Determination of Arsenical Herbicide Residues in Plant Tissues1 R. M. SACHS,~ J. L. MICEIAEL,~ I?. U. ANAS.TASIA,J and W. A. WELL+
Abstract. Paper chromatographic separation of hydroxydi- the toxicity of which may be greater than the parent methylarsine oxide (cacodylic acid), monosodium methane- arsenical (i 1). Comparativk phy?otoxicity studies in our arsonate (MSMA), sodium arsenate, and sodium arsenite was laboratories of foliar and root applications of hydroxy- achieved with the aid of four solvent systems. Aqueous ex- dimethylarsine oxide (cacodylic acid), monosodium meth- tracts of plant tissues removed essentially all the arscnicals anearsonate (MSMA), sodium arsenate, and sodium arse- applied, but mechanoiic fractionation was required before the nite revealed that cacodylic acid was the most potent extracts could be analyzed by paper chromntographic pro- of the four when all were foliarly-applied, whereas cedures. A standard nitric-sulfuric acid digestion procedure sodium arsenite was by far the most phytotoxic when all was employed for arsenic analyses, but great care was taken were root-applied (11). Hence, the need for an examina- LO avoid sulfuric-acid-induced charring by first adding rela- tion of problems of relative absorption, transport, and Lively large amounts of nitric acid to drive off chlorides present. metabolism was indicated. New methods of analysis were Depending upon the amount of chloride present, substantial developed and others modified to investigate these prob- losses of arsenic as arsine chlorides were observed if the samples lems. The methods are described in this paper. Studies charred. Five minutes in fuming sulfuric acid to completely of comparative phytotoxicity, absorption, and metab- break the carbon-arsenic bonds was another critical require- olism will appear separately (11). ment for the quantitative determination of arsenic from cacodylic acid and MSMA. The silver diethyldithiocarbamate MATERIALS calorimetric method was useful for detecting as little as 0.6 pg Seedlings of bean (Phaseolus vuIga~is L., var. Black or as much as 20 llg of arsenic per sample. Valentine), with the first trifoliolate leaves about half expanded, served as the plant tissue in all experiments. Cacodylic acid samples were about 96% pure in crys- r~I~oucx* the lone history of use of arsenicals as try- taline form, ancl MSMA was obtained as a 520/, solution. T panocides, insecticides, and herbicides, several hypoth- Reagent grade sodium arsenate5 and sodium arsenite” eses concerning the toxic effect of arsenic-containing were used to make herbicidal solutions. compounds have been proposed. Many investigations Silver diethyldithiocarbamate (herein after referred to have indicated differences in absorption or metabolic as AgDDC) dissolved in reacent grade pyridine was used conversion of the applied arsenical to other compounds, for calorimetric determination of arsenic. Reagent grade nitric and sulfuric acids were used for ‘Rcccived November 13, 1970. Y)n leave from Ihe Univ. of California. Davis. California. from digestion of plant tissues, and reagent grade solvents September 1969 lo September 1970. were used for chromatographic purposes. Whatman SPrescnt address: Weed Sci. Lab., Colorado State [Jniversity, Fort 3MM paper, in sheets or 3.8-cm strips, was used for paper Collins, Cola. chromatography; Eastman Chromagram Type 6060 cell- ‘Plant I’hysiologis& Plant Sci. Lab., Fort Detrick, F&crick, Maryland. “Obtain4 from the Fisher Chemical Company.
412 Volume 19, Issue 4 (July), 1971 SACHS ET AL. : ARSENICAL RESIDUES IN PLANT TISSUES
ulose and Type GOGI silica gel sheets were used for thin samples containing plant tissue, P-propnnol was added to layer chromatography. A Bausch and Lomb Spectronic the generation mixture to prevent excessive foaming and 20 was used for optical density measurements at 540 mu. overllow from the arsine generator. Between 0.5 and 1 ml of Z-propanol was sufficient to reduce foaming in METHODS ASD RESULTS samples containing 1 g of dried plant tissue. The addi- Arscmk detcmination. The AgDDC calorimetric method lion of 2-propanol reduced the rate of evolution of for determining arsenic was used essentially as described nascent hydrogen and, hence, the rate of formation of by Powers et al. (10) but with modified apparatus and arsine, and prolonged the generation time. Determina- elimination of the silica gel adsorber. A stable red com- Lions of arsenic in samples may not be quantitative and plex is formed between arsine and AgDDC that can be reproducible until hydrogen generation has ceased com- measured quantitatively at one of the absorption maxima; pletely, usually about 30 10 35 mill after the zinc is most often it is measured at 5400 mu. The arsine added. generator designed for this study consisted of a loo-ml We were able to detect as little as 0.G pg and as much Kjehldahl digestion flask, fitted by a serum rubber cap as 20 ug of arsenic per sample with a 5 to 15”r, variation stopper to a filter tube (used as a “scrubber”), which in of the mean. Over this range there was a linear relation- turn was fitted to a rellexed capillary tube by rubber ship between optical density (absorbance) at 540 mu and stopper (Figure 1). The down-turned end of the capillary the concentration of the arsine-AgDDC complex (Figure 2). Owing to operational errors in spectrophotometers,
Figzm 1. Arsinc generator and rack for Kjehldahl digestion flasks. Diagram drawn to scale.
tube is immersed in 5 ml of a 0.5y0 (w/v) A:gDDC-pyri- Figwe 2. Typical stantlard cnrve for arsenic determination as the dine solution. The generation mixture consisted of the arsine-AgDDC complex at 540 mp Although the line passes digested sample in 2 ml of H,SO, diluted to 40 ml with through the origin, the most rcprotlucil~le readings of optical distilled water, 2 ml of 15% potassium iodide, and 12 density arc obtained between 0.200 and 0.300 optical density drops of 40% stannous chloride in 12.5-N HCl. Fifteen units (4). minutes were allowed for reduction of pentavalent to trivalent arsenic. Then 10 ml of concentrated HCl and greatest reproducibility, i.e. the lowest variation of the approximately 4 g of granular zinc (20 mesh) were added mean, was obtained at absorbance values of 0.200 to to form nascent hydrogen, which reduced trivalent ar- 0.300, which was equivalent 1.0 4 to 6 ltg arsenic (4). senic to arsine, a hrghly volatile compound that is Whenever a new AgDDC-pyridine solution was made, trapped by the AgDDC-pyridine solution. a new standard curve was derived; this precaution elimi- The “scrubber”, which is a glass wool plug saturated nated errors due to preparation and variations in dif- with 10% lead acetate, is used as a precautionary step ferent batches of AgDDC. to trap hydrogen sulfide that may be produced by reduc- Recovery of arsenic from generation flasks was close tion of sulfuric acid in the generator. Hydrogen sulfide to 100°~O when the samples contained 20 ltg of arsenic will react with AgDDC and Interfere with the detection or less, but recovery decreased rapidly. as the arsenic of arsenic. However, reduction of sulfuric acid can be content increased (Figure 3). The declme in recovery largely avoided by making certain that its concentration may have been due to very rapid arsine evolution beyond is 5% or Iess before the reductants are added (6). the complexing capacity of the AgDDC-pyridine solution When arsenic determinations were made on undigested or to incomplete release of arstne from the generation Volume 19, Issue 4 (July), 1971 413 WEED SCIENCE
We found that, depending upon the amount of chloride present, as much as SOY0 of the arsenic present was lost if sulfuric-acid-induced charring occurred before sufficient time was permitted for nitric acid digestion. However, it was not necessary to continue adding nitric acid until a clear solution was obtained, as suggested by Milton and Waters (6). Usually 80 to 100 ml of nitric acid were suificient to remove all chloride normally present in 1 g of dried plant tissues. When this volume was reduced to a few milliliters, the flask was allowed to cool and 3 ml of sulfuric acid were added. Heating was continued until copious fumes were produced for 5 min; this was the second critical stage in the digestion procedure, part-icularly for MSMA and cacodylic acid recovery. Apparently the carbon-arsenic bonds in these compounds were not ruptured in hot nitric acid (120 C), but only in fuming sulfuric acid (170 to 190 C). No more than 5 min in fuming sulfuric acid was required, but it was a critical requirement. Several tests were run with cacodylic acid in which the fuming stage was terminated in something less than a Fipre 3. Percent recovery of arsenic as a function of the amount minute and recovery was reduced to 1 to 3Ocj,. The of arsenic added to the arsinc generator. duration of the fumiglg sulfuric stage was less critical for MSMA, but quantitatively reproducible results were flask. A recovery correction factor derived from Figure obtained only when fuming was continued for 5 min. 3 was applied when the arsenic content was in excess Extrnctio7s of filant tissues for chumafogl-afilzic nnalyses. of 20 l&g, or a por&ion of the sample coctaining less than Bean plants weighing approximately 15 g wze trea;ecl 20 kg of arsenic was used for analysis. with :50 pg of arsenic as cacodylic acid and 27 l&g of Organic arsenicals, such as cacodylic acid and MSMA, arsenic as sodium arsenate. The arsenicals were added form volatile arsine derivatives that react with AgDDC clropwise to the leaves, the solutions were allowed to to form red to brown complexes with absorption spectra dry, and the tissues then were extracted fresh or dried different from arsine-AgDDC. Chromatographic analysis in an oven at 70 C for 24 hr. Fresh plants were grouncl of these complexes on thin layer sheets of silica gel, with in a blender with distilled water to make a puree; the a pyridine-methanol (50:50) solvent system, revealed at puree was heated to boiling for 5 min and then vacuum- least two compounds different from arsine-AgDDC. Ob- filterecl through Whatman #l paper. The filtrate was servations sug,g;est that much smaller amounts of these evaporated to dryness under reduced pressure. Dried arsine derivatives are released from cacoclylic acid and plants were ground to a powder in a Wiley mill through MSMA than from inorganic arsenic, or the extinction a 20-mesh screen. Thirty milliliters of distilled water were coefficients for the AgDDC complexes are much lower added per gram of tissue. The mixture was stirred on than that for arsine-AgDDC. Digestion of the organic a hot plate, boiled for 5 min, and then vacuum-filtered arsenicals, which ruptures the carbon-arsenic bonds be- through Whatman #l paper; the filtrate was treated in fore reduction to arsine, increases the optical density at the same manner as the fresh tissues. In two experiments, 540 rnp more than eightfold for cacodylic acid and two- using three plants per arsenical treatment, the procedures fold for MSMA. described were adequate to give 70 to 99% recovery of Digestion. Plant tissues, extracts, and chromatographic applied cacodylic acid and sodium arsenate (Table 1). paper were digested by a nitric acid-sulfuric oxidation procedure to convert all arsenicals to the inorganic penta- Table 1. Recovery of cacodylic acid and inorganic at-senate from valent form. In subsequent experiments large numbers fresh dried bean seedlings. of samples had to be digested; hence, the procedures Cacodylic acid Inorganic arsenate described by Powers et al. (10) were modified to reduce Experiment ---- - the time required for each digestion. Since the digestion Fresh Dried Fresh Dried cg, (2) (%I) procedure proved to be a very critical phase of the I...... 108 l%’ investigation where substantial losses of arsenicals could 2...... 64 98 occur, it will be described in detail. Average. 74 ii: E 100 The first critical step was the addition of relatively large quantities of nitric acid and heating before sulfuric Losses occurred during transfer of the extracts and in acid was added. Milton and Waters (6) emphasized that the filtration process. nitric acid alone should be used initially to avoid reduc- Two tests with l*C-MSMA on fresh tissues yielded a ing conditions that may prevail locally, and, in the 56oj, average recovery based on 1% applied. Since ne:y- presence of chloride, cause the formatlon of volatile ligible 14C0, was trapped, substantial losses occurred m arsenic-chloride compounds. If sulfuric acid is added pieparation of the extract. The causes of sucll losses are initially, charring usually occurs as the nitric acid dis- unknown, but ion exchange of arsenicals on glassware appears; at this time the volatile chlorides may form. and paper would be expected. 414 Volume 19, Issue 4 (July), 1971 SACHS ET AL. : ARSENICAL RESIDUES IN PLANT TISSUES
The data in Table 2 show that, although slightly more Table 4. RI values for arscnicais developed on 3hIhZ paper. cacodylic acid and sodium arsenate were bound to the Cacodylic Sol~un;;g4tem Piant Arsenate Arsenite MSh4A acid Table 2. Arsenic recovered in the extract and plant residue. a lxtract Rt Rt Rt Rf - I...... 0.0-0.1 0.2-0.3 0.0 -0.0 0.4 -0.5 Fresh tissue Dried tissue I...... 7 0.0-0.1 0.0-0.1 0.0 -0.1 0.1 -0.3 Fraction --- - II...... 0.3-0.4 0.5-0.6 0.6 -0.7 0.4 -0.5 Cncodylic Cacodylic II...... 7 0.27 acid Arsenntc acid Arsenate III...... 0.2-0.24 0.6-0.7 0.6 -0.7 0.6 -0.7 III...... 7 0.25-0.35 0.25-0.35 I:Xtr*?Ct.. . . . 996 99 97 78.5 IV ...... - 0.5-0.7 0.6-0.8 0.6 -0.7 0.8 -0.9 Residue. . . . 1 1 3 1.5 Golvent systems: aData dcrivcd irom capcrilnents rcportcd in Table 1. Arcrn~cs OC both cx- 1-propanol:Nl~,OlI (7:3 v/v) periments are given. The data are the percent of total arsenic recovered; e.g. :I 2-prap~nol:lI~O-acetic a&d (10:3:0.1, V/V) III 2-propanol-llr0 (7:3, V/“) *:s-“o:i,“l ;r,,,,t x * 00 = y. arsenic in extract. IV methanol:lO-‘M NIltOIl (89, V/V)
hot water insoluble fraction in dried tissues, the amount first to separate cacodylic acid from arsenate, arsenite, bound was never more than 3oj, of the total recovered. and MSMA, which are considered as potential metab- Thus, the drying process did not fix large amounts of olites of cacodylic acid (11). The cacodylic acid fraction arsenicals to proteins or other hot water insoluble tissue was eluted with 50% methanol and rechromatographed fractions. For experiments in which analyses were made by solvent system III or IV. If residual arsenic at Ri 7 days after treatment with arsenicals, ~111 to 25% of the 0 to 0.1 was of sufficient magnitude to warrant further arsenic from sodium arsenite and arsenate was bound analyses, the paper at these regions was eluted and to the hot water insoluble fraction (11). rechromatographed in solvent II, 111, or IV. The final step in preparation for chromatographic Extracts from MSMA-treated plants were chromato- analysis was addition of 50% methanol to the vacuum- graphed first in solvent systems II, III, or IV and, follow- dried extract. This procedure eliminated large quantities ing elution of the major arsenical peak, then in solvent of water-soluble compounds that retarded the movement system I. The reason for this procedure was to insure of t!le arsenicals on paper and at times interfered with initial separation from arsenate and then arsenite. Note solvent flow across the origin. Usually, three l-ml washes that arsenite runs ahead of MSMA in solvent I (Table of 50% methanol were sufficient to suspend the residue 4). In one experiment with 14C-MSMA, the broad MSMA from 15 g of plant tissue. This suspension was centrifuged peak from solvent II was eluted and rerun in the same at 10,000 X g for 30 min; the supernatant was decanted solvent (11). Two peaks of l*C were clearly resolved by and streaked on chromatographic paper. Losses of sodium this procedure; this result suggests that both MSMA and arsenite, sodium arsenate, cacodylic acid, and MSMA the unknown compounds, presumably a histidyl-MSMA clue to methanolic fractionation were negligible (Table complex (12), were sufficiently retarded by materials at the origin to impede resolution of the two peaks. The TabZe 3. Recovery of arsenicals in 50% methanol from plant MSMA peak was retarded from R, 0.67 to 0.27 in system extractsa II. Extracts from plants treated with arsenite or arsenate Cacodylic Fraction acid X4SMA Arsenate Arsenite were chromatographed in solvent I initially to check for the presence of arsenite or organic arsenicals. The (5) (56) (%) Pg) Supernatant . . . . . 98.5 98.5 arsenical at the origin was eluted, the eluate applied to Precipitate.. .7 1.5 1.5 1 paper, and the paper developed in system III or IV. In aEleven pg of cacodyiic acid and 154 rg of the other arsenicals were added to some cases arsenite did not move from the origin in filtered, heated lant extracts; absolute methanol was added to bring the concen- tration to 50%. 5 he solutions were centrifuged and both the pellet and supernatant system I; and, hence, other solvents were required to were analyzed ior arsenic. separate the two inorganic forms.
3) and most probably due entirely to occlusion in the D ISCUSSION pellet, which was not washed. Several methods for the quantitative analysis of arsenic Analysis by @aper chronzatogmfihy. The extract from have been devised (1, 2, 6, 10, 13), but in recent years 1 g of dried plant tissue, containing as much as 160 pg silver diethyldithiocarbamate (AgDDC) calorimetric de- of arsenic, could be applied to a 15-cm-wide sheet. Con- tection has been widely recommended (1, 12) where sistent Rf values were obtained only in controlled tem- relatively expensive atomic absorption or neutron acti- perature conditions; the values quoted in Table 4 were vation equipment is not available. Powers et al. (10) were obtained at 23 to 26 C. Depending upon the solvent able to detect as little as 0.1 ppb of arsenic in organic system, solvent fronts moved 25 to 30 cm beyond the compounds by combining chromatographic adsorption origin in 4 to 8 hr. of arsines to silica gel with the AgDDC method. How- A stepwise procedure of chromatographic analysis of ever, this method required special glassware and was plant extracts was developed for each arsenical (Table too time-consuming for the large number of analyses 4). Solvent systems, henceforward, will be referred to by required in our investigations (11); hence, we simplified Roman numerals: I = 1-propanol:NH,OH (7:3, v/v); the apparatus and eliminated the silica gel adsorption II = Z-propanol:H,O:acetic acid (lO:S:O.l, v/v); III = step. We were able to detect as little as 0.6 11s of arsenic 2-propanol:H,O (7:3, v/v) (9); and IV = methanol: per 10-g sample, or 0.06 ppm. 10-s MNH,OH (8:2, v/v) (5). Solvent system I was used Calorimetric procedures proved to be less troublesome Volume 19, Issue 4 (*July), 1971 415 WEED SCIENCE than the nitric-sulfuric acid digestion step. Consider- compounds. Thus, once the elution problem is solved, able difficulties were encountered initially in obtain- anion exchange resins should be the most useful, and ing quantitative recoveries of arsenic from arsenical by far the fastest, means for separating parent arsenical standards because of inadequate or variable digestion herbicides and their metabolites from interfering ma- in nitric acid and avoidance of sulfuric-acid-induced terials in plant extracts. charring. Up to 50% of the arsenicals added were lost during digestion as volatile arsine chlorides if the chlo- ACKNOWLEDGJYIENTS ride was not first removed as nitrosyl chloride. Variable or inadequate time in fuming sulfuric acid Dr. Jerry Martin assisted in experiments testing re- was another source of error, particularly in quantitative covery of 14C-MSMA. The Ansul Chemical Company determination of cacodylic acid and MSMA. In both supplied cacodylic acid, MSMA, and 14C-MSMA. The cases the carbon-arsenic bonds were not ruptured in hot Vineland Chemical Company also supplied samples of cacodylic acid. nitric acid; in fact they were not severed until the fuming sulfuric acid stage was maintained for 5 min. If the LITERATURE CITED carbon-arsenic bonds remained intact, then arsines were 1. AMERICAN CONFERENCE OF GOVERN~~ENT INnuslaxhL HYGIENISTS. generated that formed complexes with AgDDC of dif- 1958. Manual of analytical methods-Determination of ferent absorption maxima and extinction coefficients; arsenic in air and biological materials. 1014 Broadway, thus, inadequate digestion may cause an apparent loss Cincinnati, Ohio. 20 13. 2. BABKO, A. K., 2. I. CN~LAYA, and V. F. MIRITCHENKO. 19GG. of 50 to 90% of the arsenic added as MSMA or cacodylic The determination of microquantities of arsenic with butyl- acid. rhodamine. (Transl. from Russian) Zavo. Lab. 32:270-273. Paper chromatographic procedures appeared to be 3. BRUNO, M. and V. DELLUCO. 1956. Separazione e dossaggio well adapted for fractionating cacodylic acid, MSMA, cli microquantita di arsenic0 e di fosforo. Ric. Sci. 26:3337- arsenate, and arsenite in plant extracts. Four solvent 3341. 4. FISCHER, R. B. and D. G. PETERS. 1968. Quantitative Chemical systems, I-propanol:NH,OH (7:3, v/v), 2-propanol:H,O: Analvsis. 3d Edition. Saunders Co., Philadelphia. 883 p. acetic acid (10:3:0.1, v/v), 2-propanol:H,O (7:3, v/v) and 5. MII&KOVA, V.. J. KOI~LICEK, and K. KACL. 19G8. Separation methanol:lO-“M NH,OH (8:2, v/v), permitted complete of arsenite and arsenate ions by paper chromatography. Study separatioil of t!le arsenicals from one anot!ler. Oguma of a melhanol-ammonia-water solvent system. J. Chromatogr. (8) used acidic acetone, butanol, and ethanol solvent 34284-238. 6. MILTON, R. 1;. and W. A. WATERS. 1955. Methods of quanti- systems in separating arsenate from arsenite on silica tative micro-analysis. 2d Edition. Edward Arnold, Ltd., gel thin layers. These systems have not been tested on London. 742 P . paper, although the relative movement of arsenicals ap- 7. NELSON, F. and -K. A. KRAUS. 1955. Anion-exchange studies. pears essentially the same on silica gel, cellulose layers, XVIII. Germanium and arsenic in WC1 solutions. J. Amer. and paper. The chief virtue in Oguma’s solvent systems Chem. Sot. 77:4508-4509. 8. OCUMA, K. 1967. The separation of arsenic (V) and (III) by is that arsenate moves considerably in advance of arsenite thin-layer chromatography. Talanta 14:685687. and, hence, in combination with any of our four systems, 9. OVERBY, L. R., S. F. BOCCHI~RI, and R. L. FREDERICKSON. 1965. would, provide optimal separation of the two. Chromatographic, electropboretic and ion exchange identi- Anion and cation exchange resins have been used for fication of radioactive organic and inorganic arsenicals. J. fractionation of arsenicals (3, 7, 9). Our studies with one Assoc. Of%. Agr. Chem. 48:17-22. 10. POWERS, G. W., JR., R. L. MARTIN, 1;. J. PIEHL, and J. M. anion exchange resin, Rexyn 201 (OH), indicated that all GRIFFIN. 1959. Arsenic in naphthas. Anal. Chem. 31:1589- four arsenicals could be removed almost completely from 1593. plant extracts adjusted to pH 11; however, elution with 11. SACAS, R. M. and J. L. MICHAEL. 1971. Comparative phyto- weak or strong hydrochloric acid or several other solu- toxicity among four arsenical herbicides. Weed Sci. 19: tions failed to remove more than 20 to 50% of the (In Press). 12. SCKERL, M. M. and R. E. FRANS. 1969. Translocation and MSMA and cacodylic acid and did not elute completely metabolism of MAA-*% in Johnsongrass and cotton. Weed arsenate and arsenite. The arsenic-free effluent that Sci. 17:421-427. passed through the column appeared to contain a large 13. WILKINSON, R. E. and W. S. HARDCASTLE. 1969. Plant and portion of the methanol-precipitable and pigmented soil arsenic analyses. Weed Sci. 17:536-537.
Volume 19, Issue 4 (July), 1971