| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Instituto de Química, Universidade Estadual de Campinas, Caixa Postal 6154, 13083-970 Campinas, São Paulo, Brazil
Corresponding author (airoldi{at}iqm.unicamp.br)
Received for publication November 1, 1999.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONREFERENCES |
|---|
= 
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. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONREFERENCES |
|---|
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 adsorption–desorption processes in the soil surface (Bosetto et al., 1992; Sposito, 1989).
Toxicology is concerned with the relationship among 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 systems 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 convenient 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 |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONREFERENCES |
|---|
Soil Samples
The samples of Red Latosol soil were collected from bush vegetation on the campus of the State University of Campinas, São 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 10-4 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 µW 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 (Bäckman 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 detection of the thermal events in the system (Bäckman et al., 1994). Some performance specifications are detection limit 0.15 (W, baseline noise <0.2 W) and detection sensitivity better than 2.0 x 10-4 K over a period of several days. In all experiments the samples were followed using sensitivity of 0.30 to 1.0 W V-1 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 |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONREFERENCES |
|---|
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 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.
|
Different types of catabolism are related to distinct thermal effects. The catabolism of glucose in respiration processes reported in the literature as -2814 kJ mol-1 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 power––time 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 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 mol-1 for paraquat, -1670 to -1306 kJ mol-1 for diquat, and -2239 to -539 kJ mol-1 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 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 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 mol-1 (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 (H) obtained for the agrochemical was divided by the thermal effect without any agrochemical (H') by means of the relationship:
 |
This ratio, expressed as the relative efficiency () of glucose degradation by microbial activity in the presence of agrochemical, is shown in Table 2.
|
) 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 adsorption–desorption 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 |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| The SCI Journals | Agronomy Journal | Crop Science | |||
| Vadose Zone Journal | Journal of Plant Registrations | ||||
| Journal of Natural Resources and Life Sciences Education |
Soil Science Society of America Journal |
