INTRODUCTION

Pesticides are widely used in crop production and are known to cause major environmental problems. With increasing pesticide use, questions are rising on potential effect regarding public health and environment. Pesticides pollute air, soil, water resources and contaminate the food chain. The interaction between soil components and pesticides influences the biochemical processes driven by microorganisms. The effect of Pesticides on soil microorganisms could be determined by the study of functional parameters such as carbon and nitrogen mineralization that are governed by enzymatic activities. Those activities play an important role because all biochemical transformations in soil depend on or are related to the presence of enzymes (Raith et al., 2014).

The large-scale use of pesticides has created a biotic factor which affecting every ecosystem mostly. Application of pesticides to agricultural soils may affect soil biological activity in a variety of ways (Shetty and Magu, 1998). Pesticides may affect the beneficial microorganisms in the soil which play a good role on the fertility and productivity of the agricultural soils through their effect on the biological processes such as the mineralization of organic matter and soil enzymes (El-Shahaat, 1993). Soil enzyme activity is a key feature of plant nutrients and cycling processes, and therefore measurements of specific enzyme activities may be useful in determining soil biological activity, which might be used as an index of soil fertility (Reddy et al., 1995).

Several studies were conducted to find out the effects of pesticide on soil enzymes (Burrows and Edwards, 2004; Klose and Ajwa, 2004;Gundi et al., 2007; Menon et al., 2005; Stromberger et al., 2005;Pampulha and Oliveria, 2006; Bending et al., 2007; Qian et al., 2007; Wang et al., 2007). Most of these studies conclude that pesticides at higher doses inhibit enzymatic activities.

Commercial fungicides namely dhanustin ,Dithane M-45 and Contaf are widely used for control of fungal pests in agricultural crops like groundnut. These agrochemicals may affect local metabolism or soil enzyme activities besides controlling pest problem. They may have both negative and positive effects on function of soil enzymes (Liu et al. 2008). So far little information is available on these fungicides behavior towards soil dehydrogenase, phosphatase, protease and urease.

Urease is an enzyme that catalysis the hydrolysis of urea into carbon dioxide and ammonia and is a key component in the nitrogen cycle in soils. Urease activity in soils has received a lot of attention since it was first reported by Rotini (1935), a process considered vital in the regulation of nitrogen supply to plants after urea fertilization (Makoi and Ndakidemi 2008).  Most of the referenced studies reported that herbicides and fungicides appear to have no effect (Cyconet al., 2010; Romero et al., 2010; Tejada et al., 2011; Yan et al., 2011; Bacmaga et al., 2012) or reduced effect on urease activity (Sukul, 2006; Caceres et al., 2009; Tejada, 2009). Decreased urease activity in soil due to the application of pesticides reduces urea hydrolysis, which is generally beneficial, because it helps to maintain nitrogen availability to plants (Antonious, 2003). On the contrary, the fungicides carbendazim and validamycin enhanced urease activity, respectively, up to 70 % and to 13–21 % (Qian et al., 2007; Yan et al., 2011). The urease activity appears to be either unaffected or inhibited by the addition of pesticides except carbendazim and validamycin, which tend to stimulate this enzyme activity. Thus, it is difficult to identify a clear response of this enzymatic activity to pesticides because this enzyme has received little attention during the last years.

Phosphatases are a broad group of enzymes that are capable of catalyzing hydrolysis of esters and anhydrides of phosphoric acid (Schmidt and Lawoski 1961). In soil ecosystems, these enzymes are believed to play critical roles in P cycles (Speir and Ross, 1978) as evidence shows that they are correlated to P stress and plant growth. Apart from being good indicators of soil fertility, phosphatase enzymes play key roles in the soil system (Dick and Tabatai, 1992; Dick et al., 2000).

Plants have evolved many morphological and enzymatic adaptations to tolerate low phosphate availability. This includes transcription activity of acid phosphatases, which tend to increase with high P stress (Haran et al., 2000; Baldwin et al., 2001; Miller et al., 2001; Li et al., 2002). The amount of acid phosphatase exuded by plant roots has been shown to differ between crop species and varieties, (Ndakidemi, 2006; Izaguirre-Mayoral and Carballo, 2002) as well as crop management practices (Ndakidemi, 2006; Patra et al., 1990; Staddon et al., 1998;Wright and Reddy, 2001). For instance, research has shown that legumes secrete more phosphatase enzymes than cereal (Yadav and Tarafdar, 2001). This may probably be due to a higher requirement of P by legumes in the symbiotic nitrogen fixation process as compared to cereals. In their studies, Li et al. (2004) reported that chickpea roots were also able to secrete greater amounts of acid phosphatase than maize. The ability to solubilize soil mineral elements by these phosphomonoesteraces is expected to be a higher in biologically-managed systems because of a higher quantity of organic C found in those systems. In fact, the activity of acid and alkaline phosphatases was found to correlate with organic matter in various studies (Guan 1986; Jordan and Kremer, 1994;Aon and Colaneri, 2001). Another factor that influences the rate of synthesis, release and stability of this enzyme is the soil pH (Eivazi and Tabatabai, 1977; Juma and Tabatabai, 1977; Tabatabai, 1994). For example, phosphor monoesteracesinducibility and its intensity by plant roots and micro-organisms are determined by their orthophosphate need, which is in turn affected by soil pH. It is, therefore, anticipated that management practices that induce P stress in the rhizosphere may also affect the secretion of these enzymes in the ecosystem (Ndakidemi, 2006).

Catalase is an iron porphyrin enzyme which catalase very rapid decomposition of hydrogen peroxide to water and oxygen (Nelson and Cox, 2000). The enzyme is widely present in nature, which accounts for its diverse activities in soil. Catalase activity alongside with the dehydrogenase activity is used to give information on the microbial activities in soil. Both catalase and dehydrogenase activity is very sensitive to heavy metal pollution (Naplekova and Bulavko, 1983; Perez and Gonzalez, 1987; Wilkes, 1991). Their values have been suggested to be used as a simple toxicity test (Roger and Li, 1985).

Until present, there have been few studies examining the effect of pesticides application on the ecosystem of soil enzymes activity where major crops are grown. Understanding dynamics of enzyme activity in these systems is crucial for predicting nutrient uptake. Therefore, the present study was conducted to investigate the effect of some pesticides on some soil enzymes activity in soil cultivated with wheat.

MATERIALS AND METHODS

Field experiment

The field experiment was conducted at Research and Experimental Farm, Faculty of Agriculture, Abees region - Alexandria-Egypt, during wheat growing season of 2012/2013. Wheat (Triticumastivum ) cv. MISR 2 was cultivated on November 23, 2012. Each plot was 10.5 m² (3m X 3.5m) and separated from another one by an adjacent border (1m) to minimize the pesticides drift.

The field experiment was arranged as a randomized complete blocks design and each treatment was replicated three times and the treatments were three  herbicides namely;Granstar^® 75% DF, Panther^® 55% SC,  Topik^® 15%  EC  and one fungicide; Sumi-eight^®12.5% WP, two rates of each pesticide besides the check treatment were applied as described in Table (1)and Fig (1).  The pesticides applications were applied during the growing season on Jan 19 and March 10, 2013, for fungicide. The agricultural practices were done according to the recommendations of the Egyptian Ministry of Agriculture including irrigation through surface irrigation system.

Table (1). The rates of pesticides applied during the growing season of wheat crop

Soil sampling and analysis

Soil samples were collected at the end of growing season of wheat crop at May 2013 for three soil depths, i.e. 0-5, 5-10 and 10-20 cm. The samples were collected from four points randomly and were immediately stored in sealed plastic bags and transported to the laboratory for analysis. The samples were passed through a 2-mm sieve and a composite sample was made for each soil depth and replication. Each mixed soil sample was stored at 4 °C for enzyme analysis.

Soil physical and chemical properties were determined according to routine methods (Table 2). The particle-size distribution and saturation percentage (SP) of these soil samples were estimated as described by Jackson (1973). Soil pH was measured in the soil: water suspension (1:2) using a pH meter with a glass electrode. Soil organic matter (OM) was determined using Walkley and Black method (Jackson, 1973). The calcium carbonate (CaCO₃) content was estimated using a volumetric calcium carbonate calcimeter (Nelson, 1982). The electrical conductivity of the soil water extract, 1:2 (EC_sw) was determined using an electrical conductivity meter according to Jackson (1973). Soluble calcium (Ca⁺²) and magnesium (Mg⁺²) in the saturated soil paste extract were estimated using the titration method by EDTA (Ethyline-diamine tetra acetic acid) solution. However, soluble sodium (Na⁺) and potassium (K⁺) in this extract were determined by flame photometry method (Hesse, 1998). Also,  soluble anions such as bicarbonate (HCO₃^-) and chloride (Cl^-) were determined using the titration with HCl acid (Richards, 1969) and silver nitrate solution (Jackson, 1973), respectively.

Fig. (1). Chemical structure of used pesticides in the present study

Table (2). Some physical and chemical properties of the experimental soil

Measurement of soil enzyme activity

Activities were assayed in triplicate air-dried samples as described by Guan (1986). Briefly, urease activity was determined using urea as substrate, and the soil mixture was incubated at 37°C for 24 h, the produced NH₄-N was determined by a colorimetric method, and urease activity was expressed as μg NH₄-N/g soil/h, phosphatase activity was measured using sodium phenolphthalein phosphate as a substrate, incubation at 37°C for 24 h, and the liberated phenol was determined colorimetrically, acid phosphatase activity was expressed as μg phenol/g soil/h and catalase activity was measured by mixing 2 g of soil, 40 ml of distilled water and 5 ml of 0.3% H₂O₂ were added. The mixture was shaken at 25^oC for 20 min. Then, 5 ml of 1.5 M H₂SO₄ was added and the contents were titrated with 0.1 M K₂MnO₄. Catalase activity was expressed as mg/g soil/h.

Statistical Analysis

The results were expressed as mean values and compared with respect to the check treatment. Comparisons between the test and the check were made by using analysis of variance (ANOVA). Differences between treatment means at p < 0.05 were considered as significant using Statistix 8.0 software(Statistix, 2003).

RESULTS and DISCUSION

Several factors influenced enzymatic activity. These are both natural factors (e.g., seasonal changes, geographical conditions, depth, physicochemical properties of the soil) and anthropogenic factors (such as pollution with heavy metals, organic compounds, soil acidification, pesticides, etc.). With the development of civilization, anthropogenic factors started to play the main role in soil enzymatic activity changes (Gianfreda and Bollag, 1996).

Tables (3 and 4) show the activity of studied soil enzymes i.e. phosphatase, urease and catalase as affected by some pesticides application. Phosphatase activity ranged between 333.32 and 242.44 μg phenol/g soil/h. The potential of pesticides activity significantly (p<= 0.05) differ from each other. The concentration of pesticides significantly (p<= 0.05) decreased the activity of soil enzymes in witch increasing the application rate decreased the enzyme activity as 8.61 and 11.44% for half dose and full dose, respectively as compared to control treatment (no addition of herbicides). The activity of phosphatase was significantly (p<= 0.05) increased as soil depth increased. The surface layer (0–5 cm) has the least value of activity (327.76 μg phenol/g soil/h), then increased to 350.42 μg phenol/g soil/h as soil depth increased to 10 – 20 cm depth. This is true because of pesticides transport in soil is low and did not reach more the 10 cm. 

The persistence of pesticides and their degradation products depend on how deeply they are mixed into the soil; even the most persistent compounds disappear relatively quickly when on the soil surface, yet when incorporated into the soil they are very persistent (Edwards, 1966). Generally, pesticide residues will occur in the top 15 cm of soil (Harris and Sans, 1967). This is also the region of greatest activity of soil flora and fauna in the soil ecosystem (Alexander, 1991).

Phosphatase activity, as indicated by the release of p-nitrophenol, is an index of the activity of microflora involved in soil organic phosphate decomposition. All herbicides suppressed phosphatase enzymes activities. It was demonstrated by some workers (Hoffmann and Seegerer, 1950) that phosphatase activity varies in some cases among soils. It correlates with the intensity of respiration and activity of the soil microflora.

Soil phosphatases because of their involvement in making phosphorus available to plants, need to be extensively studied in relation to pesticide use.

Results obtained from the experiment show stimulation of phosphatase activity followed by fungicide treatment at field application rate of 5 kg.ha^-1.  PRasool and Reshi (2010) declared that the fungicide mancozeb initiated stimulation under normal recommended dosage and ten times the normal dosage. In the same manner, phosphatase increased significantly in agricultural soil amended with the fungicide probineb at field application rate (Rahmansyahet.al., 2009). Enzyme activity was enhanced up to 7.5 kg.haP-1P than the controls in 10 days incubated soil samples (Srinivasuluet.al., 2012).

Soil urease has attracted a great deal of attention due to the increasing use of urea as a fertilizer. In soil, urea is rapidly hydrolyzed to ammonium carbonate by urease activity resulting in the formation of nitrate and gaseous volatilization of ammonia.

All of the studied pesticides inhibited urease activity (Table 3). Sumi-eight herbicide was more inhibition of urease activity (100.36 μg NH4-N/g soil/h) and the least one was Granstar® (172.80 μg NH4-N/g soil/h). The differences between herbicides were significant (p<= 0.05). Also, the applied concentration of Granstar®, Topik®, Panther® and Sumi-eight® significantly (p<= 0.05) inhibited the urease activity by 37.06 and 49.62% for half and full doses, respectively as compared to control treatment (no addition of pesticides). Decreased urease activity in soil due to the application of pesticides reduces urea hydrolysis, which is generally beneficial because it helps to maintain nitrogen availability to plants (Antonious, 2003).

The activity of urease was significantly (p<= 0.05) increased as soil depth increased. The surface layer (0–5 cm) has the least value of activity (17.57 μg NH4-N/g soil/h), then to 154.26 μg NH4-N/g soil/h increased as soil depth increased to 10 – 20 cm depth.

This is true because of pesticides transport in soil is low and the transport of herbicides do not reach more the 10 cm.  The inhibition of urease activity could be due to the presence of Mn and Zn ions in the pesticide, as reported by (Tabatabai, 1977).Urease has been studied more extensively relative to other soil enzymes because of its involvement in the breakdown of urea, a commonly used fertilizer (Martens and Bremner, 1997).

Also, Yang et al.(2006) showed that furadan enhanced urease activity in four soils. Rahmansyahet al. (2009), also reported an increase in urease enzyme after two weeks incubation with fungicide probineb. Same findings were found by Maleeka Begum and Rajesh, 2015). But the majority of workers stated inhibition of the enzyme in fungicide amended soils, especially at higher concentration. According to Guoet al. (2008), urease activity was inhibited at all concentrations with napropamide. Similarly, Cycon et al. (2010) noticed that activity declined with dimethomorph and mancozeb in loamy and sandy loam soils at higher concentration. Likewise, 20% decrease in activity was observed in chlorpyrifos treated soil (Tejdaet al., 2010; Wang et al., 2010).

Table (3).Average soil enzymes activity values as affected by certain pesticides (type and concentration) and soil depth

Table (4). Analysis of variance for soil enzymes activity as affected by cerain pesticides (type and concentration) and soil depth

Concerning the catalase activity, all herbicides applied to soil significantly (p<= 0.05) inhibited the catalase activity.  Granstar® herbicide was more inhibited the enzyme activity (2.48 mg/g soil/h) and the least one was Topik® (2.99 mg/g soil/h). The differences between herbicides were significant (p<= 0.05). Also, the applied concentration of Granstar®, Topik®, Panther® and Sumi-eight® significantly (p<= 0.05) inhibited the catalase activity by 27.82 and 44.63% for half and full doses, respectively as compared to control treatment (no addition of herbicides). The activity of catalase was significantly (p<= 0.05) increased as soil depth increased. The surface layer (0–5 cm) has the least value of activity (2.42 mg/g soil/h), then increased as soil depth increased to 3.13 mg/g soil/h at 10 – 20 cm depth. This is true because of pesticides transport in soil is low and the transport of pesticides do not reach more the 10 cm (Abdel-Nasser et al., 2011).

Continuous and indiscriminate use of these pesticides causes a major risk to soil health. There have been many reports of the effects of pesticides on soil enzyme activities (Anonymous, 2011; and Loganathan et al., 2002) and it has been observed that the responses of soil enzymes on different pesticides are not the same. Soil enzyme activities are more sensitive to the environment. They reflect the soil quality more quickly and directly (Srinivasulu et al., 2012). Since enzyme activity has been considered as a very sensitive indicator, any disturbance due to biotic or environmental stresses in the soil ecosystem may affect soil biological properties.

Finally, the measurement of specific enzymatic activities may contribute to understanding the metabolic processes involved in the biogeochemical cycles of nutrients. Pesticides reaching the soil may disturb local metabolism or enzymatic activities (Liu et al., 2008; Hussain et al., 2009). Negative impacts of pesticides on soil enzymes such as hydrolases, oxidoreductases, and dehydrogenase activities have been widely reported in the literature (Monkiedje and Spiteller 2002; Monkiedje et al., 2002; Menon et al., 2005; Caceres et al., 2009). There is also evidence that soil enzymes may provide valuable general information on the transformation of pesticides in soils (Gianfreda and Bollag 1994; Kalam et al., 2004; Gil-Sotres et al., 2005; Hussain et al., 2009).

Contrarily, phosphatase activity in paddy cultivated soil showed a variable pattern in response to various concentrations of triazophos after 1^st, 7^th, 14^th, 21^st and 28^th days of incubation periods. Enzyme activity significantly decreased at all the concentrations of triazophos and there was much difference in the activity compared to untreated control (Lakshmi Kalyani and Suvarnalatha Devi, 2015). Coinciding with the above, activities of two phosphatases were also inhibited (22%) in a sandy loam soil incorporated with captan fungicide at all incubations reported by Piotrowskaet.al. (2008). the decrease in enzyme activity may be ascribed to suppression of a sensitive fraction of soil biota.

It is difficult to understand the role of pesticides in disturbing the microbial communities and their enzymatic activities in soil due to divergent research findings reported in the literature. A number of factors could be responsible for those debatable results such as soil properties, chemical nature, and concentration of pesticides, biological function observed. Even if pesticides applied at recommended rates may cause slight and transient changes to populations or activities of soil microorganisms (Johnsen et al., 2001), it is obvious that long-term frequent applications of pesticides are known to interfere with the biochemical balance, which can reduce soil fertility and productivity by affecting local metabolism and enzymatic activities.

Finally, in this study, we attempt to look for general trends of enzymatic responses to pesticides, which could be useful for researchers and thus for policy decision makers. Thus, this work has allowed to future investigation for (i) identify patterns of enzymatic activity response to pesticides application, (ii) link them with the pesticides mechanisms of action, and (iii) classify the pesticides according to their stimulating, inhibiting or neutral effects on enzymatic activities. As general, application of pesticides decreased the activity of soil enzymes and this decreasing increased with increasing the pesticides application rate. Also, the decreasing of soil enzyme activity was less with increasing soil depth.