THE EFFECTS OF BIOCIDAL TREATMENTS ON

METABOLISM IN SOIL-Y

A METHOD FOR MEASURING SOIL BIOMASS

D. S. .IENKIUSON and D. S. POWLSON

Rothamstcd Experimental Station. Hal-pcndcn. Her&. AL5 2JQ

Summary--A new method for the determination of biomass in soil is described. Soil is fumigated
with CHCI, vapour. the CHCI, removed and the soil then incubated. The biomass is calculated from
the difference between the amounts of CO, evolved during incubation by fumigated and unfumigated
soil. The method was tested on a set of nine soils fl-om long-term field experiments. The amollnts
of biomass C ha- ’ in the top 23 cm of soil from plots on the Broadbalk contmuous wheat experiment
were 530 kg (unmanured plot). 590 (plot receiving inorganic fcrtihrcrs) and I I60 (plot receiving farmyard
manure). Soils that had been fallowed for I year contained less biomass than soils carrying a crop.
A calcareous woodland soil contained 1960 kg biomass C ha-‘. and an unmanured soil under permanent
grass 2020. The arable soils contained about 2”,, of their organic C in the biomass: uncultivated
soils a little more--about 3”,,.

IKTRODL’CTION \1.4TERIAI.S .A\D METHODS

From the preceding study of the effects of biocidal Soils

treatments on soil metabolism (Jenkinson and Powl-
son, 1976; Powlson and Jenkinson, 1976; Jenkinson
er al., 1976; Jenkinson, 1976) we concluded that the
flush of decomposition caused by fumigation can pro-
vide a measure of the amount of biomass in a soil.
This paper describes a practical method for measur-
ing biomass that is based on the earlier work on fumi-
gation. The new method was tested on a set of soils
of known history, selected to cover an extreme range
of systems of soil management.
In contrast to the earlier work. which was done
on small samples of carefully homogcniscd stored soil.
the work described in this paper was done on large
samples. freshly collected and taken with the mini-
mum of soil disturbance, so that the amount of bio-
mass as measured could be related to the amount
in the field. Sampling was by volume, so that biomass
could be expressed on a per hectare basis.
All the soils came from long-term experiments on
Rothamsted Experimental Farm. All were silt loams
or silty clay loams and all arc mapped as ‘Batcombe
series’. Table I gives analytical and sampling details
of the soil samples. The cropped Broadbalk soils
(&OS. 1, 3 and 5) were taken immediately after harvest
from discard areas of Section 11one of the continuous
wheat sections of the Broadbalk experiment (Johnson
and Garner, 1969). The fallowed Broadbalk soils were
taken from the fallow year of a wheat. wheat. fallow
rotation (Section 3). Jenkinson (1971) gives details of
the Broadbalk Wilderness soils: Warren and John-
ston (1964) give details of the Park Grass soils.
Sampling was to a depth of 22.9 cm with a 5 cm
dia corer in July~August. 1973. Twenty cores were
taken from each site and were placed alternately into
one of two samples. gibing two samples per site. each
of 10 cores. The samples were put through a 6.35

Table I. Description of soils used

I Bloadhalk Plot 03 hone \VhU1 h0 0 IO? I 00 0 26 2600 340

1 Hroadhalk Plot 03 hone F;,lloa X0 0 w4 O-&J 0 ?h 2610 310
3 Bwadhalk Plot 22 Farm~.mi mmu,~‘~” WhC‘l1 7X 0 239 2 47 022 ‘450 330
4 Bloadbalk Plot 22 Fdrm>cird manut-cl” Fallou 77 ,,2?X 2 :i 0 2.3 2 I70 ??(I
5 Broadhalk Plot OX NPK NeMy$“’ What ii OII? I O’Y 0 0, 2690 431
6 Hroadbalk Plot OX NPK NaMglh’ FZJllO\\ 7X 0 109 I 09 0~04 2.400 34,
7 Broadhalk W,ldcrncss NOIlL- Wooded 77 0 101 3 191’1 I) 26 I Xi0 230
X Broadhnlk Wildcrnw hone Stuhhcd 77 O?li 1 2.3 I, IX I620 320
9 Park Grass Plot 3d Nom Gr:,,s 57 0 263 3 20 NOllC 2180 I x0

cd’ 35 tonnes ha- ‘. annually.

lh’144 kg N. 32 kg P. 91 kg K. I6 kg Na and II kg Mg ha-‘. annually.
IL)Soils 7. 8 and 9 also contained. respectively. 9. 2 and 5 tonnes ha ’ of organic C m roots not passing the
6.35 cm sieve. All other soils contained negligible amounts of roots not passing the sieve.
210 D. S. JFNKINSOK
mm sieve and the weights of sieved soil, stones not
passing through the sieve and roots not passing
through the sieve measured on a spring balance. The
soils were either used on the day they were sampled
or stored overnight at 15’C: they were not allowed
to dry out.
Four portions of soil. each containing 250 g moist
soil, were taken from each sample and placed in 400
ml glass beakers; two portions were fumigated with
CHCl, and two left unfumigated. Replicate portions
of soil were taken for dry matter determinations and
for measurement of water holding capacity. Sub-sam-
ples for chemical analysis were air-dried and ground
for 3 min in a Tema disc mill. The fumigations were
done in large desiccators (30.5 cm id.) lined with
moist paper. Each desiccator contained a beaker with
50 ml of alcohol-free CHCl, (see below) and a few
anti-bumping granules. The desiccator was evacuated
until the CHCl, boiled vigorously. the tap closed and
the desiccator then left in the dark at 25‘C for 18-24
h. The beaker of CHCl, and the paper were then
removed and CHCl, vapour removed from the soil
by repeated evacuation in the desiccator. Six 3-min
evacuations, three with a water pump and three with
a high vacuum oil pump, were usually sufficient to
remove the smell of CHCl, from the soil; two ad-
ditional evacuations were then given with the high
vacuum pump. Each portion of fumigated soil was
inoculated with I g of moist unfumigated soil, mixed
in with a spatula. The unfumigated portions of soil
were not inoculated. While fumigation was in pro-
gress, the unfumigated controls were kept at 25’C in
desiccators lined with moist paper. After inoculation,
sufficient distilled water was added to each soil to
bring it to 5S”,, of its water holding capacity.
The CHCl, was purified by shaking the Analar-
grade reagent (which contains ethanol as stabilizer)
three times with 5”<, cont. H,SO,. The CHCl, was
then washed five times with water. dried over anhy-
drous K,CO, and redistilled. The purified reagent
can be stored over anhydrous K,CO, in the dark
for a few weeks.
““CO,~~C evolved by fumigated soil in @IO day period.
““Calculated from flush using a k factor of 0.5.
“’ Weights of biomass C ha-’ calculated for each of the field samples from biomass and soil weight
made on that sample alone: figs. in this column are means of two such measurements per soil.
and D. S. POWLSON
The portions of soil were incubated for IO days
at 25’C and CO, evolution measured. In addition.
the unfumigated soils were incubated for a second
IO-day period. Each beaker of soil was placed in a
wide-neck (1 1.4 cm dia) screw-top glass confectionary
jar with a volume of 3.75 1. (W.G. Glass Containers
Ltd. Staines. Middlesex), together with 100 ml of l!V
NaOH in a 250 ml beaker. The jar also contained
20 ml of distilled water to offset the drying elTect of
the alkali. The jar was closed with a rubber closure
(No. 145, Suba-Seal Works. Barnsley. Yorkshire), and
the plastic cap provided with the bottle was screwed
down over the Suba-Seal. Care must be taken to
avoid breathing into the open jars- an important
source of error with large jars such as thcsc. Blank
incubations, in which the jar contained water and
alkali but no soil, were included in each experiment.
Large jars were used to ensure that there was enough
0, for the whole incubation. In no incubation was
more than half the 0, consumed and usually the pro-
portion consumed was much less.
At the end of an incubation the NaOH was made
up to 250 ml with distilled water. A 25 ml aliquot
was placed in a 150 ml beaker with 25 ml distilled
water and four drops of a solution of carbonic anhyd-
rase. prepared by dissolving 10 mg of the pure cn;l_yc
(Sigma Chemical Corporation) in IO ml distlllcd
water. This enzyme solution can bc stored for up to
7 days in a refrigerator. Immediately after adding the
enzyme. the pH of the solution was brought to about
10 by slow addition of I Al HCl and then to X.30 by
slow addition of 0.05,V HCl, the solution being stirred
with a magnetic stirrer. Addition of carbonic anhyd-
rase improved the pH X.30 end-point by decreasing
drift. so that the titration could be done more quickly
(Underwood, 1961). The solution was then titrated
with 0.05.V HCl to pH 3.70 and the amount of CO,
evolved during incubation calculated from the volume
of acid required to bring the pH from X,3&3.70. less
that required by the blank: I ml of 0.05R; HCl is
equivalent to 0.6 mg COzmC in the NaOH solution.
Each CO, measurement in Table 2 is the mean of
two replicate incubations on each of the two soil
samples taken from each site.
less CO,-C evolved by untreated soil in I@ 20 da\. period.
mcasurcmcnts
 Metabolism
Soil pH, total N. organic C and carbonate C were
determined as previously described (Jenkinson and
Powlson, 1976). Water holding capacity was deter-
mined by Shaw’s method (1958). A conical filter fun-
nel was fitted with a small glass wool plug; a short
piece of rubber tubing which could be closed with
a clip was attached to the stem. Moist soil (50 g).
as sampled, was placed in the funnel. the clip closed.
50 ml water added, and the soil allowed to stand
for 30 min. The clip was then opened and the \,olume
of water draining in 30 min measured. The diffcrcncc
between the volume of water added and the volume
drained. plus the water previously in the soil. is taken
as the water holding capacity.
RESL’LTS A\D DISCI WON
Fumigation with CHCI, caused the usual flush of
decomposition (Table 2). Respiration in the unfumi-
gated soils was markedly less during the IO 20 day
period than during the &IO day period: for reasons
for this decline see Section 3.
The respiration measurements in Table 2 are each
the mean of four determinations: two separate
samples were taken from each site in the field and
duplicate incubations done on each of these. An
analysis of variance done on a logarithmic transform
of the data in column 5 of Table 2 shohed that the
partition of error between different ficld samples and
between replicate incubations done on the same
sample was such that. for a given number of incuba-
tions. it is better to take more field samples than to
increase the number of determinations done on each
field sample.
Because of the large initial respiration rate in the
‘untreated’ soils the flush (F) was taken as (X - J,)
where X is the COz- C evolved by fumigated soil over
the period GlO days and J’ that evolved by unfumi-
gated soil over the 1@20 day period. rather than
(X - _u) where s is the CO1 C evolved by unfumi-
gatcd soil over the &IO day period. Soil biomass (B)
was calculated from the expression B = (X - !$I,; k
was taken to be 0.5 (Jenkinson. 1976). Table 2 shows
that the amounts of biomass C in the nine soils range
from 44@2020 kg ha ‘. all to a depth of 23 cm. In
the arable soils (Nos. 1 -6) this biomass accounts for
1.7 ZOO,, of the soil organic C. The soils from the
wooded. stubbed and grassland sites (Nos. 7-9)
contain considcrabl) more biomass C than any of
the arable soils. and this biomass constitutes a larger
proportion (2.9%3,7”;,) of the total soil organic C.
These values for the amount of biomass in soil are
rather larger than previous estimates. Clark and Paul
(1970) estimated the microbial biomass (bacteria.
actinomycetes and fungi) of a Canadian grassland soil
to be 200 g dry matter/m’ to a depth of 30 cm.
Assuming that the dry matter contains 5OY, C, this
corresponds to I COO kg microbial biomass C ha- ’
to a depth of 30 cm. Dr. J. E. Satchel1 (personal com-
munication) provisionally estimated that the total
biomass (bacteria. actinomycetes. fungi and fauna) in
the litter and soil of a woodland site amounted to
200 g dry mattcr.lm’ to a depth of 35 cm.
in sod V 211
Table 2 shows that the areas of Broadbalk that
had carried a wheat crop in the year the samples
were taken (soils 1,3 and 5) contained a little more bio-
mass C per hectare than the corresponding areas that
had been fallowed for a year after growing wheat
(soils 2,4 and 6). Thus, for the unmanured plot, the bio-
mass per hectare in the fallowed area was 81% of
that in the cropped area. The corresponding figure
for the inorganic fertilizer plot was 88% and for the
farmyard manure plot 73;;,.
Soils 5-6. from the Broadbalk plot receiving
mineral fertilizers, showed a relatively small difference
in biomass between the cropped area (soil 5) and the
fallowed area (soil 6) when the results were expressed
on a per lo0 g soil basis (Table 2). Likewise. the fal-
lowed soil contained as much C and almost as much
total N as the cropped soil (Table 1). Sampling errors
arc probably the cause of this apparent similarity;
the fallowed area from which soil 6 was taken almost
certainly contained less organic C. organic N and bio-
mass than the corresponding cropped area. Table I
shows that soil 5 was more compacted than soil 6,
so that soil cores taken to a depth of 23 cm were
diluted with more subsoil than the corresponding
cores from soil 6.
The fallowed and cropped areas differed only in
the treatment they received in the year before sam-
pling: before that. both had grown wheat for many
years. Thus. it is reasonable to assume that, had the
fallowed areas been cropped in the year preceding
the collection of the soil samples, there would have
been little difference in biomass between samples l-2,
between samples 3-4 and between samples 5-6. The
comparatively small differences actually measured
show that the absence of freshly added plant material
and the extra disturbance received by the soil during
its fallow year did not result in a catastrophic loss
of biomass. The biomass in the fallowed soil is pre-
sumably made up of (a) organisms decomposing the
older and more stable parts of the soil organic matter,
(b) new biomass synthesised during the decomposi-
tion of the small quantities of plant material left from
the previous year’s crop and (c) organisms that per-
sisted in dormant states throughout the year.
The standard errors in Table 2 give a measure of
the sampling and analytical errors involved in using
the new method to measure biomass in field soils.
The measurements also contain a systematic error
arising from the use of the arbitrary k constant (see
Jenkinson. 1976) as well as systematic errors intro-
duced by difficulties associated with the determina-
tion of the respiration rate of the unfumigated soil.
In the present work. this was taken as J’, the amount
of CO,-C evolved by unfumigated soil over the 1O-20
day period. rather than s, the amount evolved over
the (&IO day period.
Respiration is stimulated by mechanical distur-
bance (Rovira and Greaten. 1957; Craswell and War-
ing. 1972). Disruption of soil aggregates kills some
soil organisms but also exposes some previously inac-
cessible substrate to microbial attack (Jenkinson, un-
published work). Thus, if a soil is subjected to disrup-
tion before fumigation. the biomass as calculated
from the relationship B = (X - ~)/0.5 will be too
712 D. S. J~NKIKSON and D. S. POWLSOU
large. because both components of the disruption-in-
duced flush in the unfumigated soil will have subsided
by the time 1%is measured. On the other hand. if the
biomass is taken as (X - .x)/0.5, it will be too small.
because mechanical disruption will have killed part
of the biomass in the unfumigated soil. making .y too
large. Limits can be set to the errors thus caused.
using the data in Table 2 for soil 9. Soil 9 is noncal-
careous and so is not subject to the additional errors
caused by bicarbonate decomposition: see below. On
the assumption that the flush caused by mechanical
disturbance is entirely due to the organisms killed
during sampling and sieving, the biomass is given by
(7OG23.5)/0.5 or 93 mg biomass C!lOO g soil. On
the assumption that this flush is caused exclusively
by the decomposition of non-biomass substrate
exposed during disturbance. the biomass is
(70%32.2)/0.5 or 76 mg. The true value will lit
between these limits.
With calcareous soils, errors are introduced
through decomposition of bicarbonate. Let p0 be the
amount of HCO;~C/lOO g soil in the fumigated soil
at the beginning of incubation. (J,,~ that after IO days
and pzo that after 20 days. Let (lo. ylo and yzo be
the corresponding amounts of HCO;-C in the unfit-
migated soil. Bicarbonate will decompose during the
initial stages of incubation because the alkali used
to absorb the CO, will cause the partial pressure of
CO, in the soil atmosphere to be lower during incu-
bation than before (see Powlson and Jenkinson, 1976)
so that. approximately /jr, > p10 1 y, ,) 1 plO. Some
HCO; will also be decomposed during fumigation
and the subsequent removal of CHCl, by repeated
evacuation. The relative constancy of the respiratory
quotient when fumigated soil is incubated. compared
with the steady dechnc in unfumigated soil (Jenkinson
and Powlson, 1976; Table I ), suggests that little
HCO; remains in fumigated soil for subsequent
release during incubation. so that q, > po. The bicdr-
bonate error in the flush (taken as X - y. as in Table
2) is then (p. - p,,,) - (y10 - y2(,). which is less than
(yO - ylo) - (y10 - q2J. At worst, the calculated bio-
mass will be too large by (s ~ ~‘)/0,5; the real error
will be much less. Bicarbonate errors are of course
most important in calcarcous soils low) in organic
matter such as soils I-2 in Table 2.
Soils should not be air-dried before fumigation: air-
drying renders some non-biomass C decomposable
(Powlson and Jenkinson. 1976) as well as killing an
appreciable fraction of the biomass. Thus, if an air-
dry soil is remoistened, one portion fumigated and
incubated and a second portion incubated without
fumigation, the COz from both fumigated and unfu-
migated soils contains a component from the killed
organisms and a component from the non-biomass
organic matter rendered decomposable. The situation
is analogous with that caused by mechanical distur-
bance, except that the effects of air-drying are much
greater than those of the rather gentle mechanical dis-
turbances considered in Section 3 above.
Again the method will not give very reliable results
on soil that has just received large additions of
decomposable organic matter. Let the evolution of
CO1 C from a fumigated soil bc X mg Ci/lOO g soil
over the G10 day period. that from the same soil
to which substrate had been added immediately
before fumigation be Z mg. that from unfumigated
soil alone s mg and from unfumigated soil plus sub-
strate I mg. Since the soils with and without substrate
contain the same amount of biomass at the moment
the substrate was added. Z - : should equal X - .x.
Figure 5 (Jenkinson and Powlson. 1976) suggests that
% - : is a little less than X - I.
The problems caused by abiotic evolution of COz,
by disturbance during sample preparation, by freshly
added substrate. etc. can largely be avoided if both
portions of soil (that to be fumigated and that not)
are given a IO day pre-incubation in the presence
of alkali. Fumigation is then done on the tenth day
and the flush taken as the difference between the
amounts of CO, evolved by the fumigated (Y) and
the unfumigatcd soil (y). both measured over the
IO-20 day period. The biomass thus measured is of
course that in the soil at the end of the pre-incuba-
tion. not that in the soil as sampled. For some appli-
cations this pre-incubation procedure is acceptable:
for others, for example the work in the present paper.
it is not.
If pm-incubation is not admissable. there is how-
ever a small modification of the experimental pro-
cedure described in the paper that will decrease bicar-
bonate errors in calcareous soils. Place a beaker con-
taining 25 g soda lime in the desiccator holding the
CHCI, and the soil to be fumigated; place the same
amount of soda lime in the desiccator holding the
unfumigatcd soil whilst fumigation of the other por-
tion of soil is in progress.
COUCI.CSIONS
In this paper the proposed method was used to
compare the effects of different systems of manage-
ment on the amounts of biomass in soils belonging
to the same soil series. It has already been used to
measure the effects of pesticides on the soil biomass
(Jenkinson and Powlson. 1970). and could be used
to follow secular changes in a soil. or to compare
mineralogically-different soils. Sorensen (1975) has
used fumigation with CHCI, as an indication of the
amount of biomass formed wlhen labelled cellulose
decomposes in a range of different soils. Using our
new value of 0.5 for k on his results. between 2- 9’5,
of the original ccllulosc C was in the soil biomass
at the end of 2 years, suggesting that the amount
of biomass synthesised per unit of cellulose (and/or
its stability once formed) differed greatly between
soils.
il~knorl,/vrly~,~l~c,r~rs-
We thank Mr. Tie Yiu Liong. vacation
student from the University of Reading, for much of the
practical work in this paper. Dr. P. J. Dart sug>vhted the
use of confcctionary .jars for the incubations. and provided
them. Mr. J. H. A. Dunwoody did the the statistical analyses.
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