The Effect of Paraquat, Difenoconazole, and Butylphenyl Methylcarbamate (BPMC) on CO2 Emissions and Phenolic Acids in Peat Soil

Pesticides are widely used in agriculture, including on peat soil. The objective of this study was to analyze the effect of the application of paraquat, difenoconazole, and butylphenyl methylcarbamate (BPMC) on CO2 emissions and concentrations of phenolic acids in a peat soil. Peat soil sample was taken in District of Pulang Pisau, Central Kalimantan. The peat soil was applied with 1.89 mg kg-1 paraquat, 1.72 mg kg-1 difenoconazole and 1.65 mg kg-1 butylphenyl methylcarbamate (BPMC), then the soil was incubated for 1, 2, 4 , 5, 7, 10, 14, 21, 26 and 30 days. The results showed that the application of pesticides on peat soil increased CO2emission, and decreased CH4 emission and phenolic acid concentrations up to 30 days of incubation. The CO2 emmisions were derived from C of degraded pesticides and from C of phenolic acids, although the oxidation reaction was not accompanied by the change of soil pH.


INTRODUCTION
The improvement of marginal land can be an option to overcome the national food security problem, and peatland is one of the potential lands that can be developed into new agricultural land (BBSDLP 2008). Peatland in Indonesia is generally formed by wood residues containing higher lignin compared to the peatland in the temperate climate area (Stevenson 1994). During anaerobic condition, degradation of peat containing high lignin will produce phenolic acids. Several phenolic acids identified in soil are vanillate acid, p-coumaric acid, p-hydroxybenzoic acid, salicylic acid, galate, sinapate, gentisate and syringic acid (Tsutsuki andKondo 1995, Sabiham 2010). A previous study showed that phenolic acids tend to be phytotoxic for plants and can inhibit the growth of plants (Stevenson 1994). Furthermore, the carbon emission from peatland has caused many problems for environment. The study of Barchia (2002) reported that the carbon emission from peaty paddy field in 78 IH Fitriyani et al.: The Effect of Paraquat, Difenoconazole and BPMC on CO 2 Emissions CO 2 ha -1 yr -1 . In addition, the study of Nurzakiah et al. (2014) reported that the CO 2 fluxes from peatland in dry season are higher than in the rainy season. The major factors influencing the CO 2 emission on peat soils are the soil water content and groundwater level.
The use of pesticides on agricultural land including peatland can control effectively weed growth, pest and plant disease. The most wellknown pesticide used in the farming activities is bipiridilium herbicide that contains paraquat as the active ingredient (Susilawati et al. 2005). The study on CO 2 emissions on peat soil applied with pesticide has not been widely conducted in Indonesia. The previous study of Bartha et al. (1967) indicated that application of high dosage of carbamate pesticide (250 mg L -1 ) on sandy loam soil in New Jersey decreased the CO 2 concentrations in the soil after 10 days of incubation. Another study by Sahid and Kasim (1994) showed that application of alachlor and metolachlor herbicides with a dosage of 20 and 150 mg L -1 on mineral soil has increased CO 2 concentration until 25 days of incubation due to the effect of microbe respiration. Both studies have given an insight to conduct the current study that focuses on the effect of pesticide application using common dosages applied by the farmers on CO 2 emissons and phenolic acid concentrations in peat agricultural soil.
The objective of this study was to analyze the effect of pesticide application on CO 2 emissons and phenolic acid concentrations in peat soil.

Study Site and Pesticide Use
The soil sample used in the current study was collected from Pulang Pisau District, Central Kalimantan Province at a depth of 0-20 cm.
Several types of pesticide were applied in this study including herbicide with paraquat as an active ingredient, a fungicide with difenoconazole, and insecticide with butylphenyl methylcarbamate (BMPC). The dosages of pesticides applied were 3 liters ha -1 Gramoxone (containing paraquat), 1 liter ha -1 Triazole (containing difenoconazole) and 1 liter ha -1 Bassa (containing BPMC). The amount of each pesticide applied in each treatment was about 20 ml, which equals to a certain dosage of active ingredient per kg peat soil, i.e. paraquat 1.89 mg kg -1 , difenoconazole 1.72 mg kg -1 , and BPMC 1.65 mg kg -1 . The incubation vessel used in this study was made from PVC tube with a diameter of 4 inches.

Research Design
The study was conducted using a Completely Randomized Design (CRD) with two factors and three replicates. The first factor was the types of pesticide, namely P = Paraquat, D = Difenoconazone, V = BPMC, and T = without pesticide. The second factor was the incubation periods, namely 01 = 1 day of incubation, 02 = 2 days, 04 = 4 days, 05 = 5 days, 07 = 7 days, 10 = 10 days, 11 = 11 days,14 = 14 days, 21 = 21 days, 26 = 26 days and 30 = 30 days of incubation. The incubation periods were determined based on pesticide retention time in soil (DT50), which could take up up to 2 weeks (EPA 1998).

Treatment Application
The experiment was conducted as follows: (1) About 264 g of peat soil (water content 316%) was placed into an incubation vessel; (2) After that, about 20 ml of pesticide was applied to the soil; (3) Then, about 10 mL NaOH solution was standardized using oxalic acid to capture CO 2 and the tube containing the NaOH solution was placed inside the vessel. The concentration of NaOH was 0.95 mol L -1 ; and (4) The incubation vessel was closed with a plastic wrap and an elastic band to make sure that there would be no air coming into the vessel. The illustration of incubation vessel was shown in Figure 1.

CO 2 Emission Measurement
Measurement of CO 2 emissions was carried out using a titration method proposed by Verstraete (1987) in Anas (1989). The measurements of CO 2 emissions were conducted at the end of each incubation period by titrating the NaOH solution with HCl solution that had been standardized previously using sodium borate. The emission of CH 4 was determined using Gas Analyzer.

Phenolic Acid Measurement
The types and concentrations of phenolic acids in the peat soil were determined at the end of each incubation period. About 5 g of soil sample was added with 25 ml of aquadest and then the suspension was shaken for 30 minutes. Then, the suspension was centrifuged at 3000 rpm for 60 minutes. About 20 ml of the supernatant was filtered using 0.45 µm millipore filter. After that, the filtrate was analyzed using HPLC (Angeles 2005). The phenolic acids including ferulic acid, sinapic acid, p-coumaric acid, vanillate acid, syringic acid andphydroxybenzoic acid were analyzed.
Initial chemical properties of the soil including organic-C content, total-N, fiber content, decomposition rate and phenolic acid concentrations were determined. The soil pH was measured before and after the incubation period.

Data Analysis
The data of CO 2 emissions were simulated using the first order kinetic equation using Sigmaplot software version 12.3 (Sugiyono 2007). In addition, the correlation test between CO 2 emission and phenolic acid concentration was performed. The first order kinetic equation used in this study is as follows: f= a(1-exp (-b*x) ) f = CO 2 emission (mg kg -1 ) a = maximum CO 2 emission (mg kg -1 ) b = CO 2 emission rate constant (hr -1 ) x = observation period (hr) exp = 2.7182

Chemical and Physical Properties of Peat Soil
The peat soil used in the current study is catergorized as oligotrophic according to its chemical properties (Hardjowigeno 1996). The chemical properties of the peat soil are presented in Table 1.
The pH of peat soil is 4.69, which is higher than the pH of natural peat soil (pH 3-4). This is probably due to the soil has been used for agricultural activities since 1980, therefore, the soil organic matter might have been leached through the drainage water, resulting in an increase of soil pH.
The organic-C content in the soil is 39.5%, which confirms that the soil is a peat soil. According to USDA classification, the soil that contains organic matter between 30% -50% is considered as Histosol (USDA 2014 properties 46%, with the colour of 10YR 3/1 and pyrophosphate index of 2, therefore, the peat is categorized as Hemic according to the criteria of peat maturity level (Lynn et al. 1974). The phenolic acids identified in the peat soil used in the current study were ferulic acid, sinapic acid, and vanillate acid (Table 2). According to Tan (1993), these phenolic acids are derived from lignin containing materials, i.e. hardwood and softwood.
On the other hand, p-hydroxybenzoic acid and pcoumaric acid were not identified in the soil sample.
The results confirmed that the peat soil used in the current study is not formed from grass residues.
The studies of Driessen (1978) and Stevenson (1994) indicated that the ombrogen peat in Indonesia contains around 64% of lignin derived from wood residues.

CO 2 Emissions After Incubation Period
The application of pesticides increased the maximum CO 2 emissions during 30 days of incubation ( Figure 2). The amount of maximum CO 2 emission in each treatment followed the order of paraquat (159.62 mg CO 2 kg -1 ) > without pesticide application/control (156.95 mg CO 2 kg -1 ) > difenoconazole (154.97 mgCO 2 kg -1 ) > BPMC  (148.61 mg CO 2 kg -1 ). The results showed that in general CO 2 emission in the soil without pesticide application (control) was higher than that in the soil applied with pesticides, except that in the soil applied with paraquat. The results could also mean that pesticide application on peat soil can reduce CO 2 emissions, except in the paraquat treatment.
According to the first order kinetic equations, the constants of CO 2 emissions (b) derived after pesticide applications are paraquat (59 × 10 -2 h -1 ) > BPMC (56 × 10 -2 h -1 ) > difenoconazole (51 × 10 -2 h -1 ) > without pesticide application/control (44 × 10 -2 h -1 ). The results showed that the organic-C degradation rate in the peat soil applied with active ingredients of pesticides varies. In addition, the results indicated that pesticide application on peat soil increased CO 2 emissions. The increase of CO 2 emissions in the soil applied with pesticides was probably due to the decomposition or oxidation of C derived from pesticides into CO 2 . The degradation periods (DT50) of each pesticide active ingredient vary. The study of Djojosumarto (2006) reported that DT50 of BPMC is 7-9 days, paraquat is 14 days and difenoconazole is 53 days. The degradation period of pesticide active ingredients was influenced by its chemical structure. Paraquat and BPMC have more simple chemical structures compared to difenoconazole, therefore, they are easily degraded, which further result in the highest maximum CO 2 emission. Even though the peat soil without pesticide application (control) releases a high amount of maximum CO 2 emission, however, the first order kinectic equation of this treatment has small constant value instead. This phenomenon may be due to the delayed rate of initial C degradation that causes the average degradation rate to be small. The amounts of CO 2 emissions derived from the degradation of the active ingredients of paraquat, difenoconazole, and BPMC were 1.39 mg kg -1 CO 2 , 1.18 mg kg -1 CO 2, and 1.16 mg kg -1 CO 2 , respectively. The total amount of CO 2 emission derived from the degradation of all the active ingredients is still lower than the CO 2 emission from the control treatment (without pesticide application) after 30 days of incubation. The results indicated that the source of CO 2 emission is not only from pesticide degradation but probably also from phenolic acid decomposition. The study of Manna et al. (2013) showed that fungicide application on paddy soil can increase CO 2 emission from the soil, however, the increase is not only due to fungicide application, but also due to microorganism activity and alkaline phosphatase activity in the soil.

CH 4 Emissions After Incubation Period
Besides CO 2 , methane (CH 4 ) is another form of carbon emission released into the atmosphere. Methane (CH 4 ) is one of the greenhouse gases that can be emitted by soil through methanogenic bacteria activity. About 80% of CH 4 is produced biologically by a group of anaerobic bacteria in a reductive environment (Ehhalt and Schmidt 1978) with a potential redox lower than -200 mV, and the bacteria grow optimally at temperature of 30-40 o C (Kirk 2004). Methane (CH 4 )will never be produced before the reduction processes of oxygen, nitrate, ferrum (III), mangan (IV) and sulfate have been finished (Smith et al. 2003). This process is mostly influenced by temperature, organic matter content, pH, humidity, and potential redox of the soil (Moore and Dalva 1993).
The CH 4 emissions from peat soil during 30 days of incubation period showed a decreasing trend in each treatment. The wet soil condition during the incubation period, although it was not stagnant, could create a perfect condition for methanogenic bacteria to grow, resulting in CH 4 emissions. In addition, the pH of peat soil (pH 4.5-5 at day 1 until 7 of measurement) was one of the supporting factors for methanogenic bacteria to grow. Especially at the beginning of incubation period, the CH 4 emissions in the soils applied with pesticides were higher than that in the control treatment, although the difference was not significant.

Phenolic Acid Concentrations After Incubation Period
The concentrations of phenolic acids in the peat soil applied with pesticides at 30 days of incubation were in general lower than that at the beginning of incubation periods (Table 4). In addition, the total concentrations of phenolic acids in the soil applied with pesticides were lower compared to that in the control treatment (without pesticide application). The decrease of phenolic acid concentrations after application of pesticides on peat soil indicated that the oxidation of C derived from phenolic acids has been occured. The types of phenolic acids identified in the peat soil applied with pesticides is various for each treatment.
In the soil with paraquat, difenoconazole and BPMC applications, all types of phenolic acids can be detected (ferulic acid, syringic acid, sinapic acid, p-coumaric acid, vanillate acid, and p-hydroxy benzoic acid), while in the soil without pesticide application, only 5 phenolic acids can be detected, namely ferulic acid, sinapic acid, pcoumaric acid, vanillate acid and p-hydroxybenzoic Table 5. Respiration of soil microorganism measured on peat soil applied with pesticides.  Average Control acid. The differences may be due to the transformation of phenolic acid into another form of phenolic acid. The different active ingredients contained in each pesticide will bring out the different phenolic acid transformation processes. The result is in accordance with the types of phenolic acids initially identified in the peat soil, i.e. ferulic acid, sinapic acid, and vanillate acid (Table 2), and subsequently after incubation, other phenolic acids were detected such as syringic acid and p-coumaric acid. According to Stevenson (1994), the transformation process of phenolic acids into different forms is mediated by microbes, then the phenolic acids are changed into quinon by phenoloxidase enzyme, and the phenolic acids will be degraded into simple organic compounds, such as humic acid and fulvic acid. The study of Gupta et al. (1986) showed that the sinapic acid degradation is mediated by Rhodotorula glutinis, which produces syringic acid, 3-methyl gallate acid and 2.6-dimetoku-1.4 benzoquinone. The study of Anwar et al. (2004) about the oxidation of peat soil using CuO (cupric oxide) observed that phydroxybenzoic acid was firstly decomposed, followed by syringic acid and vanillate acid. Similar pattern was also oberved in the current study, particularly in the peat soil without pesticide application (control treatment) and peat soil applied with paraquat. This result confirmed that the different pesticide active ingredients applied on peat soil will result in the different phenolic acid transformations. This difference is probably related to the different impacts of soil microbial activity on the different pesticide active ingredients.

Relationship between CO 2 Emission and Phenolic Acid Concentration
The coefficient of the correlation between CO 2 emission and phenolic acid concentration was 0.39 ( Figure 4). The result indicated that CO 2 emission is weakly correlated to phenolic acid concentration; the higher CO 2 emission, the lower phenolic concentration is (Sugiyono 2007). The points in the figure showed that the increase of CO 2 emission could be originated from the oxidation of phenolic acid C.
The initial concentration of sinapic acid measured in the peat soil was very high, i.e. 86.47 mg L -1 (Table 2), however, after incubation the concentrations of sinapis acid in the peat soil without pesticide application decreased up to 1.75 x 10 -2 mg L -1 (Table 4). Furthermore, the concentrations of phenolic acids in the peat soil applied with pesticides (paraquat, difenoconazole and BPMC) also decreased after incubation. According to Stevenson (1994) decomposition and degradation of phenolic acids will produce CO 2 and H 2 O as well as phenolic acid in different forms. The degradation process of phenolic acids is called demethoxylation process and the unveiling of ring structure. The unveiling of ring structure causes decomposition that produces CO 2 , CH 4 , and H 2 O, depending on soil redox potential. Therefore, we may conclude that the CO 2 emissions released by peat soil in each treatment are from degradation and decomposition of phenolic acids and the active ingredients of pesticide applied, and microorganism respiration.
The results of respiration measurements showed that the amount of CO 2 released by respiration of soil microorganism decreased during the incubation period ( Table 5). The phenomenon is probably caused by the decrease of carbon source in the soil during the incubation period. The study of Arimurti (1997) reported that the respiration of soil microorganism in mineral soils taken from several land uses in West Kalimantan is about 8.80 -11.08 mg C-CO 2 kg -1 day -1 . In addition, the study of Adhayanti (1997) showed a lower microorganism respiration measured in mineral soils taken from several land uses in Lampung, i.e. 4.57-8.57 mg C-CO 2 kg -1 day -1 . Based on the results of both studies, both mineral soil and peat soil have no significant different in microorganism respiration level.

pH of Peat Soil
The pH of peat soil applied with pesticides showed a constant trend, i.e. 4.30 -5.05 (Table 6), indicating that the application of pesticides on peat soil did not change the soil pH. The result of current study is in contrast with the study of Usui (2011) that showed that the application of herbicide (HA -ACN, (2-amino-3-chloro-1,4-naphthoquinon) 9%) on paddy soil increased the soil pH and decreased the CO 2 emissions along with the accumulation period. On the other hand, according to Bennicelli (2008), the application of the pesticide (Glyphosphat) on Histosol increased the dehydrogenase enzyme activity, pH, Eh, and CO 2 production after 42 days of incubation.

CONCLUSIONS
The application of pesticides on peat soil, especially paraquat has increased the CO 2 emissions, while the application of difenoconazole and BPMC has decreased the CO 2 emissions. In addition, the application of pesticides decreased the amount of phenolic acids in peat soils. The CO 2 emissions were expected resulting from the oxidation of phenolic acid C and the active ingredients of phenolic acid C and the active ingredients of pesticides applied. On the other hand, the application of pesticides on peat soil showed no significant effect on CH 4 emissions.