Environmental Pollution 138 (2005) 92e99
www.elsevier.com/locate/envpol

2,4-Dichlorophenoxyacetic acid (2,4-D) sorption anddegradation dynamics in three agricultural soils
Arnaud Boivin a,b,*, Samira Amellal b, Michel Schiavon b,
Martinus Th. van Genuchten aaGeorge E. Brown, Jr. Salinity Laboratory, USDA-ARS, 450 West Big Springs Road, Riverside, CA 92507-4617, USAb
Laboratoire Sols et Environnement, UMR 1120 INPL/ENSAIA-INRA 2, Avenue de la Foreˆt de Haye,
BP 172, 54505 Vandoeuvre-lès-Nancy cedex, France
Received 17 September 2004; accepted 21 February 2005

Pesticide availability varies with its residence time in soil.
Abstract
The fate and transport of 2,4-dichlorophenoxyacetic acid (2,4-D) in the subsurface is aﬀected by a complex, time-dependentinterplay between sorption and mineralization processes. 2,4-D is biodegradable in soils, while adsorption/desorption is inﬂuenced
by both soil organic matter content and soil pH. In order to assess the dynamic interactions between sorption and mineralization,
2,4-D mineralization experiments were carried using three diﬀerent soils (clay, loam and sand) assuming diﬀerent contact times.
Mineralization appeared to be the main process limiting 2,4-D availability, with each soil containing its own 2,4-D decomposers.
For the clay and the loamy soils, 45 and 48% of the applied dose were mineralized after 10 days. By comparison, mineralization in
the sandy soil proceeded initially much slower because of longer lag times. While 2,4-D residues immediately after application werereadily available (O93% was extractable), the herbicide was present in a mostly unavailable state (!2% extractable) in all threesoils after incubation for 60 days. We found that the total amount of bound residue decreased between 30 and 60 incubation days.
Bioaccumulation may have led to reversible immobilization, with some residues later becoming more readily available again to
extraction and/or mineralization.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Sorption; Mineralization; 2,4-Dichlorophenoxyacetic acid (2,4-D); Bound residues; Water quality

1. Introduction
The extensive use of pesticides in agriculture iscompromising soil and water quality. One major concern* Corresponding author. Present address: George E. Brown, Jr.
Salinity Laboratory, USDA-ARS, 450 West Big Springs Road,
Riverside, CA 92507-4617, USA. Tel.: C1 951 369 4865; fax: C1
951 342 4964.
E-mail addresses: aboivin@ussl.ars.usda.gov (A. Boivin), samira.
amellal@ensaia.inpl-nancy.fr (S. Amellal), michel.schiavon@ensaia.
inpl-nancy.fr (M. Schiavon), rvang@ussl.ars.usda.gov (M.Th. van
Genuchten).
0269-7491/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2005.02.016

is protecting contamination of water resources (Younes
and Galal-Gorchev, 2000). Sorption and degradation
are key processes aﬀecting the fate and transport ofpesticides in the environment (Linn et al., 1993; Rudel
et al., 1993). Degradation is a fundamental attenuation
process for pesticides in soil (Guo et al., 2000). This
process, catalyzed by soil microbes, is governed by bothabiotic and biotic factors. Degradation is aﬀected bya variety of interactions among microorganisms, various
soil constituents, and the speciﬁc pesticide involved.
Sorption is similarly key to controlling pesticide
advective-dispersive transport, transformation and bioaccumulation processes (Calvet, 1989).

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A. Boivin et al. / Environmental Pollution 138 (2005) 92e99

Several interactions are known to exist between
sorption and degradation (Guo et al., 2000). It is
commonly accepted that sorption limits pesticidedegradation by reducing their partitioning into the soil
liquid phase (Smith et al., 1992; Guerin and Boyd,1997). Sorbed chemicals are generally assumed to be lessaccessible to microorganisms, which preferentially or
exclusively utilize chemicals in solution. Nevertheless, aninverse relationship between sorption and degradation
does not necessarily imply that degradation of sorbed
chemicals is negligible. Reductions in the degradation
rate are often not proportional to increases in sorption(e.g., Moyer et al., 1972). Sorption is a slow, timedependent process that may proceed over a relatively
long period of time (e.g., more than 1 day) to reachequilibrium (Pignatello, 1998). With longer contact
times between the soil and the chemical, the fractionof extractable residues is expected to decrease, whileconversely the amount of bound residues should increase (Boivin et al., 2004; Mordaunt et al., 2005). Theseﬁndings suggest that aging of pesticides has an eﬀect on
sorption-desorption behavior and hence on the biological availability of pesticides in soil. Aging is known
to be one of the more critical factors governing the fate
and transport of pesticides in soils (Sharer et al., 2003;
Walker et al., 2005). Aging enhances the retention ofsorbed organic chemicals by facilitating the formation ofbound residues via chemical and/or physical nonequilibrium sorption processes, thus causing the chemical to become less susceptible to desorption anddegradation. Still, relatively few laboratory studies havebeen carried out to assess the dynamic interplay between
sorption-desorption and degradation as a function of
diﬀerent residence times in soil.
2,4-Dichlorophenoxyacetic acid (2,4-D) belongs toa group of chemicals known as phenoxy compounds,
which are potentially toxic to humans. The herbicide is
widely used to control broad leaf weeds and grasses incrops, and has been frequently detected in groundwatersupplies in Europe and North America (e.g., Goldet al., 1988; IFEN, 2004). We selected 2,4-D as a model
pesticide for our study because of its unique behavior
in soil. This chemical is both very degradable andvulnerable to leaching. For example, 2,4-D can becompletely dissipated within 20 days in various landuse types (Voos and Groﬀman, 1997). Microﬂora able
to mineralize this chemical have been found naturally
in cultivated soils (Vieublé Gonod et al., 2003). At thesame time several factors combine to make thechemical vulnerable to rapid advective-dispersive transport and leaching, such as having a relatively low
molecular mass, a limited volatilization rate (i.e.,a relatively low Henry’s constant), a negative charge
at low soil pH, and relatively high solubility. Several
studies indicate that 2,4-D sorption in soil is a functionof soil pH, and to some extent also on soil organic

matter content (e.g., Dubus et al., 2001; Spark andSwift, 2002).
The aim of this study was to assess the dynamic
interactions between 2,4-D sorption and degradation,and to identify the main factors inﬂuencing bothprocesses. Soil incubation experiments were carried out
to monitor both 2,4-D mineralization and time-dependent soil/liquid phase partitioning of 2,4-D and its
metabolites in three cultivated soils (clay, loam, sand)from initial treatment to 60 days of incubation.
Measurements were made of time-dependent 14C andcarbon dioxide release rates, the overall kinetic releaseprocess, extractable residues, and bound residues.

2. Materials and methods
2.1. Soils and herbicide used
The laboratory experiments were carried on samples
collected from the surface (0e15 cm) horizons of three
agricultural soils in Lorraine, France: a relatively ﬁnetextured vertic stagnic cambisol (further referred to as
clay soil), a medium-textured stagnic luvisol (named
loamy soil) and a coarse textured ﬂuvic stagnic cambisol(named sandy soil); (WRB, 1998). Selected physical andchemical properties of the three soils are given in Table 1.
The soils were treated with 2,4-D and [U-phenyl-14C]2,
4-D (radiochemical purity O99%; speciﬁc activity
7.7 Mbq mgÿ1). 2,4-D is a weak acidic molecule (pKa2.6) with a relative molecular mass of 221 g molÿ1 anda solubility of 23,200 mg Lÿ1 in water at pH 7, 22  C. Its
octanol/water partition coeﬃcient is 2.83 (log Kow).
2.2. Laboratory experiments
The soils were air-dried and sieved to 2 mm. Samples(50 g) of the 2 mm soil aggregates were placed in glass
dishes (6 cm diameter). Water was added to the soil
samples until approximately 80% of ﬁeld capacity. We
subsequently applied to each sample a mixture of 2,4-D
and [14C]2,4-D (59.5 kBq) in aqueous solution. The
solution was sprayed onto the soil aggregate surfaces bymeans of a micro-syringe that dispensed very small
Table 1
Selected characteristics of the three soils (surface layers, 0e15 cm) usedin this study
Soil type

Clay Loam Sand OM pHH2O C/Nb C.E.C.c(%) (%)(%) (%)a(cmol kgÿ1)

Clay soil
41
Loamy soil 31
Sandy soil 11
a

49
50
23

10
19
66

Organic matter content (%).
Organic carbon/nitrogen ratio.
cCation exchange capacity.

b

3.3
2.5
1.5

7
5.9
5.8

9.9
9.1
9.6

18.7
14.8
5.5

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A. Boivin et al. / Environmental Pollution 138 (2005) 92e99

droplets. The amount of added herbicide represented
0.75 mg per sample (15!10ÿ6 g/g sol), equivalent toa conventional ﬁeld treatment dose of 1800 g haÿ1. All
treatments involved three replicates. We also used blanktreatments (i.e., without 2,4-D), consisting of the same
50 g of soil and the same volume of water. Each soil
sample was placed in a radio-respirometer (1.5 L) that
contained ﬂasks of an aqueous sodium hydroxide
solution (0.5 M; 10 mL) and distilled water. The sodiumhydroxide was used to trap carbon dioxide evolvingfrom the soil, as well as 14C-labelled carbon dioxide
stemming from 2,4-D 14C-ring degradation. The distilled water ﬂask served to reduce water losses from thesoil by maintaining a constant humidity in the system.
The radio-respirometers were incubated in the dark at
20 G 1  C.
2.3. Release kinetics and extractable residues
Pesticide residues were extracted from the soil
immediately after application, and after 5, 10, 30, and60 days of incubation. Soil samples were rotary shaken
at 20G1  C for 20 h with aqueous calcium chloride(0.01 M, 150 ml) in polytetraﬂuoroethylene (PTFE)
centrifuge ﬂasks. The release kinetics of 2,4-D into waterat each of these extraction times was determined bymeasuring the radioactivity in the supernatant solution
after shaking for 1, 2, 5, 10, 20, 30, 60, 180, 420 and 1200
min. Each measurement was done on a 1 mL sample,
which was centrifuged at 5000!g for 20 min usinga Beckman AvantiÔ J-25 (Beckman Instruments, Inc,
Fullerton, CA). The radioactivity of the supernatantsolution was measured by liquid scintillation usinga Packard 1900 CA Tri-carb liquid scintillation analyzer(Packard Instrument Company, Medriden, CT) after
adding 10 mL of a scintillation cocktail (UltimaGoldÔ,Packard Bioscience). The counting time was 10 min,while a quench correction was made by the scintillator
analyzer after calibration.
After completing the above kinetic release studies,
the same samples were used to carry out a series oftime-dependent residues experiments using both water(0.01 M calcium chloride) to obtain readily extractable
2,4-D residues and methanol for a more comprehensive
extraction of 2,4-D residues. For these studies, the soil/
water mixtures were centrifuged at 5000!g for 20 min,the supernatant replaced with fresh aqueous calcium
chloride (0.01 M, 150 mL), and the sample shaken
as before for 20 h. This operation was repeated until
the supernatant radioactivity became less than threetimes the background noise of the scintillator analyzer(1.7 Bq). To completely extract any remaining residues,
the soil was subsequently extracted repeatedly withmethanol (100 mL) until the supernatant radioactivity
again became less than three times the background noiseof the scintillator analyzer.

2.4. Extractable residue analysis
Using only methanol, a series of soil extractions was
performed on speciﬁcally prepared additional samples
to determine the nature of extracted residues. For these
extractions, 100 mL was added to the soil samples androtary shaken at 20G1  C for 20 h in PTFE centrifuge
ﬂasks. Samples were next centrifuged at 5000!g for 20
min and the radioactivity of 1 mL of supernatantsolutions determined using liquid scintillation as before.
This process was repeated until the supernatantradioactivity became less than three times the background noise of the scintillator analyzer (1.7 Bq). Afterthe methanol extractions, the supernatants were combined and concentrated for analysis of degradation
products by high performance liquid chromatography(HPLC) using a radio-chromatography detector (Radiomatic/FLO ONE Beta e Series A-500, Packard Instrument Company, Meriden, CT). The HPLC was
equipped with a 250-4 (250 mm long, 4 mm ID) C18
Merck LichroCART column coupled to a diode-array
detector (Varian 9065, Varian BV, Middelburg, The
Netherlands). Analyses were carried out for the following conditions: a wavelength of 282 nm, injection
volumes of 80 mL, 30 min run times, 0.8 mL minÿ1
ﬂow rates, and elution with an acetonitrileCaqueous
phosphoric acid (1.0 M, pH 3) solution (ﬁrst using
a 60C40 solution by volume for 10 min, and a 65C35
solution for 20 min). Peaks were identiﬁed using
a Ultima FloÔ Packard radio-chromatography detector(Packard Instrument Company, Meriden, CT) at a ﬂow
rate of 1.2 mL minÿ1 and using counting cell volumes of
500 mL. The limit of 2,4-D detection for these conditions
was 1 Bq.
2.5. Non-extractable residues in soil
After exhaustive extraction of the residues withaqueous calcium chloride solution (0.01 M) andmethanol, the soil aggregates were air-dried andpulverized. Non-extractable 14C-residues were determined next by combustion of soil samples (300 mg)
using an Oxidizer 307 (Packard Instrument Company,
Meriden, CT). [14C]Carbon dioxide released by the
samples was trapped using Carbo-sorb EÒ (10 mL) anda scintillating solution (10 mL), while Permaﬂuor ECÒ(Packard Bioscience) was subsequently added and theradioactivity of the trapped solution determined using
liquid scintillation.
2.6. Data analysis
Descriptive statistical analyses (analysis of variance,
principal component analysis) of the data were performed with the StatisticaÒ software package, version 6(StatSoft France, Maison-Alfort, France).

A. Boivin et al. / Environmental Pollution 138 (2005) 92e99

3. Results and discussion
3.1. 2,4-D mineralization
Fig. 1 shows the amounts of 2,4-D that weremineralized during the incubation experiments. Severaltransformation pathways such as hydrolysis, methylation and ring cleavage have been suggested for 2,4-D(Roberts, 1998). Mineralization appeared to be the main
2,4-D dissipation process during the incubation experiments with our three soils. The lag phases weresigniﬁcantly diﬀerent between the three soils, although
all were followed by rapid mineralization. For the clay
soil, 25% of the applied dose was mineralized after 5days of incubation, whereas only small amounts (2e3%)
were mineralized for the other two soils. The longest lagphase was observed for the sandy soil. However, after
30e60 days of incubation, the extent of mineralizationleveled out at about 54e66%, with no signiﬁcantdiﬀerences between the three soils ( pO0.05). Other
studies have reported similar mineralization rates incultivated soils. For example, Vieublé Gonod et al.,(2003) reported relatively short lag phases followed bymineralization rates of 35e50% of the applied dose
after application of labeled 2,4-D to aggregates ofdiﬀerent sizes. The occurrence of a lag phase followed by
rapid mineralization indicates that while some 2,4-D
decomposers were initially present in the soil, their
numbers could have been the limited factor in themineralization process. Since to our knowledge no 2,4-D
previously was applied to the cultivated soils used in ourstudy, we assumed that all three soils contained natural
microﬂora able to mineralize the chemical. Soils nevertreated with 2,4-D have been known to host signiﬁcant
but low populations of microbial decomposers.
2,4-D biodegradation typically occurs either bymeans of speciﬁc degraders that use the herbicide ascarbon and nitrogen sources, or by means of cometabolism populations whose relative proportions varywith soil type (e.g., Soulas, 1993). The mineralization
rate of 2,4-D has been found to be positively correlatedMineralized amount(% applied dose)

80
70
60
50
40
30
Clay soil
Loamy soil
Sandy soil

20
10
0

0

10

20

30

40

50

60

70

Time after application (days)
Fig. 1. Amounts of 2,4-D mineralized for the three soils during 60 daysof incubation.

95

with the size of the microbial biomass or the number of
total bacteria in soil, and with soil organic matter
content (Entry et al., 1995; Voos and Groﬀman, 1997).
The mineralization of 2,4-D can involve several microbial species, which may act as a consortium so as coverdiﬀerent steps in the biodegradation process. Diﬀerences
observed in our study between the diﬀerent lag phases
could be related to the size of the populations originallypresent in the soils. The sandy soil was more acidic (pH5.8) and had a much lower organic matter content thanthe other two soils (Table 1). The sandy soil hence likely
contained fewer bacteria, and/or at least one or more
microbial species at a much lower density. Mineralization of 2,4-D may be via co-metabolism immediately
after its application, and via metabolism after microorganisms able to use 2,4-D as carbon and energy
sources have reached a critical level and become themain contributors to the mineralization process. Still,a large fraction of the 2,4-D was mineralized relatively
rapidly (within 30 days) after application to all threesoils, with a concomitant rapid decrease in its availability and hence susceptibility to leaching.
3.2. Release kinetics of 2,4-D
Fig. 2 shows the release kinetics of 2,4-D immediately
after application (T0) and after contact times of up to 60
days (T10 to T60). Immediately after application, about
70% of the applied 2.4-D was quickly released (i.e.,
within 1 h) by the clay and loamy soils, while 80% wasreleased by the sandy soil. The shapes of the release
curves indicate that as long as large amounts of residue
are available (from T0 to T10 days), the residues are able
to rapidly re-enter the solution, with a quasi-equilibrium
situation developing after only about 2 h (Fig. 2). Thissuggests that much of the 2,4-D will remain readily
available for the ﬁrst 10 days after application, andhence may be subject to possible advective transport andleaching with ﬂowing water in the soil. With longer
contact times (TR30 days), the release kinetics showthat only small amounts of 2,4-D will re-enter into thesoil solution. The limited release after 30 days likely wascaused mostly by the relatively rapid mineralization
rates in our soils, as discussed previously. However,sorption kinetics probably also played a role. For
example, the sandy soil released only small amounts of
2,4-D after 10 days of incubation (Fig. 2), even thoughmineralization at that time was still relatively low(Fig. 1). This suggests that the kinetic sorption process
may have continued for quite some time after the initial
application.
Other studies have shown that 2,4-D degradation
may only be partial, and that the main degradation
product (2,4-dichlorophenol) may be more strongly
adsorbed to soils (Soulas and Fournier, 1981).
After 10 incubation days, no additional residues were

2,4-D residues recovered (%)

96

A. Boivin et al. / Environmental Pollution 138 (2005) 92e99

100(a) Clay soil
T0

75
50

T5

25

T 10
T 30

0
0

4

8

12

16

T 60

20

24

2,4-D residues recovered (%)

Time (hours)
100(b) Loamy soil
75

T0
T5

50
25
T 10
T 30

0

0

4

8

12

16

T 60

20

24

2,4-D residues recovered (%)

Time (hours)
100(c) Sandy soil
T0

75

T5

50
25
T 10

0

T 30 T 60

0

4

8

12

16

20

24

Time (hours)
Fig. 2. Recovered residues of 2,4-D and its metabolites from (a) clay
soil, (b) loamy soil, (c) sandy soil after diﬀerent incubation periods(T0eT60 days). The recovery time studies were carried out with
a 0.01 M calcium chloride solution.

recovered from our clay and loamy soils, while theamount recovered from the sandy soil was less than 8%.
Thus, in spite of its high solubility, mineralization of
2,4-D and/or simultaneous increased sorption of theresidues caused a substantial and rapid decrease (after
10 days) in the chemical availability for the three soils.
3.3. Amount and nature of extracted 2,4-D residues
We found that after the initial application of thechemical, more than 93% of the applied 2,4-D desorbedwith water (0.01 M calcium chloride) from all three soils.

This conﬁrms that immediately after its initial application, 2,4-D sorption is reversible. This may be due inpart to its large solubility in water. With longer contact
times, the amounts of extracted residues dramaticallydecreased, and became less than 2% after 60 days
incubation.
Accessibility to 2,4-D residues was higher in the
sandy soil just after 2,4-D application and until 10 daysof incubation (Table 2). 2,4-D mineralization was slower
in this soil, which also showed less sorption because of
its lower clay and organic matter contents. We observedthat the amounts of 2,4-D residues extracted withmethanol, following extractions with water (0.01 M
calcium chloride), were very low immediately after
application, reached a peak after 10 incubation days,
and then decreased again. This would conﬁrm thatsorption of 2,4-D residues increases with longer contact
times. This seems particularly true for the sandy soil,
where 13% of the residues were desorbed with themethanol extractions after 10 contact days. The residues
extracted in this way could have been the 2,4-D itself,
and/or its degradation products that were more strongly
sorbed to the soil and not available to water extraction(0.01 M calcium chloride).
Sorption of organic substrates has been shown tooften decrease their availability to microorganisms.
Sorbed 2,4-D therefore should be less rapidly mineralized than free 2,4-D (Benoit et al., 1998; Carrizosa et al.,2000). Increased sorption over time in the sandy soil,
which means having less 2,4-D and its metabolites insolution, was expected to lead to less mineralization.
This was contrary to our results, which showed thatmineralization proceeded rapidly after the initial lag
phase (Fig. 1). Because of this and the relatively low
sorption rates in the sandy soils, we believe that theavailability of the chemical and its degradation products
was limited more by physical entrapment than byirreversible chemical bounding.
In soils, initial side chain cleavage can cause theformation of an important metabolite, 2,4-dichlorophenol (2,4-DCP), as well as small amount of various otherdegradation products (‘‘Others’’ in Fig. 3) of lower
molecular mass but generally with a higher polarity(results not shown). Analysis of the extracted products
indicated that some of the metabolites were alreadypresent very soon after 2,4-D application. Rapid biotic
and/or abiotic degradation may have generated theseproducts. It has been shown that the carbon atom of the
2,4-D molecule can contribute to bacterial cellular
protoplasm synthesis (Soulas, 1990). Neo-synthesis of
the products (bioaccumulation), with a chemical structure that diﬀers from 2,4-D, may lead to the immobilization of C from 2,4-D in the soils. The various
molecules thus created, each having a diﬀerent molecular mass, may combine to produce the 14C signals(‘‘Others’’) recorded via b detection.

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A. Boivin et al. / Environmental Pollution 138 (2005) 92e99
Table 2
2,4-D residues extracted from the three soils studied after diﬀerent incubation times
2,4-D residues extracted (% total applied) after incubation time (days) of

Clay soil
CaCl2 (0.01 M)
MeOH
Loamy soil
CaCl2 (0.01 M)
MeOH
Sandy soil
CaCl2 (0.01 M)
MeOH

0

5

10

30

60

96 (G2)0.2 (G0.1)

24 (G2)2 (G1)

2 (G2)
1.5 (G1)

2 (G1)
0.1 (G0.2)

1 (G1)
0.1 (G0.1)

93 (G2)0.1 (G0.1)

70 (G2)
1.5 (G1)

2.5 (G1)
2 (G1)

2 (G2)
0.2 (G0.1)

1.5 (G0.2)
0.1 (G0.05)

98 (G2)0.1 (G0.1)

75 (G3)2 (G1)

46 (G3)13 (G2)2.5 (G1)
0.3 (G0.1)

2 (G4)
0.05 (G0.03)

Extractions were performed with water (0.01 M calcium chloride) followed by methanol. Standard deviations are given in parentheses.

Fig. 3 shows that 2,4-D was still the main chemical
being extracted from all three soils after 5 days, while
the proportion of degradation products was relatively
low. In the sandy soil, a large proportion of theextracted residues after 10 incubation days consisted of
2,4-DCP. The relative prevalence of this metabolite
indicates that the size of at least one population, andperhaps more, of the consortium responsible for 2,4-D
mineralization was relatively small. Moreover, it supports the assumption of a signiﬁcant contribution of
non-speciﬁc microorganisms for an extended period
before the development of more eﬃcient degraders.
3.4. Dynamics of 2,4-D bound residues
formation in soil
Bound residues were already detected immediately
after application of the chemical, as shown in Fig. 4.
This may have been due to strong sorption, including
cation-bridging with bivalent metals (Dubus et al.,2001). Bound residues in the clay and loamy soils
increased rapidly after 5 days of incubation, while lessbound residues were generated in the sandy soil.

Fig. 3. Products (% of applied dose) recovered using methanol
extraction. Three groups of chemicals were examined: 2,4-D, 2,4-DCP
and ‘‘Others’’, the latter being the sum of all recorded weak b signals.
The standard deviation of each fraction was less than 5%.

The fraction of bound residues formed in the sandy soil
was found to be signiﬁcantly lower statistically( p!0.05) than in the two others soils.
Bound residue formation has been linked to thepresence of soil organic matter because of its large
number of sorption-speciﬁc sites (Pignatello, 1998).
Bounding of the residues may have contributed to theobserved decrease in the mineralization rates after 10days contact time for the loamy and clay soils. Nevertheless, no long-term quasi-equilibrium state was everreached. Fig. 4 shows that the fraction of bound residue
increased until approximately 10 incubation days in thethree soils, and then decreased again ( p!0.05). Thismeans that parts of these residues were still potentiallyavailable. We previously noted that 2,4-D degradation
produced several degradation products of diﬀerent
molecular mass. Also, 14C from 2,4-D may have been
temporally immobilized by being incorporated into theprotoplasm of some of the soil microorganisms. In
addition to chemical bounding and physical entrapment, which both should contribute to the formationof bound residue (Boivin et al., 2004), incorporation
into the constitutive parts of microorganisms (bioaccumulation) could lead to overestimation of boundresidue formation. Still, the neosynthesis compounds
have a chemical structure that diﬀers from 2,4-D, andFig. 4. Bound residues (% of applied dose) derived from 2,4-D during
60 days of contact time.

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A. Boivin et al. / Environmental Pollution 138 (2005) 92e99

4. Conclusions

Fig. 5. Results of a principal component analysis using StatisticaÒ
(projection on 1, 2 planes) for the incubation experiments using
selected soil characteristics; 77.7% of the total variability was
explained by the two selected principal component axes.

hence should not be a threat to the contamination ofwater resources.

2,4-D in the three soils was found to be readilymineralized by microorganisms. Mineralization appeared
to be the main process limiting the availability of thischemical. Hence, while this herbicide is one of the mostmobile pesticides, its very rapid mineralization (50% of
the applied dose in 10 days) lessens some of its potentiallyadverse eﬀects on the environment (groundwater andsurface water contamination). Bound residues were foundto be formed quickly in the three soils, with the amounts
slowly decreasing again near the end of the 60-day
incubation experiments, possibly due to some overestimation of the amount of 2,4-D or metabolites being taken
up by soil microorganisms (bioaccumulation). Pesticides
presenting a chemical structure similar to natural
products are biodegradable, and their use therefore seemsa reasonable compromise between crop protection andenvironmental concerns.
Acknowledgments
The authors wish to thank Richard Cherrier for his
help in improving the experimental procedures, especially the HPLC analysis.

3.5. Statistical approach of 2,4-D behavior
A principal component analysis was carried out toidentify the main factors aﬀecting 2,4-D availability.
Fig. 5 shows that incubation time was the mostimportant factor inﬂuencing 2,4-D availability, which
reﬂects the fact that mineralization and sorption areboth highly dynamic. The statistical results indicate thatwith longer contact times, 2,4-D availability decreases
considerably due to its mineralization. No signiﬁcantrelationships were found between soil characteristics (%organic matter, % clay, soil pH) and 2,4-D mineralization. We earlier showed that 2,4-D was readilymineralized irrespective of soil type, and that the only
diﬀerence between the three soils was the longer lag
phase for the sandy soil. By contrast, bound residue
formation was found to be correlated with soil type. The
amount of bound residue in the sandy soil was
signiﬁcantly lower than that in the two other soils.
Contact time was not found to have a signiﬁcant eﬀect
on bound residue formation, in part likely because of
bioaccumulation which may have caused some overestimation of the amount of bound residue after longincubation times.
The above results show that the eﬀects of aging on
2,4-D and its residues in soils is a complex process.
Overall, we found that the amount of total extractable
2,4-D residue (i.e., extractions with 0.01 M calcium
chloride, following by methanol) decreased with contact
time, while bound residue formation was probably
overestimated because of bioaccumulation.

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