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A new radiorespirometric method for measuring herbicide residues in soil: 2,4-D as a test case
The Science of the Total Environment, 123/124 (1992) 345-360 Elsevier Science Publishers B.V., Amsterdam
345
A new radiorespirometric method for measuring herbicide residues in soil: 2,4-D as a test case G. Soulas a n d B. Lagacherie INRA-Laboratoire de Microbiologie des Sols, 17, rue Sully, 21034 Dijon-cedex, France
ABSTRACT In this paper we describe the principles of an analytical procedure based on radiorespirometric measurements that could be applied to the determination of residual concentrations of 2,4-D in the soil. Basically, it consists of comparing, in two soil samples, mineralization of 2,4-D, labeled at two different specific activities. The validity of the procedure depends on the concentration of herbicide, the location of the radioactive carbon within the molecule and the possibility to distinguish several phases in the evolution of ~4CO~. The dosage has been proved more reliable at concentration levels that are consistent with agricultural practice than at lower concentrations. We also demonstrate that an appropriate mathematical treatment of the experimental data makes it possible to indentify the fraction of the mineralized radioactivity that specifically originates in the degradation of 2,4-D. This allows more realistic estimates of the amount of the parent molecule left unchanged in the soil by reducing interference by 14C-containing residuals resulting from microbial activity. We also demonstrate that carboxylic and methylenic carbon atoms of the side-chain are not assimilated with an equal efficiency. The absence of a true lag-phase indicates that the soil we have used contains a microbial population that degrades 2,4-D without requiring a preliminary period of adaptation. Key words: radiorespirometry; residues analysis; 2,4-D degradation
INTRODUCTION D e t e r m i n a t i o n o f soil residues o f the p h e n o x y herbicide 2,4-D, as well as structurally related c o m p o u n d s , has been p e r f o r m e d by different analytical methods: biological tests ( K i r k l a n d a n d Fryer, 1972; Torstensson, 1975) radiorespirometry (Norris, 1966), chemical (i.e. instrumental) m e t h o d s by s p e c t r o p h o t o m e t r y (Bandursky, 1947), T L C (Smith, 1985), G L C (Smith a n d H a y d e n , 1981) or H P L C (Skelly et al., 1977) and, m o r e recently, by i m m u n o dosage (Fleeker, 1987). All these procedures have their own advantages b u t it should be emphasized t h a t no one is able to detect all the residues actually present in the soil. As a consequence, determinations m a d e by different
346
G. SOULAS AND B. LAGACHERIE
analytical techniques may have different meanings with respect to the availability of the residues to a chemical extractant or a biological indicator. A pesticide is generally present in the soil under different physical or chemical states that correspond to different states of availability to the different chemical or biological agents used to determine their residual concentration. It can be distinguished between: -
-
-
-
-
-
the total residual amount of the chemical, the amount that is immediately or potentially available to a given biological agent. It approximates the part of the residues that is or may appear in the soil solution as a consequence of a mass action driven desorption process, the amount that is extractable by a given solvent and has no relevance with any defined physical or chemical state of the molecule.
On one hand, it is important to determine the proportion of the chemicals that remains biologically available, particularly when considered from a practical point of view. On the other hand, soil analysts and environmentalists may also be interested in looking for estimations of total residual amounts of pesticides to prevent potential hazards. In this report, we describe a new (micro)biological dosage of a herbicide, 2,4-D, in the soil. It is simple to operate, as reliable as more sophisticated instrumental determinations and can be easily interpreted in terms of total residual amount of herbicide. However, because it is based on utilization of labeled pesticides, its use is restricted to research purposes. MATERIALS AND METHODS
Methodological presentation Principle The procedure we describe is a dosage based on radiorespirometric measurements after addition of a known amount of labeled herbicide as an internal standard to duplicated soil samples that already contain residues of the same molecule either radioactive or not. This is achieved in the following way: we first prepare samples of fresh soil, called test samples, that all receive the same amount of non-labeled chemical (concentration Co). Half of the samples are also treated with radioactive material (radioactivity R0) in such a way that the final concentration remains unchanged (Co). These labeled samples are used to control mineralization of 2,4-D by following the evolution of 14CO2. At intervals, equal number of replicates of test samples are taken from each of the labeled and non-labeled series of soil for determining
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
347
At time t :
p a i r e d samples
Fresh test sample Concentration : Ct Radioactivity : 0 or Rt +
Sterile addition sample Concentration : C1 Radioactivity : R1
R'~
R'2
Amounts of mineralized radioactivity
and:
Rt = R'x - R' 2 .
R1
R' 2
Fig. 1. Descriptionof the radiorespirometricprocedure for the determinationof the residual radioactivity and concentration of 2,4-D in the soil.
the residual concentration (Ct) of herbicide which is independent of the presence of radioactivity. All are treated with a second identical amount of radioactive 2,4-D (radioactivity R~) and reincubated. Comparison between amounts of radioactivity evolved as ~4CO2 from the two types of samples, called the paired samples, makes it possible to measure the concentration of 2,4-D left in the soil just before the last addition of radioactive material. In Fig. 1 the principle of this determination is set out. In one sample the total radioactivity amounts to what is left in the soil at the end of the first incubation period (Rt, to be determined) plus what is added (R~). The radioactivity evolved as 14CO2 after reincubation is R'I. In the second sample, the corresponding mineralized radioactivity (R'2) originates in the sole radioactivity present, R~. We can write: R{ R t +
Rt =
R~
R~
(i)
R~
R { - R~
R1
(2)
348
Rt Rl R,(%) = 100 = 100 Ro Ro
G. SOUI.AS AND B. LAGACHERIE
R { - R~ R~
(3)
Technical constraints First of all, in theory, residual radioactivity (Rt) should be exclusively. present on residual untransformed herbicide. The best procedure we have found to approach such a goal is to use a pesticidal molecule with a radioactive carbon located on its most labile part to give non-labeled metabolites. For 2,4-D, it can be inferred that such a condition could be approximated by using a chain-labeled compound. However, part of the radioactive carbon is incorporated into microbial biosynthetates and waste extra-cellular products that can later contribute to the overall evolution of 14CO2. We shall examine in the next section how we have treated the data to correct this. The validity of the relation (1) supposes that both residual and added parts of the herbicide must be in the same physico-chemical state to have the same biological availability. Clearly, direct introduction of the radioactivity R1 as a solution of labeled 2,4-D must be eliminated. Rather we have found it more convenient to treat a separate fumigated-sterilized soil sample with the required amount of [~4C]2,4-D and then to mix it with the test sample. So, these additional samples, where 2,4-D has been proved to keep untransformed (Soulas et al., 1984) can all be prepared at the beginning of the experiment to give the same physico-chemical state of the pesticide as in the test samples they are mixed with. Assumption was made that microorganisms have no effect on the physico-chemical state of the remaining molecules other than lowering the soil solution concentration resulting in a progressive release of the reversibly adsorbed fraction. Treatment of the data When refering to relation (2), R{ and R~ appear to be two key experimental variables. In a first approximation they can be taken as the cumulated amounts of radioactivity evolved as 14CO2 from both paired samples when 2,4-D is supposed to have been extensively transformed. Such a procedure has been called end-point analysis. Despite its simplicity it has a major shortcoming: we have to realize that as degradation of 2,4-D proceeds a more and more significant secondary source of radioactivity comes from the turnover of the living or dead soil organic matter that becomes labeled through microbial activity. More realistic estimations of R{ and R~ have been tentatively obtained by describing total mineralization curves of radioactivity with an empirical mathematical model based on the summation of two terms (Fig. 2). The first, a modified Gompertz relation, is related to the initial phase of 14CO2 evolution exclusively linked to the degradation of the labeled herbicide. It has a limiting value, which we call the 'plateau', that reflects
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
Mineralized Radioactivity R(%)
349
End point (A+B)
S
Plateau ( A :
Time Fig. 2. Diagrammatic presentation explaining the ways of calculating the values R{ and R~ used to estimate soil residual concentrations of 2,4-D by the procedures of 'end-point' or 'plateau' analysis. The upper curve is the experimental curve used in the 'end-point' method of analysis. It has been decomposed into two parts: curve A that represents mineralization of the pesticide only and curve B that represents mineralization due to the turnover of microbial constituents.
disappearence of the pesticide. The turnover of the abiotic and biological 14C-organic components of the soil has been described by a second term that increases linearly with time. The final formulation of the model is as follows: ~---p~-3j j - e x p ( - e x p (pP~2a))l + P4t
(4)
where R(°/b) is the cumulated percentage of radioactivity evolved a s 1 4 C O 2 , P1 the maximum percentage of radioactivity originating from the herbicide, P2 the abscissa of the point of inflection, P3 the reverse of the 2,4-D mineralization constant, and P4 the rate of mineralization of 'parasitic' radioactivity. By identifying the model it was possible to calculate parameters of the model. P1 is the value of interest that has been chosen as
350 the estimate of R{ and R~ for each of the paired samples. This procedure was called 'plateau' analysis.
Reliability of the data Assessment of the reliability of the data was performed with the general method applicable when a variable X has a value x that depends on other experimentally measured variables, a, b .... for which S.D. values ~a, ~b ... are estimated by the absolute values of their differentials da, db ... By differentiating relation (3) with respect to R'1 and R'2, it can be demonstrated that:
ARt ( % ) - R'IRt(%) - R'2
JAR{ + AR~ R~,' ]
(5)
where z~R{ and z~R~ are the standard deviations of R{ and R].
Experimental protocols We have used a silty clay soil with the following characteristics: 33% clay, 52% silt, 15% sand, 1.18% organic C, 0.14% total N and 1.3% CaCO3. The pH in water was 7.8, the CEC 20.8 mequiv. 100 g-1 and the WHC 25%. The soil was partially air-dried to an appropriate water content (12-14% on a dry soil basis) making easier subsequent sieving. Only the fraction of soil aggregates between 2 and 3 mm was used to allow an easy and efficient mixing operation after test and additional soil samples have been brought together. Experiments were conducted to calibrate the analytical procedure and then to use it in kinetic studies. In each case the basic experimental protocol consisted in three steps. - - Preparation of sterile samples for addition of radioactive material: soil aggregates were divided into 10-g portions of soil placed into separate 50-ml glass vials. Water was added to bring the soil to 70% of the WHC. All samples were fumigated twice for 16 h at a weekly interval with chloroform. A week after the last fumigation each sample received 200 #1 of a solution of radioactive 2,4-D in phosphate buffer (pH 7) to give the desired soil concentration and radioactivity and a water content of 80% of the WHC. Treated samples were stored in incubation chambers at 20°C for the duration of the experiment in closed 0.5 1glass jars. These jars also contain two scintillation vials, one with 10 ml of water and one with 10 ml of a N solution of sodium hydroxide. This solution was replaced and analysed for the absence of ~4C-carbonates every 2 or 3 weeks to make sure that 2,4-D was not degraded.
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
-
-
-
-
351
Preparation of the test samples: samples of 10 g soil aggregates were distributed in 20 ml glass scintillation vials and wetted to 70% of the WHC. In the calibration experiment, half of these samples received 200/~1 of solutions of non-labeled 2,4-D to give five different concentrations and to bring the soil to 80% of the WHC. The other half of the samples received the same amounts of radioactive material. All the treated samples were used for analysis after a 1-day period of equilibration. In the kinetic experiments, all samples were treated at the same concentration, half with non-labeled 2,4-D and half with labeled 2,4-D. Treated samples were stored in incubation rooms at 20°C in 250-ml glass flasks were two other scintillation vials were also present. They contain, respectively, 10 ml of water and 5 ml of a 0.2 N solution of sodium hydroxide. When necessary, the incubation flasks were opened and the CO2 traps replaced at a frequency determined by the rate of ~4CO2 evolution from radioactive samples that were also used to follow degradation kinetics of 2,4-D. Measurement of the residual concentration in the test samples: when appropriate, an equal number of replicates of radioactive and nonradioactive test samples were mixed each with a sterile addition sample. In the calibration experiment all test samples were processed simultaneously after the 1-day equilibration period. In the kinetic experiments residual concentration was measured after different incubation periods. In all cases homogenized 20 g samples (dry soil) were wetted to 120% of the WHC and incubated again at 20°C in closed 0.5-1 glass jars in the presence of 10 ml of water and 5 ml sodium of a 0.2 N solution of sodium hydroxide in scintillation vials. Kinetics of 14CO2 were followed for a period of about 2 months after mixing.
Calibration experiment This experiment was carried out to validate the analytical procedure at two concentration levels. Only 2,4-dichlorophenoxy [2-taC]acetic acid (spec. act. of the purchased chemical: 2035 MBq mmo1-1) was used. The first group of 30 sterile samples were treated with a solution of radioactive 2,4-D to give a concentration of 0.09 mg kg -1 a.i. and a radioactivity content of 2.6 kBq. A second group of 30 samples received the same amount of radioactive material but at a concentration of 4.1 mg kg -1. In the same way, 60 test samples were first divided into 2 equal sub-groups, each comprising 6 replicates at 5 different concentrations in geometric progression to simulate residual concentrations resulting from first order kinetics of degradation: from 0.004 to 0.064 mg kg -1 in one case and from 0.26 to 4.10 mg kg -1 in the other (Table 1). At each concentration only 3 replicates received [14C]2,4-D with an amount of radioactivity linearly related to the concen-
352
o. SOULASANDB.LAGACHERIE
TABLE 1 Concentrations and radioactivities applied to the sterile and test samples in the calibration experiment
Sterile samples Test samples
Concentration (mg kg -1)
Radioactivity (kBq)
Level 1
Level 2
Level 1
Level 2
0.092 0.064 0.032 0.016 0.008 0.004
4.12 4.10 2.04 1.02 0.51 0.26
2.64 5.31 2.62 1.30 0.65 0.32
2.32 5.60 2.82 1.40 0.70 0.35
t r a t i o n o f pesticide. T h e d a y after t r e a t m e n t with the herbicide all test samples are mixed with a sterile sample a n d e v o l u t i o n o f radioactivity was m e a s u r e d o v e r a p e r i o d o f m o r e t h a n 80 days a n d the percentages o f r e c o v e r y were calculated.
Kinetic experiments T w o successive experiments have been p e r f o r m e d at two different concentration levels. F o r the p u r p o s e o f c o m p a r i s o n we have used 2,4-dichlorop h e n o x y [1-14C]acetic acid (329 M B q m m o l -~) a n d 2 , 4 - d i c h l o r o p h e n o x y TABLE 2 Concentrations and radioactivities applied to the sterile and test samples in the kinetic experiments at two concentrations. Label 1 and label 2 refer to l-14C- and 2-t4C-chain labeled 2,4D. Figures in parentheses are the number of soil samples for the corresponding treatment
Sterile samples Label 1 Label 2 Test samples Label 1 Label 2 Non-labeled
Concentration (mg kg -l)
Radioactivity (kBq)
Low level
High level
Low level
High level
0.064 (48) 0.064 (48)
4.03 (72) 3.86 (72)
0.98 0.93
2.78 2.26
0.064 (24) 0.064 (24) (48)
4.22 (36) 3.9 (36) (72)
0.99 0.93
5.67 5.40
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RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
[2-14C] acetic acid. The number of sterile samples was 96 in the first experiment with 48 with 1-14C material and 48 with 2-14C material at the uniform concentration of 0.064 mg kg -~. An equal total number of test samples was prepared and divided into 24 samples treated with [1-14C]2,4-D, 24 treated with [2-t4C]2,4-D and 48 without labeled compound. In all test samples the concentration was 0.064 mg kg -~. In the second experiment we have used the same procedure except that treatment concentration was higher ( - 4 mg kg -1) and the total number of sterile or test samples 144 (Table 2). During incubation of the test samples we have monitored degradation of 2,4-D: -
-
-
-
by conventional radiorespirometry using remaining labeled test samples not already taken for residual concentration measurements, by the mixing procedure of test and sterile samples followed by radiorespirometric measurements of the paired samples and calculation of the residual concentration by using formula (3). These estimations were made after 0, 1, 3, 6, 9, 13, 20, 38 days in the first experiment and after 1, 3, 6, 8, 10, 13, 17, 24, 31, 41, 52, 64 days in the second experiment.
TABLE 3 Percentages of recovery and corresponding S.D. values (in parentheses) estimated by two different methods in the calibration experiment Concentration (mg kg -1)
4.10 2.04 1.02 0.51 0.26 0.064 0.032 0.016 0.008 0.004
Percentage recovery End point
Plateau
99.6 100.2 97.0 100.2 105.6 102.2 99.5 93.0 101.9 102.6
(1.7) (1.0) (1.6) (3.5) (4.8) (2.5) (2.4) (4.1) (10.3) (18.1)
100.7 99.6 99.4 101.2 103.4 103.7 106.4 94.7 102.1 52.8
(4.5) (4.3) (6.4) (11.8) (28.7) (5.8) (7.4) (11.2) (26.3) (40.6)
354
G. S O U L A S A N D B . L A G A C H E R I E
0.08
0.08
A End point
B Plateau 0.06
0.06
0.04
0.04 t0
o
o
0.02
0.02
0.0
0.0 0.0
!
I
!
0.02
0.04
0.06
0
0.08
Theoretical values (mg 2,4-D / kg soil)
I
I
I
I
0.0
0.02
0.04
0.06
0.08
Theoretical values (mg 2,4-D / kg soil)
Fig. 3. Calibration curves as determined after treatment at 0.068 mg (2,4-D) k g -l (soil) by two methods of estimation of the residual concentration of 2,4-D in the soil. Concentrations are in mg (2,4-D) kg -l (soil). RESULTS A N D DISCUSSION
Calibration experiments Percentages of recovery of 2,4-D in the test samples are given in Table 3 with their corresponding S.D. values. Figures 3 and 4 give another represen5
>~
3 2
..q o
1 0 0
I
I
I
I
I
I
I
I
I
I
1
2
3
4
5
0
1
2
3
4
Theoretical values (mg 2,4-D / kg soil)
5
Theoretical values (mg 2,4-D / kg soil)
Fig. 4. Calibration curves as determined after treatment at 4.10 mg (2,4-D) kg -1 (soil) by two methods of estimation of the residual concentration of 2,4-D in the soil. Concentrations are in mg (2,4-D) kg -1 (soil).
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
355
tation of the data by relating calculated percentages of recovery to total recovery and comparing to the first bisecting line. The first obvious conclusion that can be derived from these observations is the good recovery of the chemical: all values but one are comprised between 93.05 and 106.45%. When examining S.D. values, it can be deduced that low concentrations are detected with less efficiency, probably because of a too low specific activity of the chemical we have used for our experiments. Such a conclusion is valid whenever considering dilutions of the same series of treatments or treatments at different levels of concentrations. By comparison to other biological or chemical techniques for the determination of pesticide residues in the soil, the mixing procedure seems to offer promising advantages. Besides its technical simplicity it is much more reliable and sensitive than conventional biological tests. Accuracy of the determinations is consistent with that obtained with classical chemical analysis, specially at concentration levels compatible with agricultural practice and when using the 'end-point' procedure. Comparison of the S.D. values determined for each estimation procedure is in favor of the 'end-point' method. However, it must be emphasized that estimations have been made immediately after adding the herbicide, with no other residues than the parent molecule present in the soil. Recalling that the 'plateau' method was developed to take into account non-pesticidal interfering radioactive compounds, definitive conclusions on the respective interest of both procedures cannot be drawn before they are tested in conditions of actual degradation. TABLE 4 Percentages of recovery (Rt) and corresponding standard deviations (S.D.). Values estimated by two different methods in the kinetic experiment after treatment at 0.068 mg (2,4-D) kg -~ (soil) (For label 1 and label 2 see Table 2) Time
End point
Plateau
Label 1
0 1 3 6 9 13 20 38
Label 2
Label 1
Label 2
R t (%)
S.D.
R t (%)
S.D.
R t (%)
S.D.
R t (%)
S.D.
94.5 63.7 18.4 13.1 12.1 10.5 8.2 7.4
2.0 3.1 4.2 2.7 2.2 1.4 1.6 1.2
107.4 74.3 32.6 27.9 35.5 19.6 21.5 34.3
10.6 12.5 2.7 4.5 7.6 0.9 9.1 14.6
91.0 56.5 9.6 6.7 5.5 2.6 3.4 3.7
8.5 8.5 5.9 4.6 4.6 4.0 3.9 4.1
105.1 62.6 22.4 15.3 17.0 8.5 8.9 5.2
13.3 11.1 6.2 6.3 6.8 4.0 8.1 8.9
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G. SOULASANDB. LAGACFIERIE
TABLE 5 Percentages of recovery (Rt) and corresponding standard deviations (S.D.). Values estimated by two different methods in the kinetic experiment after treatment at 4.10 mg (2,4-D) kg -1 (soil). (For label 1 and label 2 see Table 2) Time
End point
Plateau
Label 1
1 3 6 8 10 13 17 24 31 41 52 64
Label 2
Label 1
Label 2
Rt(%)
S.D.
Rt(%)
S.D.
Rt(%)
S.D.
Rt(%)
S.D.
98.8 90.3 79.0 69.3 61.4 49.1 33.8 18.5 13.2 7.7 7.1 6.2
0.5 1.2 1.6 2.2 0.4 1.0 1.1 2.1 1.2 0.6 1.0 0.4
97.8 92.0 83.1 73.2 65.8 54.1 39.7 27.2 22.3 14.7 16.4 13.1
1.5 1.2 1.5 1.3 1.8 1.9 2.6 1.9 1.1 0.3 0.4 0.5
97.1 90.0 78.2 68.1 58.7 45.7 29.9 13.7 8.4 3.3 9.5 1.3
2.0 2.4 1.8 2.0 2.4 1.5 2.1 2.8 1.5 1.1 5.7 1.0
98.9 95.8 82.6 72.5 62.0 46.5 32.6 19.1 11.4 3.5 n.d. 1.5
3.1 3.1 2.5 2.9 4.5 3.1 4.1 3.2 1.7 1.4 1.3
Kinetic experiments The results of the two successive experiments are summarized in Tables 4 and 5 and in Figs 5 and 6. Again, precision on estimates of the residual concentration is better when analysing the higher concentrations with the endpoint procedure. This is consistent with our preliminary observations from the calibration experiment. Another interesting point stems from the comparison of the degradation curves as obtained by the end-point procedure. There is a tendency for the residual concentrations to go to finite non-zero values that differ with the concentration and the position of the label on the herbicidal molecule. Such a difference is more pronounced at the lower rate of application: after 38 days of incubation only 7.4% of the chemical is detected in the soil when using [1-14C] 2,4-D as compared to 34.3% with [2-14C] 2,4-D. These figures are, respectively, 6.2% and 13.1% when higher dosage of pesticide has been ,~sed. This observation is not consistent with the classical pathway of degradation of 2,4-D that starts with the cleavage of the ether linkage leaving together both carbon atoms of the side-chain for metabolic use. However, if
120-
120-
100-
A End point
100-
100-
80-
B Plateau
C Evolution of 14C02
~, 80-
80._o 60-
60-
I v o
chain2 chain 1
•~ 4 0 -
= 60-
v o
chain2 chain
•~ 4 0 -
¢
~
~ .c_ 4 0 -
O
2O
~ 20-
20
I
I
I
I
I
I
I
0
10
20
30
40
50
60
Time (day's)
0
10
20
30
40
Time (day,s)
50
60
0
I
I
I
|
I
I
10
20
30
40
50
60
Time (days)
Fig. 5. Comparison between mineralization and degradation curves as determined by two methods of estimation of the residual concentration after application of 0.064 mg kg -] of 2,4-D. Evolved radioactivities and concentrations are expressed in percentages of initial amount of herbicide.
L~
d,
120 -
120-
2100 .> 80-
100-
A End point
100|,~ .~ 80-1
B Plateau
C Evolution of 1 4 C 0 2
~80-
.Q
~ 60. 60"
60-
~
r-
•- 40"6 20-
chain 2 chain 1
40-
o~ 20-
20-
0-
0~ I
I
0
20
I
|
4O 6O Time (days)
I
8O
:~ 40-
I
0
20
I
I
40 60 Time (days)
I
80
0I
0
20
I
I
40 6O Time (days)
I
8O
Fig. 6. Comparison between mineralization and degradation curves as determined by two methods of estimation of the residual concentration after application of 4.1 mg kg -l of 2,4-D. Evolved radioactivities and concentrations are expressed in percentages of initial amount of herbicide.
C ;> Z
.w if,
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
359
we consider mineralization data (Figs. 5C and 6C), we can see that less methylenic carbon is recovered as ~4CO2rather than carboxylic carbon. An identical observation was already made with acetate by Kassim et al. (1981) who concluded that during metabolism of acetate by the TCA cycle more of the CH3-C was used for biosynthesis. This offers a possible explanation to the difference we noted before between degradation curves corresponding to the two types of labeled compounds we have used in our experiments. Because of a greater incorporation into cell components, CH 2-14C from 2,4-D could give an increased rate of evolution of radioactivity during the late phase of mineralization in the paired samples due to the accelerated turnover of the biological radioactive carbon when the degrading biomass is returning to more natural carbon substrates. Examination of degradation curves estimated by the 'plateau' procedure gives support to this interpretation. Calculating the residual concentrations only from the fraction of the evolved radioactivity directly originating in the mineralization of the labeled herbicide makes degradation patterns nearly (low concentration) or completely (high concentration) independent of the location of the radioactive carbon on the molecule. Moreover, final concentrations are going to zero. Besides methodological discussion, some interesting considerations can be derived from the experimental results. First of all, there is a good agreement between cumulated 14CO2 evolution (Figs 5C and 6C) and [14C]2,4-D disappearance curves (Figs 5A, B and 6A, B). From this comparison it is evident that an almost constant proportion of the pesticidal carbon that has been degraded is recovered as ~4CO2. It is also confirmed that not all carbon atoms of the sidechain have the same efficiency of incorporation. Another important point lies in the absence of a true lag-phase. A 2,4-D degradation potential pre-exists in the soil we have used and can be immediately mobilized. CONCLUSION From a technical point of view, we have shown, with 2,4-D as a test case, that residual concentrations of pesticides in the soil could be advantageously determined by a biological dosage based on radiorespirometric measurements. Basically it consists in comparing mineralization of 2,4-D labeled at two different specific activities in two soil samples. The procedure has been proved the more reliable at concentration levels consistent with agricultural practice. At lower concentrations we need to reinvestigate the technique with a chemical labeled at a higher specific activity. We have also demonstrated that by an appropriate mathematical treatment it is possible to individualize the fraction of the mineralized radioactivity that specifically originates in
360
G, SOULAS AND B. LAGACHERIE
the d e g r a d a t i o n o f 2,4-D. This allows a more realistic t h o u g h less reliable estimation o f residues o f the p a r e n t molecule. F r o m a microbiological point o f view, it was d e m o n s t r a t e d t h a t carboxylic and methylenic c a r b o n a t o m s o f the side-chain were n o t assimilated at the same yield. The absence o f a true lag-phase is indicating t h a t no acclimation takes place in the soil we have used. R a t h e r , a microbial p o p u l a t i o n is present which is capable o f degrading 2,4-D immediately after its application to the soil. REFERENCES Bandursky, R.S., 1947. Spectrophotometric method for determination of 2,4-dichlorophenoxyacetic acid. Bot. Gaz., 108: 446-449. Fleeker, J., 1987. Two enzyme immunoassays to screen for 2,4-dichlorophenoxyacetic acid in water. J. Assoc. Off. Anal. Chem., 70: 874-878. Kassim, G., J.P. Martin and K. Haider, 1981. Incorporation of a wide variety of organic substrate carbons into soil biomass as estimated by the fumigation procedure. Soil Sci. Soc. Am. J., 45: 1105-1112. Kirkland, K. and J.D. Fryer, 1972. Degradation of several herbicides in a soil previously treated with MCPA. Weed Res., 12: 90-95. Norris, L.A., 1966. Degradation of 2,4-D and 2,4,5-T in forest litter. J. Forest., 475-476. Skelly, N.E., T.S. Stevens and D.A. Mapes, 1977. Isomeric-specific assay of ester and salt formulations of 2,4-dichlorophenoxyacetic acid by automated high pressure liquid chromatography. J. Assoc. Off. Anal. Chem., 60: 868-872. Smith, A.E., 1985. Identification of 2,4-dichloroanisole and 2,4-dichlorophenol as soil degradation products of ring-labeled [14C] 2,4-D. Bull. Environ. Contam. Toxicol., 34: 150-157. Smith, A.E. and B.J. Hayden, 1981. Relative persistence of MCPA, MCPB and mecoprop in Saskatchewan soils and the identification of MCPA in MCPB treated soils. Weed Res., 21: 179-183. Soulas, G., R. Chaussod and A. Verguet, 1984. Chloroform fumigation technique as a means of determining the size of specialized soil microbial populations: application to pesticidedegrading microorganisms. Soil Biol. Biochem., 16: 497-501. Torstensson, N.T.L., 1975. Degradation of 2,4-D and MCPA in soils of low pH. Eng. Environ. Qual. Saf. Suppl. 3, Pesticides, 25: 262-265.
345
A new radiorespirometric method for measuring herbicide residues in soil: 2,4-D as a test case G. Soulas a n d B. Lagacherie INRA-Laboratoire de Microbiologie des Sols, 17, rue Sully, 21034 Dijon-cedex, France
ABSTRACT In this paper we describe the principles of an analytical procedure based on radiorespirometric measurements that could be applied to the determination of residual concentrations of 2,4-D in the soil. Basically, it consists of comparing, in two soil samples, mineralization of 2,4-D, labeled at two different specific activities. The validity of the procedure depends on the concentration of herbicide, the location of the radioactive carbon within the molecule and the possibility to distinguish several phases in the evolution of ~4CO~. The dosage has been proved more reliable at concentration levels that are consistent with agricultural practice than at lower concentrations. We also demonstrate that an appropriate mathematical treatment of the experimental data makes it possible to indentify the fraction of the mineralized radioactivity that specifically originates in the degradation of 2,4-D. This allows more realistic estimates of the amount of the parent molecule left unchanged in the soil by reducing interference by 14C-containing residuals resulting from microbial activity. We also demonstrate that carboxylic and methylenic carbon atoms of the side-chain are not assimilated with an equal efficiency. The absence of a true lag-phase indicates that the soil we have used contains a microbial population that degrades 2,4-D without requiring a preliminary period of adaptation. Key words: radiorespirometry; residues analysis; 2,4-D degradation
INTRODUCTION D e t e r m i n a t i o n o f soil residues o f the p h e n o x y herbicide 2,4-D, as well as structurally related c o m p o u n d s , has been p e r f o r m e d by different analytical methods: biological tests ( K i r k l a n d a n d Fryer, 1972; Torstensson, 1975) radiorespirometry (Norris, 1966), chemical (i.e. instrumental) m e t h o d s by s p e c t r o p h o t o m e t r y (Bandursky, 1947), T L C (Smith, 1985), G L C (Smith a n d H a y d e n , 1981) or H P L C (Skelly et al., 1977) and, m o r e recently, by i m m u n o dosage (Fleeker, 1987). All these procedures have their own advantages b u t it should be emphasized t h a t no one is able to detect all the residues actually present in the soil. As a consequence, determinations m a d e by different
346
G. SOULAS AND B. LAGACHERIE
analytical techniques may have different meanings with respect to the availability of the residues to a chemical extractant or a biological indicator. A pesticide is generally present in the soil under different physical or chemical states that correspond to different states of availability to the different chemical or biological agents used to determine their residual concentration. It can be distinguished between: -
-
-
-
-
-
the total residual amount of the chemical, the amount that is immediately or potentially available to a given biological agent. It approximates the part of the residues that is or may appear in the soil solution as a consequence of a mass action driven desorption process, the amount that is extractable by a given solvent and has no relevance with any defined physical or chemical state of the molecule.
On one hand, it is important to determine the proportion of the chemicals that remains biologically available, particularly when considered from a practical point of view. On the other hand, soil analysts and environmentalists may also be interested in looking for estimations of total residual amounts of pesticides to prevent potential hazards. In this report, we describe a new (micro)biological dosage of a herbicide, 2,4-D, in the soil. It is simple to operate, as reliable as more sophisticated instrumental determinations and can be easily interpreted in terms of total residual amount of herbicide. However, because it is based on utilization of labeled pesticides, its use is restricted to research purposes. MATERIALS AND METHODS
Methodological presentation Principle The procedure we describe is a dosage based on radiorespirometric measurements after addition of a known amount of labeled herbicide as an internal standard to duplicated soil samples that already contain residues of the same molecule either radioactive or not. This is achieved in the following way: we first prepare samples of fresh soil, called test samples, that all receive the same amount of non-labeled chemical (concentration Co). Half of the samples are also treated with radioactive material (radioactivity R0) in such a way that the final concentration remains unchanged (Co). These labeled samples are used to control mineralization of 2,4-D by following the evolution of 14CO2. At intervals, equal number of replicates of test samples are taken from each of the labeled and non-labeled series of soil for determining
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
347
At time t :
p a i r e d samples
Fresh test sample Concentration : Ct Radioactivity : 0 or Rt +
Sterile addition sample Concentration : C1 Radioactivity : R1
R'~
R'2
Amounts of mineralized radioactivity
and:
Rt = R'x - R' 2 .
R1
R' 2
Fig. 1. Descriptionof the radiorespirometricprocedure for the determinationof the residual radioactivity and concentration of 2,4-D in the soil.
the residual concentration (Ct) of herbicide which is independent of the presence of radioactivity. All are treated with a second identical amount of radioactive 2,4-D (radioactivity R~) and reincubated. Comparison between amounts of radioactivity evolved as ~4CO2 from the two types of samples, called the paired samples, makes it possible to measure the concentration of 2,4-D left in the soil just before the last addition of radioactive material. In Fig. 1 the principle of this determination is set out. In one sample the total radioactivity amounts to what is left in the soil at the end of the first incubation period (Rt, to be determined) plus what is added (R~). The radioactivity evolved as 14CO2 after reincubation is R'I. In the second sample, the corresponding mineralized radioactivity (R'2) originates in the sole radioactivity present, R~. We can write: R{ R t +
Rt =
R~
R~
(i)
R~
R { - R~
R1
(2)
348
Rt Rl R,(%) = 100 = 100 Ro Ro
G. SOUI.AS AND B. LAGACHERIE
R { - R~ R~
(3)
Technical constraints First of all, in theory, residual radioactivity (Rt) should be exclusively. present on residual untransformed herbicide. The best procedure we have found to approach such a goal is to use a pesticidal molecule with a radioactive carbon located on its most labile part to give non-labeled metabolites. For 2,4-D, it can be inferred that such a condition could be approximated by using a chain-labeled compound. However, part of the radioactive carbon is incorporated into microbial biosynthetates and waste extra-cellular products that can later contribute to the overall evolution of 14CO2. We shall examine in the next section how we have treated the data to correct this. The validity of the relation (1) supposes that both residual and added parts of the herbicide must be in the same physico-chemical state to have the same biological availability. Clearly, direct introduction of the radioactivity R1 as a solution of labeled 2,4-D must be eliminated. Rather we have found it more convenient to treat a separate fumigated-sterilized soil sample with the required amount of [~4C]2,4-D and then to mix it with the test sample. So, these additional samples, where 2,4-D has been proved to keep untransformed (Soulas et al., 1984) can all be prepared at the beginning of the experiment to give the same physico-chemical state of the pesticide as in the test samples they are mixed with. Assumption was made that microorganisms have no effect on the physico-chemical state of the remaining molecules other than lowering the soil solution concentration resulting in a progressive release of the reversibly adsorbed fraction. Treatment of the data When refering to relation (2), R{ and R~ appear to be two key experimental variables. In a first approximation they can be taken as the cumulated amounts of radioactivity evolved as 14CO2 from both paired samples when 2,4-D is supposed to have been extensively transformed. Such a procedure has been called end-point analysis. Despite its simplicity it has a major shortcoming: we have to realize that as degradation of 2,4-D proceeds a more and more significant secondary source of radioactivity comes from the turnover of the living or dead soil organic matter that becomes labeled through microbial activity. More realistic estimations of R{ and R~ have been tentatively obtained by describing total mineralization curves of radioactivity with an empirical mathematical model based on the summation of two terms (Fig. 2). The first, a modified Gompertz relation, is related to the initial phase of 14CO2 evolution exclusively linked to the degradation of the labeled herbicide. It has a limiting value, which we call the 'plateau', that reflects
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
Mineralized Radioactivity R(%)
349
End point (A+B)
S
Plateau ( A :
Time Fig. 2. Diagrammatic presentation explaining the ways of calculating the values R{ and R~ used to estimate soil residual concentrations of 2,4-D by the procedures of 'end-point' or 'plateau' analysis. The upper curve is the experimental curve used in the 'end-point' method of analysis. It has been decomposed into two parts: curve A that represents mineralization of the pesticide only and curve B that represents mineralization due to the turnover of microbial constituents.
disappearence of the pesticide. The turnover of the abiotic and biological 14C-organic components of the soil has been described by a second term that increases linearly with time. The final formulation of the model is as follows: ~---p~-3j j - e x p ( - e x p (pP~2a))l + P4t
(4)
where R(°/b) is the cumulated percentage of radioactivity evolved a s 1 4 C O 2 , P1 the maximum percentage of radioactivity originating from the herbicide, P2 the abscissa of the point of inflection, P3 the reverse of the 2,4-D mineralization constant, and P4 the rate of mineralization of 'parasitic' radioactivity. By identifying the model it was possible to calculate parameters of the model. P1 is the value of interest that has been chosen as
350 the estimate of R{ and R~ for each of the paired samples. This procedure was called 'plateau' analysis.
Reliability of the data Assessment of the reliability of the data was performed with the general method applicable when a variable X has a value x that depends on other experimentally measured variables, a, b .... for which S.D. values ~a, ~b ... are estimated by the absolute values of their differentials da, db ... By differentiating relation (3) with respect to R'1 and R'2, it can be demonstrated that:
ARt ( % ) - R'IRt(%) - R'2
JAR{ + AR~ R~,' ]
(5)
where z~R{ and z~R~ are the standard deviations of R{ and R].
Experimental protocols We have used a silty clay soil with the following characteristics: 33% clay, 52% silt, 15% sand, 1.18% organic C, 0.14% total N and 1.3% CaCO3. The pH in water was 7.8, the CEC 20.8 mequiv. 100 g-1 and the WHC 25%. The soil was partially air-dried to an appropriate water content (12-14% on a dry soil basis) making easier subsequent sieving. Only the fraction of soil aggregates between 2 and 3 mm was used to allow an easy and efficient mixing operation after test and additional soil samples have been brought together. Experiments were conducted to calibrate the analytical procedure and then to use it in kinetic studies. In each case the basic experimental protocol consisted in three steps. - - Preparation of sterile samples for addition of radioactive material: soil aggregates were divided into 10-g portions of soil placed into separate 50-ml glass vials. Water was added to bring the soil to 70% of the WHC. All samples were fumigated twice for 16 h at a weekly interval with chloroform. A week after the last fumigation each sample received 200 #1 of a solution of radioactive 2,4-D in phosphate buffer (pH 7) to give the desired soil concentration and radioactivity and a water content of 80% of the WHC. Treated samples were stored in incubation chambers at 20°C for the duration of the experiment in closed 0.5 1glass jars. These jars also contain two scintillation vials, one with 10 ml of water and one with 10 ml of a N solution of sodium hydroxide. This solution was replaced and analysed for the absence of ~4C-carbonates every 2 or 3 weeks to make sure that 2,4-D was not degraded.
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
-
-
-
-
351
Preparation of the test samples: samples of 10 g soil aggregates were distributed in 20 ml glass scintillation vials and wetted to 70% of the WHC. In the calibration experiment, half of these samples received 200/~1 of solutions of non-labeled 2,4-D to give five different concentrations and to bring the soil to 80% of the WHC. The other half of the samples received the same amounts of radioactive material. All the treated samples were used for analysis after a 1-day period of equilibration. In the kinetic experiments, all samples were treated at the same concentration, half with non-labeled 2,4-D and half with labeled 2,4-D. Treated samples were stored in incubation rooms at 20°C in 250-ml glass flasks were two other scintillation vials were also present. They contain, respectively, 10 ml of water and 5 ml of a 0.2 N solution of sodium hydroxide. When necessary, the incubation flasks were opened and the CO2 traps replaced at a frequency determined by the rate of ~4CO2 evolution from radioactive samples that were also used to follow degradation kinetics of 2,4-D. Measurement of the residual concentration in the test samples: when appropriate, an equal number of replicates of radioactive and nonradioactive test samples were mixed each with a sterile addition sample. In the calibration experiment all test samples were processed simultaneously after the 1-day equilibration period. In the kinetic experiments residual concentration was measured after different incubation periods. In all cases homogenized 20 g samples (dry soil) were wetted to 120% of the WHC and incubated again at 20°C in closed 0.5-1 glass jars in the presence of 10 ml of water and 5 ml sodium of a 0.2 N solution of sodium hydroxide in scintillation vials. Kinetics of 14CO2 were followed for a period of about 2 months after mixing.
Calibration experiment This experiment was carried out to validate the analytical procedure at two concentration levels. Only 2,4-dichlorophenoxy [2-taC]acetic acid (spec. act. of the purchased chemical: 2035 MBq mmo1-1) was used. The first group of 30 sterile samples were treated with a solution of radioactive 2,4-D to give a concentration of 0.09 mg kg -1 a.i. and a radioactivity content of 2.6 kBq. A second group of 30 samples received the same amount of radioactive material but at a concentration of 4.1 mg kg -1. In the same way, 60 test samples were first divided into 2 equal sub-groups, each comprising 6 replicates at 5 different concentrations in geometric progression to simulate residual concentrations resulting from first order kinetics of degradation: from 0.004 to 0.064 mg kg -1 in one case and from 0.26 to 4.10 mg kg -1 in the other (Table 1). At each concentration only 3 replicates received [14C]2,4-D with an amount of radioactivity linearly related to the concen-
352
o. SOULASANDB.LAGACHERIE
TABLE 1 Concentrations and radioactivities applied to the sterile and test samples in the calibration experiment
Sterile samples Test samples
Concentration (mg kg -1)
Radioactivity (kBq)
Level 1
Level 2
Level 1
Level 2
0.092 0.064 0.032 0.016 0.008 0.004
4.12 4.10 2.04 1.02 0.51 0.26
2.64 5.31 2.62 1.30 0.65 0.32
2.32 5.60 2.82 1.40 0.70 0.35
t r a t i o n o f pesticide. T h e d a y after t r e a t m e n t with the herbicide all test samples are mixed with a sterile sample a n d e v o l u t i o n o f radioactivity was m e a s u r e d o v e r a p e r i o d o f m o r e t h a n 80 days a n d the percentages o f r e c o v e r y were calculated.
Kinetic experiments T w o successive experiments have been p e r f o r m e d at two different concentration levels. F o r the p u r p o s e o f c o m p a r i s o n we have used 2,4-dichlorop h e n o x y [1-14C]acetic acid (329 M B q m m o l -~) a n d 2 , 4 - d i c h l o r o p h e n o x y TABLE 2 Concentrations and radioactivities applied to the sterile and test samples in the kinetic experiments at two concentrations. Label 1 and label 2 refer to l-14C- and 2-t4C-chain labeled 2,4D. Figures in parentheses are the number of soil samples for the corresponding treatment
Sterile samples Label 1 Label 2 Test samples Label 1 Label 2 Non-labeled
Concentration (mg kg -l)
Radioactivity (kBq)
Low level
High level
Low level
High level
0.064 (48) 0.064 (48)
4.03 (72) 3.86 (72)
0.98 0.93
2.78 2.26
0.064 (24) 0.064 (24) (48)
4.22 (36) 3.9 (36) (72)
0.99 0.93
5.67 5.40
353
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
[2-14C] acetic acid. The number of sterile samples was 96 in the first experiment with 48 with 1-14C material and 48 with 2-14C material at the uniform concentration of 0.064 mg kg -~. An equal total number of test samples was prepared and divided into 24 samples treated with [1-14C]2,4-D, 24 treated with [2-t4C]2,4-D and 48 without labeled compound. In all test samples the concentration was 0.064 mg kg -~. In the second experiment we have used the same procedure except that treatment concentration was higher ( - 4 mg kg -1) and the total number of sterile or test samples 144 (Table 2). During incubation of the test samples we have monitored degradation of 2,4-D: -
-
-
-
by conventional radiorespirometry using remaining labeled test samples not already taken for residual concentration measurements, by the mixing procedure of test and sterile samples followed by radiorespirometric measurements of the paired samples and calculation of the residual concentration by using formula (3). These estimations were made after 0, 1, 3, 6, 9, 13, 20, 38 days in the first experiment and after 1, 3, 6, 8, 10, 13, 17, 24, 31, 41, 52, 64 days in the second experiment.
TABLE 3 Percentages of recovery and corresponding S.D. values (in parentheses) estimated by two different methods in the calibration experiment Concentration (mg kg -1)
4.10 2.04 1.02 0.51 0.26 0.064 0.032 0.016 0.008 0.004
Percentage recovery End point
Plateau
99.6 100.2 97.0 100.2 105.6 102.2 99.5 93.0 101.9 102.6
(1.7) (1.0) (1.6) (3.5) (4.8) (2.5) (2.4) (4.1) (10.3) (18.1)
100.7 99.6 99.4 101.2 103.4 103.7 106.4 94.7 102.1 52.8
(4.5) (4.3) (6.4) (11.8) (28.7) (5.8) (7.4) (11.2) (26.3) (40.6)
354
G. S O U L A S A N D B . L A G A C H E R I E
0.08
0.08
A End point
B Plateau 0.06
0.06
0.04
0.04 t0
o
o
0.02
0.02
0.0
0.0 0.0
!
I
!
0.02
0.04
0.06
0
0.08
Theoretical values (mg 2,4-D / kg soil)
I
I
I
I
0.0
0.02
0.04
0.06
0.08
Theoretical values (mg 2,4-D / kg soil)
Fig. 3. Calibration curves as determined after treatment at 0.068 mg (2,4-D) k g -l (soil) by two methods of estimation of the residual concentration of 2,4-D in the soil. Concentrations are in mg (2,4-D) kg -l (soil). RESULTS A N D DISCUSSION
Calibration experiments Percentages of recovery of 2,4-D in the test samples are given in Table 3 with their corresponding S.D. values. Figures 3 and 4 give another represen5
>~
3 2
..q o
1 0 0
I
I
I
I
I
I
I
I
I
I
1
2
3
4
5
0
1
2
3
4
Theoretical values (mg 2,4-D / kg soil)
5
Theoretical values (mg 2,4-D / kg soil)
Fig. 4. Calibration curves as determined after treatment at 4.10 mg (2,4-D) kg -1 (soil) by two methods of estimation of the residual concentration of 2,4-D in the soil. Concentrations are in mg (2,4-D) kg -1 (soil).
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
355
tation of the data by relating calculated percentages of recovery to total recovery and comparing to the first bisecting line. The first obvious conclusion that can be derived from these observations is the good recovery of the chemical: all values but one are comprised between 93.05 and 106.45%. When examining S.D. values, it can be deduced that low concentrations are detected with less efficiency, probably because of a too low specific activity of the chemical we have used for our experiments. Such a conclusion is valid whenever considering dilutions of the same series of treatments or treatments at different levels of concentrations. By comparison to other biological or chemical techniques for the determination of pesticide residues in the soil, the mixing procedure seems to offer promising advantages. Besides its technical simplicity it is much more reliable and sensitive than conventional biological tests. Accuracy of the determinations is consistent with that obtained with classical chemical analysis, specially at concentration levels compatible with agricultural practice and when using the 'end-point' procedure. Comparison of the S.D. values determined for each estimation procedure is in favor of the 'end-point' method. However, it must be emphasized that estimations have been made immediately after adding the herbicide, with no other residues than the parent molecule present in the soil. Recalling that the 'plateau' method was developed to take into account non-pesticidal interfering radioactive compounds, definitive conclusions on the respective interest of both procedures cannot be drawn before they are tested in conditions of actual degradation. TABLE 4 Percentages of recovery (Rt) and corresponding standard deviations (S.D.). Values estimated by two different methods in the kinetic experiment after treatment at 0.068 mg (2,4-D) kg -~ (soil) (For label 1 and label 2 see Table 2) Time
End point
Plateau
Label 1
0 1 3 6 9 13 20 38
Label 2
Label 1
Label 2
R t (%)
S.D.
R t (%)
S.D.
R t (%)
S.D.
R t (%)
S.D.
94.5 63.7 18.4 13.1 12.1 10.5 8.2 7.4
2.0 3.1 4.2 2.7 2.2 1.4 1.6 1.2
107.4 74.3 32.6 27.9 35.5 19.6 21.5 34.3
10.6 12.5 2.7 4.5 7.6 0.9 9.1 14.6
91.0 56.5 9.6 6.7 5.5 2.6 3.4 3.7
8.5 8.5 5.9 4.6 4.6 4.0 3.9 4.1
105.1 62.6 22.4 15.3 17.0 8.5 8.9 5.2
13.3 11.1 6.2 6.3 6.8 4.0 8.1 8.9
356
G. SOULASANDB. LAGACFIERIE
TABLE 5 Percentages of recovery (Rt) and corresponding standard deviations (S.D.). Values estimated by two different methods in the kinetic experiment after treatment at 4.10 mg (2,4-D) kg -1 (soil). (For label 1 and label 2 see Table 2) Time
End point
Plateau
Label 1
1 3 6 8 10 13 17 24 31 41 52 64
Label 2
Label 1
Label 2
Rt(%)
S.D.
Rt(%)
S.D.
Rt(%)
S.D.
Rt(%)
S.D.
98.8 90.3 79.0 69.3 61.4 49.1 33.8 18.5 13.2 7.7 7.1 6.2
0.5 1.2 1.6 2.2 0.4 1.0 1.1 2.1 1.2 0.6 1.0 0.4
97.8 92.0 83.1 73.2 65.8 54.1 39.7 27.2 22.3 14.7 16.4 13.1
1.5 1.2 1.5 1.3 1.8 1.9 2.6 1.9 1.1 0.3 0.4 0.5
97.1 90.0 78.2 68.1 58.7 45.7 29.9 13.7 8.4 3.3 9.5 1.3
2.0 2.4 1.8 2.0 2.4 1.5 2.1 2.8 1.5 1.1 5.7 1.0
98.9 95.8 82.6 72.5 62.0 46.5 32.6 19.1 11.4 3.5 n.d. 1.5
3.1 3.1 2.5 2.9 4.5 3.1 4.1 3.2 1.7 1.4 1.3
Kinetic experiments The results of the two successive experiments are summarized in Tables 4 and 5 and in Figs 5 and 6. Again, precision on estimates of the residual concentration is better when analysing the higher concentrations with the endpoint procedure. This is consistent with our preliminary observations from the calibration experiment. Another interesting point stems from the comparison of the degradation curves as obtained by the end-point procedure. There is a tendency for the residual concentrations to go to finite non-zero values that differ with the concentration and the position of the label on the herbicidal molecule. Such a difference is more pronounced at the lower rate of application: after 38 days of incubation only 7.4% of the chemical is detected in the soil when using [1-14C] 2,4-D as compared to 34.3% with [2-14C] 2,4-D. These figures are, respectively, 6.2% and 13.1% when higher dosage of pesticide has been ,~sed. This observation is not consistent with the classical pathway of degradation of 2,4-D that starts with the cleavage of the ether linkage leaving together both carbon atoms of the side-chain for metabolic use. However, if
120-
120-
100-
A End point
100-
100-
80-
B Plateau
C Evolution of 14C02
~, 80-
80._o 60-
60-
I v o
chain2 chain 1
•~ 4 0 -
= 60-
v o
chain2 chain
•~ 4 0 -
¢
~
~ .c_ 4 0 -
O
2O
~ 20-
20
I
I
I
I
I
I
I
0
10
20
30
40
50
60
Time (day's)
0
10
20
30
40
Time (day,s)
50
60
0
I
I
I
|
I
I
10
20
30
40
50
60
Time (days)
Fig. 5. Comparison between mineralization and degradation curves as determined by two methods of estimation of the residual concentration after application of 0.064 mg kg -] of 2,4-D. Evolved radioactivities and concentrations are expressed in percentages of initial amount of herbicide.
L~
d,
120 -
120-
2100 .> 80-
100-
A End point
100|,~ .~ 80-1
B Plateau
C Evolution of 1 4 C 0 2
~80-
.Q
~ 60. 60"
60-
~
r-
•- 40"6 20-
chain 2 chain 1
40-
o~ 20-
20-
0-
0~ I
I
0
20
I
|
4O 6O Time (days)
I
8O
:~ 40-
I
0
20
I
I
40 60 Time (days)
I
80
0I
0
20
I
I
40 6O Time (days)
I
8O
Fig. 6. Comparison between mineralization and degradation curves as determined by two methods of estimation of the residual concentration after application of 4.1 mg kg -l of 2,4-D. Evolved radioactivities and concentrations are expressed in percentages of initial amount of herbicide.
C ;> Z
.w if,
RADIORESPIROMETRIC MEASUREMENT OF HERBICIDE RESIDUES IN SOIL
359
we consider mineralization data (Figs. 5C and 6C), we can see that less methylenic carbon is recovered as ~4CO2rather than carboxylic carbon. An identical observation was already made with acetate by Kassim et al. (1981) who concluded that during metabolism of acetate by the TCA cycle more of the CH3-C was used for biosynthesis. This offers a possible explanation to the difference we noted before between degradation curves corresponding to the two types of labeled compounds we have used in our experiments. Because of a greater incorporation into cell components, CH 2-14C from 2,4-D could give an increased rate of evolution of radioactivity during the late phase of mineralization in the paired samples due to the accelerated turnover of the biological radioactive carbon when the degrading biomass is returning to more natural carbon substrates. Examination of degradation curves estimated by the 'plateau' procedure gives support to this interpretation. Calculating the residual concentrations only from the fraction of the evolved radioactivity directly originating in the mineralization of the labeled herbicide makes degradation patterns nearly (low concentration) or completely (high concentration) independent of the location of the radioactive carbon on the molecule. Moreover, final concentrations are going to zero. Besides methodological discussion, some interesting considerations can be derived from the experimental results. First of all, there is a good agreement between cumulated 14CO2 evolution (Figs 5C and 6C) and [14C]2,4-D disappearance curves (Figs 5A, B and 6A, B). From this comparison it is evident that an almost constant proportion of the pesticidal carbon that has been degraded is recovered as ~4CO2. It is also confirmed that not all carbon atoms of the sidechain have the same efficiency of incorporation. Another important point lies in the absence of a true lag-phase. A 2,4-D degradation potential pre-exists in the soil we have used and can be immediately mobilized. CONCLUSION From a technical point of view, we have shown, with 2,4-D as a test case, that residual concentrations of pesticides in the soil could be advantageously determined by a biological dosage based on radiorespirometric measurements. Basically it consists in comparing mineralization of 2,4-D labeled at two different specific activities in two soil samples. The procedure has been proved the more reliable at concentration levels consistent with agricultural practice. At lower concentrations we need to reinvestigate the technique with a chemical labeled at a higher specific activity. We have also demonstrated that by an appropriate mathematical treatment it is possible to individualize the fraction of the mineralized radioactivity that specifically originates in
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the d e g r a d a t i o n o f 2,4-D. This allows a more realistic t h o u g h less reliable estimation o f residues o f the p a r e n t molecule. F r o m a microbiological point o f view, it was d e m o n s t r a t e d t h a t carboxylic and methylenic c a r b o n a t o m s o f the side-chain were n o t assimilated at the same yield. The absence o f a true lag-phase is indicating t h a t no acclimation takes place in the soil we have used. R a t h e r , a microbial p o p u l a t i o n is present which is capable o f degrading 2,4-D immediately after its application to the soil. REFERENCES Bandursky, R.S., 1947. Spectrophotometric method for determination of 2,4-dichlorophenoxyacetic acid. Bot. Gaz., 108: 446-449. Fleeker, J., 1987. Two enzyme immunoassays to screen for 2,4-dichlorophenoxyacetic acid in water. J. Assoc. Off. Anal. Chem., 70: 874-878. Kassim, G., J.P. Martin and K. Haider, 1981. Incorporation of a wide variety of organic substrate carbons into soil biomass as estimated by the fumigation procedure. Soil Sci. Soc. Am. J., 45: 1105-1112. Kirkland, K. and J.D. Fryer, 1972. Degradation of several herbicides in a soil previously treated with MCPA. Weed Res., 12: 90-95. Norris, L.A., 1966. Degradation of 2,4-D and 2,4,5-T in forest litter. J. Forest., 475-476. Skelly, N.E., T.S. Stevens and D.A. Mapes, 1977. Isomeric-specific assay of ester and salt formulations of 2,4-dichlorophenoxyacetic acid by automated high pressure liquid chromatography. J. Assoc. Off. Anal. Chem., 60: 868-872. Smith, A.E., 1985. Identification of 2,4-dichloroanisole and 2,4-dichlorophenol as soil degradation products of ring-labeled [14C] 2,4-D. Bull. Environ. Contam. Toxicol., 34: 150-157. Smith, A.E. and B.J. Hayden, 1981. Relative persistence of MCPA, MCPB and mecoprop in Saskatchewan soils and the identification of MCPA in MCPB treated soils. Weed Res., 21: 179-183. Soulas, G., R. Chaussod and A. Verguet, 1984. Chloroform fumigation technique as a means of determining the size of specialized soil microbial populations: application to pesticidedegrading microorganisms. Soil Biol. Biochem., 16: 497-501. Torstensson, N.T.L., 1975. Degradation of 2,4-D and MCPA in soils of low pH. Eng. Environ. Qual. Saf. Suppl. 3, Pesticides, 25: 262-265.