Enzymes in the Environment: Activity, Ecology and Applications - Chapter 16 pptx

In this review, we do not present the many, sometimes contradictory, reports of effects of different management practices on soil enzyme activities that have already been reviewed in det

16 Hydrolytic Enzyme Activities to Assess Soil Degradation and Recovery

by human-induced soil degradation, and over 6% would require major capital investment

to restore its original productivity’’ (1) It is, therefore, not surprising that, among tory authorities, there is a strong desire for the development of sensitive indicators toassess soil degradation Properties that provide a snapshot assessment of the status of asoil can determine whether a management practice has had an adverse effect on soil

regula-‘‘health’’ and productivity and, better still, can predict whether a practice will have anadverse effect if it is continued This has been one of the major drivers of the worldwide

research effort on soil quality defined as ‘‘the capacity of a soil to function, within

ecosys-tem and land-use boundaries, to sustain biological productivity, maintain environmentalquality, and promote plant and animal health’’ (1) This topic has been the subject ofnumerous reviews, such as those found in the Soil Science Society of America SpecialPublications 35 (2) and 49 (3) We do not wish to enter the debate concerning a potentialrole for enzyme activity measurements in the wide soil-quality context—this topic hasalready been reviewed (4–6)—but to focus on the application of soil enzymes to scenarios

in which soil degradation is demonstrable, or at least strongly suspected to be a likelyoutcome of a particular land-management practice

In this review, we do not present the many, sometimes contradictory, reports of effects

of different management practices on soil enzyme activities that have already been reviewed

in detail (6–14) but rather use our knowledge and perceptions of soil enzymes to try tounderstand what the enzyme activity measurements are telling us about the soil and howthey can be used to assess soil degradation and recovery The scenarios we cover are soilphysical degradation as a result of human-induced factors, such as intensive cropping andsoil compaction, and soil loss from mining In this last example, there is no need to assessdegradation at all; emphasis is on rehabilitation of the land and creation of a productive

soil when the mine is closed or moved on across the landscape We also consider soilcontamination from the dumping or accidental spillage of organic and inorganic materials,e.g., hydrocarbons and heavy metals, and the application of sewage sludge and pesticides.

In order to use soil enzyme activity measurements to provide information that will enable

us to assess the extent of soil degradation or recovery, we need to recognize the limitations

of our methodology and our knowledge of the role and function of soil enzymes.Because of the diversity of life in the soil, it is probable that most known enzymescould be found in a soil sample However, the activities that have been measured arelimited to a few oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), and lyases(EC 4) (11) It is impossible to extract a significant proportion of any enzyme activityfrom soil, unlike other living systems, and activities are therefore invariably assayed insitu It is, consequently, not possible to assign activity to individual organisms or even

to particular groups of organisms The enzyme activity measured represents the sum ofcontributions from a broad spectrum of soil organisms (including plants) and also extracel-lular or abiontic enzymes (15,16) that retain their activities away from the living cell Forenzymes that do not require cofactors and that are not components of catabolic or anabolicsequences, a significant proportion of the total activity may be extracellular and any cata-lytic function performed by these particular enzymes is purely opportunistic This doesnot mean that soil organisms are unable to take advantage of this catalysis, and it may

be that such enzymes play an important role in the initial degradation of macromolecularsubstrates in soil (17,18) The most studied group of soil enzymes that are likely to have

a significant active extracellular component are the hydrolases; it is generally accepted thatthese enzymes comprise a metabolically vital intracellular fraction and an opportunisticallyactive extracellular fraction divided among several locations in the soil (19) The propor-tional size of this extracellular component is generally unknown and probably varies fromenzyme to enzyme

Most hydrolases are investigated by using artificial substrates and assay conditionsthat are quite foreign to those prevailing in soil Substrates are usually small molecules,often simple esters combining the functional group of the substrate, e.g., phosphate (forphosphatase) or glucose (forβ-glucosidase) with a chromophore, such as p-nitrophenol,

for ease of extraction from soil and ease of assay Activity normally is measured underbuffered conditions at the optimal pH for the enzyme, at enzyme-saturating substrate con-centrations, and usually at a temperature substantially greater than would generally prevail

in soil (20) The composition and molarity of the buffer are especially important, because

a buffer found suitable for some soils is not necessarily suitable for others (21) For ple, a commonly used buffer (acetate-phosphate) for assays of invertase activity inhibitedactivity in acid grassland soils and could thereby have obscured relationships of invertasewith other soil and environmental factors (22)

exam-Under suitable assay conditions, the measured activity of an enzyme such as

phos-phatase, for example, represents only the potential p-nitrophenyl phosphate-hydrolyzing

capacity of the soil It is probable that not all of the numerous phosphatases present areassayed (all may not be active against this substrate), and it is certain that the reactionrate would be much greater than the rate of phosphate production from organic phosphoruscompounds in the unamended soil It is, therefore, difficult to see how a direct causal role

in the phosphorus fertility of a soil can be ascribed to the conglomerate of phosphatase

enzymes assayed in this way Of the hydrolases, only urease and invertase are measured byusing their natural substrates, viz., urea and sucrose, respectively However, the artificialconditions used in the assay of these enzymes again preclude any direct connection be-tween measured activity and substrate hydrolysis that occurs naturally in soil Althoughstarch and cellulose are used as substrates for amylase and cellulase, the chemical formsand purity of these substrates would be very different from those found in soil.

One enzyme that has been studied extensively because of its perceived close ship with microbial activity is the oxidoreductase dehydrogenase This enzyme, or group

relation-of enzymes, is a component relation-of the electron transport system relation-of oxygen metabolism andrequires the organization of the living intracellular environment to express its activity.Consequently, dehydrogenase activity is not likely to be present in any of the extracellularcompartments occupied by the hydrolases The absence of an extracellular componentmeans that dehydrogenase activity may not be well suited to assess soil degradation be-cause it is likely to fluctuate, as does microbial activity, in response to recent managementand/or seasonal (climatic) effects (5) Although the presence of dehydrogenase activity

in soil should reflect the activity of physiologically active microorganisms, including teria and fungi (23), measured dehydrogenase activity does not correlate consistently withmicrobial activity (6) There are several possible reasons for this, including unsuitableassay conditions, the presence of extracellular phenol oxidases, and the presence of alterna-tives to the added electron acceptor (substrate) (6) These electron acceptors may be com-mon soil constituents, such as nitrate (24) or humic acids (23) It also has been found that

bac-Cu reduces apparent dehydrogenase activity, not by inhibiting the enzyme, but by ing with the assay procedure (25) These procedural artifacts raise questions about theaccuracy of dehydrogenase activity results, especially in situations in which a managementpractice may be changing the amount of a soil component or adding a xenobiotic chemicalthat may interfere with the enzyme assay In view of these concerns, and the likely suscep-tibility of dehydrogenase activity to transitory fluctuations, we focus only on the hydrolaseenzymes in this review

interfer-Obviously, at least a component of every soil enzyme has a vital metabolic role insitu, but it is most unlikely that any indication of the role(s) or even the real activity ofthe enzyme(s) under field conditions can be gained from the assay methods used Theassertion of Skujins that ‘‘obtaining a fertility index by the use of abiontic soil enzymeactivity values seems unlikely’’ (19) applies as much today as it did over 20 years ago

It is important that these considerations be acknowledged when investigating how enzymeactivities can be used to assess soil degradation and recovery

III SOIL HYDROLASE ENZYMES TO ASSESS PHYSICAL DEGRADATION OF SOILS

Soil degradation through loss of organic matter and structural integrity is a well knownoutcome of an intensive cropping regime There have been many studies comparing thechemical, physical, and biological properties of soils subjected to conventional cultivationpractices with those subjected to minimal or no tillage When comparing conventionallyploughed and no tillage plots, Klein and Koths (26) found that urease, protease, and phos-phatase activities were higher under no tillage than under ploughed treatments Dick (27)observed the same results with acid phosphatase, arylsulfatase, invertase, amidase, andurease in the top 7.5 cm of soil and concluded that changes in activity were not attributable

cultivation for up to 80 years and found that their arylsulfatase and phosphatase activitieswere considerably reduced when compared with those in native, uncultivated soils Culti-vation decreased the enzyme activities in all aggregate size fractions of a 69-year cultivated

soil and decreased the Michaelis constant (Km) and maximum reaction rate (Vm) for fatase in all cultivated soils The authors concluded that decreased arylsulfatase activity

arylsul-in the cultivated soils reflected ‘‘the reductions arylsul-in organic matter content and microbialbiomass and activity of the soil associated with land management’’ (29) They also pro-posed that clearing and cultivation of native soils result in native soil organic matter beingtransformed into more inert forms that are less likely to form complexes with either theenzyme or its substrate; this would account for increase in substrate affinity (i.e., lower

Km) in the cultivated soils Changes in enzyme activities in different aggregate size tions under cultivation regimes also have been observed by Kandeler and associates (30).The effects of three different tillage systems on the total xylanase, invertase, and alkalinephosphatase activities of the 0- to 10-cm layer of soil and also on the proportions found

frac-in different particle size fractions are illustrated frac-inFig 1 The authors also found that thereduced tillage and, especially, the conventional tillage treatments had decreased soil or-ganic C content in the coarsest (⬎200-µm) fraction This would have been the principalreason for the greatly reduced soil xylanase activity in the conventional tillage treatment,because a large proportion of this enzyme activity was located in this coarsely texturedorganic fraction (Fig 1) The other two enzymes, invertase and alkaline phosphatase, weremore closely aligned with the finer soil fractions and were less affected by tillage, althoughthe proportions in the coarsest soil fraction also were diminished (30)

Dick et al (31) examined skid-trail soils, i.e., soils compacted by dragging logs fromforestry operations, and found that compacted soils had considerably lower phosphatase,arylsulfatase, and dehydrogenase activities than the control soil, especially in the subsoil.They also showed there was a very strong correlation between the enzyme activities andsoil organic C and microbial C They concluded that a combination of physical factorsand impaired root growth was the probable reason for these compaction effects.Sulfatase activity in arctic tundra soils also was lowered significantly after vehicledisturbance (32) The wetter, depressed portions of the vehicle tracks supported morevigorous plant growth as a result of nutrient influx caused by the channeled water flow.Sulfatase activity levels in these wet areas were considered to have become depressedbecause of end product inhibition or inhibition by other ions, e.g., phosphate

Apart from the previous example, usually the main result of these, and the manyother studies (33–35), is that soil enzyme activities decline in proportion to the loss ofsoil organic matter This tendency does not provide any more information about potentialsoil degradation under a cropping regime than does the measurement of organic C alone

or any information about the short-term productivity of the soil An intensively croppedsoil with lower enzyme activity and organic matter content than those of a neighboringnative soil may, in fact, be far more productive because it has greater nutrient status Manystudies over the years have shown that, under intensive agriculture, in which nutrients can

be added from a bag, soil enzyme activities are not good predictors of soil fertility andproductivity However, it is also generally recognized that such intensive cropping prac-tices are not sustainable in the long term and that the soils become much more prone

to erosion, waterlogging, and compaction Residue-management trials have shown thatconservation tillage and organic-residue-amendment strategies maintain soil organic mat-ter and retain soil physical characteristics (26,33,36–38) Therefore, if a soil enzyme cantell us something about the location and perhaps the quality of soil organic matter in

Figure 1 Xylanase (a), invertase (b), and alkaline phosphatase (c) activities in particle-size fractions

of the 0- to 10-cm layer of a Haplic Chernozem soil (Adapted, with permission, from Ref 30.)

cropped soils, e.g., by measurement of xylanase activity in soil particle-size fractions (30),then it may be possible to use its activity as an early warning of potential structural degra-dation Changes in enzyme kinetic properties, if they reflect changes in organic matterquality (29), also may provide more information about the status of a soil than can begained from its organic matter content

DEVELOPMENT AFTER MINING

Many studies have demonstrated the decline of organic C, microbial biomass, and enzymeactivities with increasing soil depth Ross et al (39) showed the removal of 10 cm, andespecially 20 cm, of topsoil from temperate pasture plots markedly lowered activities of

a number of enzymes This finding is not at all surprising, since the top centimetres of a soilare the major loci of biological activity and organic matter What is especially interesting,however, is that removal of 10 cm of topsoil from this pasture resulted in a new topsoilwith approximately 40% less organic C, but more than 60% lower urease and phosphatase

activities, 75% lower invertase and amylase activities, and more than 80% lower cellulaseand xylanase activities; only sulfatase matched organic C with a 40% decline in activity(39) Speir et al (40) showed that organic C declined relatively linearly with depth in apasture soil, whereas most enzyme activities and soil respiratory activity and microbialbiomass fell much more rapidly in the top 15 cm than in the remainder of the soil profile(Fig 2) Here again, sulfatase activity most closely matched the decline of organic C It

Figure 2 Influence of depth on soil chemical properties and enzyme activities (Adapted, withpermission, Ref 40.)

was concluded that the carbohydrase enzymes (amylase, cellulase, invertase, xylanase,and, to a lesser extent, xylopyranosidase) may be closely related to current soil biologicalactivity and be disproportionately higher than predicted from organic C content, in thetopmost soil layer, because of improved aeration and substrate availability (40) On theother hand, urease, phosphatase, and especially sulfatase may be more related to totalorganic C because of their stabilized, extracellular, organomineral-bound component.Technologies to recover such soils after mining and methods to assess their recoveryare equally applicable to the development of landscapes reconstructed after underground,strip, or opencast mining for coal and other mineral resources It is estimated that about

1600⫻ 109 m3of mine spoils had accumulated on the Earth’s surface up to 1980 andhad increased by about 40⫻ 109m3per year by 1998 (14) Rehabilitation of these spoilsand degraded landscapes is now an integral part of mining operations in many parts ofthe world The enzymological characteristics of these constructed or ‘‘technogenic’’ soilshave been extensively reviewed (7,11,14,41,42)

Technogenic soils may have a ‘‘topsoil’’ composed of entirely subsurface materials

or the stockpiled original topsoil or some intermediate combination Stockpiling of topsoilleads to a decline of soil biological activity (14), presumably due to the lack of replen-ishment of readily degradable plant residues and to factors such as compaction and reducedaeration Speir et al (43) found that the protease, sulfatase, and urease activities of 12soils left fallow in a pot trial declined markedly over 5 months, whereas activities generallyremained unchanged or increased if the soils were planted with perennial ryegrass Thedecline in the fallow treatments was probably attributable to declining microbial activity asplant residues were degraded, leaving only more intransigent organic matter It is probable,therefore, that the initial biological activity of the topsoil of a technogenic soil, no matterhow it is constructed, is considerably lower than that of the original soil on the site Itcertainly does not have the high biological and enzymatic activities found in the verysurface layer of an undisturbed soil (40) (Fig 2)

Dick and Tabatabai (9) concluded that ‘‘in environments initially devoid of plant

or microbial life, as is often found for drastically disturbed lands, a close correlation existsbetween plant and microbial communities and the expression of enzyme activities.’’Therefore, in the early stages of recovery of land that has had the surface soil removed(e.g., after erosion or topsoil mining), or in the early stages of development of technogenicsoils from stockpiled soil and overburden materials (e.g., land reclamation after mining),

a close relationship between plant productivity and soil enzyme activity might beexpected

Ross et al (39,44) investigated the relationship between recovery of soil biochemicalproperties and plant productivity in a temperate pasture soil that had had 10 cm or 20 cm

of topsoil removed in a trial to simulate the effects of topsoil mining The rates of recovery

of invertase, amylase, cellulase, and xylanase, but not phosphatase, sulfatase, or urease,were, after 3 years, much greater than the rate of recovery of organic C (Table 1) However,after 5 years, the recovery of all properties had slowed During the early stages of restora-tion, the enzyme activities generally correlated very closely with pasture production, but

in the longer term (5 years) the activities were more closely related to the recovery oforganic C (Table 1) The comparatively rapid recovery of invertase activity also occurred

in a temperate hill pasture (45) where the original soil had eroded in slips of up to

60-cm depth Restoration of invertase activity in regenerating pasture was complete within

11 years, whereas phosphatase activity was then only about 36% of that of unerodedtopsoil (DJ Ross, TW Speir, AW West, personal communication, 1984)

Table 1 Recovery of Organic C and Enzyme Activities, and Their Correlation with Herbage(Pasture Grasses and Clover) Production, in Soil Stripped of 20 cm of Topsoil

Percentage of control (unstripped) Correlation with herbage

soil value after production, all data up to

Source: Adapted from Refs 39 and 44.

In an investigation of different replacement strategies in the construction of genic soils after simulated lignite mining, herbage yields in all replacement treatmentsreached the level of the temperate pasture control plots within 3 years, as long as the soilwas ripped to alleviate compaction (46) Biochemical activities, including those of in-vertase and sulfatase, increased rapidly in all treatments in the early stages of the trial.Invertase activity reached the level of the control soil after 3 years, and sulfatase attainedthat level in two of the three replacement treatments after 5 years In contrast, organic Ccontent had increased linearly from 47% to 76% of that of the control at the start of thetrial to 68%–92% after 5 years The correlations of organic C and invertase and sulfataseactivities with herbage yields, using all data over the 5 years of the trial, are shown inTable 2 The levels of soil invertase activity and, to a lesser extent, sulfatase activityprovided a good indication of herbage production as restoration progressed It was con-cluded that plant materials would have contributed appreciably to the rapid increase of

techno-Table 2 Correlations of Soil Organic C andInvertase and Sulfatase Activities with PastureHerbage Yields from Technogenic SoilsConstructed Using Three Soil ReplacementStrategies, Including All Data over the 5 Years

soil invertase activity Such a rapid buildup of soil biological activity and of plant ductivity is the exception rather than the rule Most investigations have shown that theenzyme activities of technogenic soils generally were considerably lower than those ofcontrol or native soils, even after 20 or more years (11,14) It is likely that optimization

pro-of factors, such as fertilizer inputs, soil aeration, drainage, and bulk density, as well asclimate, resulted in extremely favorable conditions for soil recovery in the New Zealandstudy (46)

It is interesting to speculate why there is a strong relationship between plant tivity and soil enzyme activity in at least the early stages of development of a fertile soil.Plants and nutrients in the soil are the drivers of the recovery, as plants provide C toenable the initially sparse microbial populations to proliferate The microorganisms and,

produc-to a lesser extent, the plant fragments are the principal source of the enzymes Both cellular and extracellular enzyme concentrations increase in proportion to microbial num-bers, and the extracellular enzymes are able to become bound and stabilized at the manyunoccupied binding sites in the soil As already mentioned, it is possible that an initialbuildup of an extracellular enzyme component is vital during the early stages of microbialproliferation, because such enzymes may catalyze the commencement of degradation ofthe macromolecular plant substrates (17,18) Once these mechanisms are under way, itmight be expected that the rate of recovery of soil enzyme activity would match that ofplant productivity and be proportional to the input of plant residues If nutrients and physi-cal conditions are not limiting, plant productivity drives the process toward the levels ofbiological activity found in nearby undisturbed soils with the same parent materials andchemical properties The rate of recovery of biological and enzyme activities exceeds therate of recovery of soil organic matter content As time passes and the sites for stabilization

intra-of extracellular enzymes become saturated, their concentrations may level intra-off, and creases in enzyme activity with increasing microbial numbers and organic matter contentmay then be a function of intracellular enzymes only (microbial and plant) If the soilnutrient status and physical status are not limiting, plant productivity may still drive in-creased microbial numbers and organic matter content but may no longer be related di-rectly to total soil enzyme activity

in-In soil-recovery situations, such as those described, the enzyme activities do notnecessarily need to be assigned a role in the recovery process They are merely indicatorsthat can be used to give progress reports on the rate of recovery of plant productivity andperhaps predict how long full recovery will take Some are better indicators than others;this may be a function of the enzymes themselves or it may be specific to a site, or soil,

or particular vegetation The carbohydrase enzymes, especially those involved in thebreakdown of macromolecular plant residues (e.g., xylanase), or invertase because of itsrelationship with plant materials (47), may be better predictors than the more often assayedphosphatase, sulfatase, and urease enzymes As time progresses, the activities of this lattergroup are probably more closely related to the soil organomineral components, and their(presumably) large, stabilized, extracellular component mask more subtle changes re-sulting from increasing microbial and plant production Overall, however, we do not fullyunderstand these relationships Therefore, predictions of productivity or recovery rates indegraded or technogenic soils from the assay of a single soil enzyme, or even of severalenzymes in isolation from other soil properties, would be unwise; at this stage, a predictiverole for enzymes in soil recovery is still an experimental tool

Another approach to predicting the effects of disturbance and the success of soilrehabilitation procedures has been to use a multivariate analysis technique (48) This

method uses biological properties, including the enzymes alkaline phosphatase, sulfatase,arginine deaminase, protease, invertase, and dehydrogenase, in combination with othersoil properties and is able to discriminate between soils affected by oil well drilling, surfacemining, hydrocarbon spills, and pipeline construction, and undisturbed soils from similarareas Although the reason for the choice of these particular enzymes is not clear, a dis-criminant function combining seven properties, including alkaline phosphatase and argi-nine deaminase activities, correctly classified 86% of the undisturbed soils and 70% ofthe disturbed soils This investigation, which comprised 68 soils covering five Canadiansoil groups, appears to provide a basis for reclassifying a once-disturbed soil as havingbeen remediated sufficiently to be equivalent to an undisturbed soil.

SOIL CONTAMINATION

A Contamination by Crude Oil and Oil By-Products

Because of the huge volumes of oil and its by-products that are produced, transported,and stored, there is a very serious threat of soil contamination in the vicinity of oil fields,refineries, and storage and distribution facilities The effects of oil pollution on the activi-ties of soil enzymes have been extensively reviewed by Kiss et al (14) We thereforegive only a synopsis of the data presented in that review and limit our discussion to theinteraction of oil products with enzymes and the capacity of enzyme activity measurements

to ascertain the extent of soil degradation that has occurred

Polar organic solvents such as ethanol and acetone destroy enzyme activity by tein denaturation However, nonpolar organic compounds, such as hydrocarbons, are hy-drophobic and do not interact significantly with proteins in solution In soil, crude oil andsome of the heavier oil fractions, if present in very high concentrations, may block theexpression of enzyme activity by coating organomineral and cell surfaces and therebyprevent soluble substrates reaching the enzyme molecules It may be concluded that thelighter petroleum products are not particularly inhibitory toward soil enzymes because ofthe extensive use of toluene, at concentrations up to 25% of the assay volume (19), as amicrobial inhibitor in soil enzyme assays

pro-In the research reviewed by Kiss et al (14), large amounts of crude oil were required

to cause a significant reduction of soil enzyme activities, with concentrations as high as

100 kg m⫺2 reducing invertase, protease, and phosphatase activities by 54%, 62%, and50%, respectively (49) Although the activity of most soil enzymes is adversely affected

by crude oil, urease activity often increases (14) Different responses to crude oil werealso observed in another study (50); cellulase activity declined whereas aryl-hydrocarbonhydroxylase activity increased; a shift in catabolic activity of the soil microbiota in re-sponse to the new carbon source is indicated Important findings of Samsova et al (51)were reduction of protease activity, increase in urease activity, and death of all plants oncontamination with 8% crude oil Other studies have shown that at moderate levels ofoil contamination, some enzyme activities declined and some increased, most microbialpopulations increased, but plant growth was usually impaired (14) It would seem, there-fore, that soil enzyme activities are less sensitive than plants to soil degradation by crudeoil In some instances, however, they may provide information about the potential for thesoil microorganisms to metabolize the oil and for the contaminated soil to recover fromthe pollution

Although toluene is not particularly inhibitory to soil hydrolase activities, refinedoils can inhibit urease activity In three soils, inhibition increased in the order kerosene⬍diesel⬍ motor oil ⬍ leaded gasoline, at amendment concentrations of 5%, 10%, and 25%(w/w), but only leaded gasoline at 25% resulted in more than 50% loss of urease activity(52) Amendment of soil with jet fuel at rates of 5% and 13.5% reduced the rate of fluores-cein diacetate (FDA) hydrolysis (esterase activity) (53) However, if the soil was subjected

to a bioremediation treatment (lime, fertilizers, and simulated tillage), FDA hydrolysisincreased rapidly and markedly after a 1-week lag period The reduced activity in thenonremediated soil was attributed to inhibition by jet-fuel degradation products Inhibition

by these fuel products may be caused by the aromatic, and not the aliphatic, components

of the hydrocarbon mixtures, and possibly only by benzene (54–56)

B Contamination by Heavy Metals and Metalloids

1 Inhibitory Effects of Heavy Metals in Soil

Heavy metals are toxic to living organisms primarily because of their protein-bindingcapacity and hence their ability to inhibit enzymes The cationic metals are noncompetitiveinhibitors, which bind irreversibly with sulfydryl and carboxylate groups and with histi-dine, altering protein structure and the conformation and accessibility of the enzymes’active sites The anions of metals and metalloids, e.g., As[V], W[VI], and Mo[VI], mayhave analogous structures to products and/or inhibitors of certain enzymes and are, there-fore, likely to be competitive inhibitors For example, the inhibition of soil phosphatase

by HAsO4 ⫺, WO4 ⫺, and MoO4 ⫺has been attributed to the structural similarity of theseanions to HPO4 ⫺(or H2PO4 ⫺), the product and also an inhibitor of this enzyme’s activity(57) Similarly, these anions inhibit sulfatase because HPO4 ⫺/H2PO4 ⫺ also inhibit thisenzyme (58)

In solution, cationic metal salts are effective enzyme inhibitors at very low trations However, the many metal-amendment studies (e.g., 59–65) and field studies atcontaminated sites (e.g., 66–70) have shown that inhibition of soil enzymes usually re-quires much higher heavy-metal concentrations There are two possible explanations forthis behavior:

concen-1 The physical surroundings of the soil enzymes protect them from exposure tothe metals

2 The metals are rendered less available to the enzymes by interaction with soilconstituents

The first mechanism is possible for intracellular enzymes, via mechanisms that vent metals from passing through cell membranes Protective mechanisms for extracellularenzymes appear less likely since metal ions are smaller than most enzyme substrates.However, extracellular enzymes can be protected if the site of inhibition is remote fromthe enzyme’s active site and is inaccessible to the metal ion The second mechanism is acertainty Heavy metals interact very strongly with soil inorganic and organic constituentsthrough adsorption, chelation, and precipitation reactions that render them much less avail-able Effectively, most of the metal is ‘‘locked-up,’’ and only the small amount in solubleform at the site of enzyme activity (intracellular or extracellular) is able to interact withthe enzymes La¨hdesma¨ki and Piispanen (71), using fractionation techniques, found a verymuch greater inhibitory effect of Zn and Cu salts on protease, cellulase, and amylase

pre-activities in fractions from which the clay and humus colloids had been separated outthan in the original soil One or both of the mechanisms could account for this increasedinhibition.

The capacity of a soil to protect its enzymes from inhibition by heavy metals is,therefore, a function of its ability to lock up the metals; therefore, there should be a rela-tionship of soil texture and organic matter content with enzyme inhibition In support ofthis premise, it has been shown that heavy metals caused greater inhibition of enzymeactivities and other biochemical properties in coarse-textured soils than in fine-texturedsoils (72–78) This also can be seen in the data of Tabatabai et al (57–60) and has beenattributed to the lower surface area, lower cation exchange capacity (CEC), and generallylower organic matter content of these coarse-textured soils, all of which diminish theircapacity to reduce the solubility of metal ions (72) Inhibition of enzyme activity in heavy-metal-contaminated soil should, then, reflect the ‘‘bioavailability’’ of the metals, sincethe mechanisms that are protecting soil enzymes are likely to be the same mechanismslimiting metal uptake by plants and soil organisms Therefore, soil enzyme activity may

be considered a surrogate measurement of the impact of metals on soil biota as a whole

or of their uptake by, and their toxicity to, plants Use of an enzyme activity to assesssoil degradation by heavy metals requires no knowledge of what the enzyme is doing inthe soil; it is merely an indicator or biosensor of a more general effect

This potential ecotoxicological role for soil enzymes has been investigated in several

stud-ies to determine an ecological equivalent of LD50, viz, the ecological dose 50%, ED50, of

heavy metal in soil ED50 is defined as the concentration of a toxicant that inhibits amicrobially mediated ecological process by 50% (79)

Haanstra and associates (80) developed a ‘‘logistic response model’’ to describe theobserved sigmoidal relationship between biological activity (in this instance, respiration)

and the natural logarithm of the toxicant (Ni) concentration (Fig 3) Although ED50 termined from this model was found to be a useful measure of toxicity, it provided no

de-Figure 3 The logistic response curve and the relationship describing it Parameter Y, enzyme activity; X, natural logarithm of the heavy metal concentration; c, uninhibited enzyme activity;

b, slope parameter indicating the inhibition rate and equal to 4.39/(0.1c ⫺ 0.9c); a, logarithm of concentration at which enzyme activity is half the uninhibited level (a ⫽ 0.5c); E, stochastic error

term (With permission from Ref 74.)