Application of Calorimetry to Microbial Biodegradation Studiesof Agrochemicals in Oxisols

Silvana A. M. Critter and Claudio Airoldi*

ABSTRACT different chemical compounds and can establish the con-sequences of their utilization. Control of the use of vari-Calorimetry was used to monitor the inhibitory effect caused byous chemicals can reduce the environmental effect andthe bipyridynium diquaternary salts paraquat, diquat, and phosphami-contribute to the understanding of the behavior of or-don on microbial activity in a Red Latosol soil (Oxisol). The thermal

effect was recorded on samples composed of 1.50 g of soil, 6.0 mg of ganic compounds in natural ecosystems. Researchersglucose, 6.0 mg of ammonium sulfate, and different masses of an have studied the importance and potential of the com-inhibitor ranging from zero to 8.00 mg, under a controlled moisture pounds, and their pharmaceutical and agricultural bene-content of 35%. Thermal effects of each pollutant on the degradation fit. On the other hand, various compounds are intro-curves of glucose in the soil were compared. Increasing amounts of duced directly to the soil environment and cause thethe inhibitor caused a decrease in the thermal effect from 22234 to contamination of waters and soils, for example, DDT21987 kJ mol21 for paraquat, 21670 to 21306 kJ mol21 for diquat,

[2,2-(p-chlorophenyl)-1,1,1, trichloroethane)]. Moreover,and 22239 to 2589 kJ mol21 for phosphamidon. The last xenobioticsome of these compounds are toxic or may be convertedagent caused a significant inhibitory effect on the microbial activityto hazardous products in nature. The effect of theseof the soil. The results of relative efficiency, h 5 DH/DH9, referringchemicals on microbial processes and the relationshipto the enthalpic value with (DH ) and without (DH9) agrochemical

in the soil, exhibited a significant correlation. From this correlation between microorganisms has received great attentionobtained for the ranges 2.00 to 8.00, 1.30 to 8.00, and 1.20 to 5.80 mg (Alexander, 1977, 1981).of the agrochemicals paraquat, diquat, and phosphamidon, respec- The importance of investigating properties of organictively, the following h values were calculated: 0.993 to 0.894, 0.668 compounds and their interactions in soil is related to theto 0.522, and 0.896 to 0.236, respectively, during the degradation of knowledge of microbial processes and environmentalglucose in the soil. The largest relative efficiency for paraquat implies contamination. Evaluations of the amount of organicthat this agrochemical can be metabolized by microbial activity.

compounds in soils can be obtained through estimationof the biotransformation and resistance to microbialattacks (Alexander, 1981).

Adiversity of pesticides are used on a vast and ex- Investigations showed that, in nature, microorganismspanding scale in modern agriculture, with the aim are responsible for converting organic and inorganic com-

of eliminating undesirable weeds, insects, and diseases. pounds through microbial metabolism and biosynthesis.Some of these compounds are directly applied to soil, Particular species of microorganisms convert many syn-but the great majority can reach plants and animals thetic organic chemicals or organic substrates to inor-(Pramer and Schmidt, 1959). ganic products, and chemical structure and environmen-

A serious concern of the agricultural community is tal conditions govern this activity. The application of athe increase of pesticide residues (Somich et al., 1990), given agrochemical to soils modifies the habitat, andbecause the application of these xenobiotics in soils can the transformation can cause immediate and future ef-cause damage to the ecosystem. Long-term action is fects on the community. The chemical structure, concen-required for a good preemergence herbicide to give a tration present, and persistence in the soil determinekind of sterilization. However, due to the fact that these the biological tolerance to toxic agents. On the othermolecules are stable and may accumulate, they may hand, the effect of a pollutant is evaluated by its inhibi-adversely influence microbial processes that are an es- tory action on cells, organisms, and microbial activity,sential part of the carbon, nitrogen, and sulfur cycles. which depends on the community present, concentra-Another aspect associated with pesticides is related to tion of compounds, and tolerance to exposure. The ef-their interactions with clay minerals. It has been demon- fect of toxic effects on the biodegradation of chemicalsstrated that many pesticides can be chemically and mi- in soil can be monitored by calorimetric techniquescrobiologically transformed in soil. Herbicides con- (Jolicoeur and Beaubien, 1986; Wadso, 1997).taining halogenated aliphatic acids are important weed

Investigation of chemical toxicology establishes rela-killers (Pramer and Schmidt, 1959; Tancho et al., 1992).tionships between chemical compounds and the biologi-Nevertheless, some pesticides are resistant to microbialcal structure of organisms. Biotransformations of theseattack (Somich et al., 1990) and many of them are af-compounds inside active cells are related with the struc-fected by adsorption–desorption processes in the soilture of these products and reagents and the propertiessurface (Bosetto et al., 1992; Sposito, 1989).associated with the microbial processes. Thermal effectsToxicology is concerned with the relationship amongcan monitor these bioreactions. The structure of the mol-ecules is associated with biological activity processes.

Instituto de Quımica, Universidade Estadual de Campinas, Caixa Thus, microcalorimetry is a suitable technique to followPostal 6154, 13083-970 Campinas, Sao Paulo, Brazil. Received 1 Nov. the biological activity. From its use, the power versus1999. *Corresponding author (airoldi@iqm.unicamp.br).

time curves obtained can clarify the behavior of differ-ent compounds, organisms, and cells. In biological sys-Published in J. Environ. Qual. 30:954–959 (2001).

954

CRITTER & AIROLDI: MICROBIAL BIODEGRADATION STUDIES OF AGROCHEMICALS 955

tems the thermal effect produced can be related to the nient alternative method that surpasses the more labori-ous classic microbial measurements, and also has thetoxic effects of the substance and, consequently, the

extent to which the microbial activity inhibition is advantage of being nondestructive.The present investigation reports the effects of agro-caused by the xenobiotic agent added to the system

(Drong et al., 1991; Kawabata et al., 1983). chemicals on the microbial processes in a tropical RedLatosol soil. The activity is estimated by calorimetricDuring the course of an experiment, the application of

an excessive dose of an agrochemical is not appropriate measurements of soil supplemented with glucose in thepresence of different amounts of agrochemicals such asbecause it causes death of the community. Thus, the

experiments are monitored by calorimetry with the ad- paraquat, diquat, and phosphamidon.dition of a dose that is metabolized by the biologicalsystem of microorganisms. EXPERIMENTAL

Nonspecific analytical techniques like calorimetryReagentshave an advantage in a broad range of applications.

This method has proven to be a suitable technique for Ammonium sulfate (Baker, Sao Paulo, Brazil), glucosemeasuring the microbial activity in complex systems, (Hoescht, Sao Paulo, Brazil), paraquat (1,19-dimethyl-4,49-and is able to monitor aerobic as well as anoxic meta- bipyridynium dichloride), diquat (1,19-ethylene-2,29-bipyri-

dynium dibromide), and phosphamidon (2-chloro-2-diethyl-bolic processes (Barja et al., 1997). Thus, for differentcarboyl-1-dimethyl-vinyl) were used. The agrochemicals werekinds of living systems measurement of thermal effectsobtained as standard solutions with 380, 200, and 445 gwas applied in soil, sludge, and waste water systems (Spar-dm23, respectively.ling, 1981, 1983). Recently, studies were focused on com-

paring soil microbial properties by calorimetry andSoil Samplesother methods (Raubuch and Beese, 1999).

Isothermal calorimetry applied to microbial processes The samples of Red Latosol soil were collected from bushvegetation on the campus of the State University of Campinas,when an agrochemical is added was found to be a usefulSao Paulo, Brazil, at a depth of 5 to 10 cm after removal ofmicrobiological technique, with a promising future. Thethe top surface layer. The soil was air-dried and sievedthermal effect involving glucose degradation provided(0.59 mm) to separate root fragments and large particles. Theinformation on the microbial activity of the soil microor-soil was stored in polyethylene bags at 293 6 5 K until theganisms that metabolize glucose (Airoldi and Critter,calorimetric experiments were conducted (Critter et al., 1994;1996; Wadso, 1997). However, this method does not Triegel, 1988).

support the growth of all the species of microorganisms To characterize this soil the contents of water and organicpresent in the soil. The classical microbial activity deter- matter, pH, total acidity, and total cation exchange capacitymination in soil directly measures carbon dioxide evolu- were determined, as reported elsewhere (Airoldi and Critter,tion (respirometry), biomass (by the amount of carbon 1997). Soil, ammonium sulfate, and glucose samples were

weighed on an analytical balance with a precision to 1024 g.or nitrogen mineralized), and plating count of microor-The peak area values were obtained by using a manual inte-ganisms growth (Anderson and Domsch, 1978; Jenkin-grator with a maximum error of 2%. Each measurement shownson and Powlson, 1976; Parkinson and Paul, 1982). Theis the mean of five individual determinations, given with asoil subsamples used in these determinations are de-confidence level of 61% (Airoldi and Critter, 1997; Barrosstroyed in each experiment and the conditions of investi-et al., 1999).gation are very different from that of the soil environ-

ment (Sposito, 1989).CalorimetryEach specific method for microbial activity measure-

ment has its limitations. Microscopic techniques involve The isothermal microcalorimeter used was sensitive in therange of 1 mW or better and was operated under isothermaldirect counting of only a minute part of the soil microor-conditions of the thermopile heat conduction type (Wadso,ganisms growing in plates. These require an expert re-1997). All thermal effects in the series of experiments weresearcher to distinguish between living and dead cells.measured in an isothermal calorimeter (LKB [Jarfalla, Swe-In addition, the quantity of a particular cell componentden] 2277) to determine variation enthalpy of the system.can vary considerably with growth conditions (Ander- Each thermal effect value was determined and analyzed from

son and Domsch, 1978). On the other hand, enzymatic the calorimetric curve by recording the power versus timeactivities require optically clear solutions, measured us- events. The calorimeter was calibrated by the release of electri-ing spectrophotometric methods (Bandick and Dick, cal energy in a resistor of the instrument, by passing a known1999). This problem can be overcome by microcalorime- electrical current to the calibration heater. These measure-

ments were applied to the calorimeter signal that occurredtry, which continuously quantifies the microbial activityover the same thermal effect range as the microbial growthin real time, with the incubation time being the sameprocess. Several kinds of tests and calibration processes suit-in the experiments and in actual soil conditions. Thisable for different types of experiments have been proposed,procedure is quicker than measuring separate compo-but to date there are no international standards of calibrationnent groups of microorganisms, and also nontransparent(Backman et al., 1994; Wadso, 1990).systems can be used (Barros et al., 1999). Therefore, the This instrument works as pairs on the differential heat leak

calorimetric method has been proven to be very sensitive principle and is operated in a constant temperature environ-toward changes in the microbial biomass induced, which ment, having semiconducting thermopile plates as a sensor.could not be detected by more conventional methods The calorimetric unit enclosed in a water thermostatic bath

has precise control over the isothermal conditions for the(Vandenhove et al., 1991). In summary, this is a conve-

956 J. ENVIRON. QUAL., VOL. 30, MAY–JUNE 2001

detection of the thermal events in the system (Backman etal., 1994). Some performance specifications are detection limit0.15 (W, baseline noise ,0.2 W) and detection sensitivitybetter than 2.0 3 1024 K over a period of several days. In allexperiments the samples were followed using sensitivity of0.30 to 1.0 W V21 of the recorder (Critter et al., 1994).

The thermal effect measurements were obtained by usingstainless steel ampoules with a capacity of 5.0 cm3, which werehermetically closed by teflon sealing discs aimed to controlevaporation yet allow oxygen and carbon dioxide transfer.The sample and reference were simultaneously lowered intoa thermostatic cylinder in two distinct units. After two interme-diary sequential periods of temperature equilibration, the am-poule was lowered into the definitive measuring position. Thereaction ampoule was used for the metabolic process and thereference ampoule for the basal activity of the soil. The ther-mal effect in each unit was detected and corresponded to thedifferential voltage signal from the thermopiles of the sampleand reference units. All determinations were performed inampoules charged with 1.50 g of soil and 0.80 cm3 of an aqueoussolution containing glucose, ammonium sulfate in a 1:1 propor-

Fig. 1. Chemical structure of the agrochemicals added to the soil:tion, and amounts of agrochemicals, which varied in the rangesparaquat (a ), diquat (b ), and phosphamidon (c ).2.00 to 8.00, 1.30 to 8.00, and 1.20 to 5.80 mg for paraquat,

diquat, and phosphamidon, respectively. The reference am-poule was used with 1.50 g of soil and 0.80 cm3 of distilled of the environmental nature of the studied soil. Thiswater (Critter et al., 1994). The thermal effect associated with technique was used here for measurements of the effectthe degradation was continuously recorded as a function of of the agrochemicals paraquat, diquat, and phosphami-time. The final value was calculated by comparing the inte- don on glucose degradation in soil with controlled mois-grated area of the power versus time curve, which corresponds ture and nutrients. The first two compounds are of re-to the thermal effect of the experiments and that were always markable potency and are being used extensively forcarried out at 298.15 6 0.02 K. Some calorimetric performance

weed control in agriculture as herbicides, whereas theand other specifications, details of the thermal effect measure-last one is being used as an acaricide. The respectivements, and experimental procedure have been previously de-structures of compounds are shown in Fig. 1.scribed (Wadso, 1990; Critter et al., 1994). All microbial activ-

ity determinations were monitored in duplicates using the Glucose and ammonium sulfate were added as en-calorimetric technique. ergy, carbon, and nitrogen sources. Both are oxidized

in the course of bioreactions, which are involved incatabolic and anabolic processes. The reaction productsRESULTS AND DISCUSSION of ammonium sulfate are nitrate and gaseous nitrogen aswell. Gaseous nitrogen is readily lost to the atmosphere.Ecological transformations are expected to occur in

microbial populations when a chemical compound is However, this process is of considerable importance inthe agricultural practice of microbial activity measure-introduced into the soil. The expected response of the

microbial action is its biodegradation. One of the fea- ment (Pramer and Schmidt, 1959).Different types of catabolism are related to distincttures of soil microflora is its diversity. Therefore, a very

large number of genera and species can be found in thermal effects. The catabolism of glucose in respirationprocesses reported in the literature as 22814 kJ mol21 inalmost any soil sample. The relative proportions of the

different groups are influenced by the environment and an aqueous environment, when the source of catabolicenergy consumed is totally oxidized (Gustafsson, 1991).by the capacity of microorganisms to adapt to a variety

of media. In this context, the microorganisms require In the present investigation, large quantities of para-quat, diquat, and phosphamidon were introduced to theenergy to maintain themselves and to carry out their

essential functions (Alexander, 1981; Gustafsson, 1991). soil. The thermal effect versus time for each amount ofagrochemical was calculated from the power versus timeCalorimetry can be used to quantify transformations

in energy that are nonspecific to a given kind of biologi- curve in each experiment. The peak time (PT) value isrelated to the maximum position in the power––timecal system. However, the success in interpreting the

experimental data will depend on combining the calori- curve and the thermal effect (DH) was calculated byintegration of the experimental calorimetric curve in ametric with other results obtained by the use of other

specific measurement techniques, such as biomass and convenient period of time. The enthalpic values in allcases of these experiments are exothermic in nature.carbonic gas evolution from glucose enriched with car-

bon-14 or enzymatic activity. The results of microbial degradation of glucose in thepresence of these pollutants are summarized in Table 1.Many experiments in calorimetry showed that for a

growing culture of a given microbe with a single energy The mentioned control listed in Table 1 is related tothe exponential calorimetric curve containing soil withsource, the amount of heat produced during growth is

proportional to the amount of the energy source con- a 35% moisture content, without additions of any agro-chemical, but loaded with assayed glucose. The calori-sumed (Beezer, 1980). This behavior is characteristic

CRITTER & AIROLDI: MICROBIAL BIODEGRADATION STUDIES OF AGROCHEMICALS 957

Table 1. The influence agrochemical mass on the degradation ofa sample of 1.50 g of Red Latosol soil, 6.0 mg of glucose, and6.0 of ammonium sulfate with 35% of moisture, showing thepeak time and the variation of enthalpy (DH ) of calorimetriccurves obtained at 298.15 6 0.02 K.

Mass Peak time 2DH

mg h kJ mol21

Control0.00 6 0.00 35.2 6 1.4 2495 6 49

Paraquat2.00 6 0.04 43.3 6 0.9 2482 6 993.00 6 0.06 46.7 6 0.9 2392 6 964.00 6 0.08 58.0 6 1.2 2312 6 926.00 6 0.12 65.9 6 1.3 2234 6 89

Diquat1.30 6 0.03 49.2 6 1.0 1670 6 672.70 6 0.05 50.0 6 1.0 1618 6 655.30 6 0.11 59.2 6 1.2 1621 6 656.70 6 0.13 72.5 6 1.2 1573 6 638.00 6 0.16 76.7 6 1.5 1306 6 57

Phosphamidon1.20 6 0.02 34.2 6 0.9 2239 6 902.30 6 0.05 46.8 6 0.9 2111 6 843.50 6 0.07 71.7 6 1.2 1265 6 524.70 6 0.09 95.0 6 1.9 1036 6 445.80 6 0.12 83.3 6 1.3 589 6 24

metric results of soil with 35% of moisture and agro-chemical, without glucose, did not show an exponentialcurve for the microbial activity in the period of time con-sidered.

The measurements for the agrochemicals paraquat,diquat, and phosphamidon, involving different massesadded varying from 1.00 to 8.00 mg per 1.50 g of soil, areshown in Table 1. Each determination was performed induplicate and the standard deviation was calculated.The enthalpic values (DH) decreased from 22234 to21987 kJ mol21 for paraquat, 21670 to 21306 kJ mol21

for diquat, and 22239 to 2539 kJ mol21 for phosphami-don, causing an inhibition of glucose degradation. Thepeak time was progressively increased, ranging from43.4 to 65.9 h for paraquat, 49.2 to 76.7 h for diquat,and 34.2 to 83.3 h for phosphamidon. These results showclearly that an increase in the mass of the agrochemicalcaused a shift of the peak of the curve toward a longerresponse time, accompanied by a strong reduction inenthalpy. This increase of the peak time occurred inresponse to the lengthy period of adaptation of themicroorganisms in this nutritional condition and in thehabitat of the soil, reflecting the difficulty in oxidizingthe organic substrate. On the other hand, a longer re-sponse of peak time reflects the change in the environ-mental condition of microbial growth.

The calorimetric curves of soil microorganisms werefound to be very dependent on the amount of agrochem-ical added, because a significant decrease in the enthal-pic values and an increase of peak time were observed.Figure 2 illustrates the enthalpic results of calorimetriccurves of degradation of glucose with the agrochemicals. Fig. 2. Variation of enthalpy with time for samples with 1.50 g of Red

Latosol soil, 6.0 mg of glucose, 6.0 mg of ammonium sulfate withThe variation in enthalpic values over the experimen-35% of moisture content, control (A), and variable amounts oftal period for all agrochemicals is shown in Fig. 2. Inparaquat (a ): 2.00 (B); 3.00 (C); 4.00 (D), and 6.00 (E) mg; diquatFig. 2, line A denotes the calorimetric curve of the control. (b ): 1.30 (B); 2.70 (C), 5.30 (D), 6.70 (E), and 8.00 (F) mg; and

An increase in the amount of agrochemical causes a phosphamidon (c ): 1.20 (B); 2.30 (C), 3.50 (D), 4.70 (E), and 5.80(F) mg at 298.15 6 0.02 K.decrease in the thermal effect, and when 6.00 mg of

958 J. ENVIRON. QUAL., VOL. 30, MAY–JUNE 2001

phosphamidon was applied to soil the largest reduction compounds, such as phosphamidon, can interact withthe cell proteins of the microorganisms and interruptof this effect was observed, as shown in Fig. 2c. However,

in this unfavorable condition for the microbial activity, the microbial activity, causing an increase the inhibitoryeffect. Then, the enthalpic values of the polarizable com-the microorganisms degraded glucose and after 200 h

the system reached a new stationary state of equilibrium, pounds paraquat and diquat showed a lower inhibitorythermal effect on microbial activity.as defined by a plateau in Fig. 2. The observed changes

in the curves show an obvious dependency on increasing The power versus time curves for bipyridynium di-quaternary salts are very similar in shape. The thermalagrochemical degradation. The largest inhibitory effect

is manifested with the soil containing phosphamidon, effect for diquat is lower than paraquat and the firstxenobiotic shows a higher inhibitory effect on the micro-with a large variation in enthalpy, as shown in Fig. 2c.

Nevertheless, the decrease in enthalpic values of the bial activity. Increasing amounts of phosphamidon ledto a remarkable decrease in the thermal effect, givingmicrobial activity implies a decrease in the number of

organisms (Barros et al., 1999). This behavior is in accor- a distinct behavior for the curve represented by Fig. 3(line C). The rapid decays in the enthalpic values implydance with the fact that the agrochemical could also be

metabolized, resulting in an adaptation of the microor- a stronger effect in the microbial population growth inthe soil.ganism when faced with a modification in the soil envi-

ronment. The interest in biodegradation of chemicals in a natu-ral environmental is growing. Calorimetry can be usedThe results of the total thermal effect after 200 h

of the experiment of each mass added for those three to study the effect of pollution on the growth of microor-ganisms in soil. In this process, the information on howorganic compounds added to the soil are shown in Fig.

3. The distinct structures of these compounds produced microbial species of soil cleave the aromatic moleculesof the paraquat and diquat, the contribution of the enzy-distinguishable differences in the microbial degradation

in the calorimetric curve. The characteristics of the com- matic activities, and the complexation factors of theagrochemicals have not been studied.pounds influenced the type and intensity of the toxic

In this investigation, we chose the optimum growtheffect. For this process the true characteristics can bewithout the pollutant to correspond to the control. Thisrelated to polarity and water solubility, both featuresis consistent with the fact that the thermal effect reflectsbeing related to the structure of the compounds. Thethe action of agrochemical on glucose degradation. Max-bipyridynium compounds diquat and paraquat are po-imum growth effect was obtained for actively growinglarizable and water soluble, generating their adsorptionmicroorganisms using a soil substrate with glucose (con-onto soils and clay surfaces (Hayes et al., 1972). Thesetrol). For this situation an increase in the relative effi-characteristics permit the motion of agrochemical inciency (h) may be expected in response to the energyan aqueous soil solution. Affinity to a cationic surfacesource. In this process the result of the enthalpic valueresults in an ion-exchange and/or adsorption process inin the Red Latosol soil was 22495 6 49 kJ mol21 (DH9)the soil. This fact can decrease the inhibitory effectof glucose catabolically consumed (Critter et al., 1994).on microbial activity. However, the less water-solubleIt would, however, be required to perform a microbialoptimization analysis for organisms, which is probablynot the normal state for microorganisms in soil environ-ments. In this condition, the relative efficiency of theenthalpic values for each dose of agrochemical appliedto microbial growth was estimated. The thermal effect

Table 2. Relative efficiency (h) of microbial activity as measuredby the calorimetry of glucose degradation of different agro-chemical mass divided by the effect without agrochemical.

Mass h

mgParaquat

2.00 6 0.04 0.9933.00 6 0.06 0.9574.00 6 0.08 0.9256.00 6 0.12 0.894

Diquat1.30 6 0.03 0.6682.70 6 0.05 0.6475.30 6 0.11 0.6486.70 6 0.13 0.6298.00 6 0.16 0.522

Phosphamidon1.20 6 0.02 0.8962.30 6 0.05 0.844Fig. 3. Variation of total enthalpy for samples with 1.50 g of Red3.50 6 0.07 0.506Latosol soil, 6.0 mg of glucose, 6.0 mg of ammonium sulfate with4.70 6 0.09 0.41435% of moisture content, and variable amounts of paraquat (A ),5.80 6 0.12 0.236diquat (B ), and phosphamidon (C ) at 298.15 6 0.02 K.

CRITTER & AIROLDI: MICROBIAL BIODEGRADATION STUDIES OF AGROCHEMICALS 959

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of pesticide waste and rinsate using ozone and biologically activeACKNOWLEDGMENTS soil. Environ. Sci. Technol. 24:745–749.Sparling, G.P. 1981. Microcalorimetry and other methods to assessThe authors are indebted to FAPESP for financial support

biomass and activity in soil. Soil Biol. Biochem. 13:93–98.and to CNPq for fellowships.Sparling, G.P. 1983. Estimation of microbial biomass and activity in

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