Biol Fertil Soils (1995) 19:269-279
P.C. Brookes
The use of microbial parameters in monitoring soil pollution
by heavy metals
Received: 14 December 1993
Abstract Microbial parameters appear very useful in
monitoring soil pollution by heavy metals, but no single
microbial parameter can be used universally. Microbial
activities such as respiration, C and N mineralization, bi-
ological N 2 fixation, and some soil enzymes can be mea-
sured, as can the total soil microbial biomass. Combining
microbial activity and population measurements (e.g.,
biomass specific respiration) appears to provide more
sensitive indications of soil pollution by heavy metals
than either activity or population measurements alone.
Parameters that have some form of "internal control",
e.g., biomass as a percentage of soil organic matter, are
also advantageous. By using such approaches it might be
possible to determine whether the natural ecosystem is
being altered by pollutants without recource to expensive
and long-running field experiments. However, more data
are needed before this will be possible. Finally, new appli-
cations of molecular biology to soil pollution studies
(e.g., genetic fingerprinting) which may also have value in
the future are considered.
Key words Soil pollution monitoring 9 Heavy metals
Soil microbial biomass 9 Microbial activity 9 Microbial
specific activity 9 Sewage sludge 9 Atmospheric deposi-
tion
Introduction
The soil microorganisms (collectively the soil microbial
biomass) and soil fauna mineralize both flesh organic in-
puts and the much larger pool of soil organic matter. The
fertility of natural soil ecosystems therefore depends sig-
nificantly on the rate of turnover of the soil organic mat-
ter, mediated by the soil microbial biomass. Agents that
may suppress or poison soil organisms, or change the
R C. Brookes
Soil Science Department, Rothamsted Experimental Station,
Harpenden, Herts., AL5 2JQ, UK
9 Springer-Verlag 1995
quality or quantity of organic matter (either flesh inputs
or the soil organic matter itself), can damage the func-
tioning of the soil-plant ecosystems, either in the short-
term or over much longer periods (Brookes and Verstraete
1989).
In agricultural ecosystems soil fertility can be in-
creased by applications of inorganic or organic fertilizer.
The fertility of natural ecosystems, however, depends al-
most entirely on natural microbial processes, including
N 2 fixation, the mineralization of organic forms of N, C,
P, and S, and organic matter transformations, all mediat-
ed by the soil microbial biomass. Any decline in natural
fertility resulting from pollutants entering soils will there-
fore have proportionately greater effects on natural eco-
systems.
Heavy metals as soil pollutants
Of the inorganic pollutants, by far the most important are
the heavy metals, e.g., Cu, Ni, Cd, Zn, Cr, Pb. Once they
enter soil they remain for extremely long periods, having
a half-life, depending upon the metal, of several thou-
sands of years. In practice, the metals can be removed on-
ly by removal of the soil itself, seldom a practical proposi-
tion.
Heavy metals enter soil from several sources, including
wastes from mines and smelters, atmospheric deposition
(following release of metals into the atmosphere from
metal smelting or other industries), animal manures, and
sewage sludge and, in some circumstances, inorganic fer-
tilizer. Sewage sludge, while containing useful quantities
of organic matter, N, and P, is often contaminated with
significant quantities of heavy metals, which are chelated
by the organic matter in the sludge. When the sludge de-
composes the metals are released and fixed on the soil, so
that the metals accumulate as sludge is added.
Mandatory European Union (EU) limits restrict the
amounts of metals permitted to accumulate in agricultur-
al soils. The limits are based upon known effects of met-
270

als on plant uptake and animal health. They take no ac- 99

I
count of effects on soil microorganisms or important mi-
crobial processes, e.g., organic matter or soil N dynamics.
This is because, at the time the limits were originally set,
the methods necessary to investigate the effects of metals 60
on these properties either awaited development or had not
"O
yet been tested. Recently developed methods have in-
dicated significant effects of heavy metals at concentra-
tions around, or below, current EC limits on both the
standing crop of microbial biomass and its activity. These
effects are illustrated and the suitability of soil measure-
ments for use in pollution monitoring discussed in this 0 30 60 9O
paper. Days

Fig. 1 Relationship between monitoring period and change in bio-

Assessing the significance of microbiological changes logical activity (deficit) in three categories describing the ecological
significance of injuries. From Domsch et al. (1983). Reproduced by
due to pollution by heavy metals or other agents permission of the publisher

The use of microbiological properties as indicators of soil

pollution has, in principle, much to recommend it. Mi-
June Dec June
crobes, having both mass and activity and being in inti-
mate contact with the soil microenvironment are, in many o 1000 1 1 I
ways, ideal monitors of soil pollution. Probably the best
E 800
criteria so far proposed were those o f Domsch (1980) and 0

Domsch et al. 0983). They first considered the effects of 600

C
naturally occurring stresses on soil microbial populations
and their activities, including fluctuations in temperature, = 400
>o
extremes of water potential, extremes of pH, physical dis- 200
turbances to soil (e.g., ploughing), a decreased gas ex-
change, decreased supply of nutrients, and increases in in- O 0 . . . . I = = a . I . . . . I . . . . I i . . , I . . . . I

hibitors, predators and antagonists. Any of these phe-
nomena, singly or jointly, can markedly affect both the
size and activity of the microbiological community. For
example, Domsch et al. (1983) evaluated 55 documented
fluctuations in bacterial populations under field condi-
tions and showed that depressions of up to 90~ occurred
frequently. On this basis, they considered that any stress
(or pollutant) that permitted a full recovery within 30
days of the microbiological property studied was normal,
those resulting in delays of 60 days tolerable, while those
taking longer than 60 days were considered to indicate
that further investigation was required (Fig. 1).
Cook and Greaves (1987) have given an excellent exam-
ple of the natural variation that might be expected within
a single microbiological property over a year. They moni-
tored soil CO 2 evolution directly from a field plot over
52 weeks in Oxfordshire, England, between April 1977
and May 1978. CO 2 evolution was maximal at around
950 gg cm-3 soil in the warm wet conditions in the sum-
mer of 1987 but had declined to less than half this by De-
cember of that year (Fig. 2). Similar patterns of N miner-
alization were also observed. With both properties there
was a long recovery period of 1 5 - 2 0 weeks into the next
spring, before increasing activity was observed again.
These are natural phenomena, the result of changing mi-
crobial activity presumably as a result of changing mois-
ture or temperature regimes or both, with no known extra
stress, such as a pollutant, to confound the results. The
potential of microbiological properties as indicators in
0 10 20 30 40 50 60 ,
W e e k number
Fig. 2 Carbon dioxide evolved from an Oxfordshire field over 1
year. From Cook et al. (1987). Reproduced by permission of the
publisher
soil pollution monitoring might thus appear small or
non-existent when faced with such a wide range of natu-
ral microbiological activity. I hope to show that this is not
actually the case.
Evaluation of the potential
of microbiological properties as indicators
of soil pollution monitoring
The previous example illustrates the problems of field
monitoring and interpreting the changes in the respiration
of soil microorganisms, apparently a simple characteristic
to measure, If a pollutant is also introduced into the sys-
tem, unless it has truly drastic effects, e.g., the near total
kill produced by an efficient fumigant such as chloroform
or methyl bromide, it will probably be almost impossible
to measure its effects under such conditions. It is indeed
interesting that in this field experiment, Cook and
Greaves (1987) observed that "Variation between samples
was greatest when the rainfall was heavy...". It is just at
this time that microbiological activity would be maximal,

which would also make effects due to pollutants very dif-
ficult to detect. Many other soil properties were also con-
sidered by Cook and Greaves (1987). These included soil
physical and chemical properties and conditions (e.g.,
field moisture capacity), soil temperature, meteorological
data, percent organic C, soluble organic C, pH, and bio-
logical properties (e.g., enumeration of microorganisms
by fluorescence staining), plate counts of bacteria, ac-
tinomycetes, fungi, yeast, and algae, ATP, urease, and O2
uptake. Cook and Greaves (1987) concluded that the
properties that appeared to be most responsive to envi-
ronmental change were N transformations and soil respi-
ration. However, they considered that the most striking
feature of their field data was the considerable variability
encountered even when attempts, such as multivariate
analysis, were made to overcome it.
Thus, detection of small, transient changes in micro-
bial population size or microbial activity in the laboratory
or field, as a response to a real or apparent pollutant,
should not, in the light of the large natural variations ob-
served in the field, be accorded a great deal of weight
(Domsch et al. 1983). Based on these criteria, only those
approaching or exceeding a recovery period of around
3 0 - 6 0 days should be considered critical, while over the
first 30 days at least, a depression in activity of 90% fol-
lowed by recovery still falls within the range considered
normal (Somerville et al. 1987). This theoretical frame-
work was originally developed for testing the side effects
of pesticides. However, it seems sensible to extend the
same approach to other soil pollutants, such as heavy
metals. It also seems unlikely that field monitoring of the
effects of heavy metals or other pollutants on microbial
processes will permit detection of many effects, because
of the likely variation that will be encountered. However,
once soil has been sampled, sieved, and incubated moist
at 2 0 - 2 5 ~ for several days, most microbiological activi-
ties approach some basal level and generally proceed at
relatively constant rates for considerable periods, e.g., res-
piration and N mineralization (Jenkinson 1988). Mea-
surements of both microbial biomass and microbial ac-
tivities may be conveniently measured under such condi-
tions, while recognizing the need for caution in extrapola-
tion from the laboratory to the field.
Criteria to be used in selection
of possible microbiological properties as indicators
in soil pollution monitoring
There are a number of basic criteria that a microbiologi-
cal property might be expected to fulfil as an indicator in
monitoring soil pollution by heavy metals or other pollu-
tants. (1) The property needs to be accurately and precise-
ly measurable across a wide range of soil types and soil
conditions. (2) Because a large number of samples usually
have to be analysed it is preferable that the property can
be easily and economically measured. (3) The property
needs to be of a nature that control or background mea-
surements can also be made so that the effects of the pol-
271
lutant can be precisely determined. (4) The property
needs to be sensitive enough to indicate pollution but also
sufficiently robust not to give false alarms. (5) The prop-
erty needs general scientific validity based upon reliable
and contemporary scientific knowledge. (6) It may be that
reliance upon a single property is unsafe. Two or more,
preferably independent, might be chosen. If so, their in-
terrelationships in non-polluted environments should be
known.
Possible microbiological properties as indicators
in monitoring soil pollution
Microbiological properties as indicators of soil pollution
by heavy metals fall into two main groups. The first is
those that measure the activity of the whole microbial
population, e.g., microbial respiration, soil N mineraliza-
tion. The second type measures the size of the microbial
population, at the single organism level, at the functional
group level, or at the whole population level. A third pos-
sibility is a combination of both activity and biomass da-
ta, giving specific activities of the microbial population
(see below).
The use of pure cultures of microorganisms isolated
from soil as indicators of soil pollution is rejected. Pure
cultures of isolated organisms may be atypical of their
form in soil or may undergo undetected change during
storage. Their metabolic rate may be very different from
that in soil as they are removed from their normal ecologi-
cal interactions. In view of this, interpretation of results
and extrapolation to field conditions is impossible
(Greaves et al. 1980).
Similarly the use of soil enzymes is also controversial
because the total enzymic capacity of soil is made up of
various fractions, i.e., growing microorganisms, dead
cells, extracellular enzymes associated with the clay-
humus complex (Graeves et al. 1980). However, in view of
the large amount of work done on the use of soil enzymes
as pollution indicators, further discussion is warranted.
Microbial activity measurements
Any measurements in isolation is of little use in deciding
whether an ecosystems is suffering from pollution. Mi-
crobial activity measurements are valuable in evaluating
soil pollution, but the problem is always to decide what
to compare the measurement with, i.e., what control to
use. The problem is removed if properly constructed field
experiments with fully replicated treatments are available.
A good example is the Woburn Marked Garden Experi-
ment at Rothamsted, which has provided unique data on
the effects of heavy metals on microbial biomass and mi-
crobial activity (Brookes and McGrath 1984). Interpreta-
tion os data from the natural environment is much more
difficult, as the determination of basal levels of microbial
activity is a real problem. In view of this, most or all of
the following data are from field or laboratory experi-
272
ments. However, ways must be found to study and to
monitor the natural environment.
Microbial respiration
As shown previously, microbial respiration in the field is
subject to enormous fluctuation (Cook and Greaves
1987). However, under controlled laboratory conditions
at a suitable moisture, (e.g., 4 0 - 5 0 % water-holding ca-
pacity) and temperature, (e.g., 1 5 - 2 5 ~ the respiration
of sieved (2-6.25 mm) soil can be determined accurately
and precisely (Jenkinson and Powlson 1976). This mea-
surement is widely used by microbial ecologists as an in-
dex both of microbial activity and microbial biomass it-
self. Indeed, measurement of microbial respiration forms
the basis both of the fumigation-incubation method for
measuring soil microbial biomass (Jenkinson and
Powlson 1976) and the substrate-induced respiration
method for measuring the same property (Anderson and
Domsch 1978).
However, microbial respiration appears unaffected at
heavy metal concentrations at around current EU manda-
tory limits (Brookes and McGrath 1984; Brookes et al.
1984). Only at very large concentrations is CO2 evolution
decreased. Thus Tyler (1981) reported that microbial res-
piration was decreased at more than 1000 mg kg-1 of Cu
or Zn.
N and C mineralization
It seems that mineralization rates of soil organic N, as
with microbial respiration, are unaffected at soil metal
concentrations at around current EU limits (Brookes et
al. 1984). This is supported by Doelman (1986) who re-
ported that inhibition of both N mineralization and
nitrification are inhibited at around 1000mg kg -1 Zn,
Cu, and Ni, around 1 0 0 - 5 0 0 m g k g -1 Pb and Cr, and
around 1 0 - 1 0 0 mg kg -1 Cd.
There is a general consensus that interpretations of the
effects of heavy metals on N mineralization and nitrifica-
tion are difficult for several reasons (Bfi~tth 1989; Brookes
and Verstraete 1989; Chander 1991). These include lack
of standardization of experimental procedures and varia-
tion in soil properties which may alter the relative or ab-
solute toxicity of the metal (Tyler 1981). Similarly, Dux-
bury (1985) concluded that information on the effects of
pollutants such as heavy metals on these processes is con-
flicting, and recommended caution in generalizing about
the effects of heavy metals on microbially mediated pro-
cesses in natural environments.
There is evidence that heavy metals introduced in
sewage sludge, and giving soil metal concentrations sever-
al times the current EU mandatory limits, cause accumu-
lations of soil organic matter compared with controls
treated with uncontaminated sewage sludge. Thus
Chander and Brookes (1991 c) reported that soil at Lud-
dington (15% clay) which contained Cu at 3.7 times the
current UK limit of 140 btg g-1 soil contained 32% more
organic matter than a soil treated with uncontaminated
sludge. At Lee Valley (21% clay), soil contaminated with
Zn at 3.4 times the permitted concentration contained
10% more organic matter than soil treated with uncon-
taminated sludge. Similarly, plots containing Cu at 3.8
times the limit had about 14070 more organic matter.
These results strongly suggest that the heavy metals were
decreasing the turnover rate of the organic matter,
presumably because of inhibitory effects on the microbial
biomass itself.
However, these results were obtained from well-main-
tained field experiments where all the treatments were
known and where soil was reasonably homogeneous. De-
tection of such effects in natural environments would be
extremely difficult, if not impossible, because appropriate
uncontaminated control soils would not be available.
N2 fixation
Biological fixation of atmospheric N 2 can be performed
only by specific groups of microorganisms. All biological
N 2 fixation depends upon the enzyme nitrogenase, which
occurs in three main types of microorganisms,
autotrophs, heterotrophs, and symbionts, which vary in
the potential amounts of N 2 that they can fix in tempera-
te regions.
Measurements of N 2 fixation, or more accurately
nitrogenase activity, can be made using the acetylene re-
duction assay. Nitrogenase converts acetylene to ethylene,
which may easily be measured by gas chromatography.
This provides a sensitive test for nitrogenase activity, but
the results cannot be converted directly to the quantify of
N 2 fixed. For this, measurement of conversion of 15N2 to
other forms of 15N-labelled N is required (Giller and Day
1985).
N 2 fixation has frequently been suggested as a
suitable test for pollution, particularly by heavy metals, in
soil.
Free-living heterotrophs
Rates of N 2 fixation by free-living heterotrophs in most
soils are slow and difficult to use as indicators of soil pol-
lution. Thus, Rother et al. (1982) found no relationship
Table 1 Range of potential N2 fixation in temperate soils
Organism groups a kg N fixed ha -1 year l
Free-living heterotrophic bacteria 1- 2
Cyanobacteria 5 - 30
Symbiotic associations 100 - 200
(e.g., Rhizobium-clover)
a From McGrath (1994)
 273

between acetylene reduction and heavy metal pollution, from the same experiment treated with metal-con-
even at soil metal concentrations many times in excess of taminated sewage sludge from 1942 to 1961 (contaminat-
current EC limits. ed soil) showed little surface colonization by cyano-
However, both Brookes et al. (1984) and Lorenz et al. bacteria even by day 118 of incubation, when the experi-
(1992) reported that concentrations of heavy metals close ment was terminated.
to, or less than, current maximum EC limits decreased In the uncontaminated soil there was an initial lag pe-
heterotrophic N 2 fixation by up to 90%. The reason for riod of about 14 days, then the rate of acetylene reduction
this apparent discrepancy is that in the latter, the soil wasincreased rapidly, reaching a maximum on about day 28,
incubated with glucose at up to 2000 gg g - i soil for up and declining slowly and regularly until, day 118. In con-
to 50 h before analysis. These conditions would have fa- trast, acetylene reduction had barely commenced by day
voured the developed of heterotrophic N 2 fixation and 50 in the contaminated soil. It then increased regularly
permitted a more sensitive evaluation of the effects of but much more slowly than in the uncontaminated soil,
metals on the nitrogenase enzyme system. More research and was still increasing when the experiment ended. There
is needed before this assay can be used as a standard test. was about three times more acetylene reduction in the un-
Thus, Lorenz et al. (1992) showed that some English soils, contaminated than the contaminated soil by day 118.
apparently otherwise similar to those which developed Similarly, the uncontaminated soil fixed about 10 times
measurable heterotrophic N 2 fixation, did not develop more 15N-labelled N in 24 h than did the contaminated
similar N 2 fixation activity. This difference could not be one, although no differences in soil total N content were
attributed to soil properties. They concluded that the fail- discernible between the different treatments at the end of
ure to determine standard conditions of incubation the experiment.
presently limits the usefulness of heterotrophic N 2 fixa- In a further experiment, soil was sampled at the same
tion as a biological indicator of soil pollution by heavy time and at 40-cm intervals along a gradient between the
metals. middle of an uncontaminated plot and a contaminated
plot. Concentrations of ethylenediaminetetraacetic acid
extractable Zn, Cu, Ni, and Cd increased in a curvilinear
Autotrophic N 2 fixation manner between the uncontaminated and contaminated
plots. In contrast, total acetylene reduction decreased lin-
The cyanobacteria, or blue-gree algae, contain nitro- early (Fig. 3) with increasing soil metal concentration,
genase and can grow on the soil surface. The amount of during a 60-day period. It was halved at 50 gg Zn,
N 2 that they fix in the soils of temperature ecosystems is 20 gg Cu, 2.5 gg Ni, and 3 gg total Cd. Because the soils
uncertain; estimates vary from 1 0 - 5 0 kg N ha -1 (Hen- contained all these metals, it is not possible to say which
riksson 1971; Witty et al. 1979) to nil. Field measure- metal, or metal interactions, produced the effects. How-
ments of N 2 fixation by cyanobacteria as potential mi- ever, apart from Cd, the maximum concentrations for in-
crobiological indicators of soil pollution are therefore un- dividual metals were well within individual EC maximum
likely to be profitable due to the wide variation between limits (about 30% of the soils contained Cd at more than
measurements, both temporal and spatial. For example, 3 gg Cd g - 1 soil; Fig. 3).
Witty et al. (1979) estimated (based o n C2H 2 reduction) Lorenz et al. (1992) also investigated the development
that up to 28 kg N ha -~ year -~ was fixed by cyanobac- of cyanobacteria on a gradient between an uncontaminat-
teria on soils of the Broadbalk Continuous Wheat Exper- ed and a contaminated soil from the Woburn experiment.
iment. They also reported considerable variation (up to However, instead of sampling along the middle of an un-
fivefold) in amounts of N 2 estimated to be fixed in the contaminated and a contaminated plot, as described by
same plots in different years. In contrast, when I tried to Brookes et al. (1986), they produced their gradient by
measure nitrogenase activity in situ on the soil surface of mixing the uncontaminated and contaminated soil to give
the same plots during 2 years of measurement I could de- different concentrations. They also reported inhibition of
tect nothing. the growth of blue-green algae and decreased acetylene
Despite the uncertainty in detecting N 2 fixation in the reduction in the contaminated soils, but only at the larg-
field, measurement of autotrophic N2 fixation, under est concentrations (a mixture of 83% sludge soil and 17%
standard conditions, could be a useful indicator of soil farmyard manure or in 100% sludge soil).
pollution. However, laboratory measurements reflect the The lag phase of 14 days that Lorenz et al. (1992) ob-
potential of uncontaminated and polluted soils for N 2 served for the contaminated soil was much shorter than
fixation, rather than actual fixation in the field, which is the 50 days observed by Brookes et al. (1986), and signifi-
sensitive to environmental conditions, cant differences in acetylene reduction between the least
Brookes et al. (1986) incubated moist fresh soil from (100% farmyard manure) and most metal-contaminated
the Woburn Market Garden Experiment at Rothamsted (100~ sludge) remained only until day 28, compared to
under laboratory conditions of 20~ day, 16~ night, 118 days as reported by Brookes et al. (1986).
16 h day and 50% water-holding capacity. Colonies of The major difference between the two experiments was
cyanobacteria grew rapidly on soil supplied with annual that the metal gradient of Brookes et al. (1986) was ob-
applications of farmyard manure from 1942 to 1967 (un- tained by sampling along a natural gradient between an
contaminated soil). In contrast, similarly incubated soils uncontaminated and a contaminated plot. Both were
274

300 (a) o o the light. At present, therefore, cyanobacteria appear to

250 oo y=247.6-18.5x be too sensitive to experimental conditions to provide a
~ o r=-0.78 robust indicator of heavy metal toxicity.
2O0
O 0 00

is(; 0 ~ 0 O0
Symbiotic N 2 fixation
100 00 0 0O ~
0 0 0 " 0
50 A transect o f soils across plots o f the Woburn Market
E Garden Experiment revealed a drastic decline of 50O7o or
0 t I I O ~
more in symbiotic N 2 fixation by Rhizobium legumino-
15o - (b) sarum biovar trifolii in symbiotic association with
A
t- Trifoliurn repens (white clover) in pots of soils containing
O
100 more than 334~tgZn, 99~tgCu, 27~tgNi, and
I= ~a ~ Zinc 10 ~tg Cd g-1 soil (McGrath et al. 1988). It was not possi-
r
0 ble to identify which metal, or combination of metals,
O 50
0 produced the effects. Addition of N fertilizer restored the
yields of clover on the metal-contaminated soils to those
0 ~ A A 9 1~4A9 9 9149149149 I I of clover growing in uncontaminated soil, so that the
8 7 (c) o effects were not due to direct phytotoxicity of the plants.
7
i o Rather, McGrath et al. (1988) showed that the decreased
6 [] Nickel
; ~ N 2 fixation and decreased clover yields occurred because
i
5 Dn Q 9 the nodules were not effective in fixing N2, although clo-
4 o[] o ~ n 9 ver nodulation was found in the metal-contaminated soil.
3 9 tuNa Cadmium Even when the bacteria from the ineffective nodules were
2 O~ -- OEII3--~111 isolated in the absence of heavy metals, they were still in-
~l :~ UOoO00~ i Ira-
capable of N 2 fixation on white clover. Giller et al. (1989)
0 3 6 9 12
concluded that ineffectiveness of the Rhizobium sp. in
Distance along transect (m) fixing N 2 in metal-contaminated soil was not due to
direct metal toxicity. Rather, only a single genotype of
Fig. 3 (a) Total soil N2 fixation (as C2H2 reduction) during 60 Rhizobium sp., ineffective in fixing N2, survived in the
days of incubation in soils sampled at 40-cm intervals along a metal-contaminated soil.
transect from the middle of an uncontaminated farmyard manure Although the Rhizobium-legume symbiosis is not sim-
plot to the middle of a contaminated sludge plot. (b)
Ethylenediaminetetraacetic acid (EDTA)-extractable Zn and Cu ple to work with, it could possibly be developed as a
and (e) Ni and Cd in soils sampled at 40-ca intervals along the routine indicator of soil pollution by heavy metals. The
transect. From Brookes et al. (1986). Published with permission of requirement to sample, prepare, and to measure clover dry
Pergamon Press weights, percent total N and, perhaps, ~SN-labelled N in
the plants themselves would be necessary, but could be
done.
ploughed annually. In this case the metals would have
been thoroughly dispersed throughout the soils, as more
than 30 years had elapsed between the last sludge applica-
tion and the measurements. In contrast, Lorenz et al. Adenylate energy charge ratio
(1992) produced their gradient by mixing uncontaminated
and contaminated soils in different proportions. It is like- Atkinson (1977) introduced the concept of adenylate en-
ly, therefore, that the metals would not have been so in- ergy charge (AEC):
timately distributed within the mixed soil, however care- ATP + 0.5 ADP
fully the mixing was done. Thus "islands" of uncontami- AEC = (I)
ATP + ADP + A M P
nated and metal-contaminated soil particles may well
have occurred side by side. Perhaps this is the explanation Theoretically, this charge can range from 1.0 (all ATP) to
for the differences in the results obtained in the two ex- 0.0 (all AMP). However, results from numerous laborato-
periments as all other incubation conditions were, as far ry experiments indicate that metabolically active organ-
as could be achieved, identical. isms have adenylate energy charges in the range 0.8-0.95
It may also be that cyanobacteria are very sensitive to while resting or dormant organisms have charges o f
incubation conditions in other ways. It is interesting that 0.7-0.5. Values much less than 0.4 are taken to indicate
Lorenz et aI. (t992) failed to get blue-green algae to grow a stressed or senescent population, incapable of biosyn-
even on uncontaminated Luddington soil (similar to Wo- thesis. Spores, for example, can have a charge of less than
burn in most respects). Similarly, they report that in other 0.1 (Atkinson 1977).
experiments in Sweden no cyanobacteria developed when Brookes and McGrath (1987) tested the adenylate en-
freshly sampled, uncontaminated soils were incubated in ergy charge of the soil microbial biomass to see whether

it could be used as an indicator of environmental stress
caused by heavy metals in soils from the Woburn Market
Garden Experiment. Although other microbial indices,
e.g., N 2 fixation and microbial biomass (see later), do
suggest stress-induced effects of metals on the soil micro-
flora, the charge was similar in both the uncontaminated
and contaminated soil at 0.85 and 0.89, respectively. This
suggests that the adenylate energy charge is not a valid in-
dicator of stress produced by heavy metals.
Soil enzyme activity
If enzymes are to be used as indicators, it may be impor-
tant to differentiate between exocellular and endocellular
enzymes. For example, Brookes et al. (1984) reported less
dehydrogenase activity in metal-contaminated soil than in
similar uncontaminated soil while soil phosphatase was
unaffected. Soil phosphatase can occur exocellularly as
well as within the living cell. In contrast, dehydrogenase
is active only inside intact, living cells. These results sug-
gest that dehydrogenase is a better indicator that phos-
phatase of effects of metals on soil microbial activity.
However, more recent work (Chander and Brookes
1991 a) suggests that the assay of dehydrogenase activity
itself is subject to interference from Cu in soil, because
Cu prevents the red colour development of the end prod-
uct (triphenyl formazan) from the artificial substrate
triphenyltetrazolium chloride. Thus, when Cu is added to
the soil in sludges, or in ionic form in solution, this
abiological reaction can be incorrectly interpreted as
decreased dehydrogenase activity caused by Cu. In fact,
it is simply an artefact of the method. Other common
heavy metals, e.g., Ni, Cd, Zn, do not cause this effect.
As the interference appears to be specific to Cu, dehydro-
genase activity may be of value as an indicator of other
heavy metals.
Microbial population measurements
Measuring effects of heavy metals on the microorganisms
themselves, rather than their activity, is another possible
means of monitoring soil pollution. There are two main
ways in which pollutants can act upon the microbial pop-
ulation. One is by producing direct toxic effects, i.e., kill-
ing or biochemically disabling the organisms; antibiotics
are a good example. The other way is by operating indi-
rectly, e.g., by decreasing the availability of a substrate
such as plant root exudates. Thus the decreased energy
available to the microbes could also result in a smaller
population.
There are serious problems in trying to relate in vitro
studies of heavy metals upon microorganisms to the soil
environment, and this is not considered here, unless it
provides useful background information. For example,
numerous papers refer to laboratory experiments where
heavy metals were added to microorganisms in vitro at
275
concentrations far exceeding those present in soil solu-
tion. Studies of single species or groups of microorgan-
isms in soil are also difficult because most organisms can-
not be easily isolated from soil.
With heavy metals, effects upon the microbial popula-
tion are essentially permanent, because of their toxicity
and persistence. This is illustrated by reference to effects
of heavy metals on the soil microbial biomass.
Effects of heavy metals from past applications
of sewage sludge on the soil microbial biomass
The soil microbial biomass, the living part of soil organic
matter, comprises the total mass of microorganisms that
live in soil, defined as those that have volumes of less than
5000 gm 3. Methods are now available for measuring the
biomass as a single compartment, and these have been re-
viewed elsewhere (Jenkinson and Ladd 1981; Jenkinson
1988). The fumigation-extraction procedure for measur-
ing the biomass coupled with automated analysis (Vance
et al. 1987; Wu et al. 1990) permits rapid and routine
measurements of large numbers of samples.
Biomass measurements certainly have their limitations
in soil pollution studies. Being essentially "black box"
measurements they do not permit evaluation of changes
in the community structure of the microbial population,
e.g., shifts in the fungal : bacterial ratio in soil. Neverthe-
less, they have revealed damaging effect to the microbial
population at or around current maximum EU permitted
limits of heavy metal concentrations in soil which other,
apparently more sensitive, methods did not.
Relationship between biomass C and total soil
organic C
Generally, biomass C comprises about 1-4~ of total soil
organic C (Jenkinson and Ladd 1981). Thus there is an
approximate linear relationship between these two vari-
ables, although it may vary somewhat between soils with
different physical characteristics or between soils under
different managements. For example, clay soils contain
several times more biomass than sandy soils under similar
management and climate (Jenkinson and Ladd 1981).
Also, the microbial biomass in forest and grassland soil
generally forms a larger proportion of total soil organic
matter than in arable soil (Adams and Laughlin 1981).
This accords with the statement by Jenkinson and Ladd
(1981) that situations favouring the accumulation of or-
ganic matter in soil increase both the amount of biomass
and its proportion of the total soil organic matter.
Changes in soil management cause the microbial bio-
mass to increase or decrease much faster than the total
soil organic matter content. Adams and Laughlin (1981)
reported that changing from forest or grassland to arable
management caused much greater decreases in biomass C
than total soil organic C. Similarly, Powlson et al. (1987)
276
reported that 18 years of straw incorporation in Danish
soils caused about a 4 0 - 5 0 % increase in biomass C
whereas total soil organic C only increased by 5 %. Simi-
lar results were also reported by Saffigna et al. (1989) for
Australian soils. This, and much other similar research,
supports the original idea of Powlson and Jenkinson
(1976) that the biomass is a much more sensitive indicator
of changing soil conditions than is the total soil organic
matter content, so that the biomass can serve as an early
warning of such changes long before they are detectable
in other ways.
There is accumulating evidence that heavy metals at
around, or a little in excess of, current permitted EU lim-
its also decrease the proportion of biomass C in total soil
organic matter. Thus, Brookes and McGrath (1984) re-
ported that soil from the Woburn Market Garden Experi-
ment which was hrst treated with metal-contaminated
sewage sludge more than 30 years ago (contaminated
soils) contained about half as much microbial biomass as
similar soil supplied with inorganic fertilizer or farmyard
manure (uncontaminated soils) over this period. The con-
taminated soil now contains Cu, Ni, and Zn at around
current EU limits and Cd at up to three times the limit.
The biomasses in the contaminated soils showed no corre-
lation with total soil organic C, unlike the uncontaminat-
ed soils from the same experiment. Similarly, Chander
and Brookes (1991 c) recently reported that biomass C as
a percentage of total soil organic C was twice as large
( 1 . 5 - 2 . 0 % ) in non-sludged soils or soils treated with
non-contaminated sewage sludge than in soils treated
'with sludges which were principally contaminated with
Cu or Zn ( 0 . 7 - 1 . 0 % ) in field exeperiments on Lud-
dington and Lee Valley experimental farms.
Chander and Brookes (1992) also showed that biomass
C as a percentage of total soil organic C in control soils
and in soils treated with uncontaminated sludge in an ex-
periment at Gleadthorpe ranged from 1.5 to t.6 (Fig. 4).
These values were within the range of those reported pre-
viously for other soils (see above), and are remarkably
similar to those reported by Chander and Brookes
(1991 c). The values o f this percentage in soils containing
higher concentrations of either Zn or Cu or both were
also less than half (0.4-0.7) those in control soils or in
soils treated with uncontaminated sludge (1.5-1.6).
Fig. 4 Microbial biomass C 2.0-
expressed as a percentage of 1.8-
total soil organic C in Glead- 1.6-
thorpe soils (SEM shown).
"~ 1.4-
Values given in boxes are the
total soil metal concentrations 1.2
(#g g-I soil). From Chander
and Brookes (1993). Repro- 0
.9 0.8
duced by permission of the
publisher 0 o 0.6-
iZo4- ~
o 0.2
0.0'
So far, such data have been obtained only from
carefully designed and controlled field experiments, with
full replication and containing plots that have never been
treated with sewage sludge or plots treated with uncon-
taminated sewage sludge, so tha t easy comparisons can be
made. This research should be extended to the natural en-
vironment to determine the environmental impact of
heavy metals from, for example, sewage sludge disposal,
metal mining and smelting. The collection of such data is
always beset by the difficulty of obtaining valid com-
parable analytical results from suitable uncontaminated
control soils. I suggest that the link between biomass C
and total soil organic C, discussed above, can itself consti-
tute an internal control, so that when soils deviate much
from ratios of biomass to total soil organic C that are per-
ceived as normal for the particular management, soil
type, and climate, it may be a preliminary indication that
some damage or change to the functioning of the soil eco-
system has occurred and indicate that further research
should be done. Eventually, data bases may be produced
that can provide accurate and reliable information on the
ranges of biomass, microbial activity, and organic matter
concentrations in different soil types at equilibrium under
different climatic and management regimes. If such data
bases could be produced they would be most useful in this
type of work.
So far, because no experiment in Europe has been set
up with soil contaminated with single metals at steadily
increasing concentrations, it is impossible to establish the
minimum soil metal concentrations at which effects on
the soil microbial biomass are occurring. Results reported
by Chander and Brookes (1991 c, 1992) show that both Cu
and Zn at two to four times EU limits decreased both the
amount of biomass and its proportion in soil organic
matter while similar increases in Ni had no observable ef-
fects. Similarly, Cd at twice the limit had no effect. There
was also evidence that heavy metals in sandy loam soils
affected the biomass at lower metal concentrations than
in soils with a higher clay content. These results suggest
that the most probable order of increasing metal toxicity
(in terms of current EU limits) is C u > Z n - > N i or Cd.
This, and much other evidence concerning both soil fertil-
ity aspects (Ministry of Agriculture, Food and Fisheries
and Department of Environment 1993 a) and food safety
ZZZ~
Z ZZ T iL g~ C x J
~CN
C0i,, -
r
-
~ ,~ ., N.,
~, c
ii ~ I~
: 5
F I
Control Unoontam- Ni-siudge Zn-Ni-sludge Zn-sludge Cu-sludge Zn:Cu-sludge
soil inated
sludge

and relevant health aspects (Ministry of Agriculture,
Food and Fisheries and Department of Environment
1993 b) of potentially toxic elements in sewage sludge ap-
plied to agricultural land, have recently been reviewed.
Effects of heavy metals on the specific respiration
of the biomass
It was suggested above that the relationship between bio-
mass and soil organic matter might serve as an internal
control and help overcome difficulties of interpreting
measurements made outside carefully controlled field ex-
periments. However, this idea needs further careful test-
ing before its validity can be assessed.
While measurements of soil respiration and microbial
biomass made alone can, in certain circumstances, be use-
ful indicators of environmental stress, combining the two
measurements to give amounts of CO2 evolved per unit
of biomass (biornass specific respiration) has been shown
to be a more subtle environmental indicator of stress. Be-
cause standard conditions of temperature, soil moisture,
and substrate availability are required, these measure-
ments are probably only valid under laboratory condi-
tions.
Environmental stress causes the soil microbial biomass
to direct more energy from growth into maintenance
(Killham and Firestone 1984) so that an increased propor-
tion of C taken up by the biomass will be respired as
CO> Killham (1985) therefore suggested that determina-
tion of the relative proportions of 14C-labelled glucose C
partitioned into microbial ~4C and ~4CO2 evolved might
be useful for measuring the response of the microbial bio-
mass to environmental stresses, such as pollution by
heavy metals, salinity, or changes in soil pH. Such stresses
are known to induce this response in marine microorgan-
isms (Griffiths et al. 1981). Accordingly, Killham (1985)
developed a simple bioassay procedure based on propor-
tioning ~4C-labelled glucose between biomass 14C and
14C02 evolved. He showed that for a given increase in
stress the ratio of respired ~4CO2:biomass ~4C formed
was, on average, twice at great as the magnitude of the de-
crease in either respiration alone or dehydrogenase activ-
ity.
Killham (1985) estimated the proportion of added
glucose ~4C entering the biomass indirectly because no
other method was then available. As a result of improve-
ments in methodology, biomass C can now be determined
directly in soil by fumigation-extraction during the early
decomposition of labile substrates (Ocio and Brookes
1990). This appears to be a sensitive and practical ap-
proach.
Even without any glucose amendment, biomass
specific respiration was shown to be a good indicator of
environmental stress produced by heavy metals. Thus,
Brookes and McGrath (1984) reported that rates of CO 2
evolution (gg CO2-C g-i soil) from both uncontaminat-
ed and metal-contaminated soil from the Woburn Market
277
Garden Experiment were indistinguishable when the un-
amended soils were incubated at 50% water-holding ca-
pacity and 25 ~ However, biomass specific respiration
(measured as mg C respired g-* biomass day -~) was
twice as fast in the metal-contaminated soils than in the
uncontaminated ones.
In a later experiment more total and *4C-labelled CO 2
was evolved from the contaminated than the uncontami-
nated soil (Chander and Brookes 1991b) during the first
5 days after the addition of 14C-labelled glucose and
maize (about 10 and 20%, respectively). In contrast,
about 30% less 14C-labelled biomass was synthesized per
unit of added substrate, which is in line with the findings
of Killham (1985). Similarly, Chander and Brookes
(1991d) showed that plant-derived inputs of organic ~4C
were about 20% less in the contaminated soil than the un-
contaminated soil. Also, the biomass in the contaminated
soil contained about 35% less of this 14C-labelled organ-
ic C than in the uncontaminated soil. These results indi-
cate that two mechanisms operate in causing smaller
biomasses in metal-contaminated soils. These are de-
creased C inputs from growing plants and decreased effi-
ciency of conversion of this C into new biomass C. The
latter mechanism appears to be the more important.
Applications of molecular biology to soil pollution
monitoring
Classical methods for identification of microorganisms
depend on culturing them following their isolation from
soil. More rapid techniques, such as those based upon se-
rology, have been used for decades for identifying micro-
organisms on the basis of the surface properties of their
cells. More recently, the use of monoclonal antibodies has
improved the specificity of these methods. So far, such
approaches have had only very limited applicability be-
cause of the complex and heterogeneous nature of soil.
However, attempts are now being made to combine the
use of monoclonal antibodies with other methods to iso-
late specific microbes directly from soil, e.g., antibodies
linked to magnetic beads for magnetic separation, or to
fluorochromes to facilitate flow cytometry. These might
offer great advances.
More recently, individual species and strains of micro-
organisms have both been uniquely identified by methods
exploiting differences in the nucleotide sequences of
DNA. These techniques, known colloquially as genetic
fingerprinting, have become standard in studying popula-
tion genetics. Indeed, because they are so specific, indi-
vidual organisms within a species can be identified with
certainty. They are therefore increasingly important in
crime detection, as well as playing an ever-growing role i n
microbial ecology. Using such techniques, Giller et al.
(1989) showed that isolates of Rhizobium sp. from white
clover root nodules grown on metal-contaminated soil
were virtually genetically indistinguishable whereas iso-
278
lates from similar uncontaminated soil showed the ex-
pected genetic diversity.
There are also possibilities in monitoring the survival
of introduced microorganism under field conditions.
Thus Hirsch and Spokes (1988) introduced into the field
a R h i z o b i u m sp. which had been previously selected for
chromosomally located resistance to two antibiotics fol-
lowed by insertion of a DNA sequence conferring addi-
tional antibiotic resistance. The microorganism could still
be detected 5 years later in the field on the basis of both
its antibiotic resistance markers and its unique genetic
fingerprint. Such techniques appear to have great poten-
tial for investigating microbial survival under field condi-
tions in polluted environments.
Conclusions
(1) There is no single microbiological property that is
ideal for monitoring soil pollution.
(2) A major problem exists in interpreting results, par-
ticularly because of natural fluctuations in microbial
populations and microbial activity. These fluctua-
tions are often larger than effects due to pollution.
(3) There are also problems in interpretation of environ-
mental measurements because of lack of control or
basal measurements.
(4) There appear to be big advantages in using measure-
ments that have some form of internal, control, e.g.
biomass as a percentage of soil organic matter, as it
is then easier to determine whether the functioning of
the ecosystem is being altered by factors such as
pollution.
(5) A "watching brief" should be maintained on the
newer methods of molecular biology as applied to mi-
crobial ecology. They might have considerable poten-
tial in monitoring soil pollution.
Acknowledgements I thank Dr. P. Hirsch for helpful comments on
applications of molecular biology to soil pollution monitoring and
Dr. R. Webster for useful criticism of this manuscript.
References
Adams TMcM, Laughlin RJ (1981) The effects of agronomy on the
carbon and nitrogen contained in the soil biomass. J Agric Sci
(Cambridge) 97:319-327
Anderson PJE, Domsch KH (1978) A physiological method for the
quantitative measurement of microbial biomass in soil. Soil Biol
Biochem 10:215-221
Atkinson DE (1977) Cellular energy metabolism and its regulation.
Academic Press, New York
B~dtth E (1989) Effects of heavy metals in soil on microbial process-
es and population (a review). Water Air Soil Poll 47:335-379
Brookes P, McGrath SP (1984) Effects of metal toxicity on the size
of the soil microbial biomass. J Soil Sci 35:341-346
Brookes PC, McGrath SP (1987) Adenylate energy charge in metal-
contaminated soil. Soil Biol Biochem 19:219-220
Brookes PC, Verstraete W (1989) The functioning of soil as an eco-
system. In: Soil quality assessment. State of the art report on
soil quality. Report to Commission of the European Com-
munities Directorate-General XII, Contract EV4A/0008 NL,
Brussels
Brookes PC, McGrath SP, Klein DA, Elliott ET (1984) Effects of
heavy metals on microbial activity and biomass in field soils
treated with sewage sludge. In: Environmental contamination.
CEP Consultants Ltd, Edinburgh, pp 574-583
Brookes PC, McGrath SP, Heijnen C (1986) Metal residues in soils
previously treated with sewage-sludge and their effects on
growth and nitrogen fixation by blue-green algae. Soil Biol
Biochern 18:345-353
Chander K (1991) The effects of heavy metals from past applica-
tions of sewage sludge on soil microbial biomass and microbial
activity. PhD thesis, University of Reading
Chander K, Brookes PC (1991 a) is the dehydrogenase assay invalid
as a method to estimate microbial activity in Cu-contaminated
soils. Soil Biol Biochem 23:901-915
Chander K, Brookes PC (1991 b) Microbial biomass dynamics dur-
ing the decomposition of glucose and maize in metal-contami-
nated and non-contaminated soils. Soil Biol Biochem 23:
917-925
Chander K, Brookes PC (1991 c) Effects of heavy metals from past
applications of sewage sludge on microbial biomass and organic
matter accumulation in a sandy loam and a silty loam UK soil.
Soil Biol Biochem 23:927-932
Chander K, Brookes PC (1991d) Plant inputs of carbon to metal-
contaminated soil and effects on the soil microbial biomass. Soil
Biol Biochem 23:1169-1177
Chander K, Brookes PC (1993) Effects of Zn, Cu, and Ni in sewage
sludge on microbial biomass in a sandy loam soil. Soil Biol
Biochem 25:1231 - 1239
Cook KA, Greaves MP (1987) Natural variability in microbial activ-
ities. In: Somerville L, Greaves MP (eds) Pesticide effects on soil
microflora. Taylor and Francis, London New York Philadelphia,
pp 15-43
Doelman P (1986) Resistance of soil microbial communities to
heavy metals. In: Jensen V, Kjoller A, Sorensen LH (eds) Micro-
bial communities in soil. FEMS Syrup no 33, Elsevier, Copenha-
gen London New York, pp 415-471
Domsch KH (1980) Interpretation and evaluation of data. In:
Recommended tests for assessing the side-effects of pesticides
on the soil microflora. Weed Research Organization Tech Rep no
59, Oxford, pp 6 - 8
Domsch KH, Jagnow G, Anderson TH (1983) An ecological con-
cept for the assessment of side-effects of agrochemicals on soil
micro-organisms. Res Rev 86:65-105
Duxbury T (1985) Ecological aspects of heavy metal responses in
micro-organisms. Adv Microb Ecol 8:185-235
Giller KE, Day JM (1985) Nitrogen fixation in the rhizosphere.
Significance in natural and agricultural systems. In: Fitter AH,
Atkinson D, Read DJ, Busher MB (eds) Ecological interactions
in soil, Spec Publ no 4, Br Ecol Soc. Blackwell Scientific Publi-
cations, Oxford, pp 127-147
Giller KE, McGrath SP, Hirsch PR (1989) Absence of nitrogen fixa-
tion in clover grown in soil subject to long-term contamination
with heavy metals is due to survival of only ineffective
Rhizobium. Soil Biol Biochem 21:841-848
Greaves MP, Poole N J, Domsch KH, Jagnow G, Verstraete W
(1980) Recommended tests for assessing the side-effects of pesti-
cides on the soil microflora. Tech Pep no 59, Weed Research Or-
ganization, Oxford
Griffiths RP, McNamara TM, Steven SS, Morita RY (1981) Relative
microbial activity and mineralization associated with water
masses in the Lower Cook Inlet, Alaska. J Oceanog Soc Jpn
37:227-233
Henriksson E (1971) Algal nitrogen fixation in temperate regions.
In: Biological nitrogen fixation in natural and agricultural habi-
tats. Plant and Soil, Spec Vol, pp 415-419
Hirsch PR, Spokes JR (1988) Rhizobium leguminosarum as a

model for investigating gene transfer in soil. In: Klingmt~ller W
(ed) Risk assessment for deliberate releases. Springer Verlag,
Berlin Heidelberg, pp 10-17
Jenkinson DS (1988) Determination of microbial biomass carbon
and nitrogen in soil. In: Wilson JT (ed) Advances in nitrogen
cycling in agricultural ecosystems, CAB International, Walling-
ford, pp 368-386
Jenkinson DS, Ladd JN (1981) Microbial biomass in soil: measure-
ment and turnover. In: Paul EA, Ladd JN (eds) Soil Bio-
chemistry, Vol 5. Marcel Dekker, New York, pp 415-471
Jenkinson DS, Powlson DS (1976) The effects of biocidal treat-
ments on metabolism in soil. V. A method for measuring soil
biomass. Soil Biol Biochem 8:209-213
Killham K (1985) A physiological determination of the impact of
environmental stress on the activity of microbial biomass. Envi-
ron Pollut (Ser A) 38:204-283
Killham K, Firestone M (1984) Salt stress control of intracellular
solutes in streptomycetes indigenous to saline soils. Appl Envi-
ron Microbiol 47:301-306
Lorenz SE, McGrath SP, Giller KE (1992) Assessment of free-living
nitrogen fixation activity as a biological indicator of heavy met-
al toxicity in soil. Soil Biol Biochem 24:601-606
McGrath SP (1994) Effects of heavy metals from sewage sludge on
soil microbes in agricultural ecosystems. In: Ross SM (ed) Toxic
metals in the soil-plant system. Wiley and Sons, pp 247-274
McGratb SP, Brookes PC, Giller KE (1988) Effects of potentially
toxic metals in soil derived from past applications of sewage
sludge on nitrogen fixation by Trifolium repens L. Soil BioI
Biochem 20:415-424
Ministry of Agriculture, Fisheries and Food and Department of En-
vironment (1993 a) Review of the rules for sewage-sludge appli-
cation to agricultural land. Soil fertility aspects of potentially
toxic elements. Report of the Independent Scientific Committee,
MAFF Publications, London
Ministry of Agriculture, Fisheries and Food and Department of En-
vironment (1993 b) Review of the rules for sewage-sludge appli-
cation to agricultural land. Food safety and relevant animal
health aspects of potentially toxic elements. Report of the Inde-
pendent Scientific Committee, MAFF Publications, London
279
Ocio JA, Brookes PC (1990) An evaluation of methods for measur-
ing the microbial biomass in soils following recent additions of
wheat straw and the characterization of the biomass that devel-
ops. Soil Biol Biochem 22:685-694
Powlson DS, Jenkinson DS (1976) The effects of biocidal
treatments on metabolism in soil. II. Gamma irradiation,
autoclaving, air-drying and fumigation. Soil Biol Biochem
8:179- t88
Powlson DS, Brookes PC, Christensen BT (1987) Measurement of
soil microbial biomass provides an early indication of changes
in total soil organic matter due to straw incorporation. Soil Biol
Biochem 19:159-164
Rother JA, Milbank JW, Thornton I (1982) Seasonal fluctuations
in nitrogen fixation (acetylene reduction) by free-living bacteria
in soils contaminated with cadmium, lead and zinc. J Soil Sci
33:101-113
Saffigna PG, Powlson DS, Brookes PC, Thomas GA (1989) In-
fluence of sorghum residues and tillage on soil organic matter
and soil microbial biomass in an Australian vertisol. Soil Biol
Bioehem 21:759-765
Somerville L, Greaves MP, Domsch KH, Verstraete W, Poole N J,
Van Dijk H, Anderson JPE (1987) Recommended laboratory
tests for assessing the side-effects of pesticides on the soil
microflora. In: Somerville L, Greaves MP (eds) Pesticide effects
on soil microflora. Taylor and Francis, London New York Phila-
delphia, pp 205-219
Tyler G (1981) Heavy metals in soil biology and biochemistry. In:
Paul EA, Ladd JN (eds) Soil Biochemistry, Vol 5. Marcel Dek-
ker, New York, pp 371-414
Vance ED, Brookes PC, Jenkinson DS (1987) An extraction method
for measuring microbial biomass C. Soil Biol Biochem
19:703 - 707
Witty JF, Keay PJ, Fogatt PJ, Dart PJ (1979) Algal nitrogen fixation
on temperate arable fields - the Broadbalk Experiment. Plant
and Soil 52:151-164
Wu J, Joergensen RG, Pommerening B, Chaussod R, Brookes PC
(1990) Measurement of soil microbial biomass C by fumigation-
extraction. An automated procedure. Soil Biol Biochem 22:
1167-1169