Chemosphere 45 (2001) 417±425
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Pesticide in¯uence on soil enzymatic activities F. Sannino, L. Gianfreda
*
Dipartimento di Scienze Chimico-Agrarie, Universit a di Napoli ``Federico II'', Via Universita 100, 80055 Portici, Napoli, Italy Received 9 November 2000; accepted 23 January 2001
Abstract The in¯uence of four pesticides, e.g. glyphosate, paraquat, atrazine, and carbaryl, on the activities of invertase, urease and phosphatase of twenty-two soils, numbered as 1±22, was investigated. Soils displayed a general variability of enzyme activities with invertase being more abundant than urease and phosphatase in the order listed. The addition of glyphosate and paraquat activated invertase and urease activities in several soils. Increments of invertase activity ranged from a very low increase (+4%) up to +204% in soils 11 and 14, respectively. Smaller increases were measured for urease. A general inhibitory eect (from 5% to 98%) was observed for phosphatase in the presence of glyphosate. The eects of atrazine and carbaryl on the three soil enzymes were evaluated against that exhibited by methanol, the solvent used for their solubilization. In almost all soils, atrazine further inhibited invertase activity with respect to the inhibitory eect shown by methanol. By contrast, consistent activation eects (from 61% to 10217%) were measured for urease with methanol alone and/or methanol-pesticide mixtures. Contradictory results were observed with phosphatase. Similarities found between the results obtained with enzymes in soils and those measured with synthetic enzyme complexes (e.g. free enzymes and/or clay±, organo±, and organo±clay±enzyme complexes) exposed to the same pesticides allowed some relationships between responses of soil enzymes to pesticides and soil properties to be hypothesized. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Soil enzymes; Pesticides; Clay±enzyme; Organo±enzyme; Organo±mineral complexes
1. Introduction As reviewed by Gianfreda and Bollag (1996), natural and anthropogenic factors may aect directly or indirectly the activities of enzymes in soil. Among anthropogenic factors, pesticides are of primary importance due to their continuous entry into soil environment. Pesticides may enter the soil either by direct applications (e.g. agricultural practices) or indirect applications (e.g. accidental spillage, leaks at pesticide dump sites, discharge of wastes from production facilities, or urban pollution).
*
Corresponding author. Tel.: +39-081-7885225; fax: +39081-7755130. E-mail address:
[email protected] (L. Gianfreda).
In soil, enzymes contribute to the total biological activity of the soil-plant environment under dierent states (Dick, 1995; Dick, 1997). Their catalytic eciency may be strongly in¯uenced by the composition of the surrounding in which they act as catalysts. Pesticides, usually extraneous to soil component pools, are expected to aect the behavior of enzymes (Bollag and Liu, 1990). Several investigations were devoted to study the eect of various pesticides on the activity of enzymes in soils from dierent origins. Despite the numerous reports on this topic, thoroughly reviewed and summarized by Shaer (1993), and the eorts made to ®nd reliable relationships among measured eects and properties of soils, chemical characteristics of pesticides, and/or classes of enzymes, no general conclusions were drawn. As also underlined by Shaer (1993), contradictory and somewhat confusing results are often reported about the
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action of one pesticide on the activity of a certain enzyme. Gianfreda et al. (1994, 1995) addressed this topic with a dierent methodological approach. Synthetic enzymatic systems which simulate enzymes free in soil solution and enzyme±soil colloid associations were considered and the eects of some pesticides on the activity of these enzymatic systems were studied. Then, when possible, a comparison with the results obtained on whole soils was made. The number of soils examined, however, was limited and the comparison sometimes failed to provide some general conclusions (Gianfreda et al., 1994, 1995). The scope of this work is to extend the study to a larger number of soils. The in¯uence of four pesticides, glyphosate, paraquat, atrazine, and carbaryl, characterized by dierent physico-chemical properties, on the activities of invertase, urease, and phosphatase of a great number of soils, is investigated. The results are then compared with those obtained with synthetic enzymatic systems and attempts to draw general conclusions are made. Invertase, urease, and phosphatase have been selected because of their importance in the carbon-, nitrogen-, and phosphorus-soil cycles, respectively. Soils are from dierent origins and have dierent physical and physico-chemical properties. 2. Materials and methods 2.1. Soils Soils from dierent sites of Campania (Italy), indicated as samples 1±22, were surface samples (0±20 cm), selected to give a wide range of chemical and physical properties (Table 1). Soils were sieved (0.5 mm) and stored at 4°C prior to analysis. pH was determined in a distilled water soil suspension (1-to-4 ratio), other physico-chemical properties were determined according to Nelson and Sommers (1982) and Societa Italiana della Scienza del Suolo (Violante, 2000).
(1994, 1995) and Tabatabai and Bremner (1969), respectively. Usually, 1 g of fresh soil was mixed with 5 ml of buered substrate solution in reaction ¯asks and incubated for 1 h at 30°C under continuous stirring. The following substrate concentrations and buers were used: invertase, 0.1 M saccharose in 0.1 M Na-acetate buer pH 4.65; urease, 0.2 M urea in 0.1 M Na-phosphate buer-0.001M EDTA at pH 7.0; phosphatase, 0.02 M p-nitrophenylphosphate in 0.1 M modi®ed universal buer (MUB) at pH 6.0. After incubation, the mixtures were suddenly kept in a freezer for 10 min to stop the enzymatic reaction. Urease and phosphatase samples were previously charged with 10 ml of 2 M KCl and 4 ml 0.5 M NaOH + 1 ml 0.5 M CaCl2 , respectively. After centrifugation at 3500 g for 10 min, the reaction products were determined in the supernatants. The concentrations of sugars and NH4 ions produced by saccharose and urea hydrolysis were determined by the Nelson±Somoji reagent (Nelson, 1944) and the hypochlorite-alkaline phenol method (Charney and Myrbach, 1962), respectively. Alkalization of phosphatase samples allowed p-nitrophenol (p-nitrophenylphosphate hydrolysis product) concentration to be determined by direct reading at 405 nm (molar adsorption coecient 18:5 cm 1 mmol 1 ). A unit (U) of enzyme activity was de®ned as the lmol of substrate (saccharose, urea and p-nitrophenylphosphate) hydrolyzed by 1 g of dried soil at 30°C h 1 . Control tests with autoclaved soils were carried out to evaluate the spontaneous or non-enzymatically mediated hydrolysis of substrates. Furthermore, activity assays performed in the presence of toluene demonstrated that no microbial growth was taking place during activity tests. Consequently, any additive contribution to the measured enzymatic activities due to intracellular enzymes of proliferating cells was excluded. 2.4. Phosphatase complexes
Atrazine (99% a.i.) and paraquat (>95% a.i.), analytical grade were purchased from Serva, Germany; glyphosate (99.9% a.i.) was from Monsanto, Belgium and carbaryl (>96% a.i.) was from Merck, Germany. All the other chemicals were reagent grade and were supplied by Analar, BDH Ltd, Poole, UK.
As described by Rao et al. (1996) phosphatase± montmorillonite (P±M), phosphatase±tannate (P±T), and phosphatase±Al
OHx ±tannate±montmorillonite (P±ATM) complexes were prepared at 10°C and 0.1 M Na-acetate buer pH 6.0. The complexes were resuspended in 0.1 M Na-acetate buer pH 6.0 and stored at 10°C. The residual activities of complexes were periodically measured and new complexes were prepared when the residual activity reduced by more than 20% with respect to that of the initial preparation. All the data were normalized on the same residual activity.
2.3. Enzymatic assays
2.5. Eect of pesticides
Urease (U), invertase (I), and phosphatase (P) activities were determined as described by Gianfreda et al.
The eects of pesticides and methanol (used as solvent for atrazine and carbaryl) on enzymatic activities of
2.2. Pesticides and chemicals
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419
Table 1 Selected properties of soils Soil
Sand
Silt (%)
Clay
pH (H2 O)
O.C. (%)
C.E.C. (cmol Kg 1 )
Texture
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
57.70 52.03 59.04 77.40 70.50 85.80 72.80 93.43 28.05 56.60 34.20 90.20 66.30 63.20 73.10 82.40 40.90 46.60 54.60 50.80 27.80 24.00
29.00 28.77 26.72 16.00 12.00 9.86 23.10 3.40 2.40 24.50 28.20 4.10 16.30 19.70 20.80 13.10 40.70 41.30 29.50 36.40 44.00 46.40
13.30 19.20 14.24 6.60 17.50 4.34 4.00 3.17 69.60 18.90 37.60 5.70 17.40 17.10 6.10 4.50 18.40 12.10 16.00 12.80 28.20 29.60
8.36 7.91 8.16 8.39 7.49 7.11 6.46 7.31 8.63 6.14 8.42 7.80 8.15 8.05 6.88 7.56 5.06 6.84 5.49 5.56 8.33 8.40
1.20 1.40 1.29 0.83 2.37 1.85 2.88 1.29 0.12 0.76 1.09 1.01 1.77 3.21 3.42 2.08 2.06 2.08 2.09 0.65 1.47 0.95
11.77 13.72 11.73 7.20 12.41 10.98 11.10 9.18 14.84 8.11 13.50 5.10 13.10 15.90 16.20 14.50 14.50 13.50 13.10 5.10 14.10 13.10
Silty sand Silty sand Silty sand Sand Clayey sand Sand Sand Sand Clay Silty sand Silty clay Sand Clayey sand Silty sand Sand Sand Sandy silt Sandy silt Silty sand Silty sand Sandy silt Silty clay
soils and phosphatase synthetic systems were examined under assay conditions, described above. Saturating substrate concentrations (i.e., saccharose 0.1 M in 0.1 M Na-Acetate buer pH 4.65 (Gianfreda et al., 1995); urea 0.2 M in 0.1 M Na-phosphate buer-0.001 M EDTA pH 7.0 (Gianfreda et al., 1994); p-nitrophenylphosphate 0.02 M in 0.1 M MUB at pH 6.0) were used (Tabatabai and Bremner, 1969). As demonstrated by Gianfreda et al. (1993), pesticides and methanol can interfere with the analytical methods used for evaluating the products of enzymatic reactions. For each enzyme a maximum concentration of pesticide, which assured no interference with the activity assays, was utilized (Table 2) (Gianfreda et al., 1993). Pesticide concentrations correspond to a mean application dose ranging from 40 to 200 mg kg 1 of soil. According to Gianfreda et al. (1993), kinetic studies were performed with phosphatase complexes at p-nitrophenylphosphate concentration ranging from 0.1 to 6.0 mM and in the presence of various concentrations of glyphosate. Table 2 Pesticide concentrations used for each enzyme Pesticide concentrations (mM) Enzyme
Atrazine
Carbaryl Glyphosate Paraquat
Invertase Urease Phosphatase
1.40 1.40 1.40
2.00 2.00 1.00
6.00 1.50 20.00
0.16 4.00 ±
Unless otherwise speci®ed, all reported results are averages of three replications. The data were statistically analyzed by means of error standards. Relationships between eects of pesticides on soil enzymatic activities and some physico-chemical properties of soils were established by calculating regression equations and simple linear correlation coecients (r).
3. Results and discussion The activities of invertase, urease, and phosphatase of soils are depicted in Fig. 1. Soils displayed a general variability of enzymatic activity. Moreover, soils with high activity of a certain enzyme did not show elevated values of the others. All soils showed higher levels of invertase than phosphatase and urease in the order listed. The highest invertase activities were measured in soils 13 and 14, whereas no activity was found in soils 8 and 9. Phosphatase was detected in all soils and its activity ranged from a minimum of 0.41 lmol g 1 h 1 in soil 12 to a maximum value of 7.26 lmol g 1 h 1 in soil 15. By contrast, urease enzyme was absent in several soils (1, 2, 4, 8, 9, 10 and 12) and when present showed a very low activity. The widespread presence of phosphatase and, in part of invertase, is in agreement with the indications
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the activities were correlated with the organic matter content (Gianfreda and Bollag, 1996). 3.1. Eect of glyphosate and paraquat
Fig. 1. Activities of invertase, urease, and phosphatase in soils. Standard errors ranged from 0% to 5%.
reported in the literature (Kiss et al., 1978; Speir and Ross, 1978). Invertase is known to be a very stable and persistent enzyme and its association with soil components is well documented (Kiss et al., 1978). Moreover, soil phosphatases are frequently regarded as ectoenzymes, i.e. enzymes acting outside but still linked to their cells of origin. Stable soil component-phosphatase aggregations have also been reported to contribute significantly to the overall soil phosphatase activity (Nannipieri et al., 1988). The absence of urease in many soils and its low activity in others are not easily explainable. It is usually accepted that soils exhibit appreciable urease activity. Measurable urease activities were detected even in stored and geologically preserved soils (Bremner and Mulvaney, 1978). These ®ndings led to the conclusion that native soil ureases are mainly extracellular and are particularly persistent because of their association with inorganic and organic soil colloids (Burns et al., 1972a,b; Gianfreda et al., 1992; Gianfreda et al., 1995). Other studies, however, suggested that a considerable amount of the total activity of an enzyme (including urease) in soil may be ascribed to an enzymatic fraction located either within proliferating and non-proliferating cells or attached to or contained within cell debris (Nannipieri, 1994). This enzymatic fraction does not contribute to the measured activity of a soil, because it is not easily detectable in enzyme assays as commonly conducted. Therefore, it could be assumed that such urease fractions predominate in soils under investigation in this study. Simple and multiple regression analyses were undertaken to correlate the activities of invertase, urease, and phosphatase and soil properties. They gave rise to correlation coecients ranging from 0.06 to 0.60. These values are not suciently high to establish straightforward relationships between soil properties and enzyme activities. In agreement with the ®ndings usually reported in the literature, the highest value of r was obtained when
Fig. 2 shows the eect of glyphosate on invertase, urease, and phosphatase activities of soils. The results are expressed as increments or decrements of the activities measured without pesticide (activity in the absence of glyphosate 1). The enzymes diered markedly in their response to glyphosate. Invertase and urease activities were activated in all soils except in soils 3 and 19 and soils 11 and 17, whose invertase and urease activities decreased by 23% and 33% and 37% and 43%, respectively. The extent of activation of invertase ranged from a very low increase (+4% in soil 18) to the greatest (204%) in soils 11 and 14. The highest increment for urease was detected in soil 14, whose activity increased up to 9.24-fold. A completely opposite behavior was observed with phosphatase. The activity of the enzyme was inhibited in all soils. Phosphatase activity was decreased by a minimum of 5% in soil 5 to a maximum of 98% in soil 22 and on average by 50% in other soils. A slight increase of activity (+16%) was measured only in soil 18. The eect of paraquat on the activities of invertase and urease is shown in Fig. 3. The in¯uence of the pesticide on phosphatase activity was not determined because of the complete interference of paraquat with pnitrophenol determination by the alkalization procedure (Gianfreda et al., 1993). The pesticide positively in¯uenced the activity of both the enzymes in some soils but a more pronounced activation was caused by paraquat on invertase than on urease. The increments of invertase and urease activities varied on average from 1.2- to 3.2fold and reached the greatest values, 15.4- and 4.13-fold, in soil 3 and 18, respectively. A loss of invertase activity was measured in soils 12, 19, 20, 21, and 22, where the decreases ranged from 8% to 30%. An inhibition of urease activity varying from 10% up to 80% was detected in soils 13, 14, 15, 19, 21, and 22. Some reports demonstrated that glyphosate did not aect phosphatase and urease activities of soils (Davies and Greaves, 1981; Lethbridge et al., 1981) whereas a stimulative eect was shown on invertase (Tu, 1982; Gianfreda et al., 1995). On the contrary, a slight inhibitory eect of paraquat was found in some soils on the activities of the three enzymes. No signi®cant in¯uence, however, was also registered (Shaer, 1993). The inhibition of phosphatase activity by glyphosate, observed in the majority of soils, could be attributed to the presence of a phosphoric group on the glyphosate molecule. Several ®ndings demonstrated that soil phosphatase is strongly inhibited by inorganic phosphate and phosphate fertilizers (Speir and Ross, 1978).
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421
Fig. 2. Eect of glyphosate on enzymatic activities of soils. Standard errors ranged from 0% to 5%.
Table 3 shows the eect of glyphosate and paraquat on four enzymatic model systems simulating enzymatic forms, possibly found in soil environments. Namely, free enzymes (E), clay±enzyme complexes (E±M), organo± enzyme (E±T) and organo±mineral±enzyme complexes (E±ATM). The data for invertase and urease refer to the results from Gianfreda et al. (1994) and Gianfreda et al. (1995), respectively, and are reported for comparison purposes. The data for phosphatase have been obtained in this study. The results are expressed as relative activities with respect to those determined without pesticides. The response of phosphatase systems to glyphosate is noteworthy. All the enzymatic forms except the free enzyme were inhibited by the herbicide and the decrease of activity varied from 20% to 40%. The strong decrease of activity detected for phosphatase-montmorillonite complex in the presence of glyphosate was con®rmed by kinetic studies performed at increasing glyphosate concentrations. The results demonstrated that glyphosate behaves as a typical competitive inhibitor (Segel, 1975) (Vmax decreased and Km increased by increasing glyphosate concentration, data not shown). Similar studies were also carried out on P±T and P±ATM complexes, and showed the same behavior. By contrast, invertase and urease systems behaved dierently to the presence of both pesticides, depending on their ``status''. The activities of invertase free and
Fig. 3. Eect of paraquat on enzymatic activities of soils. Standard errors ranged from 0% to 5%.
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Table 3 Eect of glyphosate and paraquat on model enzymatic systems Relative activitya Paraquat
Glyphosate Free-E E±M E±T E±ATM a
Invertase
Urease
Phosphatase
Invertase
Urease
1.80 1.10 0.98 0.93
0.93 1.03 0.99 1.20
1.05 0.61 0.79 0.59
1.23 1.10 0.85 0.92
0.96 1.79 0.95 1.03
The relative activity refers to the activity without pesticides.
immobilized on montmorillonite increased in the presence of both glyphosate and paraquat and the increment of activity was much more pronounced for the free than the immobilized form (Gianfreda et al., 1995). No signi®cant eects or very small decreases were measured when the enzyme was complexed with tannic acid or with the organo±mineral complex ATM. A detectable decrease (15%) was determined with paraquat only for the invertase±tannate complex, I±T (Gianfreda et al., 1995). A dierent behavior was shown by urease (Gianfreda et al., 1994). The activity of the enzyme markedly increased in the presence of paraquat and glyphosate when complexed to montmorillonite and to ATM, respectively. For the other two systems (e.g. free urease and urease±tannate complex), no activation or inhibition was measured (Gianfreda et al., 1994). The results shown in Table 3 seem to indicate that glyphosate and paraquat are expected to show activation eects on invertase and urease activities of soils containing high clay content, respectively. On the contrary, invertase activity of soils with high content of organic matter should respond negatively to the presence of paraquat. Moreover, the general decrease of activity measured for phosphatase complexes in the presence of glyphosate could indicate that clay, organic matter, and clay + organic matter content may contribute to enhance the inhibitory eect of the herbicide. These hypotheses appear to be supported by simple and multiple regression analyses made between the activities of soil enzymes measured in the presence of the pesticides and soil properties. The activity of soil invertase measured with glyphosate well and positively correlates with the overall level of sand + silt + clay of soil (r > 0:7), whereas no signi®cant correlations were found with organic matter or organic matter+clay contents (r 0:3). Similarly, soil urease activities measured in the presence of paraquat appear to increase by increasing soil texture (positive correlation r 0:5). No considerable relationships were found between soil properties and glyphosate eect.
3.2. Eect of methanol, atrazine, and carbaryl Fig. 4 shows the eect of methanol, atrazine, and carbaryl on invertase, urease, and phosphatase activities of soils. The results are expressed as increments or decrements of the activities assayed without methanol or pesticides. In all soils, methanol exhibited a more or less marked inhibitory eect on the invertase activity (Fig. 4) which ranged from 5% (soils 14 and 15) to 80% (soil 21). Only in soil 12, invertase activity increased by 36%. In the presence of atrazine, a further inhibitory eect was detected (on average by 48% respect to methanol) for all soils except in soils 18, 20, 21, and 22, where a slight recovery of invertase activity was observed. With carbaryl, the decrease of activity was negligible with respect to the control for soils 17, 20, 21, and 22 and increased up to 77-fold in soil 6. A dierent behavior was observed with soil urease activities (Fig. 4) which were enhanced by methanol. An inhibitory eect was measured only with soils 15 ()76%) and 19 ()37%). The addition of atrazine and carbaryl generally enhanced the activation eect exhibited by methanol on the urease of most of the soils, except in soil 19 where a further decrease of urease activity was measured. An increase up to 396-, 1344-, and 320-fold with atrazine and 6850-, 9431-, 6054-fold with carbaryl with respect to the control was observed in soils 3, 5, and 6, respectively. These results con®rm those previously obtained by the authors on urease activity of some soils (Gianfreda et al., 1994). The activation eect of methanol and the two pesticides was explained by considering a signi®cant increase in cell wall permeability or cellular lysis, and consequent increase in the accessibility of the substrate molecules to the intracellular urease (Gianfreda et al., 1994). The results obtained with soil phosphatase activities demonstrate that methanol exhibited an activation eect in all soils ranging from 3 (soil 3) to 547% (soil 10) (Fig. 4). On the contrary, the solvent inhibited enzymatic activity of two soils (8 and 12) and the decrements were )46% and )49%, respectively. Contradictory eects were observed in the presence of both atrazine and
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423
Fig. 4. Eect of methanol, atrazine, and carbaryl on enzymatic activities of soils. Standard errors ranged from 0% to 5%.
carbaryl. Both the pesticides signi®cantly magni®ed the methanol eect in soil 7, whereas no detectable in¯uence was observed in soil 14, 16, and 21. Slight increases or decreases were measured in other soils. The eect of methanol, atrazine, and carbaryl on the enzymatic model systems is reported in Table 4. As mentioned above, the results obtained with invertase and urease are from Gianfreda et al. (1994, 1995), respectively, and are reported for comparison purpose, while the data for phosphatase have been obtained in this study. Generally, there was an inhibition of enzyme activity when methanol was present in the activity assay and a partial removal of this eect was exhibited by atrazine and carbaryl. With respect to invertase, methanol inhibition ranged from 36% for the free enzyme to 57% when tannate molecules were involved in the
complex. Similar eects were displayed by the organic solvent on free urease, whereas more variable responses to the additional presence of the two pesticides were observed with immobilized invertase and urease (Gianfreda et al., 1994; Gianfreda et al., 1995). Atrazine and carbaryl partially removed the inhibitory eect of methanol on invertase free and immobilized on the matrices. An enhanced inhibition was instead shown by carbaryl on free invertase. A detectable recovery of activity was measured for invertaseATM with carbaryl. By contrast, both atrazine and carbaryl showed no or slight enhancement of methanol eect on the activity of free and immobilized urease (Gianfreda et al., 1994; Gianfreda et al., 1995). As reported by Gianfreda et al. (1994, 1995) some consideration can be made to explain the behavior of immobilized enzymes in the presence of methanol and
Table 4 Eect of methanol, atrazine, and carbaryl on model enzymatic systems Relative activitya Methanol Free-E E±M E±T E±ATM a
Atrazine
Carbaryl
Invertase
Urease
Phosphatase
Invertase
Urease
Phosphatase
Invertase
Urease
Phosphatase
0.64 0.51 0.43 0.43
0.64 0.57 0.73 0.63
0.74 0.87 0.85 0.71
0.71 0.62 0.54 0.55
0.58 0.54 0.67 0.58
0.61 0.87 0.85 0.70
0.53 0.67 0.61 0.82
0.53 0.60 0.62 0.65
0.76 0.86 0.80 0.69
The relative activity refers to the activity without pesticides.
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pesticides. In this case, it must be considered that methanol is present in the reaction mixture and it can exhibit either direct interactions (i.e. inhibition or deactivation) on enzyme molecules or more complex eects (i.e. interference with the dierent bounds between the organic and inorganic components of complexes) on the structural characteristics of the enzymatic complexes. The presence of atrazine and carbaryl further complicates the systems because of additional eects between pesticides, enzymes and matrices. In the presence of methanol, the activity of phosphatase free and immobilized on ATM was slightly reduced by 26- and 29-fold, respectively. When the enzyme was immobilized on montmorillonite or on tannic acid, a little increase of the relative activities was detected. No signi®cant eects or very small decreases were measured for the four enzymatic systems when atrazine or carbaryl was present in the reaction mixture. To establish the relationships, if any, between the eect of atrazine and carbaryl on soil enzymatic activities and physico-chemical properties simple and multiple regression analyses were performed. In general, no reliable and signi®cant (supported by high value of r) relationships were obtained; r values were usually positive but always lower than 0.5. Only with phosphatase a positive value of r (>0.8) was calculated, when the residual activities measured in the presence of carbaryl and atrazine were correlated with both clay content and pH of soils. This could indicate that the higher the clay + pH value of soils, the greater the residual activity of phosphatase and the lower the inhibition of the additives. This indication seems to be partially supported by the results shown in Table 4. In fact, phosphatase bound to montmorillonite appears to be less aected by methanol, carbaryl, and atrazine than free in solution. In conclusion, this study con®rm that pesticides may aect the activities of enzymes in soils. Since investigations were performed in vitro, under laboratory conditions, the obtained results are obviously aected by the methodologies used for evaluating and assaying enzyme activities in soil. As it is widely claimed by several authors (1, and reference herein), these methodologies do not discriminate between the various components contributing to the overall enzymatic activity of soil. Consequently, it is particularly dicult to explain a change of soil enzymatic activity in response to a certain factor, or to establish the cause±eect relationships between the applied factor and the soil enzyme activity variation. D.I.S.C.A. publication n. 192. References Bollag, J.M., Liu, S.Y., 1990. Biological transformation processes of pesticides. In: Cheng, H.H. (Ed.), Pesticides in the Soil Environment: Processes, Impacts, and Modeling.
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OHx ±montmorillonite complexes. Soil Biol. Biochem. (24), 51±58. Gianfreda, L., Rao, M.A., Violante, A., 1995. Formation and activity of urease±tannate complexes as aected by dierent species of Al, Fe and Mn. Soil Biol. Biochem. (59), 805±810. Gianfreda, L., Sannino, F., Filazzola, M.T., Violante, A., 1993. In¯uence of pesticides on the activity and kinetics of invertase, urease and acid phosphatase enzymes. Pesticide Sci. (39), 237±244. Gianfreda, L., Sannino, F., Ortega, N., Nannipieri, P., 1994. Activity of free and immobilized urease in soil: eects of pesticides. Soil Biol. Biochem. (26), 777±784. Gianfreda, L., Sannino, F., Violante, A., 1995. Pesticide eects on the activity of free, immobilized and soil invertase. Soil Biol. Biochem. (27), 1201±1208. Kiss, S., Dragan-Bularda, M., Radulescu, D., 1978. Soil polysaccharidases: activity and agricultural importance. In: Burns, R.G. (Ed.), Soil Enzymes. Academic Press, London, pp. 117±147. Lethbridge, G., Bull, A.T., Burns, R.G., 1981. Eects of pesticides on 1,3-b-glucanase and urease activities in soil in the presence and absence of fertilisers, lime and organic materials. Pesticide Sci. (12), 147±155. Nannipieri, P., Ceccanti, B., Bianchi, D., 1988. Characterization of humus±phosphatase complexes extracted from soil. Soil Biol. Biochem. (20), 683±691. Nannipieri, P., 1994. In: Pankhurst, C.E., Dounbe, B.M., Gupta, V.V.S.R., Grace, P.R. (Eds.), Soil Biota. Management in Sustainable Farming Systems. CSIRO, Adelaide, pp. 238-247.
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Tabatabai, M.A., Bremner, J.M., 1969. Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem. (1), 301±307. Tu, C.M., 1982. In¯uence of pesticides on activities of invertase, amylase and level of adenosine thriphosphate in organic soil. Chemosphere (11), 909±914. Violante, P., 2000. Metodi di analisi chimica del suolo. Franco Angeli, Roma, Italy. Liliana Gianfreda is a Professor of Agricultural Biochemistry at the University of Naples, Italy. She is the author or coauthor of more than 150 professional papers and has participated as an invited Lecturer at several National and International Conferences and Symposia. She is currently the President of the Third Commission ``Soil Biology'' of the Italian Society of Soil Sciences.
