experimental evaluation of methods to quantify dissolved organic nitrogen (don) and dissolved...

Experimental evaluation of methods to quantify dissolved organic

nitrogen (DON) and dissolved organic carbon (DOC) in soil

D.L. Jones *, V.B. Willett

School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK

Received 29 April 2005; received in revised form 3 August 2005; accepted 21 August 2005

Available online 23 September 2005

Abstract

A significant proportion of the total nutrient in soil solution can be bound to organic molecules and these often constitute a major loss from soil

to freshwater. Our purpose was to determine whether chemical extractants used for measuring inorganic N could also be used to quantify dissolved

organic nitrogen (DON) and carbon (DOC) in soil. In a range of soils, DOC and DONwere extracted with either distilled water or 2 MKCl and the

amount recovered compared with that present in soil solution recovered by centrifugal-drainage. The recovery of DON and DOC from soil was

highly dependent upon the method of extraction. Factors such as soil sampling strategy (number of samples over space and time), sample

preparation (sieving and drying), soil storage, extraction temperature, shaking time, and soil-to-extractant volume ratio all significantly affected

the amount of DOC and DON extracted from soil. To allow direct comparison between independent studies we therefore propose the introduction

of a standardized extraction procedure: Replicate samples of unsieved, field-moist soil extracted as soon as possible after collection with distilled

water, 0.5 M K2SO4 or 2 M KCl at a 1:5 w/v ratio for 1 h at 20 8C.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Amino acids; Dissolved organic matter; KCl extract; Soil quality; Soluble nitrogen; Water extract

1. Introduction

In comparison to inorganic nutrients, dissolved organic

nutrients often are the dominant elemental pool in many soils

and freshwaters and therefore represent a key component of

biogeochemical cycles (Kalbitz et al., 2000). In particular,

dissolved organic forms of nitrogen (DON) and phosphorus

(DOP) are notable since these elements often limit the

productivity of terrestrial ecosystems (Qualls and Haines,

1991a,b; Qualls and Richardson, 2003). Considerable attention

has been paid to quantify the amount and flux of dissolved

organic nutrients in freshwater and marine systems (Dafner and

Wangersky, 2002a,b; Westerhoff and Mash, 2002), however,

less work has been performed in soil. In freshwater and marine

studies, sample preparation and method of analysis can have a

significant impact on the amount of nutrient recovered (Peltzer

et al., 1996; Urbansky, 2001; Dafner and Wangersky, 2002a).

Further, the concentration of DOC and DON in water samples

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.soilbio.2005.08.012

* Corresponding author. Tel.: C44 1248 382579; fax: C44 1248 354997.

E-mail address: [email protected] (D.L. Jones).

also has been shown to vary significantly among laboratories

quantifying the same sample (Sharp et al., 1995; 2002).

Typically, biologically available nutrients are removed from

soil by shaking with a concentrated salt solution (e.g. KCl,

BaCl2, NH4Cl, NaHCO3) at a high soil weight-to-solution

volume ratio (e.g. 1:10 w/v) for short periods of time (1–3 h)

followed by separation of the solution phase by filtering or

centrifugation for subsequent analysis (Kachurina et al., 2000).

Although these methods are used widely (Sparks, 1996) they

have also drawn criticism (Dou et al., 2000). In contrast, no

such standard protocols have been validated for extracting

dissolved organic nutrients from soil, although previous studies

have indicated that extraction conditions may significantly

affect nutrient recovery (Christ and David, 1996; Chapman

et al., 1997a). Based on studies for inorganic nutrients,

recovery from soil depends on a range of factors, including

soil type, extractant type, extraction duration and extraction

temperature (Sparks, 1996). Also organic constituents con-

tained within microbial cells may be released during the

extraction procedure, which will overestimate dissolved

organic nutrient concentrations. Conversely, dissolved organic

nutrients may be degraded and lost during the extraction

procedure potentially underestimating nutrient concentrations.

In soil studies quantifying nutrient bioavailability or transport

potential, the dissolved organic nutrients can be extracted

Soil Biology & Biochemistry 38 (2006) 991–999

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D.L. Jones, V.B. Willett / Soil Biology & Biochemistry 38 (2006) 991–999992

directly from soil solution by centrifugation, circumnavigating

problems associated with chemical extraction procedures

(Giesler and Lundstrom, 1993). However, centrifugation is

unsuitable for dry soils, the processing of large numbers of

samples and requires specialized equipment. In these circum-

stances, chemical extraction procedures may be more

appropriate to quantify DOC and DON in soil.

A diverse range of laboratory extraction procedures have

been used to quantify DOC and DON in soil (Zsolnay and

Gorlitz, 1994; Suominen et al., 2003; Harrison and Bardgett,

2004; Michelsen et al., 2004). These protocols frequently differ

in their use of extraction solvent, shaking time, temperature,

soil preparation and method of analysis. The influence of these

factors on the amount of DOC and DON recovered in the soil

extract, however, remains unknown but is likely to be

substantial based upon freshwater and marine studies. Hence

there is a need to develop a standard protocol for measuring

dissolved organic nutrient concentrations in soil to allow valid

comparison between studies. The first aim of this work was to

assess the importance of soil sampling regime and soil

preparation on the quantification of DOC and DON in soil.

The second aim was to assess the influence of various

components of the extraction procedure on the recovery of

DOC and DON from soil.

2. Materials and methods

2.1. Soil sites and sampling regime

We used soil from the surface horizon at eight temperate,

oceanic locations in Wales, UK (Table 1). Soil A was collected

from under an unfertilized grassland (3853 0W 53814 0N; 410 m

elevation) with the vegetation consisting predominantly of

sheep’s fescue (Festuca ovina L.) and common bent (Agrostis

capillaris L.). Soil B was collected from underneath a mature

semi-natural English oak (Quercus robur L.) and sycamore

(Acer pseudoplatanus L.) deciduous woodland with closed

canopy and no understory vegetation (3852 0W 53814 0N; 210 m

Table 1

Selected properties of the test soils used in the method evaluation studies

Site Soil type Dominant

vegetation

Grazing Depth (cm) Hori

A. Dystric cambisol Unimproved

grassland

Sheep 2–15 Ah

B Humic cambisol Deciduous

woodland

None 0–5 O

C Leptic podzol Coniferous

forest

None 0–5 O

D Leptic podzol Deciduous

woodland

None 5–15 Ah

E Eutric cambisol Improved

grassland

Sheep 0–20 Ap

F Calcic cambisol Deciduous

woodland

None 0–15 Ah

G Eutric cambisol Improved

grassland

Sheep 0–10 Ah

H Dystric gleysol Improved

grassland

Sheep 0–15 Ahg

elevation). Soil C was collected from under a 30-year-old

mixed Sitka spruce (Picea sitchensis (Bong.) Carr.) and

European larch (Larix decidua Mill.) plantation (3852 0W

53813 0N; 180 m elevation) with closed canopy and no

understory vegetation. Soil D was collected from underneath

a mature semi-natural English oak and sycamore deciduous

woodland with moss and mixed grass understory (3852 0W

53814 0N; 200 m elevation). Soil E was collected from an

improved pasture composed predominantly of perennial

ryegrass (Lolium perenne L.) and clover (Trifolium repens L.;

3852 0W 53814 0N, 60 m elevation). Soil F was collected from

under a mixed, semi-natural mature European ash (Fraxinus

excelsior L.) and sycamore woodland with a predominantly

Allium ursinum L. understory (3848 0W 53818 0N; 30 m

elevation). Soil G was collected from a freely draining

improved pasture composed predominantly of perennial

ryegrass (4801 0W 53814 0N; 20 m elevation). Soil H was

collected from a poorly draining improved pasture composed

predominantly of perennial ryegrass and clover (4801 0W

53814 0N; 20 m elevation).

At each site, soil was collected using either a spade or 5 cm

diameter stainless steel corer. On return to the laboratory, soil

was stored field-moist at 3 8C in CO2 permeable polypropylene

bags to await analysis. Soil solution was extracted within 24 h

as described below. Soil pH and electrical conductivity were

determined in 1:1 (v/v) soil:H2O extracts. Moisture content

was determined by drying at 80 8C for 24 h. Total C and total N

were determined on sieved soil (!2 mm) with a CHN-2000

analyzer (Leco Corp., St Joseph, MI.). For the experiments

detailed below, soil samples were collected as required during

the period from March 2001 to June 2004. Properties of the

soils are listed in Table 1.

2.2. Soil solution sampling

Field moist soil (40–120 g) was broken by hand into

aggregates (0.5–5 cm diameter), large stones removed (O1 cm

diameter), and a mixed sample centrifuged (16,000 g, 30 min,

zon Texture pHH2O EC1:1

(dS mK1)

Total C

(g kgK1)

Total N

(g kgK1)

Sandy loam 4.31 0.09 119 14

– 4.27 0.15 401 23

– 4.66 0.08 453 18

Silty loam 3.9 0.1 100 12

Silty loam 5.15 0.1 54 11

Clay loam 7.01 0.12 57 8

Sandy loam 5.64 0.15 22 2

Silty loam 6.04 0.13 25 2

A: DOC

n of

DO

C (

mg

kg–1

)

600

800

1000

1200

D.L. Jones, V.B. Willett / Soil Biology & Biochemistry 38 (2006) 991–999 993

4 8C) to obtain soil solution (Giesler and Lundstrom, 1993).

Typically, 10–20 ml of soil solution was collected from each

soil sample yielding 30–60% of the soil’s total water. The

collected soil solutions were analyzed on the day of collection

or stored frozen at K20 8C to await analysis. Soil solution

collection was performed in triplicate apart from for the annual

sampling events where nine replicate samples were taken each

month.

2°C extraction of DOC (mg kg–1)

0 200 400 600 800 1000 1200

20°C

ext

ract

io

0

200

400

Water extract2M KCl extract

B: DON

2°C extraction of DON (mg kg–1)

0 20 40 60 80 100 120

20°C

ext

ract

ion

of D

ON

(m

g kg

–1)

0

20

40

60

80

100

120


C: TFAA

of T

FAA

(m

g N

kg–1

)

9

12

15

18

2.3. Extraction of DOC and DON

Unless otherwise stated, 2.5 g of field-moist soil was shaken

with 25 ml of either 2 M KCl or distilled water (1:10 w/v soil-

to-solution ratio) for 15 min in 50 cm3 polypropylene bottles

on a reciprocating shaker at a speed of 200 rev minK1. The soil

extracts were then centrifuged at 8000!g for 10 min and the

supernatant recovered and stored in polypropylene bottles in a

K20 8C freezer prior to analysis. Extractions were performed

in duplicate from each of the three sampling areas at each site;

however, the pseudoreplicates were averaged before carrying

out the statistical analysis. The following modifications to the

standard extraction procedure were done to investigate

individual factors.

1. Extractions were performed with different solvents (dis-

tilled water, 2 M KCl, 0.5 M K2SO4, 1 M NaOH or 1 M

HCl).

2. Extractions were performed at 2 or 20 8C.

3. Extracts were shaken for different times (5 min – 24 h).

4. Extractions were performed at varying soil weight-to-

extractant volume ratios (1:1 – 1:100 w/v).

5. Extractions were performed with either air-dried (30 8C,

24 h) or field-moist soil.

6. Extractions were performed on either sieved (!5, !2 or

!1 mm sieved) or unsieved soil which had been stored for

either 1, 24 h or 7 d.

7. After shaking, the extracts were either centrifuged (as

described above) or filtered (Whatman No 42) to recover

solution for analysis.

In the extractions at 2 8C, the soils and extract solutions

were pre-chilled overnight to the appropriate temperature prior

to performing the extraction.

2°C extraction of TFAA (mgN kg–1)

0 3 6 9 12 15 18

20°C

ext

ract

ion

0

3

6


Fig. 1. Impact of extractant temperature (2 or 20 8C) on the recovery of DOC

(Panel A), DON (Panel B) and total free amino acids (TFAA; Panel C) from six

soils when extracted with either distilled water or 2 MKCl. All values represent

meanGSEM (nZ4).

2.4. Investigation of temporal and spatial variability

in DOC and DON

To assess long-term temporal variability, a visually uniform

plot (2 m!6 m)wasmarked out at two field sites (Soil G and H).

Each month, nine independent random soil samples were taken

from the area and soil solution recovered by centrifugation, and

standard extractions performed with 2 M KCl and distilled

water as described above. To evaluate spatial variability, thirty

samples of soil from the same location were extracted with

either distilled water or KCl using the standard protocol

described above.


2.5. Chemical analysis

Concentrations of DOC and total dissolved N (TDN) were

determined with a Shimadzu TOC-TN analyzer (Shimadzu

Corp., Kyoto, Japan). In the annual sampling of Soils G and H,

TDN was determined by persulfate oxidation (Williams et al.,

1994). The TOC-TN analyzer and persulfate oxidation

methods gave comparable results for TDN when samples and

standard solution were analyzed by both methods (data not

presented). NHC4 in the soil solutions and extracts was

determined by the method of Mulvaney (1996) on a Skalar

autoanalyzer (Skalar Ltd, York, UK). NOK3 was determined

colorimetrically using the same Skalar autoanalyzer equipped

with Cd/Cu reduction column and NK1-napthylethylenedia-

mine chromophore. DON was calculated as the difference

between the TDN reading and the combined NHC4 and NOK

3

reading. Total amino acids were determined fluorometrically

(Jones et al., 2002).

2.6. Statistical analysis

Statistical analysis (normality testing, ANOVA with Tukey

pairwise comparison, paired t-tests; coefficient of variation,

linear regression) was performed using the computer package

Minitab 14 (Minitab Inc., State College, PA). All extraction

data is presented on a dry weight basis unless otherwise stated.

Although eight soils were examined in this study for simplicity

only representative results are presented in Section 3.

3. Results

3.1. Influence of temperature and solvent on DON and DOC

extraction from soil

Overall, extraction temperature had little effect on the

amount of DOC, DON or total free amino acids recovered in

either distilled water or KCl extracts (Fig. 1). Amino acid,

DON and DOC recovery from the soils was significantly

greater when extracted with 2 M KCl in comparison to those

extracted with distilled water (P!0.05; Fig. 1). DON typically

comprised 55G11% (meanGSEM, nZ6) of the extractable

soluble N in the soil of which approximately 11G2% was in

the form of free amino acids. These values are similar to those

in soil solutions where 45G4% of the total soluble N was

Table 2

Soluble C and N content of soil solutions from eight contrasting soils

NO�3 (mg N lK1) NHC

4 (mg N lK1) DON (mg N

A. Dystric cambisol 0.9G0.3 0.2G0.0 3.2G0.2

B. Humic cambisol 21.4G0.1 3.4G0.1 22.9G1.7

C. Leptic podzol 130.5G0.6 12.8G0.1 168.5G6.1

D. Leptic podzol 49.3G9.7 0.6G0.5 10.9G3.8

E. Eutric cambisol 65.6G6.7 0.8G0.1 16.3G5.9

F. Calcic cambisol 3.8G0.3 0.2G0.0 4.5G0.7

G. Eutric cambisol 30.4G0.9 0.7G0.1 12.2G0.3

H. Dystric gleysol 20.5G0.2 0.7G0.1 8.1G0.5

Soil solutions were recovered from the soil by the centrifugal-drainage method.

present as DON of which 9G1% of the DON was in the form

of free amino acids (Table 2).

3.2. Influence of filtration on soil DON and DOC concen-

trations in soil

After shaking the soil, the recovery of the solution phase by

either filtering (Whatman No. 42) or centrifugation had no

significant impact on the amount of DON extracted from the

soil (PO0.05; data not presented).

3.3. Influence of shaking time on DOC and DON extraction

from soil

Generally, the amount of DOC and DON recovered from the

soil gradually increased over a 24 h extraction period (Fig. 2).

The recovery of solutes in the extract solution could be

distinguished into two phases, namely an initial fast extraction

phase (0–15 min) followed by a slower extraction phase

(O15 min). On average, the instantaneous phase recovered

45G8% of that recovered in the extract solution after 24 h.

3.4. Influence of soil-to-solution ratio on DOC and DON


Above a soil weight to extractant volume rate of 0.25 g mlK1

the amount of DOC and DON recovered on a soil weight basis

remained relatively constant for both extractants (Fig. 3). After

accounting for the dilution of the soil solution by the volume of

extractant, the DOC and DON concentrations in the extracts

could be directly compared to the concentrations in soil solutions

recovered on the same day (Table 2). Although the patterns were

similar, the concentrations of both DOC and DON in the distilled

water extracts were on average 2-fold lower than recorded in the

soil solution (data not presented).

3.5. Influence of soil sieving and storage time on DOC

and DON extraction from soil

Sieving the soil (to!2 mm or!1 mm) caused a significant

increase in the recovery of DOC and DON within the distilled

water extracts in comparison to unsieved soil (P!0.05; Fig. 4).

This increase was evident both immediately after sieving

(within 1 h) and 24 h after sieving had taken place. Sieving

lK1) Amino acids

(mg N lK1)

DOC (mg C lK1) DOC-to-DON ratio

(mg C lK1)

0.11G0.01 47G2 15G1

1.01G0.03 343G15 15G1

6.46G0.10 2925G67 17G1

0.12G0.01 74G16 7G1

0.11G0.01 174G1 11G5

0.53G0.18 66G9 15G1

ND 146G3 12G1

ND 97G6 12G1

0 6 12 18 24

Ext

ract

able

DO

C (

mg

C k

g–1) E

xtractable DO

C (m

g C kg

–1)

0

50

100

150

200

250

Extraction time (hours)

0

20

40

60

80

100

DON: water extractDON: KCl extract

DOC: water extractDOC: KCl extract

Fig. 2. Influence of shaking time on the extraction of DOC and DON from a

grassland Eutric cambisol (Soil G) with either 2 M KCl or distilled water.

Symbols represent meanGSEM, nZ3.

Unsieved 5 mm sieved 2 mm sieved 1 mm sieved

Ext

ract

able

DO

C (

mg

kg–1

)

0

200

400

600

800

DOCDON

a b

c

d

e

f

Extractable D

ON

(mg kg

–1)

0

20

40

60

80

100

e

g

Fig. 4. Influence of soil sieving on the concentration of DOC and DON in

distilled water extracts of a forest Calcic cambisol (Soil F). Soil extracts were

performed within 1 h of soil sieving. Bars represent meanGSEM, nZ3.

Different letters indicate significant differences between treatments at the P!0.05 level.


caused no change in the amount of extractable NHC4 but it did

cause a significant increase in extractable NOK3 which increased

over time (P!0.05; data not presented).

Air-drying caused a 3 to 10-fold increase in the extraction of

DOC and DON from soil in comparison to soils extracted in a

field moist state (P!0.05; data not presented). Extraction of

field-moist soil with either 2 M KCl or 0.5 M K2SO4 yielded an

identical recovery of DON and DOC. Extraction with 1 M HCl

caused a 12G2-fold increase in DON and DOC recovery while

1 M NaOH caused a 57G8-fold increase in DON and DOC

A. Water extract

0

30

60

90

120

150

DOCDON

B.2 M KCl extract

Soil-to-extractant ratio (kg soil l–1extractant)

0.0 0.2 0.4 0.6 0.8 1.00

30

60

90

120

Ext

ract

able

DO

N o

r D

OC

(m

g kg

–1)

Fig. 3. Influence of soil weight-to-extractant volume ratio on the extraction of

DOC and DON from a grassland Eutric cambisol (Soil G) with either 2 M KCl

or distilled water. The legend is the same for all panels. Symbols represent

meanGSEM, nZ3.

recovery in comparison to field moist soil extracted with

distilled water (data not presented).

3.6. Influence of soil sampling intensity on DOC and DON


The soil extracts showed a high degree of variability in

concentration (Fig. 5). The coefficient of variation (CV%) for

the quality control standard for the DOC–DON analyzer was

1.5% for DOC and 1.2% for DON whilst for both the distilled

water and KCl extracts the CV% was 15G2% for both DOC

and DON. There was a positive correlation between DOC and

DON concentrations (r2Z0.83). When all the data presented in

Fig. 5 was subjected to linear regression analysis the r2 value

was 0.89. Statistical modeling in Soils E and F, alongside

others, suggests that at least 4 replicate samples of soil should

0 5 10 15 20 25 30 350

1

2

3

4

5

DOC (mgl–1)

DO

C (

mgl

–1)

2 M KCl: Soil F2 M KCl: Soil G

H2O: Soil FH2O: Soil G

Fig. 5. Variability in KCl and water extractable DOC and DON from 30

individual samples of a forest Calcic cambisol (Soil F) and grassland Eutric

cambisol (Soil G). Symbols represent experimental data points while the line

represents a linear regression analysis of all the data.


be analyzed to provide an accurate assessment of soil DOC and

DON concentrations (data not presented).

3.7. Influence of seasonality on DOC and DON concentration

in soil

The results from both grasslands for all methods indicated a

significant annual variability in DON concentrations. Typi-

cally, the amount of DON recovered varied about 5-fold from

the annual mean concentration over the year (Fig. 6). The

annual variability in DON concentration tended to be skewed

by occasional high monthly DON values. These peaks occurred

at different times of the year for the two grasslands examined

(data not presented). A similar degree of variability was

observed for NOK3 and NHC

4 as was observed for DON. In Soil

G, the amount of NOK3 relative to DON was highly dependent

upon the time of year at which the soil was sampled.

3.8. Influence of sample storage on DOC and DON extraction

from soil

Analysis of the stability of free amino acids in refrigeration-

stored KCl and distilled water extracts (after removal of the

soil) indicated no significant removal/degradation of amino

acids during a 28 d incubation period (P!0.05; data not

presented). Repeated freezing and thawing of freezer-stored

extract solutions caused the aggregation of DOC and DON in

solution producing a brown amorphous looking insoluble

material (i.e. particulate organic matter). This phenomenon

was particularly evident in soil solutions, to a lesser extent in

distilled water extracts and rarely observed in KCl extracts.

DOC and DON analysis revealed that O60% of the DOC and

DON could be lost by freeze-thaw induced precipitation (range

was 14–65% loss from solution; data not presented). If the

persulfate procedure of Williams et al. (1994) is used for DON

A. Eutric cambisol soil solution

Soi

l sol

utio

n co

nc. (

mg

N l–1

)

0

10

20

30 B. Eutric cambis

KC

l ext

ract

con

c. (

mg

N l–1

)

0

4

8

12

16

D. Dystric gleysol soil solution

Soi

l sol

utio

n co

nc. (

mg

N l–1

)

0

2

4

6

8

10

12E. Dystric gleyso

KC

l ext

ract

con

c. (

mg

N l–1

)

0

3

6

9

12

15

DON NO3– NH4

+

DON NO3– NH4

+ DON

DON

Fig. 6. Annual variability of DON, NOK3 and NHC

4 concentrations in two grassland s

zero indicates the 25th percentile, the line within the box marks the median, and the

below and above the box indicate the 10th and 90th percentiles and the symbols th

analysis, this loss can be accounted for by including the

precipitate in the digestion tube.

3.9. Comparison of soil solution and soil extract DOC and

DON concentrations

There was a positive correlation between the amount of

NOK3 in soil solution and that recovered in the extract solution

(r2Z0.97; Fig. 7). The relationship between the amount of

DOC and DON present in soil solution and that recovered in

soil extracts showed a general positive relationship. Using the

concentration relationship between the amount of NOK3 in soil

solutions and soil extracts, it appears that both water and KCl

extracts tended to overestimate the amount of DOC and DON

in soil solution. The recovery of amino acids in soil solutions

and soil extracts was similar based upon the concentration

relationship between the amount of NOK3 in soil solutions and

soil extracts (r2Z0.90).

4. Discussion

4.1. General observations

Our results show that soil sampling strategy and method-

ology have a significant impact on DOC and DON

concentrations. The differences in soil type responses to

experimental factors (e.g. shaking time, sieving, extractant)

further complicate the choice of experimental approach. This

suggests that soil extracts can only provide a comparative

estimate of soil DOC and DON concentrations. This is

unfortunate as soil chemical extraction procedures provide a

quantitative estimate of inorganic N levels in soil and the

methods have been universally adopted (Sparks, 1996).

However, comparative estimates of DOC and DON concen-

trations in soil may prove useful as demonstrated in previous

ol 2M KCl extract C. Eutric cambisol water extract

H2O

ext

ract

con

c. (

mg

N l–1

)

0

1

2

3

4

5

l 2M KCl extract F. Dystric gleysol water extract

H2O

ext

ract

con

c. (

mg

N l–1

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

NO3– NH4

+

NO3– NH4

+ DON NO3– NH4

+

DON NO3– NH4

+

oils (Eutric cambisol and a Dystric gleysol). The boundary of the box closest to

boundary of the box farthest from zero indicates the 75th percentile. Whiskers

e 5th and 95th percentiles.

A. Nitrate

Soil solution concentration (mg N l–1) Soil solution concentration (mg N l–1)

Soil solution concentration (mg l–1) Soil solution concentration (mg l–1)

0 50 100 150 200

Dis

tille

d w

ater

ext

ract

con

cent

ratio

n (m

g N

l–1)

Dis

tille

d w

ater

ext

ract

con

cent

ratio

n (m

g N

l–1)

Dis

tille

d w

ater

ext

ract

con

cent

ratio

n (m

g l–1

)

Dis

tille

d w

ater

ext

ract

con

cent

ratio

n (m

g l–1

)

0.0

2.5

5.0

7.5

10.0

D. DON

0 40 80 120 160 2000

1

2

3

4

5

B. Amino acids

0.0 0.4 0.8 1.2 1.60.00

0.03

0.06

0.09

0.12

0.15

C. DOC

0 1000 2000 3000 40000

20

40

60

80

Fig. 7. Relationship between the concentration of NOK3 , DOC, DON and free amino acids in soil solutions obtained by the centrifugal-drainage technique and in soil

distilled water extracts. The solid line in Panel A (NOK3 ) represents a linear regression fit to the experimental data. The dotted line in Panels B–D is the same linear

regression line as used in Panel A for NOK3 . Values for soil solutions represent meanGSEM (nZ3) and for distilled water extracts meanGSEM (nZ4).


studies (De Luca and Keeney, 1993; Liang et al., 1999; Ros

et al., 2003; Zhong and Makeschin, 2003; Caravaca et al.,

2005). As these studies either fail to state the extraction

methodology used, or have used different extraction tech-

niques, they cannot be readily compared. It is clear from our

results therefore that a standard extraction protocol for

measuring DOC and DON needs to be adopted to allow

comparison between different studies.

4.2. Proposal for a standard extraction protocol

for DOC and DON

For convenience, it is preferable to analyze DOC and DON

in routine extractions undertaken either for inorganic N or

microbial biomass determination in soil (Mulvaney, 1996;

Howarth and Paul, 1994). Although these standard method-

ologies differ in their use of solvent (0.5 M K2SO4 versus 2 M

KCl) and soil-to-extractant ratio (1:5 versus 1:10 w/v), our

results suggest that comparatively these factors have little

impact on the amount of DOC and DON recovered. To match

these methods, we therefore recommend the use of unsieved,

field-moist soil, an extraction shaking time of 1 h with either

0.5 M K2SO4 or 2 M KCl, temperature of approximately 20 8C

and soil-to-solution ratio of 1:5 (w/v).

Our experiments clearly show that the method of sample

preparation has a significant effect on the recovery of DON and

DOC from soil. Based upon our results we recommend that

where possible, soils should not be sieved to %2 mm and

certainly not air-dried before extraction or soil solution

recovery. Further, analysis of samples should be undertaken


as soon as possible after sample collection and preferably

within 24 h of removal from the field. Although there was little

change in DON and DOC over a 24 h soil storage period

(Fig. 4), longer storage times are known to have a significant

impact on soluble nutrient levels (Chapman et al., 1997b). To

minimize changes, we recommend that even within the field,

samples should be stored in an intact state at !5 8C. Where

possible, discernable roots should be removed before extrac-

tion. Distilled water extracts can be used to approximate the

DOC and DON in soil solution, while 2 M KCl or 0.5 M K2SO4

extracts can be used to measure the DOC and DON held in the

solution and exchange phase.

4.3. Influence of the microbial community on DOC and DON

recovery

Despite the inevitable damage to some soil microbes and

invertebrates (e.g. fungal hyphal breakage) during centrifu-

ging, this is thought to be of minor significance (Zabowski,

1989). Although chemical extracts can lyse cells, they have

little effect on NHC4 or NOK

3 recovery. We speculate this is

because microbial cells contain little inorganic N due to its

rapid transformation to organic N forms after uptake (Jennings,

1995). On the other hand, the concentration of DON and DOC

gradually increases over time during chemical extraction,

suggesting a combination of damage to microbial cells and the

gradual physical destruction of the soil allowing release from

previously inaccessible pores (i.e. physically trapped DON and

DOC). This gradual release due to physical disruption may,

however, be balanced by the microbial removal of some solutes

from solution. While fungi will be significantly disrupted by

shaking, we hypothesize that bacteria are less affected. Our

results showed that temperature had little effect on DOC and

DON concentrations in KCl and water extracts indicating that

significant biodegradation of these compounds did not occur at

higher temperatures. We speculate that this is because most

DOC and DON recovered in soil extracts is of high molecular

weight and relatively resistant to microbial attack (data not

presented). In contrast, the low molecular weight components

in solution (e.g. sugars, amino acids) are rapidly biodegraded

(data not presented). As the recovery of amino acids at 2 8C

was always greater than at 20 8C it suggests that the lower

temperatures suppressed microbial biodegradation. This is

further supported by the KCl extracts which always showed

much greater recovery of DOC and DON in comparison to

water extracts. This could also be ascribed to KCl extracts

removing amino acids held on exchange sites, however, the

very low amino acid sorption capacities of our soils make this

unlikely (Jones, 1999). To minimize the risk of microbial

contamination of samples or solute degradation we therefore

recommend the use of rapid extraction times (%1 h). Our

results show that once the extraction is complete and the water

phase has been separated from the solid phase by centrifugation

or filtration, the solutions are relatively stable for days in the

refrigerator and for months in the freezer.

4.4. Influence of soil sampling regime

Our results suggest that in some soils a single sampling

event will be insufficient to obtain an accurate representation of

a soil’s DOC and DON status. We therefore suggest that

concentrations should be monitored at different times

throughout the year depending upon the nature of the study.

This sampling should be capable of taking into account both

long-term seasonal and short-term climatic effects and the

intrinsic variability of the soil. Our experiments suggest that at

least 4 independent samples of soil should be taken at each

sampling event in order to gain a true reflection of the soil’s

DON and DOC status at any point in time.

4.5. Conclusions and recommendations

Collectively, the results support previous findings that DON

represents a major soluble N pool in soil, that its concentration

is highly soil type dependent, and that only a small proportion

of the DON is present in the form of free amino acids. Due to it

being a large potentially available N source for soil

microorganisms and plants and a major loss pathway in soil

leachates, DON and DOC should be routinely quantified

alongside NOK3 and NHC

4 . The results presented here indicate

that DON and DOC can be readily removed from soil using

standard extractants used for measuring inorganic N and

microbial biomass, however, the degree of DOC and DON

recovery appears highly dependent upon the method of sample

preparation and extraction. Any manipulation of the soil can be

expected to rapidly change its intrinsic DON, DOC and

inorganic N status. We therefore propose a standard extraction

protocol to enable comparisons to be made both within and

between studies.

Acknowledgements

This work was funded by the Natural Environment Research

Council—GANE programme, UK (DLJ).

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