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Biol Fertil Soils (1995) 19:269-279

9 Springer-Verlag 1995

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 N2 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

Soil microbial biomass 9Microbial activity 9Microbial specific activity 9Sewage sludge 9Atmospheric deposi- tion

words Soil pollution monitoring 9 Heavy metals

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

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 N2 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- 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 on these properties either awaited development or had not 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 paper.

Assessing the significance of microbiological changes due to pollution by heavy metals or other agents

The use of microbiological properties as indicators of soil

pollution has, in principle, much to recommend it. Mi- crobes, having both mass and activity and being in inti- mate contact with the soil microenvironment are, in many ways, ideal monitors of soil pollution. Probably the best criteria so far proposed were those of Domsch (1980) and Domsch et al. 0983). They first considered the effects of naturally occurring stresses on soil microbial populations and their activities, including fluctuations in temperature, extremes of water potential, extremes of pH, physical dis- turbances to soil (e.g., ploughing), a decreased gas ex- change, decreased supply of nutrients, and increases in in- 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 15-20 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

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Fig. 1 Relationship between monitoring period and change in bio- logical activity (deficit) in three categories describing the ecological significance of injuries. From Domsch et al. (1983). Reproduced by permission of the publisher

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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

at

this time that microbiological activity would be maximal,

was greatest when the rainfall was heavy

".

It is just

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 30-60 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 20-25 ~ 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., 40-50% water-holding ca- pacity) and temperature, (e.g., 15-25 ~ 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 100-500mgkg -1 Pb and Cr, and around 10-100 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 N2 can be performed only by specific groups of microorganisms. All biological N2 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 N2 that they can fix in tempera- te regions. Measurements of N2 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 N2 fixed. For this, measurement of conversion of 15N2 to other forms of 15N-labelled N is required (Giller and Day

1985).

N2 fixation has frequently been suggested as a suitable test for pollution, particularly by heavy metals, in soil.

Free-living heterotrophs

Rates of N2 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 (e.g., Rhizobium-clover)

100-

200

a From McGrath (1994)

between acetylene reduction and heavy metal pollution, even at soil metal concentrations many times in excess of

current EC limits. However, both Brookes et al. (1984) and Lorenz et al. (1992) reported that concentrations of heavy metals close to, or less than, current maximum EC limits decreased heterotrophic N2 fixation by up to 90%. The reason for this apparent discrepancy is that in the latter, the soil was incubated with glucose at up to 2000 gg g-i soil for up to 50 h before analysis. These conditions would have fa- voured the developed of heterotrophic N2 fixation and permitted a more sensitive evaluation of the effects of metals on the nitrogenase enzyme system. More research

is needed before this assay can be used as a standard test.

Thus, Lorenz et al. (1992) showed that some English soils, apparently otherwise similar to those which developed measurable heterotrophic N2 fixation, did not develop similar N2 fixation activity. This difference could not be attributed to soil properties. They concluded that the fail- ure to determine standard conditions of incubation

presently

limits the usefulness of heterotrophic N 2 fixa-

tion as a biological indicator of soil pollution by heavy

metals.

Autotrophic N2 fixation

The cyanobacteria, or blue-gree algae, contain nitro-

genase and can grow on the soil surface. The amount of

N 2 that they fix in the soils of temperature ecosystems is

uncertain; estimates vary from 10-50 kg N ha -1 (Hen- riksson 1971; Witty et al. 1979) to nil. Field measure- ments of N2 fixation by cyanobacteria as potential mi- crobiological indicators of soil pollution are therefore un- likely to be profitable due to the wide variation between measurements, both temporal and spatial. For example,

Witty et al. (1979) estimated (based on C2H 2 reduction) that up to 28 kg N ha -~ year -~ was fixed by cyanobac- teria on soils of the Broadbalk Continuous Wheat Exper- iment. They also reported considerable variation (up to fivefold) in amounts of N2 estimated to be fixed in the same plots in different years. In contrast, when I tried to measure nitrogenase activity in situ on the soil surface of the same plots during 2 years of measurement I could de- tect nothing. Despite the uncertainty in detecting N2 fixation in the field, measurement of autotrophic N2 fixation, under standard conditions, could be a useful indicator of soil pollution. However, laboratory measurements reflect the potential of uncontaminated and polluted soils for N 2

fixation, rather than actual fixation in the field, which is sensitive to environmental conditions, Brookes et al. (1986) incubated moist fresh soil from the Woburn Market Garden Experiment at Rothamsted under laboratory conditions of 20~ day, 16~ night,

water-holding capacity. Colonies of

cyanobacteria grew rapidly on soil supplied with annual applications of farmyard manure from 1942 to 1967 (un- contaminated soil). In contrast, similarly incubated soils

16 h

day and

50%

273

from the same experiment treated with metal-con- taminated sewage sludge from 1942 to 1961 (contaminat- ed soil) showed little surface colonization by cyano- bacteria even by day 118 of incubation, when the experi- ment was terminated. In the uncontaminated soil there was an initial lag pe- riod of about 14 days, then the rate of acetylene reduction increased rapidly, reaching a maximum on about day 28, and declining slowly and regularly until, day 118. In con- trast, acetylene reduction had barely commenced by day 50 in the contaminated soil. It then increased regularly but much more slowly than in the uncontaminated soil, and was still increasing when the experiment ended. There was about three times more acetylene reduction in the un- contaminated than the contaminated soil by day 118. Similarly, the uncontaminated soil fixed about 10 times more 15N-labelled N in 24 h than did the contaminated one, although no differences in soil total N content were discernible between the different treatments at the end of the experiment. In a further experiment, soil was sampled at the same time and at 40-cm intervals along a gradient between the middle of an uncontaminated plot and a contaminated plot. Concentrations of ethylenediaminetetraacetic acid extractable Zn, Cu, Ni, and Cd increased in a curvilinear manner between the uncontaminated and contaminated plots. In contrast, total acetylene reduction decreased lin- early (Fig. 3) with increasing soil metal concentration, during a 60-day period. It was halved at 50 gg Zn, 20 gg Cu, 2.5 gg Ni, and 3 gg total Cd. Because the soils contained all these metals, it is not possible to say which metal, or metal interactions, produced the effects. How- ever, apart from Cd, the maximum concentrations for in- dividual metals were well within individual EC maximum limits (about 30% of the soils contained Cd at more than 3 gg Cd g- 1 soil; Fig. 3). Lorenz et al. (1992) also investigated the development of cyanobacteria on a gradient between an uncontaminat- ed and a contaminated soil from the Woburn experiment. However, instead of sampling along the middle of an un- contaminated and a contaminated plot, as described by Brookes et al. (1986), they produced their gradient by mixing the uncontaminated and contaminated soil to give different concentrations. They also reported inhibition of the growth of blue-green algae and decreased acetylene reduction in the contaminated soils, but only at the larg- est concentrations (a mixture of 83% sludge soil and 17% farmyard manure or in 100% sludge soil). The lag phase of 14 days that Lorenz et al. (1992) ob- served for the contaminated soil was much shorter than the 50 days observed by Brookes et al. (1986), and signifi- cant differences in acetylene reduction between the least (100% farmyard manure) and most metal-contaminated (100~ sludge) remained only until day 28, compared to 118 days as reported by Brookes et al. (1986). The major difference between the two experiments was that the metal gradient of Brookes et al. (1986) was ob- tained by sampling along a natural gradient between an uncontaminated and a contaminated plot. Both were

274

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Fig. 3 (a) Total soil N2 fixation (as

days of incubation in soils sampled at 40-cm intervals along a transect from the middle of an uncontaminated farmyard manure plot to the middle of a contaminated sludge plot. (b) Ethylenediaminetetraacetic acid (EDTA)-extractable Zn and Cu and (e) Ni and Cd in soils sampled at 40-ca intervals along the transect. From Brookes et al. (1986). Published with permission of Pergamon Press

C2H2 reduction) during 60

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. (1992) produced their gradient by mixing uncontaminated and contaminated soils in different proportions. It is like- ly, therefore, that the metals would not have been so in- timately distributed within the mixed soil, however care- fully the mixing was done. Thus "islands" of uncontami- nated and metal-contaminated soil particles may well have occurred side by side. Perhaps this is the explanation for the differences in the results obtained in the two ex- periments as all other incubation conditions were, as far as could be achieved, identical. It may also be that cyanobacteria are very sensitive to incubation conditions in other ways. It is interesting that Lorenz et aI. (t992) failed to get blue-green algae to grow even on uncontaminated Luddington soil (similar to Wo- burn in most respects). Similarly, they report that in other experiments in Sweden no cyanobacteria developed when freshly sampled, uncontaminated soils were incubated in

the light. At present, therefore, cyanobacteria appear to

be too sensitive to experimental conditions to provide a

robust indicator of heavy metal toxicity.

Symbiotic N2 fixation

A transect of soils across plots of the Woburn Market

Garden Experiment revealed a drastic decline of 50O7oor more in symbiotic N2 fixation by Rhizobium legumino- sarum biovar trifolii in symbiotic association with Trifoliurn repens (white clover) in pots of soils containing more than 334~tgZn, 99~tgCu, 27~tgNi, and 10 ~tg Cd g-1 soil (McGrath et al. 1988). It was not possi- ble to identify which metal, or combination of metals, produced the effects. Addition of N fertilizer restored the yields of clover on the metal-contaminated soils to those of clover growing in uncontaminated soil, so that the effects were not due to direct phytotoxicity of the plants. Rather, McGrath et al. (1988) showed that the decreased N2 fixation and decreased clover yields occurred because the nodules were not effective in fixing N2, although clo- ver nodulation was found in the metal-contaminated soil. Even when the bacteria from the ineffective nodules were isolated in the absence of heavy metals, they were still in- capable of N2 fixation on white clover. Giller et al. (1989) concluded that ineffectiveness of the Rhizobium sp. in fixing N2 in metal-contaminated soil was not due to direct metal toxicity. Rather, only a single genotype of Rhizobium sp., ineffective in fixing N2, survived in the metal-contaminated soil. Although the Rhizobium-legume symbiosis is not sim- ple to work with, it could possibly be developed as a routine indicator of soil pollution by heavy metals. The requirement to sample, prepare, and to measure clover dry weights, percent total N and, perhaps, ~SN-labelled N in the plants themselves would be necessary, but could be done.

Adenylate

energy

charge

ratio

Atkinson (1977) introduced the concept of adenylate en- ergy charge (AEC):

AEC =

ATP + 0.5 ADP

ATP + ADP + AMP

(I)

Theoretically, this charge can range from 1.0 (all ATP) to

0.0

(all AMP). However, results from numerous laborato-

ry

experiments indicate that metabolically active organ-

isms have adenylate energy charges in the range 0.8-0.95 while resting or dormant organisms have charges of

0.7-0.5. Values much less than 0.4 are taken to indicate a stressed or senescent population, incapable of biosyn-

thesis. Spores, for example, can have a charge of less than

0.1 (Atkinson 1977).

Brookes and McGrath (1987) tested the adenylate en- 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.

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-

Methods are now available for measuring the

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 40-50% 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 of 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).

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 Cu>Zn->Ni 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

Fig. 4 Microbial biomass C expressed as a percentage of

total soil organic C in Glead- thorpe soils (SEM shown). Values given in boxes are the total soil metal concentrations (#g g-I soil). From Chander and Brookes (1993). Repro- duced by permission of the

publisher

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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 CO2 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 CO2 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 in 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 Rhizobium 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.

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