16 Hydrolytic Enzyme Activities to Assess Soil Degradation and Recovery Tom W. Speir Institute of Environmental Science and Research, Porirua, New Zealand Des J. Ross Landcare Research, Palmerston North, New Zealand I. INTRODUCTION A project of the United Nations Environmental Program on Global Assessment of Soil Degradation concluded, ‘‘Nearly 40% of all agricultural land has been adversely affected 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 regula- tory authorities, there is a strong desire for the development of sensitive indicators to assess soil degradation. Properties that provide a snapshot assessment of the status of a soil can determine whether a management practice has had an adverse effect on soil ‘‘health’’ and productivity and, better still, can predict whether a practice will have an adverse 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 environmental quality, and promote plant and animal health’’ (1). This topic has been the subject of numerous reviews, such as those found in the Soil Science Society of America Special Publications 35 (2) and 49 (3). We do not wish to enter the debate concerning a potential role for enzyme activity measurements in the wide soil-quality context—this topic has already 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 likely outcome 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 to understand what the enzyme activity measurements are telling us about the soil and how they can be used to assess soil degradation and recovery. The scenarios we cover are soil physical degradation as a result of human-induced factors, such as intensive cropping and soil compaction, and soil loss from mining. In this last example, there is no need to assess degradation at all; emphasis is on rehabilitation of the land and creation of a productive Copyright © 2002 Marcel Dekker, Inc. soil when the mine is closed or moved on across the landscape. We also consider soil contamination 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. II. ENZYMES IN SOIL—OCCURRENCE, LOCATION, AND ASSAY 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 enzymes could be found in a soil sample. However, the activities that have been measured are limited 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 activity from soil, unlike other living systems, and activities are therefore invariably assayed in situ. 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 of contributions 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. For enzymes that do not require cofactors and that are not components of catabolic or anabolic sequences, a significant proportion of the total activity may be extracellular and any cata- lytic function performed by these particular enzymes is purely opportunistic. This does not 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 macromolecular substrates 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 that these enzymes comprise a metabolically vital intracellular fraction and an opportunistically active extracellular fraction divided among several locations in the soil (19). The propor- tional size of this extracellular component is generally unknown and probably varies from enzyme to enzyme. Most hydrolases are investigated by using artificial substrates and assay conditions that 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 (for phosphatase) or glucose (for β-glucosidase) with a chromophore, such as p-nitrophenol, for ease of extraction from soil and ease of assay. Activity normally is measured under buffered 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 exam- ple, a commonly used buffer (acetate-phosphate) for assays of invertase activity inhibited activity in acid grassland soils and could thereby have obscured relationships of invertase with other soil and environmental factors (22). 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 are assayed (all may not be active against this substrate), and it is certain that the reaction rate would be much greater than the rate of phosphate production from organic phosphorus compounds 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 Copyright © 2002 Marcel Dekker, Inc. enzymes assayed in this way. Of the hydrolases, only urease and invertase are measured by using their natural substrates, viz., urea and sucrose, respectively. However, the artificial conditions used in the assay of these enzymes again preclude any direct connection be- tween measured activity and substrate hydrolysis that occurs naturally in soil. Although starch and cellulose are used as substrates for amylase and cellulase, the chemical forms and 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 relation- ship with microbial activity is the oxidoreductase dehydrogenase. This enzyme, or group of enzymes, is a component of the electron transport system of oxygen metabolism and requires the organization of the living intracellular environment to express its activity. Consequently, dehydrogenase activity is not likely to be present in any of the extracellular compartments occupied by the hydrolases. The absence of an extracellular component means 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 management and/or seasonal (climatic) effects (5). Although the presence of dehydrogenase activity in soil should reflect the activity of physiologically active microorganisms, including bac- teria and fungi (23), measured dehydrogenase activity does not correlate consistently with microbial activity (6). There are several possible reasons for this, including unsuitable assay 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 Cu reduces apparent dehydrogenase activity, not by inhibiting the enzyme, but by interfer- ing with the assay procedure (25). These procedural artifacts raise questions about the accuracy of dehydrogenase activity results, especially in situations in which a management practice may be changing the amount of a soil component or adding a xenobiotic chemical that 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 hydrolase enzymes in this review. Obviously, at least a component of every soil enzyme has a vital metabolic role in situ, but it is most unlikely that any indication of the role(s) or even the real activity of the enzyme(s) under field conditions can be gained from the assay methods used. The assertion of Skujins that ‘‘obtaining a fertility index by the use of abiontic soil enzyme activity 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 enzyme activities 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 known outcome of an intensive cropping regime. There have been many studies comparing the chemical, physical, and biological properties of soils subjected to conventional cultivation practices with those subjected to minimal or no tillage. When comparing conventionally ploughed 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, and urease in the top 7.5 cm of soil and concluded that changes in activity were not attributable to long-term pesticide application. Gupta et al. (28,29) compared soils that had been under Copyright © 2002 Marcel Dekker, Inc. cultivationforupto80yearsandfoundthattheirarylsulfataseandphosphataseactivities wereconsiderablyreducedwhencomparedwiththoseinnative,uncultivatedsoils.Culti- vationdecreasedtheenzymeactivitiesinallaggregatesizefractionsofa69-yearcultivated soilanddecreasedtheMichaelisconstant(K m )andmaximumreactionrate(V m )forarylsul- fataseinallcultivatedsoils.Theauthorsconcludedthatdecreasedarylsulfataseactivity inthecultivatedsoilsreflected‘‘thereductionsinorganicmattercontentandmicrobial biomassandactivityofthesoilassociatedwithlandmanagement’’(29).Theyalsopro- posedthatclearingandcultivationofnativesoilsresultinnativesoilorganicmatterbeing transformedintomoreinertformsthatarelesslikelytoformcomplexeswitheitherthe enzymeoritssubstrate;thiswouldaccountforincreaseinsubstrateaffinity(i.e.,lower K m )inthecultivatedsoils.Changesinenzymeactivitiesindifferentaggregatesizefrac- tionsundercultivationregimesalsohavebeenobservedbyKandelerandassociates(30). Theeffectsofthreedifferenttillagesystemsonthetotalxylanase,invertase,andalkaline phosphataseactivitiesofthe0-to10-cmlayerofsoilandalsoontheproportionsfound indifferentparticlesizefractionsareillustratedinFig.1.Theauthorsalsofoundthatthe reduced tillage and, especially, the conventional tillage treatments had decreased soil or- ganic C content in the coarsest (Ͼ200-µm) fraction. This would have been the principal reason 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 textured organic fraction (Fig. 1). The other two enzymes, invertase and alkaline phosphatase, were more closely aligned with the finer soil fractions and were less affected by tillage, although the 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 from forestry 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 and soil organic C and microbial C. They concluded that a combination of physical factors and impaired root growth was the probable reason for these compaction effects. Sulfatase activity in arctic tundra soils also was lowered significantly after vehicle disturbance (32). The wetter, depressed portions of the vehicle tracks supported more vigorous 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 depressed because 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 many other studies (33–35), is that soil enzyme activities decline in proportion to the loss of soil organic matter. This tendency does not provide any more information about potential soil 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 cropped soil with lower enzyme activity and organic matter content than those of a neighboring native soil may, in fact, be far more productive because it has greater nutrient status. Many studies 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 and productivity. 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 that conservation 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 can tell us something about the location and perhaps the quality of soil organic matter in Copyright © 2002 Marcel Dekker, Inc. 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 matter quality (29), also may provide more information about the status of a soil than can be gained from its organic matter content. IV. SOIL HYDROLASE ENZYMES TO ASSESS SOIL RECOVERY AND DEVELOPMENT AFTER MINING Many studies have demonstrated the decline of organic C, microbial biomass, and enzyme activities with increasing soil depth. Ross et al. (39) showed the removal of 10 cm, and especially 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 soil are 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 topsoil with approximately 40% less organic C, but more than 60% lower urease and phosphatase Copyright © 2002 Marcel Dekker, Inc. activities, 75% lower invertase and amylase activities, and more than 80% lower cellulase and 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 a pasture soil, whereas most enzyme activities and soil respiratory activity and microbial biomass 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, with permission, Ref. 40.) Copyright © 2002 Marcel Dekker, Inc. wasconcludedthatthecarbohydraseenzymes(amylase,cellulase,invertase,xylanase, and,toalesserextent,xylopyranosidase)maybecloselyrelatedtocurrentsoilbiological activityandbedisproportionatelyhigherthanpredictedfromorganicCcontent,inthe topmostsoillayer,becauseofimprovedaerationandsubstrateavailability(40).Onthe otherhand,urease,phosphatase,andespeciallysulfatasemaybemorerelatedtototal organicCbecauseoftheirstabilized,extracellular,organomineral-boundcomponent. Technologiestorecoversuchsoilsafterminingandmethodstoassesstheirrecovery areequallyapplicabletothedevelopmentoflandscapesreconstructedafterunderground, strip,oropencastminingforcoalandothermineralresources.Itisestimatedthatabout 1600ϫ10 9 m 3 ofminespoilshadaccumulatedontheEarth’ssurfaceupto1980and hadincreasedbyabout40ϫ10 9 m 3 peryearby1998(14).Rehabilitationofthesespoils anddegradedlandscapesisnowanintegralpartofminingoperationsinmanypartsof theworld.Theenzymologicalcharacteristicsoftheseconstructedor‘‘technogenic’’soils havebeenextensivelyreviewed(7,11,14,41,42). Technogenicsoilsmayhavea‘‘topsoil’’composedofentirelysubsurfacematerials orthestockpiledoriginaltopsoilorsomeintermediatecombination.Stockpilingoftopsoil leadstoadeclineofsoilbiologicalactivity(14),presumablyduetothelackofreplen- ishmentofreadilydegradableplantresiduesandtofactorssuchascompactionandreduced aeration.Speiretal.(43)foundthattheprotease,sulfatase,andureaseactivitiesof12 soilsleftfallowinapottrialdeclinedmarkedlyover5months,whereasactivitiesgenerally remainedunchangedorincreasedifthesoilswereplantedwithperennialryegrass.The declineinthefallowtreatmentswasprobablyattributabletodecliningmicrobialactivityas plantresiduesweredegraded,leavingonlymoreintransigentorganicmatter.Itisprobable, therefore,thattheinitialbiologicalactivityofthetopsoilofatechnogenicsoil,nomatter howitisconstructed,isconsiderablylowerthanthatoftheoriginalsoilonthesite.It certainlydoesnothavethehighbiologicalandenzymaticactivitiesfoundinthevery surfacelayerofanundisturbedsoil(40)(Fig.2). DickandTabatabai(9)concludedthat‘‘inenvironmentsinitiallydevoidofplant ormicrobiallife,asisoftenfoundfordrasticallydisturbedlands,aclosecorrelationexists betweenplantandmicrobialcommunitiesandtheexpressionofenzymeactivities.’’ Therefore,intheearlystagesofrecoveryoflandthathashadthesurfacesoilremoved (e.g.,aftererosionortopsoilmining),orintheearlystagesofdevelopmentoftechnogenic soilsfromstockpiledsoilandoverburdenmaterials(e.g.,landreclamationaftermining), acloserelationshipbetweenplantproductivityandsoilenzymeactivitymightbe expected. Rossetal.(39,44)investigatedtherelationshipbetweenrecoveryofsoilbiochemical propertiesandplantproductivityinatemperatepasturesoilthathadhad10cmor20cm oftopsoilremovedinatrialtosimulatetheeffectsoftopsoilmining.Theratesofrecovery ofinvertase,amylase,cellulase,andxylanase,butnotphosphatase,sulfatase,orurease, were,after3years,muchgreaterthantherateofrecoveryoforganicC(Table1).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 of organic 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 uneroded topsoil (DJ Ross, TW Speir, AW West, personal communication, 1984). Copyright © 2002 Marcel Dekker, Inc. 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 Property 0.5 year 3 years 5 years 3 years 5 years Organic C 41 59 66 0.68* 0.41 Urease 16 46 61 0.79** 0.73** Invertase 29 88 88 0.92*** 0.51* Amylase 34 84 101 0.70* 0.40 Cellulase 32 84 82 0.90*** 0.63** Xylanase 12 70 80 0.91*** 0.71*** Phosphatase 40 61 76 0.90*** 0.46 Sulfatase 19 48 62 0.91*** 0.47* *, **, *** ϭ P Ͻ 0.05, 0.01, 0.001, respectively. Source: Adapted from Refs. 39 and 44. In an investigation of different replacement strategies in the construction of techno- genic soils after simulated lignite mining, herbage yields in all replacement treatments reached the level of the temperate pasture control plots within 3 years, as long as the soil was 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 attained that level in two of the three replacement treatments after 5 years. In contrast, organic C content had increased linearly from 47% to 76% of that of the control at the start of the trial to 68%–92% after 5 years. The correlations of organic C and invertase and sulfatase activities with herbage yields, using all data over the 5 years of the trial, are shown in Table 2. The levels of soil invertase activity and, to a lesser extent, sulfatase activity provided 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 Table 2 Correlations of Soil Organic C and Invertase and Sulfatase Activities with Pasture Herbage Yields from Technogenic Soils Constructed Using Three Soil Replacement Strategies, Including All Data over the 5 Years of the Trial Soil replacement treatment Property 1 a 23 Organic C 0.20 0.31 0.39 Invertase 0.59** 0.75*** 0.55** Sulfatase 0.37 0.59** 0.77*** a Treatments were 1, horizon A/B/C; 2, (A ϩ B)/C; 3, (A ϩ lignite overburden (O))/B ϩ O)/C ϩ O). **, *** ϭ P Ͻ 0.01, 0.001, respectively. Source: Adapted from Ref. 46. Copyright © 2002 Marcel Dekker, Inc. soil invertase activity. Such a rapid buildup of soil biological activity and of plant pro- ductivity is the exception rather than the rule. Most investigations have shown that the enzyme activities of technogenic soils generally were considerably lower than those of control or native soils, even after 20 or more years (11,14). It is likely that optimization of factors, such as fertilizer inputs, soil aeration, drainage, and bulk density, as well as climate, resulted in extremely favorable conditions for soil recovery in the New Zealand study (46). It is interesting to speculate why there is a strong relationship between plant produc- 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 to enable the initially sparse microbial populations to proliferate. The microorganisms and, to a lesser extent, the plant fragments are the principal source of the enzymes. Both intra- 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 many unoccupied binding sites in the soil. As already mentioned, it is possible that an initial buildup of an extracellular enzyme component is vital during the early stages of microbial proliferation, because such enzymes may catalyze the commencement of degradation of the macromolecular plant substrates (17,18). Once these mechanisms are under way, it might be expected that the rate of recovery of soil enzyme activity would match that of plant 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 of biological activity found in nearby undisturbed soils with the same parent materials and chemical properties. The rate of recovery of biological and enzyme activities exceeds the rate of recovery of soil organic matter content. As time passes and the sites for stabilization of extracellular enzymes become saturated, their concentrations may level off, and in- creases in enzyme activity with increasing microbial numbers and organic matter content may then be a function of intracellular enzymes only (microbial and plant). If the soil nutrient 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 soil-recovery situations, such as those described, the enzyme activities do not necessarily need to be assigned a role in the recovery process. They are merely indicators that can be used to give progress reports on the rate of recovery of plant productivity and perhaps 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 the breakdown of macromolecular plant residues (e.g., xylanase), or invertase because of its relationship with plant materials (47), may be better predictors than the more often assayed phosphatase, sulfatase, and urease enzymes. As time progresses, the activities of this latter group 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 fully understand these relationships. Therefore, predictions of productivity or recovery rates in degraded or technogenic soils from the assay of a single soil enzyme, or even of several enzymes in isolation from other soil properties, would be unwise; at this stage, a predictive role for enzymes in soil recovery is still an experimental tool. Another approach to predicting the effects of disturbance and the success of soil rehabilitation procedures has been to use a multivariate analysis technique (48). This Copyright © 2002 Marcel Dekker, Inc. method uses biological properties, including the enzymes alkaline phosphatase, sulfatase, arginine deaminase, protease, invertase, and dehydrogenase, in combination with other soil properties and is able to discriminate between soils affected by oil well drilling, surface mining, hydrocarbon spills, and pipeline construction, and undisturbed soils from similar areas. 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% of the disturbed soils. This investigation, which comprised 68 soils covering five Canadian soil groups, appears to provide a basis for reclassifying a once-disturbed soil as having been remediated sufficiently to be equivalent to an undisturbed soil. V. SOIL HYDROLASE ENZYMES TO ASSESS 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 therefore give only a synopsis of the data presented in that review and limit our discussion to the interaction 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 pro- 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 and some of the heavier oil fractions, if present in very high concentrations, may block the expression of enzyme activity by coating organomineral and cell surfaces and thereby prevent soluble substrates reaching the enzyme molecules. It may be concluded that the lighter petroleum products are not particularly inhibitory toward soil enzymes because of the extensive use of toluene, at concentrations up to 25% of the assay volume (19), as a microbial inhibitor in soil enzyme assays. 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 Ϫ2 reducing invertase, protease, and phosphatase activities by 54%, 62%, and 50%, 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 were also observed in another study (50); cellulase activity declined whereas aryl-hydrocarbon hydroxylase 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 on contamination with 8% crude oil. Other studies have shown that at moderate levels of oil contamination, some enzyme activities declined and some increased, most microbial populations 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 crude oil. In some instances, however, they may provide information about the potential for the soil microorganisms to metabolize the oil and for the contaminated soil to recover from the pollution. Copyright © 2002 Marcel Dekker, Inc. [...]... Dekker, Inc all constants, a, b, and c, are always positive and b Ͼ c In the second, partial-inhibition, model, the inhibitor reduces the affinity of the enzyme for its substrate but does not prevent the enzyme-catalyzed reaction As the inhibitor combines with the enzyme, inhibition increases to a definite limit beyond which increasing inhibitor concentration has no further effect Therefore, the model... irreversibly with sulfydryl and carboxylate groups and with histidine, 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], may have analogous structures to products and/ or inhibitors of certain enzymes and are, therefore, likely to be competitive inhibitors For example, the inhibition of soil phosphatase... noncompetitive (Fig 4) In both instances Figure 4 Relationship between reaction rate (v) and heavy metal concentration (i) as described by the full-(—) and partial-(––) inhibition models Parameters: c 1 and c 2 , ED 50(1) , and ED 50(2) represent uninhibited rates and ED 50 values for the full- and partial-inhibition models, respectively; c 2 a/b, minimum (asymptote) for the partial-inhibition model (With... 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-binding capacity and hence their ability to inhibit enzymes The cationic metals are noncompetitive inhibitors, which bind irreversibly... As[V], in contrast, was only a moderate inhibitor of phosphatase and sulfatase activities and was ineffective against urease (77) In almost every instance in which the inhibition data fitted both models, the second model provided the better fit, implying that the inhibition was partial 3 Interpretation of Dose–Response Data There are at least five points that need to be considered when interpreting data... depending on factors such as soil type and experimental design For example, long-term field application of both the amine and ester formulations of the herbicide 2,4-D at normal agronomic rates, in two separate studies, led to the following conclusions: ‘ The effects of long-term 2,4-D application (both ester and amine) were neither ecologically significant nor did they interfere with nutrient cycling’’... activity and soil microbial respiration were reduced substantially by the ester, indicating that the ester probably interfered with nutrient cycling’’ (103) In the former study, urease and acid- and alkaline-phosphatase activities were temporarily reduced, whereas in the latter, urease activity was permanently depressed Pesticides are not designed to inhibit soil enzymes Some may indeed be enzyme inhibitors,... attributable to acidification of the soil during preincubation before assay (Fig 5) In contrast, phosphatase activity was inhibited by metals but not by acid, except in a coarse-textured soil in which acidification did result in loss of activity below pH 4 They concluded that the difference in behavior of the two enzymes might be a function of their stability to changing pH during the preincubation period Soil... short- and long-term studies, and how the complexities of multimetal-contaminated sites can be unraveled, remain challenges Laboratoryamendment studies have a role in developing an understanding of the interactions of particular heavy metals with soil enzymes, but their results should not be overinterpreted or used in ways that might lead to erroneous or misleading conclusions Detailed data on the toxicity... attributed to the lower surface area, lower cation exchange capacity (CEC), and generally lower organic matter content of these coarse-textured soils, all of which diminish their capacity to reduce the solubility of metal ions (72) Inhibition of enzyme activity in heavymetal-contaminated soil should, then, reflect the ‘‘bioavailability’’ of the metals, since the mechanisms that are protecting soil enzymes . indicating that the ester probably interfered with nutrient cycling’’ (103). In the former study, urease and acid- and alkaline-phosphatase activities were tem- porarily reduced, whereas in the. 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,. of their protein-binding capacity and hence their ability to inhibit enzymes. The cationic metals are noncompetitive inhibitors, which bind irreversibly with sulfydryl and carboxylate groups and