UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. I, No. 12, pp. 395-494, plates 6-9 April 7, 1917 CERTAIN EFFECTS UNDER IRRIGATION OF COPPER COMPOUNDS UPON CROPS* l'.v H, 11. FORBES CONTENTS page Part I. — Experimental Work 396 Introduction 396 Solid wastes 397 Soluble copper compounds 399 Distribution of copper compounds throughout the Clifton-Morenci mining and Gila River irrigated district 4iM Sources of copper 401 Processes by which copper is added to the water supply 402 Table of solubilities of ''upper compounds 403 Copper in ores and tailings from Clifton-Morenci district 405 Dissolved copper in river, irrigating and ground waters below the Clifton-Morenci district 407 Copper in soils irrigated with tailings waters 408 Miscellaneous soils unaffected by mining detritus 410 Copper in vegetation from upper Gila farms 410 Copper in vegetation from other localities Ill Copper in flesh and bones of a pig 4 1 2 Distribution of copper in plants with root systems exposed to cop- per compounds 4 13 Corn plants grown in soils containing eopper 413 Water cultures 417 Toxicity of copper solutions to plant roots in water culture 419 Stimulation effects in water cultures 422 Effects of soil upon toxicity of copper solutions 426 Irrigation experiments 428 Cultural experiments 432 Pot cultures with treated soils 432 Pot cultures with field soils 437 Pot and plot cultures 439 Field samples of soils and vegetation 440 Use of copper sulphate to kill moss in irrigating ditches II.'. Physiological observations on toxic effects of copper salts 1 I I Quantitative work I I I • Paper No. 31, Citrus Experiment Station. College of Agriculture. Uni- versity of California. Riverside, California. 396 University of California Publications in Agricultural Sciences [Vol. 1 PAGE Reactions of copper with growing points 450 Varying resistance of individual cells to copper 454 Diagnosis of copper injury 454 Part II. — General Discussion 458 Preliminary statement $ 458 Accumulations of copper 458 Possible effects upon health 460 Amounts and significance of copper in aerial vegetation 461 Amounts and significance of copper in root systems 463 Relations between amounts of copper in root systems and in- jury to plants 466 Pathological effects 467 Soil conditions relating to toxic effects of copper upon plants .... 468 Stimulation 470 Field observations .: 472 Effects of river sediments 473 Effect of cultivation upon alfalfa 474 Summary 478 Part III. — Appendix 480 Methods of analysis 480 Reagents and apparatus 480 Manipulation 480 The determination of copper in small amounts of plant ashes 483 Bibliography 487 Part I.- EXPERIMENTAL WORK INTRODUCTION The region to which the studies described in this publication more particularly relate lies in southeastern Arizona in Greenlee and Graham counties and consists, first, of the Clifton-Morenci mining district and second, of the irrigated lands along the Gila River from twenty-five to sixty miles below. The Clifton-Morenci mining district is drained by Chase Creek into the San Francisco River, which in turn empties into the Gila. From the Gila, be- ginning at a point about twenty-five miles by channel below Clifton, irrigating waters are withdrawn for the use of the rich lands extending somewhat discontinuously from above San Jose to Fort Thomas, a distance of thirty miles. For about forty years, this up-stream mining district and the irrigated lands below have developed together from small beginnings into large industries. Beginning with the initiation of smelting operations on the L917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 397 San Francisco River in 1882, comparatively small amounts of mining detritus musl have found their way into the irrigating water-supply. Following the discovery, in is!):}, of immense deposits of Low-grade sulphide ores in the district and the erec t i'ui of concentrating plants to handle them, rapidly increasing quantities of fine slimes were discharged into the stream How. becoming noticeable in the irrigating waters of Graham County about the year 1000. Following the observation of their pres- ence, various crop failures were attributed from time to time to the tailings, resulting finally in a request by the farmers of the district to the writer, for an examination of the facts relating to damage done by mining detritus to their irrigated crops. Solid W \stes Following this request, the writer began a study of the prob- lem in .May, 1904, which resulted in the publication of Bulletin 53 of the Arizona Agricultural Experiment Station. September 20, 1906. This publication established the fact that irrigating sediments, in general, may be beneficial or harmful according to their composition and physical character and to the manner of their disposition in or upon the soil. If allowed to accumulate upon the surface of the soil in the form of more or less im- pervious silt-blankets, their influence, by limiting the supply of water and air to the soil, is notably harmful. Tn the ease of the mining wastes from the Clifton-Morenci district, which are particularly plastic and "tight" in character, the damage done was found to be greater than that resulting from sediments aris ing from ordinary erosion. It was determined that the damage from these wastes, particularly to alfalfa and other crops which cannot receive constant and thorough cultivation, was of an in- creasingly serious character. The farmers of Graham County, represented by one of their number, finally brought suit against the Arizona Copper Com- pany. Limited, for discharging tailings into their irrigating water-supply. The case was decided in the District Court of Graham County in favor of the farmers, and an order was issued in November, 1907, effective May 1, 190S. restraining the mining companies from discharging "slimes, slickens or tailings" into 398 University of California Publications in Agricultural Sciences [Vol. 1 Chase Creek, the San Francisco River, or the Gila River. The case was appealed to the territorial Supreme Court where, how- ever, the decision was confirmed in March, 1909. The case was again appealed by the Arizona Copper Company to the Supreme i - v .. "y7~, Fig. 1. — General map of the Clifton-Morenci and Gila River mining and irrigation district, Arizona Court of the United States, where it was again and finally de- cided in favor of the farmers on June 16, 1913. During and since the occurrences above mentioned, large quantities of solid wastes have been impounded by the copper companies in settling basins constructed for their storage in the 1!H7| Forbes: Irrigation Effects of Copper Compounds Upon Crops 399 district. Recent investigations by the companies indicate a pos- sibility that with copper at 15 '-nits a pound these stored tailings, which average about 0.85 per cent copper, may be profitably reworked. Tn the long run. therefore, it may be found that an adjust- ment based upon a complete and impartial statement of facts relating to the tailings situation is beneficial both to the agri- cultural and to the mining interests concerned. Soluble Copper Compoi nds Following the disposition of mining detritus, there remained the problem of soluble copper compounds which, in small but continuously appreciable quantities, find their way with waste waters into the si ream-How of the region. These compounds originate in the ores of the district and are, as in the case of the carbonates, directly soluble to a slight extent in drainage waters, especially in the presence of carbon dioxide. Tn other cases, the original ores are changed through the action of air into soluble substances which then escape downstream. Sulphide ores are thus oxidized in the presence of air into soluble copper sulphate. Inasmuch as il is well known thai minute amounts of copper in solution are extremely toxic to plant roots directly exposed to them, some apprehension naturally existed as to the effects of these small amounts of copper salts escaping into 1 he water- supply of an irrigated district. Tn some respects, conditions were especially favorable here to the successful prosecution of a stud} of the foregoing ques tion. The irrigated lands are at a distance of twenty miles or more from the smelters, so thai injurious eases could not cum plicate effects upon irrigated crops, There are. also, only traces of other toxic metals to be found within the district. — more par- ticularly, arsenic, antimony, and zinc. Injurious effects due to the possible toxic action of compounds originating in the mines are therefore limited to copper. Scientific study relating to toxic effects of copper upon plaids under varying conditions has thoroughly established not only the fact that copper compounds are extremely toxic to plants when they obtain entry to their tissues, but also that various 400 University of California Publications in Agricultural Sciences [Vol. 1 agencies standing between these poisonous salts and the living plant tend to prevent injury.1 Soluble copper compounds, for instance, react with carbonate of lime, commonly abundant in soils of the arid region, to form the solid carbonates of copper. The partly decomposed silicates of these soils also precipitate soluble compounds of copper and mask their toxic character. Organic matter in the soil likewise holds large quantities of copper in comparatively harmless combinations. Through physi- cal attraction or adsorption, soluble copper compounds enter into weak combination with fine soil particles and toxic effects are thereby greatly lessened. In the presence, also, of other soluble salts, such as the various forms of "alkali" commonly found in the soils of the region, the toxicity of copper compounds is enor- mously lessened. The investigations recorded in this publication include: (1) Observations upon the distribution of copper in mining wastes, in irrigating waters, in soils and soil waters, in the plants, and in the animal life of the region. (2) The development of accu- rate methods for the determination of minute amounts of copper in all situations where they may occur. (3) Plant cultural work with waters and in soils in the presence of varying propor- tions of copper and under varying conditions. (4) A careful analytical study of the results of such cultures in order to deter- mine the symptoms of poisoning and the distribution of copper throughout poisoned plants ; and to identify, if possible, the particular parts of plants and tissues injured by copper. (5) A physiological study of plant reactions with copper. (6) Field studies for the purpose of relating the results of laboratory inves- tigations to the question of economic injury done by copper salts to irrigated crops. By reason of interruptions due to other duties, it has required a long time to mature this investigation to the point where it seems sufficiently complete for publication. This delay, however, has given perspective to the work and, especially, opportunity to verify earlier conclusions as applied to field conditions. The writer is indebted for painstaking analytical work to Messrs. R. G. Mead, Edward E. Free, Dr. W. H. Ross and i See Bibliography, pp. 487-488, references 1, 8, 14, 15, 16, 19, 34, 51. J917] Forbes: Irrigation Effects of Copper Compounds Upon Crops mi C. X. Catlin, associated with the Arizona Agricultural Experi Minit Station from time to time; and to the helpful advice of Dr. Howard S. Reed, of the Universitj of California Graduate Scl 1 of Tropica] Agriculture, in connection with the physio- logical pari of the work herein described. The publication, also, lias been criticized to its advantage by Dr. ('. B. Lipman of the I fniversil y of ( Jalifornia. DISTRIBUTION OF COPPEB COMPOUNDS THROUGH- OUT THE CLIFTON-MORENCl AND GILA RIVKI; MINING AND IRRIGATION DISTRICTS Sources of Copper The original smiivc of flic copper found in this district, according to Lindgren,2 is a Cretaceous or early Tertiary in- trusion of acidic porphyries to which, in the Clifton-Morenci district, all ore deposits may be finally referred. The original porphyries contain as little as 0.02 per cent of copper ore m the form of chalcopyrite. Under the influence of superheated waters emanating from the porphyry, this chalcopyrite, together with other metallic compounds, was carried out from the molten intrusive mass into adjoining strata and there deposited, espe cially along fissures, in the form of concentrated masses or veins of chalcopyrite and other minerals. Through erosion these de posils were afterward subjected to atmospheric oxidation, fol- lowed by downward percolation and a period of secondary enrich- ment due to numerous reactions mainly between the oxidized compounds of copper and other minerals present. In limestones and shales, these processes resulted in the formation of oxidized ores containing azurite, malachite, chryso colla, and cuprite. In porphyry, the main final result was chalcocite or copperglance, the principal constituent of the sul- phide ores of the Clifton-Morenci district. In general, therefore, the metasomatic changes associated, first, with superheated waters arising from the original intrusion of molten porphyry and. second, with meteoric waters percolating '-' V. S. Geological Survey, Professional Paper No. 43, 1005. 402 University of California Publications in Agricultural Sciences [Vol. 1 downward with oxidiziug effects through copper-bearing rocks, have brought copper from a concentration of possibly less than 0.02 per cent in the original porphyry through every degree of richness to the condition in some cases of pure copper. Processes by which Copper is Added to the Water-Supply To a slight extent, drainage waters from the ore deposits and from the mines, containing considerable amounts of copper in solution, find their way downstream. But by far the larger part of the copper which gets into the irrigating supply is derived from the ores and tailings which, in the concentrators, on the dumps, and finally in the river itself, are subjected to the action of atmospheric oxygen, and water containing carbon dioxide and various salts in solution. The residual chalcocite in tailings from sulphide ores thus reacts with oxygen from the air and yields copper sulphate in solution. This, in turn, reacts with the excess of bicarbonate of lime ordinarily contained in the waters of the San Francisco and Gila rivers. The resulting basic carbonate of copper is notably soluble in water containing carbon dioxide and certain of the various salts commonly found in river waters. The residues of carbonates of copper in oxidized ores are directly dissolved in waters containing carbon dioxide and certain soluble salts. Along with minute quantities of copper thus dissolved and carried forward, pass the solid residues discharged from the concentrators — solid wastes which find their way. unchanged, downstream and finally upon the soils of irrigated fields. At this point begins another and very important series of reactions be- tween dissolved copper compounds and the soil, tending in general to withdraw copper from its solutions and precipitate it in the form of less harmful solid compounds. These are briefly referred to above and will be discussed more in detail further on in this paper. Opposing these precipitations of copper are those solvents which tend to maintain this metal in soluble form in small quantities in the soil. Chief of these is carbon dioxide, which is always present in agricultural soils in significant quantities. Of interest in this connection is the fol- r.M, Forbes: Irrigation Effects of Copper Compounds Upon Crops 403 TABLE r Solubilities of Copper < ompounds Compound Malachite CuCO,.Cu(OH)3 Precipitated basic copper carbonate Precipitated basic copper carbonal e Precipitated basic copper carbonate Precipitated basic copper carbonate Precipitated basic copper carbonate Precipitated basic copper carbonate Precipitated basic copper carbonate Precipitated basic copper carbonate Precipitated basic copper carbonate Precipitated ba ic copper carbonate < 'opper sulphide; CuS ( !halcopyrite CuFeS3 Chalcopyrite CuFeS Malachite Chrysocolla di SiO,.n H\0 Cupric sulphide CuS < uprite Cu,0 ( lupric oxide CuO Solvent Water containing 0.12* < carbon dioxide I'u re water Water containing ti.li"; carbon dioxide Water containing 0.13% c a r b o n dioxide and 0.0195 sodium chloride Water containing 0.13% c a r b o n dioxide and 1.0% sodium chloride \\ ater i ontaining o.l i" ", c a r h O ii dioxide a nd 0.01% sodium sulphate Water containing 0.12% c a r Ii o ii dioxide and I "' , sodium sulphate w ater containing 0.12% carbon dioxide and 0.01% sod. carbonate Water containing 0.12% c a r li ii n dioxide and l.ie ; sod. carbonate Water containing 0.129$ C a r Ii ii a dioxide and I calcium sulphate Water containing 0.129? C a r b (i ii dioxide and 0.11% cale. carbonate Oxygen free water Cu dissolved parts pel million Reference E. E. Free, Journ. 29.0 31.0 Am. Chem. Soc . XXX, 9, p. 1367 1.5 E. E. Free, Journ. Am. Chem. Sue.. XXX. 9, p. 1370 E. E. Free, Journ. 34.8 Am. Chem. Soc, XXX, 9, p. L370 E. E. Free, Journ. Am. Chem. Soc, 36.0 XXX, 9, p. 1371 B. E. Free, Journ. Am. Chem. Soc, 58.0 XXX. 9, p. 1371 E. E. Free, Journ. Am. Chem. Sue.. 37.il XXX, 9, p. 1372 E. E. Free, Journ. Am. Chem. Soc. .18.0 XXX, 9, p. 1372 E. E. Free, Journ. Am. Chem. Soc, 10.0 XXX. 9, p. 1372 E. E. Free, Journ. Am. Chem. Sue.. 0.7 XXX, 9, p. L372 E. E. Free, Journ, Am. Chem. Soc, 36.0 XXX. 9, p. L372 E. E. Free, Journ. Am. ('hem. Soc, It XXX. 9, p. 1372 0.09 W. H. Ross, MSS I'u re water SodiC sulphide measurable amounts Amt. not stated ' ' insoluble in water, slightly soluble in water charged with carbon dioxide.'' "Somewhat soluble in water with carbon dioxide u ater 1 to 950,000 "Insoluble in water" " [nsoluble in water F. S. Geol. Survey Monograph XLVII, p. 1107 r. S. • leol. Survey Monograph \ LVII, p. 1106 Moissan 5, p. 1 1>7 Lindgren, U. S. u S. Prof, paper 13, p. 188 Comey, 1 >ict. Solu bilities, p. 139 Comey, Diet. Solu- bilities, p. 137 Comey, Diet. Solu- bilities, p, l.",7 406 University of California Publications in Agricultural Sciences [Vol. 1 s 3 O '_ C3 ■- c > 3 — a to 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 407 in low-grade sulphide ores (No. 3491, No. 3303), and to nearly one-half in the richer oxidized ores (No. 3492, No. 3304, No. 3439). By far the larger portion of tailings produced, however, are from low-grade sulphide ores, the wastes from which there- fore predominate, formerly imparting to river waters the whitish TABLE III Dissolved Copper in River, Irrigating, and Ground Waters below the Sample No. and date 3438 June 28, '05 3439 June 28, '05 3309 May 26, '04 3486 June 11, '05 3622 June 25, '06 3737 Feb. 22, '07 Tailings 4011 Jan. 3, '09 4029 Apr. 12 6342 Mar. 4, 3986 Jan. 2, '09 Records of Cananea C. C. Co. Jan. 4, '14 3504 Aug. 19, '05 4012 Jan. 3, '09 3526 09 16 Clifton-Morenci District Description of sample Water mixed with sulphide tailings from A. C. Co. 's mill, Clifton Water mixed with oxidized tailings from A. C. Co. 's mill, Clifton Montezuma Canal water at Solo monville; slight rise in river Montezuma Canal at Solomonville small flood Montezuma Canal at Solomonville head waters clear Montezuma Canal at Solomonville, medium flood shut out of water supply May 1, 1908 Water from Montezuma Canal at Solomonville Montezuma Canal at Solomonville Montezuma Canal at Solomonville high water Water from C. & A. Ditch, Bisbee mine waters Water from creek below concen- trator Water from Geo. Olney's well, 30 ft. deep, east of Safford, under Montezuma Canal 7000 Water from Wilson 's well, one-half mile west of Solomonville under San Jose Canal 3500 Water from University well, Tuc- son, 95 ft. deep, tapping Rillito underflow 7000 Condition and amount taken in cc. Cu found, grains Cu p.p.m 500 .0009 1.80 500 .0018 3.60 2000 .0016 .80 6000 .0015 .25 9000 .00095 .11 14000 1908. .0403 2.88 4000 .00031 .08 ! 3700 .0003 .08 1000 .00003 .03 3500 .00039 .11 .0037 less than .00001 none 2.1 .53 less than .00?. none appearance characteristic of this material. It is of interest to note in this connection that in one instance observed the tailings almost completely maintained their richness in copper between 408 University of California Publications in Agricultural Sciences [Vol. 1 Clifton and Solomonville. At Clifton, May 23, 1904, the prin- cipal discharge of sulphide tailings (3303) was observed carrying 0.93 per cent of copper. At Solomonville, three days later, the Montezuma ditch-water sediments (No. 3309), mostly of this same material, carried 0.86 per cent of copper, indicating the persistence with which the copper accompanies the wastes, with which it is associated, downstream and upon underlying irrigated lands. TABLE IV Copper in Soils Irrigated with Tailings Waters Sample No. and date 3435 May 25, '04 3434 June 10, '05 350] Aug. 19, '05 3436 June 25, '05 3437 June 25, '05 3502 Aug. 19, '05 2381 June 5, '00 3522 Oct. 25, '05 3521 Oct. 25/05 2763 Nov. 11, '01 1.S9U Apr. 20, '01 2830 Jan. 19, '00 Description of sample Top 5 in. sedimentary soil (Fred Thorstison), upper end alfalfa field west of Safford, under Montezuma Canal Top 5 in. sedimentary soil (Geo. Olney), upper end alfalfa field east of Safford, under Monte- zuma Canal Soil in place at 4 ft. depth beneath No. 3434 Top sedimentary soil (Wm. Gilles- pie), upper end of test alfalfa field west of Solomonville, under Montezuma Canal Soil in place, no sediments at sur- face of lower end of field near No. 3436 Soil in place at 4 ft. depth beneath No. 3437 Condition and weight taken, grams 12 in. from garden near Ariz., beyond tailings de- Surface Pima, posits Top 4 in. sedimentary soil upper end of alfalfa field, Station farm near Phoenix, under Grand and Maricopa canals Deep soil, no sediments, Station farm near No. 3522 Surface 12 in. from orange orchard north of Phoenix, under Ari- zona Canal Surface 15 in. from cultivated field west of Tempe, under Tempe Canal Surface 12 in. from orange orchard northeast of Phoenix, under Ari- zona Canal Cu found, Cu grams p. p.m. 96.7 water-free 96.s water-free 96.2 water-free 96.7 water-free 94 water-free 96.1 water-free 100 air-dry 95 water-free 95 water-free 100 air-dry 100 air-dry 100 air-dry .020 .0199 .0021 .0192 .0028 .001 faint trace 207 205 22 199 30 10 faint trace trace .0003 3 .0003 3 faint trace trace trace 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 409 Table III is of interest because it reveals quantities of dissolved copper in irrigating and in ground waters sufficient, under proper conditions, in water cultures, to produce toxic effects upon plants.4 It is noteworthy, however, that, following the order of the court, effective May 1, 1908, prohibiting the in- troduction of tailings into the water-supply, the amount of dis- solved copper in Montezuma canal waters greatly decreased, due to the decrease in quantity of sulphides whose oxidation affords the supply of dissolved copper. Other water-supplies also are found to contain similar amounts of copper, as the Calumet and Arizona mine waters, used for irrigation below Bisbee. As stated above, however, in the soil itself the toxic action of such copper solutions is enormously decreased. Naturally, the question arises as to the possibility of toxic effects in using such waters upon cultivated soils. This is discussed on subsequent pages. The proportions of copper (0.003 to 0.53 parts in 1,000,000 of water) found in the drainage beneath this irrigated district indicate that not all of the copper applied in irrigation remains in the soil. University well water at Tucson was observed to be free from this element. Soils Nos. 3435, 343-4, and 3436 show maximum amounts of copper, inasmuch as they are composed to a considerable extent of tailings. The soils in place beneath these sediments, Nos. 3501 and 3502, contain much less, yet noticeable amounts of copper, most of which is retained where it first comes in contact with the top soil. It is of interest to note that the surface sediments and the deep soils of the Experiment Station farm near Phoenix, Arizona, irrigated from an entirely different watershed, also con- tain small but weighable amounts of copper. This was probably derived from mines at Globe and Jerome, Arizona, whose wastes have found their way into the drainage which supplies irrigation for Salt River Valley. The quantities observed, however, three parts copper per million of soil, are negligible. Other soils from Salt River Valley also show traces of copper. i See Bibliography, p. 487, references 5, 18. 410 V niversity of California Publications in Agricultural Sciences [Vol. 1 TABLE V Miscellaneous Soils Unaffected by Mining Detritus Condition and Cu Sample No. weight taken, found, Cu and date Description of sample grams grams p. p.m. 2375 Surface 12 in. from new ground June 5, '00 near Safford, recently placed 100 under Montezuma Ditch air-dry none none 2253 Surface 12 in. university ground, 100 Jan. 3, '00 Tucson air-dry none none 3503 Surface 12 in. virgin unirrigated May 9, '05 soil, Colorado Valley bottom near 100 Yuma air-dry none none These determinations, made in widely separated localities, indicate the absence of copper in soils which are not immediately under the influence of mining detritus. TABLE VI Copper in Vegetation from Upper Gila Valley Farms Sample No. and date Description of sample 3505 Alfalfa, before blooming, from Aug. 19, '05 upper end of Geo. Olney's field east of Safford, under Monte- zuma Ditch 3512 Alfalfa from bale grown in Lay Aug. 19, '05 ton (M. B. Steele) under Monte- zuma ditch 3507 Corn in bloom, leaves only, grown Aug. 20, '05 in Layton (Jas. Welker), under Montezuma Ditch 3509 Wheat from stack, stalk and grain, Aug. 19, '05 grown in Layton (M. B. Steele), under Montezuma Ditch .'.."> 1.". Mistletoe, growing on willow 25 ft. Sept. 19, '05 above ground, one mile east of Safford, under Montezuma Ditch 3739 Alfalfa seed, crop of 1906, grown near Pima under Smithville Ditch 3741 Alfalfa seed (Wm. Gillespie), crop of 1906, grown near Solomonville, under Montezuma Ditch 3780 Shelled corn, crop of 1906, grown at Solomonville, under Monte zuma Ditch 3740 Shelled corn, crop of 1906, grown at Solomonville, under Monte zuma Ditch 3738 Shelled corn, crop of 1906, grown near Pima, under Smithville Ditch Condition and weight taken, grams i Cu found, grams Cu p.p.m. [ 1206 air-dry .0062 5.10 1 359 air-dry .0077 5.70 i 545 air-dry .0033 6.10 ] 1125 air-dry .0027 2.40 ! 1245 air-dry .0094 7.60 l 782 water-free .0026 3.33 > , 843 water-free .0023 o -o i 932 water-free .0004 .43 i 874 water-free .0008 .73 L i 1092 water-free trace trace 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 411 The prevalence of small amounts of copper in vegetation throughout this locality is shown by the figures in table VI. Samples of corn and alfalfa contained comparable quantities of copper, which, however, were exceeded by the amount found in a sample of mistletoe growing on a willow fully twenty-five feet above the ground. This is due chiefly to the perennial character of mistletoe which, therefore, has more time to accumulate cop- per. It is interesting to note also that seeds of alfalfa and corn contain less copper than corresponding foliage. Corn leaves were observed to contain 6.1 parts of copper per million parts of air-dry substance, while grain from the same locality contained from 0.73 to 0.43 parts. Alfalfa seed contained about one-half as much copper as the stalks and leaves, while wheat hay carry- ing a large proportion of grain showed a low proportion of cop- per. These facts are probably connected with transpiration, TABLE VII Copper in Vegetation from Other Localities Sample No. and date Description of sample 3508 Alfalfa hay, station farm near Aug. 25, '05 Phoenix (two samples) 3516 Alfalfa, before blooming, station Oct. 25, '05 farm near Phoenix 3515 Alfalfa hay, Colorado bottom, Oct. 4, '05 Yuma date orchard 3517 Barley hay, station farm near May, 1905 Phoenix " 3518 Corn, leaves only, station farm near Oct. 25, '05 Phoenix 3519 Corn, leaves only, grown on Rillito Oct. 14, '05 near old Fort Lowell 3529 Corn, leaves and bloom, same field Dec. 27, '05 as No. 3519 3520 Mistletoe from cottonwood 30 ft. Oct. 14, '05 above ground, old Fort Lowell, near Tucson 3989 Young (5 mos. old) alfalfa roots Dec. 31, '08 from C. & A. ranch irrigated with mine waters containing cop- per, from Bisbee 3990 Corn roots from C. & A. ranch irri- gated with mine waters contain- ing copper, from Bisbee Condition and weight taken, grams 2109 air-dry 1408 air-dry Cu found, grams .0021 .0031 Cu p.p.m. 1.00 2.20 1106 air-dry .0011 1.00 1262 air-dry none none 1304 air-dry .0002 .15 595 air-dry .0005 .84 284 air-dry .0018 6.30 1132 air-dry .0015 1.32 1160 air-dry .001 .85 2.12 air-dry .0001 47.00 16.7 air-dry .00025 15.00 412 University of California Publications in Agricultural Sciences [Vol. 1 which is maximum in leaves and quantitatively small in the fruiting parts of a plant. Additional evidence of this fact is shown in poisoned corn plants, which are discussed on a subse- quent page. Comparing the data of table VII with those of table VI. it is evident that, excluding corn and alfalfa irrigated with C. & A. mine waters, in every case except that of one sample of corn from old Fort Lowell (No. 3519) the copper in crops grown on Gila Valley farms is much in excess of that in plants coming from elsewhere for the same classes of material. The presence of appreciable amounts of copper in samples of alfalfa, corn, barley, and mistletoe also accords with the fact that the soils in which they were grown receive the drainage from copper-bearing water- sheds. The one exception, at Yuma (No. 3515) where qo trace of copper could be found either in alfalfa or in soil (No. .'5503). indicates that these alluvial river deposits, which have been sub- jected annually to the leaching action of enormous quantities of Hood waters, liave been prevented from accumulating appreciable quantities of copper. Copper in the Flesh and Bones <>f a Pig In order to follow the copper as Ear as possible in its trans- migrations, a five-months-old pig that had been born and brought up in an alfalfa pasture near Solomonville under the Montezuma Ditch, was killed and portions of the Hesh and hones were taken for examination, with the following results: Condition and Cu Sample No. weight taken, found. Cu and d.i I ■■ Description of sample grams grams p. p.m. 3779 917 May 7, '07 Liver, heart, and rib moat fresh .0053 5.78 3778 998 May 7, '07 Ribs and rib meat fresh .00006 .0(5 The largest amount of copper was found in portions of liver. heart and rib meat, only minute amounts being present in the bony material. In this connection, it is stated that about two parts of copper have been observed in one million parts of human liver; ten parts in human kidneys, and as much as fifty parts 1917 I Forbes: Irrigation Effects of tapper Compounds Upon Crops 413 in sheep's liver.5 Human food, however, is commonly contam- inated with copper compounds, which account for its presence in the human body. /// brief, the observations detailed above have shown the suc- cessive positions of copper in the original ores of the Clifton- Morenci district; in the tailings wastes from these ores, in sus- pension and in solution in river waters exposed to milling operations ; in soils irrigated with these waters ; in the ground waters beneath these soils ; in vegetation growing upon them ; and even in the animal life of the region. It is of interest to ob- serve, first, the concentration through natural processes of small amounts of copper in the original rocks into the form of rich ores ; and, second, the reversal, through human agencies, of this process, and the dilution of copper values till, in vegetation and in animal life, but traces of the metal can be detected. DISTRIBUTION OF COPPER IN PLANTS WITH ROOT SYSTEMS EXPOSED TO COPPER COMPOUNDS Corn Plants Grown in Soils Containing Copper In order to determine accurately the distribution of copper throughout a typical crop plant, thereby locating if possible the points at which injury may occur from copper compounds in the soil, three lots of corn plants were examined in detail. Two of these were grown (August 3 to November 13, 1907) in pots con- taining thirty-eight pounds of sandy loam soil very thoroughly mixed with 0.01 and 0.025 per cent of copper in the form of freshly precipitated copper carbonate (Cu(OH)._,.CuC03), made by mixing equivalent amounts of copper sulphate and sodium carbonate. The third was grown in soil containing 0.05 per cent of copper in the form of finely pulverized chalcocite. The samples were harvested with care to prevent contamina- tion with copper dust ; the root portions being washed in copper- free water saturated with carbon dioxide until the washings contained no trace of copper. Determinations of copper, as Blyth, Poisons, fourth edition, pp. 640-641. 414 University of California Publications in Agricultural Sciences [Vol. 1 usual, were made as shown under "Methods of Analysis" (see Appendix herewith). Following are the tabulated results: TABLE VIII Eleven Stalks of Corn Grown in Soil Containing 0.01 PER CENT Copper as Cu(OH)2.CuC03 (1907) No. 3869p Plant part Lower six nodes, 24 in. long Weight of sample, grams 43.4 Cu found, grams .00012 Cu p. p.m. 3.00 3869 nun. .05 3.1 mm. .01 3.3 .1 2.1 Showing no stimulation. TABLE XIV (m) Bean roots grown in distilled water with CuSO< Cu p.p.m. Elongation 48 his. 1 [eight of tops .1 2.9 mm. 87 mm. .3 1.2 91 .5 .6 85 Bean roots grown in distilled water with Cu(OH)2.CuC03 Cu p.p.m. Elongation 48 his. Height of tops .1 2.9 mm. 98 mm. .3 1. 88 .5 .6 84 Showing same behavior with CuS04 and Cu(OH)2.CuC03. 1917 J Forbes: Irrigation Effects of Copper Compounds Upon Crops 42." TABLE XIV (n) SquasJi roots grown in distilled water with CuS04 Cu p. p.m. check .01 Elongation 24 hrs. 3.6 mm. 2.8 Cu p. p. in. .05 .1 Elongation '24 hrs. 1.4 mm. 2 Squash roots grown in distilled water with Cu(OH)=.CuCOs Cu p. p.m. check .01 Elongation 24 hrs. 3.6 mm. 3.8 Cu p. p.m. .05 .1 Elongation 24 hrs. 1 .1 mm. .4 Doubtful stimulation at .01 p. p.m. TABLE XIV (o) Corn roots grown in distilled water with CuS04 Cu p. p.m. Elongation 48 hrs. check 8.7 mm. .01 10.9 Corn roots grown in distillec Cu p. p.m. Elongation 48 hrs. check 8.7 mm. .01 13.2 Cu p. p.m. .05 .1 < 'n p.p.m. .05 .1 Elongation 48 hrs. 4.7 mm. 1.5 Elongation 48 hrs. 3.3 mm. 2. These cultures, while somewhat fragmentary, afford excellenl indications of stimulating effects upon plant roots. Excluding squash, which is not satisfactory material to work with, corn and beans show consistent stimulations at very high dilutions. Meas- urements in all cases are averages of about ten observations. Experi- ment a b C e g h i j o TABLE XV Summary op Stimulation Effects Culture Corn roots Bean roots Corn roots Bean roots Corn roots Corn roots Bean roots Bean roots Corn roots Corn roots Copper salt used CutOHh.CuCO, Cu(OH),.CuCO, Cu(OH),.CuCO:i Cu(OH),.CuCO:< Cu(OH),.CuC03 Cu(OH),.CuC03 Cu(OH),.CuCO:t CuCOH^.CuCO, CuSO, Ctt(OH),.CuCOs Character and strength in copper of solution producing stimulation Well water" at .0] p.p.m. .03 .03 .05 .01-.1 Distilled water .05-.! none at .01 or above none at .01 or above .01 .01 426 University of California Publications in Agricultural Sciences [Vol. 1 Only at very high dilutions (one part of copper to from 10,000,000 to 100,000,000 of water) are accelerations of root growth observed. These occur with both corn and beans, in well water. In distilled water stimulation was observed only at the highest dilution — 1 :100,000,000. In well water stimulation was observed at from 1:100,000,000 to 1 :10,000,000— consistently with the well known fact that in presence of other soluble salts the effects of copper are lessened. EFFECTS OF SOIL UPON TOXICITY OF COPPER SOLUTIONS Of prime importance in connection with possible toxic effects of copper in soils are the various reactions (1) converting in- soluble into soluble compounds, (2) reconverting these again into insoluble combinations, and (3) modifying the toxic effects of copper salts in solution. As shown in the table of solubilities, both basic carbonate of copper and chrysocolla are soluble in carbon dioxide, forming solutions which in water cultures are highly toxic in character. Sulphides of copper are first oxidized to the sulphate, which is easily soluble : Cu2S + 50 = CuS04 + CuO For instance. 100 grams of chalcocite ore containing 3.2 per cen1 copper were shaken in a flask with 600 e.c. of water, frequently, during twenty-eight days. At the end of that time 500 c.c. of solution contained 0.0132 grams of copper. Copper sulphate then reacts in the soil to form various insoluble compounds with consequent lessening of toxic action. With calcium carbonate the following represents a reaction which may occur : 2 CuS04 + 2 CaC03 + H20 = Cu(OH)2.CuC03 + 2 CaS04 + C02 For instance, two grams of precipitated carbonate of lime were added to an excess of ten grams of copper sulphate in one liter 6 "University of Arizona well water" contains 250 p.p.m. of soluble solids, mainly sodium sulphate. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 427 of water, and digested with frequent shaking for over four months, the green precipitate being then filtered off, dried and analyzed for copper : Weight of precipitate taken 100.00 mg. Cu found 47.85 Theoretical Cu in basic carbonate 57.38 Indicating by the formula above a conversion to basic carbonate of copper of over 83 per cent of the solid carbonate of lime pres- ent. Bicarbonate of lime in solution also reacts with copper sulphate to form the basic carbonate CaH2(C03)2 -f 4 CuSCvS H,0 = 2 Cu(OH)2.CuC03 + CaS04 + 3 H2S04 + 16 H20 3 CaH2(C03)2 + 3 H2S04 = 3 CaS04 + 6 H,0 + 6 C02 The silicates of the soil, also, and particularly those of zeolitic character, react readily with soluble copper compounds to form insoluble copper silicates. Organic matter likewise combines with large amounts of copper, to form compounds of indefinite or unknown composition. As a result of all these reactions, when soils are shaken up with solutions of copper salts the latter are withdrawn from solution in large amount. Under irrigation con- ditions, where waters containing minute amounts of copper are filtered through relatively large masses of soil, this action is nearly or quite complete. Five large percolators were arranged with varying depths of TABLE XVI Percolation op Copper Solutions Through Soils Solution used Soil Depth Sandy loam 1 in. Sandy loam 5 in. Sandy loam 9 in. Sandy loam 1 in. Heavy clay containing .003% Cu 12 in. Heavy clay containing .003% Cu 12 in. Cu compound p.p.m Cu ( OH ) 2.CuC03 in CO, water 95 Cu ( OH ) 2.CuC03 in C02 water 95 Cu ( OH ) 2.CuC03 in C02 water 95 Cu ( OH ) 2.CuC03 in C02 water 56 Cu ( OH ) 2.CuC03 in C02 water 8.5 Cu in Amount of Copper in solution, percolate, percolate, p.p.m. none c.c. 2000 1500 2000 2000 600 none none .85 CuSO,.5 ILO 254 150 7.3 428 University of California Publications in Agricultural Sciences [Vol. 1 soil resting on filter paper supported by a perforated porcelain plate. Two soils, heavy clay and sandy loam, were employed ; and two copper solutions, sulphate and bicarbonate. In nearly all cases copper as basic carbonate was entirely removed from solution in percolating through as little as a single inch of sandy loam. Although appreciable amounts of copper sulphate passed out of a soil, the latter in that case itself contained a very small percentage of copper. Inasmuch as soluble copper in irrigating waters must be present ordinarily as basic carbonate, its complete withdrawal by thin layers of soil is significant in connection with irrigated crops. Irrigation Experiments A set of cultures was arranged to test the effects upon crop plants of solutions of basic copper carbonate so applied as to filter through the soil before reaching the plant roots. Six-inch Fig. 5. — Diagram of pot culture irrigated through two-inch pot inside. flower-pots were filled with sandy loam soil. In the middle of each of these pots a two-inch pot was half buried, and the plants experimented with were grown in the circles of soil between the large and small pots. These plants were irrigated by pouring the solution used into the small pot, through the bottom of which it passed, necessarily filtering through more or less soil before 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 429 reaching the plant roots. Radishes, beans, cantaloupes, cucum- bers, lettuce, peas, beets, corn, berseem, avas, onions, barley, and wheat were employed ; corn, barley, and wheat being especially successful under these conditions. All cultures were in pairs, one of each pair being irrigated with solutions of basic carbonate of copper in C02-water, and the check cultures with water only. In all other particulars — original strength of plants, exposure to light and air, and amount and time of watering — the con- ditions were identical. These cultures were carried on in a greenhouse set aside for the purpose. The experiment was begun in November and ended the following March. The solutions of basic carbonate of copper employed contained from 0 to 55 p. p.m. of copper, averaging about 20 parts, which is from 7 to 670 times as much as has been observed in the waters of the Gila River from time to time. TABLE XVn Condition at Maturity of Cultures Irrigated with Copper Solutions, as Compared with those Irrigated with Water C, copper culture; W, check. Tops C and W. The Badishes Beans C greener Lettuce Peas Beets Corn Stimulated ? C showing stronger Berseem C stimulated, earlier bloom same in appear- ance and weight C and W. About the same C and W. About the same C and W. Aver- aging the same Weighing the same, but C ap- pearing stronger C more advanced in growth, but not so heavy Roots The same, but in C roots were removed J in. from inner pot hole Equal; same number of nodules; very local ef- fect of Cu at pot hole The same except that in C roots were dead fxj in. under pot hole Both C and W having abundant nodules. No apparent damage by Cu Fewer in soil under pot hole in C, otherwise equal Equally developed, both showing strong nodule development. 430 University of California Publications in Agricultural Sciences [Vol. 1 Avas Onions Barley Wheat C stimulated, ma- tured over twice as much grain < ' stimulated matured 20% more grain Tops C and W. Same apparent growth C and W. Same general appearance The same in weight, but C matured more grain Identical appear- ance, but C ma- tured more grain Roots In C roots within $ in. of pot hole damaged. Both C and W show strong nodule develop- ment Very little local effect of Cu just under inner pot hole in C No roots in C for space of 1 x J in. under inner pot hole In C no roots under in- ner pot hole for space of 1$ x i in. In practically all cases a distinct but very local effect of copper solutions upon plant roots under the inner pot hole was observed. For a distance of a half-inch or less from the small pot hole exposed roots were dead or missing. The soil in this area was observed in two instances to contain 0.25 and 0.45 per cent copper, respectively. In one instance 80 per cent of the copper added was found in the 4:? grams of soil just under the bottom of the little pot, showing the rapidity with which copper is removed from its solutions by filtration through the soil. The tops of the cultures under consideration in no instance showed injury, but in certain cases were in a distinctly advanced condition. The amounts of copper contained in material derived from these cultures are as follows : TABLE XVII (a) Copper Content of Plants Irrigated with Copper Solutions P.p.m. of Cu in rv matter, Cu dry grams grama material Sample No. 3673 Wheat and barley tops grown in check 3675 soil containing a trace (.0025 per cent) of copper 32.90 .000100 3.04 3672 Tops of beans, peas, corn, lettuce, car- rots, cucumbers and avas grown in check soil 133.00 .000350 2.60 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 431 P. p.m. of Cu in Sample Dry matter, Cu dry No. grams grams material 3674 Tops of wheat and barley irrigated 3676 with water averaging 20 p.p.m. cop- per 30.70 .000400 13.00 Tops of beans, berseem, peas, onions, lettuce, beets, radishes, corn, avas, barley, and wheat irrigated with water averaging 20 p.p. in. copper .... 27.20 .000751 3690 Roots of same (washed in 4 per cent HC1) 8.30 .000750 27.60 90.00 In brief, even when relatively large amounts of water contain- ing excessive quantities of soluble copper were applied and the experiments so arranged that all of the copper remained in the limited volumes of soil employed, no general injury to the plants was observed, although apparently slight stimulation occurred in some cases. Prolonged irrigation with such solutions would be required to saturate the soil to a depth sufficient to seriously injure plants grown in it. \ / Fig. 6. — Wheat and barley irrigated (C) with copper solutions filtered through soil, and (W) with well water. Both show stimulated growth with copper. 432 University of California Publicatio7is in Agricultural Sciences [Vol. 1 CULTURAL EXPERIMENTS Pot Cultures with Treated Soils Pot cultures of corn, beans, and squash were also grown in soils containing copper in the form of precipitated carbonate (Cu(OH),.CuC03), finely powdered (100-mesh) chalcocite or sulphide ore, and finely powdered chrysocolla or silicate ore. Large glazed stone jars containing thirty-eight pounds of soil were used. Effects on growth were observed and the copper content of tops and of root systems was determined. The follow- ing tabulations relate to the work done in this direction, the state- ment showing the copper content of corn, bean, and squash plants expressed in parts per million of copper in dry matter. TABLE XVIII Copper Carbonate Series (1908), Beans Sample No. Culture Cu in soil, per cent Appearance and height of plants Dry matter, grams Cu found, r grams Cu p. p.m. in tops > roots Normal 3944 Beans Check 39 in. 16.6 .00022 13 4013 Beans Check .72 .00033 453 3945 Beans .01 38 17.2 .00027 16 4014 Beans .01 1.35 .00116 859 3946 Beans .025 39 15.9 .00033 21 4015 Beans .025 1.21 .00115 950 Toxic effects begin at about .035% Cu in soil Stunted 3947 Beans .05 30 13.2 .00031 23 4016 Beans .05 1.09 .00148 1358 3948 Beans .1 25 6.7 .00011 16 4017 Beans .1 1.44 .00212 1472 3949 Beans .25 14 3.7 .00009 25 4018 Beans .25 1.35 .00243 1800 3950 Beans .5 15 2.9 .0001 35 4019 Beans .87 .00147 1690 3951 Beans 1. 12 2.2 .00009 41 4020 Beans 1. .53 .00106 2000 3952 Beans 1.5 14 2.1 .00009 44 4021 Beans 1.5 .5 .00115 2300 Containing traces of copper, .0025%. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 433 Fig. 7. — Bean cultures grown in soils containing copper as precipitated carbonate, from none to 1.5 per cent Cu. TABLE XIX ( lOPPEB Carbonate Series (1907), Corn Sample No. 3870 Culture Coin Cu in soil, per cent Check5 Dry matter, grams 45.2 Cu found, grams .00(120 Cu p.p. m. in tops 4.40 > roots 3885 Corn Check4 8.8 .00035 40.00 3860 Corn .01 1 75.0 .00115 6.50 3868 . Corn .01 10.6 .00161 152.00 3865 < lorn .025 77.0 .00160 21.00 3866 Corn .025 9.2 .00670 728.00 3864 Corn .05 47.7 .00103 22.00 3867 Corn .05 4.4 .00328 745.00 3863 Corn .10 26.8 .00079 30.00 3862 ( lorn .15 9.8 .00046 47.00 3861 Corn .20 14.4 .00073 51.00 3860 Corn .30 4.6 .00110 239.00 T Containing traces of copper, .0025%. Fig. 8. — Corn cultures grown in soils containing copper as precipitated car bonate, from none to .2 per cent Cu. 434 University of California Publications in Agricultural Sciences [Vol. 1 Copper Carbonate Series (1908), Corn Sample No. 3992 3993 3994 3995 Culture Cu iu soil, per cent Corn Cheek* Corn .0] Corn .015 Corn .02 Appear- ance and Dry height matter, of plants grams Normal 43 in. 41 35 41 7.48 2.35 4.07 5.31 Cu found, grams .00058 .00049 .00171 .00397 Toxic effects begin at about .023% Cu in soil Stunted 33 in. 15 3996 3997 3998 4000 Corn Com Corn Corn .025 .05 .10 .20 22 20 4.81 .31 3.62 1.99 .00245 .00023 .00651 .00444 Cu p.p.m. roots 78.00 209.00 420.00 748.00 509.00 742.00 1798.00 2231.00 Containing traces of copper, .0025%. Copper Carbonate Series (1908), Squash Sample No. 3937 3938 3939 4026 Toxic 3940 394] Appear- Cu ance and Dry < u in soil, height matter. found, Culture percent of plants grains grams Normal Squash check 16 in. 11.2 .00016 Squash .01 16 6.3 .(10023 S,|ii;isli .025 15 9.2 .00031 Squash Chk., .01, and .025 .24 .00004 ■fleets begin at about .035% Cu in soil. Blanched and stunted .05 11 in. 3.7 .00017 .10 11 2.3 .00014 Squash Squash Cu p.p.m. tops 1 4.00 36.00 39.00 Ml. (Ml 61.00 169.00 Fig. 9. — Corn cultures grown in soils containing copper as sulphide (chalco- eite), from none to 1. per cent Cu. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 435 TABLE XX Chalcociti : Series (1908) Sample No. Culture Cu in soil, per cent Appear ance and height of plants Dry matter, grams Cu found, grams Cu p. p.m. in A tops roots Normal 3979 Corn Check * 36 in. 17.60 .00026 15.00 3979c Corn Check 4.62 .00027 58.00 3980 Corn .01 33 11.60 .00011 10.00 3980c Corn .01 2.94 .00023 78.00 3981 Corn .02 38 19.40 .00021 11.00 3981c Corn .02 6.14 .00114 186.00 3982 Corn .03 35 17.90 .00028 16.00 3982c Corn .03 6.99 .00176 252.00 3968 Corn .05 45 51.70 .00065 13.00 3978 Corn .05 6.14 .00105 171.00 Toxic effects begin at about .08% Cu in soil. Stunted 3983 Corn .10 36 in. 14.0(1 .00031 22.00 3983c Corn .10 yellow 6.08 .00625 1028.00 3984 Corn .50 8 in. 3.20 .00040 125.00 3984c Corn .50 .47 .00065 1383.00 3985 Corn 1.00 12 3.20 .00050 159.00 3985c Corn 1.00 .49 .00089 1816.00 Containing traces of copper, .00257^. The cultures described in the foregoing tables indicate sev- eral interesting facts more or less applicable to field conditions. (1) Precipitated carbonate of copper is shown to have a much more toxic effect upon corn than the finely pulverized ores of ehalcocite or chrysocolla. With the precipitated car- bonate 0.025 per cent in the soil was distinctly toxic, while with ehalcocite and chrysocolla about 0.08 per cent was required to produce an ecpial effect. Inasmuch as all of these combinations of copper may occur in a soil subject to mining detritus, a mere determination of total copper in soils containing doubtfully toxic quantities cannot convey trustworthy information as to the in- juriousness of the amounts present. Moreover, since it has been shown that in the case of pre- cipitated carbonate, and sulphate of copper, eqiiivalent quantities of these salts in solution are equally toxic, it is probable that the greater toxicity of the carbonate is due to its greater solubility under soil conditions. It is, in fact, shown in table I, "Solubili- 43(5 University of California Publications in Agricultural Sciences [Vol. 1 TABLE XXI Chrysocolla Series (1908) Sample Culture Appear- Cu anee and in soil, height per cent of plants Dry matter, grams Cu found, grams Cu p. p.m. in A No. tops roots Normal 4003 Corn Check* 32 in. 25.60 .00025 10.00 4003c Corn Cheek 6.46 .00012 19.00 4004 Corn .05 33 23.50 .00026 11.00 4004c Corn .05 6.70 .00062 93.00 Toxic effects begin at about .08% Cu in soil. Dwarfed 11 Ml.", Corn .10 30 in. 17.90 .00024 13.00 4005c Corn .10 striped 5.82 .00094 162.00 400(3 Corn .10 28 in. 10.50 .00017 16.00 4006c Corn 1.00 yellow 4.29 .00233 543.00 Containing traces of copper, .0025%. Pig. K). ('din cultures grown in soils containing copper as silicate (chryso- colla), from none to 1. per cent Cu. tics of Copper Compounds," that precipitated copper carbonate is soluble to the extent of 1.5 parts in 1,000, 000 of water, while copper sulphide is soluble to the extent of 0.09 parts of copper in 1,000,000 of water. It is most probable, also, that the finely divided condition of the precipitated carbonate is more favorable to solution, and also to reaction with the acids of plant roots. (2) Corn is seen to be distinctly more sensitive to the car- bonate of copper than either beans or squash. With corn, toxic effects appear with 0.02 per cent of copper in the soil, while with beans and squash these toxic effects do not appear until 0.0:5;") per cent of copper in the soil is reached. As is suggested 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 437 in the following pages, the physical constitution of root systems may account in part for varying degrees of sensitiveness to cop- per compounds. The presence of copper in tops and roots of check is due to 0.0025 per cent of copper in the soil which was supposed orig- inally to be free from this element. Pot Cultures with Field Soils Two field soils containing copper from irrigating waters were tested in pot culture with reference to toxic effects and Fig. 11. — Pot cultures of coin in field soils containing tailings. No. 3887, .027% Cu; no. 3888, .047% Cu ; and no copper. Cultures in field soils are slightly affected. copper content of root systems. The soils employed were from a field showing varying effects of accumulations of tailings, im- mediately southeast of Safford : Cu in soil, Sample per cent 3887 Sandy loam, surface 12 in. of soil recently put under irri- gation 027 3888 Heavy clay (tailings) mixed with sandy loam, surface 12 in., long under irrigation, much tailings 047 In these two soils, differing mainly through the addition of tailings to No. 3888, cultures of corn, beans, and squash were made, and examined for copper with the following results : 438 University of California Publications in Agricultural Sciences [Vol. 1 TABLE XXII Cultures in Tailings Soils No. Pot culture Condition 3887 Corn in sandy loam Distinctly striped 3888 Corn in sandy loam Less distinctly and tailings striped 3887 Beans in sandy loam Normal appearance 3888 Beans in sandy loam Normal appearance and tailings 3887 Squash in sandy loam Yellow and stunted 3888 Squash in sandy loam Normal appearance and tailings Cu in soil, per cent .027 Cu p. p.m. in tops .047 .027 .047 .027 .047 28.00 19.00 73.00 45.00 roots 453.00 163.00 1523.00 703.00 Pig. 12. — Showing effects of copper uie»
  • rv matter. Cu found, Cu p. p.m. in No. Sample grams grams drv matter 6332 530 tops 12.7 .00165 129.90 6331 Roots .6902 .00020 297.00 6330 16000 root tips .3664 .00103 2811.00 Amount of copper per root tip associated with slight toxic effects, .00103 -h 16000 = .000000064 gm. Total roots examined for nitrogen 2. 9223 gms. Albuminoids in roots (Alb. N. X H) 30794 Copper required to saturate albuminoids (factor 11.7%) .03603 Total Cu found 001789 Saturation f™* = 4.96% These figures show, as usual, relatively small amounts of copper in tops of plants, with large amounts in roots, increasing from coarser to finer portions, until in the root tips corn con- tains 545, peas 3428. and wheat 2811 parts per million of eopper in dry matter. For peas and wheat these are the largest propor- tions of copper thus far observed in any plant samples. When the total amount of copper found in each sample is divided by the number of root tips employed, an extraordinarily small amount of copper is found necessary to bring about toxic effects. For instance One corn root tip (terminal 3 cm.) required 000000382 gms. Cu One pea root tip (terminal 1 cm.) required 000000278 gms. Cu One wheat root tip (terminal 1 cm.) required .000000064 gms. Cu 450 University of California Publications in Agricultural Sciences [Vol. 1 Moreover, the extent to which albuminoids in affected roots are saturated with copper — only 7.99 per cent for peas and 4.96 per cent for wheat — indicates a maximum effectiveness upon roots of small amounts of the metal. Reactions of Copper with Growing Points Corn seedlings fifteen days old were fixed with cotton in tall 50-c.c. graduated Nessler tubes containing different strengths of copper sulphate in pure distilled water. The strengths of solution employed were 20, 10, 5, 2.5, and 1.25 p. p.m. There was a check culture with no copper. After three days, in all cases except the check, the roots were flaccid, showing contraction on graduations and giving biuret and ferrocyanide tests, increasing in strength from weaker to stronger concentration. An experiment with pea seedlings gave similar results, but when the quantity of pea roots was increased and weak solutions. 2.5 and 1.25 p. p.m., were employed in small quantities (20 c.c), the tests became much fainter. Severed roots of corn, also, were observed to give as good tests as roots of living plants. A large number (seventy) of severed root tips placed in a small quantity (20 c.c.) of weak solution (5 p. p.m.) gave only a faint ferrocyanide test. These observations indicate that the concentration of copper in growing points is due to ionic dissociation and migration through the semi-permeable membranes of the root systems, s rather than to transpiration. The fainter test for copper in large quantities of root material indicates lessened toxicity of dilute solutions of copper in presence of excess of root materials. Mature wheat, corn and pea plants in nutrient solutions, but not growing actively, were treated with gradually increasing amounts of copper from January 21 to February 2. as follows : Wheat, Corn, and Pea Plants, Thirty-seven Days Old, Treated with Copper in Nutrient Solution Jan. 12 1 — 2 ."> ; nutrient sol. w. 2 p.p.m. Cu. Jan. 25-28; nutrient sol. w. 4 p.p.m. Cu. Jan. 28 to Feb. 5, nutrient sol. w. 10 p.p.m. Cu. s See Bibliography, p. 488, references 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 451 Feb. 5; very faint biuret test for copper, distinct ferrocyanide test. Corn, forty-eight days old in 50 p.p.m. Cu sol., two days, gave faint biuret and ferrocyanide tests. Corn, forty-eight days old in 500 p.p.m. Cu sol., two days, gave faint tests for copper. From these observations it is evident that the nearly negative results shown are due either to nutrient salts present or to the older and therefore more quiescent material employed. To settle this question, the following experiments were made: (1) Young (ten days) wheat and corn plants were placed in copper solution in distilled water and in nutrient solutions and observed after twenty and forty hours, as follows: 2.5 p.p.m. Cu, distilled water — 20 hours 10 days old: Young wheat; flaccid?; strong biuret test; strong K4FeCy6 test 60 days old: Old wheat; not flaccid; no strong biuret test; distinct K4FeCyc test 10 days old: Young corn; flaccid; strong biuret test; strong K4FeCy6 test (10 days old : Old corn ; flaccid ; no biuret test ; old tips, faint K4FeCy6 test young tips, strong K4FeCy„ test 10 p.p.m. Cu, distilled water — 20 hours 10 days old: Young wheat; flaccid?; strong biuret test; strong K4FeCy6 test (50 days old: Old wheat; not flaccid; faint biuret test; distinct K4FeCy6 test 10 days old: Young corn; flaccid; strong biuret test; strong K4FeCyb- test 60 days old: Old corn; flaccid?; distinct biuret test; strong K4FeCy„ test 40 p.p.m. Cu, distilled water — 20 hours 10 days old: Young wheat; flaccid; strong biuret test; strong K4FeCy6 test 60 days old: Old wheat; flaccid?; faint biuret test; distinct K4FeCy6 test 10 days old: Young corn; flaccid; strong biuret test; very strong K4FeCy„ test 60 days old: Old corn; flaccid; strong biuret test; strong K„FeCy,, test The above results indicate that old roots of corn and wheat are more resistant to the penetration of copper than are the young roots. This is shown by less flaccidity in the weaker solu- tions and by the fainter tests observed. A second series with greater strengths (5, 20. and 100 p.p.m.) and longer exposure (forty-five hours) showed distinctly less differentiation than in the case of the series above given in detail. This is to be expected, inasmuch as stronger solutions must overcome resistance of roots exposed to them more quickly, and the longer time employed would likewise tend to overcome differences existing in the first few hours of the experiment. 452 University of California Publications in Agricultural Sciences [Vol. 1 (2) Young and old wheat and corn roots were placed in 10 p. p.m. Cu in distilled water and 10 p. p.m. Cu in nutrient solution. with the following results : 10 p. p.m. Cu, distilled water — 20 hours 10 days old: Young wheat; flaccid?; strong biuret test; strong K4FeCy„ test 60 days old: Old wheat; not flaccid; faint biuret test; distinct K4FeCy0 test 10 days old: Young corn; flaccid; strong biuret test; strong K4FeCy„ test 60 days old: Old corn; flaccid; distinct biuret test; strong K4FeCyi; test 10 p.p.m. Cu, neutralized nutrient solution — 20 hours 10 days old: Young wheat; not flaccid; doubtful biuret test; faint K4FeCyc test 60 days old: Old wheat; not flaccid; none or doubtful biuret test; faint K4FeCy6 test 10 days old: Young corn; not flaccid; faint biuret test; distinct K4FeCy„ test 60 days old: Old corn; not flaccid; distinct biuret test; distinct K4FeCy,, test This shows very distinctly the prevention of toxic action upon plant roots through the protective action of other solids in solu- tion. ;is already observed in water cultures by measurements of root growth. It is noteworthy in this connection that corn roots generally seem to be more sensitive to the action of copper salts than the roots of wheat or peas. In order to examine still further into the relative resistance of old and young root systems to copper salts, a solution of 5 p.p.m. Cu in distilled water was used, the time being varied from twenty to two hundred hours. The results of these obser- vations indicate that, with wheat and corn roots, the penetration of copper is distinctly more rapid in young than in old material. Peas did not give clear results. It appears from these observations, first, that the accumula- tion of copper in plant roots is distinctly due to the migration of dissociated ions into the root systems, where they are fixed by protoplasm, in which combination they are identified by means of the biuret test. Second, the presence of nutrient salts very distinctly lessens the effect of a Id p.p.m. copper solution upon sensitive young growing plant roots. Third, old quiescent plant roots developed in a nutrient solution are distinctly less sensitive to copper salts than young roots which are still actively growing. 1!»17] Forbes: Initiation Effects of Copper Compounds Upon Crops 453 The slow development of biuret tests for copper in such material after sufficient exposure to copper solutions, indicates the presence of protoplasm. It is possible that the same observations may apply to other poisons, metallic or otherwise, brought into contact with absorp- Fig. 13. — Photomicrograph of root tip of corn grown in water culture and poisoned by 1:200,000 of Cu in solution. The copper is shown as red copper ferrocyanide, which appears black in the photomicrograph. The irregular inner black line shows the penetration of the copper and also indicates sharply the differences in permeability of adjacent cells, some of which are penetrated before others. (X 80 diam.) (Photo by J. T. Barrett.) 454 University of California Publications in Agricultural Sciences [Vol. 1 tive root systems in the soil. Not only this, but it may be true that nutrient salts, as well, will be found more actively absorbed by younger and more sensitive root systems than by older ones, or by root systems which for any reason have become quiescent. This would suggest the possibility of choosing to advantage the proper time for applying substances, either to avoid injury or, as in the case of fertilizers, to secure maximum benefit from them. Varying Resistance of Individual Cells to Copper Not only do old and young roots vary as to toxic effects upon them of copper, but different degrees of resistance between individual cells in the same root and even in the same chain of cells, is clearly shown in the photomicrograph (fig. 13) of a corn root tip which has been exposed to a 1 to 200,000 solution of copper, then colored with K4FeCy0, and sectioned for obser- vation. The dark, abruptly angular line of penetration shown in the section plainly indicates that individual cells may be penetrated by copper while adjacent cells growing under pre- cisely similar physical conditions are not penetrated. If this be not due in some unseen way to morphological peculiarities of root structure, it must be due to individuality in the cells themselves, some of which must be more resistant to penetration by dilute copper solutions than others. Summing up the physiological observations relating to effects of copper upon plants, we find (1) that individual cells vary (probably) in degree of resistance to penetration by copper salts; (2) that young roots are less resistant than old roots; (3) that roots of certain species of plants (e.g. corn) are less resist- ant than roots of other species; and (4) that toxic effects may be to some extent related to the structure and distribution of root systems. DIAGNOSIS OF COPPER INJURY In the presence of toxic amounts of copper in the soil, the root systems of culture plants become harsh and crinkly with almost entire loss of root hairs. Consistent with the checking of growing points, root systems are also greatly restricted in extent, 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 455 and in feeding capacity. Individual roots are coarse, covered with thick epidermis, and are abruptly angular, apparently as a result of chemotropic contortions. Root tips are shortened and thickened and in some instances are strongly proliferated. The anatomical structures associated with these changes in form are very striking. In corn the cells of the primary cortex, in normal roots, are elongated parallel with the axis of the root, and in longitudinal tangential section measured about 74 by 30 microns. Injured cells of corn grown in soil containing 0.1 per cent copper gave longitudinal tangential sections approximately 34 by 30 microns, as shown on accompanying drawings. (See, also, plates 6, 7, and 8. HWn - <~ __ Fig. 14. — a. Tangential longitudinal section of corn root grown in soil containing .1 per cent of copper as copper sulphate, showing cells of cortex of injured rootlet, b. Tangential longitudinal section of normal corn root cells of cortex. (X ± 300 diam.) (Sections by G. F. Freeman.) These structural modifications, taken in connection with other symptoms and conditions and in the absence of other causes, such as an excess of alkali salts,9 confirm a diagnosis for copper injury in a soil of doubtful toxicity. For instance, March 4. 1916, two sets of samples of barley were collected in the district studied, and the material examined for evidence of copper in- jury, a.s follows : Lot 1. — Young barley plants from the upper end of a field midway between Safford and Solomonville, under Montezuma Canal. The soil next the ditch shows old tailings, and there are irregular areas of yellow barley immediately under the canal. 3 See Livingston, Botanical Gazette, vol. 30, no. 5, p. 229, 1900. 456 University of California Publications in Agricultural Sciences [Vol. 1 Sa^ple a. Yellow bailey plants 6343 Boots, crinkly and angular, much branched near surface. Dry weight, 3.2429 gins ; Cu, .00085 gin ; p.p.m 262 6344 Soil shaken from yellow barley roots Copper 07979% Total soluble solids (alkali) 46400 CI as NaCl 004 Sodium carbonate 008 Nitrogen 137 b. Green barley plants from near (a). 6345 Roots, smooth and straight, not much branched near sur- face. Dry weight, 1.6025 gm; Cu, .0002 gm ; p.p.m.—. 125 6346 Soil shaken from green barley roots Copper 05844% Total soluble solids (alkali) 45600 CI as NaCl 004 Sodium carbonate none Nitrogen 181 Lot 2.— Young barley plants from the upper end of a field in West Layton under Montezuma Canal. Soil near ditch known to contain tail- ings and showing spots of yellow bailey at head of field. Sample o. Yellow barley plants No. ' 6347 Roots, crinkly and angular, much branched. Dry weight, 3.2977 gins; Cu, .0014 gm ; p.p.m 425 6348 Soil shaken from roots of yellow barley plants Copper 1 113% Total soluble solids (alkali) 50 CI as NaCl 008 Sodium carbonate 008 Nitrogen 165 b. Green barley plants from near (a) 6349 Roots, smooth, straight, not much branched. Dry weight, 2.2473 gins; Cu, .0003 gm ; p.p.m 133 6350 Soil shaken from roots of green barley plants Copper 02678% Total soluble soli. Is (alkali) 40 CI as NaCl 008 Sodium carbonate 004 Nitrogen 127 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 457 Considering the above observations, we notice that the soils from which samples were taken do not contain injurious amounts of soluble salts. Their nitrogen content, also, is normal. The areas of yellow barley from which samples come are therefore not to be attributed to alkali salts, or to abnormal nitrogen content. Observation in the field, also, failed to indicate that conditions of irrigation, temperature, or light were unfavorable, these condi- tions being the same for both green and yellow samples. Excluding these considerations, therefore, we now find that there is uniformly more copper in the roots of yellow barley plants than in those of the green ones, also in the soils in which they occur. The roots of yellow plants, moreover, show the crinkly condition caused (though not exclusively) by copper when present in toxic amounts in the soil. The following state- ment summarizes these observations. Lot 1 Cu in soil Cu p. p. in per cent in roots Yellow barley .0798 262.00 Green barley .0584 125.00 Lot 2 Yellow barley .1113 425.00 Green barlev .0268 133.00 Condition of roots Crinkly and branched Straight, not branched Crinkly and branched Straight, not branched The evidence therefore indicates quite conclusively that the two yellow samples owed their color to toxic effects of copper upon the roots of the young plants. Later in the season, how- ever, no difference in mature plants, showing variations in color when young, may be observed. This must be due to the fact that as root systems penetrate more deeply into the soil they escape the surface zone of tailings, with consequent recovery from the effects of copper. 458 University of California Publications in Agricultural Sciences [Vol. 1 Part II.- GENERAL DISCUSSION PRELIMINARY STATEMENT The copper compounds, in solid form and in solution, that result from mining operations in the Clifton-Morenci district, have found their way down the San Francisco and Gila rivers to the underlying irrigated agricultural soils of Graham County in sufficient amounts to raise the question of their toxicity to crops. The largest amounts of copper in these soils are found at the heads of irrigated lands, especially where alfalfa is or has been, at which points old accumulations of tailings, laid down for the most part prior to 1908, are still to be found. Accumulations of Copper The amounts of copper accumulating in the Gila River valley soils in this way are small, the observed range being from 0.006 per cent to 0.111 per cent in surface soils and the average for eighteen soils analyzed being 0.046 per cent of copper. Irrigated soils elsewhere have been observed to contain larger quantities of copper than those above noted, for instance 1.002 per cent on the Deer Lodge River below Anaconda. Montana, with an aver- age of 0.09 per cent for eleven other samples taken in the same locality.10 These amounts of copper in a soil may or may not be toxic according to the combination in which the copper exists, the physical character of the soil and its chemical composition, climatic and moisture conditions, the crop grown, and other con- siderations which may now be discussed in order. The small amounts of soluble copper constantly coming down stream from the mines which cannot, like solid tailings, be en- tirely excluded from irrigating water supplies, are of importance because of their tendency to accumulate by reason of the fixing power for copper of silicates, carbonates and organic matter in the soil. The completeness of this fixing power of soil for copper is shown by several experiments in which solutions of copper i» IT. S. D. A. Bur. Chem., Bull. 113, p. 34, 1907. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 459 salts were percolated through one to twelve inches of soils, with little or no copper appearing in the filtrates. Under field con- ditions, therefore, this action tends to concentrate dissolved copper in irrigating water in the surface few inches of the soil. A series of samples of Montezuma Canal water taken at Solomonville affords quantitative suggestions in this connection : TABLE XXIX Copper Content of Gila River Waters pie No. date May and 26, I '04 Description River very low Amounts of Cu added in irrigation, estimated in p. p.m. of water Approx. flow of Gila River in sec. ft. 30 Approx. amts. of copper carried down stream, 1 dav lb.' 3094 Sam 3309 In tailings s 18.3 In olution .80 Total 19.1 3486 June 11, '05 Small flood .25 170 230 3622 June 25, '06 River low 1.6 .11 1.71 (Soluble only) 3737 Feb. 22 '07 Medium flood trace 2.88 ±3.0 600 9720 4011 Jan. 3, '08 2.1 .08 2.18 Tailings shut out of river May 1, 1908 4029 Apr. 12, '09 1.4 .08 1.48 6342 Mar. 4, '16* .04 .03 .07 * Following four-months shutdown of operations in Clifton-Morenci district. These figures, while somewhat meagre, seem to indicate a lessening waste of copper downstream following the restraint of tailings from the water-supply in May, 1908. This is especially true of copper in solution, due probahly to the decreased amounts of solid copper compounds in suspension from which copper in solution is derived. Assuming at the present time an average of 1 part of copper in 1,000,000 of Gila River water, four acre-feet of such water, required for one year's irrigation, would contain 10.9 pounds of copper, from which should be deducted small losses due to vegetation, drainage waters, and percolation to depths below the surface soil. Six tons of alfalfa with a copper content of 5 p. p.m. contain 0.06 lb. copper; while one acre-foot of seepage water (about the annual seepage loss) containing 0.25 p. p.m. copper would carry 0.68 lb. copper. Estimating the total loss roughly at one pound 4:60 University of California Publications in Agricultural Sciences [Vol. 1 of copper per acre a year, the net addition of copper to the soil would be approximately ten pounds, or about 0.00025 per cent. It would therefore require about forty years to accumulate 0.01 per cent of copper in the surface foot of soil. Inasmuch as, under field conditions, this is not an injurious amount, there is little likelihood, considering the district in a general way. that tbe small residues of copper now coming down stream will accumu- late to an injurious extent within a reasonable period of time. Incidentally, it is of interest to note the large total losses of copper (3094 lb. and 9720 lb. per day observed) formerly result- ing from mining operations in the district. Possible Effects upon Health With reference to the question of poisonous effects upon man and animals of dissolved copper in irrigating and well-waters, such effects, in general, are much less upon animals than upon plant life. Moore and Kellerman state, tor instance, that 0,02 gms. of copper may be absorbed daily by a man with safety.11 This amount of copper would he contained in five gallons of water containing one part per million of copper, the Largest amount of copper observed in a well-water in the district studied lieiiiLr <•. •">:'> p. p. in. It is of interest in this connection to note a belief of the copper miners of the Rio Tinto in southern Spain, where the wells are impregnated with copper, that one part of copper per million of drinking water is permissible, hut that two parts per million result in "copper colic." "" In view of experi- ments upon human subjects, however, it is more than likely that deleterious effects observed are due to associated compounds in the water. It is of importance to note that a strength of as little as one part per million of copper in pure water will de- stroy algae, which are common in clear water supplies freely exposed to light and air. This fact may he made of use in clean- ing ditches and reservoirs of aquatic growth, where the expense is not too great. The germicidal effects of small amounts of copper in waters of the district studied also have a bearing upon human health. " U. S. D. A. Bur. PI. Ind., Bull. 64, p. 23. 110 Conversation of J. W. Bennie, Clifton, Arizona. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 461 Bacilli of various species reacting upon human health are very sensitive to the action of soluble copper salts. For instance, in distilled water "one part copper in 16,000,000 parts water killed typhoid bacilli in two hours. In copper solutions made up with tap and sea water, the action was still marked, but less vigorous than in distilled water. ' ' 12 Moore and Kellerman state that one part of copper sulphate to 100,000 parts of water destroys typhoid and cholera germs in three to four hours.13 In milk supplies as little as one part of copper salts in 2,000,000 of water acts as an antiseptic against putrescent bacteria.14 It seems, therefore, that there is a possibility that the amounts of copper observed in ditch and well-waters in the district may have an antiseptic effect upon malignant germs, more particularly typhoid fever, likely to occur in the district.15 Amounts and Significance op Copper in Aerial Vegetation The amounts of copper found in aerial parts of vegetation within the district are small, ranging from a trace to 7.6 parts copper in 1,000,000 of dry matter and averaging 3.41 parts. Miscellaneous cultures in water, potted soils, and plots gave larger amounts of copper which, however, were associated in most cases with toxic effects. Table 30 (p. 462) contains a sum- mary of these data. Even allowing for errors of method and of analysis, the European figures (3) seem excessively high, although the woody character of most of the samples was for the most part very different from that of the tender crop plants of the Arizona series. Little can be said as to the toxic effects of the copper ob- served in aerial plant parts in the Arizona samples. The yellow striping of copper-poisoned corn is probably a general symptom of malnutrition to be attributed to the effect of copper upon root systems rather than upon leaves and stems. In rare instances, however, beans and squash in water culture showed dark green 12 Biochem. Jour., Aug., 1908, pp. 319-323. is IT. S. D. A. Bur. PI. Ind., Bull. 64, p. 43. 1* Jour. Ind. and Eng. Chem., Sept., 1909, p. 676. is See Bibliography, p. 487, references 3, 20, 21, 22. 462 University of California Publications in Agricultural Sciences [Vol. 1 TABLE XXX Summary of Copper Content of Aerial Vegetation Min. Max. Ave. No. of , A , samples Parts per million copper 1. Field vegetation from upper Gila.... 10 trace 7.60 3.41 Field vegetation from other sources in Arizona 9 none 6.30 1.52 2. Corn plants grown in pots .01-.05 per cent Cu 3 6.5 21.00 13.30 Tops of corn, beans and squash grown in Cu water culture 6 11.7 32.00 22.90 Tops of corn, beans, etc., irrigated with copper solutions 14.00 Beans in soils containing Cu 9 13.0 44.00 26.00 Squash ditto 5 14.0 61.00 39.00 Corn ditto 20 4.4 239.00 42.00 3. Field samples collected by Leh- mannifl 43 0 560.00 86.00 Field samples collected by Ved- rodi17 1894 26 40.0 1350.00 257.00 1895 26 10.0 680.00 151.00 patches that may possibly have been due to presence of copper, inasmuch a.s appearances of this character are sometimes noted as an effect of the application of Bordeaux mixture. Rain states, for instance, that extremely minute amounts of copper stimulate formation of chlorophyll in a cell, and therefore in- crease the formation of starch.18 Ewart, also, shows that solu- tions of copper a.s dilute as 1 to 30.000.000 prevent the action of diastase upon starch.1" It is possible, therefore, that the juices of plant tissues containing traces to 239 parts (observed) of copper in 1,000,000 of dry matter may carry sufficient of this amount in solution in the cell sap to hinder the action of enzymes upon starch, and thus prevent its normal translocation. 16 Der Eupfergehalt von Pflanzcn und Thieren in Kupfcrrcichen Gegen- den, Lehmann Archiv fur Hygiene, vol. 27, pp. 1-17, 1896. 17 Quoted in Brenchley, Inorganic Plant Poisons, p. 17, 1914. is Bain, Tenn. Agr. Exp. Sta., vol. 15, Bull. 2, p. 93, 1902. isEwert, Zeitschr. fur Pflanzenkrankh., vol. 14:3, p. 135, 1904. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 463 Amounts and Significance op Copper in Root Systems Of far more and unmistakable importance is the effect of copper on root systems of plants. Under all conditions, whether grown in water culture, in pots, plots, or as field crops, the root systems of plants contain much greater amounts of copper than do the aerial portions, as is shown briefly in the following con- densation of results : TABLE XXXI Summary of Copper Content of Tops and Eoots of Plants Cu in p. p.m. No. of , A x samples Tops Roots Ratio Coin, beans, and squash in water cul- tures, poisoned but living 3 22.00 103.00 1 to 4.7 Ditto— killed by copper 3 23.00 268.00 1 to 11.6 ( lorn grown in soil containing .01 per cent of Cu as Cu(OH)2CuC03 1 6.50 152.00 1 to 23 Corn grown in soil containing .025 per cent Cu as Cu(OH)2.CuC03 1 21.00 728.00 1 to 35 Corn grown in soil containing .05 per cent Cu as Cu2S 1 12.50 171.00 1 to 14 Bean series grown in soils containing Cu as pptd. carbonate .0025 to 1.5 per cent Cu in soil 9 26.00 1431.00 1 to 55 Corn series grown in soils containing Cu as Cu2S .01 to 1 per cent Cu in soil 7 51.00 702.00 1 to 14 Corn series grown in soils containing Cu as chrysocolla, .05 to 1 per cent Cu in soil 3 13.00 266.00 1 to 20 Corn series grown in soils containing Cu as pptd. carbonate, .0025 to .05 per cent Cu in soil 4 13.00 416.00 1 to 32 Excluding samples grown in water cultures, the roots of which were cleaned with 4 per cent HC1, probably with loss of some copper, the root systems of experimental cultures contained averages of from fourteen to fifty-five times as much copper as the aerial portions of the plants. Furthermore, fine roots of corn were found in one instance to contain about three times as much copper as coarse roots of the same sample, and, finally, the maximum amount of copper, as determined both by analysis and by observation, in water cultures, was found in the root tips 464 University of California Publications in Agricultural Sciences [Vol. 1 of plants affected by copper. Analyses of water cultures of corn, pea.s, and wheat showing slight toxic effects gave the following ratios of copper in tops, root systems, and root tips: Water cultures (u in p. p.m. showing slight , K v toxic effects Tops Roots exclusive of tips Root tips Corn 27.00 91.00 545.00 Peas 17.00 1400.00 3428.00 1680.00 Wheat 130.00 207.00 2811.00 The root tips in this material, by means of caustic potash (the biuret reaction) and potassium ferrocyanide. show the char- acteristic purple and dark-red reactions due to copper. In the former ease not only copper, but copper in combination with prol (ids, is indicated — the purple color being due to the biuret test, which identifies both copper and proteids simultaneously. Tn roots grown in water culture, and then subjected to the action of dilute copper solutions, the location of copper in a poisoned root system can he seen under a low power witli con- siderable exactness. The purple of the biurel test begins very definitely with the growing point of the root tip and fades out gradually in comet-like fashion usually within one or two millimeters distance of the tip. New growing points in process of pushing their way through the epidermis along the sides of the roots likewise give a strong but very local biuret reaction. This combination of copper (in the form of oxide) and proteids is one used for the precipitation of albuminoid nitrogen in chem- ical analysis of feeding stuffs.2" The amount of copper entering into the combination varies with proteids from various sources. As a rule, animal proteids combine with much less copper than vegetable proteids — averaging about 2.4 per cent of copper for egg albumin. Vegetable proteids combine with from 11.60 to 16.97 per cent of copper oxide and average 11.7 per cent cop- per.'1 Ordinarily, therefore, a vegetable proteid would he sat- urated with about one-ninth of its weight of copper; but its physiological activities are disarranged and the root killed by much less than the amount required to saturate the proteid. 20 See Bibliography, p. 488, reference 48. 21 Mann, Chemistry of the proteids, p. 305. 1917 I Forltts: Irrigation Effects of Copper Compounds Upon Crops 465 For instance, in samples of wheat and pea roots grown in water culture, it was found by means of nitrogen and copper deter- minations, using the factor 11.7 per cent copper for saturation of albuminoids, that in wheat roots 4.96 per cent of the copper required for saturation was present and in pea roots 7.99 per cent. It appears, therefore, first, that copper attacks plant proteids at the most delicate and vulnerable points in the whole plant organization — the growing points of the root systems; and, sec- ond, that a small proportion of the copper required for complete reaction is sufficient to kill the protoplasm at these points. Again, it is to be observed that, especially in the seedling stages of growth, the number of growing points is small so that only extremely minute amounts of copper are required to arrest the growth of root tips, the spread of root systems and the nutrition of the plant. Inasmuch, also, as plants vary greatly in the physical struc- ture and the physiological activity of their root systems, includ- ing the number, delicacy and absorptiveness of their growing points, it is not unlikely that the varying sensitiveness to copper salts of different plants, and of the same plant at different ages, may be explained by these observations. Corn, for instance, the most sensitive plant worked with, is characterized in its seedling stages by a small number of vigorously absorptive growing points. By means of the more delicate dark-red potassium ferro- cyanide test, copper may usually be traced through the vessels of the root systems for considerable distances, showing that it is through these channels that small amounts of the metal finally reach the steins and leaves. Here the maximum amounts of copper are found in the (inter and upper portions of the plant, where evaporation is most active, and where the greatest residuum of copper therefore occurs. The potassium xanthate (yellow) and hydrogen sulphide (brown) tests also reveal copper in root structures but are not so satisfactory for this purpose as potas- sium ferroeyanide. (See plate 9.) The above described reactions, which are so conspicuous in water-cidture material killed by copper, are very obscure or imperceptible in roots grown in soils containing copper. The 466 University of California Publications in Agriculture - oes [Vol.1 first material, however, is dead and more nearly saturated with copper: while living roots from soil culture, with proteids com- bined to bu1 a small per cenl of their capacity fur cupper, do not give satisfactory color tests. These reactions, therefore, do no1 serve for qualitative determinations of toxic effects in field material. Relations Between Amoi m- of Coppeb i\ Root Systi m- \m> Km BY l" Pi INTS An efforl tu establish relations between the amounts of copper in parts per million <>t' dry matter in pool systems, ami toxic effects as shown in the condition of aerial portions of the plant, was only partiallj successful; hut a sufficient number of observations on samples of sufficient size produced under care- fully regulated conditions would probably establish such rela- tions. In the tahles shown on the preceding pages there is a fair degree of agreemenl between the meml each experi- mental series, the copper found in root systems increasing in most cases with the amount of copper in the soils of each particu- lar series of cultures. Tn the case of beans and corn Lrro\\ n in cul- tures containing copper in the form of precipitated carbonate, beans show a somewhal higher resistance to toxic effects ami also contain larger amounts of copper in the pool systems throughoul the series. The conditions under which the samples were grown seem to have, within limits, more effecl upon the copper content of root systems than the amounts , • copper in the soil, as is indicated in the following tabular statemenl TABLE XXXII Toxii Concentrations op Copper in Soi ■ i: E bms Co r rool Points .it v, stem »1 poinl i m •'!' which to] • bowing point showing effi to* ffecta Culture p.p.m c r "i Corn. seven samples from field soils 12 :>t }r,<; Corn in field plots containing Cu as sulphate 04% 296 at .0595 Corn in pot cultures containing Cu as carbonate 023% 748 at .0 509 at .02595 Roans in pot cultures contain- ing Cu as carbonate 035% 950 at .02595 1358 at .0595 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 467 In this statement, for instance, field samples of corn roots grown in soil containing 0.07 per cent of copper contained only 42 p. p.m. of copper in dry matter, while a plot sample grown in soil containing .025 per cent of copper contained 245 p. p.m. of copper in dry matter, and corn grown in pot culture containing 0.02 per cent of copper in soil contained 748 p. p.m. of copper in dry matter. These differences may be due to the coarser root systems of plot and field-grown samples, this condition being associated with relatively small amounts of copper in dry matter. In view of the great labor involved in preparing root samples for analysis and the very variable results obtained from copper determinations made upon such material, there seems to be little hope of estab- lishing satisfactory ratios of copper to dry matter for the pur- pose of determining that a sample of field material has been injured by copper. It is probable, however, that for comparative purposes, pot cultures of field soils conducted under uniform and carefully regulated conditions, with standard plants of known behavior, may yield figures of comparative value in de- termining the character, toxic or otherwise, of a soil containing copper. Corn is an excellent summer-growing plant for the pur- pose, inasmuch as it shows toxic effects easily, grows rapidly, and affords abundant root materials for analytical determinations. For winter cultures, wheat serves well. Both plants are repre- sentative of standard crops for the region under discussion. Pathological Effects Pathological effects in tops and roots may confirm to a con- siderable extent, the fact that a plant has been poisoned by cop- per. The lengthwise yellow striping of corn and wheat leaves due to toxic amounts of copper is not distinctive since the same appearances may result from various other conditions in- ducing malnutrition, such as those mentioned on a preceding page. Usually, however, careful observation will identify or eliminate these other disturbing factors. Root systems grown in coppered soils are also conspicuously injured, being stunted in growth, of harsh and crinkly texture 468 University of California Publications in Agricultural Sciences [Vol. 1 and (in the case of corn) showing characteristic proliferated root tips. The epidermis is thick and rough and the cells in longi- tudinal tangential section contract from the oblong toward the circular form. Here, again, other factors, such as alkali salts in excess, may lead to similar appearances ; and these must be eliminated in a diagnosis of copper injury. Soil Conditions Relating to Toxic Effects of Copper upon Plants Certain conditions favor, others oppose the toxic action of copper under field conditions, the general tendency being to modify or do away with toxic effects, where the amounts of copper are not excessive. Carbon dioxide in the soil, alone and in conjunction with cer- tain salts (NaCl, Na2S04) tends to form solutions of basic cop- per carbonate. Carbonates (Na2C03,CaC03) lessen the solubil- ity of basic copper carbonate in carbon dioxide and, therefore, the toxicity of copper compounds in soils containing these carbonates.22 Coarse, sandy soils favor toxicity by permitting free move- ment of solutions and because the withdrawal in them of copper from solution by physical and chemical reactions is minimum.23 Tlie character of I In com pound of copper to which roots are exposed is important. In pot cultures of precipitated carbonate of copper, of sulphide in the form of chalcocite pulverized to go through a 100-mesh sieve, and of silicate in the form of chryso- colla pulverized to 100-mesh, toxic effects appeared with corn as follows : Pot culture of corn; Cu in form of pptd. carbonate — showing toxic effects at .023% Cu in soil Pot culture of corn; Cu as chalcocite, 100-mesh — showing toxic effects at .08% Cu in soil Pot culture of corn; Cu as ehrysocolla, 100-mesh — showing toxic effects at .08% Cu in soil The precipitated carbonate is not only more soluble in car- bon dioxide than in chalcocite, but also more easily acted upon 22 See Bibliography, p. 487, reference 12. 23 See Bibliography, reference 18. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 4(>9 by the acids of plant roots than chalcocite or, probably, ehryso- colla. Under field conditions, copper in tailings is originally mostly in the form of sulphides, chiefly chalcocite, which oxidizes only slowly to sulphate in presence of water and air. Chalcocite, 3.2 grams, shaken up with 600 c.c. of water, and air, for twenty- eight days, yielded only 16 mg. of soluble copper. The soluble sulphate in contact with silicates and carbonates of the soil is converted to insoluble forms. The process is gradual and the amount of soluble copper present at any one time is small. The tilth of the soil is significant. A pot culture very thoroughly mixed with 0.1 per cent of copper as carbonate re- sulted in badly poisoned plants containing about four times as much copper in root systems as in a lumpy mixture of soil con- taining the same amount of copper. The heavy tailings clay, with which copper is chiefly associated in the district studied, tends to remain in lumps and masses, thus minimizing toxic- effects of contained copper compounds. In water cultures toxic effects of copper salts are lessened by salts contained in well-water or in nutrient solutions. This is due, in part, to the presence of other ions, the effect of which is to decrease the ionization of copper salts, with consequent decrease in toxicity. This observation applies to soil-water solutions which contain considerable amounts of alkali salts. It is of interest in this connection to note that certain combinations of salts representing complete mineral nutrients exert maximum antitoxic action to copper salts ;24 and that therefore a fertile soil containing maximum amounts of plant nutrients will tend to minimize toxic effects of copper. Antagonistic solutions, so called, involving copper, may also account for a decrease in toxicity. By reason of a property of the semipermeable membranes of root systems, ions may be either more readily or less readily allowed to penetrate. When pene- tration is decreased through the addition of ions of other soluble salts this salt is said to be antagonistic in character. Copper is thus antagonized by sodium and potassium salts, of which the soluble salt content of the soil is chiefly composed.25 2* A. Le Eenard, Essai sur la valeur antitoxique de 1 'aliment complet et incomplet. Abstracted in Science n. s. vol. 28, no. 712, p. 236, 1908. 25 See Bibliography, p. 488, references 35-44, 52. 470 University of California Publications in Agricultural Sciences [Vol. 1 Physical attractions, or adsorptive effects, also account for a very considerable lessening of the amount of dissolved copper salts, in contact with soil particles. Jensen, for instance, finds that a dilute copper solution is ten times as toxic in the free con- dition as when it is mixed with an artificial quartz soil, that is to say, the quartz reduces the toxic effects about nine-tenths. In- asmuch as the reduction in toxicity is a function of the solid surface to which the soluble salts are exposed, the finer the state of division of a soil the more will be the adsorption and the less will be the toxic effects of a stated copper solution.26 The age of plant roots markedly affects their susceptibility to copper salts. Young and tender roots, containing large amounts of protoplasm, are much more quickly and easily poisoned than old and comparatively fibrous structures contain- ing a small proportion of protoplasmic materials. This may be due to differences in the thickness of cell walls protecting the cell contents from outside substances ; it may be due to a different degree of permeability of the protoplasm of older roots to copper salts; or it may be due to lessened reactivity due to changed chemical character. In any case, this observation indicates a distinctly greater resistance to copper in soils, of older, more fibrous, and possibly intrinsically more resistant root systems. Different species of plants also show varying degrees of resistance to copper salts. In pot cultures, peas are distinctly more re- sistant to precipitated carbonate of copper than corn. Different plants of the same species also show a certain amount of indi- viduality with reference to absorption of copper. Stimulation Not only do the various influences described above lessen the toxic effects of copper upon plants, but it is possible, also, that the amounts of copper may be decreased in the field to the point at which stimulating effects occur. As shown in the dis- cussion of water cultures on preceding pages, extreme dilutions of copper salts in distilled water, for instance, 1 part to 100,- 26 G. H. Jensen, Botanical Gazette, vol. 43, p. 11, Jan., 1907. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 471 000,000, caused increased growth of root tips growing in these solutions. This observation accords with those of some other experimenters, not only with copper solutions but with solutions of various other metals, and bears a certain analogy to stimulat- ing effects upon animals observed with very small amounts of poisons, such as arsenic and strychnine. Stimulation was also observed in the case of certain pot cultures watered with dilute copper solution in such a way that these solutions were filtered through a thin layer of soil before they reached the plant roots. Under these conditions a portion of the root systems must come in contact with extremely dilute copper solutions residual from the reactions of copper salts with the soil. As in the case of water cultures, these extremely dilute solutions must have ex- erted the stimulating effects which were apparent in several cultures made in this manner. In the case of pot cultures also, in which stated amounts of copper were uniformly mixed throughout the soil, apparent stim- ulation of growth was occasionally observed ; for instance, with 0.01 per cent of copper in the form of precipitated carbonate in a culture of corn. A satisfactory explanation of stimulation effects is not avail- able. It is to be supposed that stimulation in a soil culture in which copper sulphate is used may be explained by the action of the S04 ion upon the soil in releasing plant food for the use of the plant. However, such stimulation is seen in water cultures where this does not occur. Lipman27 has observed that under certain conditions the nitrifying flora of soils is stimulated by salts of copper, zinc, iron and lead. Such stimulation, through increased elaboration of nitrates, may account for the behavior of cultures showing increased growth. Stimulation effects, there- fore, which undoubtedly occur both in water and in soil cultures, are perhaps due to more than one different cause — to chemical and bacterial agencies in soils, and to a pathological disturbance in water cultures.28 Taking into account the very minute amounts of copper salts with which stimulated growth is associated, and the very gradual -' Lipman, C. B., and Burgess, P. S., Univ. Calif. Publ. Agr. Sci., vol. 1, no. 6, pp. 127-139, 1914. as See Bibliography, pp. 487-488, references 2, 4, 27, 33, 53, 45. 472 University of California Publications in Agricultural Sciences [Vol. 1 addition of copper to new ground that may occur through irri- gating waters, it is not impossible that in favorable situations an actual increase in vegetable growth in the field due to copper may take place ; but it is not possible in the field to prove this supposition because of many other factors, the effects of which prevent trustworthy observation. Field Observations In view of the many factors influencing results in the field, some leading towards toxic copper effects, some opposing toxic effects, and still others pointing to the possibility of stimulated growth, it is of interest, finally, to refer to field conditions as they have existed in irrigated lands under the Clifton-Morenci mines for the twelve years during which the district has been under observation. At the beginning of this period, in 1904, considerable accumulations of copper-bearing tailings were evi- dent, more particularly at the heads of alfalfa fields, where they sometimes attained a thickness of as much as ten inches or more. These blankets usually thinned out and disappeared between 100 and 200 feet from the head ditches, leaving crops in lower por- tions unaffected. Deposits of river sediments were observed in other irrigated districts not affected by mining detritus. The growth of alfalfa was more depreciated by the denser and thicker tailings blankets; and yellow foliage of young grain and young corn was considerably in evidence in tailings, but not as an effect of ordinary sediments. In 1908, the tailings were impounded, and some of the best farmers began the practice of cultivating alfalfa to break up the old accumulations, incorporate them with the soil, and secure better penetration of water and air to the roots of the crop. Following this procedure the stunted growth at the heads of alfalfa lands has considerably but not yet en- tirely recovered. Patches of yellow young barley, wheat, and oats are still to be observed on old tailings deposits; but as the plants become older they become normal in appearance, and yield apparently normal crops. These observations, which may be repeated many times in the course of a day's reconnaissance in the district, from May to September for alfalfa, and February 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 473 to May for grain, may be explained by the following consider- ations : The wedge-shaped deposit of tailings indicated in the diagram (fig. 15) at first so obstructed access of water and air to alfalfa root-systems that only stunted development was pos- sible either of roots or tops. With an annual cultivation of this blanket and the incorporation of river sediments and better penetration of irrigating waters, deleterious effects tend to dis- appear and the crop again approaches normal. Similar land when plowed for grain contains most of the copper associated with old tailings at the surface of the soil. Young grain, therefore, with shallow and susceptible root sys- irCiiti^---^ »^^ri^^^iA;j-'-.-v4ir Fig. 15. — Diagram showing behavior of root systems under influence of tailings blanket. terns, at first, if ever, shows effects of copper in the soil, recovering as root systems penetrate to greater depths where they encounter uncontaminated soil. Effects of River Sediments With reference to the further trend of copper effects upon vegetation in the district, assuming the permanent exclusion of solid tailings but a constant addition of about one part of copper to one million of irrigating water used, it is of interest to take into account the diluting effect of river sediments upon copper compounds in the district. In four acre-feet of Gila River water, these sediments will amount to about eighty tons per acre a year,29 of which amount the ten pounds of copper contributed in irrigating waters is only 0.006 per cent. -'» Forbes, R. H., Ariz. Agr. Exp. Sta. Bull. 53, p. 61. 474 University of California Publications in Agricultural Sciences [Vol. 1 Irrigating sediments alone, therefore, considered in their general relation to amounts of copper which cannot be prevented from reaching irrigated fields, are sufficient in quantity to re- duce ultimately the amounts of copper observed below 0.01 per cent in the soils of this district. Since 0.01 per cent is a safe minimum, river sediments, alone, incorporated with the soil are probably sufficient to ameliorate gradually existing accumula- tions of copper salts and to take care of further contributions in soluble form which cannot at present be avoided. Effect of Cultivation upon Alfalfa Finally, it is of interest to observe the improvement in a field of alfalfa, in the district studied, between the years 1!)0."> and 1916. June 23, 1905, the writer carefully measured, cut and weighed a representative plot of alfalfa in William Gillespie's field near Solomonville, Arizona. This field was suffering from an accu- mulation of tailings, the depreciation in yield at the upper ends of alfalfa lands being conspicuously evident. Following the exclusion of tailings from the irrigating supply in 1908, and witli a cultivation each winter witli a disk or a spring-tooth harrow, the condition of the field gradually improved until, June 13, 1916, the writer returned and again measured, cut, and weighed the identical plot of alfalfa that had shown bad effects eleven years before. Following are the data, with diagrams, relating to these two cuttings of alfalfa, which are representative for the district within which tailings were deposited. 1. Alfalfa seriously affected b>/ taiUngs, Jinn 23, 190J. Three lands in William Gillespie's field east of house, near Solomonville, under Montezuma Ditch, out of Gila River. A good stand of alfalfa five years old. Heavy adobe soil; field never disked. The three lands observed were, over all, 95 feet wide, and divided into plots 100 feet long from top to bottom of field. Ten feet next the ditch was discarded because of banks and bare spots, and the extreme lower portion of the field because of roadways. A portion of plots 6 and 7 was discarded on account of Johnson grass. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 475 Observations were made June 23, 1905, on the second cutting, just beginning to bloom, the field having been irrigated twice since the last cutting. After stirring and raking, the yield of dry hay was weighed June 24. Weather very hot and dry. Fol- lowing are the data relating to this series : Plot Dimen- sions in feet Height of alfalfa, inches Yield of plot, pounds Tons per acre Depth of tailings on plot, inches Condition of surface soil at time of cutting 1 95x100 19 240 .69* 1*-3| Dust-dry and somewhat cracked 2 95x100 20 340 .87* 1 -2 Dry and badly cracked 3 95x100 23-25 570 1.31 £-H Dry, cracked at upper end 4 95x100 24 595 1.36 l-i Moist, not cracked 5 95x100 23 550 1.26 f-i Moist, not cracked 6 60x100 28 400 1.41* *-i Moist, not cracked 7 60x100 27 430 1.48* i- I Moist, not cracked * Corrected for thin stand and trash. 2. Alfalfa slightly affected by tailings, June 13, 1916. The same three lands, continuously in alfalfa since 1905. A perfect stand, thin spots reseeded by means of a seed crop in 1915. The field had been spring-tooth harrowed each winter for about ten years, especially at heads of lands, to break up the tailings blanket and secure better penetration of irrigating water. As in 1905, ten feet next the ditch was discarded, also the extreme lower portion of the field. Johnson grass had nearly entirely disappeared. Observations were made June 13, 1916, on the second cutting, just beginning to bloom, the field having been irrigated twice since the last cutting. After raking and piling, the dry hay was hauled and weighed June 17. The weather was moderately hot and dry; and conditions generally the same as those under which the crop was cut in 1905. Following are the data for the second series of observations: 476 University of California Publications in Agricultural Sciences [Vol. 1 Plot Dimen- sions in feet Height of alfalfa, inches Yield of plot, pounds Tons per acre Appearance of tailings Condition of soil at time of cutting 1 95x100 36-21 875 2.00 Distinct Surface dusty, drier soil 2 95x100 22-34 857 1.96 Distinct Surface dusty, drier soil 3 95x100 34-36 972 2.22 Slight Moist throughout 4 95x100 30-36 910 2.08 None Moist throughout 5 95x100 31-33 900 2.05 None Moist throughout 6 95x100 28-36 860 1.96 None Moist throughout 7 95x100 34-37 870 1.98 None Moist throughout 8 95x100 33-36 910 2.08 Nunc Moist throughout Comparing these two statements, and illustrating them by means of the following diagram (fig. 16), it is evident that the depreciation in yield observed in the upper plots in 1905 has dis- appeared in 1916, the yields on the last date being practically uniform from top to bottom of the field. Effects of tailings are still plainly visible in plots 1 and 2 in spots and patches of short alfalfa, compensated for, however, by areas of stimulated growth apparently due to seepage from the adjacent ditch. The yield of the field as a whole is also much improved due to cultivation and reseeding of the field. In brief it may now be stated that, following the exclusion of tailings from the irrigating waters of this locality, it has been found possible, in this carefully observed case, to overcome the deleterious effects of tailings deposits upon alfalfa, slowly but almost entirely, in about ten years. Thus, co-operation between miners, in restraining tailings from irrigating streams, and those farmers who cultivate their alfalfa intelligently, effectually disposes of the most serious prob- lem that has arisen in connection with copper-mining detritus. The chemical composition of tailings, in fact, would indicate that, as in the case of humid region subsoils, when they arc en- riched by the addition of organic matter and nitrogen, and filled with bacterial life, they may make very good soil. Following is a statement of the composition of four representative samples of ores and tailings, with reference to potash and phosphoric acid : 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 477 P Hs 6 S < c S5 ^, cd B i_. "• 5 as co CD QTO CO p" o &P S EL CD Hi M ri «C l-1 S3" « CD - r+ — £ - r- Hi O 1/2 o ~ r-t~ - o a. — . p p j: — 5! TJ T> — 13 a ?r p CD a p To C -. — r-f — O Ha < '-■ CD ~ <: jo - cc CD t* — - ■c Pi O oo o ■"* Hi < < V y vo Hi — I — - - — CD CO P W CO Ditch n Discard — t 00 H 95 Co H CO CD H ro H 4^ GO .— . . — EOo1 On +^cr>03oro-NcT)ooo5 OOOOOOOOOOo:, =< TJ ro CO Id o CJ1 \ \ \ \ \ \ \ 1 \ 1 j 1 0 1 01 1 CD n ^ Discard I 4 __ 5< z ^0O 5 o ><= 9 tv> 2 £ 3 CD H 95' Co ro -c to rn ro r o H o ■n J> *> i- ■n > ro r o "n CD > H ~D r~ £ H fn c_ ro c o z en m H Co rn _u CD Lo CD H -4 lo CO H CO ro o Oo H Discard 478 University of California Publications in Agricultural Sciences [Vol. 1 Sample No. Potash K,0 Phosphoric acid PA Nitrogen N 3491 Sulphide ore .64% .11% Doubtful 3492 Oxidized ore .44 .11 3438 Sulphide tailings .79 .29 traces 3439 Oxidized tailings .67 .12 These ores and the tailings derived from them are rich in potash, and contain unexpectedly large amounts of phosphoric acid ; but nitrogen is almost nil. SUMMARY 1. Copper is shown, as a direct effect of the Clifton-Morenci mining operations in Arizona, to be distributed throughout water- supplies, soils, the vegetable and the animal life of an under- lying irrigated district. 2. Smaller amounts of copper are found elsewhere in the State where the drainage basin includes mining operations or ore-bearing areas. 3. Individual plants grown in water cultures or in soil con- taining copper show a comparatively small, and probably not injurious, accumulation of copper in the aerial portions of the plants ; but the root systems, carefully cleansed of externally adhering copper, contain relatively great amounts. 4. Copper in root systems, as shown by the biuret test, is largely in combination with plant proteids, especially at the growing points of root systems and near vicinity. The place and nature of the reaction accounts for the extreme toxicity of copper salts to plants. The varying sensitiveness of plants to copper salts may possibly be explained in part by the number and disposition of exposed growing points. 5. Conditions favoring toxicity of copper compounds are the presence of carbon dioxide and certain soluble salts which assist in forming copper solutions that come into contact with plant roots ; coarse, sandy soils favoring free access of copper solutions to plant roots and minimizing the withdrawal of copper from solution by adsorption ; and the presence of copper in the form of the more soluble precipitated carbonate. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 479 6. Conditions opposing toxicity of copper compounds are the presence of copper in the form of chrysocolla and chalcocite ; adsorption through contact with finely divided soil particles ; reactions with carbonates, silicates, and organic matter tending to precipitate copper from its solutions ; the presence of certain soluble salts in the soil that overcome toxic action; and increased resistance of old plant roots. 7. The stimulation by copper of vegetative growth in pot and water cultures has been observed. Stimulated growth of crops under field conditions is a possibility. 8. Pot cultures may be used for comparative determinations of toxic effects upon plants of copper in soils, if conducted under rigidly uniform conditions. The copper content and the physio- logical response to copper of such material will be much greater than for similar cultures grown under plot or field conditions. 9. Copper injury in field soils containing doubtfully toxic amounts of copper may be diagnosed by a combination of symp- toms. Facts which indicate such injury in a soil containing 0.] per cent of copper (more or less) are: yellow tops (for winter grains) in absence of other conditions that cause yellow tops; crinkly root systems (in absence of excessive amounts of alkali salts) ; and a high copper content in dry matter of root systems. Combined evidence of this character, which may be observed in the district studied, indicates toxic copper effects. 10. Field observations before and following the exclusion of tailings from the irrigating water-supply indicate that conditions in the district studied are gradually improving, due to the culti- vation of alfalfa and to the incorporation of river sediments with accumulations of tailings. Noticeable toxic effects in the field exist only where the roots of young, growing crops are exposed to surface soils containing maximum amounts of copper. The general tendency in the district is probably toward decreasing rather than increasing percentages of copper in irrigated soils. 11. Methods of analysis have been developed for the purpose of determining reliably small amounts of copper in vegetative material, particularly in root sj^stems of plants grown in soils containing copper. 480 University of California Publications in Agricultural Sciences [Vol. 1 Part III.-APPEND1X METHODS OF ANALYSIS With the Collaboration of E. E. Free and Dr. W. H. Ross Freedom of samples, especially vegetation, from contamina- tion with adhering copper; and accurate methods for determin- ing minute amounts of copper in sediments, soils, waters and vegetation, are vital to the integrity of the work recorded in this publication. Unusual care was taken to perfeel methods for preparation of samples, especially roots grown in media containing copper; and refined manipulation in the determination of copper reduced the limit of error to approximately .00001 gram, or .01 milligram. Reagents and Apparatus Distilled water of three derivations was used: | 1 | University of Arizona well water very slowly distilled through a block-tin worm; (2) the same, redistilled from glass; and (3) University of Arizona well water distilled from glass. Nitric (ihd sulphuric acids from Baker & Adamson were used. Ammonia and II s employed were passed through two wash bottles. Blank determinations from time to time with reagents em- ployed gave no trace of copper, thus insuring results obtained by means of them. Copper was determined by electrolysis, in minute amounts according to the manipulation of E3 E. Free The balance used was a No. 2112 Eimer and Amend short- beam assa\ balance, "distinctly sensitive to 1 200 milligram." .M VXIITI.ATIOX Ores and ladings. — 1-2 gms. were digested with a mixture of 8 Co.. II.\<>. and :» c.c. IK'I on a hot plate, then 1 c.c. II so, :il\<\<>\ and evaporated to H2S04 fumes (method used in old Dominion laboratory at Globe, and Copper Queen at Bisbee). Took up with water, tillered, neutralized with ammonia, then added 2 c.c. II so, and a \'cw drops of Il\o and electrolyzed. Soils. — Soils were examined by two methods; (a) 100 gms. soil was treated with a mixture of 80 C.C UNO and 20 c.c. II, SO, and digested in a porcelain dish on a hot plate to sulphuric fumes; digested with 200 c.c. water, filtered, washed "1» to about 500 r.r.. evaporate, | to '-'nil c.C. precipitated iron with ammonia, filtered, washed with about odd c.c. water, alkaline filtrate reduced by evaporation, acidified faintly with IK'I and H,S passed for half an hour. The faint black precipitate was i Electrolytic, determination of minute quantities of copper 12th (Jen meeting Am. Electrochem. Soc, October 17 19, L907. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 481 allowed to settle several hours, then filtered, and the precipitate, including filter, digested with 5-10 c.c. HN03 and water until copper was dissolved, solution filtered, a few drops of H2S04 added, evaporated to fumes, and copper determined by elec- trolysis with addition of 5-25 drops of HNO,. (b) 200 gms. soil was digested as above with HNO, and H2S04, evaporated to fumes of H2S04, digested with water, filtered and washed up to 500 c.c, made alkaline with ammonia and made up to 1000 c.c. After settling, 500 c.c. or 100 gms. aliquot, was filtered off and copper determined as in (a). Waters. — Waters were evaporated to dryness, the residue digested with sulphuric acid and water, filtered hot, excess of H2S04 evaporated, filtered into platinum dish, a few drops of HNO, added, and electrolyzed. Vegetation. — Air-dried samples were burned in a small sheet- iron stove, the iron of which was found to contain no trace of copper. Two samples of mistletoe, difficult to burn, were reduced in a new muffle in gasoline assay furnace. The charred and partly burned material was moistened with water, and concen- trated HNO, added (100 to 200 c.c.) until effervescence ceased, digested until in plastic condition, diluted with hot water and filtered. Evaporated bulky filtrate to dryness, took up with water and HNO.,, filtered (getting rid of much organic matter), added about 20 c.c. H,S04, evaporated to H2SO+ fumes, driving off all but about 5 c.c. H2S04, added water, filtered off insolubles, made up filtrate to about 500 c.c, passed H2S, and proceeded as usual for copper. The completeness of the extraction of copper from vegetation by the above method was verified as follows: The extracted. charred residue from 2 lb. 8 oz. of dry corn leaves and blooms in which 1.32 parts Cu per million was found (Sample 3529) was removed from filter paper after washing, moistened with H2S04 and additionally burned in a porcelain dish, being finally reduced, after again moistening with H2S04, in a platinum dish in the muffle. The resulting pink ash was then fused with three parts of dry Na2CO, (Kahlbaum) and poured on clean porcelain. The fusion was soaked in water with addition of H2S04, evapor- ated nearly to dryness, filtered from insoluble portion (lime, salts, etc.), again evaporated and filtered, and a third time the same, finally driving off excess of H.SO, and electrolyzing as usual. A black precipitate of carbon but no Cu was obtained, the same being true of a blank determination on the Na2(JO, used. Roots of plants grown in water cultures or in soils must be most thoroughly cleansed of externally adhering copper, since this will introduce excessive errors where the content of copper is small. Three methods of preparing roots for copper determina- tion were employed : 1. Roots grown in water cultures containing copper were dipped for about ten seconds in 4 per cent HC1, immediately 482 University of California Publications in Agricultural Sciences [Vol. 1 washed in copper-free water and dried. Careful observation in- dicated that adhering copper salts deposited from water solution were completely removed by this treatment. It is probable that the acid penetrates plant tissues somewhat in the time employed and removes some copper. The results are, therefore, probably severely conservative. 2. Roots grown in soil cultures containing copper cannot be safely cleansed with HC1, which does not readily dissolve silicates and sulphides of copper, and which cannot be allowed to remain in contact with plant roots for more than a few seconds. Carbon dioxide in water was finally selected as a mild, slow but finally effective solvent for the purpose. Samples of roots were first very thoroughly washed in copper-free well-water, then placed in five-liter jars with ground glass covers, a stream of washed C02 passed, the jars shaken and treatment with C02 repeated until the water was saturated, then allowed to stand witli occasional shaking for twenty-four hours. The solution was then siphoned or filtered off and the treatment repeated until, on evaporating the bulky filtrates, no more copper was found. To prevent putrefaction during long-continued washings, a pinch of thymol was added to each washing. From nine to thirty-one washings were found necessary to cleanse plant roots thoroughly, the process being laborious and time-consuming. When the sample yielded no more copper to wash waters it was dried, burned and copper determined according to the method for small amounts in plant ashes. Following is a record of washings for examples of roots cleaned by this process: (1) Corn roots grown in a pot culture of soil containing 0.01 per cent of copper as basic carbonate. Quantital h e bj U2H test electrolysis First wash distinct Fifth wash distinct Ninth wasli doubtful 1 liter of filtrate no Cu (2) Corn roots grown in a pot culture of soil containing 0.05 pei- cent copper as Cu2S. Quantitative by electrolysis Tenth wash 2 litres of filtrate .00006 gm. Cu (3) Barley roots from field soil containing tailings. Quantitative bj electrolysis First wash 2.433 litres of filtrate .00035 gm. Cu Second wash 2.531 .00012 Fifth wash 2.22 .00009 Sixth wash 2.41 .00004 Seventh wash 2.00 .00002 Eleventh wash 2.00 .00000 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 483 (4) Coarse roots of field corn grown in soil containing tail- ings. H2S test Quantitative by electrolysis Twenty-fifth wash distinct Twenty-ninth wash .00005 gra. Cu Thirty-first wash .00000 Samples vary as to number of washings required to remove the last trace of copper, but the definiteness with which, finally, copper usually ceases to be extracted by C02 water indicates completeness of the operation. This is further emphasized by the comparatively large amounts of copper which are then found in root systems thus cleansed. 3. A third method of preparing roots for copper determin- ation, involving less labor than by washing in C02 water, is as follows : Cleanse roots thoroughly in clean water with a camel- hair brush, dry, burn and weigh the ash, then estimate total copper. Determine copper in soil shaken from sample, assume ash as all soil and deduct copper in this amount of soil from total copper found in ash. Results by this method are low, but not seriously in error if sample is thoroughly washed. Pts. Cu Dry per Example matter Ash Gms. Cu million .Sample 2a grown in soil containing 0.05% copper .3561 gm. 10.84% .000115 322 Ash in sample .0386 Copper in ash as- sumed as soil .000019 Net copper assumed .000096 270 The correction introduced reduces parts per million of copper from 322 to 270, which latter figure is conservative in character. Of the three methods above described, No. 2 is undoubtedly most exact, but is extremely laborious and time-consuming. THE DETERMINATION OF COPPER IN SMALL AMOUNTS OF PLANT ASHES The ash is placed in a platinum dish without previous pulver- ization and moistened with concentrated sulphuric acid in suf- ficient quantity to bring all parts of the ash in intimate contact with the acid. The material is then thoroughly stirred and heated on a sand bath until fumes of SO< begin to come off, then allowed to cool and a sufficient quantity of hydrofluoric acid added to bring the acid in contact with the whole mass, then allowed to stand for at least half an hour and again heated until 484 University of California Publication* in Agricultural Sciences [Vol.1 S03 fumes come oft'. The material is now washed into a casserole, moistened with sulphuric and nitric acids and digested at a low heat for at least one hour. The heal is then increased until S03 fumes are again driven oft. The mass is moistened with three to four times its bulk <>!' distilled water and digested at a gentle heat from one to two hours, filtered hot and then the lil- trate and washings evaporated almost to dryness, thus driving oft the exeess of sulphuric acid. The resulting residue is taken up with hot water and again filtered to separate the solution from precipitated calcium sulphate. This evaporation and filtration may have to he repeated one. two or three times in order to gel the solution sufficiently free from calcium sulphate. The final filtrate, which contains the copper, is then diluted to about 1">I) to 200 c.c. in a tall beaker, a small quantity of hydrochloric acid is added and hydrogen sulphide passed until the solution is thoroughly saturated. During the hydrogen sulphide precipi- tation there should he no nitric acid or nitrates presenl in the solution. A large quantity of organic matter is also disadvan- tageous and may be avoided by evaporating the solution several times to dryness with nitric and sulphuric acids, finishing finally with an evaporation with sulphuric acid alone in order to drive off all t races of nit ric acid. Tlie precipitate from the treatment with hydrogen sulphide is filtered off. washed with water saturated with hydrogen sulphide and digested with a small quantity - to 5 C.C. of nitric acid in a casserole. The digestion should he begun cold and the heal era dually increased. If the digestion is begun at a high tempera ture the sulphur formed by the decomposition of the copper sulphide will form a film of molten sulphur around the granules of copper sulphide, and this tends to prevent their solution in nitric acid. The precipitate after digestion in nitric acid should he a clear green or else a yellow. If there is any trace of dark Color, brown or black, it means that either organic matter has been precipitated with the copper sulphide precipitate, which is extremely unlikely, or else that the above-mentioned sulphur film has formed around some of the particles of copper sulphide preventing their solution in the nitric acid. If the latter he the case, the determination may still he saved h\ placing the precipitate in a platinum dish and heating over a gentle flame until the sulphur is volatilized. The residue of copper sulphide or of copper oxide may then he digested in nitric acid. The digestions in nitric acid should not he carried to a heat high enough to decompose the copper nitrate Formed by the solution of copper sulphide. After digestion in nitric acid and the evaporation of an\ large excess of nitric acid, the residue is taken up in hoi wafer, acidified to contain 2 4 per cent nitric acid and filtered into a large platinum dish. • , to V2 c.c. of sulphuric acid is added. and the solution elect rolvzed with a voltage of from 2 to L" 9 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 485 volts and a current not greater than one ampere. The voltage may be higher than 2i/> volts if necessary but should not be high enough to raise the current beyond the limit given. The elec- trolysis should be continued at least three hours and preferably nine to twelve hours. The dish is, of course, the cathode. When the electrolysis is complete the electrolyte is washed out of the dish by means of the sucking-bottle and the dish is thoroughly washed with distilled water-. In ease the deposit of copper on the dish is spongy and loosely adherent it is not safe to wash out the electrolyte. In this case the copper should be redissolved and the electrolysis repeated, using a little more sulphuric acid. If the copper still refuses to come down in adherent form the addition of 2 to 5 c.c. of a one per cent solution of gelatine will often assist the precipitation. In ease of a stubborn refusal of the copper to give an adherent deposit it is necessary to dissolve it, evaporate to dryness with sulphuric acid, and reprecipitate with hydrogen sulphide, continuing the process from this point as before. If the copper refuses to come down at all the trouble is probably an excess of acid in the solution. This may be corrected by the addition of a few drops of ammonia. The concentration of acid in the solution must lie between one and five per cent. At least a small part of this should be sulphuric acid as nitric acid will be destroyed in the course of the electrolysis if it alone is present, and the solution may become alkaline (from NH4OH), which will prevent proper precipitation. Chlorides and organic salts, such as acetates and tartrates, should be carefully avoided. The resulting deposit of copper will probably contain traces of carbon and possibly of platinum. In order to eliminate these and at the same time precipitate copper upon an electrode more suitable for accurate weighing, a second electrolysis is made, using this time the dish as anode and using as cathode a small spiral of platinum wire suspended from a hook of silver (or platinum) wire which in turn is connected to the battery. The electrolysis should also be conducted in nitric and sulphuric acid solution and what is said above as to obtaining satisfactory deposits applies with equal force here. In this case, however, owing to the small surface area of the cathode, it is necessary to work with very much smaller currents than were used in the first electrolysis. The maximum current to be used must be so adjusted by trial as to give bright and adherent deposits. l-100th ampere and 1.8 volts is a good current for the purpose. It is well to use as the source of current for this electrolysis four Edison- Lalande cells and to have in the circuit a resistance of from 30 to 80 ohms. This gives an electromotive force at the dish of about 1.8 volts. Two determinations may be run in parallel. In this case it is not permissible to use a gelatine solution in order to secure satisfactory deposits, as the copper will be slightly contam- inated with gelatine and the obtained weight will be too high. 486 University of California Publications in Agricultural Sciences [Vol. 1 The electrolysis should be run at least nine hours. When com- pleted, the electrolyte should be washed out as before without breaking the current, the electrode lifted from the solution, dis- engaged from the supporting hook, and washed and dried by dipping successively in water, alcohol and ether and placing in a desiccator over sulphuric acid. After having remained in the desiccator for an hour the electrode is ready for weighing. Weighings should be made on an assay (button) balance adjusted to maximum sensibility. After weighing, the copper is removed from the electrode by dipping in concentrated nitric acid, and the electrode cleaned and dried by dipping successively in dis- tilled water, alcohol and ether and placing in a desiccator. It is again weighed as before and the difference of the two weights gives the copper obtained. The electrolyte (from each electrolysis) which has been washed out of the dish by means of the suction flask, is evapor- ated to dryness taken up with water, acidified with nitric acid and tested for copper by electrolyzing, using the point of platinum wire as cathode. In this way any possible loss of copper by incomplete precipitation in either of the electrolyses is prevented. If any copper is found in this check test it should be dissolved from the platinum wire, added to the solution ob- tained by dissolving the copper from the small electrode, and the electrolysis repeated in order to get the true weight. In case a quantity of copper too small to be weighed is obtained its identity as copper may be most easily established by electrolyzing it onto the point of a platinum wire as described above. In these electrolyses with the platinum wire as cathode the current must, of course, be kept low in order to obtain satis- factory deposits. If this precaution is observed the deposit on the platinum wire will be of a brilliant red color and easily dis- tinguishable as copper. If the deposit is brownish or blackish its identity as copper may be established by the green flash when the point of the wire is held in the colorless flame of the Bunsen burner, particularly if the wire has been first dipped in hydro- chloric acid. Nitric acid must not be used, as nitric acid itself will give a green flash in the Bunsen burner flame The reagents used in the above process should all be tested as to freedom from copper. The water used should be doubly distilled and, at least the second time, from glass. All utensils should be cleaned by boiling in nitric acid. Care must also be taken to conduct the operations in rooms free from dust which might possibly contain copper. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 487 BIBLIOGRAPHY 1 AderhoM, K., Zur Frage der Wirkung des Kupfers auf die Pflanze. Ber. deut. bot. Ges., vol. 24, p. 112, 1898. 2 Bain, S. M., The action of copper on leaves, Tenn. Agr. Exp. Sta., vol. 15, Bull. 2, 1902. 3 Barker, B. T. P., and Gimingham, C. T., The action of Bordeaux mixture on plants, Ann. Appl. Biol., vol. 1, no. 1, pp. 9-21, May, 1914. * Brenchley, W. E., Influence of copper sulphate and manganese sul- phate upon the growth of barley, Ann. Bot., vol. 24, no. 95, p. 571, 1910. 5 Brenchley, W. E., Inorganic plant poisons and stimulants, pp. 15-35. (1914.) 6 Burrill, T. J., Effect of copper on bitter-rot spores, Illinois Exp. Sta. Bull. 118, p. 569, Sept., 1907. 7 Clark, H. W., and Gage, S. DeM., On the bactericidal action of copper, Jour. Infect. Dis., Supplement no. 2, Feb., 1906. s Collier, Peter, Influence of copper compounds in soils upon vege- tation, N. Y. Agr. Exp. Sta. Bull. 41, pp. 35-43, April, 1892. 9 Coupin, H., Sur la toxicite des sels de cuivre a l'egard des vegetaux superieurs, Compt. rend., Acad. Sci., vol. XCCVII, p. 400, 1898. io Devaux, H., Fixation of metals by cell membrane (Tr.), ibid., vol. 138, p. 58, 1901. ii Ewert, Dr., A chemical-physiological method for determining small amounts of copper in extreme dilution (Tr.), Zeitsch. f. Pflanzenkrankh., vol. 14, p. 135, 1904. i2 Free, E. E., The solubility of precipitated basic copper carbonate in solutions of carbon dioxide, Jour. Am. Chem. Soc, vol. 30, no. 9, p. 1366, Sept., 1908. is Free, E. E., The electrolytic determination of minute quantities of copper, Proc. Am. Electrochem. Soc, Oct. 17-19, 1907. 14 Freytag, Dr., Die schadliche Bestandtheile des Huttenrauchs der Kup- fer- Blei- und Zink-Hutten und ihre Beseitigung, Landw. Jahrb., vol. 11, 1882. 15 Haselhoff, E., Concerning the injurious effect of water containing copper sulphate and copper nitrate upon soil and plants (Tr.), Landw. Jahrb., vol. 21, p. 263, 1892. is Hattori, H., Studies upon the effect of copper sulphate upon certain plants, Jour. Coll. Sci., Univ. Tokio, vol. 15, pt. Ill, 1901. 17 Havwood, J. K., Injury to vegetation and animal life by smelter wastes, U. S. D. A., Bur. Chem. Bull. 113 (revised), July, 1910. is Jensen, G. H., Toxic limits and stimulation effects of some salts and poisons in wheat, Bot. Gaz., vol. 43, p. 11, Jan., 1907. 19 Kirchner, O., ttber die Beeinflussung der Assimilationstatigkeit von Kartoffelpflanzen durch Bespritzung mit Kupfervitriol Kalkbriihe, Zeitschr. f. Pflanzenkrankh., vol. 18, Heft 2, p. 65, 1908. 20 Kraemer, Henry, The copper treatment of water, Am. Jour. Pharm., vol. 76, no. 12, p. 574, Dec, 1904. 21 Kraemer, Henry, The use of metallic copper for the purification of drinking water, ibid., vol. 78, no. 3, p. 140, March, 1906. 22 Kraemer, Henry, The use of copper in destroying typhoid organisms, and the effects of copper on man, ibid., vol. 77, no. 6, p. 265, June, 1905. 23 Lehmann, H. B., Der Kupfergehalt von Pflanzen und Thieren in kupferreichen Gegenden, Archiv. fiir Hygiene, vol. 27, p. 1, 1896. 2* Lipman, C. B., and Wilson, F. H., Toxic inorganic salts and acids as affecting plant growth, Bot. Gaz., vol. 55, no. 6, p. 409, 1913. 488 University of California Publications in Agricultural Sciences [Vol. 1 25 Lipman, C. B., and Burgess, P. S., The effect of copper, zinc, iron and lead salts on ammonification and nitrification in soils, Univ. Calif. Publ. Agr. Sci., vol. 1, no. 6, pp. 127-139, March, 1914. 27 Livingston, B. E., Chemical stimulation of a green alga, Contr. N. Y. Bot. Garden, no. 63, 1905. '-'8 Long, J. H., The physiological significance of some substances used in the preservation of food, Science, n.s., vol. 37, no. 950, p. 401, March, 1913. -"■' Luckey, Use of copper sulphate in fertilizer, U. S. P. 838036, 1906. so Miani, D., uber die Einwirkung von Kupfer auf des Wachsthum lebender Pflanzenzellen, Ber. deutsch Bot. Ges., vol. 19, p. 461, 1901. 31 Michigan Acad. Sci., The toxic action of copper sulphate upon cer tain algae in the presence of foreign substances, Seventh Report, p. 48, 1905. 32 Moore, Geo. T., and Kellerman, Karl F., A method of destroying or preventing the growth of algae and certain pathogenic bacteria in water supplies, U. S. D. A., Bur. PI. Ind., Bull. 64, May, 1904. ss Ono, H., Tiber die Wachsthumsbesehleunigung einiger Algen uud I'il/e durch chemische Reize, Jour. Coll. Sci. Univ. Tokio, vol. 13, p. 141, 1900. 34 Otto, R., Investigations concerning the behavior of plant roots towards solutions of copper salts (Tr.), Zeitschr. f. Pflanzenkrankh., Bd. ::. 1S93. ■"•■"' Osterhout, W. J. A'., The decrease <>f permeability due to certain bivalent kations, Bot. Gaz., vol. 59, no. 4, p. 317, April, 1915. 36 Osterhout, W. J. V., Extreme alterations of permeability without injury, ibid., vol. 59, no. 3, p. 242. 37 Osterhout, W. .1. Y., Protoplasmic contractions resembling plas molvsis which are caused by pure distilled water, ibid., vol. 55, no. 6, p. 146, June, 1913. 38 Osterhout, W. J. A'., Quantitative researches <>n the permeability of plant cells, Plant World, vol. 1(5, p. 129, May, 1913. 3» Osterhout, W. J. V., Permeability of protoplasm to ions and the theory of antagonism. 40 Osterhout, W. .1. V., On the nature of antagonism, Science, n.s., vol. 42, no. 1050, p. 255, February, 1915. 41 Osterhout, W. J. V., Extreme toxicity of sodium chloride and its prevention by other salts, Jour. Biol. Chem., p. 363, March, L906. *2 Osterhout, W. .1. Y., Plants which require sodium, Bot. Gaz., vol. 54, no. 6, p. 532, December, 1912. i3 Osterhout, W. J. Y.. Organization of the cell with reaped to per meability, Science, n.s., vol. 38, no. 977, p. lux, September, 1913. ** Osterhout, W. J. Y.. Vitality and injury as quantitative concep tions, ibid., vol. 40, no. 1031, p. 488, October, 1914. '■'• Porchet, F., and Chouard, 10., De I'action des sels de cuivre sur les vegetaux, Bull, de In Muriethienne, vol. 33, 1905. 46 Phillips, F. C, Absorption of metallic oxides hv plants, Chem. News, vol. 46. p. 22 1, March, 1882. 47 Reed, H. S., Abstract of Essai sur le valeur antitoxique de I 'aliment complet et incomplet, by A. l>e Renard, Science, n.s., vol. 28, no. 712, August, 1908. is Ritthausen, II., Verbindungen der Proteinstoffe mit Kupferoxyd, Jour, f. prakt. Chem., Bd. 5, p. 215, 1872. »'' Ritthausen, II., Verbindungen ricv Eiweisskorper mit Eupferoxyd, ibid., Bd. 7, p. 361, 1873. so Rumm, Ueber die Wirkung der Kupferpraparate, etc., Ber. dent. Bot. Ges., vol. 11, p. 79, 1893. '•' Schander, R., Ueber die physiologische Wirkung der Kupfervitriol Kalkbriihe, Landw. Jahrb., vol. 33, p. 517, 1904. •"^Stiles and Jorgensen, [., Studies in permeability, Ann. Bot., vol. 29, no. 115, p. 349, July, 1915. 1917] Forbes: Irrigation Effects of Copper Compounds Upon Crops 489 53 Stockberger, W. W., Effect of some toxic solutions on mitosis, Bot. Gaz., vol. 49, p. 416, June, 1910. r>4 Sullivan, E. C, Precipitation of natural silicates, Econ. Geol., vol. 1, no. i, p. 67, 1907. 35 Vedrodi, V., Der Kupfer als Bestandtheil der Sandboden und unserer Kulturpflanzen, Chem. Zeitung, vol. 17, p. 1932, December, 1893. r,,i Viala, On the action of certain toxic substances upon the vine (Tr.), Revue de Viticulture, nos. 3 and 5, 1894. r,T Spaeth, B. A., The vital equilibrium, Science, n. s., vol. 43, no. 1110, p. 502, April, 1916. PLATE 6 Fig. 1. — Eoot system of corn plant injured by 0.1 per cent of copper added as copper sulphate to the soil. Fig. 2. — Normal corn root grown in similar soil containing no copper. (Photos by G. F. Freeman.) [490] < CD EC o OJD o > o CO of CD < GD r> GL < but) . PLATE 7 Fig. 1. — Individual roots of corn injured by 0.1 per cent of copper added as copper sulphate to the soil. Fig. 2. — Individual root of corn, normal. (Photos by G. F. Freeman.) [492] UNIV. CALIF. PUBL. AGR SCI. VOL. 2 [FORBES] PLATE 7 Fig. 1 Fig. 2 I ♦ » PLATE 8 Fig. 1. — Thickened rootlets and proliferated root tips of corn injured by 0.1 per cent of copper added as copper sulphate to the soil. (X 3 diam.) Fig. 2. — Fine roots and root tips of corn, normal. ( X 3 diam.) (Photos by G. F. Freeman.) [494] UNIV. CALIF. PUBL. AGR. SCI. VOL 2 [FORBES] PLATE 8 Fig. Pie. 2 PLATE 9 Corn root-tips killed in a solution of 1 part copper to 100,000 of water, and colored by means of (1) caustic potash, which gives the violet biuret reaction, identifying both copper and protein; (2) hydrogen sulphide, brown; (3) potassium xanthate, yellow; and (4) potassium ferrocyanide, red. ( X ± 30 diam.) UNIV. CALIF. PUBL. AGR. SCI. VOL [FORBES] PLATE 9 !