Potential for denitrification at depth below long-term grass swards

~~717(~~12~

POTENTIAL

FOR DENITRIFICATION AT DEPTH LONG-TERM GRASS SWARDS S. C. JARVIS and

AFRC

Soil Biol. Eiochz. Vol. 26,No. 12,pp. 1629-1636, 1994 Else&z scienceLtd. Printedin Great Britain

BELOW

D. J. HATCH

Institute of Grassland and Environmental

Research, North Wyke, Okehampton EX20 2SB, England

~A~ceFted I9 May 1994) Summary-The denitrification potential (PDN) of soil and chalk samples taken to depth (down to > 6 m) below a number of long-term grassland and arable managements was determined under controlled laboratory conditions. Cut and grazed grassland (either grass-clover with no fertilizer N or grass with 420 kg N ha-‘) and a continuous arable management were examined. PDN rates were always greater in the top soil samples and declined progressively with depth. However, even at depths of 6 m, significant PDN was demonstrated under all treatments. This was enhanced bv the addition of available C. There were apparent differences between the previous management treatments particularly in the upper soil samples, but these were also found at greater depths in some cases. PDN was always less in the samples from the grassxlover swards than in comparable samples from fertilized swards. Adding extra labile C had little or no affect on PDN in the grass-clover samples. Calculation of PDN on an area basis indicated that even in the absence of added C, and even in those treatments with low PDN rates, there was substantiai potential for denitrifi~tion. Adding C increased the overall potential by, on average, nearly Z-fold for the grassland samples and by over 4-fold for samples from the long-term arable system. Even if only a small proportion of this potential was achieved in practice, this has major significance for decreasing the amount of NO, reaching aquifers.

INTRODUCTION

The recent introduction of EC standards for NO; concentrations in drinking and other waters (EC, 1980) has stimulated much interest in the controls over the movement of NO; through soil profiles to water bodies. There is considerable evidence accumulated over recent years that, under intensive managements, grassland swards used to support grazing livestock may be responsible for considerable amounts of excess NO;-N leaching from the rooting zone into aquifers (Ryden et al., 1984; Macduff et al., 1990a). A review (Jarvis, 1993) of recent leaching studies on a variety of soil types under a range of conditions has indicated that the equivalent of up to 45% of the annual fertilizer N addition may be leached from highly-fertilized, grazed grassland. The movement of such quantities of N results in NO; concentrations in the percolating water which are considerably in excess of the EC standard. Even with fertilizer N additions which represent average rates for intensive lowland agriculture in the U.K. (i.e. ea. 200 kg), NOT-N concentrations in leachates may be well above the recommended standard of 11.3 mg 1-l (Schofield et al., 1993). It is often assumed that NO; moving in this way is non-reactive and will therefore be transported to the saturated zone or confined aquifer without change and at a rate that is dependent on the factors controlling water movement. If this is the case, then

unless dilution caused by dispersion takes place, the initial NO; concentration in the soil solution would be transmitted through to the stored water. There is, however, evidence from a number of studies which suggests that there may be microbial effects which influence the final concentrations of NO; in the ground water. Denitrification, i.e. the dissimilatory reduction of NO; to dinitrogen (N2) and nitrous oxide (N,O) gases, at depth has been suggested as being a significant mechanism which may reduce the loading of NO; in the saturated zone (Gillham, 1991). For this to occur, appropriate distributions of bacteria (facultative anaerobes) are required with a source of soluble C to provide an energy source. Many aquifers will contain organic materials transported from the soil, and whilst these will be small in comparison with those in the upper soil layers, there will also be communities of bacteria present (Wood et al., 1991). Denitritication has been demonstrated in a number of aquifers (Trudell et al., 1986; Gillham, 1991), although the magnitude and significance of this activity have not been quantified. There is also the possibility that denitrification may also be an important removal mechanism as NO, moves through the unsaturated zone (Lind and Eiland, 1989; Lehn-Reiser et al., 1990). Whilst it is likely that aerobic conditions exist in parts of this zone (much more than in a confined aquifer for example), the physical structure of the profile will be such that much of it will be anaerobic.

I629

S. C.

1630

JAWS

and D. J. HATCH

High concentrations of NO; can move from grassland soils to the unsaturated zone: with fertilizer rates of 400 kg N ha-‘, concentrations substantially greater than 50 mg N I-’ have been found in leachates from grazed grassland (Macduff et al., 1990b). These soils may also, because of their high contents of organic matter (as compared with arable soils), provide a ready supply of mobile C to allow significant denitrification to occur. Grazing animals may be particularly important because both C and N are cycled in relatively mobile forms in their excreta. Denitrification is known to occur in parts of the soil profile which are not usually examined (Jarvis et ai., 1991). but in situ measurements in the less accessible, deeper parts of the profile, are difficult to undertake. However, samples of the matrix abstracted from the profile and then taken back to the laboratory for investigation under controlled environment conditions can provide an indication of the extent of the denitrifying population and current energy supply. Whilst not providing quantification of absolute rates, such information is an important step in defining the overall effects of existing and changing agricultural managements on the long term behaviour of NO;. In our studies we therefore examined the potential that existed for denitrification to occur to a depth of up to 6-7 m below a well-drained soil overlying chalk which had received defined and contrasting managements for long periods. MATERIALS

AND METHODS

Experimental site Samples were collecting from old experimental treatment areas on the IGER farm at Hurley, Berkshire in SE England, which had formed the basis for a number of recent investigations into the effects of grassland management on N transformations and losses from grassland. Earlier studies by Ryden ef al. (1984) had indicated that some of the treatments involved in this experimental regime, had resulted in major fluxes of NO; from the swards down to at least 6 m. The main experimental area was on a freely drained loam of the Frilsham series (a typical argillic brown earth or orthic uvisol). The top-soil (to 20 cm depth) contained, on an oven dry basis, 10.4% clay, 2.3% organic C, 0.27% organic N and had a pH of 65 and a CEC of 202 equivalents kg-‘. The soil texture changes from a calcareous sandy loam to a non-calcareous sandy clay at 40-75 cm and overlies chalk (upper cretaceous, upper chalk) or chalky head which is present at a depth of 75100cm. The previous treatments were based on rectangular plots (i.e. 0.123 ha, 56 x 22m) in which swards had been either grazed or cut and removed, and which had had a range of N inputs. The swards had been established in 1976 with treatments imposed up to, and including, 1990: no further fertilizer inputs had been made after that time and herbage was cut and

removed from all plots during 1991. The areas we investigated were the cut and grazed treatments of ryegrass swards which had had 420 kg fertilizer N ha-’ and grass-clover swards with no fertilizer N. The cut swards were harvested at appropriate times each year to provide material to be conserved as silage. Grazing was with young steers under a rotational management, i.e. 1 week grazing plus 3 weeks recovery. Appropriate P, K and lime regimes had been maintained throughout to sustain sward production. As well as the four grassland managements, a long-term (since 1954) arable treatment which had been in continuous spring barley since 1962, but sited on a neighbouring experiment on the same soil type was also sampled: this had received 156 kg N ha-’ yr-’ since 1960. Sample collection Samples for the determination of potential denitrification (PDN) were collected from the experimental areas during two periods as follows: (a) July 1991, cut and grazed grass swards +420 fertilizer N ha-‘, and cut and grazed mixed grass-clover swards (0 fertilizer N) and (b) March 1992, cut and grazed grass swards f420 kg N ha-’ and the longterm continuous arable treatment. Samples were collected at random from within the central area of the 0.123 ha plots with a 5 cm power-driven flighted auger (Mobil Drilling Co.. Indianapolis, U.S.A.) mounted on the rear of a Land Rover. Sampling was arranged so that the following depths were obtained wherever possible: O-0.5, 0.5-1.0, 1.0-2.0, 2.0-4.0, 4.0-6.0 and >6.0 (but ~7) m. On a number of occasions large flints and flinty horizons within the chalk prohibited access to greater depth and drillings stopped at a shallower depth. Great care was taken to avoid cross contamination between samples with careful cleaning of the auger between each depth and between each drilling site. Soil and chalk samples were collected from each drilling position in their separate horizons into polythene bags and transported to the laboratory for immediate investigation and analysis in the case of the first sampling period, or stored at 2-4‘C in a controlled temperature room prior to examination in the case of the latter period. Duplicate drillings were taken in each treatment plot wherever possible. Laboratory measwements The contents of each polythene bag, i.e. from each depth interval, were thoroughly mixed by hand and stones removed from soils to provide an integrated sample. Chalk samples were obtained as consolidated slurries and needed little mixing. In some cases the profile horizons did not coincide with the designated depths but the materials were kept within the depth categories even where profile boundaries had been crossed. Duplicate sub-samples were taken from each separated horizon from each drilling for the determination of water contents (gravimetrically) and

Denitrification potential at depth mineral N contents (by extraction in 2 M KC1 and analysis of the extract for NO;- and NH,I by flow injection analysis). Determination of PDN was based on the method described by Bijay-Singh et al. (1989). With a knowledge of the water contents, the equivalent of 15 g dry sample was weighed into 70 ml serum bottles to provide duplicate incubations for each depth sample from each drilling. Amendments were then added to each bottle to adjust the soil or chalk sample to 90% of a predetermined saturated water holding capacity (WHC) for that particular depth. Many of the chalk samples were already at >90% saturation and were therefore allowed to dry gently in a cooled incubator at 4°C so that aqueous additions could be made. Each sample of soil or chalk in the incubation vessel was then amended with NO; [in solution as Ca(NO,),] to provide 100 pg added NOT-N g-’ dry soil. This was added in either the presence or absence of an added energy source supplied as glucose at the rate of 1OOyg C g-’ dry soil. Additional water was then supplied as necessary to bring all samples to the designated water contents. Once all the additions had been made to the bottles, they were flushed with N, and stoppered with an airtight stopper (Suba-Seal No. 21). 2 ml of the head space atmosphere were then removed by syringe and replaced with 2 ml acetylene (to provide a headspace concentration of approx. 10%) to inhibit the reduction of N,O to N,. The bottles were then placed in a cooled incubator in the dark at 20°C. At each of 2, 24, 48 and 96 h after the start of incubation, a 2.5 ml sample was taken from the head space atmosphere via a syringe fitted with a stopcock: on each occasion this was replaced with 2.5ml N,. The stored headspace gas samples were analysed for their N,O contents using a gas chromatograph with an electron capture detector. Wherever possible this was immediately after collection, otherwise the samples and standard N,O samples of known concentrations were stored (for no longer than 2 days) in their syringes for later analysis. Any decay in concentration of the standard samples (which was never > lO-15%) was used to apply appropriate corrections to each storage batch. Denitrification rates were

Table 1. Mineral N contents

(pgg“

calculated on the basis of NzO accumulated over the various incubation periods, account being taken of that removed in the head space samples taken at the previous sampling occasion. N,O held in solution was calculated by using the Bunsen coefficient. RESULTS

Mineral N contents of the soil and chalk samples (Table 1) varied with previous management and depth. Previous measurements on the same treatments by Ryden et al. (1984) indicated marked effects of cutting or grazing and similar trends, although not so distinct, were also shown in our study. In general, contents were low under grass-clover, cut fertilized grass and arable managements but were greater under the grazed fertilized grass: samples from the single drilling in the grazed 420 N treatment in March had exceptionally high NH: contents. The NO; added to the incubations (100 fig N g-’ dry soil) was always considerably in excess of that naturally present: a relatively uniform substrate concentration was therefore available in all samples from all treatments and depths. In any case, the amounts present were considerably in excess of the apparent maximum cumulative PDN found in any of the treatments. In many instances there was a deciine in PDN rate over the final period (48-96 h) of incubation (see Fig. 1). Because this may reflect conditions that are no longer at their optima for denitrification, general discussion of the results is confined to the data up to and including that for the 48 h incubation. However, a typical example of the patterns of changes in PDN rates with time and of the cumulative PDN over the whole measurement period for a single treatment is shown in Figs 1 and 2. PDN rates have been calculated for each incubation period between successive samplings. The data were examined for differences by analysis of variance (ANOVA): the two sampling periods were analysed separately. Typically PDN was always greater with the top soil samples and declined progressively with depth (P < 0.001). At the lowest depth sampled, i.e. >6 m, and in the absence of added C,

dry sample) of samples taken to depth below a freely draining for duplicate drillings Fertilized

GraSs-&Wf Cut* J%pth Cm) O-OS OS-I.0 I .O-2.0 2.&4.0 4.w.o 16.0

1631

GXX.XJ

a*

bt

Arable?

bt

a$

NH;

NO,

NH:

NO;

NH;

NO,

NH:

NO;

NH:

NO;

4.3 I .6 0.7 0.3 0.2 0.4

3.1 I.1 1.5 1.0 0.9 1.2

4.1 2.2 1.1 0.8 1.0 1.3

4.6 2.5 1.8 1.8 2.0 1.6

4.2 2.9 1.9 2.6 2.9 2.7

5.1 2.9 3.7 2.9 4.4 3.4

0.5 0. I 0.1 0.7 -

22.2 6.9 4.8 1.0 -

5.5 1.4 14.6 13.7 1.3 -

6.1 4.0 6.5 8.0 7.8 -

*Sampled in July (1991). TSampled in March (1992). $Single drilling only.

Data are the means

grass

Cut Grazed*

soil in SE. England.

NH:

NO;

NH:

NO,

0.7
17.6 9.3 12.6 2.2 2.8 -

0.1
5.0 2.4 2.0 0.1 0.1 -

S. C. JARVISand D. J. HATCH

1632

I

O-2 HOURS

2-24 HOURS

0.60

0.60

0.50

0.50

0.40

0.40

0.30

0.30

0.20

0.20

0.10

0.10

0.00

0.00

24-40 HOURS

I

49-96 HOURS

Fig. 1. Potential denitrification (PDN) rates (pg N g-’ dry sample h-’ ) by samples taken to various depths below a fertilized, cut grass sward on a freely draining soil in SE. England. Data are for the G-2, 2224, 2448 and 48-96 h periods after the start of incubation and are the means for duplicate incubations from each of duplicate drillings. Samples were incubated in the presence of acetylene and added NO; and with (NC) or without (N) added C and were from 0 to 0.5 m (m, N: W, NC), 0.5 to 1.0 m ( 2 to 4 m (0, N: 0, NC) and >6 m (a, N: 0, NC). Bars indicate SEDs for all treatments and depths over each sampling period.

PDN never exceeded 0.014pg N g-l soil h-‘. As demonstrated in the fertilized cut grass sward example (Figs 1 and 2), PDN at most depths increased with time during each of the first three incubation periods indicating a build up of microbial activity especially with added C: without added C PDN declined during the final periods (Fig. 1). There was no increase with time in the case of the deepest

sample without added C. Added C had a marked stimulatory effect (P < 0.001) on PDN in all treatments even in those parts of the profile where a ready supply of mobile C may have been expected. The data indicate that there had been little cross-contamination between samples because any major transfer of either bacteria or C from the upper to lower parts of the profile during sampling would have enhanced

Denitrification potential at depth

0

24

48

96

INCUBATION TIME (h)

Fig. 2. Cumulative potential denitrification (PDN) (pg N g-l dry sample) by samples taken to various depths below a fertilized cut grass sward on a freely draining soil in SE. England. Data are for a 48 h incubation period and are the means for duplicate incubations from each of duplicate drillings. Samples were incubated in the presence of acetylene and added NO, and with (NC) (solid symbols) or without (N) (open symbols) added C and were from 0 to 0.5m(O,N:~,NC),0.5to1.0m(~,N:~,NC),2to4m (A, N: A, NC) and >6 m (V, N: ‘I, NC). Bars indicate SEDs for all treatments at each time.

the PDN of the deeper samples to a much greater extent than found. In all the treatments there was at least a 100% increase in PDN, and often considerably more, when C was supplied.

1633

It was clear that the maximum rates of PDN were achieved with added C over the 24-48 h period. Data from this period are therefore taken to represent the maximum potential for denitrification from each sample. Tables 2 and 3 show data for all treatments either as hourly rates for the 24-48 h incubation period or as cumulative PDN over 48 h. A number of effects and differences are illustrated. The effect of added C is shown clearly in all cases except in the upper horizons of the grazed fertilized grass (especially at the second sampling date). In most treatments the effects of C increased with depth; this was less obviously so with the arable soil where C had a relatively constant effect with depth. As well as the effects of C, there were also marked differences between the different field treatments: although this was shown most clearly in the upper samples there were also effects at depth in some cases. PDN was always less in the samples from the grass-clover swards than in the comparable samples from the fertilized swards. This was particularly marked in the upper soil horizon where PDN rate was over seven times greater with the fertilized grass than with the grass-clover sward: even at 24 m depth where differences had diminished, the average rate for the fertilized swards was over two times greater than that for grass-clover. The PDN rates in the samples from the cut grass-clover sward, either with or without added C, were very similar to those from the arable soil without added C especially at O-O.5 m depth. It is remarkable that adding extra C to the upper parts of profiles from grass-clover swards had little or no effect on PDN even though rates were at a relatively low level in both cut and grazed treatments. There was little effect of C added to the upper horizons

Table 2. Potential denitrification rates (pg N g-’ dry sample h-‘) by samples taken to depth below freely draining soil in SE. England. Data are for the 24-48 h period after the start of incubation and are the means for duplicate incubations from each of the duplicate drillings. Samples were incubated in the presence of acetylene and added NO; and with (NC) or without (N) added C Fertilized grass Grass-clover Depth (m) W.5

Cut

Grazed

Cut’

Grazed*

I*

IV

r*i

11t

Arable?

N NC

0.070 0.059

0.029 0.070

0.257 0.541

0.158 0.729

0.338 0.387

0.447 0.394

0.065 0.647

0.5-1.0

N NC

0.035 0.053

0.007 0.028

0.131 0.255

0.098 0.215

0.158 0.200

0.119 0.289

0.037 0.329

I s&2.0

N NC

0.001 0.014

0.030 0.092

0.069 0.172

0.064 0.148

0.008 0.088

0.032 0.106

0.042 0.213

2.W.O

N NC

0.013 0.038

0.003 0.128

0.010 0.126

0.012 0.043

0.014 0.079

0.039 0.347

0.017 0.147

4.0-6.0

N NC

0.01 I 0.045

0.003 0.071

0.005 0.012

-

-

0.029 0.219

0.035 0.207

>6.0

N NC

0.009 0.035

0.010 0.078

0.006 0.065

-

-

0.014t 0.092t

-

SED 5 B 5 11 ‘Sampled in July (1991). iSampled in March (1992). iSingle drilling only. @ED for all treatments and depths for samples from July (1991) = 0.0446. llSED for all treatments and depths for samples from March (1992)= 0.1487.

l!

11

S. C. JARVISand D. J. HATCH

1634

Table 3. Cumulative potential denitrification (pg N g-’ dry sample) by samples taken to depth below freely draining soil in SE. England. Data are for a 48 h incubation period and are the means for duplicate incubations from each of duplicate dtillings. Samples were incubated in the presence of acetylene and added NO; and with (NC) or without (N) added C Fertilized Gi-ass-clOVer Depth (m)

grass Grazed

Cut I’

11t

1*i

11t

Arablet

w.5

N NC

2.08 2.38

4.03 5.94

II.30 19.41

7.01 26.31

14.00 15.80

18.06 18.40

3.81 22.94

0.5-l .o

N NC

I .49

I .98

I .93 2.49

5.71 IO.91

3.21 6.91

5.09 6.63

6.12 12.04

2.68 10.86

N NC

0.97 I .4s

2.04 4.10

3.25 5.70

2.55 4.17

0.52 2.84

2.80 5.43

I .65 6.44

4.43 12.97

I .22 7.16 2.01 7.86

I O-2.0

2.e4.0

cut*

Grazed’

N

0.47

0.29

0.45

0.52

0.62

NC

I.41

4.15

3.63

I.17

2.69

4.0-6.0

N NC

0.47 1.34

0.24 2.18

0.21 0.43

-

I.91 7.18

>6.0

N NC

0.35 I .02

0.55 2.29

0.23 1.74

-

0.577 2.70t

§

5

I

SED

11

-

P

8

*Sampled in July (1991). tSampled in March (1992). iSingle drilling only. @ED for all treatments and depths for samples from July (1991) = 1.335. l/SED for all treatments and depths for samples from March (1992) = 4.279.

where the fertilized grass had been grazed: effects increased with cutting. In the absence of C, for both pasture systems, the cumulative PDN rates were greater in the grazed than in the cut swards in the soil section of the profile: where C had been added, this difference extended to greater depth. There was no apparent effect of time of sampling: similar rates and patterns of change were apparent in both sets of samples from fertilized grass whether collected in summer or in early spring although it is possible that there may have been greater activity to greater depth in the spring sample from the grazed treatment.

DISCUSSION

Potential denitrification measurements provide an indication only of what might happen under field conditions and are unlikely to ever represent actual rates. Nevertheless, they can yield useful information on trends and effects on soil, environment and management and they have been used on a wide range of soil types from a wide range of ecosystems to provide information on the denitrification process (BijaySingh et al., 1989; Staley et al., 1990). Although procedures are available for isolating and modifying water samples from depth and re-injecting these for subsequent re-collection and determination of denitrification (Gillham, 1990), these are difficult and expensive and the only practical option in many cases is to provide a measure of potential. This is particularly the case where a number of treatments are to be examined and some degree of replication provided. The PDN rates we measured for the soil component of the profiles were of the same order to those

found in previous studies of agricultural soils. Bijay Singh et al. (1989) found rates of 0.31 /Ig N g-’ h-’ in soil samples (O-10cm) from the same arable experiment used in our study. In their case, however, a sample from an older grass-clover pasture (31 yr) than we studied but on the same soil type, had a much higher PDN rate (1.46 pg N g- ’ h- ’ ) than found by us. Other studies of New Zealand ryegrass-clover pasture soils (Limner and Steele, 1982) found PDN rates which were equivalent to 1.2 pg N g-’ h-‘, again considerably greater than the value for the grass
Denitrification potential at depth oping after ca 48 h and a declining rate after 75 h (Limner and Steele, 1982). The initial linear phase has been assumed to reflect the activity of denitrifying enzymes present in the samples when collected, followed by a phase which has been attributed to steady state synthesis of denitrifying enzymes by existing micro-organisms (Limner and Steele, 1982). The decline in rate over the present 48-96 h period has already been noted. NO; concentration has little effect on PDN over a wide range of concentrations (Ryden and Lund, 1980; Limner and Steele, 1989) and it can be calculated that at 48 h at least 75% of the added NO, was still present and it is also unlikely that available C would have been limiting when this was added. Acetylene depletion (by microbial activity or removal of head space gases) may have reduced effectiveness in stopping reduction of N,O to N,. Under the highly-reducing conditions of the incubation system there would have been every opportunity for this to occur with the result that N,O production would have been smaller and apparent denitrification reduced. This is unlikely to have been an effect during the 2448 h period since PDN rate increased over this time when C was added. An alternative approach to avoid this problem would have been to measure the rate of NO; disappearance, but DeCatanzaro and Beauchamp (1985) have concluded that N,O accumulation is the more sensitive approach. The difference between treatments is of interest and some importance. Other studies have made comparisons across broad categories of conditions, e.g. managed vs natural-semi-natural soils (Kroeze et al., 1989) tilled and grassland soils (Limner and Steele, 1982; Bijay Singh et d., 1989) but few have looked at different managements within those categories. Differences between fertilizer- and legume-based managements (Svensson et al., 1985) have been noted before and higher PDN with the fertilized crops have been related to enhanced energy availability and NO; abundance. Under the conditions of our study any differences in the availability of native mobile C and NO; would have been removed because of the additions made. Under field conditions, over the long-term, differing supplies of C and NO; substrate may have resulted in differing background populations of denitrifying micro-organisms to provide the contrasting PDN rates found, not only between arable and grassland soils, but also between the different categories of grassland. Many other studies (Svensson et al., 1985; Staley et al., 1990) have emphasized the importance of C rather than NO; in determining PDN and this is likely to have been a key factor in determining the difference between the treatments we investigated. The very large potential that has been demonstrated for denitrification to occur in the soil and the underlying strata on this free-draining site may be of major significance in determining the fate of NO; leaking from soils under intensive agriculture. Other

1635

studies have demonstrated the existence of this potential in other circumstances. For example, all of the soils within a 569 km* German catchment area with water tables at 8, < 3 and 1.5m had significant PDN in their unsaturated zones (Lehn-Reiser et al., 1990). Where attempts have been made to examine PDN in aquifers, some important differences have been shown which reflect the effect that the unsaturated zone has had (Gillham, 1991). Thus where water tables were shallow (i.e. ~2-3 m), denitrification in the aquifer was thought to be an important and persistent process over significant areas and time. Within deeper aquifers there was no evidence of denitrification, and it was assumed that most of the labile C was oxidized in the unsaturated zone before reaching the water table. Availability of C is important for denitrification in all sectors of the system and although the potential for considerable activity exists under the various managements on this site, labile C will determine actual activity, as shown in other studies (Lind and Eiland, 1989). There is good evidence to support the presence of denitrifiers in aquifers; activity has been demonstrated in sediment samples taken from depths of up to 260 m in the U.S.A. (Francis et al., 1989) and in a confined Lincolnshire aquifer in limestone (Parker and James, 1985). Therefore there would seem to be little problem in appropriate denitrifiers moving through and into appropriate components of the unsaturated zone. However, within these zones, denitrification is only likely to occur in localized microsites of appropriately low redox potential (which will be widely available) and in which soluble organic C is present. Expression of any degree of the potential demonstrated with the present soil-chalk strata will therefore be determined by the mobility and availability of labile organic C. It is clear that, at least at the times of sampling from our site, C supplies at most depths below the rooting zone were restrictive, even under those soils under managements which had promoted a long-term build up of soil organic C and PDN activity. Little is known about the forms of labile C and the controls over its transport, transformation characteristics and its availability for microbial utilization. It can be calculated, assuming a bulk density of 1.42 g cm-’ for the soil (Tyson et al., 1990) and 1.62 g cm-’ for chalk (British Geological Survey, pers. commun.), that even in the absence of added C, and even in the systems with lowest PDN rates there is apparent potential for high rates of NO; loss at least down to 7 m ranging from 35 to over 200 kg N ha-’ d-’ in the system we studied. Adding C increased the overall potential by, on average, nearly 2.5-fold for grassland and by over 4.5-fold for the long-term arable systems. It is probable that at any one time only relatively small proportions of the denitrification potential demonstrated in our study will be active and the calculated reduction of NO, per unit area will be much smaller. Nevertheless, even

1636

S. C. JARVIS and D. J. HATCH

at very low rates, on a regional basis, the process is likely to be of major significance and a greater knowledge is required to enable better management and policy decisions relating to potential pollution from agriculture to be made. The fact that (a) the potential for denitrification exists, and (b) this differs markedly, especially in the upper layers, under different managements may offer the opportunity to reduce NO, loading by changing agricultural practices. The possibility is made even more attractive by the observation that the ratio of N,O:N, may decrease when denitrification activity occurs at depth (Rolston et al., 1976) thus reducing another potential environmental

problem. Acknowledgements-We are grateful to N. PritchardGordon for assistance in collecting and processing samples and to A. Rook for statistical advice. The work described was funded by Ministry of Agriculture, Fisheries (London), in part by an Open Contract.

and Food

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