United States Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park NC 27711 xvEPA Research and Development EPA-600/S1-82-010 Nov 1982 Project Summary Speciation of Arsenic Compounds in Water Supplies Kurt J. Irgolic The objectives of this project were to develop and testanalytical methods that would allow the chemical form (i.e. valence state or compound) of arsenic in drinking waters to be determined, and to use the methods to analyze samples of drinking water from sources where adverse health effects in consumers had been attri- buted to arsenic. Analytical techniques were developed for the determination of arsenate (differential pulse polaro- graphy), for inorganic and organic arsenic compounds (high pressure liquid chromatography with graphite furnace atomic absorption spectro- metry as element-specific detector) and for the detection of arsenocho- line, arsenobetaine, and iodoarsines (mass spectrometry). These tech- niques, inductively coupled argon plasma emission spectrometry, and hydride generation/DC-helium arc emission were used for the character- ization of water samples from Utah, Alaska, Antofagasta, Taiwan and Nova Scotia. The total arsenic con- centration ranged from 18 ppb to 8 ppm with arsenite/arsenate ratios between 0.007 and 3.4. No organic arsenic compounds were detected in any of the water samples. The trace elements Al, B, Ba, Ca, Cu, Fe, Li, Mg, Mn, Na, P, S, Si and Sr were present in most of the water samples. The results show that the various physiological effects observed in populations ex- posed to the arsenic-containing water supplies could not be caused by arsenic compounds other than ar- senite or arsenate. Other trace ele- ments acting in concert with arsenite and/or arsenate might produce these symptoms. However, sufficient data are not yet available to evaluate these hypotheses. This Project Summary was devel- oped by EPA's Health Effects Re- search Laboratory. Research Triangle Park. NC, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction Arsenic is an element possessing a rich chemistry Inorganic and organic arsenic compounds may contain tri- valent arsenic The trivalent arsenic compounds are generally more toxic than the pentavalent derivatives. Many inorganic and organic arsenic com- pounds are linked in a cycle with chemical and biologically mediated reactions changing the compounds into each other The input of arsenic into this cycle is supplied by weathering of arsenic-containing rocks, human use, and disposal of various arsenic com- pounds ------- Since arsenic is ubiquitous, man consumes small amounts of arsenic compounds with the food he eats and the water he drinks. Life developed in the presence of arsenic. Therefore, organisms are expected to tolerate a certain, not yet clearly defined, dosage of arsenic. Certain geographically limited groups oT people have taken arsenic compounds into their systems over extended periods of time. These arsenic compounds are present in their drinking water supplies The most famous localities where arsemc-con- tainmg waters have been consumed are certain regions in Taiwan and the city of Antofagasta in Chile Hyperpigmenta- tion, skin cancer, vascular problems and other ailments have been attributed to the arsenic present in the drinking water Other groups, such as the people in Fallen, Nevada, have been exposed to similar arsenic levels in their drinking water without any ill effects This project was undertaken in order to determine the arsenic compounds and other trace elements present in arsenic- containing water supplies and to check whether these drinking water supplies contain the same or different arsenic compounds. Thus far, arsenite, arsenate, methy- larsmic acid, dimethylarsinic acid, methylarsines, arsenobetame, trimeth- ylarsoniolactic acid and arsenic-contain- ing lipids have been identified in environmental samples. Preservation of Arsenic Compounds in Aqueous Solutions Water samples generally cannot be analyzed for trace elements immediate- ly after collection. Several hours or even several days elapse between collection and analysis. During this time the chemical nature of a trace element, such as arsenic, can change. Trace elements can be lost by volatization and/or can be adsorbed on container walls. The absorption of many metal ions and of phosphate ions has been studied, but arsenic was rarely included. Disagreements exist in the literature as to the extent of loss of arsenic from solutions stored in various containers. Even less certainty exists about the conditions under which various arsenic compounds can be preserved Experi- ments have shown that arsenite, arsenate and dimethylarsinic acid are not adsorbed from 1 ppm solution on the walls of Cubitainers* (soft polyethylene containers manufactured by Kimberly) Walls of Pyrex containers removed approximately one percent of the arsenic from the solutions. Ascorbic acid at a concentration of 1 mg/mL has been found to prevent the oxidation of arsenite to arsenate in distilled water solutions at room temperature On the basis of these results most water samples were collected and stored in Cubitainers and some of the samples were preserved by adding ascorbic acid Development of Analytical Techniques for the Determination of Arsenic Compounds Whereas adequate methods for the determination of total arsenic concen- trations do exist, the choice of tech- niques for the estimation of arsenic compounds is limited All the methods available for the speciation of arsenic compounds at the time this project was initiated had severe limitations. The methods were applicable only to the determination of arsenite, arsenate, methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, ethyl-, propyl- and butylarsonic acid, and the arsmes obtainable from these com- pounds. Non-volatile arsenic com- pounds and arsenic compounds not reducible to volatile arsmes could not be determined with the existing methods. Therefore, an analytical system with an element-specific detector had to be developed that was capable of separat- ing volatile and non-volatile arsenic compounds in complex matrices. The development efforts produced a high pressure liquid chromatography- Hitachi Zeeman graphite furnace ato- mic absorption system, a differential pulse polarographic method for the determination of arsenite and arsenate and the elucidation of the mass spectral behavior of organylarsenic acids, or- ganyl lodoarsmes, arsenocholine and arsenobetaine. Hitachi Zeeman Graphite Furnace Atomic Absorption Spectrometer as an Element- Specific Detector for High Pressure Liquid Chroma tograph y Liquid chromatography and specially high pressure liquid chromatography (HPLC) with the great resolving power of its microparticulate columns are poten- 'Mention of tradenames or commercial products does not constitute endorsement or recommen- dations for use tially the best techniques for the simultaneous detection and determina- tion of arsenic compounds A water sample may contain many substances in addition to arsenic compounds. The common detectors will not respond specifically to arsenic compounds. The identification of arsenic-containing fractions is, therefore, difficult if not impossible unless element-specific detectors with high sensitivity are available A graphite furnace atomic absorption spectrometer (GFAA) com- bines the advantage of element-speci- ficity with high sensitivity for many elements An HPLC-GFAA analytical system was developed employing a Hitachi Zeeman GFAA with a sample valve, an injector, and associated electronics to control the analysis sequence The HPLC-GFAA system has func- tioned almost flawlessly during the past three years Aliquots of the column effluent are automatically transferred into the graphite cup of the GFAA for analysis The time interval between consecutive analyses can be made within 30 seconds. The Hitachi Zeeman GFAA Model 170-70 has a detection limit for arsenic of 10 picograms. This sensitivity is, of course, retained in the HPLC-GFAA system for each injection. Upon migration through the chromato- graphic column the arsenic compounds are separated and spread out into bands. Aliquots of 40 fjL are withdrawn from the effluent. The 40 ^L aliquots taken from the center of the band must each contain at least 10 picograms of arsenic. The detection limit of the HPLC- GFAA system is, therefore, strongly dependent on the degree of band spreading. Conditions have been found which allow the separation of inorganic arsenic (arsenite and arsenate), arsen- ocholine and arsenobetaine employing a C-18 reverse phase column, organ- ylsulfonates as countenons and mix- tures of water/acetonitrile/acetic acid as the mobile phase. Arsenite, arsenate, methylarsonic acid and dimethylarsinic acid were similarly separated using water/methanol mixtures saturated with tetraheptylammonium nitrate as the mobile phase. The HPLC-GFAA system, of course, is neither limited to the analyses of the arsenic compounds listed above nor to compounds contain- ing only arsenic. ------- Differential Pulse Polarographic (DPP) Determination of Arsenate and Arse nit e Arsenite is reducible at the dropping mercury electrode and can be deter- mined polarographically at concentra- tions as low as 0 3 ppb. Arsenate is polarographically inactive under these conditions. Addition of polyhydroxy compounds to an acidic solution of arsenate makes arsenate reducible Among 11 polyhydroxy compounds, D- mannitol at 0.5 M concentration in 2 0 M aqueous perchloric acid produced the largest reduction peak for arsenate The DPP curve of arsenate under these conditions is characterized by maxima at -0 55 V and -075 V Above an As (arsenate) concentration of 500 ppb a current maximum appears at -0 59 V, which increases in intensity with increasing concentration The peak at -0 55 V merges into the current maxi- mum and becomes a shoulder at As(arsenate) concentrations of approx- imately 5 ppm The rather low intensity peak between-0 Wand-0.8 V might be obscured at low arsenate concentra- tions by the solvent breakdown and at high arsenate concentrations by the current maximum The arsenite reduc- tion wave in 2 0 M perchloric acid solution shifts from -0.425 V to -0 34V upon addition of mannitol When arsenite and arsenate are present in solution, the arsenate reduction peak at -0 55 V can be used for the determination of arsenate with some confidence only when the con- centration of As(arsemte) is between 100 ppb and approximately 500 ppb, the current is not lower than 2 /uA and the arsenate concentration is equal to or higher than the arsenite concentration If these conditions are not fulfilled, arsenite must be oxidized to arsenate by cenumflV) ammonium nitrate. Excess cenum(IV) must be reduced with hy- droxylamme hydrochlonde Arsenate is then determined in the presence of mannitol using the peak at -0 55 V. Arsenite is determined in another aliquot of the sample in the absence or presence of mannitol. The arsenate concentration in the sample is obtained as the difference between the total arsenic concentration and the Asfarse- nite) concentration The detection limits for arsenate under these conditions are 6 ppb at the 95 percent confidence level Mass Spectrometry of Organylarsonic Acids, Diorgan ylarsinic A cids, Organyliodoarsmes, Arsenocholine and Arsenobetaine Organic arsenic compounds could perhaps be determined by mass spectro- metry in the residues obtained by evaporation of the water samples Therefore, the mass spectral behavior of several organic arsenic compounds was studied At probe temperatures between 110°C and 250°C required to obtain satisfactory mass spectra, organylar- sonic acids, RAs03H2, and diorganylar- smic acids, R2AsOOH, formed anhy- drides and decomposed The products of these thermal reactions were then ionized and fragmented yielding com- plicated mass spectra with many peaks at m/e values higher than those expected for the molecular ions A detailed investigation of the spectra of five arsonic acids and nine arsinic acids indicated that mass spectrometry was of little value for the identification of arsonic acids, but can be used to establish the presence of diorganylar- smic acids in the residues from water samples Exact mass measurements by high resolution mass spectrometry might be necessary to distinguish arsenic-containing from arsenic-free ions Organyliodoarsmes, RnAsU-n (n=1,2), are much more volatile than arsinic or arsonic acids and are easily prepared by treating these acids with hydnodic acid All of the 14 organyliodoarsmes investi- gated gave intense molecular ion peaks Fragmentation proceeded by loss of alkyl groups, iodine and hydrogen abstraction Organyliodoarsmes are well suited for the mass spectrometric identification of organic arsenic com- pounds which can be converted to iodoarsmes Arsenocholine chloride, [(CH3)3AsCH2 CHgOHJCI, and arsenobetame chloride, [(CH3)3AsCH2COOH] Cl, produce rich mass spectra which do not contain molecular ion peaks The highest mass peaks correspond to (CH3);3AsCH2CH20 and (CH3)2AsCH2COOH, which were formed by thermal cleavage of HCI and CHsCI from the arsonmm salts In spite of the absence of molecular ions, mass spectrometry can provide an indication of the presence of arsenocholme and/or arsenobetaine in a sample. Analysis of Water Samples Samples of arsenic-containing drink- ing water supplies selected by the EPA project officer were collected and shipped to College Station as quickly as possible Total arsenic concentrations and the concentrations of arsenite and arsenate were determined by several methods Each water sample was checked for the presence of methylated arsenic compounds and other organic arsenic derivatives Water samples from Hmckley, Utah, Delta, Utah, Barefoot Site, Alaska, Mauer Site, Alaska, Antofagasta, Chile, Yenshei, Taiwan; Hartlm Site, Nova Scotia; and Sullivan Site, Nova Scotia were invest- igated Graphite furnace atomic absorption spectrometry, differential pulse polaro- graphy, high pressure liquid chromato- graphy with a GFAA as an element- specific detector, the hydride genera- tion technique with a DC-helium arc detector, and inductively coupled argon plasma emission spectrometry were employed for the determination of concentrations of total arsenic, and trace elements The samples were collected and stored in Cubitamers or Pyrex glass containers Unpreserved samples and samples preserved with ascorbic acid or nitric acid were analyzed The analyses were carried out as soon as possible after receipt of the samples The water samples had total arsenic concentrations in the range of 18 ppb to 8 ppm The arsenite/arsenate ratios were in the range of 0 007 to 3 4 (Table 1) No indications of the presence of methylated arsenic compounds, which are reducible to methylarsme or di- methylarsme, have been found Exper- iments with the high pressure liquid chromatograph/graphite furnace atomic absorption spectrometer system, which would provide information about the presence of organic arsenic compounds not reducible to methylarsmes, detected only arsenite and arsenate Comparison of total arsenic concentrations with the sum of the arsenite and arsenate concentrations placed an upper limit on the concentrations of any other arsenic compounds which might be present These upper limits were m most cases in the low ppb range The other trace elements found in these water samples by ICP are also listed m Table 1. There were no signifi- cant concentrations of these elements ------- Table 1. Summary of Total Arsenic, Arsenite, Arsenate and Trace Element Concentrations in Drinking Water Samples^ Hinckley Delta Barefoot]1^ Antofagasta Antofagasta /Wauertt Untreated Treated Yenshei I Yenshet II Nova Scotia 1 Nova Scotia 2 Total As Arsenite Arsenate Arsenite/ Arsenate Ratio Al B Ba Be Ca Cu Fe K Li Mg Mn Na P Pb S Si Sr Ti V Zn 0 18 0010 0 18 006 12 0026 — 324 008 — — — 7 83 — 233 — — * 734 0 10 — — — 002 0010 0010 -7 0 007 0045 — 154 026 0007 — — 665 — 65 — 7 77 * 753 052 — — — 3 7 24 07 34 006-050 — 0 77 — 180-291 — 29-53 12 — 52-113 042 37-47 027 — 28 11 0 047 0008 0 15 0 14 45-60 0 35-4 6 01-43 0 06-0 54 — 023 — 200-309 — 25 13-118 0007 55-139 056-0 76 35-50 — ~ 775 77 0 060 0009 0 77 030 0 75 0016 0 74 002 008 28 0008 74 200 0003 0 11 133 0 62 74 0002 102 034 — « 38 7 028 — — 0 70 047" 0003 047 0007 3 7 2,5 0008 — 203 0007 030 732 064 75 0006 703 026 — • 363 029 — — 0 JO 0 85 0023 084 003 — — — 43 — 046 7 0 0007 86 — 250 49 — 05 34 005 — — — 7 7 0024 1 08 002 057 — — 775 — 0.50 736 007 23 — 796 23 — 75 34 020 — — — SO 45 35 7 3 » * * » » 0008 <0. 1 » * » 24 60 * * * * * <0 7 » * 063 037 032 1 0 » * » * * 027 <0 7 » * * <0 7 50 * + * * * <09 * * "Not determined '**As in treated water is normally less than 100 ppb See text for discussion '[The concentrations are given in ppm ^Results from two different samples collected one year apart in these water supplies with the exception of beryllium found in the untreated Antofagasta sample. The various physiological effects observed in populations exposed to these arsenic-containing drinking water supplies (Table 2) could have been caused by the presence of varying amounts of arsenite and arsenate It is also conceivable that one or more of the trace elements present m the water supplies acted in concert with arsenicto cause the observed effects More samples need to be analyzed and the results of these analyses correlated with epidemiological studies before a definite statement can be made about the interactions of trace elements with arsenite or arsenate The chromatographic work on the fluorescent compounds in the Taiwan well waters strongly suggests the presence of alkaloids, such as D- lysergic acid, ergometrme and calciferol Additional experiments (preparative chromatography, mass spectrometry) could not be carried out because of insufficient samples Table 2. Sampling Location Arsenic-Containing Water Supplies and Their Physiological Manifestations in Man Taiwan (37 villages) Chile (Antofagasta) Bskersfield, CA. Fallon. NV Type of Water and Range of the Total Arsenic Concentration Symptoms Observed in the Pop- ulation Artesian well waters used for 45 years, arsenic leached from geologic deposits, 0 017- 1.097 ppm; median 0 5 ppm Drinking water supply since 1958, 0 8 ppm before water treatment, 0 1 after water treatment Community drinking water supply, 0.3-0 7 ppm Drinking water, 0.1 ppm. Melanosis, keratosis, skin cancel 15% prevalence among males over age 60, normal incidence 2-3% Melanosis, hyperkeratosis, vascular manifestations: myocardialischemia, hemipleglia with occlusion of the carotid artery, mesenteric arterial thrombosis, pneumonia No adverse effects reported * No known adverse physiological effects. * *(n a study of the arsenic exposure of populations in Bakersfield, Ca , and Fallon, Nv., by a questionnaire designed to elicit information about arsenic related symptoms and diseases, very few symptoms were reported The incidence of these symptoms was not significantly different from control populations not exposed to arsenic 4 ------- Recommendations The use of methods such as the hydride generation technique; differential pulse polarography, high pressure liquid chromatography with sensitive, element-specific detectors and colori- metnc methods for determining con- centrations of total arsenic and arsenic compounds, and the availability of simultaneous, inductively coupled argon plasma emission spectrometers for trace element determinations and of ion chromatography for anion analyses make the thorough characterization of water samples a relatively easy and not too-time-consuming task. Additional arsenic-containing water samples must be analyzed in support of or in prepara- tion for epidemiological studies. Exper- ience has shown that analysis by one method is not sufficient to produce reliable results. At least two independent techniques should be used for the determination of arsenic compounds. Kurt J. Irgolic is with Texas A&M University, College Station, TX 77843. Frederick C. Kopfler is the EPA Project Officer (see below). The complete report, entitled "Speciation of Arsenic Compounds in Water Supplies, "(Order No. PB 82 -257 817; Cost: $ 12.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Health Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 27711 •&U. S. GOVERNMENT PRINTING OFFICE: 1982/659-095/553 ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 S Kt S 230 $ DEARBOKN SlRtET CHICAGO it ------- |