Effects of Freeze-Thaw and Soil Structure on Nitrous Oxide Produced in a Clay SoilEric van Bochove,* Danielle Prevost, and France Pelletier

ABSTRACTFreezing and thawing have been shown to cause significant soil

physical and biological changes. The increase in denitrification follow-ing thawing may be attributed to the diffusion of organic substratesnewly available to denitrifiers from disrupted soil aggregates. Theobjective of this study was to evaluate the effect of freezing andthawing on N,O production in a clay soil under contrasting croprotations and tillage practices. Laboratory experiments were con-ducted in soil slurries to favor substrate diffusion, in macroaggregatefractions separated by wet sieving to characterize the biologicallyactive soil organic matter (SOM) pool, and in undisturbed soil coresto simulate field conditions. In slurries, a freezing and thawing cycleincreased denitrification rates by 32%. Soil slurries from no-tillageunder rotation (NT-R) exhibited denitrification rates 92% higherthan those from conventional till under continuous cereal (CT-C).Macroaggregates fractions (0.25-2 and 2-5 mm) from both manage-ment systems increased their rates of C mineralization and denitrifica-tion activity by 95% following freezing, but the increases tended tobe greater (57%) in small than in large macroaggregates. Higher ratesof denitrification (55%) found in both aggregate fractions of NT-Rsystem were attributed to the higher mineralizable organic C content.Undisturbed soil cores sampled in November showed increased N2Oproduction by 220% after thawing. This thawing effect was also signifi-cantly higher in cores from NT-R than in those from CT-C.

NITROUS OXIDE is a radiactively active atmospherictrace gas with a long lifetime (150 yr) and is cur-

rently accounting for 2 to 4% of total greenhouse warm-ing potential (Watson et al., 1992). Cultivated soils arethought to be a major source of anthropogenic N2O(Duxbury et al., 1993). Although the critical environ-mental factors responsible for N2O emissions are wellunderstood, sources and sinks of N2O in agriculturalsoils are not yet fully quantified. One reason for thisincomplete information is the lack of measurementsin various agricultural systems (Mosier et al., 1996),especially during winter (Rover et al., 1998). Bremneret al. (1980) estimated that 6 to 21% of the annual N2Oflux from agricultural land occurred during thawingof topsoil in spring. In cold temperate climates (e.g.,Canada and northern Europe), some studies reportedsignificant N2O losses from cultivated soils followingfreeze-thaw cycles in spring (Nyborg et al., 1997;Wagner-Riddle and Thurtell, 1998; Rover et al., 1998)or even during winter and snowmelt from unfrozen soils(van Bochove et al., 1996,2000). The processes involvedin N2O emissions during freeze and thaw are unclear,although several factors have been investigated (Roveret al., 1998). In Quebec, Canada, seasonal variations indenitrification enzyme activities (DEA) were observed

Soils and Crops Research and Development Centre, Agriculture andAgri-Food Canada, 2560 Hochelaga Blvd., Sainte-Foy, QC, CanadaG1V 2J3. Received 26 Oct. 1999. Corresponding author (vanbochovee@em.agr.ca).

Published in Soil Sci. Soc. Am. J. 64:1638-1643 (2000).

and significant DEA were measured in the coldestmonths of the year (Pelletier et al., 1999).

Thawing causes the disruption of soil structure, mostlyof macroaggregates, and enhances microbial activity dueto the release of organic C from plant and microbialdetritus (Christensen and Christensen, 1991). The rela-tionships between disruption of soil macroaggregatesand availability of readily mineralizable substrates fordenitrification may play an important role in the poten-tial of a soil to produce N2O during a freeze-thaw event.Consequently, management-induced changes in aggre-gation may modify the denitrification potential of a soil.In fact, different long-term soil cropping and tillagepractices have significant effects on the water-stableaggregate distribution (WSA) of macroaggregates (Tis-dall and Oades, 1980; Elliott, 1986; Angers et al., 1993b).Among these practices, no-till increases aggregate sta-bility and C and N content (Cambardella and Elliott,1993; Angers and Carter, 1996). Stable macroaggregatesare relatively enriched in labile C of recent origin (El-liott, 1986; Puget et al., 1995).

However, reduced tillage also reduces total soil poros-ity (Pagliai and De Nobili, 1993) and increases soil mois-ture content (Rice and Smith, 1982), a factor known torestrict O2 diffusion through soil. The presence of ananaerobic microsite at the center of soil aggregates iscritical for the occurrence of denitrification (Sexstoneet al., 1985).

Under circumstances of specific soil managementpractices, freeze-thaw cycles may cause physical disrup-tion of soil structure, diffusion of soluble C, and changesin soil porosity that may promote denitrification andepisodic N2O emissions. Under the Quebec climate, infall, agricultural soils undergo overnight freeze and thawcycles. We performed various incubations in the labora-tory to investigate the effect of freeze-thaw cycles onN2O production in soils under contrasting agriculturalpractices. Three types of incubations were carried outas follows: (i) soil slurries to determine denitrificationactivities in conditions that remove substrate diffusionconstraints, (ii) fractions of macroaggregates to charac-terize the biologically active pool of SOM that maybe associated with N2O production, (iii) soil cores toevaluate N2O production in conditions where diffusionis a function of the undisturbed soil structure.

MATERIALS AND METHODSField Site and Soil Sampling

Cultivated plots were located at the Agriculture and Agri-Food Canada Experimental Farm at La Pocatiere, Quebec,

Abbreviations: CT-C, conventional till under continuous cereal;DEA, denitrification enzyme activity; DR, denitrifying rate; GC, gaschromatography; NT-R, no-tillage under rotation; OM, organic mat-ter; SOM, soil organic matter; WSA, water-stable aggregate distri-bution.

1638

VAN BOCHOVE ET AL.: FREEZE-THAW AND SOIL STRUCTURE EFFECTS ON NITROUS OXIDE 1639

on a Kamouraska clay (Orthic Humic Gleysol). The long-termexperimental plots were arranged in a split-plot design withfour replicates. Crop sequence treatment was the main plottreatment and tillage practice was the subplot treatment(Angers et al., 1993a). Two treatments with contrasted crop-ping-tillage combination treatments were selected: (i) no-till-age with a 2-yr barley (Hordeum vulgare L.)-red clover (Trifo-lium pratense L.) rotation (NT-R) in the second year ofrotation and (ii) conventional till moldboard plowing withcontinuous barley (CT-C).

For incubation in slurries and on macroaggregates, threereplicate soil samples (0-5 cm depth) were cored randomlyfrom each plot on 5 Oct. 1995.

For incubation of the undisturbed soil structure, soil coreswere collected in three replicates from each plot at the 0- to5-cm depth on two sampling dates, 5 Oct. and 27 Nov. 1995.Stainless steel corers (i.d., 11 cm; height, 10 cm) were sharp-ened at one end and soil cores were collected with a driversystem that allows a precise core of 5-cm depth.

Incubation ExperimentsDenitrification Rate in Slurries

Sieved soil samples (6 mm) were frozen at — 12°C for 20to 24 h and then thawed for 25 min. Unfrozen controls werekept at 4°C before incubations started. After freezing-thawingsimulation, denitrifying rate (DR.) was determined on foursubsamples of each soil sample. The DR incubations wereconducted at 25°C under anaerobic conditions in the presenceof 10% acetylene (QEy. Assay solution contained 10 mMNO3~ to provide nonlimiting electron acceptor. Glucose andchloramphenicol were not added to the solution (modifiedfrom Martin et al., 1988). The C2H2 was purified by bubblingsuccessively through solutions of H2SO4 (2 M) and deionizedwater (Nanopure series 851 system, Barnstead/Thermolyne,Dubuque, IA) to remove traces of acetone. Gas samples werecollected in vacutainers (Vacutainer Brand, Becton Dickinsonand Co., Rutherford, NJ) at 0.5,2, and 4 h^after the incubationvessels were sealed. Incubation vessels were not shaken duringincubation to minimize aggregates breaking.

Incubations of Macroaggregate FractionsPrior to macroaggregate fractionation by wet sieving, soil

samples were gently passed through a 6-mm sieve and keptat 4°C in crush-resistant air-tight containers. Field-moist soilsamples were wetted under vacuum and constant head at 5 cmH2O during 30 min to minimize slaking of aggregates duringwet sieving following the method of Le Bissonnais (1989).The separatibn into two macroaggregate size fractions (0.25-2and 2-5 mm) was performed using an apparatus similar to thatdescribed by Bourget and Kemp (1957); operating conditionswere 3.7-cm stroke at 29 cycles s"1 during 10 min. After wetsieving, macroaggregate fractions were equilibrated underconstant head at 10 cm H2O. Soil water content was deter-mined gravimetrically on each fraction, small macroaggregates(0.42 kg kg"1) were wetter than the large ones (0.30 kg kg"1).Aggregate fractions were divided into subsamples to measure(i) N2O and CO2 production during anaerobic incubation fordenitrification following a freeze-thaw simulation and (ii) min-eralization rate of organic C during aerobic incubation.

Anaerobic Incubations for Denitrification. Macroaggre-gates were confined in polyvinyl chloride cores (i.d., 5 cm;height, 3 cm) that were placed in 125-mL air-tight jars beforefreezing treatment at — 12°C for 18 to 24 h. Unfrozen soilcontrols were kept at 4°C. After 20 min for thawing, the head-space of frozen and unfrozen jars was flushed with N2, and

C2H2 (10%, v/v) was injected in jars to measure denitrification.Anaerobic N2O and CO2 accumulations were monitored dur-ing 3 h at 25°C using gas chromatography (GC) as de-scribed below.

Aerobic Incubations for Carbon Mineralization. AerobicCO2 production was estimated during 27 d of incubation at25°C. To characterize the biologically active SOM (Zibilske,1994), the CO2 evolved was trapped in NaOH and the excessNaOH was titrated with standardized HC1 after addition ofBaQ2 and phenolphtalein. The pool of mineralizable C (C0,mg CO2-C kg"1) and the corresponding mineralization coeffi-cient (k, d~!) were estimated by fitting the data for the cumula-tive mineralized CT (mg CO2-C kg"1) with a first-order kineticmodel using nonlinear regression

CT = C0[l - exp(-fc)]

Incubations of Undisturbed Soil CoresUndisturbed soil core incubations were performed in stain-

less steel pipes (i.d. 11 cm, height 10 cm) closed at each endwith a neoprene sealed cover and equipped with independentrecirculation gas systems. Unfrozen control cores were keptat 4°C. Frozen cores were put at -20°C for 18 h and thenthawed at 25°C during 1 h before incubations started. Thecore headspace volume was estimated using pure Kr (Germon,1980). The core and headspace atmosphere was recirculatedduring incubation by using sealed aquarium pumps (Elite 799,Mansfield, MA) operating at 10.35 X 103 Pa and air outputof 1200 cm3 min"1. Soil cores were successively incubatedduring 3.5 h under aerobic (ambient air) and anaerobic (90%N2 + 10% C2H2, v/v) conditions, with the gas recirculationsystem. Gas samples were collected periodically (30 min)through rubber septa inserted in the cover and analyzed forN2O and CO2 by GC.

Gas Analysis

At sampling time, the air from incubation flasks orcores was sampled with syringes and drawn directly fromthe syringes into evacuated vials (7.5 mL) with Vacu-tainer septa (10-mL Vacutainers). The N2O and CO2concentrations were analyzed by gas chromatography(Hewlett Packard 5890 series II, Wilmington, DE)equipped with an automatic sample injector system(CTC Analytics, Zwingen, Swirzerland). The operatingsetup and conditions for CO2 were: a Porapak Q column(Chrbmatographic Specialties, Brockville, QN) (50°C)used with N2 as a carrier gas (40 mL min"1), CO2 wasreduced to CH4 in a Ni catalyst tube coupled to theflame ionization detector (250°C). The operating setupand conditions for N2O were: a Porapak Q precolumn(0.91 m by 0.32 cm diam.) coupled to an analytical col-umn (1.83 m by 0.32 cm diam.) used with Ar/CH4 ascarrier gas (0.95:0.05 m3 m"3, 45 mL min"1), and anelectron capture detector (250°C). The detection limitand precision were, respectively, 77 X 10~6 mol mol"1

and 16 X 10~6 mol mol"1 for CO2 analysis, and 70 X10"9 mol mol"1 and 14 X 10~9 mol mol"1 N2O analysis.

The Kr concentrations were analyzed by gas chroma-tography (Hewlett Packard 5890). The operating condi-tions for Kr were: thermal conductivity detector(110°C), Porapak Q column (i.d. 0.32 cm; length 6 m),and carrier gas He (25 mL min"1).

1640 SOIL SCI. SOC. AM. J., VOL. 64, SEPTEMBER-OCTOBER 2000

Statistical AnalysesAll statistical analyses were performed using SAS (SAS

Institute, 1988). The data from DR assays were analyzed statis-tically using the ANOVA procedure. The data from incuba-tion on macroaggregate fractions were first checked for homo-geneity using Bartlett's test and then logarithmicallytransformed before ANOVA analysis. The C0 and k valuescalculated for the replicates of each aggregate size fractionand each treatment were analyzed using ANOVA. The datafrom the dynamic soil cores incubations were first checkedfor homogeneity using the Bartlett test and then analyzedusing a nonparametric multiple comparison test, PROCRANK (Montgomery, 1984; Conover, 1980).

RESULTS AND DISCUSSION

Effects of Freeze-Thaw and Agriculture Practiceon Denitrifying Rate in Slurries

Both freeze-thaw and agricultural practices had asignificant effect on DR (Fig. 1). Denitrification rateswere higher in frozen-thawed than in unfrozen treat-ments. In general, it is assumed that N is not limitingduring anaerobic slurry incubation of agricultural soils(Myrold and Tiedje, 1985). Moreover, the addition ofNO3 to the slurries of the present study assures thatC substrate and/or denitrifying enzymes were the onlylimiting factors for denitrification. We also assume thatbreaking of aggregates was minimized under experi-mental conditions for DR. Therefore, the burst of deni-trification after thawing probably resulted from the re-lease of organic C substrates previously sequestered inaggregates that are disrupted by freezing or from micro-bial biomass and microfauna that are killed by the freez-ing process (Soulides and Allison, 1961; Bullock et al.,1988). The increases of 32% in denitrification followingthawing were similar for both agricultural practices. De-

0.25

30.15

0.05

0.00CT-C NT-R

Fig. 1. Effect of thawing and agriculture practices on denitrifying rate(DR) in soil slurries of conventional till continuous barley (CT-C)and no-tillage—barley-red clover rotation (NT-R) treatments. Ag-ricultural practices and freeze treatments indicate significant differ-ences (P < 0.0005 and 0.05); interaction between treatments wasnot significant. Vertical bars represent standard error of four rep-licates.

nitrification rates were 92% higher under NT-R thanunder CT-C. Because N was not limiting in slurry incu-bations, the higher denitrification rates in soils fromNT-R may be due to greater concentrations of mineral-izable organic C in soils from this conservation practicethan in CT-C. Both no-till and red clover are agricul-tural practices known to increase the concentrations ofmineralizable C and mineralizable N in soil (Odell etal., 1984; Sheaffer and Barnes, 1987). Although not sig-nificant, data collected from our experimental plots inSeptember 1995 showed higher levels of organic C andtotal N in NT-R than in CT-C (Nicole Bissonnette,2000, personal communication). Furthermore, on thesame plots, Angers et al. (1993a) observed significantincreases in microbial biomass C and hot water-extractable and acid-hydrolyzable carbohydrates due tothe soil management and to a lesser extent to the rota-tion after 4 yr of no-till. The differences in concentra-tions of organic mater (OM) labile forms were alsogreater for the NT than for the CT in 1995. This is inagreement with other studies showing that soil conserva-tion management systems (e.g., reduced tillage, rota-tion) result in higher aggregate stability and organicmatter content than conventional systems (Angers etal., 1993b; Beare et al., 1994). Higher rates of denitrifica-tion in soils from NT-R may also be attributed to differ-ences in enzymes present in samples at the beginningof incubation, as the DR assay (adapted from Martinet al., 1988) was initially designed to show.

Effects of Freeze-Thaw and Agriculture Practiceon Denitrification in Macroaggregates

Freeze-thaw treatment and agricultural managementsystems showed significant effects on denitrification inmacroaggregates (Fig. 2). Denitrification rates were95% higher in frozen than in unfrozen macroaggregates(P = 0.01), and these increases were equivalent for bothmanagement systems. Macroaggregates from the NT-Rmanagement system exhibited denitrification rates 55%higher than those from the CT-C system (P = 0.002).There was no significant interaction between thawingand agricultural management treatments, but the in-crease in denitrification following freeze-thaw was 57%higher in small (0.25-2 mm) than in large (2-5 mm)macroaggregates (P = 0.07). Our results showed that asingle short freezing and thawing cycle on macroaggre-gates increased denitrification activity by 95%. Thehighly significant relationship between denitrificationand CO2 emissions (R2 = 0.94, n = 24) confirms thatthe burst of denitrification activity may be related to asubstantial supply of organic C. This is in accordancewith Bijay-Singh et al. (1988) who concluded that in thefield, microaerophilic mineralizable C may often be alimiting factor for denitrification. Carbon renderedavailable for denitrification by freeze-thaw may origi-nate from organic matter previously sequestered in ag-gregates that are broken down by freeze-thaw cycle(Soulides and Allison, 1961; Bullock et al., 1988) andfrom organisms that are killed by the freezing process(Christensen and Christensen, 1991). Forces disruptingaggregates are probably a result of ice crystals ex-

VAN BOCHOVE ET AL.: FREEZE-THAW AND SOIL STRUCTURE EFFECTS ON NITROUS OXIDE 1641

panding in pores between particles, interrupting parti-cle-to-particle bonds and effectively breaking the aggre-gates into smaller aggregates (Bullock et al., 1988).

Results obtained from C mineralization tests in thelaboratory show some of the same tendencies as thoseobserved during denitrification incubations. Indeed, thepool of mineralizable C (C0) was on average 65% higherin NT-R than in CT-R macroaggregates (P = 0.05) and45% higher in small than in large macroaggregates (P =0.10; Table 1). The higher C0 found in both aggregatefractions of the NT-R management system may be re-sponsible for the higher rates of denitrification observedin this system than in the CT-C system. Some of thevariations in the distribution of C0 between managementsystems is more likely attributable to tillage practicesrather than to legume in the rotation because legumesdid not significantly increase the labile OM contents inthe soil (Nicole Bissonnette, 2000, personal communi-cation). In no-tillage practices, particulate organic mat-ter would be incorporated within macroaggregates,whereas in conventional tillage, aggregates are moresubject to disruption, releasing aggregate-protected or-ganic matter for mineralization (Beare et al., 1994). Al-though there were differences in the pool sizes of miner-alizable C (C0), the calculated C mineralization rates(/c) did not vary within management systems (P > 0.30),which may be due to differences in biological activity,soil organic matter availability, and/or stage of humifi-cation.

The effect of freeze-thaw on denitrification activityvaried with aggregate size fractions, which showed a40% higher denitrification rate within small than withinlarge macroaggregates. We also measured a water con-tent 40% higher in small macroaggregates compared

Table 1. Pool of mineralizable C (C0), mineralization rate (£),and half-life time (tm) of soil organic matter associated withmacroaggregates from two management systems.

Aggregatesize fractions C0

0.25-2 mm2-5 mm

0.25-2 mm2-5 mm

mg COr-C kg 'Conventional tillage-continuous barley

280210

No-tillage-rotation (NT-R)490320

kd-'

(CT-C)4334

3641

tin

d

2116

1719

with large macroaggregates. Because the wet-sievingmethod used to separate macroaggregate fractions mayinterfere with C0 distribution by removing a part ofthe labile organic C and by modifying water content(Christensen and Christensen, 1991), wet sieving mayinfluence the denitrification rates following freeze-thawof macroaggregate fractions. This possibility is corrobo-rated by Satricka and Benoit (1995), who observed thatsurface resistance of soil aggregates to breakdown byfreezing impact declines as aggregate size and soil watercontent increases. However, Seech and Beauchamp(1988), using a dry sieving method to separate aggre-gates, found that denitrification potential was relatedto mineralizable C and was greater in small (<0.25 and0.25-0.5 mm) than in large aggregates. This suggeststhat our results obtained using the wet-sieving methodusing a prewetting procedure are in concordance withresults obtained by dry-sieving. Although denitrificationrates can be reduced by using wet sieving, two-thirdsof the difference between the fractions persisted afterwashing, suggesting that differences of denitrification

Frozen Unfrozen Frozen UnfrozenFig. 2. Effect of freezing on denitrification rates in 0.25- to 2- and 2- to 5-mm macroaggregate fractions from continuous barley under conventional

till (CT-C) and a barley-red clover rotation under no-till (NT-R) management systems.

1642 SOIL SCI. SOC. AM. J., VOL. 64, SEPTEMBER-OCTOBER 2000

Table 2. Nonparametric multiple comparison test on ranks ofN2O and CO2 emissions vs. freezing-thawing cycle and agricul-tural practices from data obtained from undisturbed soil cores.

Agriculturalpractices

(CT-Cvs.NT-R)t

Incubation

Aerobic (October 95)Aerobic (November 95)Anaerobic (October 95)

N2O

NSt

NS

C02

ttt

Freezing-thawingeffect (frozen vs.

unfrozen)

N2O

NS*

NS

CO2

NS**

*, t Significant at the 0.05 and 0.005 probability levels between freezingand tillage treatments, respectively.

t CT-C is conventional tillage-continuous barley; NT-R is no-tillage-rotation.

activity-in aggregates of various sizes were primarilyrelated to intrinsic sample properties (Christensen andChristensen, 1991).

Effects of Freezing-Thawing on Nitrous Oxideand Carbon Dioxide Emissions in Soil CoresUnder aerobic conditions, agricultural practices and

freezing-thawing cycle showed a significant effect onN2O emission from soil sampled in November but notfrom that sampled in October (Table 2, Fig. 3a). Inundisturbed soils obtained in November, as observed inslurries, the NT-R treatment exhibited N2O production

N20 DCT-C Frozen and thawedIE Unfrozen CT-C0NT-R Frozen and thawed

i Unfrozen NT-R

CO,

2.00 T

0.00

AnaerobicOctober 95

Fig. 3. Effect of freezing-thawing cycle and agricultural practices onN2O and CO2 emissions in undisturbed soil cores under aerobic(ambient air) and anaerobic (N; + 10% C;H:, v/v) incubations.

significantly higher (=500%) than those from CT-C,indicating that environmental factors conducive to deni-trification were more likely present in the late autumn(November). Among the critical environmental factors,volumetric soil water content, which regulates O2 avail-ability to denitrifiers, averaged 39% in soil cores sam-pled in November and 28% in those sampled in October.In November, N2O emission also increased (=220%)similarly in both agricultural practices after a freezing-thawing cycle. This is in agreement with other studiesshowing that the thawing of a soil can cause temporalincrease in N2O production by denitrification (Chris-tensen and Tiedje, 1990). Measurements of N2O madeunder aerobic (ambient air) conditions represented thenet emission of N2O evolved from soils, whereas mea-surements in anaerobic conditions and in the presenceof acetylene represented the potential of N2O plus N2production by denitrification.

Furthermore, under anaerobic conditions, N2O pro-duction in soil cores collected in October showed thetendency (results not significant) to produce more(=138%) N2O in NT-R than in CT-C soils (Fig. 3a). Itis known that O2 is the dominant factor limiting denitrifi-cation in agricultural soils, but C availability could alsolimit denitrification when anaerobic zones are present(Firestone and Davidson, 1989).

Production of CO2 from soil cores was significantlyaffected by agricultural practices and by freezing-thawing cycle, except for the aerobic incubation in Octo-ber (Table 2, Fig. 3b). Despite the lack of effects ofagricultural practices on N2O and CO2 emissions in Oc-tober, the pattern of Fig. 3b suggests that differencesin available C occurred in October. In general, CO2production, which is due to microbial respiration, fol-lowed the same trend as N2O production (Fig. 3a). Bothgases were higher in NT-R than in CT-C soils, probablyreflecting the higher mineralizable C (not shown) thatcan support denitrification. There was also a burst ofCO2 production following thawing, which supports theincrease in microbial activity, such as denitrification.

CONCLUSIONSSoils under no-tillage-barley-clover rotation practice

(NT-R) showed higher capacities to denitrify than thosefrom conventional till-continuous culture (CT-C).These soil properties (e.g., available C and sufficientlydepleted O2) were reflected in the levels of N2O pro-duced from undisturbed soils when conditions for deni-trification were not limiting. This study also showedthat a freezing-thawing cycle increases denitrificationactivity, which may result in higher net N2O emissionsfrom soils.

Our results showed that a single short freezing andthawing cycle caused a burst of denitrification in macro-aggregates. This burst was sustained by C mineralizationfrom organic matter released by disruptive forces in-duced by freezing, and it was higher in small than inlarge macroaggregates. The greater effect of freezingon small aggregates was probably due to their higherwater content. The no-tillage and rotation system(NT-R) resulted in a greater pool of mineralizable C

VAN BOCHOVE ET AL.: FREEZE-THAW AND SOIL STRUCTURE EFFECTS ON NITROUS OXIDE 1643

in macroaggregates, and stimulated denitrification ratesin the topsoil compared with a conventional tillage withcontinuous barley system (CT-C).

The effects of freezing and thawing cycles should beincluded in simulation models of N2O emissions for cli-matic zones where freezing of the topsoil is a frequentevent. Understanding of exact mechanisms and quantifi-cation of the fundamental relationships between micro-structure with denitrification activity and organic matteravailability will improve the accuracy of model outputs.

ACKNOWLEDGMENTSThis study was supported by the GHG Initiative of Agricul-

ture and Agri-Food Canada. We wish to thank Dr. D.A.Angers and Dr. C. Chenu for their valuable comments at thebeginning of this study, as well as G. Levesque and Dr. H.Benmoussa for their work in the laboratory. The technicalassistance of Brigitte Patry is also gratefully acknowledged.We thank Dr. D.A. Angers and Dr. B.H. Ellert for valuablecomments on earlier version of this manuscript.