Tissue accumulation and distribution of arsenic compounds in three marine fish species: relationship to trophic position

In this study the accumulation and distribution of arsenic compounds in marine fish species in relation to their trophic position was investigated. Arsenic compounds were measured in eight tissues of mullet Mugil cephalus (detritivore), luderick Girella tricuspidata (herbivore) and tailor Pomatomus saltatrix (carnivore) by high performance liquid chromatography–inductively coupled plasma‐mass spectrometry. The majority of arsenic in tailor tissues, the pelagic carnivore, was present as arsenobetaine (86–94%). Mullet and luderick also contained high amounts of arsenobetaine in all tissues (62–98% and 59–100% respectively) except the intestines (20% and 24% respectively). Appreciable amounts of dimethylarsinic acid (1–39%), arsenate (2–38%), arsenite (1–9%) and trimethylarsine oxide (2–8%) were identified in mullet and luderick tissues. Small amounts of arsenocholine (1–3%), methylarsonic acid (1–3%) and tetramethylarsonium ion (1–2%) were found in some tissues of all three species. A phosphate arsenoriboside was identified in mullet intestine (4%) and from all tissues of luderick (1–6%) except muscle. Pelagic carnivore fish species are exposed mainly to arsenobetaine through their diet and accumulate the majority of arsenic in tissues as this compound. Detritivore and herbivore fish species also accumulate arsenobetaine from their diet, with quantities of other inorganic and organic arsenic compounds. These compounds may result from ingestion of food and sediment, degradation products (e.g. arsenobetaine to trimethylarsine oxide; arsenoribosides to dimethylarsinic acid), conversion (e.g. arsenate to dimethylarsinic acid and trimethylarsine oxide by bacterial action in digestive tissues) and/or in situ enzymatic activity in liver tissue. Copyright © 2001 John Wiley & Sons, Ltd.     

Arsenic bioaccumulation and species in marine polychaeta

Published whole tissue arsenic concentrations in polychaete species tissues range from 1.5–2739 µg arsenic/g dry mass. Higher mean total arsenic concentrations are found in deposit‐feeding polychaetes relative to non‐deposit‐feeding polychaete species collected from the same locations. However, mean arsenic concentrations at some of the locations are skewed by the high arsenic concentrations of Tharyx marioni. There appears to be no direct correlation between sediment arsenic concentrations and polychaete arsenic concentrations. Arsenic bioaccumulation by polychaetes appears to be more controlled by the physiology of the polychaetes rather than exposure to arsenic via ingested material or the prevailing physiochemical conditions. Arsenic concentrations in polychaete tissues can vary greatly. Most polychaete species contain the majority of their arsenic as arsenobetaine (57–98%), with trace concentrations of inorganic arsenic (<1%) and other simple methylated species (<7.5%). However, this is not always the case, with unusually high proportions of arsenite (57%), arsenate (23%) and dimethylarsinic acid (83–87%) in some polychaete species. Arsenobetaine is probably accumulated by polychaetes via organic food sources within the sediment. The presence of relatively high proportions of phosphate arsenoriboside (up to 12%) in some opportunistic omnivorous Nereididae polychaete species may be due to ingestion of macroalgae, benthic diatoms and/or phytoplankton. Consideration of the ecology of individual polychaete species in terms of their habitat type, food preferences, physiology and exposure to arsenic species is needed for the assessment of arsenic uptake pathways and bioaccumulation of arsenic. Future research should collect a range of polychaete species from a wide variety of uncontaminated marine habitats to determine the influence of these ecological factors on total arsenic concentrations and species proportions. Copyright © 2005 John Wiley & Sons, Ltd.     

Ion chromatography–inductively coupled plasma mass spectrometry determination of arsenic species in marine samples

Arsenic speciation analysis in marine samples was performed using ion chromatography (IC) with inductively coupled plasma mass spectrometry (ICP‐MS) detection. The separation of eight arsenic species, viz. arsenite, monomethyl arsonic acid, dimethylarsinic acid, arsenate, arsenobetaine, tetramethylarsine oxide, arsenocholine and tetramethylarsonium ion was achieved on a Dionex AS4A (weaker anion exchange column) by using a nitric acid pH gradient eluent (pH 3.3 to 1.3). The entire separation was accomplished in 12 min. The detection limits for the eight arsenic species by IC–ICP‐MS were in the range 0.03–1.6 µ g l^?1, based on 3σ of the blank response (n = 6). The repeatability and day‐to‐day reproducibility were calculated to be less than 10% (residual standard deviation) for all eight species. The method was validated by analyzing a certified reference material (DORM‐2, dogfish muscle) and then successfully applied to several marine samples, e.g. oyster, fish muscle, shrimp and marine algae. The low power microwave digestion was employed for the extraction of arsenic from seafood products. Copyright © 2004 John Wiley & Sons, Ltd.     

Speciation and health risk considerations of arsenic in the edible mushroom Laccaria amethystina collected from contaminated and uncontaminated locations

Samples of the edible mushroom Laccaria amethystina, which is known to accumulate arsenic, were collected from two uncontaminated beech forests and an arsenic-contaminated one in Denmark. The total arsenic concentration was 23 and 77 μg As g⁻¹ (dry weight) in the two uncontaminated samples and 1420 μg As g⁻¹ in the contaminated sample. The arsenic species were liberated from the samples using focused microwave-assisted extraction, and were separated and detected by anion- and cation-exchange high-performance liquid chromatography with an inductively coupled plasma mass spectrometer as arsenic-selective detector. Dimethylarsinic acid accounted for 68–74%, methylarsonic acid for 0.3–2.9%, trimethylarsine oxide for 0.6–2.0% and arsenic acid for 0.1–6.1% of the total arsenic. The unextractable fraction of arsenic ranged between 15 and 32%. The results also showed that when growing in the highly arsenate-contaminated soil (500–800 μg As g⁻¹) the mushrooms or their associated bacteria were able to biosynthesize dimethylarsinic acid from arsinic acid in the soil. Furthermore, arsenobetaine and trimethylarsine oxide were detected for the first time in Laccaria amethystina. Additionally, unidentified arsenic species were detected in the mushroom. The finding of arsenobetaine and trimethylarsine oxide in low amounts in the mushrooms showed that synthesis of this arsenical in nature is not restricted to marine biota. In order to minimize the toxicological risk of arsenic to humans it is recommended not to consume Laccaria amethystina mushrooms collected from the highly contaminated soil, because of a genotoxic effect of dimethylarsinic acid observed at high doses in animal experiments. © 1998 John Wiley & Sons, Ltd. No Abstract.     

Biodegradation of arsenosugars in marine sediment

In the marine environment, arsenic accumulates in seaweed and occurs mostly in the form of arsenoribofuranosides (often called arsenosugars). This study investigated the degradation pathways of arsenosugars from decaying seaweed in a mesocosm experiment. Brown seaweed (Laminaria digitata) was placed on top of a marine sediment soaked with seawater. Seawater and porewater samples from different depths were collected and analysed for arsenic species in order to identify the degradation products using high‐performance liquid chomatography–inductively coupled plasma mass spectrometry. During the first 10 days most of the arsenic found in the seawater and the shallow sediment is in the form of the arsenosugars released from the seaweed. Dimethylarsenoylethanol (DMAE), dimethylarsinic acid (DMA(V)) and, later, monomethylarsonic acid (MMA(V)) and arsenite and arsenate were also formed. In the deeper anaerobic sediment, the arsenosugars disappear more quickly and DMAE is the main metabolite with 60–80% of the total arsenic for the first 60 days besides a constant DMA(V) contribution of 10–20% of total soluble arsenic. With the degradation of the soluble DMAE the solubility of arsenic decreases in the sediment. The final soluble degradation products (after 106 days) were arsenite, arsenate, MMA(V) and DMA(V). No arsenobetaine or arsenocholine were identified in the porewater. Copyright © 2005 John Wiley & Sons, Ltd.     

Simultaneous separation of 17 inorganic and organic arsenic compounds in marine biota by means of high-performance liquid chromatography/inductively coupled plasma mass spectrometry

A method using high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) has been developed to determine inorganic arsenic (arsenite, arsenate) along with organic arsenic compounds (monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide, tetramethylarsonium ion and several arsenosugars) in fish, mussel, oyster and marine algae samples. The species were extracted by means of a methanol/water mixture and a dispersion unit in 2 min, with extraction efficiencies ranging from 83 to 107% in the different organisms. Up to 17 different species were determined within 15 min on an anion-exchange column, using a nitric acid gradient and an ion-pairing reagent. As all species are shown in one chromatogram, a clear overview of arsenic distribution patterns in different marine organisms is given. Arsenobetaine is the major compound in marine animals whereas arsenosugars and arsenate are dominant in marine algae. The method was validated with CRM DORM-2 (dogfish muscle). Concentrations were within the certified limits and low detection limits of 8 ng g(-1) (arsenite) to 50 ng g(-1) (arsenate) were obtained.     

Arsenic Speciation Analysis in Solutions Treated with Zeolites

Experiments have been carried out to study the behaviour of organoarsenicals treated with zeolites by means of speciation analysis. IC-ICP-MS was applied to identify and quantify arsenite, arsenate and the following organoarsenicals: monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), tetramethylarsonium bromide (TMA⁺), arsenobetaine (AsB) and arsenocholine (AsC). Zeolites loaded with ferrous ions did not significantly increase the retention of inorganic arsenic species compared to the native zeolites, while there was a ten-fold removal of arsenate relating to arsenite. The formation of As(V) and DMA in the leachates containing clinoptilolites and mordenites was confirmed in the presence of natural and synthetic zeolites. Arsenobetaine and arsenocholine yielded higher levels of arsenate than the methylated species.     

Inductively coupled plasma mass spectrometry: Application to the determination of arsenic species

The application of ion-pair reversed phase chromatography (HPLC) and inductively coupled plasma mass spectrometry to the determination of six species of arsenic is described: arsenious acid (As^III), arsenic acid (As^V), monomethylarsinic acid (MMA), dimethylarsinic acid (DMA), arsenocholine (AsC) and arsenobetaine (AsB) in marine biota and in natural fresh water. The coupling conditions of HPLC-ICP-MS are given and also the evaluation of the extraction procedure applied to determine these species in marine organisms. The limits of detection are between 6 and 25 g.l^–1.     

Comparison of extraction procedures for arsenic speciation in environmental solid reference materials by high‐performance liquid chromatography–hydride generation‐atomic fluorescence spectroscopy

Water and ‘soft’ extractions (hydroxylammonium hydrochloride, ammonium oxalate and orthophosphoric acid) have been studied and applied to the determination of arsenic species (arsenite, arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in three environmental solid reference materials (river sediment, agricultural soil, sewage sludge) certified for their total arsenic content. The analytical method used was ion exchange liquid chromatography coupled on‐line to atomic fluorescence spectroscopy through hydride generation. Very low detection limits for arsenic were obtained, ranging from 0.02 to 0.04 mg kg^?1 for all species in all matrices studied. Orthophosphoric acid is the best extractant for sediment (mixed origin) and sludge samples (recent origin) but not for the old formation soil sample, from which arsenic is extracted well only by oxalate. Both inorganic forms (As(III) and As(V)) are significant in all samples, As(V) species being predominant. Moreover, organic forms are found in water extracts of all samples and are more important in the sludge sample. These organic forms are also present in the ‘soft’ extracts of sludge. Microwave‐assisted extraction appears to minimize the risk of a redox interconversion of inorganic arsenic forms. This study points out the necessity of combining direct and sequential extraction procedures to allow for initial arsenic speciation and to elucidate the different mineralogical phases–species associations. Copyright © 2002 John Wiley & Sons, Ltd.     

The separation of dimethylarsinic acid,methylarsonous acid,methylarsonic acid,arsenate and dimethylarsinous acid on the Hamilton PRP‐X100 anion‐exchange column

In order to separate the potential arsenite metabolites methylarsonous acid and dimethylarsinous acid from arsenite, arsenate, methylarsonic acid and dimethylarsinic acid, the pH‐dependent retention behaviour of all six arsenic compounds was studied on a Hamilton PRP‐X100 anion‐exchange column with 30 mM phosphate buffers (pH 5, 6, 7, 8 and 9) containing 20% (v/v) methanol as mobile phase and employing an inductively coupled plasma atomic emission spectrometer (ICP–AES) as the arsenic‐specific detector. Baseline separation of dimethylarsinic acid, methylarsonous acid, methylarsonic acid, arsenate and dimethylarsinous acid was achieved with a 30 mmol dm⁻³ phosphate buffer (pH 5)–methanol mixture (80:20, v/v) in 25 min. Arsenite is not baseline‐separated from dimethylarsinic acid under these conditions. Copyright © 1999 John Wiley & Sons, Ltd.     

Formation of arsenobetaine from arsenocholine by micro-organisms occurring in sediments

As one of the experiments to pursue marine circulation of arsenic, we studied microbiological conversion of arsenocholine to arsenobetaine, because arsenocholine may be a precursor of arsenobetaine in these ecosystems. Two culture media, 1/5 ZoBell 2216E and an aqueous solution of inorganic salts, were used in this in vitro study. To each medium (25 cm³) were added synthetic arsenocholine (0.2%) and about 1 g of the sediment, and they were aerobically incubated at 25°C in the dark. These conversion experiments were performed in May and July 1990. In both seasons, two or three metabolites were derived in each mixture. These metabolites were purified using cation-exchange chromatography. Their structures were confirmed as arsenobetaine, trimethylarsine oxide and dimethylarsinic acid by high-performance liquid chromatography, thin-layer chromatography, FAB mass spectrometry and a combination of gas-chromatographic separation with hydride generation followed by a cold-trap technique and selected-ion monitoring mass spectrometric analysis. From this and other evidence it is concluded that, in the arsenic cycle in these marine ecosystems, as recently postulated by us, the pathway arsenocholine → arsenobetaine → trimethylarsine oxide → dimethylarsinic acid → methanearsonic acid → inorganic arsenic can be carried out by micro-organisms alone.     

Degradation of a tetramethylarsonium salt by microorganisms occurring in sediments and suspended substances under both aerobic and anaerobic conditions

Microbial degradation of a tetramethylarsonium salt during incubation at 25°C was investigated under both aerobic and anaerobic conditions. Two media (1/5 ZoBell 2216E and inorganic salt medium), added with the sediments or suspended substances as the sources of the microorganisms, were used. Degradation of the tetramethylarsonium salt occurred only in the ZoBell medium: under anaerobic conditions, trimethylarsine oxide and dimethylarsinic acid were derived with the sediments, and dimethylarsinic acid with the suspended substances, the salt degrading more rapidly with the former than with the latter. Small amounts of two metabolites, trimethylarsine oxide and inorganic arsenic(V), was also derived in the aerobically incubated ZoBell medium added with the suspended substances. This result means that the tetramethylarsonium salt is degraded to inorganic arsenic, which is the starting material for arsenic circulation in marine ecosystems, via trimethylarsine oxide and dimethylarsinic acid.     

Organoarsenic compounds in plants and soil on top of an ore vein

Plants and soil collected above an ore vein in Gasen (Austria) were investigated for total arsenic concentrations by inductively coupled plasma mass spectrometry (ICP‐MS). Total arsenic concentrations in all samples were higher than those usually found at non‐contaminated sites. The arsenic concentration in the soil ranged from ∼700 to ∼4000 mg kg⁻¹ dry mass. Arsenic concentrations in plant samples ranged from ∼0.5 to 6 mg kg⁻¹ dry mass and varied with plant species and plant part. Examination of plant and soil extracts by high‐performance liquid chromatography–ICP‐MS revealed that only small amounts of arsenic (<1%) could be extracted from the soil and the main part of the extractable arsenic from soil was inorganic arsenic, dominated by arsenate. Trimethylarsine oxide and arsenobetaine were also detected as minor compounds in soil. The extracts of the plants (Trifolium pratense, Dactylis glomerata, and Plantago lanceolata) contained arsenate, arsenite, methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, the tetramethylarsonium ion, arsenobetaine, and arsenocholine (2.5–12% extraction efficiency). The arsenic compounds and their concentrations differed with plant species. The extracts of D. glomerata and P. lanceolata contained mainly inorganic arsenic compounds typical of most other plants. T. pratense, on the other hand, contained mainly organic arsenicals and the major compound was methylarsonic acid. Copyright © 2002 John Wiley & Sons, Ltd.     

Quantitative Speciation of Arsenic in Water and Sediment Samples from the Mokolo River in Limpopo Province,South Africa

The Mokolo River is disposed to environmental contaminants such as arsenic (As) due to its proximity to several anthropogenic activities. Speciation of As in water and sediment samples from Mokolo River is crucial to evaluate the level and distribution of As in the river and underlying sediment since toxicity depends on its chemical forms. In this study, As species in water and sediment were determined by developing a new method for sediment extraction. Effective microwave-assisted extraction of As species in sediment samples was achieved using 0.3?M (NH₄)₂HPO₄ and 50?mM EDTA, which showed no species interconversion during extraction. The chromatographic separation and detection of As(III), dimethylarsinic acid (DMA), monomethylarsonic acid, and As(V) in water and sediment samples were achieved by coupling to high-performance liquid chromatography to inductively coupled plasma mass spectrometry. Baseline separation of four As species was achieved in 12?min using gradient elution with 10 and 60?mM NH₄NO₃ at pH 8.7 as the mobile phase. The analytical figures of merit and validation of analytical procedures were assessed and adequate performance and percentage recoveries ranging from 81.1 to 102% for water samples and 73.0–92.0% for sediments were achieved. The As species concentration in water and sediment samples was found to be in the range of 0.304–4.99?µg?L^?1 and 74.0–92.0?ng?g^?1, respectively. DMA was not detected in both water and sediment samples.     

Determination of inorganic arsenic and organic arsenic compounds in marine organisms by hydride generation/cold trap/gas chromatography— mass spectrometry

The behavior of arsenite, methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, dimethyl-R-arsine oxides, and trimethyl-R-arsonium compounds (R = carboxymethyl, 2-carboxyethyl, 2-hydroxyethyl) toward sodium borohydride and hot aqueous sodium hydroxide was investigated. The arsines obtained by sodium borohydride reduction of the undigested and digested solutions were collected in a liquid-nitrogen cooled trap, separated with a gas chromatograph, and detected with a mass spectrometer in the selected-ion-monitoring mode. The investigated arsenic compounds were stable in hot 2 mol dm^?3 sodium hydroxide except arsenobetaine [trimethyl(carboxymethyl)arsonium zwitterion] that was converted to trimethylarsine oxide, and dimethyl(ribosyl)arsine oxides that were decomposed to dimethylarsinic acid. Hydride generation before and after digestion of extracts from marine organisms allowed inorganic arsenic, methylated arsenic, arsenobetaine, and ribosyl arsenic compounds to be identified and quantified. This method was applied to extracts from shellfish, fish, crustaceans, and seaweeds.     

Arsenobetaine and other arsenic species in mushrooms

Arsenic species in arsenic accumulating mush- rooms (Sarcosphaera coronaria, Laccaria amethystina, Sarcodon imbricatum, Entoloma lividum, Agaricus haemorrhoidaius, Agaricus placomyces, Lycoperdon perlatum) were determined. HPLC/ICP MS and ion-exchange chromatogra- phy–instrumental neutron activation analysis (NAA) combinations were used. The remarkable accumulator Sarcosphaera coronaria (up to 2000 mg As kg^?1 dry wt) contained only methylarsonic acid, Entoloma lividum only arsenite and arsenate. In Laccaria amethystina dimethylarsinic acid was the major arsenic compound. Sarcodon imbricatum and the two Agaricus sp. were found to contain arsenobetaine as the major arsenic species, a form which had previously been found only in marine biota. Its identification was confirmed by electron impact MS.     

Excretion patterns of arsenic and its metabolites in human saliva and urine after ingestion of Chinese seaweed

There are no reports in scientific literature on arsenic species in human saliva after seaweed exposure. The present article reports for the first time the regular excretion patterns of arsenic in the saliva of volunteers with one-time ingestion of Chinese seaweed. Total arsenic and speciation analyses were carried out by high-performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC-ICP-MS). Results show that the excretion time of total arsenic in saliva is a trifle earlier than that in urine, total arsenic in human saliva also shows a regular excretion pattern like that in urine within 72 h after exposure to seaweed. For speciation analysis, four species, including the major dimethylarsinic acid (DMA) species, were detected in urine prior to seaweed intake. Six species were detected in urine after seaweed ingestion, including DMA, methylarsonic acid (MMA), oxo-dimethylarsinoylethanol (oxo-DMAE), thio-dimethlyarsenoacetate (thio-DMAA), arsenite (As^III) and arsenate (As^V). In saliva samples, three species were found before seaweed ingestion, with the major peak identified as As^III. After consumption, the kinds of arsenic metabolites in saliva were less than those in urine. The major species was inorganic arsenic (iAs As^III+As^V), followed by DMA, MMA and a trace amount of oxo-DMAE. Taken together, the present study suggests that saliva assay can be used as a potential tool for understanding the regular excretion pattern of total arsenic after seaweed ingestion. Whether or not it’s an efficient tool for assessing arsenic metabolites in humans exposed to seaweed requires further investigation.     

Conversion of arsenobetaine to dimethylarsinic acid by arsenobetaine-decomposing bacteria isolated from coastal sediment

Arsenobetain [(CH₃)₃As⁺CH₂COO^-]-containing growth media (1/5 ZoBell 2216E and solution of inorganic salts) were inoculated with two bacterial strains, which were isolated from a coastal sediment and identified as members of the Vibro-Aeromonas group, and incubated under aerobic and anaerobic conditions. Arsenobetaine was converted to a metabolite only under aerobic conditions. This arsenic metabolite was identified as dimethylarsinic acid [(CH₃)₂AsOOH] by hydride generation/cold trap/GC MS/SIM analysis and high-performance liquid-chromatographic behaviour. The conversion pattern shown by these arsenobetaine-decomposing bacteria (that is, arsenobetaine → dimethylarsinic acid) was fairly different from that shown by the addition of sediment itself as the source of arsenobetaine-decomposing micro-organisms (that is, arsenobetaine → trimethylarsine oxide → inorganic arsenic). This result suggests to us that various micro-organisms, including the arsenobetaine-decomposing bacteria isolated in this study, participate in the degradation of arsenobetaine in marine environments.     

Field Enhanced Sample Injection for the CE Determination of Arsenic Compounds Using Successive Multiple Ionic Polymer Layer Coated Capillaries

A capillary electrophoresis method using indirect UV detection has been applied to the determination of arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid and dimethylarsinic acid. The arsenic species were successfully separated in a successive multiple ionic polymer layer coated capillary. On-line sample preconcentration of arsenic compounds were performed by employing field enhanced sample injection. A baseline separation was achieved in a basic background solution of 10 mM 2,6-pyridinedicarboxylic acid at pH 10.3. The precision of migration time was 1.2–2.4% RSD and peak height was 8.1–12.9% RSD. The limits of detection at a S/N ratio of 3 for the four arsenic compounds were found to be 20–70 ppb, which are comparable to other on-line preconcentration techniques. The enhancement factor was improved by 230–1,500-fold.

     

Arsenic Compounds in Terrestrial Organisms I: Collybia maculata,Collybia butyracea and Amanita muscaria from Arsenic Smelter Sites in Austria

Three mushroom species from two old arsenic smelter sites in Austria were analyzed for arsenic compounds. The total arsenic concentrations were determined by ICP–MS. Collybia maculata contained 30.0 mg, Collybia butyracea 10.9 mg and Amanita muscaria 21.9 mg As kg⁻¹ dry mass. The arsenic compounds extracted with methanol/water (9:1) from the dried mushroom powders were separated by HPLC on anion-exchange and reversed-phase columns and detected by ICP-MS using a hydraulic high-pressure nebulizer. In Collybia maculata almost all arsenic is present as arsenobetaine. Collybia butyracea contained mainly arsenobetaine (8.8 mg As kg⁻¹ dry mass) and dimethylarsinic acid (1.9 mg As kg⁻¹). Amanita muscaria contained arsenobetaine (15.1 mg As kg⁻¹), traces of arsenite, dimethylarsinic acid and arsenate, and surprisingly arsenocholine (2.6 mg As kg⁻¹) and a tetramethylarsonium salt (0.8 mg As kg⁻¹). © 1997 by John Wiley & Sons, Ltd.