Pest Management Science Pest Manag Sci 56:607±614 (2000)

Factors influencing rates of degradation of anarylamide and a benzoic acid in subsoilsPeter Nicholls,* Andrew T Campbell and Roger H WilliamsIACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK

(Rec

* CoContContCont

# 2

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 INTRODUCTIONA 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

topsoils, there is much less information for subsoils.1,2

Nevertheless, the potential of micro-oganisms in deep

locations to degrade organic compounds has been

studied3,4 and pesticide degradation in subsurface soil

reviewed.5 The in¯uence of depth in soil on biodegra-

dation has been discussed for triazines6 and for

aldicarb.7 The degradation of herbicides in deep

sediments,8 the unsaturated zone9 and in aquifer

material10,11 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

eived 25 February 2000; accepted 27 March 2000)

rrespondence to: Peter Nicholls, IACR-Rothamsted, Harpenden, Hertract/grant sponsor: Shell Internationale, The Hague, The Netherlandsract/grant sponsor: Biotechnology and Biological Research Councilract/grant sponsor: Ministry of Agriculture, Fisheries and Food

000 Society of Chemical Industry. Pest Manag Sci 1526±498X/2

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.

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

s AL5 2JQ, UK

000/$17.50 607

Figure 1. Structures of compounds used in study.

P Nicholls et al

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 EXPERIMENTAL2.1 ChemicalsN-(4-Nitrophenyl)propanamide (Fig. 1) was synthe-

sised by stirring a solution of 4-nitroaniline

(100mmol) and propionyl chloride (50mmol) in

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

(70�30, by volume).

2.2 SoilsSamples 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)propa

Site

Soil properties

Depth

(cm)

Organic carbon

(%)

H2O a

(%)

FC b

(%)

Woburn 0±30 0.52 11.8 15.0

60±100 0.08 7.2 9.3

200±220 0.11 8.9 11.7

Whittlesford 0±25 1.26 13.2 20.1

100±125 0.45 9.03 20.5

160±200 0.09 15.0 20.8

Shuttleworth 0±30 0.99 12.5 14.1

60±100 0.05 11.0 12.4

190±210 0.03 4.7 7.9

Park Farm 0±30 27.1 38.3 n/a

200 0.4 n/a n/a

a Soil-water content during incubation.b Field capacity.

608

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 ÿ50cm of water (ÿ50kPa).

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.

2.3 Analysis by high-pressure liquidchromatographyAliquots (20ml) of solutions or extracts for analysis

were injected into a high-pressure liquid chromato-

graph (HPLC) and eluted (1.5ml minÿ1) with

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 long�4.6mm

ID) packed with Lichrosorb ODS (10mm) was used,

preceded by a guard column of Lichrosorb ODS RP18

(4mm�4mm). 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

namide

Sorption

pH

Sand

(%)

Silt

(%)

Clay

(%)

Kd

(litrekgÿ1)

Koc

(litrekgÿ1)

6.57 84 7 9 0.87 167

6.87 89 4 7 0.27 338

7.00 92 2 6 0.23 209

7.54 58 26 16 1.10 89

7.73 41 39 20 0.25 55

8.02 23 56 21 0.18 201

4.95 76 15 9 0.66 67

4.95 82 18 0 0.14 280

5.64 93 1 6 0.07 233

5.98 72 9 19 Ð Ð

7.56 98 0 2 Ð Ð

Pest Manag Sci 56:607±614 (2000)

Degradation rates of an arylamide and a benzoic acid in subsoils

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.

2.4 Hydrolysis of N-(4-nitrophenyl)propanamide inaqueous buffer solutionSolutions of different pH were prepared in distilled

water using the following buffers or salts: pH 0.34

(0.5M HCl), pH 1.5 (KCl:HCl), pH 5.05 (CH3COO-

Na:CH3COOH), pH 6.93 (Na2HPO4:NaH2PO4), pH

8.74 (Na2B4O7:KOH), pH 11.2 (KCl:KOH) and pH

13.04 (KOH). A stock solution of N-(4-nitrophenyl)-

propanamide (0.5ml 2mg mlÿ1) 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.

2.5 Degradation rates and sorption in unsterilisedsoilsSoil/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 litreÿ1) in

calcium chloride (0.01M) in Pyrex centrifuge tubes

(30ml). After centrifugation at 1700rev minÿ1 for

10min, the concentration of the compound remaining

in solution was determined by HPLC.

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

the soil samples at a concentration of 8±10mg kgÿ1

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.

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)

aqueous solution of 4-nitrobenzoic acid (0.2mg mlÿ1;

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;

0.4mg mlÿ1) 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

1000rev minÿ1 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.

2.6 Degradation in partially sterilised soilSoil 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

of sodium azide in water (10ml; 24g litreÿ1) 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.

2.7 Degradation rates of different initialconcentrations of 4-nitrobenzoic acidThe compound was previously incubated at a con-

centration of 10mg gÿ1 soil on a fresh weight basis.

Additional incubations at 100, 1.0 and 0.1mg gÿ1 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 exposedto 4-nitrobenzoic acidWoburn 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.

609

Table 2. Rate of hydrolysis of N-(4-nitrophenyl)propanamidein buffer solution at 10°C

pH First-order rate constant (hÿ1) Half-life (h)

0.34 9.4�10ÿ3 74

1.50 6.5�10ÿ4 1060

7.00 1.0�10ÿ7 7�106a

11.20 3.1�10ÿ4 2300

13.04 8.2�10ÿ2 10

a Value obtained by extrapolation.

P Nicholls et al

3 RESULTS3.1 Degradation and sorption to soils of N-(4-nitrophenyl)propanamide3.1.1 Sorption to soils of N-(4-nitrophenyl)propanamideThe 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 Kd values

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.

3.1.2 Rates of hydrolysis of N-(4-nitrophenyl)propanamide in buffer solutionsHydrolysis 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

Table 3. Rates of degradation at 10°C of N-(4-nitrophenyl)propanamide in soil (9mg gÿ1)

Soil

Woburn loamy sand

Whittlesford sandy loam

Shuttleworth sandy loam

a A lag-phase was observed

50% of the parent compound

610

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.

3.1.3 Degradation of N-(4-nitrophenyl)propanamide inunsterilised soilsThe 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.

3.1.4 Degradation of N-(4-nitrophenyl)propanamide inpartially sterilised soilsTreatment 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

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,

Depth (cm)

Unsterilised Sterilised

k1 (hÿ1) t1/2 (h) k1 (hÿ1) t1/2 (h)

0±30 0.066 11 0.017 42

60±100 Ð 46a 0.0046 150

190±220 Ð 105a 0.0002 3620

0±25 0.15 4.4 0.072 8

100±125 0.15 4.4 0.051 13

160±200 Ð 136a 0.00005 13100

0±30 0.060 12 0.005 147

60±100 0.012 57 0.005 150

180±210 Ð 120a 0.0002 3600

in these experiments and so here the values quoted are the time at which only

remained.

Pest Manag Sci 56:607±614 (2000)

Figure 2. Degradation of N-(4-nitrophenyl)propanamide (9mg gÿ1 soil) at10°C in Shuttleworth sandy loam from different soil depths: (&) 0–30cm,(*) 30–60cm, (~) 60–100cm, (&) 180–210cm.

Figure 3. Degradation of N-(4-nitrophenyl)propanamide (9mg gÿ1 soil) at10°C in Shuttleworth sandy loam from different soil depths after azidesterilisation: (&) 0–30cm, (*) 30–60cm, (~) 60–100cm, (&) 180–210cm.

Figure 4. Degradation of 4-nitrobenzoic acid (10mg gÿ1 soil) at 10°C inWoburn loamy sand from different soil depths: (&) run 1, (*) run 2, (~) run3.

Degradation rates of an arylamide and a benzoic acid in subsoils

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 acid3.2.1 Degradation of 4-nitrobenzoic acid in aerobictopsoils and subsoilsThe 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

Table 4. Degradation of 4-nitrobenzoicacid in Woburn soil at 10°C

Depth

(cm)

Initial concen

(mggÿ1

Non-sterilised

160±200 10

160±200 1

160±200 0.1

160±200 10a

Azide-sterilised

0±30 10

80±120 10

160±200 10

a Data refer to degradation of seconb Slow degradation with no lag-phas

Pest Manag Sci 56:607±614 (2000)

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.

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

tration

)

Duration of lag-phase

(h)

Lag-phase t1/2

(h)

Rapid phase t1/2

(h)

130±170 700 3

70±80 >2000 8

50±80 >2000 4

27 29 2

Ð Ð >2000b

Ð Ð >2000b

Ð Ð >2000b

d application.

e.

611

Table 5. Degradation at 10°C of 4-nitrobenzoicacid (9mg gÿ1) in Shuttleworth clay loam

Depth (cm)

Duration of lag-phase

(h)

Lag-phase t1/2

(h)

Rapid phase t1/2

(h)

Non-sterilised

0±30 30±40 167 3

160±200 100±130 1500 2

Azide-sterilised

0±30 Ð Ð >2000a

160±200 Ð Ð >2000a

a Slow degradation with no lag-phase.

Table 6. Degradation of 4-nitrobenzoic acid(9mg gÿ1) in Whittlesford soil at 10°C

Depth (cm)

Duration of lag-phase

(h)

Lag-phase t1/2

(h)

Rapid phase t1/2

(h)

Non-sterilised

0±40 45±60 118 5

140±180 80 96 3

Azide-sterilised

0±40 Ð Ð >2000a

140±180 Ð Ð >2000a

a Slow degradation with no lag-phase.

Figure 5. Degradation of 4-nitrobenzoic at 10°C in Park Farm saturatedsand from 200cm depth underlying peat, effect of initial concentration:(&) run 1, (&) run 2, (^) run 3.

P Nicholls et al

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.

3.2.2 Degradation of 4-nitrobenzoic acid in Park FarmsoilIn 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-nitrobenzoic acid in the saturated subsoil taken from

2m depth at Park Farm gave rather variable results. At

10mg gÿ1 soil, a mean lag-phase of duration about

220h (and having t1/2 165h) was followed by a rapid

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

612

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 initialconcentrations of 4-nitrobenzoic acidOn 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

10mg gÿ1 soil. Breakdown during the lag-phase at both

the lower initial concentrations of 1.0 and 0.1mg gÿ1

soil, was apparently slower (t1/2>2000h) than at 10mg

gÿ1 soil, but this was probably due to the dif®culty of

Pest Manag Sci 56:607±614 (2000)

Table 7. Degradation of 4-nitrobenzoicacid in Park Farm soil at 10°C

Depth (cm)

Initial concentration

(mggÿ1)

Duration of lag-phase

(h)

Lag-phase t1/2

(h)

Rapid phase t1/2

(h)

0±30 10 0 Ð 8

200 100 >300 176 Ð

200 10 ca 220 165 9

200 1 ca 140 79 2

200 0.1 ca 140 42 9

200 10a Ð Ð 27b

a Data refer to degradation of second application.b No lag-phase observed.

Degradation rates of an arylamide and a benzoic acid in subsoils

estimating the rate from the data. Incubation of lower

concentrations of 4-nitrobenzoic acid (1.0 and

0.1mg gÿ1) 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

(Table 7). On increasing the initial concentration to

100mg gÿ1 in Park Farm soil, a single phase of

degradation lasting at least 300h with a half-life of

176h was observed. These results are again consistent

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.

3.2.4 Degradation rates in subsoils previously exposed to4-nitrobenzoic acidTreating 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.

Figure 6. Degradation of 4-nitrobenzoic acid (10mg gÿ1 soil) at 10°C inWoburn loamy sand from 160–200cm depth. First application: (&) run 1,(*) run 2, (~) run 3; second application: .

4 DISCUSSION AND CONCLUSIONSEven 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

found by others,7,12±14 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.

Pest Manag Sci 56:607±614 (2000)

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

sandy subsoil. Lag-phases15,16 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

and Christenesen11. 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

concentration of 100mg gÿ1, 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

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

compound.9±11

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

as the soil incubations studied here.4,17,18 Both aerobic

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

®ndings indicate that subsoils have appreciable capa-

city to degrade organic compounds over a wide range

of concentrations, and this may mitigate the leaching

of such compounds to aquifers.

ACKNOWLEDGEMENTSWe 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.

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