ABSTRACT THE BASIS FOR ENHANCED PHYTOTOXICITY OF ATRAZINE-PHOSPHATE COMBINATIONS by Charles Frederick Stolp This study confirmed the occurrence of an inter- action between atrazine (2-chloro-4-ethylamino—6— isopropylamino-gftriazine) and phOSphate which produces a synergistic reduction in plant growth not due to in- creased herbicide uptake or altered mineral composition. The objective of this study was to find the basis of this interaction. Of the five crop species examined, corn (§§a_may§ L.), peas (Pisum sativum L.), sorghum (Sorghum vulgare L.), and soybeans (Glycine max Merril) showed greater reduction in plant growth following atrazine treatment if high levels of phosphate were also present in the root treatment solution. Only in the case of peas was the high level, 10—2 M phosphate, in the treatment solution significantly phytotoxic by itself. Barley (Hordeum 1 Charles Frederick Stolp vulgare L.) did not show the atrazine-phOSphate combina- tion effect evident in corn, peas, sorghum, and soybeans. The enhanced phytotoxicity of atrazine in the presence of phosphate appeared to be related to a syner- gistic increase in respiration of corn, peas, sorghum, and soybean plants receiving the combination treatment. The atrazine induced inhibition of photosynthesis in these species was not synergistically enhanced by the additional presence of phosphate. Although the trends were not statistically sig— nificant at the 5% level, at the 10—5 M atrazine level, increasing levels of phosphate tended to reduce total atrazine uptake during the treatment period. For corn, peas, and soybean this trend was reversed at the 10'.4 M atrazine level. The percent of chloroform—soluble metabolites, including the parent atrazine, increased with increasing atrazine and phosphate levels in corn and peas. If this effect were of sufficient magnitude it could explain en— hanced phytotoxicity. The presence of phosphate with the atrazine treatment reduced the metabolism of atrazine to a methanol-insoluble residue and increased the 2 Charles Frederick Stolp accumulation of hydroxylated metabolites in corn, peas, and soybeans. It is difficult to envision how this ef- fect could explain the altered phytotoxicity unless the assumptions on the phytotoxicity of these metabolites are erroneous or this block in metabolism also affected the rate of conversion of atrazine to hydroxylated meta- bolites. There did not appear to be any effect of phos- phate on atrazine metabolism by sorghum. Atrazine did not affect the uptake or distribu- tion of 32P by corn. The enhanced phytotoxicity of atrazine in the presence of high levels of phosphate can best be explained by an interaction on the rate of reSpiration. The in— creased respiration could be due to an interaction effect on the reSpiration apparatus or by an increase in the internal concentration of atrazine resulting from a slight increase in uptake and a slight decrease in metab— olism of atrazine in the presence of high levels of phosphate. These alternatives are not mutually exclu- sive. THE BASIS FOR ENHANCED PHYTOTOXICITY OF ATRAZINE-PHOSPHATE COMBINATIONS BY Charles Frederick Stolp A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1970 ACKNOWLEDGEMENT The author wishes to greatfully acknowledge the assistance of Dr. Donald Penner in the conduct of this study and in the preparation of the manuscript. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENT . . . . . . . . . . . . . . . . . . ii LIST OF TABLES. . . . . . . . . . . . . . . . . . . V LIST OF FIGURES . . . . . . . . . . . . . . . . . . Vii INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3 szriazine Herbicide Selectivity. . . . . . . . 3 §7Triazine Herbicide Action . . . . . . . . . . 6 Metabolism of §7Triazine Herbicides . . . . . . 15 §7Triazine Herbicide-Phosphate Interactions . . 19 MATERIALS AND METHODS . . . . . . . . . . . . . . . 23 Culture Technique and Growth Measurements . . . 23 Measurement of Respiration and Photosynthesis . 25 Extraction of l4C—Atrazine and Its Metabolites . . . . . . . . . . . . . . . . . 28 Separation and Identification of l4C-Atrazine and Its Metabolites . . . . . . . . . . . . . 3O Assay of Atrazine Effect on 32P Uptake of Corn. 32 iii TABLE OF CONTENTS (Cont.) Page RESULTS AND DISCUSSION. . . . . . . . . . . . . . . 36 Atrazine—Phosphate Effects on Plant Growth. . . 36 Atrazine-Phosphate Combination Effects on Plant Respiration and Photosynthesis. . . . . . . . 45 Atrazine—Phosphate Combination Effects on Atrazine Uptake and Metabolism. . . . . . . . 54 . 2 . . . AtraZine Effect on 3 P Uptake and Distribution in Corn . . . . . . . . . . . . . . . . . . . 68 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 72 LITERATURE CITED. . ... . . . . . . . . . . . . . . 75 iv Table 10. 11. LIST OF TABLES Atrazine-phOSphate combination effects on soybean growth. . . . . . . . . . . . . Atrazine-phosphate combination effects on barley growth . . . . . . . . . . . . . . Atrazine-phosphate combination effects on sorghum growth. . . . . . . . . . . . . . Atrazine—phosphate combination effects on pea growth. . . . . . . . . . . . . . . . Atrazine—phosphate combination effects on corn growth . . . . . . . . . . . . . . . Atrazine—phOSphate combination effects on reSpiration and photosynthesis of corn. Atrazine-phosphate combination effects on respiration and photosynthesis of pea . . Atrazine—phOSphate combination effects on respiration and photosynthesis of sorghum Atrazine-phosphate combination effects on respiration and photosynthesis of soybean Atrazine-phosphate combination effects on atrazine uptake and metabolism in corn. . Atrazine-phOSphate combination effects on atrazine uptake and metabolism in pea . . Page 40 41 42 43 44 50 51 52 53 61 62 LIST OF TABLES (Cont.) Table 12. 13. 14. 15. 16. 17. 18. Atrazine—phosphate combination effects on atrazine uptake and metabolism in sorghum Atrazine—phosphate combination effects on atrazine uptake and metabolism in soybean Atrazine-phOSphate combination effects on the composition of the chloroform-soluble metabolite fraction of pea. . . . . . . . Atrazine-phOSphate combination effects on the composition of the chloroform-soluble metabolite fraction of sorghum. . . . . . Atrazine-phosphate combination effects on the composition of the chloroform-soluble metabolite fraction of soybean. . . . . . The effect of assay procedure on 14C-atrazine recovery during extraction procedure. . The effect of atrazine on 32P uptake. . . . vi Page 63 64 65 66 67 68 71 Figure LIST OF FIGURES Page Atrazine metabolism in higher plants as postulated by Lamoureux §E_§l, (l7). . . . 20 Plastic plant chamber used in CO analysis . 27 32P treatment of corn in aerated solution culture for uptake and distribution study. 34 2 Atrazine-phosphate combination effects on the growth of 7-day-old corn . . . . . . . 37 The effect of atrazine on 32P uptake of corn plants grown in 10"2 M phosphate. Autoradiograph on left shows plants which received atrazine treatment at 10“4 M. Autoradiograph on right shows plants which received no atrazine . . . . . . . . 69 The effect of atrazine on 32P uptake of corn plants growh without phosphate. Autoradiograph on left shows plants which received atrazine treatment at 10-4 M. Autoradiograph on the right shows plants which received no atrazine . . . . . . . . 7O vii INT RODUCT ION There have been several reports of interactions between herbicides and phosphate. One of the best docu- mented is a synergistic reduction of plant growth by atrazine (2-chloro—4—ethylamino—6-isopropylamino—sf triazine) and phosphate (l,2,9,32). Atrazine is a widely used herbicide of great economic importance. It is often applied in a band at the same time as fertilizer application resulting in the close proximity between the herbicide and fairly high concentrations of phOSphate fertilizer. Since it has been shown that atrazine and phosphate in the same medium may have a synergistic effect, it is possible that early seedling growth is reduced. If the reduction was limited to weed species, this could enhance weed control in the rows; but if the crOp species was also affected, it would be advantageous to alter the fertilization or weed con- trol program. With the present emphasis on environmental qual— ity, plans for recycling nutrients found in wastes have 1 been suggested. The high phosphate content of some of these wastes could result in situations where a phosphate— herbicide interaction could occur. It is important to know the basis of this inter- action in order to reduce a potential loss of herbicide selectivity or to eXploit the interaction by applying less herbicide and reducing herbicide residue. It is the purpose of this work to study several systems which could be causal factors of the interaction. LITERATURE REVIEW §7Triazine Herbicide Selectivity Most of the studies on the selectivity of g: triazine herbicides indicate that uptake and transloca— tion of the herbicides have little effect on their selec- tivity. Moreover, the sensitivity of isolated chloro— plasts to triazines measured as inhibition of the Hill reaction is found to be the same for tolerant and sensi- tive Species (21). The ability of tolerant plant species to metabolize the toxic compounds to a non-toxic form appears to account for their tolerance. Using the present knowledge about triazine action and metabolism, Shimabukuro and Swanson (28) have devel- Oped a model for selectivity. The site of triazine action is believed to be the chlorOplast. §7Triazine metabolism is thought to occur in the cytOplasm. The chlorotriazines are very lipid soluble and can readily penetrate the lipid-rich chlorOplasts of both tolerant and susceptible Species. Atrazine present in the leaves 3 of treated plants will enter the chlorOplasts and accu- mulate until an equilibrium exists with atrazine in the cytOplasm. Hydroxylated derivatives, however, are lipid insoluble so they are excluded from the site of herbicide action. Shimabukuro and Swanson (28) have shown that when leaf tissue of sorghum was treated with 14C-atrazine, the percent of label in the cytOplasm increased with time as did oxygen evolution. In order to maintain the equil- ibrium of atrazine between chloroplasts and cytOplasm, atrazine was removed from the site of action as the com— pound was metabolized in the cytoplasm. Plants subse- quently recovered photosynthetic ability. It is not known if the hydroxylated atrazine de- rivatives have the molecular structure necessary for in- hibiting the Hill reaction. But since these compounds may not reach the site of action this is of little conse- quence. The literature is not without some controversy regarding gftriazine herbicide selectivity. Wheeler and Hamilton (34) eXposed wheat, corn, and sorghum to atrazine in solution culture. Following prolonged treatment, they extracted the plant material and determined the amount of unaltered atrazine spectrOphotometrically. Tolerant species were found to accumulate leaf concentrations of herbicide which were comparable to those found in sensi- tive species at the point of acute toxicity. This sug— gested that more is involved in selectivity than simply detoxication. They offered the hypotheses that corn leaves may bind the herbicide at inactive sites within the corn leaf tissue or prevent a particular series of reactions which mediate the acute toxicity. Even though the tolerant Species did not Show acute toxicity symptoms, a severe growth inhibition was observed. Preliminary observations indicated that photo— synthesis was also inhibited in these Species. If this is true, it might Simply be an overloading of the detoxi- cation mechanism. The "tolerant" species were not behav— ing as tolerant plants. They differed from the suscep- tible species only in the degree to which they Showed acute toxicity symptoms. Their data in (34) Showed that wheat accumulated 9.0 ppm atrazine from a 1.0 ppm solution in 6 days. Corn and sorghum accumulated 4.0 ppm and 9.5 ppm reSpec— tively in 20 days from a 10.0 ppm solution. These data suggested active accumulation in wheat versus exclusion and/Or metabolism by corn and sorghum. Their work did not seem to exclude metabolism to non—toxic compounds as the primary basis for triazine selectivity. §7Triazine—Herbicide Action Extensive work has been reported in the litera- ture on the action of the triazines (11,13). Most of the work has been done on simazine (2—chloro-4,6-bis (ethylamino)-§ftriazine) and atrazine, the two compounds most widely used in weed control, although the action of other triazines has been studied (11,13). Since the action of the gftriazines, particularly the chlorinated triazines, is generally the same, their action, selectivity and degradation can be considered as a group. It is generally accepted that the §ftriazines inhibit the photolysis of water depriving the chlorOphyll system of electrons necessary for photosynthesis. This inhibition of the Hill reaction has been studied Spectro- photometrically as change in absorbance with the reduction of potassium ferricyanide or Janus Green. The simazine concentration necessary for 50% inhibition (I50) of this reaction on isolated chlorOplastS was 7 X 10—7 M (11). The chlorophyll concentration in the solution was much higher than the herbicide concentration. Moreland and Hill (21) using simazine found that at the I the 50' molar ratio of chlorophyll to herbicide was 5.3. This value was much higher than the ratios for most of the other inhibitors they tested. They suggest that sima— zine is fairly Specific in its action. However Since not a great deal is known about the photolysis of water, the exact molecular Site of §7triazine action remains unknown. Related to the loss of reducing power is the increase in fluorescence observed when photosynthesis inhibitors like the triazines are added to chlorophyll. The fluorescence results from the release of energy not captured by the carbon—reduction cycle. The pF 50 agrees fairly well with the pI determined using the 50 decreased rate of oxygen evolution. The §7triazines have also been shown to inhibit non—cycic photOphosphorylation, but the cycic photophos— phorylation system was not inhibited (ll). Uncoupling occurred only at triazine concentrations 100 times greater than that necessary to inhibit the Hill reaction. A consequence of the inhibition of the Hill reac— tion is the inhibition of the reduction of NADP. The production of NADPH as well as ATP by the photosynthetic system is indiSpensable for the subsequent assimilation of C02. It is easy to measure this decrease in CO2 assim- ilation by isolated chloroplasts or intact plants in the light. Labeling studies with 14CO2 Show that 14C incor- poration into sucrose in the presence of triazine herbi— cides is severely inhibited. Zweig t 21, (37) found that 99% of photosynthetic COz—fixation could be blocked by atrazine in excised bean leaves. The triazines did not, however, inhibit dark fixation of C02. Zweig (36) found no effect on PEP-carboxylase-catalyzed COZ-fixation. Zweig _t__l, (37) Showed no effect of atrazine on the formation of aspartic or glutamic acids and other com— . . . 14 pounds assoc1ated Wlth the TCA cycle. Comparing C - labeling of these compounds, they found no difference between their dark control, atrazine in the dark, or atrazine in the light. Couch and Davis (8) obtained similar results. In one case (18) simazine treatment of Norway Spruce (Picea abies) at 20 ppm stimulated photofixation of 14CO2 but one would expect that the site of action for this effect was not at the level of the photo reactions themselves. Several studies indicate that the triazine herbi— cides do not affect carbohydrate metabolism, particularly the enzymes involved in starch synthesis. Ashton _t.al. (4) showed that cessation of growth of atrazine-treated algae (Chlorella vulgaris) could be overcome by the addi— tion of exogenous glucose. This, plus the fact that glucose can prevent the atrazine—induced starch loss from plant cells, indicates that atrazine does not prevent the use of this exogenous energy source. Sucrose can also prevent starch loss (11). This is a fairly strong indi- cation that atrazine acts at the level of monosaccharide synthesis without affecting subsequent carbohydrate metabolism. Several studies have involved examination of atrazine-treated plants with an electron microsc0pe. Changes in the fine structure of barnyardgrass chloro- plasts have been observed by Hill §t_§l, (16). Ultra- structural changes occurred before any macrosc0pic changes were evident. They could be seen after 2 hours of treat— ment with 2, 5, 10, and 20 ppm of atrazine. The degrada— tion of the chloroplast began as swelling of the 10 inter—granal fret system followed by swelling and disrup— tion of granal discs. In the more advanced stages of degradation, the membranes of the grana and chloroplast envelope were ruptured. They also observed a decrease in the number of starch grains present in the treated cells as treatment time exceeded four hours. Mitochon— dria appeared normal throughout the experiments, irre- spective of concentration or length of treatment. Ashton §£_al, (5,6) made Similar observations using bean leaves but a 30—hour delay period was some— times necessary before changes were evident. The aforementioned studies were designed to Show triazine herbicide effects on light systems. Ashton (3) using Kanota oats and red kidney beans studied the rela— tionship between light and the toxicity symptoms of atra- zine. Toxicity symptoms develOped in the light but not in the dark and the degree of acute toxicity was Shown to be a function of light intensity and quality. The action Spectrum of atrazine injury correSponded to the absorption Spectrum of chlorophyll, indicating that this pigment was involved in the expression of toxicity symp- tims. Ashton concluded that light was necessary for the expression of toxicity symptoms and suggested that atrazine ll toxicity was not due to the compound p§£_§§ but rather due to a secondary substance formed by some mechanism involving atrazine and light. Ashton (3) and Sweetser and Todd (30) came to similar conclusions for monuron (3-(prchlorophenyl)-l,l—dimethylurea). Ashton _§_§l, (5,6) suggested that a free radical might mediate the toxicity symptoms while Sweetser and Todd (30) thought there might be an accumulation of a toxic photosynthetic intermediate. No further evidence has been produced to support either hypothesis. Several other effects of the triazines have been reported. Atrazine and simazine inhibited the growth of tobacco callus tissue in the dark, and in a medium con- taining sucrose (11). The ISO was 10-6 M, almost 20 times the concentration necessary to inhibit photosyn- thesis. Such results could explain why phytotoxicity is observed with some tissues in the dark. Ebert and Mfiller (11) suggest atrazine might affect plant hormones but the possibility that the action could be an enzymes involved in tissue growth should not be excluded. Ries _£._l, (23) have studied the effect of sima- zine on protein and nitrogen metabolism. Rye was grown at 22°/l7°C or l7°/12°C day/hight temperatures. At 12 optimum concentrations of simazine, protein accumulation increased to levels as high as 79% above controls. There was no change in protein quality. Treated plants Showed a slight increase in respiration with no change in respir- atory quotient (R0). The dry weight of treated plants was somewhat lower than for the control plants although the fresh weights were similar. Nitrate reductase ac- tivity increased in treated plants. The above observa- tions held for plants grown on nitrate but not for plants receiving ammonia as the nitrogen source. The magnitude of the response decreased as the nitrate concentration approached the optimal nutritional level. Pea plants grown in simazine contained 40% more protein in the seeds than non—treated plants. In Similar studies, Tweedy and Ries (31) found an increase in the efficiency of nitrate utilization at low nitrate concen- tration and low temperature in treated versus non-treated plants. Ebert and Van Assche (12) worked with soybean callus tissue grown on agar which contained kinetin. Atrazine increased growth measured as an increase in fresh weight over controls. The optimum range of concen— tration was 10-8 to 10_18 M. 13 §7Triazines have also been reported to affect the Size of leaves and the diameter of the stems. Darker leaves as a result of higher chlorophyll and nitrogen have also been observed. In some cases, treatment can delay senescence. These observations led Ebert and Van Assche (12) to study the effect of atrazine on auxin. They reasoned that if atrazine inhibited peroxidase ac- tivity, IAA degradation would be inhibited. Enhancement of peroxidase activity on the other hand would result in lower IAA levels. They incubated decapitated oat coleOp— tiles for 9 hours in atrazine solutions of various con- centrations, then transferred the coleoptiles into fresh . . . 14 14 treatment solutions which contained IAA—l- C. CO2 was trapped over a 14-hour period and counted as a mea- sure of enzyme activity. Peroxidase activity was in- —6 —8 . —10 creased at 10 to 10 M atraZine. Between 5 x 10 -21 . . . . . and 0.5 x 10 M atraZine, the range in which auXin—like effects were observed, enzyme activity was inhibited. While it is very difficult to imagine significant biolog- ical activity at herbicide concentrations less than one molecule per milliliter, it is possible that the effects might be real in the higher range of concentrations. 14 Good (13) studied several inhibitors of the Hill reaction in order to correlate activity with chemical structures. Although he felt a more diverse group of triazines needed to be studied before activity could be reliably predicted from molecular structure, he did make two tentative generalizations. First, chlorine at the 2-position resulted in a more active herbicide than a methoxy group substitution. Secondly, the presence of two imino hydrogens gave greater phytoxicity than the presence of only one, which in turn resulted in greater activity than none at all. Moreland and Hill (21) ob— tained Similar results. They concluded that the chlorine and imino hydrogens were important for spacial orienta- tion. They found no apparent correlation between water solubility and herbicidal activity. Solubilities were 5, 8.6, 10, and 3200 ppm in water for simazine, propazine (2-chloro—4,6-bis(isopropylamino—gftriazine), chlorazine (2—chloro-4,6-bis(diethylamino)-§ftriazine), and methoxy— simazine (2—methoxy—4,6-bis(isopropylamino)-§7triazine) respectively. The order of activity is simazine > prOpazine > methoxysimazine > chlorazine. 15 Metabolism of §7Triazine Herbicides The most extensive studies on the metabolism of triazine herbicides have been done on atrazine and sima— zine. The first metabolites to be isolated and identified were the hydroxylated derivatives of simazine and atrazine, 2—hydroxy—4,6-bis(ethylamino)—§ftriazine and 2-hydroxy-4- ethylamino—6—isoprOpylamino—gftriazine, respectively (20). This conversion was found to be non-enzymatically cata— lyzed by the cyclic hydroxamate, 2,4—dihydroxy-3-keto-7- methoxy-1,4—benzoxazine (benzoxazinone). Montgomery and Freed (20) cited evidence of fur— ther breakdown of the triazine molecule. They reported that 14CO2 was given off by corn plants treated with 14C- _atrazine or l4C—Simazine although they could not Show this with labeled prometryne (2—methylmercapto-4,6-bis (isoprOpylamino)—§ftriazine) or propazine. They proposed a metabolism scheme whereby hydroxylation at the 2- position was followed by cleavage of the ring to form 14CO2 and a substituted biguanide. The biguanide was then thought to be hydrolized to form a biuret or a sub- stituted guanidine and a substituted urea. 16 Hamilton (14) studied the tolerance of plant species to the chloro—gftriazines in relation to their benzoxazinone content. He found that the ability of ex— cised roots to metabolize triazines to their hyroxy de- rivatives was directly related to their benzoxazinone content. Of the species tested rye, corn, and wheat con— tained benzoxazinone but only corn was tolerant. Sorghum, another tolerant species, contained no benzoxazinone. Clearly, non-enzymatic conversion of chloro-sftriazines to their hydroxy derivatives could not completely explain selectivity. Following a report that the soil fungus, Asper- gillus fumigatus, Fres, was able to dealkylate simazine, Shimabukuro_;§__l. (27) isolated and identified metabo— lites of atrazine from mature pea plants, Pisum sativum. Employing infra-red spectroscopy and thin layer chroma- tography they identified the compound 2-chloro-4-amino- 6—iSOpropy1amino—grtriazine (Compound I) and designated 2—chloro—4—amino-6-ethylamino—Sftriazine as Compound II. Another metabolite in addition to hydroxyatrazine was detected but was not identified. In a subsequent series of papers Shimabukuro and his co-workers (l7,25,26,27,28,29) further elucidated l7 atrazine metabolism. Compound I was found to be a major atrazine metabolite in both the roots and Shoots of pea. Only a small amount of atrazine was metabolized to water- soluble and methanol—insoluble compounds. Hydroxyatrazine was detected after 48 hours indicating that very little metabolism beyond dealkylation occurred in this period of time. The herbicidal action of Compound I was compared to atrazine. No difference at 10_7 and 10"6 M concentra— tions was observed but at 10"5 M, Compound I was less toxic as measured by the reduction in root and Shoot dry weight. The ISO for Compound I, measured as inhibition of the Hill reaction on isolated pea chloroplasts was 4.6 x 10-5 M or 23 times greater than the ISO for atrazine at 2.0 x 10.6 M (28). Shimabukuro concluded that accumula- tion of this less toxic atrazine derivative was respon- sible for the intermediate sensitivity of pea plants to atrazine. Shimabukuro (25) next studied atrazine metabolism in corn, sorghum, pea, wheat, and soybean. He found that all of these could metabolize atrazine by Nfdealkylation. Corn and wheat, because they contained benzoxazinone could also convert atrazine to hydroxyatrazine. Subsequent 18 metabolism resulted in the formation of more polar com- pounds and other compounds found in the methanol-insoluble residue. Contrary to the results of Montgomery and Freed (20) there was no evidence for cleavage of the triazine ring. Susceptible species contained more unaltered atra- zine than the tolerant ones. In sorghum, a tolerant Species, atrazine content decreased with time. The in— soluble residue increased with time at the expense of atrazine and unidentified water soluble compounds. Shimabukuro (26) compared the metabolism of atra— zine in corn and sorghum. Sorghum, as Shown before (25), could metabolize atrazine by Nfdealkylation but in this experiment, Compound II was also isolated. Corn formed both Compounds I and II in addition to hydroxyatrazine. Two additional water-soluble metabolites were also iden— tified. These were 2—hydroxy—4—amino-6—iSOpropylamino- §ftriazine (Hydroxycompound I) and 2-hydroxy—4-amino—6— ethylaminojs-triazine (Hydroxycompound II). These could be formed either by Nrdealkylation of hydroxyatrazine or by hydroxylation of Compound I or II. Shimabukuro and co—workers (17) have subsequently identified a new pathway for atrazine metabolism in l9 sorghum involving the formation of §f(4—ethylamino-6- isoprOpylamino-Z—sftriazino)glutathion and y-L-glutamyl- Sr(4-ethylamino-6—isoprOpylamino—Z—§ftriazino)-L-cysteine. Two additional water—soluble metabolites were formed after treatment for extended periods of time but they have not yet been identified. The most complete scheme presently available for atrazine metabolism is Shown in Fig. 1 (17). Montgomery §t_§l, (19) have Shown that hydroxy— simazine is metabolized to Hydroxycompound II. §7Triazine Herbicide— Phosphate Interactions Several workers have observed that high levels of phOSphate caused increased phytotoxicity of several herbi- cides. Upchurch _§_§1, (32) studied 12 herbicides and found statistically significant herbicide—phOSphate inter- action for diuron [3—(3,4—dichlorophenyl)-l,l—dimethyl urea], amitrole (3—amino—gftriazole), CDAA (Njgfdiallyl- 2—chloroacetamide) and simazine. Adams (1) suggested that the herbicide simazine reduced the amount of phosphorus required to cause salt toxicity. Dhillon §§_§l, (9) found that simazine at 20 ppm 20 /OH 0H 0H N N (CH 3)ZCHNH NHC2H5 (CH3)2CHNH KN/UNHZ 112 N J: IJIHIHCZHE ‘) T T Cl Cl N¢’*\CN NJ¢4\\N (CH3)2CHNH‘K NJ NH2 HgN ”KN/“NH‘EHS “K\\\ N5¢Si\N ////a (CH 3)ZCHNH K H} NHCZHS u///(At:azine) o o «H 9H lo! HO-C-CH2-CH2-C-N-CH-C-N-CH2-C-OH CH 82 \ s .r H” N (CH3)2CHNH LQENl/J NHC2H5 ? J. \ o o 0 \ ll NH I HO-C-CH-CHZ-CH2-C-N-0H-é-0H ‘ \ 0H N s 4¢*\. N N H . r (CH5)2CHNH JUN ) NIICZHS Fig. l.--Atrazine metabolism in higher plants as postulated by Lamoureux gt a}, (17). 21 decreased the rate of phosphorus uptake and accumulation in roots and subsequently increased its translocation from roots to stem and needles of red pine seedlings. More recently Adams (2) studied the effects of phosphorus and atrazine treatments on the mineral compo- sition of soybeans. He concluded that phosphorus appeared to increase the sensitivity of soybeans to atrazine. Doll §t_al, (10) studied the influence of herbi- cide and phosphate combinations on the root absorption of amiben and atrazine. They concluded that the herbicide- phosphate interaction they observed was not due to in- creased uptake of either of the herbicides in the presence of phOSphate by roots of corn, soybean, squash, or pig— weed. Penner (22) found that inhibition of phytase ac— tivity by amiben and atrazine was enhanced by high but not phytotoxic levels of phosphate in the culture medium. Herbicide—phosphate interactions could result from any one of several possibilities. AS Adams first sug— gested (l) the herbicide could make the plant more sen— sitive to phosphate salt toxicity. Conversely, as he has later concluded, phOSphate could make the plant more sen— sitive to herbicide injury. The possibility that the 22 herbicide affects the uptake of phosphorus or vice versa has already been investigated and does not appear to ex- plain the interaction. Effects on the status of other nutrients have also been studied and there seems to be little need to pursue that alternative further. If phosphate is affecting the herbicide phyto- toxicity it could do so either by acting at the site of herbicide action in the plant or by influencing the fate of the herbicide itself in the plant. This latter pos— sibility could be particularly significant in those cases where the basis of selectivity is detoxication by tolerant plant Species. The examination of these two possibilities is consistent with the main objective of the thesis, namely to determine the basis for the atrazine-phosphate interaction. MATERIALS AND METHODS Culture Technique and Growth Measurements Corn (Zea mays L., var. 400-F26), soybeans (Glycine max Merril), va. Hark and barley (Hordeum vulgare L. var. Larker) were planted, ten seeds per cup, in washed quartz sand. The sand was placed in 6 oz. styrafoam hot cups with drainage out the bottom. These cups were in turn placed within 10 oz. wax cups with drain holes cut in the side to maintain a constant level of solution when filled to run-out. Treatment solutions were added every two days for the first several days and daily when necessitated by higher rates of transpiration. All plants in the study were grown in controlled environment chambers. Light intensity at the top of the cups was.2000 foot candles from mixed fluorescent and in— candescent sources. The photoperiod was 16 hours of light and 8 hours of darkness. The temperature was held con- stant at 30°C for corn and soybeans and 20°C for barley. 23 24 The experimental design in the study determining atrazine-phosphate interaction on corn, soybean, and barley growth was a 4 x 4 factorial. There were two replications and the experiment was repeated a second time. Atrazine concentrations were 10-4, 10-5, 10—6 and 0 M. Phosphate as an equimolar mixture of mono- and dibasic potassium phosphate was added at 10_2, 10-3, 10-4, and 0 M. Solutions containing phosphate were na- turally buffered at a pH of 6.8. The other solutions were adjusted to a pH of 6.8 to 7.0. No additional nu- trients were added to the solutions. Plants were harvested after 7, 8, and 9 days for corn, barley, and soybean, respectively. At this age, growth inhibition by several treatments was evident by visual observation. The plants were removed from their cups and the sand was washed from the roots. Roots and shoots were separated and the remains of the seed or, in the case of soybean, the cotyledons, were removed and discarded. Total Shoot height was measured and the plants counted. Root and shoot were oven dried separately and weighed. Peas, Pisum sativum L., var Progress No. 9 and sorghum, Sorghum vulgare L., var. Forage Sorghum Hybrid, 25 were planted in a 3 X 3 factorial design and grown at 20 and 30°C, respectively. The lowest concentrations of . . —6 —4 atraZine and phosphate, i.e., 10 M and 10 M respec- tively, were eliminated. A modified Hoagland's No. 1 nutrient solution without phosphate was added to all solutions. Pea and sorghum plants were harvested at 14 and 8 days respectively. There was a total of four rep- lications of peas and five of sorghum. Measurement of Respiration and Photosynthesis Atrazine—tolerant corn and sorghum, intermediately susceptible peas, and susceptible soybeans plants grown as previously described were used in this study. Two repli— cations of each species were treated in a 3 x 3 factorial design. Concentrations of atrazine, phosphate, and nu- trients were the same as for peas and sorghum. In addi— . 14 . . . tion, C-atraZine was added to those solutions which contained unlabeled atrazine. The solutions contained 14 . . . 5pc of C—atraZine per liter. A small aliquot of each treatment solution was counted so all of the final data could be corrected for concentration differences. 26 Plants were grown 7, 8, 10, and 14 days for corn, soybeans, sorghum, and peas respectively. On the day of harvest, respiration and photosynthesis was determined for the plants in each cup using a Beckman Infra—red Analyzer Model IR 215. The experimental apparatus consisted of an Open- flow system which was Operated at a constant flow rate (500 ml/min) measured at the exit port of the analyzer. Air from a compressed air tank passed through tygon tub- ing into the controlled environment chamber where it entered the plant treatment chamber (Fig. 2). This chamber was made from clear plastic pipe, sealed on both ends. The pipe was cut near the base and the base fitted with a Sleeve. The union between the two parts of the chamber was sealed with masking tape. After passing over the plant, air moved through tygon tubing to the outside of the growth chamber. It was dried by passing it through tubes of CaSO4 dessicant which was changed as necessary. The dried air was then analyzed for CO2 content. The apparatus was calibrated by zeroing with ni- trogen. Half scale deflection was adjusted to compressed air passed directly through the analyzer. The difference 27 Fig. 2.——Plastic plant chamber used in CO analysis. 2 28 between 0 and half-scale deflection was assumed to be 0.3%.C02. Distances of deflection on the chart were determined as ppm per unit and the data was calculated as ug CO per min per unit area or weight. 2 After respiration and photosynthetic rates had been determined, plants were removed from the growth chamber and cut off at the crown. The leaves were traced on notebook paper and total leaf area was determined using a planimeter. After the plants were measured they were frozen on dry ice and placed in a freezer until all the pots had been harvested. The plant material was freeze-dried, weighed, and held for extraction of 14C— atrazine and its metabolites. Extraction of l4C-Atrazine and Its Metabolites The extraction procedure for atrazine and its metabolites was essentially that used by Shimabukuro (24). The freeze—dried plant material was homogenized with 30 m1 of 95% methanol. The extract was filtered and the residue re—extracted twice with an additional 20 m1 of methanol. The filtrate was placed in a hot water bath 29 at 70°C and Shaken gently for 15 minutes. A portion of the residue was folded in a preweighed piece of black paper for combustion and assay of the methanol-insoluble 14C—atrazine metabolites. The methanol extract was evaporated under vacuum in a flash evaporator leaving the aqueous fraction. This was washed from the boiling flask and centrifuged for 15 min at 13,300 x g. The pellet was washed twice by re- suspending it in water and then recentrifuging. A 0.5 ml aliquot of the supernatant fluid was removed for counting. The aqueous fraction was then washed five times with equal volumes of chloroform. The chloroform soluble fraction which includes unaltered atrazine and Compounds I and II was evaporated to dryness under vacuum. The residue was removed from the boiling flask with 10 ml of absolute methanol and placed in a vial for the determina— tion of radioactivity. The samples now dissolved in methanol were evap— orated to dryness under nitrogen. The residue was redis— solved in 1 ml of absolute ethanol. A 0.5 ml aliquot was removed for the determination of radioactivity. The re— maining sample was saved for spotting thin-layer chromato- graph (TLC) plates. 30 Control extractions were done using weighed amounts of untreated plant material. A known amount of 4C-atrazine was added to this material when it was first homogenized. Two samples of each Species were then carried through the aforementioned extraction procedure. Two additional samples of corn were extracted as outlined by Shimabukuro (25). The only additional step involved boiling the aqueous extract for five minutes before the chloroform wash to inactivate the benzoxazinone. Separation and Identification of 4C-Atrazine and Its Metabolites Unaltered atrazine and its chloroform soluble metabolites were separated by TLC on 250 u thick Silica gel H plates developed in benzene:acetic acid (50:4, . l4 . v/V). Six samples and a pure C—atraZine standard were run on each plate. After they had been developed, the plates were divided into zones and the Silica gel scraped into vials for the determination of radioactivity. The two plates with extracts from corn were scraped in 10 zones of 1.5 cm each. All other plates were scraped into 13 zones including the origin and 7 l-cm zones from the 31 lower half of the plate and five 1.5-cm zones from the remaining upper portion. Positive identification of the metabolite remain- ing at the origin was determined by multiple thin-layer chromotography. Three samples of corn extract containing almost solely metabolites that remained at the origin were Spotted and developed in the same solvent system as above. The origin was scraped and the gel washed with ethanol to remove the radioactivity. The resulting solu- tion was dried under nitrogen and redissolved in 0.1 ml ethanol, half of which was re-spotted on a second plate which was then developed in benzene:acetic acidzwater, 50:50:3 (v/V/V). Hydroxyatrazine ran at Rf 0.27 in this system. Plates were scraped into 1 cm zones, and placed in vials for counting. Quantitative determination of methanol-insoluble atrazine metabolites were made by the Schoeninger combus- tion method of Wang and Willis (33). The papers contain- ing the plant residue were dried and weighed and placed in sealed vacuum flasks. The flasks were evacuated and refilled three times with pure oxygen to atmospheric pres- sure. The samples were then ignited with a beam of infra- red light. 32 Twenty ml of ethanol—ethanolamine (2:1) was in- jected into the flask through a serum cap in the top. After stirring.20 minutes were allowed for this mixture to absorb the 14CO2 in the flask. A 5 ml aliquot was removed for counting. Fifteen ml of scintillation solu- tion containing 5 grams PPO and 0.3 grams of dimethyl POPOP per liter of toluene were added to each vial. The scrapings from the TLC plates were first dissolved in 0.5 ml of absolute ethanol before 15 ml of the scintillation solution was added. This scintillation solution containing 5 grams PPO and 0.5 grams POPOP per liter of 30% absolute ethanol in toluene. Two different Packard Tri-carb counters were used. Samples were counted either at 14% gain or 24% gain on channel II. Window settings were 50-1000. Gain on chan— nel I was 4%. .All samples were corrected for quenching using the channels ratio method. Assay of Atrazine Effect on 32P Uptake by Corn To check the effect of atrazine on phosphate up- take and distribution an experiment was designed, adding 33 32 . P to the culture solutions. Corn plants were grown as previously described in a 2 x 2 factorial with two repli— cations. The concentrations of atrazine and phosphate —4 —2 . were 0 and 10 M and 0 and 10 M respectively. Culture solutions at this point contained only unlabeled phos- phorus. When the plants were seven days old, they were removed from the cups and the sand was washed off the . . 32 roots. Five representative plants were selected for P treatment. These were placed into 100 ml of treatment . . . 32 solution in wax cups to which had been added 0.1 mc P as KH2P04. The plants were placed back into the con— trolled environment chamber at 30°C for 6 hours. Solu- tions were aerated as Shown in Fig. 3. After the treatment period, the plants were re— moved from the solutions and the roots rinsed four times with distilled water. The plants were frozen on dry ice and then freeze dried and prepared for autoradiography. The plant material was found to contain large 32 . amounts of P and several exposure times were necessary to obtain good pictures. Final exposure times were 16 . . —2 . hours for treatments containing 10 M phosphate in solu- tion and 40 minutes for the treatments without phosphorus. 34 . 32 . . Fig. 3.-- P treatment of corn in aerated solution culture for uptake and distribution study. 35 The difference in time was due to a dilution of 32P when unlabeled phosphate was present. Labeled phosphate was determined quantitatively using the methods of White and Ellis (35). Treatments were made in duplicate for autoradiography and two samples were taken from each of these making four replications. Two or three plant Shoots were removed from the blotter paper and ground in a Wiley mill (40 mesh screen). The plant material was collected in preweighed crucibles which were then reweighed. The material was placed in a muffle oven for 6 hours at 400°C. The material was allowed to cool and 10 ml of 1 N HNO3 was added to each crucible. The acid was evaporated Slowly on a hot plate and the crucibles heated again for 10 minutes at 400°C. The res- idue was redissolved in 10 m1 2 N HCl and these solutions transferred to scintillation vials for counting without additional scintillators using Cerenkov radiation. A Packard Tri-carb Scintillation Spectrometer was used for counting the samples. The gain setting was 50%. Quenching was assumed to be uniform so data was calculated as counts per minute per unit weight of plant material. RESULT S AND D ISCU SS ION Atrazine-PhOSphate Effects on Plant Growth Typical injury symptoms of atrazine-phOSphate combinations on corn are Shown in Fig. 4. Plants in the combination treatments were shorter than other plants and often were malformed. They were usually lighter in color showing typical atrazine injury. Average values for plant height, shoot weight, and root weight on a per plant basis are given in Tables 1 through 5. Duncan's Multiple Range Test was used to compare means when an analysis of variance Showed them to be Significant at the 5% level. Two basic patterns can be seen in the data. In the Species susceptible to atrazine injury, soybeans and barley (Tables 1 and 2), injury was due to atrazine with no effect of phosphate concentration on barley growth and only small effects on soybean growth. For this reason barley was not used after the initial growth experiment. 36 37 CORN 7 days O)d Atrni "t Fig. 4.-—Atrazine—phOSphate combination effects on the growth of 7-day—old corn. 38 In soybeans, the combined inhibition by atrazine and phosphate tended to be more than additive for plant height and shoot weight. There was a significant syner- gistic height reduction for atrazine and phosphate at —6 —2 . . . . 10 M and 10 M respectively and a synergistic inter— action approaching Significance at 10_5 M atrazine and —2 10 M phOSphate. Using the same comparisons as above, sorghum,a tolerant plant, was shown to behave similarly to soybeans and barley, but to a lesser degree (Table 3). Atrazine . -4 -5 . . . at either 10 or 10 M Significantly reduced plant height, shoot and root weight to levels below that of the control. Plants receiving phosphate without atrazine were different from the control only in reduced root weight. Except for root weight the values of the com- bined inhibition effectsmere more than additive at the high atrazine concentration. Peas follow a second pattern in which phOSphate injury is readily apparent (Table 4). Atrazine alone at the high concentration was not Significantly different from the control. The combination treatment was not Sig— nificantly more toxic than the phosphate treatment alone, 39 however, the injury was more than additive for plant height. The observed decrease in root weight for the combination treatment was approximately additive. Neither atrazine nor phosphate alone caused a significant decrease in corn height compared with the controls (Table 5). Combined inhibition with phosphate at 10-2 M and atrazine at 10-4 or 10_5 M appeared to be more than additive although these combination treatments were not significantly different from the phosphate treatment alone. Shoot weights Showed no significant differences between treatments. Root weight, as in sor- ghum and peas, was more sensitive to atrazine than shoot weight and the combination treatment Showed only additive effects of the atrazine and phosphate treatments. The results of these preliminary experiments Show that an atrazine—phosphate interaction does occur and can be measured at least for corn, sorghum, peas, and soybean. Consequently these Species were used in subse- quent eXperimentS to further study this interaction. 40 TABLE l.——Atrazine-phosphate combination effects on soy— bean growth.l Atrazine Phosphate Height Shoot Root concen— concen- mm weight weight tration tration mg mg 10—4 M 10’2 M 38 a2 17 a 9 a -4 —3 10 M 10 M 50 ab 21 ab 20 abc -4 —4 10 M 10 M 44 ab 20 ab 19 abc 10’4 M o 54 abc 25 abc 20 abc -5 -2 10 M 10 M 61 bc 27 abcd 12 ab —5 -3 10 M 10 M 97 def 32 bcd 22 abcd -5 -4 10 M 10 M 86 d 29 abcd 24 bcde 10"5 M o 89 def 28 abcd 22 abcd —6 —2 10 M 10 M 60 abc 32 bcd 25 bcde —6 —3 10 M 10 M 111 f 39 def 28 cdef —6 —4 10 M 10 M 96 ef 34 cde 30 cdefg 10"6 M o 99 def 33 cde 36 defg o 10’2 M 75 cd 47 f 37 efg o 10"3 M 94 def 45 ef 36 defg o 10"4 M 88 def 34 de 43 g 0 0 91 def 38 def 42 fg 1Values are the mean of 4 replications with 10 plants per replication. Means with common letters are not Significantly different at the 5% level by the Duncan Multiple Range Test. 41 TABLE 2.--Atrazine—phosphate combination effects on barley growth. Atrazine Phosphate Height Shoot Root concen— concen— mm weight weight tration tration mg mg 10"4 M 10'2 M 80 ab2 8 ab 2 a 10’4 M 10'3 M 73 a 7 ab 2 a 10’4 M 10’4 M 66 a 6 a 2 a -4 10 M 0 83 abcd 8 ab 2 a —5 —2 10 M 10 M 100 bcd 9 b 4 b —5 -3 10 M 10 M 102 cd 9 b 4 b -5 —4 10 M 10 M 99 bcd 8 ab 4 b 10'5 M 0 99 bcd 8 ab 4 b —6 -2 10 M 10 M 103 cd 9 b 4 b -6 —3 10 M 10 M 103 cd 9 b 4 b -6 —4 10 M 10 M 104 cd 9 b 5 b 10'6 M 0 103 cd 8 ab 5 b 0 10"2 M 105 cd 14 c 9 d 0 10’3 M 118 d 16 d 8 cd —4 0 10 M 110 d 13 c 7 c 0 0 107 d 12 c 8 cd 1Values are the mean of 4 replications with 10 plants per replication. Means with common letters are not Significantly different at the 5% level by the Duncan Multiple Range Test. 42 TABLE 3.-—Atrazine—phosphate combination effects on sor— ghum growth. Atrazine Phosphate Height Shoot Root concen- concen- mm weight weight tration tration mg mg 10'4 M 10'2 M 75 a2 9 a 3 a —4 -3 10 M 10 M 90 ab 10 a 4 a —4 10 M 0 100 ab 11 a 4 a —5 —2 10 M 10 M 100 ab 13 a 4 a —5 -3 10 M 10 M 119 bc 13 a 4 a 10"5 M 0 104 ab 12 a 5 a 0 10’2 M 124 bc 19 b 8 b 0 10"3 M 160 d 21 b 8 b 0 0 138 cd 18 b 11 c 1Values are the mean of 4 replications with 10 plants per replication. 2Means with common letters are not significantly different at the 5% level by the Duncan Multiple Range Test. 43 TABLE 4.-—Atrazine—phOSphate combination effects on pea growth. Atrazine Phosphate Height Shoot Root concen- concen- mm weight weight tration tration mg mg 10'4 M 10’3 M 25 a2 23 a 21 a —4 -3 10 M 10 M 42 bcd 42 abc 27 a 10'4 M 0 45 bcd 45 bcd 33 ab —5 —2 10 M 10 M 38 bc 40 abc 32 ab —5 -3 10 M 10 M 42 bcd 47 bcd 30 ab 10’5 M 0 45 bcd 44 bcd 28 a 0 10"2 M 35 ab 36 ab 27 a 0 10'3 M 52 d 59 cd 49 c 0 0 49 cd 64 d 43 bc 1Values are the mean of 4 replications with 10 plants per replication. Means with common letters are not significantly different at the 5% level by the Duncan Multiple Range Test. 44 TABLE 5.——Atrazine—phosphate combination effects on corn growth.l Atrazine Phosphate Height Shoot ' Root concen— concen- mm weight weight tration tration mg mg 10‘4 M 10‘2 M 69 a2 54 32 ab —4 -3 10 M 10 M 127 cde 69 32 ab —4 -4 10 M 10 M 98 abc 61 27 a 10'4 M 0 120 bcde 69 34 abc -5 -2 10 M 10 M 79 ab 53 33 abc -5 -3 10 M 10 M 149 e 81 47 bcd -5 -4 10 M 10 M 139 cde 74 44 bcd 10'5 M 0 137 cde 67 46 bcd —6 -2 10 M 10 M 103 abcd 77 47 bcd —6 -3 10 M 10 M 138 cde 76 52 d —6 -4 10 M 10 M 145 de 73 48 cd 10"6 M 0 132 cde 70 38 abcd 0 10'2 M 102 abcd 71 45 bcd 0 10'3 M 146 de 82 . 48 cd 0 10"”4 M 143 de 77 53 d 0 0 145 de 74 50 d 1Values are the mean of 4 replications with 10 plants per replication. 2 . . . . . Means With common letters are not Significantly different at the 5% level by the Duncan Multiple Range Test. 45 Atrazine-Phosphate Combination Effects on Plant Respiration and Photosynthesis Plant height, Shoot weight, and leaf area were three measurements available for evaluating the atrazine- phosphate combination effects on plant growth in this study. Since there were only two replications of these treatments, it was more difficult to obtain statistically significant results. The statistically Significant re— sults of the preliminary experiments reported here and in past research (l,2,9,32) are considered to be evidence enough that an interaction does occur. The growth response of plants used for the photo- synthesis and respiration studies supplied with phosphate, atrazine or the combination of the two is reported in Tables 6 to 9. The phosphate level has a significant effect on leaf area, plant height, and dry weight of corn plants. At 10.5 M, it promoted growth as measured by leaf area and plant height, whereas these values were significantly lower than the controls at the 10-2 M phos— phate level (Table 6). The first response suggests growth stimulation of phosphate-starved plants, the second phosphate toxicity. This phosphate stimulation 46 occurred at all atrazine levels for area, height, and weight. There was no atrazine toxicity to plant growth at 10-5 M and only limited toxicity at 10-4 M Showing up as a height reduction. Although the differences were not significant at the 5% level, the combination treat— ment of high levels of both atrazine and phosphate re— sulted in a more than additive inhibition. Respiration of corn plants was not affected by phosphate alone. Atrazine only Slightly inhibited res— piration at the 10'.2 M level. The combination treatment almost doubled the respiration rate above its eXpected value. This depletion of energy reserves would be ex- pected to cause a reduction in plant growth. Net photosynthesis was reduced by both atrazine and phosphate at their highest levels. In combination at those concentrations, they tend to have less than additive effects. The differences between means were not Significant for total photosynthesis. There, too, atrazine at high concentration tended to reduce photo- synthesis but the reduction due to the combination treat- ment was less than expected. Only the 10-4 M concentration of atrazine Showed a significant inhibitory effect on the dry weight of peas 47 grown for respiration and photosynthesis studies (Table 7). The leaf area and plant height were not affected by atra— zine. Phosphate by itself tended to reduce leaf area and plant weight but not significantly. Plant height was un— effected. Combination treatments Showed only additive inhi- bition at the highest concentrations of atrazine and phosphate. The significant interaction at 10.5 M atra- zine, 10-2 M phosphate did not Show up in the preliminary experiment. The present result is partly due to the death of one replication. The inhibitory effect of the combination on growth can be partially explained in peas on the basis of a Sig- nificant synergistic promotion of respiration (Table 7). The observed respiration rate was approximately two to four times that which would have been expected if the ef- fects were additive. There was no Significant promotion of respiration by phosphate alone while atrazine alone reduced rather than increased reSpiration. The rate of photosynthesis in treated peas indi- cates that atrazine was very inhibitory and that phosphate level had very little if any effect (Table 7). 48 Neither phosphate nor atrazine alone caused a sig— nificant reduction in sorghum growth (Table 8). The com- bination treatment using high atrazine and phosphate levels was more toxic than phosphate at 10-2 M by itself as indicated by plant height, area, and weight, and more toxic than the high atrazine level treatment as indicated by plant height. In all three cases, the growth in the above combination treatment was significantly less than the control. The combined inhibition by atrazine and phosphate at the high concentrations tended to be more than additive for area and weight and were Significantly more than additive for height. With sorghum, there was no significant effect of phosphate or atrazine level on respiration rate (Table 8). Plants receiving the combination treatment at high concen— trations respired at a rate almost three times that of the control, a more than additive effect. Net photosynthesis was reduced whenever atrazine was present in the nutrient solution (Table 8). There was also a reduction in net photosynthesis as the phosphate concentration increased. The same was true for total photosynthesis where atrazine level had a significant 49 inhibitory effect. The phosphate level did not affect total photosynthesis. In this part of the experiment, soybeans showed less injury from atrazine than in Table 1. There was a promotion of plant growth by phosphate at 10-3 M for height and area and at 10-2 M for weight (Table 9). How- ever, high phOSphate levels tend to reduce growth. In- creasing atrazine level Significantly decreased plant height, weight, and leaf area. The growth inhibition by high atrazine and phosphate combination tended to be more than additive. Atrazine at 10_4 M in the nutrient solution Sig- nificantly increased soybean respiration (Table 9). At the high atrazine level there was no inhibitory interac— tion with phosphate. The respiration rate in the presence 5 M atrazine and 10—2 M phosphate in the nu- of both 10- trient solutions was twice that of the atrazine effect alone and 3 to 4 times that of the phosphate effect alone. 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Emma 000m Icwocoo Icmocoo Hmuoe uwz aoaumuammwm uanm panda Imam mama wumnmm0Qm mawwmuufl H 1') H fl M .EDQmHom ca mameQGMMOHOQm 0cm cowumnflmmwn no mvommmm COHQMGHQEOO mumnmmOQmechmuufiII.m Manda 53 .umoa mmcdm mamauasz cmocdo mQu mQ ambma mm mQu um unmuommap wapcmoamaamam no: 0H0 mnmuuwa SOEEoo Quaz memo: N .mpmmm 0a Eoum Umumaaaumm mmcaanmom pwcamucoo QoaQS mcoaumoaammu 03» we came an» mum m09a0> a 0>.0 00.0 vam an 0m m 0 I Nw.0 00.0 nun N0 0N 0 z mIoa I No.0 m0.0 mvaa av 0N m z NI0a I 00.0 00.0 0 wow Q Nb 0 0m Q m I 0 a0.0 m0.0 w 0am Q on Q 0m 0 v I z mloa 0a.0 00.0 Q mama 0 mm 0 ha 0 N I z ¢I0a mambma mo wommmm mmmum>¢ 0N.a 00.0 0 00m 0Q 08 0Q mm 0Q h 0 0 0m.a 00.0 Qm mmv 0Q 05 0Q mm 0 0a 2 mtoa 0 00.0 00.0 m Nmm oQ m0 o 00 0Q m z NI0a 0 mm.0 00.0 Q0 000 o 00 0Q hm 0Q 0 0 2 mIoa Na.0 00.0 Qm amv 0Q 05 0Q0 0N Q0 0 z mI0a z mI0a mh.a 0a.0 Qm mama Qm mm QM 0N Q0 m z NI0a z mIoa 00.0 00.0 Q 005a Qm N0 0 ma Q0 N 0 z I0a 0m.0 00.0 Qm puma Q0 00 0 ON QM m z mI0a z WIoa 00.0 00.0 Q voba w 0N n ma NM 0 z NI0a z ¢I0a NEU\caE\NOO 0: NEU\caE\NOU m: N . BE 05 _ NED coaumuu acaumuv mameQSNMOQOQm mammnucwmouozm m\aaa\ 00 m: uQmamQ uQmam3 womb 000m Icmocou Invocoo amuoe umz coaumnammmm Madam unwam Imam mama 0QMQQMOQN 0caumnu< a IHIJ '1') l I) .mqmeNOm ca mameuahmoQOQm 0S0 soaumuammmu co “comma SeaumnaQEoo mQMQmmosmImzaNmuumtI.m mamma 54 Atrazine—Phosphate Combination Effect on Atrazine Uptake and Metabolism The total amount of l4C-atrazine uptake during the treatment period is reported in Tables 10 to 13 for corn, peas, sorghum and peas, respectively. The amount of the 14C associated with the respective metabolic frac- tions is also presented for these crOps in Tables 10 to 13. In corn, the chloroform-soluble fraction which contained the unchanged atrazine and small amounts of dealkylated metabolites tended to increase with increas- ing phosphate levels (Table 10). Increasing the concen- tration of atrazine in the culture solution also tended to cause an increase in that fraction, suggesting an overload of the detoxication mechanism. The phosphate treatment also contributed to this effect suggesting that high levels of phosphate might interfere with atrazine metabolism. Supporting this conclusion is the observation that the combination of high atrazine and phosphate re- sulted in a significant synergistic reduction in the amount of methanol-insoluble metabolites, the insoluble 55 residue assumed to be least toxic to the plants. There appears to be a reciprocal relationship between these compounds and the hydroxylated metabolites. However, since neither is toxic to plants it is difficult to en- vision how blocking the pathway between these two might be detrimental. It would be interesting to know how the concentration of hydroxylated metabolites subsequently affects the rate of atrazine metabolism to these same compounds. High concentration of hydroxylated compounds could Shift the equilibrium in the direction of unaltered atrazine. In corn, it appears that the interaction can best be explained by increased plant respiration and reduction in the rate of atrazine detoxication, specifically to a methanol—insoluble residue. At the 10-4 M atrazine level, increasing the phosphate level tended to increase atrazine uptake (Table 10). Whereas at the 10-5 M atrazine level the opposite effect was apparent. However, none of the changes were significant and undoubtedly did not wholly account for the observed increase in phytotoxicity at the higher phOSphate level. 56 Atrazine metabolism by peas followed a pattern Similar to that of corn. The level of chloroform-soluble compounds tended to increase with increasing atrazine level, again suggesting an overloading effect on the de— toxication system (Table 11). The lowest value was again for the low atrazine treatment alone. The reciprocal re— lationship between hydroxylated compounds and methanol— insoluble metabolites was also apparent as in corn. The least conversion to methanol—insoluble meta- bolites occurred in the combination treatments with 10—2 M phosphate. An increase in the available level of phos- phate significantly promoted atrazine uptake at 10-4 M atrazine whereas increasing the phosphate level reduced the level of atrazine uptake at the 10—5 M atrazine con- centration. Here the plants most affected had the lowest concentration of atrazine in the tissues. The conclusions with respect to peas are similar to those for corn. The interaction seems to be the re- sult of a promotion of respiration possibly enhanced by a disruption of atrazine metabolism. There was no signif— icant difference between treatments in the composition of the chloroform-soluble fraction as shown in Table 14, al- though the hydroxylated metabolites increased with 57 increasing levels of phosphates. This trend was Similar to that observed for the hydroxylated metabolites in the initial separation (Table 11). There was very little difference between the treatment means in sorghum for the distribution of 14C— labeled compounds in the three metabolite fractions (Table 12). Chloroform-soluble metabolites tended to be slightly higher in plants grown in 10—4 M atrazine solu- tion than in plants grown in 10"5 M atrazine solution but the level of phosphate did not either increase or decrease the total amount of compounds in this fraction. Hydroxy— lated metabolites make up a slightly lower amount of the total compounds present in plants grown on 10_4 M compared with 10-5 M atrazine. Phosphate level again had no ef- fect. The amount of methanol—insoluble metabolites was not influenced by either atrazine or phosphate level. An analysis of the makeup of the chloroform- soluble fraction Shows that there was no significant dif- ference between treatment means for atrazine or the hydroxylated metabolites running at the origin (Table 15). There were Significant differences between treatment means for the dealkylated metabolites. The combination - - —2 treatments of atraZine at 10 4 M and phosphate at 10 M 58 and 10—3 M contained larger amounts of these dealkylated metabolites than either treatment with atrazine alone. The small magnitude of the difference makes this statis- tically Significant result of doubtful importance as a causal factor to the observed decrease in plant growth. Atrazine uptake by sorghum was not linear with concentration in the culture medium (Table 12). Whereas there was a tenfold difference in atrazine concentration between the first three and second three treatments, there is only a three-fold difference in atrazine uptake. At any given level of atrazine, phosphate had no Signif- icant effect on atrazine uptake, although it tended to increase uptake at the 10_4 M atrazine level. The failure of uptake to be linear suggests that it may be an active process in sorghum. Atrazine metabolism data for soybean clearly re- flects the limited ability of this species to convert atrazine to other compounds (Table 13). Although there are no Significant differences between the treatment means for chloroform—soluble compounds, a higher per- centage of chloroform—soluble compounds remained in soy- bean plants than in the other Species tested. There were no significant differences between the means for the 59 percentage of methanol—insoluble metabolites. The rela— tive amount of hydroxylated metabolites is highest in the plants receiving both high atrazine and phOSphate but the lowest relative amount is found in plants receiving 10-5 M atrazine in combination with 10“3 M phosphate. Only these two extremes are significantly different from each other. There were no Significant differences between treatment means for the component metabolites of the chloroform-soluble fraction (Table 16). AS with sorghum, the metabolism study did not produce any information to explain the atrazine phosphate interaction on plant growth. Atrazine uptake by soybean was linear with con- centration. Phosphate at 10—2 M inhibited the uptake of atrazine from a solution containing 10—5 M and 10-4 M atrazine compared with phosphate at lower levels (Table 13). The combination treatment of 10—2 M phosphate and 10-4 M atrazine was reduced in terms of plant growth compared to the other two treatments containing 10_4 M atrazine despite the lower atrazine uptake of these plants in the combination treatment. Thus a phOSphate affect on atrazine uptake did not explain the atrazine- phosphate interaction on growth. 60 Table 17 Shows the effect of the assay procedure 14 . . . on C—atraZine recovery during the extraction procedure. . l4 . Because a fairly constant amount of C-atraZine was associated with each milligram of dry material extracted, . . l4 . the weight of each sample influenced C—atra21ne re- covery. The label found in the insoluble fraction prob- ably represents absorption rather than conversion during the extraction procedure. Since label in methanol- insoluble metabolites generally increases on a unit weight basis as sample weight increases, the data in Tables 10 through 13 represent a real conversion by the plant to insoluble residue. Recovery of total label appears to be satisfac- tory. Multiple thin-layer chromatography of the metab— olites remaining at the origin Showed most of the radio- activity to be present in hydroxyatrazine. Two other distinct metabolites of unknown identity were also present. 61 bananas: :mocso mQu >Q a0>0a wm 0Qu um ucmummmaw mauamoamacmam uo: mum muouuma GOEEOU Qua3 mammz .umme QOCMM N .mpmmm 0a Scum pmumcashwm mmcaapmwm pwcamucoo QoaQ3 mcoaumoaamwu N we came onu 0H0 m0§a0>a 0a Nb Qm ma Nmm 0 I «a N0 Q NN Nvm 2 mIoa I va on 0 ma amv E NI0a I 0 aa 00 Q am pm I z mI0a ma an 0 ha who I E vIoa mam>ma mo uommwm ommum>d 0 QB Nb Q 0a 00 0 2 mIoa aa Q0 00 Q aN 00 z mI0a z mIoa ma 0 me Q NN 00 z NI0a 2 mI0a aa Qm Nb Q ha vnm 0 2 aqua aa 0 m0 Q mN 0am z mI0a z vloa 0a Q mm m m avm z mI0a z vloa abpou mo unmoumm amuou mo ucmonmm amuou mo . coaumup moaumuu mmuaaOQmumE mmuaaOQMQME usmouwm macammu uQmamS muw m\m: Icwucoo Icmocoo maQsaOMIEHomouoaQU Umumamxouwhm 0aQ5aOmca mxmumd baaNMHQN oumnmmonm ocaumuaa a .cuoo ca EmaaOQmume paw mxwumd mcaumuum do muommmm coaumcaQfioo 0QMQmmonmlmcaNmuuma wm wQu um ucoummmaw haucmoamacmam pom mum mnmuuoa COEEOU Qua3 mammz N .mvmom 0a Eoum vmumcaahom mmcaapmwm pmcamucoo QoaQB mcoaumoaamwu N mo.cmmE mQu mum mmaam>a 0 0m mv 0Na 0 I 0 av am 0Na 2 mI0a I m 00 am aha z NI0a I 0 0 mm av Q 00 I z mloa m 0v vv 6 vNN I z vI0a ma0>oa mo uowmmm mmmum>m v mm av 0Q0 on 0 z mloa aa av mv Q0 00 E mIoa z mIoa 0 mm mm 0 av z NI0a z mI0a 0 vv 0v 0Q Nha 0 z vI0a n 0v vm 00 00a 2 mI0a 2 vI0a aa a0 0N NU aom z mI0a z vI0a aMQOQ No #000800 . annoy mo unwoumm_ : amuou.mo _ coaumuu coaumuu mwuaaOQdumfi mwuaaOQmumfi unmoumm mspawmu uQma03 who m\0: Icwonoo Icmocoo maQsaOMIEHomonoaQU pmumamxoupwm oaQsaowca mxmumfi mcaumuum mHMQmmOQm mcaumuud a ilr [I I 'II 1' ..mmm.ca EmaaOQmume.Ucmn0x0ums;mcanmuum:couwuommmmzqoaumcaQEoo.QQMQmmonImcaumuuma mm mQu #0.»:0H0mmac waucmoamacmam uo: mum mumuuma SOEEOU Qua3 mammz N .mpwmm 0a Eoum pmumcashmm mmcaapmom pwcamuaoo Qans MSOaumoaammu N no :00& 0Q» mum mosam>a 0 a0 mm mvv 0 I N am vm mum z mIoa I 0 00 mm aNm z NI0a I 0 0 N0 mm Q v0N I z mI0a 5 mm mm 0 von I 2 vI0a ma0>wa mo uommmm womum>¢ 0 m0 0N m 00N 0 z mIoa 0 00 mm m mmN z mI0a z mIoa m 00 mm m NON z NI0a z mIoa h hm 0m Qm 00m 0 z vI0a N am vm Q 0mm 2 mI0a z vI0a 0 mm mm NQ 0mm 2 mI0a z vI0a amuou mo ucwoumm amuou mo unmoumm amuou mo coaumuu coaumuu mmuaaOQmuwe mwuaaOQmumE ucmouwm mspammn uQ0a03 >HU m\m: Iawoaoo Icooqoo maQSaOMIEM0moHoaQU 00u0a>xoupwm maQsaomca wxmumfi wcanmuum mgmnmmocm maaNMHufl a .Edcbuom Ca EmaaOQmumE paw mxmums mcaNmnum co muowmmm coaumcaQEoo wQMQQMOQmImcaNMHQNII.Na mamas 64 .umoe mmcmm mamauasz CMUSDQ 0Q» mQ am>ma wm mQu um ucmnwmmap maucmoamacmam no: mum mumuuma GOEEOU Qua3 mammz N .mpwwm 0a Eonm wmumcasumm mmcaapmmm pmcamucoo Quanz mcoaumoaamou N 00 Game 0Qu mum mosam> a 0N QM 0v Nv amN 0 I MN Q 0m mv abN z mI0a I ma 0 0v Nm v0N z NI0a I 0 aN 0v 0m Q mv I z mI0a 0a 0m Nv m MNv I z vI0a ma0>0a mo uoommw mmmumbd ha Qm bv mm m vm 0 z mIoa 0N m 0N vv 0 vm z mIoa z mloa ma Q0 0v mm 0 0m 2 NI0a z mIoa 0a Qm vm Nv oQ 00v 0 z vI0a ha Qm Nm am 0 mmv z mI0a z vI0a aN Q Nm 0N NQ th z mI0a z vI0a amuou mo ucwoumm amuou mo unmoumm amuou mo coapmuu ceaumup mmuaaOQmumE mmaaaOQmuQE Dawouwm onwamwu UQmams mun m\m: Icmocoo Icmocoo maQsaOMIEHOMOHOaQU pmumamxoupmm waQsaomca mxmumd meanmuufi wDMQQMOQm mcanmuum 'I II. a .amemom ca EmaaOQmums can mxmnmb mcaucuum co muommmw coaumcaQEoo mHMQQMOQmImQaNMHumII.ma mamma 65 m: cmxmu mcaNMHQMIU va amuou mo vamp me a.awm mo coapomum muaaOQmumE .mvmmm 0a Eoum Umpmcafiumm mmcaawmmm Wmsamucoo Qoan mcoaumoaammu N 00 Gama 0Q» mum mmsam>a 0.0 0.N 0.a 0 I 0.a 0.0 N.N z mI0a I a.a v.v 0.a z NI0a I 0 m.a a.m v.a I z mIoa HOH mow wom | 2 VIC.” mam>ma mo uowmmm mmmum>¢ 0.a 0.N 0.0 0 z mloa m N v v 0 N z Mloa z mIoa m 0 a m 0 0 2 NI0a z mloa 0.0 0.m m.N 0 z vI0a . . . z 2 0 0 m m 0 a mloa vI0a 0 a 0 m a m z NI0a 2 vI0a mmuaaOQmumfi 00u0awxamwa mmuaaOQmumE Umumamxouwwm wcaumnu< coaumuucmocoo GOaHMHucwocoo 0u0Qmm0Qm mcaumnum maQDaOmIEHomouoaQo wQu mo coauamomEoo mQu ab muowmmw coaumcaQEoo wumnmmosmImcaNmuu¢ll.va mqmfle .um09 00:00 0amauasz smocso 0Qu >Q a0>0a 00 0Q» um uc0n0mmaw maucmoamaamam #0: 0mm mn0uu0a SOEEoo QuaB 0:002 66 - 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TABLE l7.--The effect of assay procedure on C-atraZine recovery during extraction procedure.1 Percent of label in fraction: Species Chloroform Chloroform Insoluble Percent of total soluble . _. insoluble residue2 14C recovered Corn 45 34 21 77 Sorghum 68 29 3 99 Soybean 60 33 7 86 Pea 65 22 13 81 1Values are the mean of two replicate extractions. 2Variability is due to the amount of material extracted. A constant amount of label, 0.045 disintegrations per minute per milligram dry weight of extracted material was absorbed. Atrazine Effect on 32P Uptake and Distribution in Corn Autoradiographs from the phosphate uptake study are Shown in Figures 5 and 6. Figure 5 Shows plants which received 10—2 M phosphate with and without atrazine. Phosphate is seen to be distributed uniformly throughout the plants in both treatments with no apparent difference between treatments. Shown in Figure 6 are plants which did not receive phosphate. These plants receiving or not receiving atrazine also Show a uniform phosphate distri— bution with little apparent difference between treatments. 69 E N omCHNMHufl 0G flmyflmuwh £0033 0000am 03030 0:000 00 000000a000000< .z_vI0a 00 000500000 00000000 00>a000u Q0033 0000am 030:0 000a 0o 000000a000009< .000000000 I0a 0a 03000 0000am 0000 no 0M00ms m NM 00 00000000 no 000000 0&91I.0 .0am II. I D 70 .00000000 00 00>00000 £00£3 00000m 03030 0£m00 000 00 £m00m000000050 .2 0100 00 0000800000 00000000 00>00000 300:3 000009 03030 0000 00 £000m000000000 .000000000 050:003 0300m 00000m 0000 00 0M00m5 mam 00 00000000 00 000000 0591:.0 .m0m 71 It was not possible to compare phosphate uptake with and without phosphate in solution. Phosphate uptake was no . . . —2 longer linear w1th concentration at 10 M. Table 18 shows the data for actual amounts of 32F present in the plant material. As the t-test indicates there are not significant differences at the 5% level between plants treated and not treated with atrazine. The differences are approaching significance in the phosphate-treated plants but it does not appear that effects on phOSphate uptake can explain the atrazine- phosphate interaction. TABLE 18.--The effect of atrazine on 32F uptake.l Atrazine Phosphate 32F concentration concentration counts/minute 10’4 M 10'2 M 432 -2 O 10 M 652 —4 10 M O 7458 O O 6923 1Values are the mean of 4 samples each containing 2 or 3 corn shoots. SUMMARY AND CONCLUSIONS Of the five crop species examined, corn, peas, sorghum, and soybeans showed greater reduction in plant growth following atrazine treatment if high levels of phosphate were also present in the treatment solution. Only in the case of peas was the high level, 10.2 M phOSphate, in the treatment solution significantly phyto— toxic by itself. Barley did not show the atrazine— phosphate combinations effect evident in corn, peas, sorghum, and soybeans. The enhanced phytotoxicity of atrazine in the presence of phOSphate appeared to be related to a syner- gistic increase in respiration of corn, peas, sorghum, and soybean plants receiving the combination treatment. The atrazine induced inhibition of photosynthesis in these species was not synergistically enhanced by the additional presence of phosphate. Although the trends were not statistically sig— nificant at the 5% level, at the 10-5 M atrazine level, increasing levels of phOSphate tended to reduce total 72 73 atrazine uptake during the treatment period. For corn, peas, and soybean this trend was reversed at the 10.4 M atrazine level. The percent of chloroform-soluble metabolites, including the parent atrazine, increased with increasing atrazine and phosphate levels in corn and peas. If this effect were of sufficient magnitude it could explain en- hanced phytotoxicity. The presence of phosphate with the atrazine treatment reduced the metabolism of atrazine to a methanol-insoluble residue and increased the accumu— lation of hydroxylated metabolites in corn, peas, and soybeans. It is difficult to envision how this effect could explain the altered phytotoxicity unless the assump— tion on the phytotoxicity of these metabolites are erron— eous or this block in metabolism also affected the rate of conversion of atrazine to hydroxylated metabolites. There did not appear to be any affect of phosphate on atrazine metabolism by sorghum. Atrazine did not affect the uptake or distribu- tion of 32P by corn. The enhanced phytotoxicity of atrazine in the presence of high levels of phosphate can best be explained by an interaction on the rate of respiration. The 74 increased respiration could be due to an interaction effect on the respiration apparatus or by an increase in the internal concentration of atrazine resulting from a slight increase in uptake and a slight decrease in metabolism of atrazine in the presence of high levels of phosphate. These alternatives are not mutually ex- clusive. LITERATURE C ITED Adams, R. S. 1965. Phosphorus fertilization and the phytotoxicity of simazine. Weeds 13: 113-116. Adams, R. S. and W. G. Espinoza. 1969. Effect of phosphorus and atrazine on mineral composition of soybeans. J. Agr. Food Chem. 17: 818—822. Ashton, F. M. 1965. Relationship between light and toxicity symptoms caused by atrazine and monuron. Weeds 13: 164-168. Ashton, F. M., T. Bisalputra, and E. B. Risley. 1966. Effect of atrazine on Chlorella vulgaris. Amer. J. Bot. 53: 217-219. Ashton, F. M., E. M. Gifford Jr., and T. Bisalputra. 1963. Structural changes on P, vulgaris induced by atrazine. I Histological changes Bot. 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