4.1 Degradation of chlorpyrifos under natural conditions
The experiment was conducted to understand the effect of chlorpyrifos degradation in coarse textured laterite soil under natural conditions. The results showed that the amount of chlorpyrifos reduced remarkably in the soil within a 60 days’ period. Earlier studies reported that the biological active period of chlorpyrifos in soil ranges from 20 days to a few months (Awasthi and Prakash 1998).
The results showed that chlorpyrifos content was lost from the soil with progress of incubation period and there was less persistence of chlorpyrifos in the coarse textured laterite soil. The experimental soil is categorized as laterites and coarse textured sandy loam with low clay (3.32%) content and low organic carbon content (0.36%) which has inherently low retention capacity. Hence the persistence of chlorpyrifos in the study soil was found to be less. The persistence of chlorpyrifos was found to be lower in mineral soil (Gebremariam et al. 2012). Various factors that determine the persistence of organophosphates such as chlorpyrifos in soil include organic matter, clay content and iron and aluminium oxyhydroxide of soil (Weber 1972).
The degradation of chlorpyrifos under natural conditions was found to be effective and rapid in coarse textured sandy loamy soil. Tashiro and Kuhr (1978) found out that chlorpyrifos has a degradation half-life of 7–16 days in sandy loam soil, whereas in clayey soil it was up to 120 days (Freed et al. 1979). At the 60th day of natural degradation, the chlorpyrifos content was analyzed and found that there was only 34.76 per cent reduction in chlorpyrifos content within 60 days and however, no complete disappearance of the compound was observed in the experimental soil. Chishti et al. (2013) confirmed that complete degradation or 100 percent recovery of the active ingredient of chlorpyrifos was not possible during the degradation process. The application of chlorpyrifos showed significant effect on microbial biomass carbon in soil and reflected a decrease on the 60th day of incubation. The soil microbial biomass carbon was reduced during incubation period compared to initial content recorded at start of experiment. This decrease in the microbial biomass carbon might be due to the lowering of microbial population immediately after application of chlorpyrifos. This might be due to the harmful effect of the compound on microbial activities in soil during chlorpyrifos degradation under natural conditions. Chu et al. (2008) reported that chlorpyrifos has an inhibitory effect on microbial population on 7th day of incubation.
As the degradation progressed, the chlorpyrifos compound transformed into its products, such as chloride, phosphate, sulphate and nitrate ions which were increased during the incubation period. Increase in the phosphate ion was noticed at the 60th day of incubation period and it was not found to be significant during natural degradation process. Increase in the phosphate ions could be due to breakage of phospho-ester bond in the chlorpyrifos compound. Tang et al. (2011) confirmed that hydrolysis and oxidation of chlorpyrifos leads to release of phosphate ions to the soil. Chloride and sulphate ions were found to be significant at the 60th day of incubation period. During the process of degradation, homolytic cleavage of chlorine atoms in the chlorpyrifos compound was responsible for the release of chloride ions to the soil (Feng et al. 1997). Effects of chlorpyrifos application on sulphate ions in soil were found to be significant and an increase in the sulphate ion was observed. This might be due to desulphurization of chlorpyrifos compound into chlorpyrifos oxon during the degradation process. Effects of chlorpyrifos application on nitrate ions in soil were found to be non-significant, however an increase in the nitrate ions were observed. This could be due to mineralization of ammonium ions into nitrate ions. Significant decrease in soil pH was noticed after the application of chlorpyrifos indicating adverse effect on soil health.
4.2 Effect of treatments on chlorpyrifos degradation
The results showed that the physical, chemical and biological agents imposed as treatments in the degradation experiment, could significantly reduce chlorpyrifos content in soil during the 60 days incubation period in comparison to the natural degradation process. During the period of incubation, application of microbial inoculants in combination of Pseudomonas fluorescens and Trichoderma viride showed the highest degradation of chlorpyrifos, followed by Pseudomonas fluorescens and Trichoderma viride within 60 days period. The highest degradation of chlorpyrifos exhibited by microbial inoculants might be due to the synergistic effect of microbes in soil as reported by Sasikala et al. (2012). Fungal population introduces minor structural changes to the pesticide molecule and convert into nontoxic metabolites, releasing them back to soil and they would be further degraded by bacterial species through cometabolism and mineralization (Alvarenga et al. 2015).
Chlorpyrifos degradation was higher in soil with bacterial inoculant (Pseudomonas fluorescens) than fungal inoculant (Trichoderma viride). These findings were contradictory to the previous report that chlorpyrifos application caused the short-term inhibitory effect on bacterial population which recovered to initial population with progress of chlorpyrifos degradation whereas the fungal population stimulated after the application of the compound (Pandey and Singh 2004). Lowest degradation was observed in control which proves that degradation of chlorpyrifos is fastened under the influence of different treatments, particularly the biological treatments in coarse textured laterite soil.
Combinations of microbial inoculants as well as a single inoculant were proved as the best treatments for fastening the degradation of chlorpyrifos. Sariwati et al. (2017) reported that mixed cultures of microorganism proved more efficient in degradation of pesticide like DDT than single cultures. Lakshmi et al. (2008) found out that Pseudomonas fluorescens has the ability to degrade the chlorpyrifos to 75–87%. Jayaraman et al. (2012) reported that strains of Trichoderma viride and Trichoderma harzianum were efficiently used for the degradation of chlorpyrifos. In the present study the combination of Pseudomonas fluorescens and Trichoderma viride were used and it was evident from the data that combination of microbes performed well as degraders of chlorpyrifos.
Enzymes play an important role in the degradation of chlorpyrifos in soil. During the degradation process, several enzymes are released by the microbes, which would speed up the degradation of the pesticide. The superior effect of microbial inoculants on chlorpyrifos degradation would be attributed to enhanced enzymatic activity in the soil as compared to physical and chemical treatments. The dehydrogenase activity in the soil was found to be significant with respect to treatments. Soil treated with microbial inoculants of Pseudomonas fluorescens recorded the highest dehydrogenase activity followed by soil with Trichoderma viride and combination of Pseudomonas fluorescens and Trichoderma viride. According to Jastrzębska (2011) dehydrogenase enzyme was very sensitive to chlorpyrifos and its activity decreased in presence of chlorpyrifos content. In our study, we found that activity of dehydrogenase in biological treatments was comparable with initial activity estimated at start of the experiment. This indicated that the dehydrogenase activity was maintained in biological treatments even in presence of chlorpyrifos at 60th day of incubation. However, this trend was not observed in chemical and physical treatments.
Phosphatase activity in the soil was found to be significantly increased with respect to treatment application. At 60th day, maximum activity was recorded in treatment with Trichoderma viride, followed by soil treated with a combination of Pseudomonas fluorescens and Trichoderma viride while the lowest activity was recorded in control. The change in phosphatase activity of the chlorpyrifos treated soil in each treatment was different. Phosphatase activity of physical and chemical treatments recorded lower values. Decline in the phosphatase activity could be due to inhibitory effect of chlorpyrifos or its metabolites directly on phosphatase enzyme (Das and Mukherjee 1999). According to Aziz et al. (2021), amended and unamended chlorpyrifos contaminated treatments showed decreased phosphatase activity up to 15th day and thereafter it stabilized and slowly increased at the end of incubation. Significant recovery of enzymatic activities might be due to removal of chlorpyrifos through degradation (Valle et al. 2006). Accelerating effect of biological treatments consisting of microbial inoculants on phosphatase activities might be due to the introduction of more microbial communities. This will enhance the production of extracellular enzymes which was capable of degrading the chlorpyrifos (Tejada et al. 2009). Hydrolysis of variety of organic phosphomonoesters was catalyzed by the phosphatase enzyme and has been widely used for the degradation of organophosphorus pesticides (Kanekar et al. 2004).
Effect of treatments on urease activity in soil was found to be significant at the 60th day. Treatment with microbial inoculants in combination of Pseudomonas fluorescens and Trichoderma viride recorded the maximum activity of urease. Urease activity was lower in soil treated with chemicals. T1 (control) and T3 (Fenton reagent) recorded the lowest urease activity at 60th day. Inhibitory effect of chlorpyrifos on urease activity was noticed by Wang et al. (2010) which could be due to inhibitory effects of chlorpyrifos oxon formed during the degradation.
There was decrease in the soil pH in all the treatments imposed during degradation. Bisht et al. (2019) reported that pH of soil was reduced significantly during the degradation of pesticides such as chlorpyrifos. The lowering of the soil pH might be due to dehalogenation of the compound during its degradation, which leads to the formation of acids. Treatment with combination of physical agents (Sunlight + Ultra violet) showed higher soil pH which was followed by biological treatments consisting of microbial inoculants. The treatment with Trichoderma viride recorded higher values among the biological treatments. It was observed that addition of microbes did not lower the soil pH to a greater extent when soil was incubated with chlorpyrifos content in laterite soil. Treatment with chemical agents (Hydrogen peroxide- + Fenton reagent) recorded lowest pH values at the 60th day of incubation. The decrease in pH in soil with chemical treatments might be due to application of H2O2 and Fe2SO4 leading to the formation of acids. Under the acidic condition hydroxyl free radicals formed from the Fenton reagent, led to increase in the H+ ions in the soil and thus reduced the pH of soil. It is evident from the study that though chemical agents are effective degraders of chlorpyrifos, there was drastic reduction in soil pH, which could further acidify the laterite soil leading to deterioration of soil health.
The degradation of chlorpyrifos released its products into the soil during the degradation experiment. The chloride ions in soil were found to be increased though non-significant, during the degradation process. Highest chloride ions were observed in soils treated with microbial combination of P. flourescens and T. viride followed by chemical and physical treatments whereas control showed the lowest. According to Feng et al. (1997), release of chloride ion was stoichiometrically and concurrently related to removal of chlorpyrifos. During the degradation of chlorpyrifos, dehalogenation takes place which leads to increased levels of chloride ions in soil. A significant increase in the phosphate ions were observed during the degradation of chlorpyrifos. The phosphate content recorded in natural degradation experiment was similar to the values recorded under the influence of different treatments. Tang et al. (2011) confirmed that hydrolysis and oxidation of chlorpyrifos led to the release of phosphate ions to the soil. Adelowo et al. (2015) reported that organophosphates were used by microorganisms as a source of phosphate ions for their growth and metabolism during the degradation process.
Higher content of sulphate ions was observed during the degradation process. Chlorpyrifos compounds undergo hydrolytic desulphurization which led to the formation of chlorpyrifos oxon (Nolan et al. 1984). Martin (1966) reported that sulphur containing pesticides release the sulphate ions to the soil during the degradation.
Treatments with microbial inoculants showed a higher amount of sulphate ions in soil than chemical and physical treatments. Chlorpyrifos treated soil with combination of microbial inoculants (Pseudomonas fluorescens + Trichoderma viride) showed the highest sulphate ions in soil whereas soil treated with UV light showed the minimum sulphate content.
Nitrate ions also showed an increase in content during chlorpyrifos degradation. Treatments with Trichoderma viride as biological treatment and sunlight as physical treatment showed highest nitrate ions in soil. Treatments with combination of microbial inoculants (Pseudomonas fluorescens + Trichoderma viride) had lower nitrate content on comparison with T. viride and UV light. Earlier, Affam and Chaudhuri (2013) observed increased nitrate content during the photocatalytic degradation of chlorpyrifos.
Among the treatments biological treatments consisting of microbial inoculants showed the highest removal of chlorpyrifos followed by chemical and physical treatments. Hydroxyl radicals in the Fenton reagent might be responsible for the degradation of organic compounds whereas iron acts as catalyst and speeds up the degradation process. Marican and Duran-Lara (2018) reported that the combination of hydrogen peroxide and iron salts in the Fenton reagent act as a strong oxidizing agent and led to the formation of hydroxyl radicals which could oxidize the organic molecules. Physical degradation was a less effective method among the different treatments, imposed in the study which was in agreement with earlier work by John and Shaike (2015).