Inorganic arsenic in seaweed: a fast HPLC-ICP-MS method without coelution of arsenosugars.

Seaweed is becoming increasingly popular in the Western diet as consumers opt for more sustainable food sources. However, seaweed is known to accumulate high levels of arsenic-which may be in the form of carcinogenic inorganic arsenic (iAs). Here we propose a fast method for the routine measurement of iAs in seaweed using HPLC-ICP-MS without coelution of arsenosugars that may complicate quantification. The developed method was optimised using design of experiments (DOE) and tested on a range of reference materials including TORT-3 (0.36 ± 0.03 mg kg-1), DORM-5 (0.02 ± 0.003 mg kg-1), and DOLT-5 (0.07 ± 0.007 mg kg-1). The use of nitric acid in the extraction solution allowed for the successful removal of interferences from arsenosugars by causing degradation to an unretained arsenosugar species, and a recovery of 99 ± 9% was obtained for iAs in Hijiki 7405-b when compared with the certified value. The method was found to be suitable for high-throughput analysis of iAs in a range of food and feed matrices including Asparagopsis taxiformis seaweed, grass silage, and insect proteins, and offers a cost-effective, fast, and robust option for routine analysis that requires minimal sample preparation. The method may be limited with regards to the quantification of dimethylarsenate (DMA) in seaweed, as the acidic extraction may lead to overestimation of this analyte by causing degradation of lipid species that are typically more abundant in seaweed than other marine matrices (i.e. arsenophospholipids). However, the concentrations of DMA quantified using this method may provide a better estimation with regard to exposure after ingestion and subsequent digestion of seaweed.

Lipophilic arsenic compounds in the cultured green alga Chlamydomonas reinhardtii.

In this study, arsenic (As) speciation was investigated in the freshwater alga Chlamydomonas reinhardtii treated with 20 µg/L arsenate using fractionation as well as ICP-MS/ESI-MS analyses and was compared with the known As metabolite profile of wild-grown Saccharina latissima. While the total As accumulation in C. reinhardtii was about 85% lower than in S. latissima, the relative percentage of arsenolipids was significantly higher in C. reinhardtii (57.0% vs. 5.01%). As-containing hydrocarbons and phospholipids dominated the hydrophobic As profile in S. latissima, but no As-containing hydrocarbons were detectable in C. reinhardtii. Instead for the first time, an arsenoriboside-containing phytol (AsSugPhytol) was found to dominate the hydrophobic arsenicals of C. reinhardtii. Interestingly, this compound and its relatives had so far been only found in green marine microalgae, open sea plankton (mixed assemblage), and sediments but not in brown or red macroalgae. This compound family might therefore relate to differences in the arsenic metabolism between the algae phyla.

Role of complex organic arsenicals in food in aggregate exposure to arsenic.

For much of the world's population, food is the major source of exposure to arsenic. Exposure to this non-essential metalloid at relatively low levels may be linked to a wide range of adverse health effects. Thus, evaluating foods as sources of exposure to arsenic is important in assessing risk and developing strategies that protect public health. Although most emphasis has been placed on inorganic arsenic as human carcinogen and toxicant, an array of arsenic-containing species are found in plants and animals used as foods. Here, we 2evaluate the contribution of complex organic arsenicals (arsenosugars, arsenolipids, and trimethylarsonium compounds) that are found in foods and consider their origins, metabolism, and potential toxicity. Commonalities in the metabolism of arsenosugars and arsenolipids lead to the production of di-methylated arsenicals which are known to exert many toxic effects. Evaluating foods as sources of exposure to these complex organic arsenicals and understanding the formation of reactive metabolites may be critical in assessing their contribution to aggregate exposure to arsenic.

Arsenobetaine and thio-arsenic species in marine macroalgae and herbivorous animals: Accumulated through trophic transfer or produced in situ?

Arsenobetaine (AB) and thio-arsenoribosides were measured in common macroalgae species (8 phaeophyta, 4 rhodophyta and 2 chlorphyta), along the Australian south east coast line. As well, arsenic species profiles were measured for two common marine herbivores, the sea urchin Centrostephanus rodgersii and the fish Odax cyanomelas that graze on these macroalgae to understand if trophic transfer of these species would account for their presence in marine herbivores. AB was found in seven of the fourteen macroalgae species investigated but does not contributed significantly to any of the macroalgae arsenic content (0.01-1.2µg/g). AB was found in only two of the brown macroalgae and all the red and green macroalgae (with the exception of Corallina officinalis). Thio-arsenic species were found sporadically, but not in high concentrations in any of the macroalgae investigated. AB present in macroalgae is likely to be associated with epiphytic organisms while thio-arsenoribosides are likely to be produced by decaying parts of damaged macroalgae. A laboratory feeding experiment in which the herbivorous gastropod, Austrocochlea constricta, was fed macroalgae containing thio-arsenoribosides for a 24hr period every three days showed that these are readily accumulated over a short period. Thio-arsenoribosides in herbivores are therefore probably obtained through trophic transfer. Some AB is also obtained through trophic transfer; however, the presence of trimethylated arsonioribosides, a hypothesized precursor of AB formation in herbivores, suggests that some AB is produced within herbivores from the transformation of arsenoribosides accumulated from their diet.

Arsenosugar extracted from algae: Assessment of countercurrent chromatography for isolation.

Arsenosugars are the predominant species of arsenic in most seaweed. The analysis of these compounds is hampered by the lack of calibration standards needed in their unambiguous identification and quantification. This affects the availability of reliable information on their potential toxicity, which is scarce and controversial. Knowing the potential of centrifugal partition chromatography (CPC) as a preparative separation technique applied to a number of natural compounds, the aim of this work is to investigate the feasibility of CPC in the case of isolation and purification of arsenosugars from algae extracts. Several biphasic solvents systems have been studied to evaluate the distribution of the As species. Given the physical characteristics of these compounds, the presence of strong acids, the formation of ionic pairs or the presence of salts at high ionic strength have been considered. System 1-BuOH/EtOH/sat.(NH4)2SO4/water at a volume ratio 0.2:1:1:1 originates adequate distribution constants of analytes that allows the required separation. The total arsenic content and the arsenic speciation of the eluted solutions from CPC were analyzed by ICP-MS and IC-ICP-MS, respectively. The developed CPC procedure allows us to obtain of the three arsenosugars with a purity of 98.7 % in PO4-Sug, 90.4 % in SO3-Sug and 96.1 % in SO4-Sug.

Arsenic speciation in low-trophic marine food chain - An arsenic exposure study on microalgae (Diacronema lutheri) and blue mussels (Mytilus edulis L.).

Microalgae and blue mussels are known to accumulate undesirable substances from the environment, including arsenic (As). Microalgae can biotransform inorganic As (iAs) to organoarsenic species, which can be transferred to blue mussels. Knowledge on As uptake, biotransformation, and trophic transfer is important with regards to feed and food safety since As species have varying toxicities. In the current work, experiments were conducted in two parts: (1) exposure of the microalgae Diacronema lutheri to 5 and 10 µg/L As(V) in seawater for 4 days, and (2) dietary As exposure where blue mussels (Mytilus edulis L.) were fed with D. lutheri exposed to 5 and 10 µg/L As(V), or by aquatic exposure to 5 µg/L As(V) in seawater, for a total of 25 days. The results showed that D. lutheri can take up As from seawater and transform it to methylated As species and arsenosugars (AsSug). However, exposure to 10 µg/L As(V) resulted in accumulation of iAs in D. lutheri and lower production of methylated As species, which may suggest that detoxification mechanisms were overwhelmed. Blue mussels exposed to As via the diet and seawater showed no accumulation of As. Use of linear mixed models revealed that the blue mussels were gradually losing As instead, which may be due to As concentration differences in the mussels' natural environment and the experimental setup. Both D. lutheri and blue mussels contained notable proportions of simple methylated As species and AsSug. Arsenobetaine (AB) was not detected in D. lutheri but present in minor fraction in mussels. The findings suggest that low-trophic marine organisms mainly contain methylated As species and AsSug. The use of low-trophic marine organisms as feed ingredients requires further studies since AsSug are regarded as potentially toxic, which may introduce new risks to feed and food safety.

Arsenosugar extracted from algae: Isolation by anionic exchange solid-phase extraction.

Obtaining reliable speciation data for evaluating dietary exposure, and increasing understanding of arsenic biochemistry in algae, are hindered by the availability of suitable standards of arsenosugars, the major species in these types of samples. Moreover, chemical syntheses of such compounds have been reported to be complex and tedious. The aim of this work was to investigate the feasibility of the anionic exchange SPE cartridges (SAX and WAX) as an easy and quick alternative for the isolation and preconcentration of arsenosugars. Two commercial silica-based SPE cartridges strong anion exchange sorbent (DSC-SAX) and weak anion exchange sorbent (DSC-NH2) were compared for the SPE of three arsenosugars (PO4-Sug, SO3-Sug and SO4-Sug). The effect of pH, ionic strength, type of salt and elution solvent on the elution protocols of these arsenosugars are studied. Eluted solutions from SPE were analyzed by ICP-MS for total arsenic content and IC-ICP-MS for the study of arsenic speciation. The developed SPE procedure allows to obtain a solution containing the three arsenosugars isolated from other arsenic species with recoveries over 75% for SO3-Sug and SO4-Sug, whereas for PO4-Sug were around 45%.

Metabolic and residual characteristic of different arsenic species contained in laver during mouse digestion.

Laver is one of the major arsenic contributors to human diets. The study on metabolic and residual characteristic of each arsenic species contained in laver is important to scientifically assess the intake risk of arsenic in the laver. The metabolic and residual characteristic of main arsenic species in laver, namely arsenate [As(V)], dimethylarsinic acid [DMA(V)] and two arsenosugars, was investigated by mouse experiments in this study. The results showed that the intake of higher-dose laver did not lead to a notable increase of As(V) concentration in mouse muscle/organs and feces. In contrast, DMA(V) excretion in feces and DMA(V) residue in muscle/organs showed a close correlation with laver-dose intake. Most DMAsSugarMethoxy was translated into other arsenic species and then was together excreted out via mouse feces; two dominant arsenic species, arsenosugar DMAsSugarMethoxy and DMAsSugarPhosphate, were not detected in mouse muscle/organs after 20-Day or 30-Day feeding whether in lower-dose laver groups containing 1/36 (mass ratio) of the laver in mouse feed or higher-dose laver groups containing 1/6 (mass ratio) of the laver in mouse feed. About 65-77% of total arsenic digested by mouse was excreted out via feces; only 0.12-0.78% of it was accumulated in mouse organs/muscle. The results of this study provided valuable knowledge for comprehending the stability and metabolic characteristics of different arsenic species from Fujian laver in vivo, also for more scientifically assessing the intake risk of arsenic in laver.

Intake of food supplements based on algae or cyanobacteria may pose a health risk due to elevated concentrations of arsenic species.

Despite the health benefits of food supplements (FS) based on algae or cyanobacteria, the elevated arsenic (As) concentrations in these FS may raise a health concern. In the present study 33 FS containing algae or cyanobacteria were collected and As (species) were analysed to estimate consumer exposure. Based on hazard and exposure data, potential risks were evaluated using inorganic arsenic (Asi) and the potentially toxic As fraction (Astot minus arsenobetaine (AB)). Astot concentrations were in the range 0.053-57 mg/kg with highest concentrations in FS containing brown algae. Asi concentrations were in the range <0.02-4.7 mg kg-1. A large part of As in FS containing algae or cyanobacteria was identified as potentially toxic AsSugars species. Negligible amounts of AB were detected. According to a tentative risk evaluation, the intake of Asi related to all FS collected was of no health concern for the general population. In 8 out of 33 of the analysed FS, however, the Asi concentration was of concern for population groups with increased cancer risks. If all As species except the non-toxic AB were taken into consideration, only 26 out of 33 of the FS showed 'no concern' for the general population, while for the other 7 FS a potential health risk was identified. This study indicates the need to obtain more data on toxicity of AsSugars and to develop limits for As (species) in FS.

Determination of total arsenic and hydrophilic arsenic species in seafood.

Marine organisms are vital sources of staple and functional food but are also the major dietary route of human exposure to total arsenic. We surveyed the total arsenic content and the mass fractions of hydrophilic arsenic species from five different marine food types cutting across the food chain from microalgae, macroalgae, bivalve clam, crustaceans and finfish. Total arsenic was determined using inductively coupled plasma-mass spectrometry (ICP-MS) while arsenic speciation analysis was performed using high-performance liquid chromatography (HPLC) coupled to ICP-MS as the detector. The total arsenic contents ranged from 133 ± 11 ng/g to 26,630 ± 520 ng/g. The mass fractions of inorganic arsenic (iAs), arsenobetaine (AsB), dimethylarsinic acid (DMA), and the four commonly occurring arsenosugars (AsSugars) are reported. Extractable hydrophilic arsenic species accounted for 10 % (aquacultured shrimp) to 95 % (kelp) of the total arsenic. DMA was established to be a byproduct of the decomposition of AsSugars in acid extracts of samples known to contain these species.

Arsenic species and their transformation pathways in marine plants. Usefulness of advanced hyphenated techniques HPLC/ICP-MS and UPLC/ESI-MS/MS in arsenic species analysis.

The growing popularity of algae as a foodstuff around the world raises concern for the safety of this food type with respect to arsenic content in algae. The need for determination of total arsenic content and arsenic speciation in algae food has become an important issue. In this paper we have developed a complete analytical procedure for arsenic determination in algae products comprised of 1) total arsenic (tAs) determination in native algae samples after digestion, 2) extraction of As species with the use of two extraction methods with three extracting agents, 3) extracted total arsenic (extracted tAs) determination in algae extracts, 4) bespoke As speciation, 4) mass balance estimation based on extracted tAs and bespoke As speciation results, 5) unknown arsenic (uAs) species identification. Two advanced hyphenated techniques, HPLC/ICP-MS and UPLC/ESI-MS/MS, were employed along with the HPLC/ICP-MS method validation. Total As content in edible algae samples was found to range from (19.28 ± 0.45) mg kg-1 up to (72.6 ± 2.7) mg kg-1. Bespoke arsenic speciation of edible algae samples has revealed the presence of some known inorganic and simple organic As compounds such as As(III) from

Investigation of hydride generation from arsenosugars - Is it feasible for speciation analysis?

Hydride generation (HG) from arsenosugars (dimethylarsinoylribosides) in batch and flow injection modes was studied. Its efficiency was found higher in H2SO4 medium than in HCl and higher in the batch mode than in the flow injection mode. To increase the efficiency in the flow injection mode a new generator with two inlets of NaBH4 solution was designed. This modified generator was interfaced between a HPLC column and an atomic fluorescence detector. The arsenosugars studied yielded HG efficiencies in the range 13-30% most probably due to a complicated mechanism of HG. While the mechanism included a formation of two structures of the analyte-borane-complexes, only one of them can lead to a formation of volatile arsanes (dimethylarsane, methylarsane, and arsane were identified).

Arsenic biotransformation by a cyanobacterium Nostoc sp. PCC 7120.

Nostoc sp. PCC 7120 (Nostoc), a typical filamentous cyanobacterium ubiquitous in aquatic system, is recognized as a model organism to study prokaryotic cell differentiation and nitrogen fixation. In this study, Nostoc cells incubated with arsenite (As(III)) for two weeks were extracted with dichloromethane/methanol (DCM/MeOH) and the extract was partitioned between water and DCM. Arsenic species in aqueous and DCM layers were determined using high performance liquid chromatography - inductively coupled plasma mass spectrometer/electrospray tandem mass spectrometry (HPLC-ICPMS/ESIMSMS). In addition to inorganic arsenic (iAs), the aqueous layer also contained monomethylarsonate (MAs(V)), dimethylarsinate (DMAs(V)), and the two arsenosugars, namely a glycerol arsenosugar (Oxo-Gly) and a phosphate arsenosugar (Oxo-PO4). Two major arsenosugar phospholipids (AsSugPL982 and AsSugPL984) were detected in DCM fraction. Arsenic in the growth medium was also investigated by HPLC/ICPMS and shown to be present mainly as the inorganic forms As(III) and As(V) accounting for 29%-38% and 29%-57% of the total arsenic respectively. The total arsenic of methylated arsenic, arsenosugars, and arsenosugar phospholipids in Nostoc cells with increasing As(III) exposure were not markedly different, indicating that the transformation to organoarsenic in Nostoc was not dependent on As(III) concentration in the medium. Our results provide new insights into the role of cyanobacteria in the biogeochemical cycling of arsenic.

Algae as nutritional and functional food sources: revisiting our understanding.

Global demand for macroalgal and microalgal foods is growing, and algae are increasingly being consumed for functional benefits beyond the traditional considerations of nutrition and health. There is substantial evidence for the health benefits of algal-derived food products, but there remain considerable challenges in quantifying these benefits, as well as possible adverse effects. First, there is a limited understanding of nutritional composition across algal species, geographical regions, and seasons, all of which can substantially affect their dietary value. The second issue is quantifying which fractions of algal foods are bioavailable to humans, and which factors influence how food constituents are released, ranging from food preparation through genetic differentiation in the gut microbiome. Third is understanding how algal nutritional and functional constituents interact in human metabolism. Superimposed considerations are the effects of harvesting, storage, and food processing techniques that can dramatically influence the potential nutritive value of algal-derived foods. We highlight this rapidly advancing area of algal science with a particular focus on the key research required to assess better the health benefits of an alga or algal product. There are rich opportunities for phycologists in this emerging field, requiring exciting new experimental and collaborative approaches.

Bioaccessibility and degradation of naturally occurring arsenic species from food in the human gastrointestinal tract.

Humans are exposed to organic arsenic species through their diet and therefore, are susceptible to arsenic toxicity. Investigating the transformations occurring in the gastrointestinal tract will influence which arsenic species to focus on when studying metabolism in cells. Using a physiologically based extraction test, the bioaccessibility of arsenic species was determined after the simulated gastrointestinal digestion of rice, seaweed and fish. Pure standards of the major arsenic species present in these foodstuffs (arsenic glutathione complexes, arsenosugars and short chain fatty acids) were also evaluated to assess the effect of the food matrix on bioaccessibility and transformation. Approximately 80% of arsenic is released from these foodstuffs, potentially becoming available. Hydrolysis and demethylation of arsenic glutathione complexes and arsenosugars standards was observed, but no transformations occurred to arsenosugars present in seaweed. Demethylation of MA and DMA from rice occurs increasing the amount of inorganic arsenic species available for metabolism.

In vitro toxicological characterization of two arsenosugars and their metabolites.

SCOPE: In their recently published Scientific Opinion on Arsenic in Food, the European Food Safety Authority concluded that a risk assessment for arsenosugars is currently not possible, largely because of the lack of relevant toxicological data. To address this issue, we carried out a toxicological in vitro characterization of two arsenosugars and six arsenosugar metabolites. METHODS AND RESULTS: The highly pure synthesized arsenosugars, DMA(V) -sugar-glycerol and DMA(V) -sugar-sulfate, investigated in this study, as well as four metabolites, oxo-dimethylarsenoacetic acid (oxo-DMAA(V) ), oxo-dimethylarsenoethanol (oxo-DMAE(V) ), thio-DMAA(V) and thio-DMAE(V) , exerted neither cytotoxicity nor genotoxicity up to 500 µM exposure in cultured human bladder cells. However, two arsenosugar metabolites, namely dimethyl-arsinic acid (DMA(V) ) and thio-dimethylarsinic acid (thio-DMA(V) ), were toxic to the cells; thio-DMA(V) was even slightly more cytotoxic than arsenite. Additionally, intestinal bioavailability of the arsenosugars was assessed applying the Caco-2 intestinal barrier model. The observed low, but significant transfer rates of the arsenosugars across the barrier model provide further evidence that arsenosugars are intestinally bioavailable. CONCLUSION: In a cellular system that metabolizes arsenosugars, cellular toxicity likely arises. Thus, in strong contrast to arsenobetaine, arsenosugars cannot be categorized as nontoxic for humans and a risk to human health cannot be excluded.