Effects of Paraquat on Selected Microbial Activities in Soil E.A. SMrrH and C.I. MAYFIELD Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
Abstract. Paraquat, applied as Gramoxone, to a nonamended sandy loam soil at five times the suggested field application rate (10 lb/A ~ 115/xg/cm 2) increased the numbers of bacteria, actinomycetes, and fungi during a 14-day incubation at 25~ This increase was attributed to the use of compounds in the Gramoxone formulation rather than the use of paraqnat. Treatment at one and five times the normal rate reduced CO2 evolution by 44% and 67%, respectively, in soil amended with 2% glucose during a 12-flay incubation. Similar treatments reduced CO~ evolution in 1% straw-amended 9 soil by 39% and 58%, respectively, during a 28-day incubation. Cellulose decomposition of cotton duck containing 13 and 176 ~g of paraquat per milligram of material was inhibited for 15 and 28 days, respectively, in soil containing a large population of cellulolytic microorganisms. A concentration of 5000/~g/gm of paraquat was necessary to inhibit nitrification in soil by 44% druing a 28-day incubation at 20~ Paraquat inhibited C2H2 reduction in artificial aggregates of soil amended with 2% glucose and incubated anaerobically at 25~ Nitrogenase activity in aggregates was inhibited by 43 % and 52% at concentrations of 580 and 720 ~g/gm of paraquat respectively. The inhibitory effects of the herbicide were reduced when soil was amended with organic matter in the form of peat or straw. The availability of paraquat controlled the toxicity of the herbicide to soil microorganisms.
Introduction T h e d i p y r i d y l i u m h e r b i c i d e s , p a r a q u a t a n d d i q u a t , w e r e i n t r o d u c e d as effective nonselective herbicides, and a large amount of information has since been d o c u m e n t e d d e s c r i b i n g t h e i r p h y t o t o x i c m o d e o f a c t i o n [3, 7, 28]. A l t h o u g h p a r a q u a t h a s b e e n s h o w n to b e t o x i c to n o n t a r g e t o r g a n i s m s , s u c h a s t h e soil f a u n a Collembola, Acari, andHomoptera [6, 18], o t h e r i n v e r t e b r a t e s a n d fish [5, 23], a n d e x p e r i m e n t a l l a b o r a t o r y a n i m a l s a n d m a n [2, 8, 21], t h e r e is a l a c k o f i n f o r m a t i o n p e r t a i n i n g to t h e effects o f t h e h e r b i c i d e o n soil m i c r o o r g a n i s m s . It h a s b e e n s u g g e s t e d t h a t the r e c o m m e n d e d field a p p l i c a t i o n rates o f G r a m o x o n e (0.5 to 2 . 0 l b / A ) w i l l h a v e no a p p r e c i a b l e effects o n m i c r o b i a l a c t i v i t i e s that are i m p o r t a n t to soil fertility [ 2 5 , 26]. In t h e p r e s e n t s t u d y , the effects o f v a r i o u s c o n c e n t r a t i o n s o f p a r a q u a t o n s e l e c t e d m i c r o b i a l a c t i v i t i e s in soils arc e x a m i n e d .
Materials and Methods
Soils. In this study two types of soil were used. Sandy clay loam was collected from the surface 10 cm of an uncultivated grassland site in Waterloo County, Ontario. Its pH was 7.4 and it conMicrobial Ecology 3,333-343 (1977) 9 1977 by Springer-Verlag New York Inc.
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E . A . Smith and C. I. Mayfleld
tained 0.06% nitrogen (oven-dried at 105~ to constant weight). Sandy loam soil (St. Bernard sandy loam) was collected from the plow-layer (0 to 15 cm) of a pasture on the Macdonald College Farm, Quebec. Its pH was 7.2 and it contained 0.23% nitrogen (oven-dry basis). The soils were stored at 4~ during the experimental period.
Paraquat. Paraquat used in this investigation was obtained directly from Chipman Chemicals Ltd., Stoney Creek, Ontario as the 'Gramoxone' formulation. It contained 240 gm/liter paraquat (99.9% pure 1,1'-dimethyl-4,4'-dipyridilium dichloride) as the active ingredient. Numbers of microorganisms. Fifty-gram samples of the sandy loam soil were sprayed with the field application rate (F.A.R. = 0.5 to 2.0 lb/A) of Gramoxone. The moisture content of the soil was adjusted to 25% with distilled water. After 1 and 14 days of incubation at 20~ estimates of the numbers of fungi, actinomycetes, and aerobic heterotrophie bacteria were obtained by the pour-plate method, using Czapek Dox agar (NaNO3, 2.0 gm; KCI, 0.5 gm; FeSO4'7HzO, 0.01 gm; K2SO4, 0.35 gm; sucrose, 30 gm; magnesium glycerophosphate, 0.5 gin; agar, 15 gm; distilled water, 1000 ml; pH 5.5), starch casein agar (casein, 2.0 gm; NaC1, 2.0 gm; KzSO4, 2.0 gm; MgSO4.7H20, 0.05 gm; CaCOa, 0.02 gin; FeSO4-7H20, 0.01 gm; soluble starch, 10 gm; agar, 15 gm; distilled water, 1000 ml) and nutrient agar, (BBL, Cockeysville, Maryland) respectively, as isolation media. Five plates at each dilution were incubated at 25~ and the numbers of microorganisms per gram of soil were determined. COs evolution from glucose- and straw-amended soil. Ninety-gram samples of the sandy loam soil amended with 2% glucose (w/w) or straw (23.6 mesh/cm) were placed in 500-ml flasks (125 cm 2 surface area) and sprayed with the equivalent of one and five times the F.A.R. of Gramoxone. Paraquat adsorbed onto ground straw was added to soil samples to yield a concentration of 500 /zg/gm. Distilled water was used in the controls and nonamended samples were included. Fivemilliliter portions of a 0.2 M KOH solution were pipetted into 10-ml beakers suspended from rubber stoppers and the flasks were plugged to form an air-tight system. Three flasks at each treatment were incubated at 25~ and the micromoles of COz evolved from the glucose- and straw-amended samples were determined by titration at 2- and 7-day intervals, respectively, using methyl red indicator. Cellulose decomposition. Cotton duck (Dominion Textiles Ltd., Toronto, Ontario, Canada) strips (2 x 5 cm), which had been soaked for 12 hr in Gramoxone solution containing various concentrations of paraquat, were placed (three strips at each concentration) in 25-ml burettes. Strips soaked in distilled water served as controls. Paraquat was eluted from the strips with saturated NH4CI solution at a flow rate of 1 ml/min and after 1 hr of soaking, 100 ml of the eluate were analyzed [11]. Thirty strips at each concentration were incubated in randomly selected trays (30 • 30 x 15 cm) containing sandy loam soil and the amount of cellulose decomposition (i.e., cotton duck degradation) during a 31-day incubation at 25~ was determined according to the Textile Test Methods [24]. The strip at each concentration, which showed an intermediate amount of degradation, was photographed after 5, 10, 15, 20, 23, 28, and 31 days of incubation. Nitrification. Fifty-gram samples of the sandy clay loam soil containing 80 p,g/gm of NH4"N, 0.2 gm of Ca(OH)~, and 625, 1250, 2500, 5000, or 10000/xg/gm of paraquat as Gramoxone were placed in 250-ml flasks. Nonamended and paraquat-free samples were included. The moisture content of each sample was adjusted to 30% with distilled water. Three flasks at each treatment were sealed with parafilm and incubated at 25~ The amounts of NOa--N were determined, according to the method of Jackson [ 10], at 7-day intervals during a 28-day incubation. Cfl-lrreduction assays. Sandy loam soil at a moisture content of 25% and containing 2% glucose (w/w) was moulded into 1-gm aggregates with a spatula. Aggregates containing 290, 720, 1400, and 2900/tg/gm of paraquat as Gramoxone were also prepared. Three aggregates at each treatment were placed in 25-ml flasks. The flasks were capped, evacuated, and backfilled with N2 and were incubated at 25~ for 48 hr. Three flasks were prepared for each treatment. The C2Hz reduction as-
Effect of Paraquat on Soil Bacteria
335
says were performed to determine the effect of paraquat on nitrogenase synthesis, according to the method of Mayfield and Aldworth [15]. To study the effect of paraquat on nitrogenase activity, as opposed to nitrogenase synthesis, paraquat was added to aggregates amended with 2% glucose after the 48-hr incubation period. Gramoxone solutions were injected onto soil aggregates with a l-ml syringe to yield concentrations of 290, 580, 1400, and 2900/.tg/gm of paraquat. The C~H~reduction assays were performed after 1 and 4 hr of incubation at 25~ and the rate was determined as the difference between the two time points. C2H2 reduction assays were also performed on aggregates containing 2900 p.g/gm of paraquat but amended with 2% glucose plus various levels of straw or peat (26.3 mesh/cm) and on aggregates containing 5900/zg]gm of paraquat adsorbed onto Zerolit 225 cation-exchange resin (BDH Chemicals Ltd., Poole, England). After a 48-hr incubation and following the l hr CzH2-reduction assays, 1 gm of resin was added to some flasks of soil containing 2900/xg/gm of paraquat in order to adsorb some of the herbicide. Results
Soil, treated with one and five t i m e s the suggested F . A . R , o f G r a m o x o n e , s h o w e d no significant increases in the n u m b e r s o f fungi, a c t i n o m y c e t e s or bacteria during a 24-hr incubation period. A f t e r 14 days h o w e v e r , the n u m b e r o f aerobic heterotrophic bacteria increased f r o m 1.6 • 10 a to 4 . 4 x 10X0/gm in soil treated w i t h five t i m e s the F . A . R . o f G r a m o x o n e (Table 1). T h e n u m b e r s o f fungi and a c t i n o m y c e t e s also s h o w e d nearly a 100-fold increase. T h e l e v e l s o f CO2 e v o l v e d f r o m sandy l o a m soil a m e n d e d w i t h 2% g l u c o s e w e r e significantly l o w e r w h e n paraquat was added to the soil. T r e a t m e n t s o f one and five t i m e s the F . A . R . r e d u c e d COs e v o l u t i o n by 4 4 % and 6 7 % , r e s p e c t i v e l y , during a 12-day incubation (Table 2). C o n s i d e r a b l y l o n g e r incubation periods w e r e r e q u i r e d to detect CO2 e v o l u t i o n f r o m soil a m e n d e d with 1% straw. A f t e r 28 days, soil sprayed with one and five t i m e s the F . A . R . o f G r a m o x o n e p r o d u c e d 39% and 57% less COz than did untreated soil and the level o f CO2 e v o l v e d f r o m soil a m e n d e d with straw was m o r e than t w i c e that o f n o n a m e n d e d soil (Table 3).
Table 1 Effect of Paraquat on Numbers o f Some Microorganisms in Soil Incubation (days)
Paraquat applied
1 1 1 14 14 14
0 1 x F.A.R. a 5 x F.A.R. 0 1 x F.A.R. 5 x F.A.R.
N u m b e r o f m i e r o o r g a n i s m s / g m soil Fungi 3.8 x 6.7 x 5.4 x 4.1 x 8.4">< 1.2 x
al • F.A.R. = field application rate (2.0 lb/A).
lO s l0 s l0 s l0 s 10 6 10 7
Actinomycetes 5.3x 4.1 x 3.6 x 5.2 x 7.2x 1.3 x
l0 s l0 s l0 s l0 s 10 s 10 7
Bacteria 1.6x 1.5 x 2.0 x 1.2x 6.0x 4.4x
10 a 10 a l0 s 10 8 10 9 10 x~
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E. A. Smith and C. I. Mayfield
Table 2 Effect o f Paraquat on C02 Evolution from 2% Glucose-A mended Sandy Loam Soil Incubation
CO2 evolved (/.tmol)
(days)
Untreated a
1 x F.A.R.
5 • F.A.R.
Nonamended b
2 4 6 8 10 12
44 71 126 162 184 226
9 39 64 73 111 122
6 24 9 51 55 70
6 10 25 31 34 39
aNo paraquat. bNo glucose, no paraquat.
Increasing the level of organic matter in soil has been found to increase the rates and amounts o f CO2 evolution [4]. Cotton duck fabric served as an excellent source o f cellulose for cellulolytic microflora. Noticeable colonization o f the cotton duck was observed after a 5-day incubation at 25~ The concentrations o f the herbicide, which inhibited cellulase activity as indicated by the colonization of the strips, are shown in Figure 1. After 5 days of incubation in soil containing a large population of cellulolytic microorganisms, no visible colonization of the treated strips had occurred. Growth was noticeable on the strips containing 5 and 9 ~ g / m g of paraquat after a 10-day incubation, and after 15 days colonization on the fringes of the strips containing 13 and 1 9 / z g / m g of paraquat was observed. A 28-day incubation was required before microorganisms could attack strips containing 176 p,g/mg of paraquat. If watering of the plots leached paraquat from the strips, then the concentrations listed may be overestimates o f the actual inhibitory concentrations. Table 3 Effect o f Paraquat on C02 Evolution from 1% Straw-A mended Sandy Loam Soil CO 2 evolved (/amol) Incubation (days)
Straw only
1 x F.A.R.
5 • F.A.R.
Paraquats o a k e d straw a
Nonamended b
7 14 21 28
8 31 44 71
10 32 37 42
13 23 29 31
35 48 67 91
6 17 22 29
aparaquat-soaked straw was added to soil to yield 500 t~g of paraquat per gram of soil. bNo straw, no paraquat.
Effect of Paraquat on Soil Bacteria
337
Fig. 1. Effect of paraquat on cellulose decomposition. Dark areas on strips of cotton duck fabric represent colonization.
Paraquat applied to soil to yield 625/xg/gm caused only a 10% reduction in the levels of NO3--N after a 28-day incubation at 25~ and 5000/zg/gm was necessary to inhibit nitrification by 44% (Table 4). Paraquat at 720 p.g/gm of soil, suppressed C2H2 reduction in artificial soil aggregates by more than 50% (Table 5). Still higher concentrations of the herbicide were required to inhibit nitrogen-
338
E. A. Smith and C. I. Mayfield
Table 4 Effect of Paraquat on Nitrification in Soil Paraqua t 0ag/gm soil)
N O 3-- Na (/.tg/gm soil)
Percent Inhibition
0 625 1250 2500 5000 10000
170 150 120 95 28 10
0 12 30 44 84 94
aDetermined after a 28-day incubation at 25~
ase a c t i v i t y ; 1400 / z g / g m i n h i b i t e d CzHz r e d u c t i o n by 6 8 % in a g g r e g a t e s a m e n d e d with 2% glucose (Table 5). W h e n the capacity o f the soil for adsorbing paraquat was increased by adding o r g a n i c matter in the f o r m of peat or straw, the h e r b i c i d e c o n c e n t r a t i o n , w h i c h p r e v i o u s l y totally i n h i b i t e d a c t i v i t y ( 2 9 0 0 / z g / g m ) , s h o w e d d e c r e a s e d inhibitory effects (Table 5). Soil a g g r e g a t e s a m e n d e d with 1% peat and c o n t a i n i n g 2900 ~ g / g m of paraquat, s h o w e d almost a 30-fold increase in C2H2 reduction activity as c o m p a r e d to n o n a m e n d e d aggregates c o n taining the same l e v e l o f herbicide.
Table 5 Effect o f Paraquat (Gramoxone) on C2H 2 Reduction in Artificial Aggregates o f Soil A mended with 2 ~ Glucose and the Effect o f Organic Matter and Herbicide Availability under Anaerobic Conditions Paraquat /ag/gm soil
C2 H2 reduced (nmol hr-1 gm-1 soil dry weight) a
b
0 290 580 720 1400 2900
130 95(27)* 75(43)* 42(68)* 2(99)*
146 86(41)* 70(52)* 28(81)* 9(99)*
23800 e
-
-
a 175 a 0.09a(99) * 181(3)*
b [ 157,176,171] f ---[0.6(99)*,10(94)*, 17(89)*g -
* (% inhibition) a Paraquat added before 48-hr incubation (nitrogenase synthesis). b Paraquat added after 48-hr incubation (nitrogenase activity). c Soil amended with 1 gm of cation-exchange resin. d Resin added after 48-hr incubation to adsorb herbicide. e Resin-bound paraquat. f Nonamended (157), 2% straw-amended (176) and 1% peat-amended (171). g Nonamended (0.6), 2% straw-amended (10) and 1% peat-amended (17).
Effectof Paraquaton Soil Bacteria
339
The availability of the herbicide seemed to be the critical factor in controlling its toxicity. Aggregates amended with 2% glucose and containing 2900 /.Lg/gm of paraquat demonstrated no C2H2 reduction activity but soil containing 23,800 /xg of resin-bound paraquat had virtually normal activity (Table 5). Paraquat bound to cation-exchange resin was not available to the microorganisms. The resin was not toxic to the microbial population and the addition of resin to soil after paraquat treatment did not alleviate the inhibitory effects of the compound.
Discussion Foliage-applied Gramoxone reaches the soil through drippage and overspray. Estimates of the numbers of selected microorganisms in the soil following paraquat application indicated that there may not be an immediate response to the herbicide. However, during a 14-day exposure period, the numbers of actinomycetes, fungi, and bacteria increased about 100-fold. The microorganisms probably did not utilize the paraquat, the active ingredient in Gramoxone, as a source of carbon or nitrogen, but it is likely that other components of the Gramoxone formulation were utilized. Bacterial decomposition of surface active agents such as those used as wetting agents in herbicide preparations has been described [22]. Similar increases in numbers of actinomycetes and bacteria during an 8-day incubation in paraquat treated soil have been noted [9]. Paraquat, at concentrations as low as 12.5 ~g/gm has been found to inhibit CO2 evolution in soil containing Sclerotium rolfsii and a level of 500 ~g/gm of paraquat in soil almost completely eliminated respiration of this microorganism during the first 14 days of incubation [ 19]. These results show that the herbicide can inhibit metabolic activities of microorganisms in situ at concentrations far below the capacity of the soil for binding paraquat. In these experiments the response of the entire microbial population was assessed, and concentrations of the herbicide used were those that were necessary to inhibit the most paraquat resistant microoganisms. The F.A.R. of Gramoxone (2 lb/A) inhibited CO2 evolution by 44% in sandy loam soil during a 28-day incubation at 25~ The observed increases in the numbers of microorganisms in paraquat treated soil, as determined by plate count, may represent an overestimate of the actual increases because microorganisms, which may have been severely inhibited in the presence of the herbicide, may recover from the effects of the herbicide in nutrient media. Cellulose decomposition was found to be a paraquat resistant process. A level of 9 / z g of paraquat per milligram of cotton duck fabric was required to inhibit cellulase activity for 15 days in soil containing a large population of cellulose-decomposing microorganisms. Although the cellulolytic microflora
340
E.A. Smith and C. I. Mayfield
were not isolated and identified, the herbicide was able to inhibit a large portion of the population, since it is well known that many species of bacteria, actinomycetes, and fungi are capable of degrading cellulose both aerobically and anaerobically [1]. The effects of various inhibitory compounds on nitrification in soils has been described previously [13]. Physical characteristics of soils, such as aggregate size, moisture content, and oxygen availability, will affect the process of nitrification and will also influence the availability of potentially inhibitory chemicals [ 14, 20]. Various pesticides, which do not bind tenaciously to soil components, may inhibit soil processes at field application rates due to their availability. In contrast, paraquat applied to soil to yield 5000 p,g/gm inhibited nitrification by only 44% during a 28-day incubation. Nitrification, therefore, would not appear to be a microbial process that would be adversely affected by the herbicide. These findings agree with the results reported by other workers. [25, 26]. Tenacious binding of the herbicide by soil components [12, 27] required that concentrations as high as 2900/xg/gm of paraquat be used to inhibit C2H2 reduction activity by 99%. It may be possible for microbial activities to be affected, even though the compound is bound to soil components. Adsorption of paraquat and bacteria onto the surfaces of soil particles would facilitate contact of the herbicide by microorganisms. Thus, concentrations of paraquat that would saturate all of the binding sites of a soil would not be necessary to inhibit microbial activities. Higher concentrations of paraquat were required to inhibit nitrogenase activity compared with nitrogenase synthesis in artificial aggregates of soil amended with 2% glucose. These results may be explained on the basis of bacterial numbers. After 48 hr at 25~ a large population of N2 fixing bacteria had been cultivated, and as a result, more herbicide was needed to inhibit their activ-. ity. Organic matter has been found to play an important role in soil adsorption of paraquat [17]. Increasing the organic matter content of soil increased the capacity of the soil for binding paraquat by supplying additional adsorption sites. Osgerby [16] found that higher levels of paraquat were required in soils with a high organic matter content than soils containing less organic matter to achieve the same level of weed control. The inhibitory concentration of 2900/zg/gm of paraquat was not as harmful to the N2 fixing microorganisms in soil amended with organic matter in the form of peat or straw. The availability of the herbicide may have been reduced through binding onto the organic material, or the organic matter supplied additional nutrients, which may have permitted the bacteria to overcome the inhibitory effects of the herbicide; the organic matter significantly stimulated nitrogenase synthesis in the control samples.
Effect of Paraquat on Soil Bacteria
341
The adsorption capacity and cation-exchange capacity of soil will determine the amount of herbicide available and therefore inhibitory to paraquatsensitive microorganisms. Paraquat-saturated cation-exchange resin added to soil to yield 2 3 , 8 0 0 / x g / g m had no noticeable effect on C2H2 reduction, indicating that the herbicide was not available in this form. Generally, the levels of paraquat required to inhibit various microbial processes in soil were higher than those that would be encountered in the field except in isolated areas receiving increased amounts of paraquat through drippage and overspray. Adsorption o f the herbicide by binding sites in soil effectively reduces the toxicity of paraquat to microorganisms. It is therefore unlikely that the concentrations of G r a m o x o n e that are presently being used (0,5 to 2,0 Ib/A) for weed control will have any long-term deleterious effects on nitrification or cellulose decomposition in soil. However, decreased levels of respiration and the sensitivity of asymbiotic, anaerobic, Na fixing microorganisms to paraquat suggests that injurious effects may be observed in areas receiving herbicide treatments. Although paraquat-free Gramoxone formulation was not investigated in this study, it is unlikely that the low levels o f wetting agents present in Gramoxone will contribute significantly to the herbicide's toxicity. Since extremely low doses of paraquat are lethal to green foliage, if increased care is exercised in application o f the herbicide, lower application rates m a y prove to be efficient in killing weeds and reducing toxicity o f the chemical to nontarget organisms.
Acknowledgements This study was supported by a grant from the Pesticides Advisory Committee, Ontario Ministry of the Environment.
References Alexander, M. (ed.) 1961. Microbiology of cellulose. In: Introduction to Soil Microbiology. pp. 163-180. John Wiley and Sons, New York. 2.
Almog, G. and Tal, E. 1967. Death from paraquat after subcutaneous injection. Br. Med. J. 3: 721.
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Baldwin, B.C., Dodge, A.D. and Harris, N. 1968. Recent studies of the mode of action of the bipyridylium herbicides. Proc. Br. Weed Control Conf. 10: 630-644.
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Bingeman, C.W., Wagner, J.E. and Martin W.P. 1953. The effect of the addition of organic materials on the decomposition of an organic soil. Soil Sci. Soc. Am. Proc. 17: 34-38.
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Crosby, D.G., and Tucker R.K. 1966. Toxicity of aquatic herbicides to Daphnia magna. Sc&nce 154: 289-290.
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Edwards, G.A. 1965. Some side effects resulting from persistent insecticides. Ann. Appl. Biol. 55: 329.
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Farrington, J.A., Ebert, M., Land, E.J. and Fletcher, K. 1973. Bipyridylium quoternary salts and related compounds. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of the bipyridyl herbicides. Biochim. Biophys. Acta. 314: 372-381.
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Favarel-Garriques, J.C. 1972. Acute pulmonary fibrosis resulting from the ingestion of herbicides containing paraquat. Bull. Physiopathol. Respir. 8: 1289-1292.
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Funderburk. H.H. Jr., and Bozarth, G.A. 1967. Review of the metabolism and decomposition of diquat and paraquat. J. Agric. Fd. Chem. 15: 563-567.
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Jackson, M.L. (ed.) 1970. Nitrate determination. In: Soil Chemical Analysis, pp. 197--201. Prentice Hall, New Jersey.
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Jealott's Hill Research Station. 1972. Determination of Residues in Soil; Spectrophotometric Method. Bracknell Berkshire, England, I.C.I. Plant Protection Limited.
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Knight, B.A.G., and Tomlinson, T.E. 1967. The interaction of paraquat (1,1'-dimethy-4, 4'dipyridylium dichloride) with mineral soils. J. Soil Sci. 18: 233-243.
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Lees, H , and Quastel, J.H. 1946a. Biochemistry of nitrification in soil. I. Kinetics of the effects of poison on soil nitrification as studied by the soil perfusion technique. Biochemistry 40: 803-814.
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Lees, H., and Quastel, J.H. 1946b. Biochemistry of nitrification in soil. I1. The site of soil nitrification. Biochemistry 40: 814-823.
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Mayfield, C.I., and Aldworth, R.L. 1974. Acetylene reduction by nonsymbiotic bacteria in artifical soil aggregates amended with glucose. Can. J. Microbiol. 20: 877-881.
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Osgerby, J.M. 1973. Processes affecting herbicides action in soil. Pestic. Sci. 4: 247-258.
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Radelli, L., and Fusi, P. 1968. Adsorption and desorption of dipyridyl cations by the organic colloids of soil. Agrochimica 12: 558-566.
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Raporporti, E.H., and Cangioli, G. 1963. Herbicides and soil fauna. Pedobiologia, 2: 235238.
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Rodriguez-Kabana, R., Curl, E.A. and Funderburk, H.H. Jr. 1967. Effect of paraquat On growth of Sclerotium rolfsii in liquid culture and soil. Phytopathology 57:911-915.
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Seifert, J. 1964. Influence of size of soil structural aggregates on degree of nitrification. Fol. Microbiol. 9: 347.
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Sinow, J. and Wei, E. 1973. Ocular toxicity of paraquat. Bull. Environ. Contain. Toxicol. 9: 163-168.
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Skinner, F.A. 1959. Decomposition of anionic surface active agents by bacteria. Nature (London), 183: 548-549.
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Surber, E.W., and Pickering, Q.H. 1962. Acute toxicity of Endothal, diquat, hyamine, Dalapon and Silvex to fish. Progr. Fish-Cult. 24: 164-171.
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Thornburg, R.P., and Tweedy, J.A. 1973. A rapid procedure to evaluate the effect of pesticides on nitrification. WeedSci. 21: 397-399.
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Tu, C.M., and Bollen, W.B. 1968. Effect of paraquat on microbial activities in soils. Weed Res. 8: 28-37.
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Weed, S.B., and Weber, J.B. 1969. The effect of cation-exchange capacity on the retention of diquat ~+ and paraquat 2+ by three layer-type clay minerals. I. Adsorption and release. Soil Sci. Soc. Am. Proc. 33: 379-382.
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Wilkins, R.J., and Tetlow, R.M. 1970. The effect of diquat and paraquat used as desiccants on the moisture content of maize for silage. WeedRes. 10: 288-292.
Abstract. Paraquat, applied as Gramoxone, to a nonamended sandy loam soil at five times the suggested field application rate (10 lb/A ~ 115/xg/cm 2) increased the numbers of bacteria, actinomycetes, and fungi during a 14-day incubation at 25~ This increase was attributed to the use of compounds in the Gramoxone formulation rather than the use of paraqnat. Treatment at one and five times the normal rate reduced CO2 evolution by 44% and 67%, respectively, in soil amended with 2% glucose during a 12-flay incubation. Similar treatments reduced CO~ evolution in 1% straw-amended 9 soil by 39% and 58%, respectively, during a 28-day incubation. Cellulose decomposition of cotton duck containing 13 and 176 ~g of paraquat per milligram of material was inhibited for 15 and 28 days, respectively, in soil containing a large population of cellulolytic microorganisms. A concentration of 5000/~g/gm of paraquat was necessary to inhibit nitrification in soil by 44% druing a 28-day incubation at 20~ Paraquat inhibited C2H2 reduction in artificial aggregates of soil amended with 2% glucose and incubated anaerobically at 25~ Nitrogenase activity in aggregates was inhibited by 43 % and 52% at concentrations of 580 and 720 ~g/gm of paraquat respectively. The inhibitory effects of the herbicide were reduced when soil was amended with organic matter in the form of peat or straw. The availability of paraquat controlled the toxicity of the herbicide to soil microorganisms.
Introduction T h e d i p y r i d y l i u m h e r b i c i d e s , p a r a q u a t a n d d i q u a t , w e r e i n t r o d u c e d as effective nonselective herbicides, and a large amount of information has since been d o c u m e n t e d d e s c r i b i n g t h e i r p h y t o t o x i c m o d e o f a c t i o n [3, 7, 28]. A l t h o u g h p a r a q u a t h a s b e e n s h o w n to b e t o x i c to n o n t a r g e t o r g a n i s m s , s u c h a s t h e soil f a u n a Collembola, Acari, andHomoptera [6, 18], o t h e r i n v e r t e b r a t e s a n d fish [5, 23], a n d e x p e r i m e n t a l l a b o r a t o r y a n i m a l s a n d m a n [2, 8, 21], t h e r e is a l a c k o f i n f o r m a t i o n p e r t a i n i n g to t h e effects o f t h e h e r b i c i d e o n soil m i c r o o r g a n i s m s . It h a s b e e n s u g g e s t e d t h a t the r e c o m m e n d e d field a p p l i c a t i o n rates o f G r a m o x o n e (0.5 to 2 . 0 l b / A ) w i l l h a v e no a p p r e c i a b l e effects o n m i c r o b i a l a c t i v i t i e s that are i m p o r t a n t to soil fertility [ 2 5 , 26]. In t h e p r e s e n t s t u d y , the effects o f v a r i o u s c o n c e n t r a t i o n s o f p a r a q u a t o n s e l e c t e d m i c r o b i a l a c t i v i t i e s in soils arc e x a m i n e d .
Materials and Methods
Soils. In this study two types of soil were used. Sandy clay loam was collected from the surface 10 cm of an uncultivated grassland site in Waterloo County, Ontario. Its pH was 7.4 and it conMicrobial Ecology 3,333-343 (1977) 9 1977 by Springer-Verlag New York Inc.
334
E . A . Smith and C. I. Mayfleld
tained 0.06% nitrogen (oven-dried at 105~ to constant weight). Sandy loam soil (St. Bernard sandy loam) was collected from the plow-layer (0 to 15 cm) of a pasture on the Macdonald College Farm, Quebec. Its pH was 7.2 and it contained 0.23% nitrogen (oven-dry basis). The soils were stored at 4~ during the experimental period.
Paraquat. Paraquat used in this investigation was obtained directly from Chipman Chemicals Ltd., Stoney Creek, Ontario as the 'Gramoxone' formulation. It contained 240 gm/liter paraquat (99.9% pure 1,1'-dimethyl-4,4'-dipyridilium dichloride) as the active ingredient. Numbers of microorganisms. Fifty-gram samples of the sandy loam soil were sprayed with the field application rate (F.A.R. = 0.5 to 2.0 lb/A) of Gramoxone. The moisture content of the soil was adjusted to 25% with distilled water. After 1 and 14 days of incubation at 20~ estimates of the numbers of fungi, actinomycetes, and aerobic heterotrophie bacteria were obtained by the pour-plate method, using Czapek Dox agar (NaNO3, 2.0 gm; KCI, 0.5 gm; FeSO4'7HzO, 0.01 gm; K2SO4, 0.35 gm; sucrose, 30 gm; magnesium glycerophosphate, 0.5 gin; agar, 15 gm; distilled water, 1000 ml; pH 5.5), starch casein agar (casein, 2.0 gm; NaC1, 2.0 gm; KzSO4, 2.0 gm; MgSO4.7H20, 0.05 gm; CaCOa, 0.02 gin; FeSO4-7H20, 0.01 gm; soluble starch, 10 gm; agar, 15 gm; distilled water, 1000 ml) and nutrient agar, (BBL, Cockeysville, Maryland) respectively, as isolation media. Five plates at each dilution were incubated at 25~ and the numbers of microorganisms per gram of soil were determined. COs evolution from glucose- and straw-amended soil. Ninety-gram samples of the sandy loam soil amended with 2% glucose (w/w) or straw (23.6 mesh/cm) were placed in 500-ml flasks (125 cm 2 surface area) and sprayed with the equivalent of one and five times the F.A.R. of Gramoxone. Paraquat adsorbed onto ground straw was added to soil samples to yield a concentration of 500 /zg/gm. Distilled water was used in the controls and nonamended samples were included. Fivemilliliter portions of a 0.2 M KOH solution were pipetted into 10-ml beakers suspended from rubber stoppers and the flasks were plugged to form an air-tight system. Three flasks at each treatment were incubated at 25~ and the micromoles of COz evolved from the glucose- and straw-amended samples were determined by titration at 2- and 7-day intervals, respectively, using methyl red indicator. Cellulose decomposition. Cotton duck (Dominion Textiles Ltd., Toronto, Ontario, Canada) strips (2 x 5 cm), which had been soaked for 12 hr in Gramoxone solution containing various concentrations of paraquat, were placed (three strips at each concentration) in 25-ml burettes. Strips soaked in distilled water served as controls. Paraquat was eluted from the strips with saturated NH4CI solution at a flow rate of 1 ml/min and after 1 hr of soaking, 100 ml of the eluate were analyzed [11]. Thirty strips at each concentration were incubated in randomly selected trays (30 • 30 x 15 cm) containing sandy loam soil and the amount of cellulose decomposition (i.e., cotton duck degradation) during a 31-day incubation at 25~ was determined according to the Textile Test Methods [24]. The strip at each concentration, which showed an intermediate amount of degradation, was photographed after 5, 10, 15, 20, 23, 28, and 31 days of incubation. Nitrification. Fifty-gram samples of the sandy clay loam soil containing 80 p,g/gm of NH4"N, 0.2 gm of Ca(OH)~, and 625, 1250, 2500, 5000, or 10000/xg/gm of paraquat as Gramoxone were placed in 250-ml flasks. Nonamended and paraquat-free samples were included. The moisture content of each sample was adjusted to 30% with distilled water. Three flasks at each treatment were sealed with parafilm and incubated at 25~ The amounts of NOa--N were determined, according to the method of Jackson [ 10], at 7-day intervals during a 28-day incubation. Cfl-lrreduction assays. Sandy loam soil at a moisture content of 25% and containing 2% glucose (w/w) was moulded into 1-gm aggregates with a spatula. Aggregates containing 290, 720, 1400, and 2900/tg/gm of paraquat as Gramoxone were also prepared. Three aggregates at each treatment were placed in 25-ml flasks. The flasks were capped, evacuated, and backfilled with N2 and were incubated at 25~ for 48 hr. Three flasks were prepared for each treatment. The C2Hz reduction as-
Effect of Paraquat on Soil Bacteria
335
says were performed to determine the effect of paraquat on nitrogenase synthesis, according to the method of Mayfield and Aldworth [15]. To study the effect of paraquat on nitrogenase activity, as opposed to nitrogenase synthesis, paraquat was added to aggregates amended with 2% glucose after the 48-hr incubation period. Gramoxone solutions were injected onto soil aggregates with a l-ml syringe to yield concentrations of 290, 580, 1400, and 2900/.tg/gm of paraquat. The C~H~reduction assays were performed after 1 and 4 hr of incubation at 25~ and the rate was determined as the difference between the two time points. C2H2 reduction assays were also performed on aggregates containing 2900 p.g/gm of paraquat but amended with 2% glucose plus various levels of straw or peat (26.3 mesh/cm) and on aggregates containing 5900/zg]gm of paraquat adsorbed onto Zerolit 225 cation-exchange resin (BDH Chemicals Ltd., Poole, England). After a 48-hr incubation and following the l hr CzH2-reduction assays, 1 gm of resin was added to some flasks of soil containing 2900/xg/gm of paraquat in order to adsorb some of the herbicide. Results
Soil, treated with one and five t i m e s the suggested F . A . R , o f G r a m o x o n e , s h o w e d no significant increases in the n u m b e r s o f fungi, a c t i n o m y c e t e s or bacteria during a 24-hr incubation period. A f t e r 14 days h o w e v e r , the n u m b e r o f aerobic heterotrophic bacteria increased f r o m 1.6 • 10 a to 4 . 4 x 10X0/gm in soil treated w i t h five t i m e s the F . A . R . o f G r a m o x o n e (Table 1). T h e n u m b e r s o f fungi and a c t i n o m y c e t e s also s h o w e d nearly a 100-fold increase. T h e l e v e l s o f CO2 e v o l v e d f r o m sandy l o a m soil a m e n d e d w i t h 2% g l u c o s e w e r e significantly l o w e r w h e n paraquat was added to the soil. T r e a t m e n t s o f one and five t i m e s the F . A . R . r e d u c e d COs e v o l u t i o n by 4 4 % and 6 7 % , r e s p e c t i v e l y , during a 12-day incubation (Table 2). C o n s i d e r a b l y l o n g e r incubation periods w e r e r e q u i r e d to detect CO2 e v o l u t i o n f r o m soil a m e n d e d with 1% straw. A f t e r 28 days, soil sprayed with one and five t i m e s the F . A . R . o f G r a m o x o n e p r o d u c e d 39% and 57% less COz than did untreated soil and the level o f CO2 e v o l v e d f r o m soil a m e n d e d with straw was m o r e than t w i c e that o f n o n a m e n d e d soil (Table 3).
Table 1 Effect of Paraquat on Numbers o f Some Microorganisms in Soil Incubation (days)
Paraquat applied
1 1 1 14 14 14
0 1 x F.A.R. a 5 x F.A.R. 0 1 x F.A.R. 5 x F.A.R.
N u m b e r o f m i e r o o r g a n i s m s / g m soil Fungi 3.8 x 6.7 x 5.4 x 4.1 x 8.4">< 1.2 x
al • F.A.R. = field application rate (2.0 lb/A).
lO s l0 s l0 s l0 s 10 6 10 7
Actinomycetes 5.3x 4.1 x 3.6 x 5.2 x 7.2x 1.3 x
l0 s l0 s l0 s l0 s 10 s 10 7
Bacteria 1.6x 1.5 x 2.0 x 1.2x 6.0x 4.4x
10 a 10 a l0 s 10 8 10 9 10 x~
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E. A. Smith and C. I. Mayfield
Table 2 Effect o f Paraquat on C02 Evolution from 2% Glucose-A mended Sandy Loam Soil Incubation
CO2 evolved (/.tmol)
(days)
Untreated a
1 x F.A.R.
5 • F.A.R.
Nonamended b
2 4 6 8 10 12
44 71 126 162 184 226
9 39 64 73 111 122
6 24 9 51 55 70
6 10 25 31 34 39
aNo paraquat. bNo glucose, no paraquat.
Increasing the level of organic matter in soil has been found to increase the rates and amounts o f CO2 evolution [4]. Cotton duck fabric served as an excellent source o f cellulose for cellulolytic microflora. Noticeable colonization o f the cotton duck was observed after a 5-day incubation at 25~ The concentrations o f the herbicide, which inhibited cellulase activity as indicated by the colonization of the strips, are shown in Figure 1. After 5 days of incubation in soil containing a large population of cellulolytic microorganisms, no visible colonization of the treated strips had occurred. Growth was noticeable on the strips containing 5 and 9 ~ g / m g of paraquat after a 10-day incubation, and after 15 days colonization on the fringes of the strips containing 13 and 1 9 / z g / m g of paraquat was observed. A 28-day incubation was required before microorganisms could attack strips containing 176 p,g/mg of paraquat. If watering of the plots leached paraquat from the strips, then the concentrations listed may be overestimates o f the actual inhibitory concentrations. Table 3 Effect o f Paraquat on C02 Evolution from 1% Straw-A mended Sandy Loam Soil CO 2 evolved (/amol) Incubation (days)
Straw only
1 x F.A.R.
5 • F.A.R.
Paraquats o a k e d straw a
Nonamended b
7 14 21 28
8 31 44 71
10 32 37 42
13 23 29 31
35 48 67 91
6 17 22 29
aparaquat-soaked straw was added to soil to yield 500 t~g of paraquat per gram of soil. bNo straw, no paraquat.
Effect of Paraquat on Soil Bacteria
337
Fig. 1. Effect of paraquat on cellulose decomposition. Dark areas on strips of cotton duck fabric represent colonization.
Paraquat applied to soil to yield 625/xg/gm caused only a 10% reduction in the levels of NO3--N after a 28-day incubation at 25~ and 5000/zg/gm was necessary to inhibit nitrification by 44% (Table 4). Paraquat at 720 p.g/gm of soil, suppressed C2H2 reduction in artificial soil aggregates by more than 50% (Table 5). Still higher concentrations of the herbicide were required to inhibit nitrogen-
338
E. A. Smith and C. I. Mayfield
Table 4 Effect of Paraquat on Nitrification in Soil Paraqua t 0ag/gm soil)
N O 3-- Na (/.tg/gm soil)
Percent Inhibition
0 625 1250 2500 5000 10000
170 150 120 95 28 10
0 12 30 44 84 94
aDetermined after a 28-day incubation at 25~
ase a c t i v i t y ; 1400 / z g / g m i n h i b i t e d CzHz r e d u c t i o n by 6 8 % in a g g r e g a t e s a m e n d e d with 2% glucose (Table 5). W h e n the capacity o f the soil for adsorbing paraquat was increased by adding o r g a n i c matter in the f o r m of peat or straw, the h e r b i c i d e c o n c e n t r a t i o n , w h i c h p r e v i o u s l y totally i n h i b i t e d a c t i v i t y ( 2 9 0 0 / z g / g m ) , s h o w e d d e c r e a s e d inhibitory effects (Table 5). Soil a g g r e g a t e s a m e n d e d with 1% peat and c o n t a i n i n g 2900 ~ g / g m of paraquat, s h o w e d almost a 30-fold increase in C2H2 reduction activity as c o m p a r e d to n o n a m e n d e d aggregates c o n taining the same l e v e l o f herbicide.
Table 5 Effect o f Paraquat (Gramoxone) on C2H 2 Reduction in Artificial Aggregates o f Soil A mended with 2 ~ Glucose and the Effect o f Organic Matter and Herbicide Availability under Anaerobic Conditions Paraquat /ag/gm soil
C2 H2 reduced (nmol hr-1 gm-1 soil dry weight) a
b
0 290 580 720 1400 2900
130 95(27)* 75(43)* 42(68)* 2(99)*
146 86(41)* 70(52)* 28(81)* 9(99)*
23800 e
-
-
a 175 a 0.09a(99) * 181(3)*
b [ 157,176,171] f ---[0.6(99)*,10(94)*, 17(89)*g -
* (% inhibition) a Paraquat added before 48-hr incubation (nitrogenase synthesis). b Paraquat added after 48-hr incubation (nitrogenase activity). c Soil amended with 1 gm of cation-exchange resin. d Resin added after 48-hr incubation to adsorb herbicide. e Resin-bound paraquat. f Nonamended (157), 2% straw-amended (176) and 1% peat-amended (171). g Nonamended (0.6), 2% straw-amended (10) and 1% peat-amended (17).
Effectof Paraquaton Soil Bacteria
339
The availability of the herbicide seemed to be the critical factor in controlling its toxicity. Aggregates amended with 2% glucose and containing 2900 /.Lg/gm of paraquat demonstrated no C2H2 reduction activity but soil containing 23,800 /xg of resin-bound paraquat had virtually normal activity (Table 5). Paraquat bound to cation-exchange resin was not available to the microorganisms. The resin was not toxic to the microbial population and the addition of resin to soil after paraquat treatment did not alleviate the inhibitory effects of the compound.
Discussion Foliage-applied Gramoxone reaches the soil through drippage and overspray. Estimates of the numbers of selected microorganisms in the soil following paraquat application indicated that there may not be an immediate response to the herbicide. However, during a 14-day exposure period, the numbers of actinomycetes, fungi, and bacteria increased about 100-fold. The microorganisms probably did not utilize the paraquat, the active ingredient in Gramoxone, as a source of carbon or nitrogen, but it is likely that other components of the Gramoxone formulation were utilized. Bacterial decomposition of surface active agents such as those used as wetting agents in herbicide preparations has been described [22]. Similar increases in numbers of actinomycetes and bacteria during an 8-day incubation in paraquat treated soil have been noted [9]. Paraquat, at concentrations as low as 12.5 ~g/gm has been found to inhibit CO2 evolution in soil containing Sclerotium rolfsii and a level of 500 ~g/gm of paraquat in soil almost completely eliminated respiration of this microorganism during the first 14 days of incubation [ 19]. These results show that the herbicide can inhibit metabolic activities of microorganisms in situ at concentrations far below the capacity of the soil for binding paraquat. In these experiments the response of the entire microbial population was assessed, and concentrations of the herbicide used were those that were necessary to inhibit the most paraquat resistant microoganisms. The F.A.R. of Gramoxone (2 lb/A) inhibited CO2 evolution by 44% in sandy loam soil during a 28-day incubation at 25~ The observed increases in the numbers of microorganisms in paraquat treated soil, as determined by plate count, may represent an overestimate of the actual increases because microorganisms, which may have been severely inhibited in the presence of the herbicide, may recover from the effects of the herbicide in nutrient media. Cellulose decomposition was found to be a paraquat resistant process. A level of 9 / z g of paraquat per milligram of cotton duck fabric was required to inhibit cellulase activity for 15 days in soil containing a large population of cellulose-decomposing microorganisms. Although the cellulolytic microflora
340
E.A. Smith and C. I. Mayfield
were not isolated and identified, the herbicide was able to inhibit a large portion of the population, since it is well known that many species of bacteria, actinomycetes, and fungi are capable of degrading cellulose both aerobically and anaerobically [1]. The effects of various inhibitory compounds on nitrification in soils has been described previously [13]. Physical characteristics of soils, such as aggregate size, moisture content, and oxygen availability, will affect the process of nitrification and will also influence the availability of potentially inhibitory chemicals [ 14, 20]. Various pesticides, which do not bind tenaciously to soil components, may inhibit soil processes at field application rates due to their availability. In contrast, paraquat applied to soil to yield 5000 p,g/gm inhibited nitrification by only 44% during a 28-day incubation. Nitrification, therefore, would not appear to be a microbial process that would be adversely affected by the herbicide. These findings agree with the results reported by other workers. [25, 26]. Tenacious binding of the herbicide by soil components [12, 27] required that concentrations as high as 2900/xg/gm of paraquat be used to inhibit C2H2 reduction activity by 99%. It may be possible for microbial activities to be affected, even though the compound is bound to soil components. Adsorption of paraquat and bacteria onto the surfaces of soil particles would facilitate contact of the herbicide by microorganisms. Thus, concentrations of paraquat that would saturate all of the binding sites of a soil would not be necessary to inhibit microbial activities. Higher concentrations of paraquat were required to inhibit nitrogenase activity compared with nitrogenase synthesis in artificial aggregates of soil amended with 2% glucose. These results may be explained on the basis of bacterial numbers. After 48 hr at 25~ a large population of N2 fixing bacteria had been cultivated, and as a result, more herbicide was needed to inhibit their activ-. ity. Organic matter has been found to play an important role in soil adsorption of paraquat [17]. Increasing the organic matter content of soil increased the capacity of the soil for binding paraquat by supplying additional adsorption sites. Osgerby [16] found that higher levels of paraquat were required in soils with a high organic matter content than soils containing less organic matter to achieve the same level of weed control. The inhibitory concentration of 2900/zg/gm of paraquat was not as harmful to the N2 fixing microorganisms in soil amended with organic matter in the form of peat or straw. The availability of the herbicide may have been reduced through binding onto the organic material, or the organic matter supplied additional nutrients, which may have permitted the bacteria to overcome the inhibitory effects of the herbicide; the organic matter significantly stimulated nitrogenase synthesis in the control samples.
Effect of Paraquat on Soil Bacteria
341
The adsorption capacity and cation-exchange capacity of soil will determine the amount of herbicide available and therefore inhibitory to paraquatsensitive microorganisms. Paraquat-saturated cation-exchange resin added to soil to yield 2 3 , 8 0 0 / x g / g m had no noticeable effect on C2H2 reduction, indicating that the herbicide was not available in this form. Generally, the levels of paraquat required to inhibit various microbial processes in soil were higher than those that would be encountered in the field except in isolated areas receiving increased amounts of paraquat through drippage and overspray. Adsorption o f the herbicide by binding sites in soil effectively reduces the toxicity of paraquat to microorganisms. It is therefore unlikely that the concentrations of G r a m o x o n e that are presently being used (0,5 to 2,0 Ib/A) for weed control will have any long-term deleterious effects on nitrification or cellulose decomposition in soil. However, decreased levels of respiration and the sensitivity of asymbiotic, anaerobic, Na fixing microorganisms to paraquat suggests that injurious effects may be observed in areas receiving herbicide treatments. Although paraquat-free Gramoxone formulation was not investigated in this study, it is unlikely that the low levels o f wetting agents present in Gramoxone will contribute significantly to the herbicide's toxicity. Since extremely low doses of paraquat are lethal to green foliage, if increased care is exercised in application o f the herbicide, lower application rates m a y prove to be efficient in killing weeds and reducing toxicity o f the chemical to nontarget organisms.
Acknowledgements This study was supported by a grant from the Pesticides Advisory Committee, Ontario Ministry of the Environment.
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Farrington, J.A., Ebert, M., Land, E.J. and Fletcher, K. 1973. Bipyridylium quoternary salts and related compounds. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of the bipyridyl herbicides. Biochim. Biophys. Acta. 314: 372-381.
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Mayfield, C.I., and Aldworth, R.L. 1974. Acetylene reduction by nonsymbiotic bacteria in artifical soil aggregates amended with glucose. Can. J. Microbiol. 20: 877-881.
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Osgerby, J.M. 1973. Processes affecting herbicides action in soil. Pestic. Sci. 4: 247-258.
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Radelli, L., and Fusi, P. 1968. Adsorption and desorption of dipyridyl cations by the organic colloids of soil. Agrochimica 12: 558-566.
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Rodriguez-Kabana, R., Curl, E.A. and Funderburk, H.H. Jr. 1967. Effect of paraquat On growth of Sclerotium rolfsii in liquid culture and soil. Phytopathology 57:911-915.
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Seifert, J. 1964. Influence of size of soil structural aggregates on degree of nitrification. Fol. Microbiol. 9: 347.
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Sinow, J. and Wei, E. 1973. Ocular toxicity of paraquat. Bull. Environ. Contain. Toxicol. 9: 163-168.
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Skinner, F.A. 1959. Decomposition of anionic surface active agents by bacteria. Nature (London), 183: 548-549.
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Surber, E.W., and Pickering, Q.H. 1962. Acute toxicity of Endothal, diquat, hyamine, Dalapon and Silvex to fish. Progr. Fish-Cult. 24: 164-171.
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Weed, S.B., and Weber, J.B. 1969. The effect of cation-exchange capacity on the retention of diquat ~+ and paraquat 2+ by three layer-type clay minerals. I. Adsorption and release. Soil Sci. Soc. Am. Proc. 33: 379-382.
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