenic, and therefore faster excretion from the human body, was always believed to be a detoxification process. Recently, MA(III) was discovered by Aposhian et al. [30] to be present in urine of humans exposed to inorganic arsenic. Both MA(III) and DMA(III) were reported by Le et al. [31] in human urine from a person 4 h after administration of 300 mg sodium 2,3-dimercapto-1-propane sulfonate. Subsequent work on cultured cells showed that the two trivalent organoarsenic
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compounds are at least as toxic as the inorganic arsenic compounds. Moreover, these two trivalent arsenicals are proposed by Mass et al. [32] to have a very high genotoxicity. These new findings raise the question of whether the methylation of arsenic in the human body is really a detoxification process. There are only a few studies on arsenic speciation in blood. The reasons for this are on the one hand the short half-life of arsenic in blood and on the other hand the difficulties in the determination of arsenic compounds in blood or its compartments. Shibata et al. [33] found AB in serum. In a Belgian study, AB and DMA were detected in uremic patients [34]. Due to the high affinity of arsenic to cysteine, which is present in high concentrations in the keratin of the hair, arsenic is accumulated in hair. In contrast to urinary arsenic, which is comparable to a photograph (3 –4 days back), the arsenic concentration in hair is a kind of film that, depending on the length of the hair (growth typically 1 cm per month), can provide information about previous exposure. Total arsenic concentration in the hair of unexposed persons seldom exceeds 200 mg As/kg. In hair samples of exposed people 10 times higher values are not unusual [5]. Arsenic speciation in hair is difficult because no appropriate extraction technique is available. The few studies, thus far have revealed that dimethylated species are present. 31.3
STABILITY OF ARSENIC COMPOUNDS
Whereas for total arsenic determinations the sample matrix and often concurrently the arsenic compounds have to be destroyed, a destruction of the sample matrix is not needed for speciation analysis. For speciation analysis, the arsenic compounds must be extracted from the sample. The extraction step should be quantitative but must not destroy any arsenic compounds present. When the question is raised whether the extraction process itself has already altered the species information, one has to reply certainly with “yes”. It is very unlikely that the different arsenic compounds are “swimming” freely in the cytosol without being attached to other cell compartments. As long as different extraction methods (the milder the better) produce similar results with respect to the determined species, we can assume that these compounds were originally present in the sample and are not operationally defined results. When enzymatic digestion is used for destruction of the organic matrix (as often is in case for selenium) and thereafter the fractions (selenoaminoacids) of the proteins are determined, it is difficult to speak about speciation analysis, because the macromolecule (selenium containing compound) is definitely destroyed [35]. Unfortunately, in situ determination of arsenic compounds at environmental concentrations is not possible with the techniques available at the moment. Xray absorption fine structure spectroscopy has shown to be a possible way of direct speciation analysis, albeit high arsenic concentrations are required as recently described by Langdon et al. [36].
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Storage of samples in a freeze-dried status over an inert atmosphere, as well as freezing of the samples are often employed. Some detailed information about stabilities of arsenic compounds is given below. 31.3.1 Arsenite and arsenate These two arsenic compounds are commonly found in our environment. As(V) is the dominant arsenic compound in seawater. In marine biota, As(III) as well as As(V) have been reported as minor species, with the exception of the marine alga Hizikia fusiforme, in which As(V) can be found at high concentrations (up to 50%). In the terrestrial ecosystem these two arsenic compounds contribute substantially to the total arsenic concentration. In glass manufacturing, As2O3 is often employed for clearing the glass. This might pose a significant problem when glassware (also autosampler vials) are used for speciation analysis of As(III) and As(V) at trace concentrations. As(III) and As(V) are easily interconverted and, therefore, are often found together in real samples. Under oxidising conditions, As(V) is the thermodynamically favoured form and generally found in environmental samples. Under reducing conditions, As(III) is thermodynamically favoured. Arsenites are more soluble and, therefore, more mobile than arsenates. Inskeep et al. [37] recently described the nonequilibrium behaviour of the As(V)/As(III) couple. Arsenites are observed in oxic environments and arsenates persist in anoxic systems. Slow kinetics and/or biological phenomena were invoked to explain the apparent lack of thermodynamic equilibrium. Stock standard solutions of As(V) prepared from Na2HAsO4·7H2O and As(III) prepared from NaAsO2 at a concentration of 1 g As/l can be stored at 48C without any changes. In high alkaline solutions (pH . 12), As(III) is oxidised to As(V) as described by Kuehnelt et al. [38]. At low concentrations (up to 5 mg As/l), we sometimes observe in our laboratory a complete interconversion of As(III) to As(V) or vice versa for aqueous standard solutions. The standard solutions are filled in closed (crimbed) polypropylene autosampler vials and stored overnight. These phenomena cannot be observed all the time. The samples are always prepared from the same stock solutions with the same water quality. A partial oxidation/ reduction at higher concentrations was not observed thus far. These observations make clear that is very difficult to determine the exact concentrations of As(III) and As(V) at low concentrations. In a recently published article by Bednar et al. [39], it was shown that addition of EDTA for complexation of metals present in natural water can be successfully applied for preserving the species information until analysis in the laboratory. Extraction of spiked As(III) and As(V) from a plant matrix using pressurised liquid extraction (also known as accelerated solvent extraction, ASE) in a temperature range from 60 to 1808C revealed that As(V) was stable up to 1508C whereas at 1208C only ,70% of the As(III) were recovered [40]. Surprisingly, no correlation was found between As(III) diminution and
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an increase of As(V). Vela et al. [41] determined the arsenic speciation in carrots using ASE at 1008C and water as a solvent. It was found that the ASE extraction procedure did not cause any redox or interconversion reactions among the arsenic species. Lindemann et al. [42] examined the stability of As(III) and As(V) (in the presence of Se, Sb, and Te species) in water and urine (NIST 2670 normal level) as well as in extracts of fish (NRCC DORM-2) and soil (NIST 2710) under various storage conditions. Surprisingly, it was found that storage of an aqueous solution at 2 208C for 30 days resulted in a substantial loss of As(III), whereas at 3 and 208C quantitative recovery could be obtained. The recovery of As(V) was not influenced by the storage temperature. In a urine matrix spiked with As(V) as well as As(III) both were quantitatively recovered after 5 days of storage at 38C. Spiked fish extracts as well as spiked soil samples could be stored for 3 days without significant change of the spiked As(III) and As(V) concentrations. When aqueous arsenic standard solutions were added to the solid fish and soil material before the extraction procedure, quantitative recovery was obtained for the fish matrix, whereas only 70% of the spiked As(V) and 40% of the spiked As(III) were recovered from the soil. As no additional arsenic species were detected in the chromatograms, an incomplete extraction was blamed for the findings, although no arsenic determinations of the extraction residue had been performed. The authors concluded that extraction of species from solid material and species transformation is still the Achilles heel in speciation analysis. In an earlier study, Palacios et al. [43] systematically investigated the stability of several arsenic species in deionised water and urine. The authors did not consider As(III) in their study because “…it is oxidised in almost all conditions to As(V)”. In deionised water and urine samples, As(V) was stable for 67 days at temperatures of 2 20, 48C, and room temperature. In a preceding study, these authors found immediate conversion of As(III) to As(V) when spiked to urine. These findings are somewhat surprising, as As(III) then should never be detectable in urine samples. Montperrus et al. [44] found that microwave assisted extraction minimises the risk of interconversion of the inorganic arsenic species because of the shorter time needed for complete extraction as compared to conventional shaking. From the above, one can clearly see that no general recipe can be given to preserve the species information. In the case where the As(III) concentration has to be determined in addition to As(V), the stability must be evaluated in the sample matrix to be investigated. For many studies, it is not really very important to distinguish between these two forms as the toxicity of both compounds is very similar and the sum of both is a good measure of exposure. 31.3.2 Methylarsonous acid and dimethylarsinous acid With the more or less clear discovery of the key metabolites of MA(III) and DMA(III) in human urine, interest in studying metabolism and health effects of
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these two arsenic compounds is increasing. In recent work by Gong et al. [45], the stability of these two compounds in deionised water and urine was systematically investigated. Storage of MA(III) in deionised water at 220 and 48C resulted in a slight oxidation of about 10% to MA after 4 months. At room temperature (258C), 15% of MA(III) were already oxidised after 3 days and 80% after ,20 days. The situation was even worse, when the stability of MA(III) in the urine matrix was investigated. At room temperature, complete conversion of MA(III) to MA occurred within 1 week. At 2 20 and 48C an oxidation of , 30% to MA was evident. After 30 days at 48C, about 98% was converted to MA. The stability of MA(III) was better at 2 208C (time days/% converted: 30/60, 60/80, 110/90). DMA(III) was found to be very unstable in water as well as in the urine matrix. DMA(III) was quantitatively oxidised to DMA in the water matrix after 10 days at 258C, after 13 days at 48C, and after 15 days at 2 208C. In urine, DMA(III) was quantitatively converted to DMA after 90 min at 258C. It took 12 and 17 h to convert DMA(III) quantitatively to DMA at 4 and 2 208C, respectively. This work was performed at high concentrations (100 mg As/l) usually not detectable in urine samples of exposed people. One can easily imagine that at a natural concentration level the situation might be even worse. Thus far, this is the only systematic investigation of the stability of the trivalent arsenicals. Storage at lower temperature, e.g., shock freezing in liquid nitrogen, or storage in an inert atmosphere, e.g., N2, or even freeze-drying might improve the stability, especially that of DMA(III). 31.3.3 Methylarsonic acid and dimethylarsinic acid In contrast to the trivalent forms, MA and DMA are stable arsenic compounds. Storage of stock standard solutions (1 g As/l) over 1 year in polyethylene containers at 48C did not show any changes of these species. A change of the species, even at low concentrations as mentioned for As(III) and As(V), was not detected. Palacios et al. [43] found MA to be stable in deionised water for 67 days at room temperature, 48C, and 2 208C when stored together with As(V), DMA, AB, and AC. In the same study, an increase of the DMA concentration was observed at 48C and room temperature. As a possible explanation, a conversion of AB and AC to DMA was suggested. In a urine matrix, MA as well as DMA were stable at 48C and room temperature for 67 days. Lindemann et al. [42] found good stability of MA and DMA in extracts of four different matrices (water, urine, fish, and soil) over 30 days at 220, þ 3, and 208C. Schmidt et al. [40] investigated the thermal stability (60 –1808C) of MA and DMA using pressurised solvent extraction with water as the extractant. The recoveries were minus 10% for MA and minus 2 20% for DMA at 1808C as compared to the values obtained at 608C. Detailed information about whether the compounds have been lost or decomposed was not given. Heating MA and DMA in nitric
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acid at various temperatures revealed that MA is stable up to 1408C and DMA up to 2008C [46]. MA and DMA are relatively stable arsenic compounds. No special requirements are necessary for extracting them as long as the conditions are not too harsh. 31.3.4 Arsenobetaine, arsenocholine, trimethylarsine oxide, and the tetramethylarsonium ion AB, the dominant arsenic compound in marine animals, is very stable. It can be stored at 48C in a refrigerator for years unless microbiological transformation is excluded. Khokiattiwong et al. [47] found that AB is demethylated to dimethylarsionylacetate by microorganisms. Mu¨rer et al. [48] found that AB as well as AC decompose in the presence of hydrogen peroxide when exposed to daylight, AC being the more unstable arsenic compound. Upon heating in nitric acid, AB and AC are converted quantitatively to trimethylarsine oxide (TMAO), as shown by Goessler and Pavkov [46] which then needs about 1 h heating at 3008C for complete conversion to As(V). Similar results were found by Slejkovec et al. [49] and Wasilewska et al. [50]. When roasting a piece of lobster, AB is converted to TETRA, as shown by Hanaoka et al. [51]. Palacios et al. [43] reported that AC disappears from an aqueous solution after 3 days at room temperature (oxidised to AB). In the same work, AB was found to be converted to DMA after 67 days. These findings are not in agreement with our experience. As well, Lindemann et al. [42] found AB to be stable for 30 days when spiked to four different matrices (water, urine, fish, and soil) at 2 20, 3, and 208C. AC is stable up to 1508C when pressurised solvent extraction with water was employed [40]. At 1808C, only 75% recovery was obtained. Kirby and Maher [52] reported that AB, AC, and TETRA were not changed when water– methanol mixtures and microwave assisted heating were used for extracting these arsenicals from fish matrices. AB, TMAO, and TETRA are stable arsenic compounds when they are not exposed to light in an oxidative environment. AC is rather labile and has to be treated with care. Quaternary arsonium compounds, such as AB and AC, decompose to TMAO when heated in alkaline solutions (2 M NaOH) at , 958C for 3 h [53]. 31.3.5 Arsenosugars Arsenosugars, the dominant arsenic compounds in marine algae, have not been studied too much from their chemical point of view due to a lack of synthetic standards. Aqueous solutions of the four major arsenosugars (Fig. 31.2) tend to be relatively stable, with the exception of arsenosugars 2 and 4, which might be converted to arsenosugar 1 [54]. In early work by Edmonds and Francesconi [12], it was reported that arsenosugars decompose to DMA upon heating with
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hydrochloric acid. Recently, Gamble et al. [55] found that arsenosugars do not decompose to DMA, but form the base arsenosugar without aglycone when treated in 78 mM hydrochloric acid or nitric acid. This base arsenosugar (5-deoxy-5-dimethylarsinoyl-D -ribose) was already prepared by Edmonds et al. [56] upon treatment of the analogous methylriboside with HCl. Arsenosugars are certainly more labile than AB. The use of harsher (acidic) conditions for higher extraction yields must be avoided in order to determine which arsenosugars are really present in a sample. Because risk assessment should be based on the “real” arsenic compounds present, further research for better characterisation of the arsenosugars is needed.
31.4
EXTRACTION OF ARSENIC COMPOUNDS FROM ENVIRONMENTAL SAMPLES
Thus far, several articles have been published with the goal of finding the “ideal” method to make the arsenic compounds present in a solid sample available for analysis. Usually, the following points are considered: † † † †
The The The The
extractant extractant extractant extractant
should completely penetrate the sample. should be a solute for the all the arsenic compounds. must not change the speciation. must be compatible with the analytical method chosen.
In the field of arsenic speciation in biological samples, water and water/methanol mixtures are certainly the most common extractants. For extraction of nonpolar arsenicals, acetone or chloroform is sometimes employed. Easy handling, good sample penetration, high solubility for the common arsenicals, reasonable stability of the arsenic compounds, and excellent compatibility with the analytical methods are the reasons for using these extractants. In the case of methanol interfering with the chromatographic separation, it can be easily evaporated. To improve extraction yields, mechanical agitation, vortexing, and sonication are often applied. Pressurised liquid extraction (ASE) at elevated temperatures as well as microwave assisted heating were recently shown to increase extraction efficiencies. Due to the high arsenic concentrations in marine biota, extraction methods are often compared for fish or algal reference materials. DORM-1 and DORM-2 (defatted dogfish muscle) from the National Research Council of Canada (NRCC) are well characterised for their arsenic compounds and, therefore, serve as a “model matrix”. Moreover, the compounds present in this CRM are easily extracted, most probably due to the defatted matrix. Extraction with water/methanol (1 þ 9) at a ratio 1:99 (solid material to extractant) and shaking for 14 h extracted 93% of the arsenic present in DORM-2 [57]. A slightly higher extraction yield of 97% was obtained by Mattusch and
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Wennrich [58] after threefold extraction of DORM-2 with water/methanol (1 þ 1) and sonication for 30 min. Londesborough et al. [59] released only 82% of the arsenic present in DORM-2 after shaking for 2 h with water. Microwave heating to 70–758C with methanol/water (1 þ 1) for 5 min quantitatively released arsenic (103 ^ 2%) from DORM-2 [52]. McKiernan et al. [60] compared pressurised liquid extraction with sonication and recovered 94 and 92% using acetone and water/methanol (1 þ 1), respectively. Recently, Kuehnelt et al. [61] used 1.5 M phosphoric acid (commonly used as extractant for soil samples), methanol/water (9 þ 1), or water for extracting the arsenicals from DORM-2 at room temperature by shaking for 14 h. The recoveries obtained were 94, 92, and 87%, respectively. The results indicate that methanol improves the extraction yields of arsenic compounds, especially AB, from marine animals. Although there is no clear proof for the role of AB in marine animals, it has been suggested that it is utilised in a manner similar to gylcine betaine, an important osmolyte, as speculated by Gailer et al. [62]. The usually high extraction yields for AB imply that it is not bound to any cell compartment and support the speculations that it functions as an osmolyte and is therefore free in the cytosol. When liver tissues of marine mammals were extracted with methanol/water (9 þ 1) and mechanical agitation for 14 h, the extraction yields ranged from 44 to 77% [63]. The fact that a procedure extracting practically 100% from a muscle tissue is only capable of extracting up to 77% from a liver tissue clearly shows that no common extraction procedure is available thus far. Besides extraction of arsenicals from marine animals, much effort has been devoted to the extraction of the arsenic compounds present in marine algae. Kuehnelt et al. [61] used 1.5 M phosphoric acid, methanol/water (9 þ 1), or water for extracting the arsenicals from the brown alga Hijiki fuziforme at room temperature by shaking for 14 h. The methanol/water mixture extracted only 33% of the total arsenic, whereas the same procedure extracted 92% from DORM-2. With pure water, 62% and with 1.5 M phosphoric acid, 76% were extractable. The concentrations of the arsenic compounds present in the different extracts were about the same with the exception of arsenosugar 1, which was best extracted with phosphoric acid, and of As(III) of which only ,10% were extractable with methanol/water. Yoshinaga et al. [64] also found water to be more effective for removing As(V) from algal material. When investigating mushrooms with high concentrations of inorganic arsenic, Byrne et al. [22] obtained better extraction yields with water as compared to water/methanol mixtures. Extraction yields above 85% were obtained by McSheehy et al. [65] when seaweed samples were sonicated for 3 h with water/methanol (1 þ 1) and then sonicated with water/methanol (9 þ 1) for 3 h and 20 min. Only a few systematic investigations have been conducted for the extraction of arsenic species from terrestrial plants. Vela et al. [41] successfully employed pressurised liquid extraction to release arsenic from freeze-dried carrots. For most of the analysed samples, extraction yields above 80% were obtained. Moreover, it is
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important to mention that the authors did not observe any species transformation. Helgesen and Larsen [66] used methanol/water (1 þ 9) and microwave assisted heating for 8 min at 708C for extracting arsenic compounds from carrots. Extraction yields obtained ranged from 46 to 69%. In a recent publication by Heitkemper et al. [67] quantitative extraction of arsenic compounds from SRM 1568a rice flour (NIST) was reported using pressurised solvent extraction at room temperature or 1008C with water or water/methanol mixtures, but only 36% was extracted from a natural rice sample. The extraction efficiency for this rice sample was improved to 92%, only when hydrolysing the sample with 2 M trifluoroacetic acid at 1008C for 6 h. Arsenic compounds present in apples were extracted with water/ methanol (1 þ 1) and 6 h sonication. The extraction yield was ,70% [68]. A further improvement in the extraction yield to 80–90% was obtained when an enzymatic treatment with amylase was performed prior to the solvent extraction with water/methanol. The amylase treatment successfully broke up the starch structure of the apple matrix. Extraction of arsenic and its compounds from soil and sediments usually gives low yields. As extractants, mineral acids are commonly employed because with water or water/methanol mixtures, less than 10% are extractable. Demesmay and Olle [69] used hydrochloric acid/nitric acid mixtures (with magnetic stirring or microwave solubilisation) for the extraction of arsenic compounds from a lake sediment sample. Quantitative extraction yields were obtained but As(III) was quantitatively oxidised to As(V). MA and DMA were stable under these conditions. The oxidation of As(III) was avoided when the arsenic compounds were extracted with 0.3 M orthophosphoric acid and magnetic stirring. With microwave assisted solubilisation, about 10–30% of the As(III) was oxidised to As(V). Quantitative recovery of the arsenic compounds was also obtained with 0.3 M ammonium oxalate at pH 3. Vergara Gallardo et al. [70] extracted a soil (NIST SRM 2709, NIST), a river sediment (CRM 320, BCR), and a sewage sludge (CRM 007-040, RT) with orthophosphoric acid at three concentrations (0.3, 1, 3 M) using microwave solubilisation. Quantitative extraction yields were obtained for the sediment and the sludge sample, irrespective of the orthophosphoric acid concentration used, whereas the yield for the soil sample did not exceed 62%. A partial conversion of As(III) to As(V) was observed with increasing orthophosphoric acid concentration for the sediment sample, whereas the opposite was observed for the sludge sample. Moreover, it was reported for the sludge sample that 90% of the As(III) was converted to As(V) after 4 h when the 3 M extract was stored. Neutralisation or dilution of the extract ensures stability of As(III) for several hours. For the soil sample, an improvement in the extraction yield was obtained with increasing orthophosphoric acid concentration. A 0.2 M ammonium oxalate solution was reported by Montperrus et al. [44] to extract .80% of the total arsenic present in this soil sample. In classical extraction schemes, ammonium oxalate was used to dissolve crystalline iron oxides.
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31.5
CONCLUSIONS
Frequently scientific publications imply that speciation analysis is nowadays already routinely performed. This might be true for the determination step (chromatographic separation and detection) of the analysis, although there are only a few round robin exercises that have generated good agreement between different laboratories. The sample storage and sample preparation step is often completely neglected in these publications. It is well known that the sampling process, together with sample storage and preparation accounts for more than 80% of the total error of an analytical result. In our opinion, there much research remains to be done to improve the extraction efficiencies without changing the compounds. The use of microwave assisted extraction at low power settings, or the employment of pressurised liquid extraction for releasing arsenic compounds from a solid matrix, can certainly improve the situation with respect to extraction efficiencies, less time consumption, and species preservation. Nevertheless, the extraction procedures have to be optimised with respect to the sample matrix and the arsenic compounds present in the sample (unfortunately there is no ultimate extraction procedure available thus far). For the routine determination of arsenic compounds in solid samples of any matrix, much research has to be done to obtain accurate and comparable results, necessary for legislators to set appropriate limits for human health and to save our environment. The “lipid soluble” arsenic (the fraction of arsenic that is not extracted with polar solvents), in particular, has been treated as a stepchild until now and will need a lot more attention in the future.
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R.M. Allesch, Arsenik. Ferd. Kleinmayr, Klagenfurt, 1959, pp. 239 –250. J.M. Azcue and J.O. Nriagu, Arsenic: historical perspectives. In: J.O. Nriagu (Ed.), Arsenic in the Environment. Part I: Cycling and Characterisation. Wiley, New York, 1994, pp. 1–15. J.O. Nriagu, Arsenic poisoning through the ages. In: W.T. Frankenberger (Ed.), Environmental Chemistry of Arsenic. Marcel Dekker, New York, 2002, pp. 1 –26. Arsenic in drinking water 2001 update. National Academy Press, Washington, DC, 2001. D. Chakraborti, M.M. Rahman, K. Paul, U.K. Chowdhury, M.K. Sengupta, D. Lodh, C.R. Chanda, K.C. Saha and S.C. Mukherjee, Talanta, 58 (2002) 3. Handbook of Chemistry and Physics 67th edn. 1986– 1987. CRC Press, Boca Raton, 1986. A.J. Jones, The arsenic content of some of the marine algae. In: C.H. Hamshire (Ed.), Yearbook of Pharmacy, Transactions of the British Pharmaceutical Conference. Churchill, London, 1922, pp. 388–395. A.C. Chapman, Analyst, 51 (1926) 548.
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H. Yamauchi, K. Takahashi, M. Mashiko, J. Saitoh and Y. Yamamura, Appl. Organomet. Chem., 6 (1992) 383. S.S. Tao and P.M. Bolger, Food Addit. Contam., 16 (1999) 465. J.S. Edmonds, K.A. Francesconi, J.R. Canon, C.L. Raston, B.W. Skelton and A.H. White, Tetrahedron Lett., 18 (1977) 1543. J.S. Edmonds and K.A. Francesconi, Nature, 289 (1981) 602. K.A. Francesconi and D. Kuehnelt, Arsenic compounds in the environment. In: W.T. Frankenberger (Ed.), Environmental Chemistry of Arsenic. Marcel Dekker, New York, 2002, pp. 51–94. T. Kaise, S. Watanabe and K. Itoh, Chemosphere, 14 (1985) 1327. K.A. Francesconi, R. Tanggaar, C.J. McKenzie and W. Goessler, Clin. Chem., 48 (2002) 92. K.A. Francesconi and J.S. Edmonds, Oceanogr. Mar. Biol. Annu. Rev., 31 (1993) 111. K.A. Francesconi and J.S. Edmonds, Adv. Inorg. Chem., 44 (1997) 147. P. O’Neill, Arsenic. In: B.J. Alloway (Ed.), Heavy Metals in Soils, 2nd edn. Blackie Academic & Professional, London, 1995, pp. 105 –121 (and references therein). D.C. Chilvers and P.J. Peterson, Global cycling of arsenic. In: T.C. Hutchinson and K.M. Meema (Eds.), Lead, Mercury, Cadmium and Arsenic in the Environment. Wiley, London, 1987, pp. 279– 301. W.T. Frankenberger and M. Arshad, Volatilization of arsenic. In: W.T. Frankenberger (Ed.), Environmental Chemistry of Arsenic. Marcel Dekker, New York, 2002, pp. 360–380, (and references therein). W.R. Cullen and K.J. Reimer, Chem. Rev., 89 (1989) 713. A.R. Byrne, Z. Slejkovec, T. Stijve, L. Fay, W. Goessler, J. Gailer and K.J. Irgolic, Appl. Organomet. Chem., 9 (1995) 305. D. Kuehnelt, W. Goessler and K.J. Irgolic, Appl. Organomet. Chem., 11 (1997) 289. D. Kuehnelt, W. Goessler and K.J. Irgolic, Appl. Organomet. Chem., 11 (1997) 459. A. Geiszinger, W. Goessler, D. Kuehnelt, K.A. Francesconi and W. Kosmus, Environ. Sci. Technol., 32 (1998) 2238. Z. Slejkovec, A.R. Byrne, T. Stijve, W. Goessler and K.J. Irgolic, Appl. Organomet. Chem., 11 (1997) 673. E.H. Larsen and T. Berg, Trace element speciation and international food legislation—a codex alimentarius position paper on arsenic as contaminant. In: L. Ebdon, L. Pitts, R. Cornelis, H. Crews, O.F.X. Donard, P. Quevauviller (Eds.), Trace element speciation for environment, food and health. Cambridge: RSC, 2001, pp. 251–260 (and references therein). Environmental Health Criteria 224. Arsenic and Arsenic Compounds. 2nd edn. World Health Organisation, Geneva, 2001. M. Vahter, Variation in human metabolism of arsenic. In: W.R. Chappell, C.O. Abernathy, R.L. Calderon (Eds.), Arsenic exposure and health effects. Amsterdam: Elsevier, 1999, pp. 267–279 (and references therein). V.H. Aposhian, E.S. Gurzau, X.C. Le, A. Gurzau, S.M. Healy, X. Lu, M. Ma, L. Yip, R.A. Zakharyan, R.M. Maiorino, R.C. Dart, M.G. Tirus, D. Gonzalez-Ramirez, D.L. Morgan, D. Avram and M.M. Aposhian, Chem. Res. Toxicol., 13 (2000) 693. X.C. Le, X. Lu, W.R. Cullen, V. Aposhian and B. Zheng, Anal. Chem., 72 (2000) 5172. M.J. Mass, A. Tennant, R.C. Roop, W.R. Cullen, M. Styblo and D.J. Thomas, Chem. Res. Toxicol., 14 (2001) 305. Y. Shibata, J. Yoshinaga and M. Morita, Appl. Organomet. Chem., 8 (1994) 249. X. Zhang, R. Cornelis, J. De Kimpe, L. Mees, V. Vanderbiesen, A. De Cubber and R. Vanholder, Clin. Chem., 42 (1996) 249.
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D.M. Templeton, F. Ariese, R. Cornelis, L.-G. Danielsson, H. Muntau, H.P. Van Leeuwen and R. Lobinsky, Pure Appl. Chem., 72 (2000) 1453. C.J. Langdon, A.A. Meharg, J. Feldmann, T. Balgar, J. Charnock, M. Farquhar, T.G. Piearce, K.T. Semple and J. Cotter-Howells, J. Environ. Monit., 4 (2002) 603. W.P. Inskeep, T.R. McDermott and S. Fendorf, Arsenic (V)/(III) cycling in soils and natural waters: chemical and microbial processes. In: W.T. Frankenberger (Ed.), Environmental Chemistry of Arsenic. Marcel Dekker, New York, 2002, pp. 183–215 (and references therein). D. Kuehnelt, W. Goessler and K.J. Irgolic, The oxidation of arsenite in aqueous solutions. In: C.O. Abernathy, R.L. Calderon and W.R. Chappell (Eds.), Arsenic: Exposure and Health Effects. Chapman & Hall, London, 1997, pp. 45– 54. A.J. Bednar, J.R. Garbarino, J.F. Ranville and T.R. Wildeman, Environ. Sci. Technol., 36 (2002) 2213. A.-C. Schmidt, W. Reisser, J. Mattusch, P. Popp and R. Wennrich, J. Chromatogr. A, 889 (2000) 83. N.P. Vela, D.T. Heitkemper and K.R. Stewart, Analyst, 126 (2001) 1011. T. Lindemann, A. Prange, W. Dannecker and B. Neidhard, Fresenius J. Anal. Chem., 368 (2000) 214. M.A. Palacios, M. Gomez, C. Camara and M.A. Lopez, Anal. Chim. Acta, 340 (1997) 209. M. Montperrus, Y. Bohari, M. Bueno, A. Astruc and M. Astruc, Appl. Organomet. Chem., 16 (2002) 347. Z. Gong, Y. Lu, W.R. Cullen and X.-C. Le, J. Anal. At. Spectrom., 16 (2001) 1409. W. Goessler and M. Pavkov, Analyst, 128 (2003), 796. S. Khokiattiwong, W. Goessler, S.N.R. Cox and K.A. Francesconi, Appl. Organomet. Chem., 15 (2001) 481. A.J.L. Mu¨rer, A. Abildtrup, O.M. Poulsen and J.M. Christensen, Analyst, 117 (1992) 677. Z. Slejkovec, J.T. van Elteren and U.D. Woroniecka, Anal. Chim. Acta, 443 (2001) 277. M. Wasilewska, W. Goessler, M. Zischka, B. Maichin and G. Knapp, J. Anal. At. Spectrom., 17 (2002) 1121. K. Hanaoka, W. Goessler, H. Ohnu, K.J. Irgolic and T. Kaise, Appl. Organomet. Chem., 15 (2001) 61. J. Kirby and W. Maher, J. Anal. At. Spectrom., 17 (2002) 838. S. Maeda, H. Wada, K. Kumeda, M. Onue, A. Okhi, S. Higashi and T. Takeshita, Appl. Organomet. Chem., 1 (1987) 456. A.D. Madsen, W. Goessler, S.N. Pedersen and K.A. Francesconi, J. Anal. At. Spectrom., 15 (2000) 657. B. Gamble, P.A. Gallagher, J.A. Shoemaker, X. Wei, C.A. Schwegel and J.T. Creed, Analyst, 127 (2002) 781. J.S. Edmonds, Y. Shibata, K.A. Francesconi, J. Yoshinaga and M. Morita, Sci. Total Environ., 12 (1992) 321. W. Goessler, D. Kuehnelt, C. Schlagenhaufen, Z. Slekovec and K.J. Irgolic, J. Anal. At. Spectrom., 13 (1998) 183. J. Mattusch and R. Wennrich, Anal. Chem., 70 (1998) 3649. S. Londesborough, J. Mattusch and R. Wennrich, Fresenius J. Anal. Chem., 363 (1999) 577.
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J.W. McKiernan, J.T. Creed, C.A. Brockhoff, J.A. Caruso and J.M. Lorenzana, J. Anal. At. Spectrom., 14 (1999) 607. D. Kuehnelt, K.J. Irgolic and W. Goessler, Appl. Organomet. Chem., 15 (2001) 445. J. Gailer, K.A. Francesconi, J.S. Edmonds and K.J. Irgolic, Appl. Organomet. Chem., 9 (1995) 341. W. Goessler, A. Rudorfer, E.A. Mackey, P.R. Becker and K.J. Irgolic, Appl. Organomet. Chem., 12 (1998) 491. J. Yoshinaga, Y. Shibata, T. Horiguchi and M. Morita, Accred. Qual. Assur., 2 (1997) 154. S. McSheehy, M. Marcinek, H. Chassaigne and J. Szpunar, Anal. Chim. Acta, 410 (2000) 71. H. Helgesen and E.H. Larsen, Analyst, 123 (1998) 791. D.T. Heitkemper, N.P. Vela, K.R. Stewart and C. Westphal, J. Anal. At. Spectrom., 16 (2001) 299. J.A. Caruso, D.T. Heitkemper and C. B’Hymer, Analyst, 126 (2001) 136. C. Demesmay and M. Olle, Fresenius J. Anal. Chem., 357 (1997) 116. M. Vergara Gallardo, Y. Bohari, A. Astruc, M. Potin-Gautier and M. Astruc, Anal. Chim. Acta, 441 (2001) 257.
Sample preparation for arsenic speciation Walter Goessler and Doris Kuehnelt
31.1
INTRODUCTION
For many centuries arsenic was known to be the element with two faces. On the one hand, arsenic, or rather its compounds, were used in special formulations for curing various diseases, as described by Allesch [1]. As an example Salvarsan (arsphenamine) was the only medicine for curing syphilis until the discovery of penicillin [2]. On the other hand, arsenic (arsenic trioxide) was known as the king of poisons because of its lack of smell and taste. Recently, the use of arsenic as poison through the ages was reviewed by Nriagu [3]. The acute toxic properties of arsenic—or rather its compounds—are certainly the reasons for all the research efforts devoted to this element in the past. Nowadays, millions of people, especially in West Bengal and Bangladesh, are exposed long-term to high levels of inorganic arsenic via the drinking water. Such exposure causes an increased risk of skin, bladder, and kidney cancer. In addition to this elevated risk of cancer, cardiovascular and neurological effects have also been attributed to the exposure to inorganic arsenic [4]. Today, this human tragedy is the driving force for many research projects focussing on arsenic, as recently reviewed by Chakraborti et al. [5]. For simplification, in this chapter the various ions deriving from arsenous acid [As(III)] and arsenic acid [As(V)] will not be distinguished according to their degree of protonation. For example, the ions deriving from arsenic acid 22 (H3AsO4), dihydrogen arsenate (H2AsO2 4 ), hydrogen arsenate (HAsO4 ), and arsenate (AsO32 ) will always be referred to as arsenate [As(V)]. Methylarsonic 4 acid (MA), methylarsonous acid [MA(III)], dimethylarsinic acid (DMA), and dimethylarsinous acid [DMA(III)] will also not be distinguished according to their degree of protonation but will be, in contrast to As(V) and As(III), referred to as the acids. Comprehensive Analytical Chemistry XLI Mester and Sturgeon (Eds.) q 2003 Elsevier B.V. All rights reserved
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31.2
OCCURRENCE AND DISTRIBUTION OF ARSENIC IN THE ENVIRONMENT
Although arsenic is only ranked 51st in elemental abundance in the earth’s crust, it is ubiquitous in our environment, albeit at low concentrations [6]. About 30 arsenic compounds have been identified thus far. The chemical structures of the most common are listed in Figs 31.1 and 31.2. Although recent findings have revealed that there is not much difference in the arsenic speciation of the marine and the terrestrial environment, these two ecosystems are discussed separately. 31.2.1 Marine environment Besides the importance of arsenic in the forensic sciences, due to its abuse as suicidal and homicidal poison, the fact that high arsenic concentrations were detected in seafood forced researchers to look into detail which form of arsenic is present. Although the arsenic concentration in seawater is usually below 2 mg As/l, Jones [7] found high arsenic concentrations in marine algae (5 – 94 mg As/kg dry mass). He speculated that different forms of arsenic must exist in the marine environment, because no toxic effects could be observed after consumption of algae with arsenic concentrations up to , 100 mg As/kg dry mass. Four years later, in 1926, Chapman [8] completed a systematic study of arsenic concentrations in marine algae and fish and concluded the presence of a more or less complex arsenic containing organic compound of low toxicity. Today the average arsenic intake of a person living in Japan, a country with a diet typically rich in seafood, was estimated by Yamauchi et al. [9] to be 273 mg As/day. In contrast, in the United States the mean total arsenic intake is ,50 mg As/day [10]. It is obvious that, especially in countries with seafood rich diets, high pressure from the public encouraged researchers to identify the arsenic compounds present in seafood because of the bad reputation of arsenic. In 1977, Edmonds et al. [11], an Australian research team, identified for the first time the arsenic compound present in the Western rock lobster to be arsenobetaine (AB). Subsequent work revealed that AB is the dominant arsenic compound in all marine animals. About 4 years later, Edmonds and Francesconi [12] identified arsenosugars (dimethylarsinoylribosides) as the dominant arsenic compounds in brown kelp. Presently, 16 arsenosugars have been identified in marine algae, but only four (Fig. 31.2) are usually present at high concentrations. Typical total arsenic concentrations in marine animals and plants including algae, fish, and crustaceans, range from 0.5 to 50 mg As/ kg (wet mass). Data have been recently compiled by Francesconi and Kuehnelt [13]. With the identification and the subsequent synthesis of AB, toxicological studies revealed that AB is not metabolised in the human body and, therefore, does not pose any risk to humans. The acute toxicity (LD50 mouse oral) of AB
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Fig. 31.1. Chemical structures of the most common environmental arsenic compounds (abbreviations used in the text in brackets).
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Fig. 31.2. Chemical structures of the four most common arsenosugars.
was determined by Kaise et al. [14] to be .10 g/kg body mass. This means that AB is about three orders of magnitude less acute toxic than As(III) and As(V). Thus far, only limited toxicity data are available for arsenosugars. Difficulties in the synthesis of these compounds on a large scale are certainly to blame for this lack of data. Nevertheless, authorities assume that no risk for humans occurs after consumption of marine algae. In a recent study conducted by Francesconi et al. [15], it was shown that after a single oral dose of arsenosugar 1 (Fig. 31.2), up to 12 metabolites could be detected in the urine of the consumer. About half of the metabolites have thus far not been identified. Further research is necessary to identify these unknown metabolites to ensure exclusion of any risk to humans. For further, more detailed information about arsenic in the marine environment, two review articles published by Francesconi and Edmonds [16,17] are highly recommended. More recent findings about arsenic in the marine environment have been summarised by Francesconi and Kuehnelt [13].
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31.2.2 Terrestrial environment Whereas the marine environment has been studied intensively, less research effort has been devoted to the terrestrial ecosystem because of the lack of appropriate analytical techniques to handle the low arsenic concentrations. Typical arsenic concentrations in rocks range from 0.5 to 2.5 mg As/kg. In areas with high volcanic activity, such as Japan or Mexico the soil arsenic concentrations can be at least one order of magnitude higher than the average soil arsenic concentration of less than 10 mg As/kg [18]. Apart from the arsenic input into the biosphere through weathering processes and volcanic activities, the human contribution has been estimated to be 40% of the total arsenic input [19]. The use of arsenic containing insecticides and herbicides is forbidden nowadays in many countries. Arsenic compounds are still in use as wood preservatives, in glass the industry, and in farming for protection against parasitic diseases. Additionally, there is a big demand for arsenic in the electronic industries for semiconductor production. Besides the anthropogenic input of arsenic into our environment, microbiological activities are responsible for annually releasing ,2 £ 107 kg of arsenic from land surfaces into the atmosphere, as noted by Frankenberger and Arshad [20]. For a long time it was thought that arsenic compounds in the terrestrial environment were restricted to As(III), As(V), MA, DMA, and trimethylarsine [21]. The more complex arsenic compounds such as AB and arsenosugars, seemed to be a privilege of marine biota. With improvement of analytical methods, especially with the use of ICP-MS as an arsenic-specific detector, Byrne et al. [22] identified AB for the first time in terrestrial mushrooms in 1995. Two years later, arsenocholine (AC) and the tetramethylarsonium cation (TETRA) were discovered by Kuehnelt et al. [23,24] in the mushroom Amanita muscaria collected at a former arsenic roasting facility. After these reports several research groups devoted their efforts to the analysis of terrestrial samples for arsenic compounds. Arsenoriboses were detected for the first time in earthworms by Geiszinger et al. [25]. Nowadays, it is well accepted that identical arsenic compounds are present in marine and in terrestrial biota, albeit at lower concentrations and different concentration ratios. Typical arsenic concentrations in terrestrial plants from uncontaminated sites seldom exceed 20 mg As/kg (wet mass). As an exception, mushrooms have to be mentioned, which may contain several mg As/kg (wet mass) [26]. 31.2.3 Humans Humans are exposed to arsenic via air, food, and drinking water. Except from burning arsenic rich coals and workplace exposure in certain industries, exposure to humans via air is minor. Total arsenic concentrations derived from food as well as drinking water are certainly the most important sources of exposure. In the case of food, one has to keep in mind that only seafood
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(sometimes mushrooms) contributes substantially to the total arsenic burden. Fortunately, only a few percent of the arsenic present in seafood is inorganic arsenic [27]. The highest health risk for humans is certainly the consumption of arsenic contaminated water, because almost all the arsenic is present either as As(V) or As(III). Although the World Health Organisation [28] has defined a drinking water limit of 10 mg As/l to be safe, there are still some countries in the world having a limit of 50 mg As/l. The usefulness of discussing which limits are safe is questionable, especially when even the higher limits are exceeded in some countries, such as Bangladesh due to the geological situation. Tube wells with arsenic concentrations in the mg As/l range are not uncommon, especially in West Bengal and Bangladesh where millions of people suffer from chronic arsenicism which is manifested in elevated skin and internal cancer rates [5]. Usage of surface water as drinking water is not an alternative because of the bacteriological situation. High temperatures and high humidity in this part of the world are ideal for bacterial growth. Other actions, such as collection of rain water or labelling safe tube wells (arsenic concentration ,50 mg As/l) green and the ones with arsenic concentrations above 50 mg As/l, red have not been very successful until now. Many researchers are still looking for an appropriate solution that is on the one hand affordable and on the other hand acceptable for the people living in these areas. Humans excrete most ingested arsenic via the kidneys. Urinary arsenic concentrations of unexposed people are typically below 10 mg As/l [29]. The ratio of the excreted arsenic compounds is typically 60– 80% DMA, 10 –30% inorganic arsenic [sum of As(III) and As(V)], and 10–20% MA. These numbers can be significantly changed by recent (3 –4 days) seafood consumption. Recently, it was shown that there appears to be a polymorphism in arsenic methylation. Whereas Andean people from north-west of Argentina have low percentages of MA (, 2%), in their urine, people from Taiwan have an average relative MA concentration of 27% [29]. The biomethylation of inorganic arsenic is considered to occur via reduction of As(V) to As(III), addition of a methyl group from S-adenosylmethionine, reduction of the formed MA to MA(III), and further transfer of a methyl group to give DMA. Still, little is known about the enzymes involved in this process. The liver seems to be an important site for arsenic methylation, but methylation has been documented in other tissues as well. The organic forms (DMA and MA) are less reactive with tissue constituents, less toxic, and faster excreted in the urine than As(III) and As(V). As(III) was found to be the arsenic compound interacting most with tissue [29]. The methylation of arsenic, and therefore faster excretion from the human body, was always believed to be a detoxification process. Recently, MA(III) was discovered by Aposhian et al. [30] to be present in urine of humans exposed to inorganic arsenic. Both MA(III) and DMA(III) were reported by Le et al. [31] in human urine from a person 4 h after administration of 300 mg sodium 2,3-dimercapto-1-propane sulfonate. Subsequent work on cultured cells showed that the two trivalent organoarsenic
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compounds are at least as toxic as the inorganic arsenic compounds. Moreover, these two trivalent arsenicals are proposed by Mass et al. [32] to have a very high genotoxicity. These new findings raise the question of whether the methylation of arsenic in the human body is really a detoxification process. There are only a few studies on arsenic speciation in blood. The reasons for this are on the one hand the short half-life of arsenic in blood and on the other hand the difficulties in the determination of arsenic compounds in blood or its compartments. Shibata et al. [33] found AB in serum. In a Belgian study, AB and DMA were detected in uremic patients [34]. Due to the high affinity of arsenic to cysteine, which is present in high concentrations in the keratin of the hair, arsenic is accumulated in hair. In contrast to urinary arsenic, which is comparable to a photograph (3 –4 days back), the arsenic concentration in hair is a kind of film that, depending on the length of the hair (growth typically 1 cm per month), can provide information about previous exposure. Total arsenic concentration in the hair of unexposed persons seldom exceeds 200 mg As/kg. In hair samples of exposed people 10 times higher values are not unusual [5]. Arsenic speciation in hair is difficult because no appropriate extraction technique is available. The few studies, thus far have revealed that dimethylated species are present. 31.3
STABILITY OF ARSENIC COMPOUNDS
Whereas for total arsenic determinations the sample matrix and often concurrently the arsenic compounds have to be destroyed, a destruction of the sample matrix is not needed for speciation analysis. For speciation analysis, the arsenic compounds must be extracted from the sample. The extraction step should be quantitative but must not destroy any arsenic compounds present. When the question is raised whether the extraction process itself has already altered the species information, one has to reply certainly with “yes”. It is very unlikely that the different arsenic compounds are “swimming” freely in the cytosol without being attached to other cell compartments. As long as different extraction methods (the milder the better) produce similar results with respect to the determined species, we can assume that these compounds were originally present in the sample and are not operationally defined results. When enzymatic digestion is used for destruction of the organic matrix (as often is in case for selenium) and thereafter the fractions (selenoaminoacids) of the proteins are determined, it is difficult to speak about speciation analysis, because the macromolecule (selenium containing compound) is definitely destroyed [35]. Unfortunately, in situ determination of arsenic compounds at environmental concentrations is not possible with the techniques available at the moment. Xray absorption fine structure spectroscopy has shown to be a possible way of direct speciation analysis, albeit high arsenic concentrations are required as recently described by Langdon et al. [36].
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Storage of samples in a freeze-dried status over an inert atmosphere, as well as freezing of the samples are often employed. Some detailed information about stabilities of arsenic compounds is given below. 31.3.1 Arsenite and arsenate These two arsenic compounds are commonly found in our environment. As(V) is the dominant arsenic compound in seawater. In marine biota, As(III) as well as As(V) have been reported as minor species, with the exception of the marine alga Hizikia fusiforme, in which As(V) can be found at high concentrations (up to 50%). In the terrestrial ecosystem these two arsenic compounds contribute substantially to the total arsenic concentration. In glass manufacturing, As2O3 is often employed for clearing the glass. This might pose a significant problem when glassware (also autosampler vials) are used for speciation analysis of As(III) and As(V) at trace concentrations. As(III) and As(V) are easily interconverted and, therefore, are often found together in real samples. Under oxidising conditions, As(V) is the thermodynamically favoured form and generally found in environmental samples. Under reducing conditions, As(III) is thermodynamically favoured. Arsenites are more soluble and, therefore, more mobile than arsenates. Inskeep et al. [37] recently described the nonequilibrium behaviour of the As(V)/As(III) couple. Arsenites are observed in oxic environments and arsenates persist in anoxic systems. Slow kinetics and/or biological phenomena were invoked to explain the apparent lack of thermodynamic equilibrium. Stock standard solutions of As(V) prepared from Na2HAsO4·7H2O and As(III) prepared from NaAsO2 at a concentration of 1 g As/l can be stored at 48C without any changes. In high alkaline solutions (pH . 12), As(III) is oxidised to As(V) as described by Kuehnelt et al. [38]. At low concentrations (up to 5 mg As/l), we sometimes observe in our laboratory a complete interconversion of As(III) to As(V) or vice versa for aqueous standard solutions. The standard solutions are filled in closed (crimbed) polypropylene autosampler vials and stored overnight. These phenomena cannot be observed all the time. The samples are always prepared from the same stock solutions with the same water quality. A partial oxidation/ reduction at higher concentrations was not observed thus far. These observations make clear that is very difficult to determine the exact concentrations of As(III) and As(V) at low concentrations. In a recently published article by Bednar et al. [39], it was shown that addition of EDTA for complexation of metals present in natural water can be successfully applied for preserving the species information until analysis in the laboratory. Extraction of spiked As(III) and As(V) from a plant matrix using pressurised liquid extraction (also known as accelerated solvent extraction, ASE) in a temperature range from 60 to 1808C revealed that As(V) was stable up to 1508C whereas at 1208C only ,70% of the As(III) were recovered [40]. Surprisingly, no correlation was found between As(III) diminution and
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an increase of As(V). Vela et al. [41] determined the arsenic speciation in carrots using ASE at 1008C and water as a solvent. It was found that the ASE extraction procedure did not cause any redox or interconversion reactions among the arsenic species. Lindemann et al. [42] examined the stability of As(III) and As(V) (in the presence of Se, Sb, and Te species) in water and urine (NIST 2670 normal level) as well as in extracts of fish (NRCC DORM-2) and soil (NIST 2710) under various storage conditions. Surprisingly, it was found that storage of an aqueous solution at 2 208C for 30 days resulted in a substantial loss of As(III), whereas at 3 and 208C quantitative recovery could be obtained. The recovery of As(V) was not influenced by the storage temperature. In a urine matrix spiked with As(V) as well as As(III) both were quantitatively recovered after 5 days of storage at 38C. Spiked fish extracts as well as spiked soil samples could be stored for 3 days without significant change of the spiked As(III) and As(V) concentrations. When aqueous arsenic standard solutions were added to the solid fish and soil material before the extraction procedure, quantitative recovery was obtained for the fish matrix, whereas only 70% of the spiked As(V) and 40% of the spiked As(III) were recovered from the soil. As no additional arsenic species were detected in the chromatograms, an incomplete extraction was blamed for the findings, although no arsenic determinations of the extraction residue had been performed. The authors concluded that extraction of species from solid material and species transformation is still the Achilles heel in speciation analysis. In an earlier study, Palacios et al. [43] systematically investigated the stability of several arsenic species in deionised water and urine. The authors did not consider As(III) in their study because “…it is oxidised in almost all conditions to As(V)”. In deionised water and urine samples, As(V) was stable for 67 days at temperatures of 2 20, 48C, and room temperature. In a preceding study, these authors found immediate conversion of As(III) to As(V) when spiked to urine. These findings are somewhat surprising, as As(III) then should never be detectable in urine samples. Montperrus et al. [44] found that microwave assisted extraction minimises the risk of interconversion of the inorganic arsenic species because of the shorter time needed for complete extraction as compared to conventional shaking. From the above, one can clearly see that no general recipe can be given to preserve the species information. In the case where the As(III) concentration has to be determined in addition to As(V), the stability must be evaluated in the sample matrix to be investigated. For many studies, it is not really very important to distinguish between these two forms as the toxicity of both compounds is very similar and the sum of both is a good measure of exposure. 31.3.2 Methylarsonous acid and dimethylarsinous acid With the more or less clear discovery of the key metabolites of MA(III) and DMA(III) in human urine, interest in studying metabolism and health effects of
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these two arsenic compounds is increasing. In recent work by Gong et al. [45], the stability of these two compounds in deionised water and urine was systematically investigated. Storage of MA(III) in deionised water at 220 and 48C resulted in a slight oxidation of about 10% to MA after 4 months. At room temperature (258C), 15% of MA(III) were already oxidised after 3 days and 80% after ,20 days. The situation was even worse, when the stability of MA(III) in the urine matrix was investigated. At room temperature, complete conversion of MA(III) to MA occurred within 1 week. At 2 20 and 48C an oxidation of , 30% to MA was evident. After 30 days at 48C, about 98% was converted to MA. The stability of MA(III) was better at 2 208C (time days/% converted: 30/60, 60/80, 110/90). DMA(III) was found to be very unstable in water as well as in the urine matrix. DMA(III) was quantitatively oxidised to DMA in the water matrix after 10 days at 258C, after 13 days at 48C, and after 15 days at 2 208C. In urine, DMA(III) was quantitatively converted to DMA after 90 min at 258C. It took 12 and 17 h to convert DMA(III) quantitatively to DMA at 4 and 2 208C, respectively. This work was performed at high concentrations (100 mg As/l) usually not detectable in urine samples of exposed people. One can easily imagine that at a natural concentration level the situation might be even worse. Thus far, this is the only systematic investigation of the stability of the trivalent arsenicals. Storage at lower temperature, e.g., shock freezing in liquid nitrogen, or storage in an inert atmosphere, e.g., N2, or even freeze-drying might improve the stability, especially that of DMA(III). 31.3.3 Methylarsonic acid and dimethylarsinic acid In contrast to the trivalent forms, MA and DMA are stable arsenic compounds. Storage of stock standard solutions (1 g As/l) over 1 year in polyethylene containers at 48C did not show any changes of these species. A change of the species, even at low concentrations as mentioned for As(III) and As(V), was not detected. Palacios et al. [43] found MA to be stable in deionised water for 67 days at room temperature, 48C, and 2 208C when stored together with As(V), DMA, AB, and AC. In the same study, an increase of the DMA concentration was observed at 48C and room temperature. As a possible explanation, a conversion of AB and AC to DMA was suggested. In a urine matrix, MA as well as DMA were stable at 48C and room temperature for 67 days. Lindemann et al. [42] found good stability of MA and DMA in extracts of four different matrices (water, urine, fish, and soil) over 30 days at 220, þ 3, and 208C. Schmidt et al. [40] investigated the thermal stability (60 –1808C) of MA and DMA using pressurised solvent extraction with water as the extractant. The recoveries were minus 10% for MA and minus 2 20% for DMA at 1808C as compared to the values obtained at 608C. Detailed information about whether the compounds have been lost or decomposed was not given. Heating MA and DMA in nitric
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acid at various temperatures revealed that MA is stable up to 1408C and DMA up to 2008C [46]. MA and DMA are relatively stable arsenic compounds. No special requirements are necessary for extracting them as long as the conditions are not too harsh. 31.3.4 Arsenobetaine, arsenocholine, trimethylarsine oxide, and the tetramethylarsonium ion AB, the dominant arsenic compound in marine animals, is very stable. It can be stored at 48C in a refrigerator for years unless microbiological transformation is excluded. Khokiattiwong et al. [47] found that AB is demethylated to dimethylarsionylacetate by microorganisms. Mu¨rer et al. [48] found that AB as well as AC decompose in the presence of hydrogen peroxide when exposed to daylight, AC being the more unstable arsenic compound. Upon heating in nitric acid, AB and AC are converted quantitatively to trimethylarsine oxide (TMAO), as shown by Goessler and Pavkov [46] which then needs about 1 h heating at 3008C for complete conversion to As(V). Similar results were found by Slejkovec et al. [49] and Wasilewska et al. [50]. When roasting a piece of lobster, AB is converted to TETRA, as shown by Hanaoka et al. [51]. Palacios et al. [43] reported that AC disappears from an aqueous solution after 3 days at room temperature (oxidised to AB). In the same work, AB was found to be converted to DMA after 67 days. These findings are not in agreement with our experience. As well, Lindemann et al. [42] found AB to be stable for 30 days when spiked to four different matrices (water, urine, fish, and soil) at 2 20, 3, and 208C. AC is stable up to 1508C when pressurised solvent extraction with water was employed [40]. At 1808C, only 75% recovery was obtained. Kirby and Maher [52] reported that AB, AC, and TETRA were not changed when water– methanol mixtures and microwave assisted heating were used for extracting these arsenicals from fish matrices. AB, TMAO, and TETRA are stable arsenic compounds when they are not exposed to light in an oxidative environment. AC is rather labile and has to be treated with care. Quaternary arsonium compounds, such as AB and AC, decompose to TMAO when heated in alkaline solutions (2 M NaOH) at , 958C for 3 h [53]. 31.3.5 Arsenosugars Arsenosugars, the dominant arsenic compounds in marine algae, have not been studied too much from their chemical point of view due to a lack of synthetic standards. Aqueous solutions of the four major arsenosugars (Fig. 31.2) tend to be relatively stable, with the exception of arsenosugars 2 and 4, which might be converted to arsenosugar 1 [54]. In early work by Edmonds and Francesconi [12], it was reported that arsenosugars decompose to DMA upon heating with
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hydrochloric acid. Recently, Gamble et al. [55] found that arsenosugars do not decompose to DMA, but form the base arsenosugar without aglycone when treated in 78 mM hydrochloric acid or nitric acid. This base arsenosugar (5-deoxy-5-dimethylarsinoyl-D -ribose) was already prepared by Edmonds et al. [56] upon treatment of the analogous methylriboside with HCl. Arsenosugars are certainly more labile than AB. The use of harsher (acidic) conditions for higher extraction yields must be avoided in order to determine which arsenosugars are really present in a sample. Because risk assessment should be based on the “real” arsenic compounds present, further research for better characterisation of the arsenosugars is needed.
31.4
EXTRACTION OF ARSENIC COMPOUNDS FROM ENVIRONMENTAL SAMPLES
Thus far, several articles have been published with the goal of finding the “ideal” method to make the arsenic compounds present in a solid sample available for analysis. Usually, the following points are considered: † † † †
The The The The
extractant extractant extractant extractant
should completely penetrate the sample. should be a solute for the all the arsenic compounds. must not change the speciation. must be compatible with the analytical method chosen.
In the field of arsenic speciation in biological samples, water and water/methanol mixtures are certainly the most common extractants. For extraction of nonpolar arsenicals, acetone or chloroform is sometimes employed. Easy handling, good sample penetration, high solubility for the common arsenicals, reasonable stability of the arsenic compounds, and excellent compatibility with the analytical methods are the reasons for using these extractants. In the case of methanol interfering with the chromatographic separation, it can be easily evaporated. To improve extraction yields, mechanical agitation, vortexing, and sonication are often applied. Pressurised liquid extraction (ASE) at elevated temperatures as well as microwave assisted heating were recently shown to increase extraction efficiencies. Due to the high arsenic concentrations in marine biota, extraction methods are often compared for fish or algal reference materials. DORM-1 and DORM-2 (defatted dogfish muscle) from the National Research Council of Canada (NRCC) are well characterised for their arsenic compounds and, therefore, serve as a “model matrix”. Moreover, the compounds present in this CRM are easily extracted, most probably due to the defatted matrix. Extraction with water/methanol (1 þ 9) at a ratio 1:99 (solid material to extractant) and shaking for 14 h extracted 93% of the arsenic present in DORM-2 [57]. A slightly higher extraction yield of 97% was obtained by Mattusch and
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Wennrich [58] after threefold extraction of DORM-2 with water/methanol (1 þ 1) and sonication for 30 min. Londesborough et al. [59] released only 82% of the arsenic present in DORM-2 after shaking for 2 h with water. Microwave heating to 70–758C with methanol/water (1 þ 1) for 5 min quantitatively released arsenic (103 ^ 2%) from DORM-2 [52]. McKiernan et al. [60] compared pressurised liquid extraction with sonication and recovered 94 and 92% using acetone and water/methanol (1 þ 1), respectively. Recently, Kuehnelt et al. [61] used 1.5 M phosphoric acid (commonly used as extractant for soil samples), methanol/water (9 þ 1), or water for extracting the arsenicals from DORM-2 at room temperature by shaking for 14 h. The recoveries obtained were 94, 92, and 87%, respectively. The results indicate that methanol improves the extraction yields of arsenic compounds, especially AB, from marine animals. Although there is no clear proof for the role of AB in marine animals, it has been suggested that it is utilised in a manner similar to gylcine betaine, an important osmolyte, as speculated by Gailer et al. [62]. The usually high extraction yields for AB imply that it is not bound to any cell compartment and support the speculations that it functions as an osmolyte and is therefore free in the cytosol. When liver tissues of marine mammals were extracted with methanol/water (9 þ 1) and mechanical agitation for 14 h, the extraction yields ranged from 44 to 77% [63]. The fact that a procedure extracting practically 100% from a muscle tissue is only capable of extracting up to 77% from a liver tissue clearly shows that no common extraction procedure is available thus far. Besides extraction of arsenicals from marine animals, much effort has been devoted to the extraction of the arsenic compounds present in marine algae. Kuehnelt et al. [61] used 1.5 M phosphoric acid, methanol/water (9 þ 1), or water for extracting the arsenicals from the brown alga Hijiki fuziforme at room temperature by shaking for 14 h. The methanol/water mixture extracted only 33% of the total arsenic, whereas the same procedure extracted 92% from DORM-2. With pure water, 62% and with 1.5 M phosphoric acid, 76% were extractable. The concentrations of the arsenic compounds present in the different extracts were about the same with the exception of arsenosugar 1, which was best extracted with phosphoric acid, and of As(III) of which only ,10% were extractable with methanol/water. Yoshinaga et al. [64] also found water to be more effective for removing As(V) from algal material. When investigating mushrooms with high concentrations of inorganic arsenic, Byrne et al. [22] obtained better extraction yields with water as compared to water/methanol mixtures. Extraction yields above 85% were obtained by McSheehy et al. [65] when seaweed samples were sonicated for 3 h with water/methanol (1 þ 1) and then sonicated with water/methanol (9 þ 1) for 3 h and 20 min. Only a few systematic investigations have been conducted for the extraction of arsenic species from terrestrial plants. Vela et al. [41] successfully employed pressurised liquid extraction to release arsenic from freeze-dried carrots. For most of the analysed samples, extraction yields above 80% were obtained. Moreover, it is
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important to mention that the authors did not observe any species transformation. Helgesen and Larsen [66] used methanol/water (1 þ 9) and microwave assisted heating for 8 min at 708C for extracting arsenic compounds from carrots. Extraction yields obtained ranged from 46 to 69%. In a recent publication by Heitkemper et al. [67] quantitative extraction of arsenic compounds from SRM 1568a rice flour (NIST) was reported using pressurised solvent extraction at room temperature or 1008C with water or water/methanol mixtures, but only 36% was extracted from a natural rice sample. The extraction efficiency for this rice sample was improved to 92%, only when hydrolysing the sample with 2 M trifluoroacetic acid at 1008C for 6 h. Arsenic compounds present in apples were extracted with water/ methanol (1 þ 1) and 6 h sonication. The extraction yield was ,70% [68]. A further improvement in the extraction yield to 80–90% was obtained when an enzymatic treatment with amylase was performed prior to the solvent extraction with water/methanol. The amylase treatment successfully broke up the starch structure of the apple matrix. Extraction of arsenic and its compounds from soil and sediments usually gives low yields. As extractants, mineral acids are commonly employed because with water or water/methanol mixtures, less than 10% are extractable. Demesmay and Olle [69] used hydrochloric acid/nitric acid mixtures (with magnetic stirring or microwave solubilisation) for the extraction of arsenic compounds from a lake sediment sample. Quantitative extraction yields were obtained but As(III) was quantitatively oxidised to As(V). MA and DMA were stable under these conditions. The oxidation of As(III) was avoided when the arsenic compounds were extracted with 0.3 M orthophosphoric acid and magnetic stirring. With microwave assisted solubilisation, about 10–30% of the As(III) was oxidised to As(V). Quantitative recovery of the arsenic compounds was also obtained with 0.3 M ammonium oxalate at pH 3. Vergara Gallardo et al. [70] extracted a soil (NIST SRM 2709, NIST), a river sediment (CRM 320, BCR), and a sewage sludge (CRM 007-040, RT) with orthophosphoric acid at three concentrations (0.3, 1, 3 M) using microwave solubilisation. Quantitative extraction yields were obtained for the sediment and the sludge sample, irrespective of the orthophosphoric acid concentration used, whereas the yield for the soil sample did not exceed 62%. A partial conversion of As(III) to As(V) was observed with increasing orthophosphoric acid concentration for the sediment sample, whereas the opposite was observed for the sludge sample. Moreover, it was reported for the sludge sample that 90% of the As(III) was converted to As(V) after 4 h when the 3 M extract was stored. Neutralisation or dilution of the extract ensures stability of As(III) for several hours. For the soil sample, an improvement in the extraction yield was obtained with increasing orthophosphoric acid concentration. A 0.2 M ammonium oxalate solution was reported by Montperrus et al. [44] to extract .80% of the total arsenic present in this soil sample. In classical extraction schemes, ammonium oxalate was used to dissolve crystalline iron oxides.
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31.5
CONCLUSIONS
Frequently scientific publications imply that speciation analysis is nowadays already routinely performed. This might be true for the determination step (chromatographic separation and detection) of the analysis, although there are only a few round robin exercises that have generated good agreement between different laboratories. The sample storage and sample preparation step is often completely neglected in these publications. It is well known that the sampling process, together with sample storage and preparation accounts for more than 80% of the total error of an analytical result. In our opinion, there much research remains to be done to improve the extraction efficiencies without changing the compounds. The use of microwave assisted extraction at low power settings, or the employment of pressurised liquid extraction for releasing arsenic compounds from a solid matrix, can certainly improve the situation with respect to extraction efficiencies, less time consumption, and species preservation. Nevertheless, the extraction procedures have to be optimised with respect to the sample matrix and the arsenic compounds present in the sample (unfortunately there is no ultimate extraction procedure available thus far). For the routine determination of arsenic compounds in solid samples of any matrix, much research has to be done to obtain accurate and comparable results, necessary for legislators to set appropriate limits for human health and to save our environment. The “lipid soluble” arsenic (the fraction of arsenic that is not extracted with polar solvents), in particular, has been treated as a stepchild until now and will need a lot more attention in the future.
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