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A comparison of the breakdown of the triazine herbicides cyanazine, atrazine and simazine in soils and in maize
PESTICIDE
HIOCHEMISTRY
AND
A Comparison
of the
Cyanazine,
Breakdown
Atrazine I<. Shell
!& 153-161
PHYSIOLOGY,
I.
and G.
BEYNON,
Research
Ltd.,
Received
January
of the
Simazine STOYDIN
Woodstock
in Soils AND
Agric~~ltural
21, 1972;
Triazine
accept,ed
A. 2\‘.
and
in Maize
WRIGHT
Research May
Herbicides
Cenlre
3, 1972
The breakdown of three triazine herbicides, cyanazine [Blades, 2-chloro+(ethylamino)-6-(l-cyar~o-l-methylethylamino)-S-triazine], atrazine [2-chloro4-(ethylamine)-6-(isopropylamino)-S-triazine] and simazine [2-chloro-4,6-bis-(ethylamino)S-triazine] in soil, maize sap and maize grown in treated soils has been studied using radioisotope techniques. Atraaine and simazine degraded in soils at a slower rate than did cyanazine. With the former two compounds hydrolysis of the chlorine atom t)o hydroxyl predominated although dealkylation reactions were also evident. With cyanazine, hydrolysis of the nitrile group occurred more rapidly than that of the chlorine at,om. Atrazine was degraded in maize sap at a slower rate than was cyanazine under comparable conditions and only hydrolytic reactions were evident. Atrazine was converted to its hydroxy analogue following hydrolysis of the chlorine atom. Cyanazine was converted by hydrolysis of the chlorine atom and of the nitrile grollp to a carboxy group. When maize plants were grown in soil, chlorotriazines and hydroxy triazines including dealkylated derivatives were present following cyanazine treatment. The chlorotriazines present were probably taken up as such from the soil. Residues in the plants were higher following atrazine and simazine treatment and hydroxy triazines or conjugates predominated although there was also some evidence again for dealkylation reactions.
ucts of [‘*C]cyanazine in soils of different types and in maize grown in treated soils has been described previously (2) and is summarised in Fig. 1. Hydrolytic reactions to form (II), (III) and (IV) predominat’ed in soils. In maize plants dealkylation reactions occurred to a greater extent and the breakdown products in plants were mainly (II), (IV), (VI) and (VIII) together with polar material. The polar material could be hydrolysed with acid to give (IV) and (VIII) and could have been present as conjugates with glutathione or L-cysteinrb similar to those identified in plants following trttatment with atrazine (3, 4). Conjugates with L-cysteine have also been detected in the urine of rats treated with cyanazincb (5).
INTRODUCTION
The herbicide cyanasine, Bladex’ 2-chloro4- (ethylamino)-6-(1-cyano - 1 - methylethylamino) -S-triazine previously known as WL 19805 or SD 15418, shows promise for the control of weeds in cereals, beans, peas and potatoes. It is less persistent (1) than some other triazines such as atrazine and simazine and is of particular use in crops such as maize when it is wished to minimise the problems that can arise from the carry ovel of herbicide residues into subsequent seasons with consequent damage to susceptible rotation crops. The identification of the breakdown prod1 Shell
Registered
Trade
Mark. 153
Copyright All rights
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
154
BEYNON,
IV)
STOYDIN
AND
WI)
Mai*e Soil-trace
(VII)
Maize Sail4race
(Ill Soil Maize
WRIGHT
Maize
Soil hots
only)
WlllJ Soil Maize
WA Soil 1 Conjugates of IIV) and PJIIII in Maize
OX) Soil-trace
FIG,
1. The
breakdown
of cyanazine in soils and in maize grown in
treated
soils.
Degradation of the triazine ring in atraThe metabolism of atra,zine and simazine has been studied extensively and has been zine and its hydroxy analogue has been reviewed (6). N-Dealkylation reactions are shown (9, 10) to be slow in soils, plants or evident as well as hydrolysis of the chlorine animals. However, the degradation is more atom or conjugation reaction of the chloro- rapid in the N-dealkylated analogue, ammetriazine with glutathione or cysteine. Hyline, and in cyanuric acid and the timedrolytic reactions predominate in soils limiting step in the complete degradation of whereas dealkylation, hydrolytic and con- atrazine is probably the dealkylation reacjugation reactions occur in animals and tion. plants. It has been suggested (7) that dealIn the present work the breakdown of kylation reactions are more evident in plants cyanazine has been compared with that of that are susceptible to triazine herbicides. atrazine and simazine in soils, in maize The hydrolytic reactions in plants have grown in treated soil and in the sap of maize been shown (8) to be catalysed by a natu- plants. Experiments were undertaken under rally occurring substance (2,4-dihydroxysimilar conditions and at the same time 7-methoxy-l , 4-benzoxazin-3-one). The hy- with the three compounds so that a comdrolytic reactions contribute significantly to parison of their behaviour could be readily the detoxification of atrazine only when the made. herbicide is introduced to the plant through EXPERIMENTAL the roots (4). The formation of glutathione and cysteine conjugates is now considered RadiolabeledHerbicides (4) to be a more important factor than the Ring-labeled [W]cyanazine of specific achydrolytic reactions in the resistance of tivity 10 nCi/pg was available as described maize to atrazine. previously (2). Ring-labeled [14C]simazineof
COMPARATIVE
BREAKDOWN
OF
TRIAZINE
Simazine
$pccific activity 4.1 nCi/pg was available as a byproduct of the particular synthesis that was used for the [‘*C]cyanazine. Ring-labeled [14C]atrazine of specific activity 7 nCi/fig was supplied by Degussa Forschung Chemie Organisch, Hanau/Main, W. Germany. All of the compounds were at least 99.5% radiochemically pure. Preparatiori
cm1 Treatment of Maize Sap
\‘oung maize plants (Zea mays L., Dekalb XL 45) 7 days after emergence were chopped, cooled to -70°C and powdered in a pestle and mortar. As the plants warmed up the sap was expressed by hand through a muslin bag. The expressed sap was centrifuged at 17,000 y before use. [l*C]Cyanazine and [‘*C]atrazine were added to t.he sap in acetone solution (< 1% of final volume) and the mixtures were left atI ambient temperature (23’C). The sap \\-as not sterile and t,he studies were limited to a 24-hr period before microorganisms had mult’iplicd. The> sap was not, extracted but aliquots (100 ~1) were applied directly to thin layer plates.
HEBBICI!)ES
Ih-
SOILS
.4ND
\JAlZE
I .sT,
cm drep) above a &cm layer of &ones of up to approximately 1 cm diam which w(‘rc present to assist t’he drainage of the pots. Nine maize serds (Zea mays I,., (iolden Bant’am) were sown in each pot. The s(ledS were covered with 1 cm of sifted soil and thp soil surface was treated with [14C]tLyanazine. [‘*C]atrazine or [‘*C]simazine by applying 9 mg of the compound dissolvrd in 10 ml of acctontt to a pot. This \vas equivalent, to an overall application of 1.5 kg/ha. The trclated soil was covered with a further 1 cm of soil and the surface was watered. Some six seeds germinated per pot and all but, one plant werf’ sampled 32 days after sowing by cutting off at, soil level. The remaining plant was sampled when thr male flower \vas formed, 70 days after the sowing and soil treatment. Soil sampleswere>taken 32 and 70 days after treatment. Plant sampleswer(’ cut, up with a pair of scissors and were extra&d in a Sorvall Omni-mixer by homogenising with 20 y5’v, ;v water in methanol, using 4-10 ml of solvent for each 1 g of plant, followed by filtration t’hrough a sintered glass disc and rinsing the residuum with met,hanol. Thr leaf residuum at, 70 days from treatment with cyanazine was reextracted in a Soxhlet apparat’us with water for 16 hr. Soil samples were tbxtractcd with 20 “0 v/v water in met.hanol using 2 ml of solvfbnt to 1 g of soil followed by filtration and washing the residuum with mct’hanol. Before further examination the ext,rac*ts \vertx c*oncentrated by rotary evaporation at .iO-tiO°C using a water pump vacuum. The aqueous solution that. remained was shaken with rhloroform and th(> two phases \v(‘rf’ rxamincd separately. Examinatior, of the Extracts ad Residua
A local soil (Sand 54 % of oven dry weight, Silt 24 %, Clay 20%, Organic Matter 2 %, pH 7.3) was treated with fertiliser and was filled into flower pots (2.5 cm diam and 25
The amounts of radioactivity in the solutions were checkrd at all stages t’o ensure that there were no unexplained losses.Measurements were made with a Packard Tri-
156
BEYNON,
STOYDIN
Carb 4000 Counter using a dioxane-based scintillator. The radioactivity remaining in crops and soils after extraction was determined by combustion to produce 14C02 which was adsorbed in a basic scintillator. The aqueous and organic phases and other extracts were examined by thin layer chromatography on silica gel I?254 ready coated plates (Merck). The components from the organic phases were usually eluted initially with acetone-chloroform (40: 60 v/v) and the components in the aqueous phases were usually eluted initially with ethyl acetate-water-formic acid (70: 10: 10 v/v). “C-Components were located on the plates using a Desaga radiodetector. The separate components were desorbed using acetone for components originally in the organic phase and using 20% v/v water in acetone for components originally in the aqueous phase. The components were examined further using a range of elution systems and these systems and the reference compounds have been described previously (2). The components were chromatographed, mixed with unlabeled reference compounds and, normally, at least six elution systems were used for each identification. The exact coincidence of the shape and the position of the radioactive spot with that of the unlabeled reference compound in all of the systems used was required for identification. Some of the components were examined chromatographically before and after treatment with diazomethane. RESULTS
AND
DISCUSSION
Soil
The compounds identified in soils 32 and 70 days after treatment with cyanazine, atrazine or simazine are indicated in Tables 1 and 2. These compounds were identified by thin layer chromatography as indicated above. Added confirmation of the identity of the carboxylic acid (III) was obtained by
AND
WRIGHT
treating the radiocomponent with diazomethane which converted it to a derivative with the same thin layer chromatographic properties as the methyl ester prepared in the same way from the reference compound (III). Confirmation of the identity of the hydroxy acid (IV) was also obtained by treatment with diazomethane. In this case a mixture of similar products was formed in low yield from both the radiocomponent and the reference compound (IV). One of the product’s was probably the methyl ester and the other the 2-methyl ether of the methyl ester. The breakdown products of cyanazine in soil were similar to those found in previous studies (2). The molecule was changed by hydrolysis of the nitrile group successively to amide and acid and hydrolysis of the 2-chloro group also occurred. No “hydroxy cyanazine” as such was detectable, the initial breakdown apparently being at the nitrile group. The major component present after 32 days was the acid (III), and after 70 days appreciable amounts of (III) were present together with the hydroxy acid (IV), much of the unextracted residue being probably due to the latter. As in the previous studies (2), there was relatively little dealkylation in the soil, although compound (IX) was formed in small amounts. In contrast, atrazine and simazine were considerably more persistent, and the residues of the parent compounds 70 days after application were nearly 12 times greater than those remaining from cyanazine. The breakdown of atrazine and simazine was mainly by hydrolysis of the chlorine atom to the hydroxy analogues, together with some dealkylation to compounds (IX) and (XI). These are reactions in common for all the compounds and, in fact, compound (IX) was formed in small amounts from all three herbicides. The lower persistence of cyanazine compared with that of atrazine and simazine must be due to the presence of the
COMPAR.4TIVE
BREAKDOWN
OF
TRIAZINE
HERBICIDES
TABLE
loam basis
treated at 1.5 kg/ha (near 20y0 wt water)
after maize as sampled.
SOILS
seed (cv.
Golden
produrta
in
Bantam)
maize
(a) In plant compound
Cl
=O.Ol
CZH,
I I
\
Cl Cl Cl Cl Cl Cl
IX II x.
6 XI Unidentified polar Residue unextracted ous methanol Total
8 component with aqua 8-
(b) Parent
cm layer)
IX II III IV XI x Unidentified polar Residue unextracted oua methanol Total
and soil a/ 3,~ rln~/s
was sown.
[‘“Cl cyanazine
In soil (O-3 compound
1 .i7
MAIZE
Soil
residues on
/ Residue expressed as ppm of parent , compound equivalent to the radioactivity detected following application of the compound indicated
Component
Parent
.~PJD
1
lienidtres of cyanazine, atrazine and sinrazine and their breakdown after application at sowing Medium wet. weight
IN
H CzHs
CzHs H H H
I\ =
-
Cl
component 5 with aqua e-
CLH, C(CH,),CN H C(CH&CONH2 C @H&H C(CH&CONH2 H --
Cl Cl Cl OH Cl Cl -
CJib H C(CH&CONH2 C(CH&COOH C(CHa)&OOH H C(CH&H -
Q Some II may have been included in this fraction. b Probably mainly 2-hydroxy analogues of simazine and atrazine. c The component is not an expected metabolite of the particular d Probably IV and VIII.
I
PC1 atrazine
l’Y>! simazine
0.02
0.09
or.03
Total 0.02 0.01 c <0.03
<;.01 2.48” 0.43
0.74
3.06
1.58
0.19
1.65
I .7ti
0.10 0.18 1.56a <0.20 (0.02
0.06 ( c c
C(CH&CN or C(CH&H
CzHs CzHs CzHs C&L H H
I
L parent
compound.
(’
i.04
<0.02 0.17 0.2Ob 0.72 2.86
3.05
158
BEYNON,
STOYDIN
AND
studies there was no loss of radioactivity from the sap solution. In the maize sap, 22 hr after treatment with cyanazine, two metabolites were detected by thin layer chromatography. One was the hydroxy acid (IV) and its identity was confirmed by methylation as described above. The other component was present in similar amounts to the first and was converted into it on treatment with cold M hydrochloric acid. These mild conditions are
nitrile group in cyanazine which is more easily attacked than the chlorine atom. It is not apparently due to any change in the reactivity of the chloro group, since the total residues of chlorotriazines, as opposed to parent herbicides, present in the three soils are similar after 70 days. Plant Sap The persistence of cyanazine and atrazine in maize sap is shown in Fig. 2. In these Residues
wet
of cyanazine,
Medium weight
loam treated basis (near
WRIGHT
TABLE 2 and simazine and their breakdown products at 70 days after application at sowing at 1.5 kg/ha after maize seed (cv. Golden Bantam) 13% wt water) as sampled,
atrazine
in soils
under
was sown.
maize Soil
sampled
residues
on
Residue expressed as p m of parent compound in 0- P..5 cm soil layer” equivalent to the radioactivity detected following application of the compound indicated
- ___- R’
-
--
R2
Parent
compound
C&L
Cl
[‘“Cl
R3 --
C(CH&CN
,:yanazine ___-
_-
0.03
[‘“Cl atrazine
[‘T] simazine
0.40
0.43
0.:3 c
o.‘lo
C(C:s&H or Cd& V IX II III X VI XI IV VIII polar compo-
Unidentified nents Residue unextracted aqueous methanol Total
H
Cl Cl Cl Cl Cl Cl Cl OH OH -
Cd% CzHs Cd% H H H
C&L H -
-
with
-
-
-
C(CHMN H C(CH,)#ONE C(CH&COOI C(CH&H C (CH&CONE H C(CH,)zCOOI C(CH,)nCOOI -
12 I 12
I I
0.05 0.02 0.23
C
o.“lo
c c
0.:3 c
o.“lo c
0 .&.
0 .EofY
0.49
0.34
I
1.11
-
1.03
0 About 75% of 2-hydroxy analogue of atrazine and 20yo of the des-N-ethyl analogue of this with traces of the des-N-isopropyl analogue. * Mainly 2-hydroxy analogue of simazine. c See footnote to Table 1. d Residues in the 7.5-15-cm layer were 10% or less of the residues in the 0-7.5.cm layer and contained the same metabolitee in similar ratios.
(‘OMPARATIVE
BREAKDOWN
OF
TRIAZINE
HERBICIDES
IX
SOILS
ANI)
;\IAIZE
I Xl
FIG. 2. The breakdown of W-cyanazine and W-atrazine in expressed maize sap, at .W’C. (a) “C-ah-azinc at 10 pglml. (b), (c), (d) WY-cyanazine at 160,bO and 10 pg/ml respectively.
probably insufficient to hydrolyse glutathione or cysteine conjugates and the component was probably a salt or sugar conjugate of the carboxylic acid. No other components were detected in the sap 22 hr after treatment. However, another metabolite was detected in small amounts (below 1% of the applied activity) in the sap 4 hr after treatment with cyanazine but not at later times. It did not correspond to any of the compounds in Fig. 1. The hydroxy analogue of atrazine was the only metabolite detected in the sap treated with atrazine. The degradation of atrazine was slower than that of cyanazine at similar concentrations. Neither cyanazine nor atrazine was dealkylated under these conditions and there was no evidence for the formation of glutathione or cysteine conjugates. These results suggest that cyanazine was attacked initially at the nitrile group whereas in atrazine attack is at the chlorine atom. Therefore the reaction mechanism with eyanazine is probably fundamentally different from t,hat of
atrazine, where hydrolysis has been shown (8) to be catalysed by a naturally occurring compound, 2,4-dihydroxy-7-methoxy-l ,+ benzoxazin-a-one. The rate of hydrolysis of cyanazine in maize sap was far greater t,han the rate in buffered aqueous systems (11) and it is likely that the hydrolysis is vatalysed by plant substances. Plants The compounds identified in the maize plants 32 and 70 days after treatment with cyanazine, atrazine or simazine are shown in Tables 2 and 3. Components were ident.ified by thin layer chromatography as indicsated above and confirmation of the identity of compounds such as (IV) and (VIII) was obtained by methylation and further chromatography. The materials referred t#o in Table 2 as (IV) or (VIII) in free and conjugated forms were a mixture of similar amounts of the free compound and of a conjugat,e which released the free compound on heating with 6 M hydrochloric acid (2 hr at 90°C). These conjugates are possibly
160
BEYNON,
STOYDEN
AND
glutathione or cysteine conjugates and this may well apply also to the unidentified polar material that is indicated in Tables 2 and 3. In addition to the unidentified polar material the plants contained radioactive compounds
which were not extracted with methanol. These may well be conjugates although the conjugates of atrazine with methionine or cysteine can be extracted from plants with methanol (3).
TABLE Residues
of cyanazine,
Medium
loam
atrazine
treated
and simazine
at 1.5 kg/ha
-
WRIGHT
3
and their breakdown application at sowing
after
the
maize
seed
(cv.
products
in maize
sampled
70 days
Golden
Bantam)
was sown.
after
Structure
R’ N Component
A
Residue expressed as ppm of parent compound equivalent to the radioactivity detected following application of the compound indicated
‘N
R2HN lNANHR3 --
-
I
R’
--___ WI
R2
WI atrazine -
cyanazine
R3
Leaf
T
[‘“Cl
-
Stem
Leaf
Stem
Leaf
Stem
(0.01
0.02
<0.03
o.iI2 c
<0.03
c c
.-i.01 c
oc42
Tztal
1.12
=
c
c
0.18
3.03
0.22
2.12
0.20
0.01
1.31
0.09
0.49
0.03
0.19
4.41
0.32
2.81
0.23
Parent
compound
CzHz
Cl
C(CHd&N
simazine
-.
or C(CHdaH
V IX II X VI XI IV (free and conjugated) VIII (free and conjugated) Unidentified polar components Unextracted residue* Total Total
-
residue calculated weight basis
Cl Cl Cl Cl Cl Cl HO
H CJL H H H C,H,
$I, C(CHd&N H C(CH&CONH2 C(CH&H C(CH,)zCONH2 H C(CH&COOH
HO
H
C(CHa)&OOH
-
-
-
-
-
-
CzHs
-
<:o.
0.17 2.55
on a whole
100
plant
-! 0.78
-
c 0.05
C
cf.02 C
-I
-I
C
C
c
0."16 c
Tztal
c
=
1.36
-
a No unidentified material was detected in this sample but the presence of VIII could prevent the detection of small amounts of other components. b In the cyanazine study this refers to the residue remaining in the plant after extraction with aqueous material followed by hot water. In the atrazine and simazine study this refers to the residue remaining after extraction with aqueous methanol. c See Footnote to Table 1.
CORIPARATIVE
BREAKDOWN
OF
TRIAZINE
HERBICIDES
IN
SOILS
AND
MAIZE
161
Plantti grown in soils treated with cydifferent components which could occur in anazine contained residues of the chlorotrithe field and not to be an indication of thclir axines (II) and (VI) as well as those of the absolute values. hydroxy triazines (IV) and (VIII). DealkylaREFERENCES tion reactions are slow in the soil so it is 1. T. Chapman, D. Jordan, 11. H. Payne, W. .I. likely that (II) was converted to (VI) in the Hughes and R. H. Schieferst,ein, Proc. 9th plant and not in the soil. Based on the studies Bit. Weed Cont. Conf. 1018 (1968). with maize sap, maize plants are likely to 2. K. I. Beynon, G. Stoydin and 8. N. Wright,, c*onvert’ cyanazine directly to (IV). Thus the Pestic. Ski., in press. 3. G. L. Lamoureux, K. H. Shimabukuro, H. 11. amide (II) present in plants grown in treated Swanson and D. S. Frear, J. Agr. Foorl soil was probably t,aken up by the plant as Chem. 18,81 (1970). su<+h. 4. R. II. Shimabukuro, II. 1~. Swanson and W. (.‘. The total residuesin the maize grown with Walsh, Plant Physiol. 46, 103 (1970). 5. D. H. Hutson, E. C. Hoadley, M. H. Grifiths soil treated with cyanazine were lower than and C. Donninger, J. Agr. Food Chem. 18, those grown in soils treated with atrazine or 507 (1970). simazine. The difference was greater at 32 6. E. Knuesli, D. Berrer, G. Dupuis, and II. days t’han at 70 days after treatment. FurEsser, in “Degradation of Herbicides” thermore, the plants grown in soils treat,ed (P. C. Kearney and 1~. D. Kaufman, Eds.), Marcel Dekker Inc., New York, 1969. with at,razine and simazine contained mainly 7. R. H. Shimabukuro, Plant Ph,ysiol. 42, 1269 residues of t’he hydroxy derivatives. (1967). The residues in the present work were de8. W. Roth, C. R. Acad. Sci. 245,942 (1957). tected in plants grown indoors in boxes with 9. K. Goswami and R. E. Green, Environ. Sti. consequent restrictions on the space for the Technol. 5,426 (1971). root,s and in the absence of wind and rain. 10. G. Dupuis, T. Laanio, P. Marbach and H. 0. Esser, VIIth International Congress of Thus, t#heyare likely to be much larger than Plant Protection, Paris 21-25th Sept,ember, will o(*cur in the field and they should be 1970.Summariesof papersp. 711. considered to be an indication of the type 11. N. P. H. Brown and C. G. L. Furmidge, Pestic. and t,hr rcblative amounts of residues of t’he Sci., in press.
HIOCHEMISTRY
AND
A Comparison
of the
Cyanazine,
Breakdown
Atrazine I<. Shell
!& 153-161
PHYSIOLOGY,
I.
and G.
BEYNON,
Research
Ltd.,
Received
January
of the
Simazine STOYDIN
Woodstock
in Soils AND
Agric~~ltural
21, 1972;
Triazine
accept,ed
A. 2\‘.
and
in Maize
WRIGHT
Research May
Herbicides
Cenlre
3, 1972
The breakdown of three triazine herbicides, cyanazine [Blades, 2-chloro+(ethylamino)-6-(l-cyar~o-l-methylethylamino)-S-triazine], atrazine [2-chloro4-(ethylamine)-6-(isopropylamino)-S-triazine] and simazine [2-chloro-4,6-bis-(ethylamino)S-triazine] in soil, maize sap and maize grown in treated soils has been studied using radioisotope techniques. Atraaine and simazine degraded in soils at a slower rate than did cyanazine. With the former two compounds hydrolysis of the chlorine atom t)o hydroxyl predominated although dealkylation reactions were also evident. With cyanazine, hydrolysis of the nitrile group occurred more rapidly than that of the chlorine at,om. Atrazine was degraded in maize sap at a slower rate than was cyanazine under comparable conditions and only hydrolytic reactions were evident. Atrazine was converted to its hydroxy analogue following hydrolysis of the chlorine atom. Cyanazine was converted by hydrolysis of the chlorine atom and of the nitrile grollp to a carboxy group. When maize plants were grown in soil, chlorotriazines and hydroxy triazines including dealkylated derivatives were present following cyanazine treatment. The chlorotriazines present were probably taken up as such from the soil. Residues in the plants were higher following atrazine and simazine treatment and hydroxy triazines or conjugates predominated although there was also some evidence again for dealkylation reactions.
ucts of [‘*C]cyanazine in soils of different types and in maize grown in treated soils has been described previously (2) and is summarised in Fig. 1. Hydrolytic reactions to form (II), (III) and (IV) predominat’ed in soils. In maize plants dealkylation reactions occurred to a greater extent and the breakdown products in plants were mainly (II), (IV), (VI) and (VIII) together with polar material. The polar material could be hydrolysed with acid to give (IV) and (VIII) and could have been present as conjugates with glutathione or L-cysteinrb similar to those identified in plants following trttatment with atrazine (3, 4). Conjugates with L-cysteine have also been detected in the urine of rats treated with cyanazincb (5).
INTRODUCTION
The herbicide cyanasine, Bladex’ 2-chloro4- (ethylamino)-6-(1-cyano - 1 - methylethylamino) -S-triazine previously known as WL 19805 or SD 15418, shows promise for the control of weeds in cereals, beans, peas and potatoes. It is less persistent (1) than some other triazines such as atrazine and simazine and is of particular use in crops such as maize when it is wished to minimise the problems that can arise from the carry ovel of herbicide residues into subsequent seasons with consequent damage to susceptible rotation crops. The identification of the breakdown prod1 Shell
Registered
Trade
Mark. 153
Copyright All rights
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
154
BEYNON,
IV)
STOYDIN
AND
WI)
Mai*e Soil-trace
(VII)
Maize Sail4race
(Ill Soil Maize
WRIGHT
Maize
Soil hots
only)
WlllJ Soil Maize
WA Soil 1 Conjugates of IIV) and PJIIII in Maize
OX) Soil-trace
FIG,
1. The
breakdown
of cyanazine in soils and in maize grown in
treated
soils.
Degradation of the triazine ring in atraThe metabolism of atra,zine and simazine has been studied extensively and has been zine and its hydroxy analogue has been reviewed (6). N-Dealkylation reactions are shown (9, 10) to be slow in soils, plants or evident as well as hydrolysis of the chlorine animals. However, the degradation is more atom or conjugation reaction of the chloro- rapid in the N-dealkylated analogue, ammetriazine with glutathione or cysteine. Hyline, and in cyanuric acid and the timedrolytic reactions predominate in soils limiting step in the complete degradation of whereas dealkylation, hydrolytic and con- atrazine is probably the dealkylation reacjugation reactions occur in animals and tion. plants. It has been suggested (7) that dealIn the present work the breakdown of kylation reactions are more evident in plants cyanazine has been compared with that of that are susceptible to triazine herbicides. atrazine and simazine in soils, in maize The hydrolytic reactions in plants have grown in treated soil and in the sap of maize been shown (8) to be catalysed by a natu- plants. Experiments were undertaken under rally occurring substance (2,4-dihydroxysimilar conditions and at the same time 7-methoxy-l , 4-benzoxazin-3-one). The hy- with the three compounds so that a comdrolytic reactions contribute significantly to parison of their behaviour could be readily the detoxification of atrazine only when the made. herbicide is introduced to the plant through EXPERIMENTAL the roots (4). The formation of glutathione and cysteine conjugates is now considered RadiolabeledHerbicides (4) to be a more important factor than the Ring-labeled [W]cyanazine of specific achydrolytic reactions in the resistance of tivity 10 nCi/pg was available as described maize to atrazine. previously (2). Ring-labeled [14C]simazineof
COMPARATIVE
BREAKDOWN
OF
TRIAZINE
Simazine
$pccific activity 4.1 nCi/pg was available as a byproduct of the particular synthesis that was used for the [‘*C]cyanazine. Ring-labeled [14C]atrazine of specific activity 7 nCi/fig was supplied by Degussa Forschung Chemie Organisch, Hanau/Main, W. Germany. All of the compounds were at least 99.5% radiochemically pure. Preparatiori
cm1 Treatment of Maize Sap
\‘oung maize plants (Zea mays L., Dekalb XL 45) 7 days after emergence were chopped, cooled to -70°C and powdered in a pestle and mortar. As the plants warmed up the sap was expressed by hand through a muslin bag. The expressed sap was centrifuged at 17,000 y before use. [l*C]Cyanazine and [‘*C]atrazine were added to t.he sap in acetone solution (< 1% of final volume) and the mixtures were left atI ambient temperature (23’C). The sap \\-as not sterile and t,he studies were limited to a 24-hr period before microorganisms had mult’iplicd. The> sap was not, extracted but aliquots (100 ~1) were applied directly to thin layer plates.
HEBBICI!)ES
Ih-
SOILS
.4ND
\JAlZE
I .sT,
cm drep) above a &cm layer of &ones of up to approximately 1 cm diam which w(‘rc present to assist t’he drainage of the pots. Nine maize serds (Zea mays I,., (iolden Bant’am) were sown in each pot. The s(ledS were covered with 1 cm of sifted soil and thp soil surface was treated with [14C]tLyanazine. [‘*C]atrazine or [‘*C]simazine by applying 9 mg of the compound dissolvrd in 10 ml of acctontt to a pot. This \vas equivalent, to an overall application of 1.5 kg/ha. The trclated soil was covered with a further 1 cm of soil and the surface was watered. Some six seeds germinated per pot and all but, one plant werf’ sampled 32 days after sowing by cutting off at, soil level. The remaining plant was sampled when thr male flower \vas formed, 70 days after the sowing and soil treatment. Soil sampleswere>taken 32 and 70 days after treatment. Plant sampleswer(’ cut, up with a pair of scissors and were extra&d in a Sorvall Omni-mixer by homogenising with 20 y5’v, ;v water in methanol, using 4-10 ml of solvent for each 1 g of plant, followed by filtration t’hrough a sintered glass disc and rinsing the residuum with met,hanol. Thr leaf residuum at, 70 days from treatment with cyanazine was reextracted in a Soxhlet apparat’us with water for 16 hr. Soil samples were tbxtractcd with 20 “0 v/v water in met.hanol using 2 ml of solvfbnt to 1 g of soil followed by filtration and washing the residuum with mct’hanol. Before further examination the ext,rac*ts \vertx c*oncentrated by rotary evaporation at .iO-tiO°C using a water pump vacuum. The aqueous solution that. remained was shaken with rhloroform and th(> two phases \v(‘rf’ rxamincd separately. Examinatior, of the Extracts ad Residua
A local soil (Sand 54 % of oven dry weight, Silt 24 %, Clay 20%, Organic Matter 2 %, pH 7.3) was treated with fertiliser and was filled into flower pots (2.5 cm diam and 25
The amounts of radioactivity in the solutions were checkrd at all stages t’o ensure that there were no unexplained losses.Measurements were made with a Packard Tri-
156
BEYNON,
STOYDIN
Carb 4000 Counter using a dioxane-based scintillator. The radioactivity remaining in crops and soils after extraction was determined by combustion to produce 14C02 which was adsorbed in a basic scintillator. The aqueous and organic phases and other extracts were examined by thin layer chromatography on silica gel I?254 ready coated plates (Merck). The components from the organic phases were usually eluted initially with acetone-chloroform (40: 60 v/v) and the components in the aqueous phases were usually eluted initially with ethyl acetate-water-formic acid (70: 10: 10 v/v). “C-Components were located on the plates using a Desaga radiodetector. The separate components were desorbed using acetone for components originally in the organic phase and using 20% v/v water in acetone for components originally in the aqueous phase. The components were examined further using a range of elution systems and these systems and the reference compounds have been described previously (2). The components were chromatographed, mixed with unlabeled reference compounds and, normally, at least six elution systems were used for each identification. The exact coincidence of the shape and the position of the radioactive spot with that of the unlabeled reference compound in all of the systems used was required for identification. Some of the components were examined chromatographically before and after treatment with diazomethane. RESULTS
AND
DISCUSSION
Soil
The compounds identified in soils 32 and 70 days after treatment with cyanazine, atrazine or simazine are indicated in Tables 1 and 2. These compounds were identified by thin layer chromatography as indicated above. Added confirmation of the identity of the carboxylic acid (III) was obtained by
AND
WRIGHT
treating the radiocomponent with diazomethane which converted it to a derivative with the same thin layer chromatographic properties as the methyl ester prepared in the same way from the reference compound (III). Confirmation of the identity of the hydroxy acid (IV) was also obtained by treatment with diazomethane. In this case a mixture of similar products was formed in low yield from both the radiocomponent and the reference compound (IV). One of the product’s was probably the methyl ester and the other the 2-methyl ether of the methyl ester. The breakdown products of cyanazine in soil were similar to those found in previous studies (2). The molecule was changed by hydrolysis of the nitrile group successively to amide and acid and hydrolysis of the 2-chloro group also occurred. No “hydroxy cyanazine” as such was detectable, the initial breakdown apparently being at the nitrile group. The major component present after 32 days was the acid (III), and after 70 days appreciable amounts of (III) were present together with the hydroxy acid (IV), much of the unextracted residue being probably due to the latter. As in the previous studies (2), there was relatively little dealkylation in the soil, although compound (IX) was formed in small amounts. In contrast, atrazine and simazine were considerably more persistent, and the residues of the parent compounds 70 days after application were nearly 12 times greater than those remaining from cyanazine. The breakdown of atrazine and simazine was mainly by hydrolysis of the chlorine atom to the hydroxy analogues, together with some dealkylation to compounds (IX) and (XI). These are reactions in common for all the compounds and, in fact, compound (IX) was formed in small amounts from all three herbicides. The lower persistence of cyanazine compared with that of atrazine and simazine must be due to the presence of the
COMPAR.4TIVE
BREAKDOWN
OF
TRIAZINE
HERBICIDES
TABLE
loam basis
treated at 1.5 kg/ha (near 20y0 wt water)
after maize as sampled.
SOILS
seed (cv.
Golden
produrta
in
Bantam)
maize
(a) In plant compound
Cl
=O.Ol
CZH,
I I
\
Cl Cl Cl Cl Cl Cl
IX II x.
6 XI Unidentified polar Residue unextracted ous methanol Total
8 component with aqua 8-
(b) Parent
cm layer)
IX II III IV XI x Unidentified polar Residue unextracted oua methanol Total
and soil a/ 3,~ rln~/s
was sown.
[‘“Cl cyanazine
In soil (O-3 compound
1 .i7
MAIZE
Soil
residues on
/ Residue expressed as ppm of parent , compound equivalent to the radioactivity detected following application of the compound indicated
Component
Parent
.~PJD
1
lienidtres of cyanazine, atrazine and sinrazine and their breakdown after application at sowing Medium wet. weight
IN
H CzHs
CzHs H H H
I\ =
-
Cl
component 5 with aqua e-
CLH, C(CH,),CN H C(CH&CONH2 C @H&H C(CH&CONH2 H --
Cl Cl Cl OH Cl Cl -
CJib H C(CH&CONH2 C(CH&COOH C(CHa)&OOH H C(CH&H -
Q Some II may have been included in this fraction. b Probably mainly 2-hydroxy analogues of simazine and atrazine. c The component is not an expected metabolite of the particular d Probably IV and VIII.
I
PC1 atrazine
l’Y>! simazine
0.02
0.09
or.03
Total 0.02 0.01 c <0.03
<;.01 2.48” 0.43
0.74
3.06
1.58
0.19
1.65
I .7ti
0.10 0.18 1.56a <0.20 (0.02
0.06 ( c c
C(CH&CN or C(CH&H
CzHs CzHs CzHs C&L H H
I
L parent
compound.
(’
i.04
<0.02 0.17 0.2Ob 0.72 2.86
3.05
158
BEYNON,
STOYDIN
AND
studies there was no loss of radioactivity from the sap solution. In the maize sap, 22 hr after treatment with cyanazine, two metabolites were detected by thin layer chromatography. One was the hydroxy acid (IV) and its identity was confirmed by methylation as described above. The other component was present in similar amounts to the first and was converted into it on treatment with cold M hydrochloric acid. These mild conditions are
nitrile group in cyanazine which is more easily attacked than the chlorine atom. It is not apparently due to any change in the reactivity of the chloro group, since the total residues of chlorotriazines, as opposed to parent herbicides, present in the three soils are similar after 70 days. Plant Sap The persistence of cyanazine and atrazine in maize sap is shown in Fig. 2. In these Residues
wet
of cyanazine,
Medium weight
loam treated basis (near
WRIGHT
TABLE 2 and simazine and their breakdown products at 70 days after application at sowing at 1.5 kg/ha after maize seed (cv. Golden Bantam) 13% wt water) as sampled,
atrazine
in soils
under
was sown.
maize Soil
sampled
residues
on
Residue expressed as p m of parent compound in 0- P..5 cm soil layer” equivalent to the radioactivity detected following application of the compound indicated
- ___- R’
-
--
R2
Parent
compound
C&L
Cl
[‘“Cl
R3 --
C(CH&CN
,:yanazine ___-
_-
0.03
[‘“Cl atrazine
[‘T] simazine
0.40
0.43
0.:3 c
o.‘lo
C(C:s&H or Cd& V IX II III X VI XI IV VIII polar compo-
Unidentified nents Residue unextracted aqueous methanol Total
H
Cl Cl Cl Cl Cl Cl Cl OH OH -
Cd% CzHs Cd% H H H
C&L H -
-
with
-
-
-
C(CHMN H C(CH,)#ONE C(CH&COOI C(CH&H C (CH&CONE H C(CH,)zCOOI C(CH,)nCOOI -
12 I 12
I I
0.05 0.02 0.23
C
o.“lo
c c
0.:3 c
o.“lo c
0 .&.
0 .EofY
0.49
0.34
I
1.11
-
1.03
0 About 75% of 2-hydroxy analogue of atrazine and 20yo of the des-N-ethyl analogue of this with traces of the des-N-isopropyl analogue. * Mainly 2-hydroxy analogue of simazine. c See footnote to Table 1. d Residues in the 7.5-15-cm layer were 10% or less of the residues in the 0-7.5.cm layer and contained the same metabolitee in similar ratios.
(‘OMPARATIVE
BREAKDOWN
OF
TRIAZINE
HERBICIDES
IX
SOILS
ANI)
;\IAIZE
I Xl
FIG. 2. The breakdown of W-cyanazine and W-atrazine in expressed maize sap, at .W’C. (a) “C-ah-azinc at 10 pglml. (b), (c), (d) WY-cyanazine at 160,bO and 10 pg/ml respectively.
probably insufficient to hydrolyse glutathione or cysteine conjugates and the component was probably a salt or sugar conjugate of the carboxylic acid. No other components were detected in the sap 22 hr after treatment. However, another metabolite was detected in small amounts (below 1% of the applied activity) in the sap 4 hr after treatment with cyanazine but not at later times. It did not correspond to any of the compounds in Fig. 1. The hydroxy analogue of atrazine was the only metabolite detected in the sap treated with atrazine. The degradation of atrazine was slower than that of cyanazine at similar concentrations. Neither cyanazine nor atrazine was dealkylated under these conditions and there was no evidence for the formation of glutathione or cysteine conjugates. These results suggest that cyanazine was attacked initially at the nitrile group whereas in atrazine attack is at the chlorine atom. Therefore the reaction mechanism with eyanazine is probably fundamentally different from t,hat of
atrazine, where hydrolysis has been shown (8) to be catalysed by a naturally occurring compound, 2,4-dihydroxy-7-methoxy-l ,+ benzoxazin-a-one. The rate of hydrolysis of cyanazine in maize sap was far greater t,han the rate in buffered aqueous systems (11) and it is likely that the hydrolysis is vatalysed by plant substances. Plants The compounds identified in the maize plants 32 and 70 days after treatment with cyanazine, atrazine or simazine are shown in Tables 2 and 3. Components were ident.ified by thin layer chromatography as indicsated above and confirmation of the identity of compounds such as (IV) and (VIII) was obtained by methylation and further chromatography. The materials referred t#o in Table 2 as (IV) or (VIII) in free and conjugated forms were a mixture of similar amounts of the free compound and of a conjugat,e which released the free compound on heating with 6 M hydrochloric acid (2 hr at 90°C). These conjugates are possibly
160
BEYNON,
STOYDEN
AND
glutathione or cysteine conjugates and this may well apply also to the unidentified polar material that is indicated in Tables 2 and 3. In addition to the unidentified polar material the plants contained radioactive compounds
which were not extracted with methanol. These may well be conjugates although the conjugates of atrazine with methionine or cysteine can be extracted from plants with methanol (3).
TABLE Residues
of cyanazine,
Medium
loam
atrazine
treated
and simazine
at 1.5 kg/ha
-
WRIGHT
3
and their breakdown application at sowing
after
the
maize
seed
(cv.
products
in maize
sampled
70 days
Golden
Bantam)
was sown.
after
Structure
R’ N Component
A
Residue expressed as ppm of parent compound equivalent to the radioactivity detected following application of the compound indicated
‘N
R2HN lNANHR3 --
-
I
R’
--___ WI
R2
WI atrazine -
cyanazine
R3
Leaf
T
[‘“Cl
-
Stem
Leaf
Stem
Leaf
Stem
(0.01
0.02
<0.03
o.iI2 c
<0.03
c c
.-i.01 c
oc42
Tztal
1.12
=
c
c
0.18
3.03
0.22
2.12
0.20
0.01
1.31
0.09
0.49
0.03
0.19
4.41
0.32
2.81
0.23
Parent
compound
CzHz
Cl
C(CHd&N
simazine
-.
or C(CHdaH
V IX II X VI XI IV (free and conjugated) VIII (free and conjugated) Unidentified polar components Unextracted residue* Total Total
-
residue calculated weight basis
Cl Cl Cl Cl Cl Cl HO
H CJL H H H C,H,
$I, C(CHd&N H C(CH&CONH2 C(CH&H C(CH,)zCONH2 H C(CH&COOH
HO
H
C(CHa)&OOH
-
-
-
-
-
-
CzHs
-
<:o.
0.17 2.55
on a whole
100
plant
-! 0.78
-
c 0.05
C
cf.02 C
-I
-I
C
C
c
0."16 c
Tztal
c
=
1.36
-
a No unidentified material was detected in this sample but the presence of VIII could prevent the detection of small amounts of other components. b In the cyanazine study this refers to the residue remaining in the plant after extraction with aqueous material followed by hot water. In the atrazine and simazine study this refers to the residue remaining after extraction with aqueous methanol. c See Footnote to Table 1.
CORIPARATIVE
BREAKDOWN
OF
TRIAZINE
HERBICIDES
IN
SOILS
AND
MAIZE
161
Plantti grown in soils treated with cydifferent components which could occur in anazine contained residues of the chlorotrithe field and not to be an indication of thclir axines (II) and (VI) as well as those of the absolute values. hydroxy triazines (IV) and (VIII). DealkylaREFERENCES tion reactions are slow in the soil so it is 1. T. Chapman, D. Jordan, 11. H. Payne, W. .I. likely that (II) was converted to (VI) in the Hughes and R. H. Schieferst,ein, Proc. 9th plant and not in the soil. Based on the studies Bit. Weed Cont. Conf. 1018 (1968). with maize sap, maize plants are likely to 2. K. I. Beynon, G. Stoydin and 8. N. Wright,, c*onvert’ cyanazine directly to (IV). Thus the Pestic. Ski., in press. 3. G. L. Lamoureux, K. H. Shimabukuro, H. 11. amide (II) present in plants grown in treated Swanson and D. S. Frear, J. Agr. Foorl soil was probably t,aken up by the plant as Chem. 18,81 (1970). su<+h. 4. R. II. Shimabukuro, II. 1~. Swanson and W. (.‘. The total residuesin the maize grown with Walsh, Plant Physiol. 46, 103 (1970). 5. D. H. Hutson, E. C. Hoadley, M. H. Grifiths soil treated with cyanazine were lower than and C. Donninger, J. Agr. Food Chem. 18, those grown in soils treated with atrazine or 507 (1970). simazine. The difference was greater at 32 6. E. Knuesli, D. Berrer, G. Dupuis, and II. days t’han at 70 days after treatment. FurEsser, in “Degradation of Herbicides” thermore, the plants grown in soils treat,ed (P. C. Kearney and 1~. D. Kaufman, Eds.), Marcel Dekker Inc., New York, 1969. with at,razine and simazine contained mainly 7. R. H. Shimabukuro, Plant Ph,ysiol. 42, 1269 residues of t’he hydroxy derivatives. (1967). The residues in the present work were de8. W. Roth, C. R. Acad. Sci. 245,942 (1957). tected in plants grown indoors in boxes with 9. K. Goswami and R. E. Green, Environ. Sti. consequent restrictions on the space for the Technol. 5,426 (1971). root,s and in the absence of wind and rain. 10. G. Dupuis, T. Laanio, P. Marbach and H. 0. Esser, VIIth International Congress of Thus, t#heyare likely to be much larger than Plant Protection, Paris 21-25th Sept,ember, will o(*cur in the field and they should be 1970.Summariesof papersp. 711. considered to be an indication of the type 11. N. P. H. Brown and C. G. L. Furmidge, Pestic. and t,hr rcblative amounts of residues of t’he Sci., in press.