In Vitro Evaluation of Ferrihydrite as an Enterosorbent for Arsenicfrom Contaminated Drinking Water

J. F. Taylora, A. Robinsona, N. Johnsona, A. Marroquin-Cardonaa, B. Brattina, R. Taylora,and T. D. Phillipsa,*aDepartment of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas,USA

AbstractArsenic (As) is a toxic trace element found in groundwater due to natural and industrial processes.Exposure has been linked to cancers of the bladder, lungs, skin, kidneys, nasal passages, liver, andthe prostate. Arsenic in drinking water is a problem in many countries, notably Bangladesh andTaiwan. The purpose of this research was to utilize binding isotherms, a simulated gastrointestinal(GI) model, and the adult Hydra bioassay to evaluate ferrihydrite’s potential to bind As and serve asa potential enterosorbent for As found in drinking water. A variety of clay minerals and synthesizediron oxides including ferrihydrite were screened for their ability to bind As(III), as sodium arsenite,and As(V), as sodium arsenate. After ferrihydrite was demonstrated to be the most effective sorbentfor both As species, adsorption isotherms were performed. All isotherm data were fit to the Langmuirequation to determine adsorption capacity (Qmax). Ferrihydrite bound 96% of As(III) and 97% of As(V) in the screening studies and had a Qmax of 1.288 mol/kg for As(III) and 0.744 mol/kg for As(V).Using a simulated GI model, ferrihydrite was found to effectively adsorb As(V) and As(III) in thestomach and intestine. Ferrihydrite (0.25% w/w) protected adult hydra at levels up to 200 times theminimal effective concentration (MEC) for As(III) and up to 2.5 times the MEC for As(V). Theseexperiments confirm that ferrihydrite is a high capacity sorbent of As, and that it is effective atremoving As in a simulated GI model. These results suggest that ferrihydrite could be used as apotential enterosorbent for As found in drinking water. Future work will focus on verifyingferrihydrite’s safety and efficacy in vivo.

IntroductionArsenic (As) is a toxic trace element found in groundwater due to natural and industrialprocesses. Health effects of chronic As exposure from groundwater include skin lesions, skincancer, bladder cancer, kidney cancer, lung cancer, neurological effects, hypertension andcardiovascular disease, pulmonary disease, peripheral vascular disease, and diabetes mellitus(1). Inorganic As, the most common form of As in groundwater, is usually found in one of twoforms, arsenate or arsenite, hereafter referred to as As(V) and As(III), respectively, with As(V) being more prevalent in oxidized environments and As(III) more common in reducedenvironments like those found in groundwater. Relatively slow redox transformations allowboth species to commonly be found in both environments (2). The current WHO guideline andEPA legal limit for As is 10 ug/L in drinking water.

*Corresponding author: Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University,4458 TAMU, College Station, TX 77843, USA. Tel.: (979)845-6414. Fax: (979)862-4929. E-mail address: tphillips@cvm.tamu.edu,(T.D. Phillips).

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Published in final edited form as:Environ Sci Technol. 2009 July 15; 43(14): 5501–5506.

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Surface adsorption is important to As mobility and bioavailability in the environment. Ironoxides and oxy-hydroxides, particularly ferrihydrite (also referred to as hydrous ferric oxide)have a high capacity for adsorption of both As(V) and As(III). Ferrihydrite’s adsorptioncapacity for both arsenic species is higher than more crystalline iron oxides and oxy-hydroxideslike goethite and soil minerals such as kaolinite, montmorillonite, and illite (3,4). The Asbinding reaction with ferrihydrite is pH dependent with As(V) being adsorbed in largerquantities at lower pHs, and As(III) at higher pHs. The two species are equally adsorbed atapproximately pH 6–7.5 (5). At higher pHs, As(V) was found to steadily decrease in amountadsorbed while As(III) bound remained high at increasing pH’s up to about 10 (5).

Geophagy, the intentional consumption of earthy substances (e.g. clay), has been observed inanimals and humans from numerous continents (6). Incidental consumption of soil has beeninvestigated as a potential source of As exposure, particularly in children (7–9). Currently Asin soil is assessed by using total digests of soils and using As concentration values to determinerisks of exposure from contaminated soils (10). Recent research, however, suggests that arsenicfound in soil has limited bioavailability (11–13) and, importantly, studies on arsenate loadedferrihydrite showed that over 95% of the arsenate remained adsorbed to ferrihydrite throughouta simulated GI model (14).

A current strategy for reducing dietary exposure to toxicants such as mycotoxins is the additionof sorbents or binding agents in the diet. These sorbents when added to food or feed serve tosorb the toxins in the GI tract and thus reduce the bioavailability of the toxin (15–18). Previouswork has shown that NovaSil clay, a calcium montmorillonite, can reduce the adverse effectsof exposure to aflatoxin in a variety of animal models. NovaSil and similar montmorillonitesand smectite clays have been shown to protect against the adverse effects of dietary aflatoxinsin a variety of animals including rodents, chicks, turkey poults, ducklings, lambs, pigs, minkand trout (reviewed in 19). More recently, NovaSil has been shown to be safe and e ffectivein an aflatoxin-exposed human population in the West African country of Ghana (20,21). Thefact that much of the As adsorbed to contaminated soil is limited in its bioavailability suggeststhat soil minerals or iron oxides have the potential to protect humans from As found in drinkingwater by using them as enterosorbents. To our knowledge, the potential use of soil minerals oriron oxides and iron oxy-hydroxides as enterosorbents for protection from As has not beentested. Given the success of a similar strategy for mycotoxins, we feel it warrants investigation.For this reason, the objectives of this work were to identify the best potential binding agent forAs and to evaluate its potential use as an enterosorbent for As in vitro.

Materials and MethodsChemical Reagents and Sorbents

All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and were at leastACS reagent grade. Goethite was obtained from Sigma, while kaolinite, muscovite,clinoptilolite, attapulgite, halloysite, and SWy-2 were obtained from the Clay MineralRepository (Purdue University, Indiana). Low pH montmorillonite (LPHM) and NovaSil wereobtained from BASF corporation, Jackson, MS. Goethite, 2-line ferrihydrite (hereafter referredas ferrihydrite), and magnetite were prepared according to Schwertmann and Cornell, 2000(22). All iron oxides were air dried in a fume hood and ground and sieved to < 100 µm. Ironoxides were verified by X-ray diffraction, and their surface areas were measured after degassingat 110°C for 2 hours (5) using a multi-point BET analysis with N2 adsorption on a MicromeriticsASAP 2020. CP-LPHM was prepared according to Lemke et al. (23). SWy-2 was exchangedat 100% of its CEC with L-cysteine ethyl ester, L-cysteine dimethyl ester, and thiamine tomake SW-CYSTE, SW-CYSTI, and SW-THIAM, respectively, using previously establishedmethods (24).

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Arsenic Screening ProcedureStock solutions of 9.375 and 37.5 ppm As(V) and As(III), respectively, were prepared in waterfrom sodium metarsenite and sodium arsenate, respectively. The final pH of each solution wasadjusted to 7. Into 15 mL polypropylene tubes, 4 mL of the stock solution of each specie ofarsenic was added along with 1 mL of a 5 mg/mL slurry of sorbent for a final concentration of1 mg/mL sorbent and 7.5 ppm As(V) or 30 ppm As(III). The final concentrations were basedon previous work showing that 1 mg/mL ferrihydrite in a total of 5 mL solution bound 98%of 7.5 ppm As(V) in water (25) and from Raven et al. (5) who found the adsorption maximaof As(III) on ferrihydrite to be nearly 4 times higher than As(V) at higher pH values (5). Thetubes were then agitated at 1000 rpm on a shaker at 25°C for 4 hours. They were thencentrifuged at 2500 rpm, and 2.5 mL of the supernatant was saved for arsenic analysis. Eachtest was done in triplicate. All As analysis was done by inductively-coupled plasma-opticalemission spectroscopy (ICP-OES).

Arsenic and Iron AnalysisSample extracts were diluted 1:1 with a solution containing deionized water, 2% nitric acid (v/v) and 10% hydrochloric acid (v/v) in order to match the sample matrix to the standards usedto calibrate the instrument. Samples were then analyzed for As and Iron (Fe) using a SpectroCirOS axial ICP-OES (Spectro AI, Fitchburg, MA) employing a modified version of USEPAMethod 200.7 (26). The diluent was analyzed as an independent sample, which showed notraces of As or Fe at the levels measured in the samples. The instrument was calibrated usinga series of standards (As 100 ppm, Fe 500 ppm) at approximately twice the concentration insolution observed in the highest test sample for As and above all Fe samples. The calibrationwas verified against independent standards and the calibration blank before the analytical run,approximately every ten samples during the run, and at the end of the analytical run. Ytterbiumwas employed as an internal standard.

Arsenic Isotherm ProcedureIsotherms were run using modifications of methods previously reported (27,5). Stock solutionsof 125 ppm As(III) and As(V) were prepared in water from sodium metarsenite and sodiumarsenate, respectively. The final pH of each solution was adjusted to 7. From these stocksolutions of As(III) or As(V), a series of dilutions were added to 15 mL polypropylene tubesalong with 1 mL of a 5 mg/mL suspension of sorbent for a final volume of 5 mL and finalsorbent concentration of 1 mg/mL. Final concentrations of the respective arsenic species were100, 50, 25, 10, 5, 2.5, and 1 ppm. Each dilution was prepared in triplicate. The tubes werethen agitated at 1000 rpm on a shaker at 25°C for 4 hours. They were then centrifuged at 2500rpm for 20 min, and 2.5 mL of the supernatant was saved for arsenic analysis via ICP-OES. .Binding data were fit to the Langmuir equation using Table 2 Curve software (27).

Adult Hydra BioassayThe adult Hydra bioassay was carried out as previously described (28,29). Hydra vulgaris wereobtained as a gift from E. Marshall Johnson, Jefferson Medical College (Philadelphia, PA) andwere cultured in shallow trays containing media consisting of 1.0 mM calcium chloride, 0.45mM TES buffer, and 0.012 mM EDTA, pH 7 at 18°C. These conditions ensure asexualreproduction of Hydra, which aids in the reliability and reproducibility of this assay. For assays,only adult Hydra were used. For each concentration of As(III) or As(V), 4.0 mL of media, withor without As, was placed in a small Petri dish at 18°C; all assays with As were done withoutEDTA in the media. Adult Hydra were observed for signs of toxicity at 0, 4, 20, 28, 44, 68 and92 hours. The tulip stage has been validated as the toxic endpoint of the assay (28,29). Theminimal effective concentration (MEC) at 92 h was determined for As(III) and As(V) througha series of assays, consisting of three phases. The first phase involved exposing adult Hydra

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to whole log concentrations of each As specie to determine the range of toxicity apparent within92 h. The lowest concentration of As resulting in the toxic endpoint, MEC, was carried forwardto the second phase of testing. In phase II of testing, the highest and lowest concentrations ofAs were within one log unit of one another. The MEC from the second phase of testing wasused as the highest concentration in the phase III testing. The results from the phase III testwere used to confirm those of the previous assays. To evaluate ferrihydrite’s ability to protectHydra, 0.25% ferrihydrite was added to each dish 24 hours prior to the addition of Hydra, andthe assay was carried out as previously indicated.

Arsenic Adsorption to Ferrihydrite in a Simulated GI ModelAs adsorption on ferrihydrite was evaluated in a simulated GI tract. We used a previouslydescribed method with slight modifications as follows (14,30). To simulate the binding of Asto ferrihydrite in the stomach, 1.5g of ferrihydrite was added to 600 mL of water in a water-jacketed beaker at 37°C containing 0, 10, 25, 50 or 100 ppm As(V) or As(III) at pH 1.8 usingconcentrated HCl. The solution was stirred with an overhead stirrer at approximately 100 rpmwhile argon was bubbled through the solution. The total time for the stomach phase was 2hours. To simulate the conditions in the intestine, the pH of the solution was raised to 6.5 bythe addition of saturated NaHCO3. The total time in the intestine phase was 4 hours with thetotal time for both phases equaling 6 hours. Aliquots (40 mL) were taken at the end of eachphase, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was filtered through a0.45 µm nylon filter and analyzed for Fe and As by ICP-OES.

Results and DiscussionFerrihydrite proved to be the most effective sorbent in the screening experiments, binding95.9% and 97.2% of the As(III) and As(V) in solution (Figure 1). Attapulgite and clinoptiloliteboth have very large surface areas with small pores, but neither is an effective sorbent for As(III) or As(V) when compared to the iron oxides and ferrihydrite. The two montmorilloniteminerals, NovaSil and SWy-2, adsorbed virtually none of the As(V), but bound 9.93% and9.34%, respectively of the As(III). The exchanging of SWy-2 with sulfur containing organiccations greatly increased the adsorption of both As species. SW-CYSTE, SW-CSYSTI, andSW-THIAM all showed a ffinity for As(III) and As(V) suggesting that they could be effectivein removing As from water. They have previously been shown to be effective at removingheavy metals from water including mercury (24). However, this is the first report of these typesof clay-based composites having efficacy for As. The exchanging of low pH montmorillonite(LPHM) with cetyl pyridinium (CP) increased the binding of As(V) when compared to theother montmorillonites. Since we exceeded a 1:1 stoichiometry for exchange, this effect couldbe due to an excess of the organic cations on the clay surface which have been shown to resultin a positively charged clay. The positively charged clay may then attract As(V), which wouldbe in the H2AsO4

− or HAsO42− state at neutral pH. As(III) exists as H3AsO3 at neutral pH

which could explain CP-LPHM’s lack of affinity for As(III). It was clear from these screeningexperiments that ferrihydrite had a much higher adsorption capacity for both As species thanany of the other sorbents tested, including two goethites and magnetite.

Binding isotherms for As(III) and As(V) further illustrated ferrihydrite’s ability to adsorb As(Figure 2). Ferrihydrite had a maximum adsorption capacity (Qmax) and affinity (Kd) of 1.288and 2.7×103 mol/kg and 0.745 and 5.27×104 mol/kg for As(III) and As(V), respectively.Ferrihydrite’s high capacity for As adsorption has been demonstrated previously (5). As(V)and As(III) both form innersphere (or bidentate) complexes on the surface of ferrihydrite(31–34). The large binding capacity of ferrihydrite is due to a high surface area, calculated as273 m2 /g by multi-point BET. This finding is consistent with previous characterization of this

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material indicating that the surface area of synthetic ferrihydrite ranges between 200–320m2 /g (22).

Due to the pH dependent nature of the As-Fe binding reaction, As binding to ferrihydrite wasdemonstrated using a simulated GI model. This GI model has been used to evaluate the potentialbioavailability of As from contaminated soil and As loaded ferrihydrite (14,30). Moreover, ithas been positively correlated with in vivo results in pigs (30). In the present study, experimentswere conducted to evaluate ferrihydrite’s ability to sorb As from the GI tract to simulate anexposure to contaminated drinking water with subsequent, or prior treatment with ferrihydriteas an enterosorbent. The results showed that ferrihydrite retains significant affinity for both As(III) and As(V) with observed maximum binding of 0.212 mol/kg and 0.194 mol/kg,respectively, in the simulated stomach (Figure 3). These values were lower than what wasfound in isotherms which could be a result of the optimal pH for As(V) binding being around4, while the optimal pH for As(III) is above 7 (5). Small changes in sorption could result fromAs(III) being oxidize to As(V) by the conditions of the simulated stomach. However, oxidationof As(III) in the simulated stomach (pH 1.8 under anoxic conditions) would be unlikely giventhe duration of our study, i.e., 2 hr. Nakazawa and Hareyama noticed no observable As(III)oxidation in aqueous media at pH 1.8 after 5 days(35). Observed maximum bound values forAs binding to ferrihydrite in the simulated intestine were 0.335 mol/kg and 0.266 mol/kg forAs(III) and As(V), respectively (Figure 3). Raven et al. showed that ferrihydrite has similaraffinities for both As(III) and As(V) from pH 6–7.5. These values are also similar to thosefound for mycotoxin binders which have been shown to be effective in vivo (15,16).

Along with As, the quantity of Fe that passed through the 0.45 µm filter was measured.Similarly to Beak et al (14), we chose to use a 0.45 µm filter to mimic the 0.5 µm size cutoffof particles capable of entering epithelial cells. This would approximate the Fe exposure tocells that is either dissolved Fe (likely in the form of Fe3+) or colloidal ferrihydrite remainingin suspension after centrifugation. The levels of Fe in the simulated stomach decreased withincreasing As(V) concentration, similar to what was previously reported (14); however, therewas no clear trend of Fe concentration with respect to increasing As(III) concentration (Figure4 and Figure 5). The maximum Fe levels seen in the stomach phase reached 7.57 ppm and 8.97ppm with initial As(V) concentration of 10 ppm and initial As(III) concentration of 10 ppm,respectively. Concentrations of Fe seen in the simulated intestine were very low for allconcentrations tested and for both As(III) and As(V). The highest Fe concentration measuredin the intestine was 0.12 ppm. The recommended daily intake of Fe is 18 mg, with the maximumtolerable daily Fe intake being 40 mg for children and 45 mg for adults (36). The maximumexpected Fe available for absorption from each test with a total of 1.5g of ferrihydrite was 5.4mg. This dose (1.5g) of ferrihydrite was chosen for the GI model based on prior work withNovaSil clay in an aflatoxin-exposed group from Ghana (20). This ferrihydrite level throughoutthe GI model experiments represented the dose if enterosorption therapy is given twice dailyfor a total of 3g/day. Any enterosorption therapy including ferrihydrite would have to be closelymonitored, as some individuals are sensitive to Fe or suffer from hemochromatosis which couldcause a toxic buildup of excess iron (37).

Ferrihydrite was also evaluated for its ability to protect a small aquatic organism, Hydravulgaris, that is very sensitive to As. The genetic homogeneity of this organism has been strictlymaintained in culture to facilitate the reproducibility of the MEC for diverse toxins includingchlorinated phenols (28), heavy metals (38), estrogenic compounds (39), and organophosphatenerve agents (29). The body wall of Hydra consists of a trilaminar structure and a hollow tubecalled the gastric cavity or gut. The Hydra gut contains a mouth (hypostome) and aboral porethat expels digested materials and fluids which is in equilibrium with the surrounding mediaand toxins. The Hydra assay has been reported to accurately predict the safety and efficacy of

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potential enterosorbents (40,41) prior to animal studies and has been utilized along with an invitro GI model (42) for screening purposes.

The toxic endpoint of the Hydra bioassay has been validated as the tulip stage, which representsthe point at which the Hydra will not recover if placed in control media. The minimal effectiveconcentration (MEC) for As(V) and As(III) were 90 ppm and 1 ppm, respectively (Figure 6).The addition of 0.25% w/w ferrihydrite protected the Hydra at As levels of 250 ppm and 200ppm for As(V) and As(III), respectively. Additionally, ferrihydrite was not toxic to the hydra,unlike other sorbents that are lethal to this sensitive organism (40,41). Ferrihydrite alsoprotected the Hydra from As toxicity at levels that were predicted from isothermal analysis.

The current treatments for arsenic from either an acute or chronic exposure include chelationtherapy with analogs of dimercaprol (BAL), oral binders such as charcoal, or agents thatpromote methylation and elimination of As via the urine. Other than remediating current watersources or finding alternative sources, no current treatment exists for reducing exposure to Asfrom drinking water (43). A previous study found no difference in placebo and groups given2,3-dimercaptosuccinic acid for treatment of chronic arsenicosis from drinking water (44). Arecent toxicity profile for As by ATSDR suggested “phosphate” binders and compounds withsulfhydryl groups need further investigation for their potential as treatment for As toxicity(43). Our work demonstrates that ferrihydrite has a high capacity for both species of inorganicAs, was effective in a simulated GI model, and protects a susceptible organism from As toxicity.Previous work that focused on evaluating As bioavailability from contaminated soil, suggestslimited bioavailability from the soil, possibly due in part to surface adsorption to soil minerals(12,13). Concentrations of As found in contaminated wells from Bandgladesh and Taiwan havebeen shown to range from 50–6700 ppb (45); our tests demonstrate that ferrihydrite may offerprotection from As toxicity at even higher levels of contamination. Our work shows thatferrihydrite could offer protection from As, either long or short term, but future work is neededto verify safety and efficacy in vivo. Studies are ongoing to evaluate ferrihydrite’s safety andefficacy in a rodent model.

AcknowledgementsThis research was supported by NIEHS P42-ES04917.

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Figure 1.Initial As(V) concentration was 5 ppm. Initial As(III) concentration was 30 ppm, and sorbentconcentration was 1 mg/mL. Ferrihydrite bound 97.2% of the As(V) in solution, and theexchanging of SWy-2 resulted in increased binding from 0.0% for SWy-2 to 10.5%, 22.45%,and 30.1% for SW-CYSTE, SW-CYSTI, and SW-THIAM, respectively. In As(III) screenings,ferrihydrite, bound 95.9% of the As(III) in solution while the exchanging of SWy-2 with S-containing groups increased the binding of As(III) from 9.3% to 18.1%, 32.9%, and 24.8% forSW-CYSTE, SW-CYSTI, and SW-THIAM, respectively. Key: 1 Attapulgite, 2 Clinoptilolite,3 CP-LPHM, 4 Goethite (Sigma), 5 Halloysite, 6 Muscovite, 7 NS, 8 SWy-2, 9 Ferrihydrite,10 Goethite (synthetic), 11 Magnetite, 12 SW-CYSTE, 13 SW-CYSTI, 14 SW-THIAM. Errorbars represent ± 1 standard deviation.

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Figure 2.Isotherms performed at initial pH 7 verified ferrihydrite’s high binding capacity for botharsenate and arsenite. The high capacity of ferrihydrite for As(III) and As(V) is demonstratedby Qmax values of 1.288 and 0.745 mol/kg, respectively. Error bars represent ± 1 standarddeviation.

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Figure 3.Ferrihydrite binds both As(V) and As(III) at a high capacity in a GI model. Observed maximumbound values for As(III) binding to ferrihydrite were 0.211 mol/kg and 0.335 mol/kg for thesimulated stomach and intestine, respectively. For As(V), the observed maximum bound valueswere 0.194 mol/kg and 0.266 mol/kg for the simulated stomach and intestine, respectively.These results confirm that ferrihydrite exhibits high binding capacity at low pH for both Asspecies and maintains high capacity throughout the simulated intestine. Error bars represent ±1 standard deviation.

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Figure 4.In the simulated stomach, measured iron generally decreased as initial As(V) concentrationincreased suggesting that the increased surface coverage of As(V) on ferrihydrite offeredprotection from acid dissolution. Iron levels were low throughout the simulated intestine. Errorbars represent ± 1 standard deviation.

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Figure 5.In the simulated stomach, measurable iron was found with all initial As(III) concentrationswith no clear pattern of iron concentration. Iron in solution was very low in the simulatedintestine at all initial As(III) concentrations. Error bars represent ± 1 standard deviation.

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Figure 6.The results show that As(III) and As(V) are lethal to Hydra at 1 ppm versus 90 ppm,respectively. Ferrihydrite protected Hydra from toxicity of both species of As and was not toxicat 0.25% w/w.

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