Factors influencing rates of degradation of an arylamide and a benzoic acid in subsoils

Article abstract of DOI:10.1002/1526-4998(200007)56:7<607::AID-PS189>3.0.CO;2-E

The kinetics of fundamental reactions (hydrolytic, oxidative and reductive) involved in the degradation of organic compounds such as pesticides in subsoils were investigated using the model compounds N-(4- nitrophenyl)propanamide and 4-nitrobenzoic acid. The rates of hydrolysis of N-(4-nitrophenyl)propanamide were also measured in aqueous buffers, hydrolysis being extremely slow at neutral pH; its degradation in three soils was by microbially mediated hydrolysis, being very much faster than aqueous hydrolysis at the same pH. Rates of degradation of N-(4- nitrophenyl)propanamide in subsoils were initially up to thirty times slower than those in topsoil, and in some subsoils degradation showed a marked lag- phase of between 72-144 h. For 4-nitrobenzoic acid, a similar lag-phase of slow degradation, followed by a phase of rapid degradation, was observed in both topsoils and subsoils. Remarkably, the rapid phases of degradation in subsoils often approached rates occurring in the corresponding topsoil. No reduction of the nitro group on either compound was observed, even in a water-saturated subsoil. Sometimes there were differences in the length of the lag-phases measured for replicate samples of subsoils; also, application of lower concentrations of 4-nitrobenzoic acid generally gave rise to shorter lag-phases. Partial sterilization of soils by azide greatly slowed breakdown of both compounds, confirming the important role of microbial degradation. Such behaviour is consistent with the variable build-up of populations of micro-organisms able to degrade the compound, smaller populations being able to deal rapidly with the lower concentrations. After applying a second dose of 4-nitrobenzoic acid to soil, degradation was rapid but initially not as fast as the final rates during breakdown of the first treatment. Hence, soil may only partially retain the ability to degrade previously applied xenobiotics. Nonetheless it is noteworthy that, even in deep subsoils, indigenous microbial populations can rapidly adapt to degrade certain small organic molecules. (C) 2000 Society of Chemical Industry.

Full text of DOI:10.1002/1526-4998(200007)56:7<607::AID-PS189>3.0.CO;2-E

Pest Management Science
Pest Manag Sci 56:607±614 (2000)
Factors influencing rates of degradation of an
arylamide and a benzoic acid in subsoils
Peter Nicholls,* Andrew T Campbell and Roger H Williams
IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Abstract: The kinetics of fundamental reactions (hydrolytic, oxidative and reductive) involved in the
degradation of organic compounds such as pesticides in subsoils were investigated using the model
compounds N-(4-nitrophenyl)propanamide and 4-nitrobenzoic acid. The rates of hydrolysis of N-(4-
nitrophenyl)propanamide were also measured in aqueous buffers, hydrolysis being extremely slow at
neutral pH; its degradation in three soils was by microbially mediated hydrolysis, being very much
faster than aqueous hydrolysis at the same pH. Rates of degradation of N-(4-nitrophenyl)propanamide
in subsoils were initially up to thirty times slower than those in topsoil, and in some subsoils
degradation showed a marked lag-phase of between 72±144h. For 4-nitrobenzoic acid, a similar lag-
phase of slow degradation, followed by a phase of rapid degradation, was observed in both topsoils and
subsoils. Remarkably, the rapid phases of degradation in subsoils often approached rates occurring in
the corresponding topsoil. No reduction of the nitro group on either compound was observed, even in a
water-saturated subsoil. Sometimes there were differences in the length of the lag-phases measured
for replicate samples of subsoils; also, application of lower concentrations of 4-nitrobenzoic acid
generally gave rise to shorter lag-phases. Partial sterilization of soils by azide greatly slowed
breakdown of both compounds, con®rming the important role of microbial degradation. Such
behaviour is consistent with the variable build-up of populations of micro-organisms able to degrade
the compound, smaller populations being able to deal rapidly with the lower concentrations. After
applying a second dose of 4-nitrobenzoic acid to soil, degradation was rapid but initially not as fast as
the ®nal rates during breakdown of the ®rst treatment. Hence, soil may only partially retain the ability
to degrade previously applied xenobiotics. Nonetheless it is noteworthy that, even in deep subsoils,
indigenous microbial populations can rapidly adapt to degrade certain small organic molecules.
#
2000 Society of Chemical Industry
Keywords: degradation; micro-organisms; subsoil; arylamide; benzoic acid
1
INTRODUCTION
organic matter, and so the factors that most in¯uence
the fate of organic contaminants in topsoils are much
reduced in subsoils. The important factors determin-
ing breakdown and leaching of chemicals in subsoils
need to be established so that models can be further
developed to predict the risks of contamination of
groundwater.
A good understanding of the behaviour of organic
compounds in subsoils is required to interpret the
results of monitoring programmes for contaminants in
ground and surface waters. Although there is a broad
understanding of how organic compounds behave in
1,2
topsoils, there is much less information for subsoils.
Nevertheless, the potential of micro-oganisms in deep
locations to degrade organic compounds has been
The objective of this work was to investigate some
fundamental reactions (hydrolytic, oxidative and
reductive) undergone by organic compounds in sub-
soils and the factors that in¯uence the rates of such
reactions. The ®rst model compound, N-(4-nitro-
phenyl)propanamide, was chosen because it can be
hydrolysed and/or reduced. Hydrolysis reactions are of
particular interest because they are important degra-
dation processes in subsoils and in aquifers, and can
potentially occur abiotically as well as by microbially
mediated processes. The second compound, 4-nitro-
benzoic acid, was chosen because it has no function-
ality susceptible to hydrolysis, though it can be
3,4
studied and pesticide degradation in subsurface soil
5
reviewed. The in¯uence of depth in soil on biodegra-
6
dation has been discussed for triazines and for
7
aldicarb. The degradation of herbicides in deep
8
sediments, the unsaturated zone and in aquifer
9
10,11
material
has been investigated. Leaching of non-
ionised compounds in topsoils is limited by sorption,
which occurs primarily on soil organic matter.
Degradation of most compounds is mediated by soil
micro-organisms, whose populations are low in soils
low in organic matter. Subsoils generally contain little
*
Correspondence to: Peter Nicholls, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Contract/grant sponsor: Shell Internationale, The Hague, The Netherlands
Contract/grant sponsor: Biotechnology and Biological Research Council
Contract/grant sponsor: Ministry of Agriculture, Fisheries and Food
(
Received 25 February 2000; accepted 27 March 2000)
#
2000 Society of Chemical Industry. Pest Manag Sci 1526±498X/2000/$17.50
607

P Nicholls et al
Bedfordshire were taken from depths down to 2.2m.
Soils were sampled using either a Jarrett augur or a
Humax electric corer. The latter machine packages
soil samples into PVC cylinders (each of length 25cm
and diameter 5.5cm) by means of an inner stainless-
steel sleeve, which minimises the risk of contamination
of soils taken from deeper layers by soil from upper
layers. Soils were sieved (4mm) and their water
contents determined, and they were stored brie¯y in
sealed polythene bags at 10°C. Field capacity was
measured at a pressure of � 50cm of water (� 50kPa).
At Park Farm in Norfolk, a peat soil overlays a light
sandy soil. Topsoil was sampled with a Jarrett augur,
and saturated soil down to 3m depth was obtained
using a rigid PVC pipe inserted into a pre-drilled hole
and attached to a suction pump. Prior to sampling, the
pipe was partially sterilised by rinsing with methanol.
Saturated samples of slurried soil were stored as
necessary under nitrogen at 10°C before use. The
properties of all the soils (Table 1) were measured by
the Soil Survey and Land Research Centre, Silsoe,
UK.
Figure 1. Structures of compounds used in study.
microbially degraded by both oxidative and reductive
processes. Rates of degradation of the two compounds
were measured in three topsoils and subsoils of
different pH together with a peat overlying a satu-
rated-sand subsoil. Both compounds were chosen to
have rates of degradation in subsoils suf®ciently fast to
be studied under laboratory conditions, as microbial
activity in incubated soils can become depleted with
time. The effect of changing the applied concentration
of 4-nitrobenzoic acid was investigated, and rates of
degradation were measured in soils receiving repeated
applications. The role of microbial breakdown of both
compounds was investigated using partial sterilisation
of soil with sodium azide.
2
2
EXPERIMENTAL
.1 Chemicals
2.3 Analysis by high-pressure liquid
chromatography
N-(4-Nitrophenyl)propanamide (Fig. 1) was synthe-
sised by stirring solution of 4-nitroaniline
100mmol) and propionyl chloride (50mmol) in
Aliquots (20ml) of solutions or extracts for analysis
were injected into a high-pressure liquid chromato-
a
�
1
graph (HPLC) and eluted (1.5ml min ) with
(
diethyl ether for 30min. The reaction mixture was
washed with aqueous hydrochloric acid (1M), the
product being obtained by rotary evaporation of the
ethereal layer. N-(4-Nitrophenyl)propanamide and
commercially obtained 4-nitrobenzoic acid (Fig. 1)
were puri®ed by recrystallisation from methanol:water
methanol‡water (70‡30, by volume) for N-(4-
nitrophenyl)propanamide or methanol‡water‡
acetic acid (70‡30‡1, by volume) for 4-nitrobenzoic
acid. A stainless steel column (25cm longÂ4.6mm
ID) packed with Lichrosorb ODS (10mm) was used,
preceded by a guard column of Lichrosorb ODS RP18
(4mmÂ4mm). Detection was by UV spectrophoto-
metry at 326nm for N-(4-nitrophenyl)propanamide
and 260nm for 4-nitrobenzoic acid. Peaks for the
parent compounds and their respective aniline reduc-
tion products were well separated and no interference
was noted from soil co-extractives. The compounds
(70‡30, by volume).
2.2 Soils
Samples of loamy sand from Woburn, Bedfordshire, of
calcareous sandy loam from Whittlesford, Cambridge
and of an acidic sandy loam from Shuttleworth,
Table 1. Soil properties and sorption coefficients for N-(4-nitrophenyl)propanamide
Soil properties
Sorption
a
b
Depth
(cm)
Organic carbon
(%)
H O
FC
(%)
Sand
(%)
Silt
Clay
(%)
Kd
(litrekg
Koc
(litrekg )
2
�
1
� 1
Site
(%)
pH
(%)
)
Woburn
0±30
0.52
0.08
0.11
1.26
0.45
0.09
0.99
0.05
0.03
27.1
0.4
11.8
7.2
15.0 6.57
9.3 6.87
11.7 7.00
20.1 7.54
84
89
92
58
41
23
76
82
93
72
98
7
4
9
7
0.87
0.27
0.23
1.10
0.25
0.18
0.66
0.14
0.07
Ð
167
338
209
89
6
0±100
2
00±220
8.9
2
6
Whittlesford
Shuttleworth
0±25
13.2
26
39
56
15
18
1
16
20
21
9
1
1
00±125
60±200
0±30
9.03 20.5 7.73
55
15.0
20.8 8.02
14.1 4.95
12.4 4.95
7.9 5.64
201
67
12.5
11.0
4.7
6
0±100
0
280
233
Ð
1
90±210
6
Park Farm
0±30
38.3
n/a
n/a
n/a
5.98
7.56
9
19
2
200
0
Ð
Ð
a
Soil-water content during incubation.
Field capacity.
b
608
Pest Manag Sci 56:607±614 (2000)

Degradation rates of an arylamide and a benzoic acid in subsoils
�
1
were quanti®ed by comparing peak heights with
standards, and a precision of 1.4% RSD was obtained.
Recoveries of both compounds from soils and soil-
slurry were >90% and results reported were not
corrected for recovery.
aqueous solution of 4-nitrobenzoic acid (0.2mg ml
;
10ml). Untreated samples were prepared similarly.
Samples of Park Farm topsoil were incubated,
sampled, extracted and analysed as for the other
topsoils.
Soil slurry (40ml) from Park Farm subsoil (depth
2m) was placed in Duran bottles, such that the soil
sediment was covered by a layer of water approxi-
mately 5mm deep so as to maintain anoxic conditions.
An aqueous solution of 4-nitrobenzoic acid (1.0ml;
2
.4 Hydrolysis of N-(4-nitrophenyl)propanamide in
aqueous buffer solution
Solutions of different pH were prepared in distilled
water using the following buffers or salts: pH 0.34
�
1
(0.5M HCl), pH 1.5 (KCl:HCl), pH 5.05 (CH COO-
Na:CH COOH), pH 6.93 (Na HPO :NaH PO ), pH
0.4mg ml ) was added. The contents of each bottle
were mixed and sparged with oxygen-free nitrogen for
5min; the bottles were then tightly capped and
incubated at 10°C. At intervals, sample bottles were
taken and their contents acidi®ed to pH 2.0 with
concentrated hydrochloric acid (1.5ml) and extracted
by shaking for 2h with water-saturated ethyl acetate
(40ml). The resulting emulsion was treated with
calcium chloride (100mg) and centrifuged at
3
3
2
4
2
4
8
.74 (Na B O :KOH), pH 11.2 (KCl:KOH) and pH
2 4 7
1
3.04 (KOH). A stock solution of N-(4-nitrophenyl)-
�
1
propanamide (0.5ml 2mg ml ) in methanol was
added to each solution (100ml) in stoppered conical
¯
asks and maintained at 10°C in the dark. Duplicate
samples taken at intervals were analysed directly by
HPLC. Solutions of pH 11.2 and 13.04 were
protected from atmospheric carbon dioxide with a
trap of potassium hydroxide pellets. The pH of the
solutions was checked at monthly intervals.
�
1
1000rev min for 10min. Aliquots of supernatant
liquid were reduced to dryness by rotary evaporation
and the residues then redissolved in HPLC elution
solvent and analysed as described for the other soils.
2.5 Degradation rates and sorption in unsterilised
soils
Soil/water distribution coef®cients were measured for
each depth at room temperature by gently shaking the
air-dried soil (10g) for 10min with a solution of N-(4-
nitrophenyl)propanamide (20ml; 25mg litre ) in
calcium chloride (0.01M) in Pyrex centrifuge tubes
2.6 Degradation in partially sterilised soil
Soil from Woburn, Whittlesford or Shuttleworth
(1.0kg) was sieved and divided into treated and
control samples. One sample (500g) was treated with
N-(4-nitrophenyl)propanamide or 4-nitrobenzoic acid
as described above. After mixing by sieving, a solution
�
1
�
1
(
30ml). After centrifugation at 1700rev min for
0min, the concentration of the compound remaining
�
1
1
in solution was determined by HPLC.
of sodium azide in water (10ml; 24g litre ) was
added and the soil mixed again by passing twice
through a 2-mm sieve. The soil samples were incu-
bated and soil samples taken for analysis as described
above.
Sieved soil from Woburn, Whittlesford or Shuttle-
worth (ca 1.5kg) was divided into three sub-samples.
Aqueous solutions of N-(4-nitrophenyl)propanamide
or 4-nitrobenzoic acid were incorporated into two of
�
1
the soil samples at a concentration of 8±10mg kg
soil (oven-dry basis) and mixed by sieving. The
remaining sample was prepared similarly but as an
untreated control. The soil samples were placed in 1-
litre amber-glass jars with loose-®tting screw caps. The
water content was adjusted, using glass-distilled water,
to its value at collection. The samples were incubated
at 10°C in the dark. Duplicate sub-samples (20g)
were taken immediately after preparation and then at
intervals, and were extracted by shaking for 2h with
methanol (20ml) for N-(4-nitrophenyl)propanamide
or with methanol‡water‡acetic acid (80‡20‡1 by
volume; 20ml) for 4-nitrobenzoic acid, followed by
centrifugation. The concentration of N-(4-nitro-
phenyl)propanamide or of 4-nitrobenzoic acid in the
supernatant solutions was determined by HPLC as
described above. Calcareous Whittlesford soils re-
quired acidi®cation (1 drop of concentrated sulfuric
acid) prior to analysis by HPLC.
2.7 Degradation rates of different initial
concentrations of 4-nitrobenzoic acid
The compound was previously incubated at a con-
�
1
centration of 10mg g soil on a fresh weight basis.
�
1
Additional incubations at 100, 1.0 and 0.1mg g were
done in the Woburn and Park Farm subsoils, and the
usual procedure was followed with one amendment. In
order to improve the detection of the compound in
soils treated at the lower initial concentrations, the
supernatant solution obtained from the extracted soil
was concentrated by drying an aliquot (10ml) using a
rotary evaporator and dissolving the residue in HPLC
elution solvent (1.0ml).
2.8 Degradation rates in soils previously exposed
to 4-nitrobenzoic acid
Woburn and Park Farm subsoils were treated with the
test compounds and incubated as described above.
When degradation was complete (con®rmed by
analysis), the soil was re-treated with 4-nitrobenzoic
acid and subsequent degradation was monitored.
The Park Farm subsoil samples required different
incubation and extraction procedures because of their
¯ooded condition. Sub-samples (20g) of the peaty
topsoil from a depth of 0±30cm were treated with an
Pest Manag Sci 56:607±614 (2000)
609

P Nicholls et al
Table 2. Rate of hydrolysis of N-(4-nitrophenyl)propanamide
in buffer solution at 10°C
2
). The extrapolated half-life at pH 7, estimated from
rates at lower and higher pH, was 790 years, indicating
that chemical hydrolysis does not contribute to the
degradation in soil, which was ®ve orders of magnitude
faster.
�
1
pH
First-order rate constant (h
)
Half-life (h)
0
1
7
.34
9.4Â10�
3
4
7
4
2
74
�
.50
.00
6.5Â10
1060
�
�
6a
1.0Â10
7Â10
3.1.3 Degradation of N-(4-nitrophenyl)propanamide in
unsterilised soils
1
1.20
3.04
3.1Â10
2300
10
1
8.2Â10�
The only product of degradation, identi®ed by HPLC
co-chromatography with an authentic standard, was 4-
nitroaniline, which in turn was degraded quite slowly,
so that a mass balance could be made. The only
reaction occurring in aerobic subsoils was hydrolysis,
with reduction of the nitro group not being observed.
Fastest degradation in topsoil occurred in the
alkaline Whittlesford soil which also contained the
most organic carbon of the tested soils (Table 3).
Rates of degradation in soils taken from 2m depth
were similar for all three soil types. Rates of degrada-
tion decreased with depth to about 1/30th of that in
topsoil for the Whittlesford soil and to about 1/10th of
that in topsoil for the Shuttleworth and Woburn soils.
Lag-phases of about 80±100h were observed in some
deeper soils, such as that from Shuttleworth, before a
phase of quite rapid degradation (Fig 2). Lag phases
were longer as depth increased from 60±100 to 180±
210cm. Dual-phase kinetics were observed consis-
tently in replicate samples and on soil sampled at
different dates from the same site.
a
Value obtained by extrapolation.
3
3
RESULTS
.1 Degradation and sorption to soils of N-(4-
nitrophenyl)propanamide
.1.1 Sorption to soils of N-(4-nitrophenyl)propanamide
3
The availability of a compound in soil solution to
biological organisms or for leaching is an inverse
function of its sorption to soil solids. Sorption of N-(4-
nitrophenyl)propanamide (Table 1) was proportional
to organic carbon content, which decreased with soil
depth. In the deepest samples, sorption was very weak.
Values for Koc estimated from the measured K values
d
were greater in subsoils (below 30cm depth) than in
topsoil. Unless the sorptive strength of organic matter
in subsoils is much greater than in topsoil (which is
unlikely), an appreciable proportion of sorption in
subsoils is probably on the soil minerals. Rates of
degradation are correlated with coef®cients of sorption
for some soils and compounds because, as sorption
increases, less of the compound is available for
degradation by micro-organisms. Sorption measure-
ments con®rmed that sorption by subsoils was weaker
than that for topsoils and that strong interactions with
minerals in subsoils did not occur to greatly reduce
availability for degradation.
3.1.4 Degradation of N-(4-nitrophenyl)propanamide in
partially sterilised soils
Treatment of soil with sodium azide increased its pH
by up to about 0.5 units, which might slightly in¯uence
rates of degradation (Table 3, Fig 3). Sterilisation
decreased rates of degradation to between 1/2 and 1/
100th of those in unsterilised soil. The greatest effect
3.1.2 Rates of hydrolysis of N-(4-
nitrophenyl)propanamide in buffer solutions
in topsoil was for the acidic Shuttleworth soil (Fig 3)
and the least was for the alkaline Whittlesford soil. In
contrast, for soils taken from 2m depth, the greatest
effect was on the Whittlesford and the least on the
Shuttleworth soil. Breakdown rates were similar in the
Shuttleworth and neutral Woburn soils for the
sterilised and also for the non-sterilised treatments,
Hydrolysis of N-(4-nitrophenyl)propanamide in solu-
tion was too slow to measure at pH values close to
neutral with no measurable degradation occurring at
pH 7.0 after 3.5 months at 10°C. However the
hydrolysis was both acid and base catalysed (Table
Unsterilised
Sterilised
�
1
� 1
Soil
Depth (cm)
k1 (h
)
t1/2 (h)
k1 (h
)
t1/2 (h)
Woburn loamy sand
0±30
0.066
Ð
11
0.017
0.0046
0.0002
0.072
0.051
42
150
a
6
0±100
46
a
1
90±220
0±25
Ð
105
3620
8
Whittlesford sandy loam
Shuttleworth sandy loam
0.15
0.15
Ð
4.4
4.4
1
1
00±125
60±200
0±30
13
a
136
0.00005
0.005
13100
147
0.060
0.012
Ð
12
57
6
0±100
0.005
150
a
1
80±210
120
0.0002
3600
a
Table 3. Rates of degradation at 10°C of N-(4-
A lag-phase was observed in these experiments and so here the values quoted are the time at which only
50% of the parent compound remained.
�
1
nitrophenyl)propanamide in soil (9mg g
)
610
Pest Manag Sci 56:607±614 (2000)

Degradation rates of an arylamide and a benzoic acid in subsoils
�
1
Figure 2. Degradation of N-(4-nitrophenyl)propanamide (9mg g soil) at
0°C in Shuttleworth sandy loam from different soil depths: (&) 0–30cm,
*) 30–60cm, (~) 60–100cm, (&) 180–210cm.
�
1
1
Figure 4. Degradation of 4-nitrobenzoic acid (10mg g soil) at 10°C in
Woburn loamy sand from different soil depths: (&) run 1, (*) run 2, (~) run
(
3.
degradation, followed by a phase of very rapid
degradation with a DT50 of 1±3h in all soils. The
duration of the lag-phase increased with depth, from
40h in topsoil to 120±160h in subsoil. The lag-phase
was presumed to be caused by the increase of
populations of soil microbes able to degrade 4-
nitrobenzoic acid. Once the population reached a
threshold size, very rapid degradation occurred. The
increase of the duration of the lag-phase with increas-
ing depth may be due to the initial population of
suitable micro-organisms being less at depth. This
would also account for the slower rate of degradation
during the lag-phase in deeper soils.
�
1
Figure 3. Degradation of N-(4-nitrophenyl)propanamide (9mg g soil) at
0°C in Shuttleworth sandy loam from different soil depths after azide
sterilisation: (&) 0–30cm, (*) 30–60cm, (~) 60–100cm, (&) 180–
10cm.
1
In Whittlesford and Shuttleworth topsoils, degrada-
tion of 4-nitrobenzoic acid followed similar kinetics to
those in Woburn topsoil: an initial lag-phase of 30±
60h during which the half-life of the compound was
118±170h, followed by a phase of rapid degradation
with a half-life of 3±5h (Tables 5 and 6). The subsoils
from both sites showed a longer lag-phase followed by
a rapid phase with a half-life of 2±3h. The half-lives
during the lag-phase in Whittlesford subsoil and
topsoil were similar, whilst in Shuttleworth subsoil
the half-life was much longer than that in topsoil. The
behaviour of 4-nitrobenzoic acid was therefore gen-
erally similar in the aerobic soils from the three
different locations, for both topsoils and subsoils.
There was some variation in the duration of the lag-
phase for different samples of subsoils measured under
2
which implies that micro-organisms that catalyse the
hydrolytic reaction can be similarly ef®cient in soils of
different pH. Degradation was slowest in the sterilised
Whittlesford subsoil which was predominantly com-
prised of calcium carbonate. Degradation closely ®tted
single-phase ®rst-order kinetics.
3
.2 Degradation in soils of 4-nitrobenzoic acid
3.2.1 Degradation of 4-nitrobenzoic acid in aerobic
topsoils and subsoils
The degradation kinetics of 4-nitrobenzoic acid in
Woburn soil consisted of two distinct phases (Fig 4
and Table 4): initially there was a lag-phase of slow
Depth
cm)
Initial concentration Duration of lag-phase Lag-phase t
1
Rapid phase t1/2
(h)
1/2
�
(
(mgg
)
(h)
(h)
Non-sterilised
1
1
1
1
60±200
60±200
60±200
60±200
10
1
130±170
70±80
50±80
27
700
>2000
>2000
29
3
8
4
2
0.1
a
10
Azide-sterilised
±30
0±120
60±200
b
0
10
10
10
Ð
Ð
Ð
Ð
Ð
Ð
>2000
b
8
>2000
>2000
b
1
a
b
Table 4. Degradation of 4-nitrobenzoic
acid in Woburn soil at 10°C
Data refer to degradation of second application.
Slow degradation with no lag-phase.
Pest Manag Sci 56:607±614 (2000)
611

P Nicholls et al
Duration of lag-phase
(h)
Lag-phase t1/2
(h)
Rapid phase t1/2
(h)
Depth (cm)
Non-sterilised
0
±30
60±200
Azide-sterilised
±30
60±200
30±40
167
3
2
1
100±130
1500
a
0
Ð
Ð
Ð
Ð
>2000
>2000
a
1
Table 5. Degradation at 10°C of 4-nitrobenzoic
�
1
a
acid (9mg g ) in Shuttleworth clay loam
Slow degradation with no lag-phase.
Duration of lag-phase
(h)
Lag-phase t1/2
(h)
Rapid phase t1/2
(h)
Depth (cm)
Non-sterilised
0
1
±40
45±60
80
118
96
5
3
40±180
Azide-sterilised
a
0
1
±40
Ð
Ð
Ð
Ð
>2000
a
40±180
>2000
Table 6. Degradation of 4-nitrobenzoic acid
�
1
a
(9mg g ) in Whittlesford soil at 10°C
Slow degradation with no lag-phase.
similar conditions (Fig 4). The variation was observed
for all subsoils including those from Park Farm (Fig 5).
The precise time of onset of the rapid phase was thus
dif®cult to predict. This may have been caused by
different initial populations of degrading micro-organ-
isms in different samples, despite the relatively large
size of those samples (0.5±1kg). The variation was
probably not great enough to have been caused by
adaptation of micro-organisms by mutation. Results
are given as the mean of replicate determinations.
Partial sterilisation of Woburn soil with sodium
azide eliminated the lag-phase and greatly slowed the
degradation of 4-nitrobenzoic acid (Table 4). Half-
lives were>2000h in all soils, con®rming the im-
portant role of microbial activity. Partial sterilisation
had similar effects in Whittlesford and Shuttleworth
topsoils and subsoils (Table 5 and 6). Degradation
closely ®tted single-phase ®rst-order kinetics.
incubation, during which time the half-life was 640h.
This slow breakdown could be due to the sterilisation
process being less effective in saturated soils, or due to
chemical breakdown of the compound. Reducing
conditions prevailed in the subsoil but 4-aminobenzoic
acid, the reduction product of 4-nitrobenzoic acid, was
not detected.
3.2.3 Degradation rates in subsoils of different initial
concentrations of 4-nitrobenzoic acid
On lowering the applied concentration of 4-nitroben-
zoic acid in Woburn subsoil, the lag-phase was
shortened, as was the half-life during this phase; the
subsequent rapid phase had a half-life of 3±8h (Table
4). The half-life of the compound during the rapid
phase of degradation was similar at 0.1, 1.0 and
�
1
10mg g soil. Breakdown during the lag-phase at both
�
1
the lower initial concentrations of 1.0 and 0.1mg g
soil, was apparently slower (t >2000h) than at 10mg
1
/2
�
1
3.2.2 Degradation of 4-nitrobenzoic acid in Park Farm
soil
g
soil, but this was probably due to the dif®culty of
In the peaty topsoil from Park Farm, a single phase of
rapid degradation occurred with a half-life of 8h
(Table 7 and Fig 5). This is consistent with there being
much organic matter in this peaty soil (27.1%) and
therefore a large microbial population. Incubation of
4
2
1
2
-nitrobenzoic acid in the saturated subsoil taken from
m depth at Park Farm gave rather variable results. At
0mg g
soil, a mean lag-phase of duration about
20h (and having t1/2 165h) was followed by a rapid
�
1
phase of breakdown with a half-life of 9h (Table 7).
The latter is very similar behaviour to that in the
aerobic subsoils at the same concentration.
Little breakdown occurred in Park Farm topsoil
treated with sodium azide. Partial sterilisation of the
saturated subsoil gave no lag-phase during 1100h
Figure 5. Degradation of 4-nitrobenzoic at 10°C in Park Farm saturated
sand from 200cm depth underlying peat, effect of initial concentration:
(&) run 1, (&) run 2, (^) run 3.
612
Pest Manag Sci 56:607±614 (2000)

Degradation rates of an arylamide and a benzoic acid in subsoils
Initial concentration Duration of lag-phase Lag-phase t1/2 Rapid phase t1/2
�
1
Depth (cm)
(mgg
)
(h)
(h)
(h)
0
00
00
00
00
00
±30
10
100
10
0
Ð
176
165
79
8
Ð
9
2
2
2
2
2
>300
ca 220
ca 140
ca 140
Ð
1
2
0.1
a
10
42
9
b
Ð
27
a
b
Table 7. Degradation of 4-nitrobenzoic
acid in Park Farm soil at 10°C
Data refer to degradation of second application.
No lag-phase observed.
estimating the rate from the data. Incubation of lower
concentrations of 4-nitrobenzoic acid (1.0 and
Breakdown of 4-nitrobenzoic acid showed similar
kinetics, though the possibility of abiotic transforma-
tion would be limited to reduction of the nitro group
which was again not observed, even in an anaerobic
�
1
0
.1mg g ) in saturated subsoil from Park Farm
produced rather variable results perhaps due to the
dif®culty of analysis at such low concentrations, but
the duration of the lag-phase tended to be shorter
15,16
sandy subsoil. Lag-phases
were well marked and,
in contrast to the behaviour of N-(4-nitrophenyl)pro-
panamide, occurred even in most of the topsoils;
indeed the lag-phases were shorter in topsoils than
subsoils. Breakdown after the lag-phase was very
rapid, with half-lives of 1 to 8h. Azide sterilisation of
samples of the three aerobic topsoils and subsoils
essentially completely stopped breakdown. This im-
pact of sterilisation on breakdown, observed to varying
degree for both compounds, con®rmed the dominant
role of microbial processes in the degradation in soil.
Behaviour of 4-nitrobenzoic acid in the anaerobic
Park Farm subsoil was remarkably similar to that in
the aerobic subsoils indicating that different soils
presumably containing different populations of
micro-organisms can nonetheless similarly degrade
xenobiotics, as was also found for mecoprop by Heron
(
Table 7). On increasing the initial concentration to
�
1
1
00mg g
in Park Farm soil, a single phase of
degradation lasting at least 300h with a half-life of
76h was observed. These results are again consistent
1
with a small population of microbes growing to a
threshold size beyond which rapid degradation occurs,
the lower concentrations of 4-nitrobenzoic acid
requiring a smaller population to cause rapid break-
down.
3
4
.2.4 Degradation rates in subsoils previously exposed to
-nitrobenzoic acid
Treating Woburn or Park Farm subsoils with two
applications of 4-nitrobenzoic acid demonstrated that
the soil only partially retained the ability to degrade
this compound (Tables 4 and 7, and Fig 6). The
second dose of 4-nitrobenzoic acid was degraded in
Woburn soil with lag-phases intermediate in length
between those occurring in topsoil and those in
previously untreated subsoil.
11
and Christenesen . Application of lower concentra-
tions of 4-nitrobenzoic acid generally gave rise to
shorter lag-phases and indeed, over 300h of incuba-
tion, no lag-phase was observed for the highest applied
�
1
concentration of 100mg g , although substantial
degradation was occurring. Lag-phases in the degra-
dation of 4-nitrobenzoic acid at practical concentra-
tions in all the soils, except the peaty topsoil from Park
Farm, were relatively short and fairly reproducible. On
applying a further treatment of 4-nitrobenzoic acid to
loamy sand subsoil previously treated, breakdown of
4
DISCUSSION AND CONCLUSIONS
Even in subsoils, degradation of N-(4-nitrophenyl)-
propanamide was considerably faster than aqueous
hydrolysis at the same pH, and this emphasises the
importance of microbial degradation. In aerobic
subsoils from the greatest depths of around 2m,
degradation was initially up to thirty times slower than
in topsoils despite sorption being low (giving high
availability in soil water) at depth; this is consistent, as
7,12±14
found by others,
with there being least organic
matter and microbial activity in these subsoils.
However lag-phases of about 30±150h occurred in
the subsoil incubations of N-(4-nitrophenyl)propana-
mide followed by more rapid degradation, this effect
being most marked for the deeper subsoil samples.
Such breakdown was primarily by microbially
mediated hydrolysis, with no reduction of the nitro
group observed. Partial sterilisation with azide of the
soils slowed degradation, most markedly for the
deepest soil samples.
� 1
Figure 6. Degradation of 4-nitrobenzoic acid (10mg g soil) at 10°C in
Woburn loamy sand from 160–200cm depth. First application: (&) run 1,
(*) run 2, (~) run 3; second application:
.
Pest Manag Sci 56:607±614 (2000)
613

P Nicholls et al
the compound remained enhanced. These observa-
tions indicate that a time interval is required for the
growth of that fraction of the microbial community
with the innate ability to break down the
3 Johnson AC and Wood M, Microbial potential of sandy aquifer
material in the London basin. Geomicrob J 10:1±13 (1992).
4
Moorman TB, Adaptation of micro-organisms in subsurface
environments, in Enhanced Biodegradation of Pesticides in the
Environment, ed by Racke KD and Coats JR, ACS Symp Ser
462, American Chemical Society, Washington DC, USA. pp
167±179 (1990).
9±11
compound.
The kinetics of the observed breakdown of 4-
nitrobenzoic acid do not indicate that Monod-type
induction of enzymes was occurring. Also, the fair
agreement between replicate incubations and the short
lag-phases indicate that mutation was not responsible
for the lag-phase. The pattern of breakdown is,
however, indicative of microbial growth, lag-phases
being a common feature of batch culture systems such
5
Helweg A, Degradation of pesticides in subsurface soil, in
Proceedings of the International Symposium on Environmental
Aspects of Pesticide Microbiology. ed by Anderson JPE, Arnold
D, Lewis F and Torstensson L, Sigtuna, Sweden. pp 249±265
(1997).
6 Kruger EL, Somasundaram L, Kanwar RS and Coats JR,
1
4
Persistence and degradation of C-atrazine and deisopropyla-
trazine as affected by soil depth and moisture conditions.
Environ Toxicol Chem 12:1959±1967 (1993).
4,17,18
as the soil incubations studied here.
Both aerobic
7
8
9
Ou L-T, Rao PSC, Edvardsson KSV, Jessup RE, Hornsby AG
and Jones RL, Aldicarb degradation in sandy soils from
different depths. Pestic Sci 23:1±12 (1988).
and saturated subsoils thus can contain micro-organ-
isms capable of ef®ciently degrading certain small
organic molecules such as might arise from the
metabolism of pesticides. Whilst not all such xenobio-
tics might be so readily degraded, nonetheless these
Stolpe NB and Shea PJ, Alachlor and atrazine degradation in a
Nebraska soil and underlying sediments. Soil Sci 160:359±370
(
1995).
Issa S and Wood M, Degradation of atrazine and isoproturon in
®
ndings indicate that subsoils have appreciable capa-
the unsaturated zone. Pestic Sci 55:539±545 (1999).
city to degrade organic compounds over a wide range
of concentrations, and this may mitigate the leaching
of such compounds to aquifers.
10 Wood M, Harold J, Johnson A and Hance RJ, The potential for
atrazine degradation in aquifer sediments, in Pesticides in Soils
and Water: Current Perspectives, ed by Walker A, BCPC,
Farnham, Surrey, UK, pp 175±182 (1991).
1
1 Heron G and Christensen TH, Degradation of the herbicide
mecoprop in an aerobic aquifer determined by laboratory
batch studies, Chemosphere 24:547±557 (1992).
ACKNOWLEDGEMENTS
We are grateful to Shell Internationale, The Hague,
The Netherlands, for funding the project and to Drs IJ
Graham-Bryce, RH Bromilow and TR Roberts for
their encouragement. We thank Novartis, Whittle-
sford and Shuttleworth Agricultural College for
providing sites for soil sampling. IACR-Rothamsted
receives grant-aided support from the Biotechnology
and Biological Research Council and the Ministry of
Agriculture, Fisheries and Food.
12 Fournier J-C, Soulas G and Parekh NR, Main microbial
mechanisms of pesticide degradation in soils, in. Soil Ecotox-
icology, ed by Tarradellas J, Bitton G and Rossel D, CRC Press
Inc, Boca Raton, USA, pp 85±116 (1997).
1
3 Moorman TB and Harper SS, Transformation and mineralisa-
tion of metribuzin in surface and subsurface horizons of a
Mississippi delta soil. J Envir Qual 18:302±306 (1989).
1
4
1
4 Helweg A, Degradation and adsorption of
C-mecoprop
(
MCPP) in surface soils and in subsoil. In¯uence of tem-
perature, moisture content, sterilization and concentration on
degradation. Sci Total Envir 132:229±241 (1993).
1
5 Audus LJ, The biological detoxication of 2,4-dichlorophenoxy-
acetic acid in soil. Plant Soil 2:31±36 (1949).
REFERENCES
16 Hurle K, lag-phase studien mit DNOC und 2,4-D im boden.
Proc Eur Weed Res Coun Symp Herbicides-Soil, pp 151±162
(1973).
1
Nicholls PH, Organic contaminants in soils and groundwaters, in
Organic Contaminants in the Environment. Environmental Path-
ways and Effects. ed by Jones KC, Elsevier Applied Science,
London, UK, pp 87±133 (1992).
17 Spain JC, Microbial adaptation in aquatic ecosystems, in
Enhanced Biodegradation of Pesticides in the Environment, ed by
Racke KD and Coats JR, ACS Symp Ser 426, American
Chemical Society, Washington DC, USA, pp 181±190 (1990).
18 Brock TD and Madigan MT, Biology of Microorganisms, 5th edn,
Prentice-Hall Inc, New Jersey, USA, pp 184±187 (1984).
2
Campbell AT, Nicholls PH and Bromilow RH, Factors
in¯uencing rates of degradation of an arylamide in subsoils.
in Pesticides in Soils and Water: Current Perspectives ed by Walker
A, BCPC, Farnham, Surrey, UK, pp 155±161 (1991).
614
Pest Manag Sci 56:607±614 (2000)