Application of Calorimetry to Microbial Biodegradation Studies

of Agrochemicals in Oxisols
Silvana A. M. Critter and Claudio Airoldi*
ABSTRACT

different chemical compounds and can establish the consequences of their utilization. Control of the use of various chemicals can reduce the environmental effect and
contribute to the understanding of the behavior of organic compounds in natural ecosystems. Researchers
have studied the importance and potential of the compounds, and their pharmaceutical and agricultural benefit. On the other hand, various compounds are introduced directly to the soil environment and cause the
contamination of waters and soils, for example, DDT
[2,2-(p-chlorophenyl)-1,1,1, trichloroethane)]. Moreover,
some of these compounds are toxic or may be converted
to hazardous products in nature. The effect of these
chemicals on microbial processes and the relationship
between microorganisms has received great attention
(Alexander, 1977, 1981).
The importance of investigating properties of organic
compounds and their interactions in soil is related to the
knowledge of microbial processes and environmental
contamination. Evaluations of the amount of organic
compounds in soils can be obtained through estimation
of the biotransformation and resistance to microbial
attacks (Alexander, 1981).
Investigations showed that, in nature, microorganisms
are responsible for converting organic and inorganic compounds through microbial metabolism and biosynthesis.
Particular species of microorganisms convert many synthetic organic chemicals or organic substrates to inorganic products, and chemical structure and environmental conditions govern this activity. The application of a
given agrochemical to soils modifies the habitat, and
the transformation can cause immediate and future effects on the community. The chemical structure, concentration present, and persistence in the soil determine
the biological tolerance to toxic agents. On the other
hand, the effect of a pollutant is evaluated by its inhibitory action on cells, organisms, and microbial activity,
which depends on the community present, concentration of compounds, and tolerance to exposure. The effect of toxic effects on the biodegradation of chemicals
in soil can be monitored by calorimetric techniques
(Jolicoeur and Beaubien, 1986; Wadso, 1997).
Investigation of chemical toxicology establishes relationships between chemical compounds and the biological structure of organisms. Biotransformations of these
compounds inside active cells are related with the structure of these products and reagents and the properties
associated with the microbial processes. Thermal effects
can monitor these bioreactions. The structure of the molecules is associated with biological activity processes.
Thus, microcalorimetry is a suitable technique to follow
the biological activity. From its use, the power versus
time curves obtained can clarify the behavior of different compounds, organisms, and cells. In biological sys-

Calorimetry was used to monitor the inhibitory effect caused by

the bipyridynium diquaternary salts paraquat, diquat, and phosphamidon 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
glucose, 6.0 mg of ammonium sulfate, and different masses of an
inhibitor ranging from zero to 8.00 mg, under a controlled moisture
content of 35%. Thermal effects of each pollutant on the degradation
curves of glucose in the soil were compared. Increasing amounts of
the inhibitor caused a decrease in the thermal effect from 2234 to
1987 kJ mol1 for paraquat, 1670 to 1306 kJ mol1 for diquat,
and 2239 to 589 kJ mol1 for phosphamidon. The last xenobiotic
agent caused a significant inhibitory effect on the microbial activity
of the soil. The results of relative efficiency, H/H, referring
to the enthalpic value with (H ) and without (H) agrochemical
in the soil, exhibited a significant correlation. From this correlation
obtained for the ranges 2.00 to 8.00, 1.30 to 8.00, and 1.20 to 5.80 mg
of the agrochemicals paraquat, diquat, and phosphamidon, respectively, the following values were calculated: 0.993 to 0.894, 0.668
to 0.522, and 0.896 to 0.236, respectively, during the degradation of
glucose in the soil. The largest relative efficiency for paraquat implies
that this agrochemical can be metabolized by microbial activity.

diversity of pesticides are used on a vast and expanding scale in modern agriculture, with the aim
of eliminating undesirable weeds, insects, and diseases.
Some of these compounds are directly applied to soil,
but the great majority can reach plants and animals
(Pramer and Schmidt, 1959).
A serious concern of the agricultural community is
the increase of pesticide residues (Somich et al., 1990),
because the application of these xenobiotics in soils can
cause damage to the ecosystem. Long-term action is
required for a good preemergence herbicide to give a
kind of sterilization. However, due to the fact that these
molecules are stable and may accumulate, they may
adversely influence microbial processes that are an essential part of the carbon, nitrogen, and sulfur cycles.
Another aspect associated with pesticides is related to
their interactions with clay minerals. It has been demonstrated that many pesticides can be chemically and microbiologically transformed in soil. Herbicides containing halogenated aliphatic acids are important weed
killers (Pramer and Schmidt, 1959; Tancho et al., 1992).
Nevertheless, some pesticides are resistant to microbial
attack (Somich et al., 1990) and many of them are affected by adsorptiondesorption processes in the soil
surface (Bosetto et al., 1992; Sposito, 1989).
Toxicology is concerned with the relationship among

Instituto de Qumica, Universidade Estadual de Campinas, Caixa

Postal 6154, 13083-970 Campinas, Sao Paulo, Brazil. Received 1 Nov.
1999. *Corresponding author (airoldi@iqm.unicamp.br).
Published in J. Environ. Qual. 30:954959 (2001).

954

CRITTER & AIROLDI: MICROBIAL BIODEGRADATION STUDIES OF AGROCHEMICALS

tems the thermal effect produced can be related to the

toxic effects of the substance and, consequently, the
extent to which the microbial activity inhibition is
caused by the xenobiotic agent added to the system
(Drong et al., 1991; Kawabata et al., 1983).
During the course of an experiment, the application of
an excessive dose of an agrochemical is not appropriate
because it causes death of the community. Thus, the
experiments are monitored by calorimetry with the addition of a dose that is metabolized by the biological
system of microorganisms.
Nonspecific analytical techniques like calorimetry
have an advantage in a broad range of applications.
This method has proven to be a suitable technique for
measuring the microbial activity in complex systems,
and is able to monitor aerobic as well as anoxic metabolic processes (Barja et al., 1997). Thus, for different
kinds of living systems measurement of thermal effects
was applied in soil, sludge, and waste water systems (Sparling, 1981, 1983). Recently, studies were focused on comparing soil microbial properties by calorimetry and
other methods (Raubuch and Beese, 1999).
Isothermal calorimetry applied to microbial processes
when an agrochemical is added was found to be a useful
microbiological technique, with a promising future. The
thermal effect involving glucose degradation provided
information on the microbial activity of the soil microorganisms that metabolize glucose (Airoldi and Critter,
1996; Wadso, 1997). However, this method does not
support the growth of all the species of microorganisms
present in the soil. The classical microbial activity determination in soil directly measures carbon dioxide evolution (respirometry), biomass (by the amount of carbon
or nitrogen mineralized), and plating count of microorganisms growth (Anderson and Domsch, 1978; Jenkinson and Powlson, 1976; Parkinson and Paul, 1982). The
soil subsamples used in these determinations are destroyed in each experiment and the conditions of investigation are very different from that of the soil environment (Sposito, 1989).
Each specific method for microbial activity measurement has its limitations. Microscopic techniques involve
direct counting of only a minute part of the soil microorganisms growing in plates. These require an expert researcher to distinguish between living and dead cells.
In addition, the quantity of a particular cell component
can vary considerably with growth conditions (Anderson and Domsch, 1978). On the other hand, enzymatic
activities require optically clear solutions, measured using spectrophotometric methods (Bandick and Dick,
1999). This problem can be overcome by microcalorimetry, which continuously quantifies the microbial activity
in real time, with the incubation time being the same
in the experiments and in actual soil conditions. This
procedure is quicker than measuring separate component groups of microorganisms, and also nontransparent
systems can be used (Barros et al., 1999). Therefore, the
calorimetric method has been proven to be very sensitive
toward changes in the microbial biomass induced, which
could not be detected by more conventional methods
(Vandenhove et al., 1991). In summary, this is a conve-

955

nient alternative method that surpasses the more laborious classic microbial measurements, and also has the
advantage of being nondestructive.
The present investigation reports the effects of agrochemicals on the microbial processes in a tropical Red
Latosol soil. The activity is estimated by calorimetric
measurements of soil supplemented with glucose in the
presence of different amounts of agrochemicals such as
paraquat, diquat, and phosphamidon.
EXPERIMENTAL
Reagents
Ammonium sulfate (Baker, Sao Paulo, Brazil), glucose
(Hoescht, Sao Paulo, Brazil), paraquat (1,1-dimethyl-4,4bipyridynium dichloride), diquat (1,1-ethylene-2,2-bipyridynium dibromide), and phosphamidon (2-chloro-2-diethylcarboyl-1-dimethyl-vinyl) were used. The agrochemicals were
obtained as standard solutions with 380, 200, and 445 g
dm3, respectively.

Soil Samples
The samples of Red Latosol soil were collected from bush
vegetation on the campus of the State University of Campinas,
Sao Paulo, Brazil, at a depth of 5 to 10 cm after removal of
the top surface layer. The soil was air-dried and sieved
(0.59 mm) to separate root fragments and large particles. The
soil was stored in polyethylene bags at 293 5 K until the
calorimetric experiments were conducted (Critter et al., 1994;
Triegel, 1988).
To characterize this soil the contents of water and organic
matter, pH, total acidity, and total cation exchange capacity
were determined, as reported elsewhere (Airoldi and Critter,
1997). Soil, ammonium sulfate, and glucose samples were
weighed on an analytical balance with a precision to 104 g.
The peak area values were obtained by using a manual integrator with a maximum error of 2%. Each measurement shown
is the mean of five individual determinations, given with a
confidence level of 1% (Airoldi and Critter, 1997; Barros
et al., 1999).

Calorimetry
The isothermal microcalorimeter used was sensitive in the
range of 1 W or better and was operated under isothermal
conditions of the thermopile heat conduction type (Wadso,
1997). All thermal effects in the series of experiments were
measured in an isothermal calorimeter (LKB [Jarfalla, Sweden] 2277) to determine variation enthalpy of the system.
Each thermal effect value was determined and analyzed from
the calorimetric curve by recording the power versus time
events. The calorimeter was calibrated by the release of electrical energy in a resistor of the instrument, by passing a known
electrical current to the calibration heater. These measurements were applied to the calorimeter signal that occurred
over the same thermal effect range as the microbial growth
process. Several kinds of tests and calibration processes suitable for different types of experiments have been proposed,
but to date there are no international standards of calibration
(Backman et al., 1994; Wadso, 1990).
This instrument works as pairs on the differential heat leak
principle and is operated in a constant temperature environment, having semiconducting thermopile plates as a sensor.
The calorimetric unit enclosed in a water thermostatic bath
has precise control over the isothermal conditions for the

956

J. ENVIRON. QUAL., VOL. 30, MAYJUNE 2001

detection of the thermal events in the system (Backman et

al., 1994). Some performance specifications are detection limit
0.15 (W, baseline noise 0.2 W) and detection sensitivity
better than 2.0 104 K over a period of several days. In all
experiments the samples were followed using sensitivity of
0.30 to 1.0 W V1 of the recorder (Critter et al., 1994).
The thermal effect measurements were obtained by using
stainless steel ampoules with a capacity of 5.0 cm3, which were
hermetically closed by teflon sealing discs aimed to control
evaporation yet allow oxygen and carbon dioxide transfer.
The sample and reference were simultaneously lowered into
a thermostatic cylinder in two distinct units. After two intermediary sequential periods of temperature equilibration, the ampoule was lowered into the definitive measuring position. The
reaction ampoule was used for the metabolic process and the
reference ampoule for the basal activity of the soil. The thermal effect in each unit was detected and corresponded to the
differential voltage signal from the thermopiles of the sample
and reference units. All determinations were performed in
ampoules charged with 1.50 g of soil and 0.80 cm3 of an aqueous
solution containing glucose, ammonium sulfate in a 1:1 proportion, and amounts of agrochemicals, which varied in the ranges
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 ampoule was used with 1.50 g of soil and 0.80 cm3 of distilled
water (Critter et al., 1994). The thermal effect associated with
the degradation was continuously recorded as a function of
time. The final value was calculated by comparing the integrated area of the power versus time curve, which corresponds
to the thermal effect of the experiments and that were always
carried out at 298.15 0.02 K. Some calorimetric performance
and other specifications, details of the thermal effect measurements, and experimental procedure have been previously described (Wadso, 1990; Critter et al., 1994). All microbial activity determinations were monitored in duplicates using the
calorimetric technique.

RESULTS AND DISCUSSION

Ecological transformations are expected to occur in
microbial populations when a chemical compound is
introduced into the soil. The expected response of the
microbial action is its biodegradation. One of the features of soil microflora is its diversity. Therefore, a very
large number of genera and species can be found in
almost any soil sample. The relative proportions of the
different groups are influenced by the environment and
by the capacity of microorganisms to adapt to a variety
of media. In this context, the microorganisms require
energy to maintain themselves and to carry out their
essential functions (Alexander, 1981; Gustafsson, 1991).
Calorimetry can be used to quantify transformations
in energy that are nonspecific to a given kind of biological system. However, the success in interpreting the
experimental data will depend on combining the calorimetric with other results obtained by the use of other
specific measurement techniques, such as biomass and
carbonic gas evolution from glucose enriched with carbon-14 or enzymatic activity.
Many experiments in calorimetry showed that for a
growing culture of a given microbe with a single energy
source, the amount of heat produced during growth is
proportional to the amount of the energy source consumed (Beezer, 1980). This behavior is characteristic

Fig. 1. Chemical structure of the agrochemicals added to the soil:

paraquat (a ), diquat (b ), and phosphamidon (c ).

of the environmental nature of the studied soil. This

technique was used here for measurements of the effect
of the agrochemicals paraquat, diquat, and phosphamidon on glucose degradation in soil with controlled moisture and nutrients. The first two compounds are of remarkable potency and are being used extensively for
weed control in agriculture as herbicides, whereas the
last one is being used as an acaricide. The respective
structures of compounds are shown in Fig. 1.
Glucose and ammonium sulfate were added as energy, carbon, and nitrogen sources. Both are oxidized
in the course of bioreactions, which are involved in
catabolic and anabolic processes. The reaction products
of ammonium sulfate are nitrate and gaseous nitrogen as
well. Gaseous nitrogen is readily lost to the atmosphere.
However, this process is of considerable importance in
the agricultural practice of microbial activity measurement (Pramer and Schmidt, 1959).
Different types of catabolism are related to distinct
thermal effects. The catabolism of glucose in respiration
processes reported in the literature as 2814 kJ mol1 in
an aqueous environment, when the source of catabolic
energy consumed is totally oxidized (Gustafsson, 1991).
In the present investigation, large quantities of paraquat, diquat, and phosphamidon were introduced to the
soil. The thermal effect versus time for each amount of
agrochemical was calculated from the power versus time
curve in each experiment. The peak time (PT) value is
related to the maximum position in the powertime
curve and the thermal effect (H) was calculated by
integration of the experimental calorimetric curve in a
convenient period of time. The enthalpic values in all
cases of these experiments are exothermic in nature.
The results of microbial degradation of glucose in the
presence of these pollutants are summarized in Table 1.
The mentioned control listed in Table 1 is related to
the exponential calorimetric curve containing soil with
a 35% moisture content, without additions of any agrochemical, but loaded with assayed glucose. The calori-

CRITTER & AIROLDI: MICROBIAL BIODEGRADATION STUDIES OF AGROCHEMICALS

957

Table 1. The influence agrochemical mass on the degradation of

a sample of 1.50 g of Red Latosol soil, 6.0 mg of glucose, and
6.0 of ammonium sulfate with 35% of moisture, showing the
peak time and the variation of enthalpy (H ) of calorimetric
curves obtained at 298.15 0.02 K.
Mass
mg
0.00 0.00
2.00
3.00
4.00
6.00

0.04
0.06
0.08
0.12

1.30
2.70
5.30
6.70
8.00

0.03
0.05
0.11
0.13
0.16

1.20
2.30
3.50
4.70
5.80

0.02
0.05
0.07
0.09
0.12

Peak time

h
Control
35.2 1.4
Paraquat
43.3 0.9
46.7 0.9
58.0 1.2
65.9 1.3
Diquat
49.2 1.0
50.0 1.0
59.2 1.2
72.5 1.2
76.7 1.5
Phosphamidon
34.2 0.9
46.8 0.9
71.7 1.2
95.0 1.9
83.3 1.3

kJ mol1
2495 49
2482
2392
2312
2234

99
96
92
89

1670
1618
1621
1573
1306

67
65
65
63
57

2239
2111
1265
1036
589

90
84
52
44
24

metric results of soil with 35% of moisture and agrochemical, without glucose, did not show an exponential
curve for the microbial activity in the period of time considered.
The measurements for the agrochemicals paraquat,
diquat, and phosphamidon, involving different masses
added varying from 1.00 to 8.00 mg per 1.50 g of soil, are
shown in Table 1. Each determination was performed in
duplicate and the standard deviation was calculated.
The enthalpic values (H) decreased from 2234 to
1987 kJ mol1 for paraquat, 1670 to 1306 kJ mol1
for diquat, and 2239 to 539 kJ mol1 for phosphamidon, causing an inhibition of glucose degradation. The
peak time was progressively increased, ranging from
43.4 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 show
clearly that an increase in the mass of the agrochemical
caused a shift of the peak of the curve toward a longer
response time, accompanied by a strong reduction in
enthalpy. This increase of the peak time occurred in
response to the lengthy period of adaptation of the
microorganisms in this nutritional condition and in the
habitat of the soil, reflecting the difficulty in oxidizing
the organic substrate. On the other hand, a longer response of peak time reflects the change in the environmental condition of microbial growth.
The calorimetric curves of soil microorganisms were
found to be very dependent on the amount of agrochemical added, because a significant decrease in the enthalpic values and an increase of peak time were observed.
Figure 2 illustrates the enthalpic results of calorimetric
curves of degradation of glucose with the agrochemicals.
The variation in enthalpic values over the experimental period for all agrochemicals is shown in Fig. 2. In
Fig. 2, line A denotes the calorimetric curve of the control.
An increase in the amount of agrochemical causes a
decrease in the thermal effect, and when 6.00 mg of

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 with
35% of moisture content, control (A), and variable amounts of
paraquat (a ): 2.00 (B); 3.00 (C); 4.00 (D), and 6.00 (E) mg; diquat
(b ): 1.30 (B); 2.70 (C), 5.30 (D), 6.70 (E), and 8.00 (F) mg; and
phosphamidon (c ): 1.20 (B); 2.30 (C), 3.50 (D), 4.70 (E), and 5.80
(F) mg at 298.15 0.02 K.

958

J. ENVIRON. QUAL., VOL. 30, MAYJUNE 2001

phosphamidon was applied to soil the largest reduction

of this effect was observed, as shown in Fig. 2c. However,
in this unfavorable condition for the microbial activity,
the microorganisms degraded glucose and after 200 h
the system reached a new stationary state of equilibrium,
as defined by a plateau in Fig. 2. The observed changes
in the curves show an obvious dependency on increasing
agrochemical degradation. The largest inhibitory effect
is manifested with the soil containing phosphamidon,
with a large variation in enthalpy, as shown in Fig. 2c.
Nevertheless, the decrease in enthalpic values of the
microbial activity implies a decrease in the number of
organisms (Barros et al., 1999). This behavior is in accordance with the fact that the agrochemical could also be
metabolized, resulting in an adaptation of the microorganism when faced with a modification in the soil environment.
The results of the total thermal effect after 200 h
of the experiment of each mass added for those three
organic compounds added to the soil are shown in Fig.
3. The distinct structures of these compounds produced
distinguishable differences in the microbial degradation
in the calorimetric curve. The characteristics of the compounds influenced the type and intensity of the toxic
effect. For this process the true characteristics can be
related to polarity and water solubility, both features
being related to the structure of the compounds. The
bipyridynium compounds diquat and paraquat are polarizable and water soluble, generating their adsorption
onto soils and clay surfaces (Hayes et al., 1972). These
characteristics permit the motion of agrochemical in
an aqueous soil solution. Affinity to a cationic surface
results in an ion-exchange and/or adsorption process in
the soil. This fact can decrease the inhibitory effect
on microbial activity. However, the less water-soluble

compounds, such as phosphamidon, can interact with

the cell proteins of the microorganisms and interrupt
the microbial activity, causing an increase the inhibitory
effect. Then, the enthalpic values of the polarizable compounds paraquat and diquat showed a lower inhibitory
thermal effect on microbial activity.
The power versus time curves for bipyridynium diquaternary salts are very similar in shape. The thermal
effect for diquat is lower than paraquat and the first
xenobiotic shows a higher inhibitory effect on the microbial activity. Increasing amounts of phosphamidon led
to a remarkable decrease in the thermal effect, giving
a distinct behavior for the curve represented by Fig. 3
(line C). The rapid decays in the enthalpic values imply
a stronger effect in the microbial population growth in
the soil.
The interest in biodegradation of chemicals in a natural environmental is growing. Calorimetry can be used
to study the effect of pollution on the growth of microorganisms in soil. In this process, the information on how
microbial species of soil cleave the aromatic molecules
of the paraquat and diquat, the contribution of the enzymatic activities, and the complexation factors of the
agrochemicals have not been studied.
In this investigation, we chose the optimum growth
without the pollutant to correspond to the control. This
is consistent with the fact that the thermal effect reflects
the action of agrochemical on glucose degradation. Maximum growth effect was obtained for actively growing
microorganisms using a soil substrate with glucose (control). For this situation an increase in the relative efficiency () may be expected in response to the energy
source. In this process the result of the enthalpic value
in the Red Latosol soil was 2495 49 kJ mol1 (H)
of glucose catabolically consumed (Critter et al., 1994).
It would, however, be required to perform a microbial
optimization analysis for organisms, which is probably
not the normal state for microorganisms in soil environments. In this condition, the relative efficiency of the
enthalpic values for each dose of agrochemical applied
to microbial growth was estimated. The thermal effect
Table 2. Relative efficiency () of microbial activity as measured
by the calorimetry of glucose degradation of different agrochemical mass divided by the effect without agrochemical.

Mass
mg

Fig. 3. Variation of total enthalpy for samples with 1.50 g of Red

Latosol soil, 6.0 mg of glucose, 6.0 mg of ammonium sulfate with
35% of moisture content, and variable amounts of paraquat (A ),
diquat (B ), and phosphamidon (C ) at 298.15 0.02 K.

2.00
3.00
4.00
6.00

0.04
0.06
0.08
0.12

1.30
2.70
5.30
6.70
8.00

0.03
0.05
0.11
0.13
0.16

1.20
2.30
3.50
4.70
5.80

0.02
0.05
0.07
0.09
0.12

Paraquat
0.993
0.957
0.925
0.894
Diquat
0.668
0.647
0.648
0.629
0.522
Phosphamidon
0.896
0.844
0.506
0.414
0.236

CRITTER & AIROLDI: MICROBIAL BIODEGRADATION STUDIES OF AGROCHEMICALS

(H) obtained for the agrochemical was divided by

the thermal effect without any agrochemical (H) by
means of the relationship:
H/H
This ratio, expressed as the relative efficiency () of
glucose degradation by microbial activity in the presence of agrochemical, is shown in Table 2.
Table 2 shows distinct values of relative efficiency ()
in the degradation with the increase of the amount of
agrochemical, reflecting a distinct tolerance toward the
inhibitor. The values allowed relative measurements
to be made in microbial degradation of glucose with
agrochemicals. The paraquat, diquat, and phosphamidon assayed over a range of agrochemical additions
from 2.00 to 6.00, 1.30 to 8.00, and 1.20 to 5.80 mg,
caused a decrease in in the range of 0.993 to 0.894,
0.668 to 0.522, and 0.896 to 0.236, respectively. In such
conditions, the comparison of results showed the effects
of the agrochemicals in the soil. The decrease in the
values with the incorporation of different compounds
implies the decrease in microbial activity. Then, thermal
effects showed a significant tolerance of the populations
of microorganisms to bypiridinium compounds, within
the amounts of the agrochemicals studied.
Microorganisms may contribute to the destruction of
agrochemicals, but this cannot be considered to be the
major pathway of degradation due to the other reactions
that occur in the soil, such as adsorptiondesorption
processes of the agrochemical, redox reactions, and enzymatic activity. In this case, to determine if microorganisms play an important role in agrochemical degradation
the soil would require an autoclaved soil and the agrochemical would have to be incorporated in both autoclaved and non-autoclaved samples, providing a system
of degradation of the processes monitored by the microbiological technique.
ACKNOWLEDGMENTS
The authors are indebted to FAPESP for financial support
and to CNPq for fellowships.

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