Kinetics of Biological Treatment of Low Level Pesticide Wastewater

Pesticides are chemical substances intended to protect food crops and livestock from pests in order to promote agricultural productivity and protect public health. Contamination of soil, air and water and threat to human and animal health are the major constraints in the use of pesticides. Treatment of pesticide contaminated water is, therefore, paramount. Biological treatment provides the most economical option when compared to other treatment methods. The aim of the study was to develop a safe and effective in the farm biological treatment for low level agricultural pesticide wastewater. The degradation of the fungicide captan was evaluated under batch and continuous modes of operation with a retention time of 15 days. The initial cell number (30.1×10 cells/mL) in the soil water mixture first declined with time during the 24 h reaching 15.6×10 and 11.1×10 cells/mL in the batch and continuous bioreactors, respectively. This was due to the inhibitory effect of pesticide on some of the soil microbial species that had less tolerance to captan at the initial concentration of 144 mg L. Then, the microbial population started growing, reaching its maximum after 5 and 12 days from the start in the batch and continuous bioreactors, respectively. The lag period and the specific growth rate for the batch bioreactor were 22 h and 0.096 h, respectively. A captan degradation efficiency of 89.6% was achieved after 10 days in the continuous bioreactor compared to a degradation efficiency of 100% after 5 d in the batch bioreactor. This study showed that the effluent from the continuous bioreactor has a captan concentration of 12 mg L which is not acceptable for livestock water according to Health Canada Guidelines. A half life of 52 h is observed in the batch bioreactor.


INTRODUCTION
Pesticides provide the primary means for controlling organisms (fungi, bacteria, mites, insect's rodents, nematodes and undesirable plants) that compete with man for food and fibre or cause injury to man, livestock and crops. They are classified based on the pest they control into several classes: fungicides, bactericides, acaricides, insecticides, rodenticides, nematicides and herbicides. The worldwide pesticide expenditures in 2007 were 39.4 billion dollars accounting for 2.37 billion kg of pesticide. Pesticide expenditures account for 13-22% of the total cost of production per hector. However, for every dollar spent on pesticide farmers receive an additional of $4-33 in revenue from increased yield, depending upon crop rotation and year of production (Horowitz and Lichtenberg, 1993;Osteen and Livingstion, 2007).
Pesticides played a vital role in increasing agricultural production and permitting the economic production of wide ranges of vegetable, fruit, cereal, forage, fibre and oil crops which now constitute a large part of successful agricultural industry in many countries. They lower crop losses, increase revenue to farmers because of the additional marketable yield obtained with their use and thus lower the cost of production per unit output (Horowitz and Lichtenberg, 1993). Other benefits of pesticides includes: (a) reduced uncertainty of crop loss from pests, (b) increased profit to farm input suppliers (machinery, fertilizer, chemicals and seed companies) from increased sale, (c) benefit to consumers through decreased price of raw foods or improved quality of food products and (d) benefit to Science Publications AJES society as whole (farmers, consumers, farm suppliers, food processors) from increased employment opportunities and expanded export of food products (Oerke and Dehne, 2004;Cooper and Dobson, 2007).
However, pesticides are toxic chemicals that can adversely affect people, pets, livestock, wildlife and desirable plants in addition to the pests they are intended to destroy (Centner, 1998;Wilson and Tisdell, 2001;Ridgway et al., 1978). Pesticide residues remain in the containers and application equipment after pesticides are applied to target areas. These residues are removed from applicators by rinsing with water resulting in the formation of a toxic wastewater that represents a disposal problem for many farmers (Kearney et al., 1988). Currently, disposal of pesticide wastewater is carried out by several methods including (a) land cultivation, (b) dumping on land, in ditches, in lagoons and in soil pits and in extreme cases in sewers and streams near the rinsing operation, (c) use of evaporation ponds and (d) land filling (Al Hattab and Ghaly, 2012). These methods of disposal are totally unsafe, as the surface run off will reach streams, rivers and lakes and the infiltration of the wastewater into the local soil will eventually end up in ground water. The ecological impact of unsafe disposal of pesticides can be significant depending on the type and concentration of pesticides in the wastewater. Therefore, pesticide containing wastewater must be properly treated. The treatment methods currently used for pesticide containing wastewater include (a) incineration, (b) chemical treatment such as O 3 /Uv oxidation, Fenton oxidation and hydrolysis (c) physical treatment using inorganic and organic absorbents and (d) biological treatment such as phytoremediation, composting and bioaugmentation. These treatment methods either require land or are expensive and suffer from variability of effectiveness (Balestra and Misaghi, 1997;Winterlin et al., 1989;Al Hattab and Ghaly, 2012).
The aim of the study was to develop a safe and effective on farm biological treatment for low level agricultural pesticide wastewater and to evaluate its mode of operation (batch vs continuous).

EXPERIMENTAL APPARATUS
The pesticide treatment system ( Fig. 1) consisted of pesticide contaminated wastewater feeding system, 2 bioreactors (batch and continuous) and 2 effluent collection tanks.

Bioreactors
Two identical bioreactors (each of approximately 15.7 L volume) were constructed from Plexiglas material and designed to hold 10 L each of soil-water-pesticide mixture plus a head space. Each bioreactor was constructed of 1.0 cm thick Plexiglas cylinder of 20 cm diameters and 50 cm height. The bottom of the cylinder was made of 1.0 cm thick circular Plexiglas plate of 20 cm diameter which was glued to the cylinder. The top lid was made of 1.0 cm thick circular plate of 22 cm diameter which was secured into the cylinder using six stainless steel screws and wing nuts. The lid had three 1.5 cm diameters holes: one in the center for the mixing shaft and the other two holes were used for the wastewater input and air exhaust. The reactor contents were mixed using as 55 rpm induction motor (Japan Servo Company, Tokyo, Japan) which was mounted on the reactor lid and connected to a 1.5 cm diameter mixing shaft. A 15 cm diameter propeller was placed on the shaft at 2 cm from the diffuser. An air inlet port was provided at the bottom of each cylinder and was fitted with 20 mm PVC elbow. An air diffuser (Dynamic Aqua-Supply Ltd., Sydney, Canada) was attached to the elbow inside the cylinder. The diffuser diameter and height were 15 and 25 mm, respectively. The other end of the elbow was connected to Tygon tubing which connected the diffuser to the air supply unit that consisted of an air compressor (3/4 HP Shanborn model MCIFC 75-715), a pressure regulator (Model ZFMQ000PR, Millipore Pressure Regulator, Massachusetts, USA) and a flow meter (Model 60648, Cole Parmer, Chicago, Illinois, USA).

Feeding System
The wastewater feeding system consisted of storage tank and a distribution manifold with a set of three values. A 100 L plastic tank was used to store the waste water. The tank was fitted with a stirring paddle driven by a 1/12 HP electric motor (Model 5SCP10FG17AX, General Electric, Mississauga, Ontario) mounted on the tank cover. A feeding and ventilation ports were also provided on the tank cover. A 5 cm ball valve was connected to the fitting on the tank from one side and to the manifold from the other side. The other two valves on the manifold controlled the flow into the bioreactors.

Collection Tanks
Two 25 L plastic carboys (Cat No.02-961B) Fishers Scientific, Montreal, Quebec) were used to collect the effluent from the bioreactors.

Pesticide
The fungicide Captan 80-WP (C 9 H 8 ClNO 2 S) was chosen for this experiment because it is one of the most heavily used pesticide in the Province of Nova Scotia. The properties and structure of captan 80-WP are shown in Table 1. It is classified as a protectant eradicant fungicide and is one of the most used pesticides today due to its effectiveness in controlling a wide variety of fungal diseases ( Table 2). The formulation of this pesticide is a microfine wettable powder containing 80% active ingredient (75.5% N-[trichloromethyl)thio]-4-cyclohexene-1,2-dicarboaimi de and 4.3% related derivatives). The balance (20%) is made of mineral dust and wetters dispersants. This formulation is preferred by many growers since it minimizes visible residue and thus provides an excellent vegetable and fruit finish.

Soil
The soil used in this experiment was obtained from an agricultural field in Stewiack, Nova Scotia. It was obtained from a field where 50 kg/acre of urea, 75 kg/acre of 17-17-17 NPK fertilizers and 1500 kg of liquid manure were applied annually. The top trash coves of the soil was scraped away and the soil was removed by a shovel from the top 30 cm and placed in a heavy duty (16 m thick) Polyethylene bag and transported to the waste management laboratory. The soil was used as a source of a mixed microbial culture.

Experimental Protocol
Two experiments were carried out: a batch experiment which was run for 15 days and a continuous experiment which was run with a retention time of 15 days. Eighteen grams of captan dust (14.4 g active material) were added into the water (100 L) in the feeding tank to provide a captan concentration of 144 mg L −1 in the wastewater. The initial soil:water ratio in the bioreactors was 1:3. The soil (0.75 L) was placed in the bioreactors and the water (2.25 L) was then added. The batch bioreactor was filled up to the 10 L level. For the continuous bioreactor, the feeding pump was adjusted to provide a flow rate of 0.46 mL/minute in order to achieve the 15 day retention time. The mixing motor and the air compressor were turned on. The air flow rate was set at 10 L −1 min -1 (1 v/v/min) using the flow meter. Samples were collected from each bioreactor on a daily Science Publications AJES basis and vacuum filtered using a coarse filter paper (P8 Grade, Fisher Scientific, Canada) to remove any soil particles. The filtered samples were used for plate count and pesticide analyses.

Plate Count
The plate count was performed on the initial soil water mixture before the addition of pesticide and on the samples collected from the bioreactors. For each sample, 5 test tubes were filled with 9.9 ml of pepton. A 1:100 dilution was prepared by pipetting 0.1 mL of the initial dilution into the first tube. This was then mixed well and 0.1 mL was placed on an agar lined Petri dish and spread over the entire surface using a glass wand. The wand was cleaned with reagent alcohol and flamed before each use. From the diluted tube, a 0.1 mL was transferred to another tube contains 9.9 mL pepton to produce a dilution of 1:1000. The mixture was mixed well and 0.1 mL was transferred into another Petri dish. The same procedure was repeated to produce final dilutions of 1: 100, 1:1000, 1:10000, 1:10000, 1:100000 and 1:1000000. Three plates of each dilution were carried out. All the inoculated Petri dishes were then placed in a temperature control incubator (Model 2020, Sheldon Manufacturing Inc, Oregon, USA) at 35°C. The plates were examined after 24h and the dilution that produced reasonable countable number of calories were selected. The total colonies on each Petri dish were calculated using a plate counter (Model 7-901, Fisher Colony Counter, New York, USA). The population was then determined by multiplying the number of colonies by the dilution factor.

Pesticide Analysis
Three ml of the sample were mixed with 3 mL of the hexane/ether solvent (95:5) in a test tube. The test tube was then caped and heated at 115°C for 30 min followed by a cooling period to a temperature below 0°C. When the sample was frozen, the liquid solvent was withdrawn from the top. 2 µL of this sample were then used for injection into a gas chromatographer (5890 series II, Hewlett Packard, California, USA). The initial and final concentrations of captan in each unit were determined. The chromatograph was calibrated by injecting 1.0 µL of the extracted captan mixture into the 25 m X 0.2 mm capillary column. 1.0 µL of the extracted sample was then injected into the column. The column temperature was first maintained at 40°C for three minutes and then increased at the rate of 10°C per minute until a temperature of 270°C was attained. The column was then maintained at 270°C for five minutes. The injection port was set at 25°C while the flame inonization detector was set at 250°C. Helium was used as a carrier gas at a flow rate of 1.2 mL −1 min.

Microbial Growth
The growth of the mixed microbial population in the batch and continuous bioreactors are shown in Fig. 2. The initial cell number in the soil water mixture in both bioreactors was 30.1×10 6 cells/mL. The number of cells first declined with time during the first 24 h reaching 15.6×10 6 cells/mL in the batch bioreactor and 11.1×10 6 cells/mL in the continuous bioreactor. This was due to the inhibitory effect of pesticide on some of the soil microbial species that had less tolerance to captan at the initial concentration of 144 mg L −1 . The lower value (29% lower) observed in the continuous bioreactor compared to that of the batch bioreactor was due to the loss of microbes from that bioreactor with the effluent. Wainwright and Pugh (1975) reported declined trend of bacterial population (from 16.5×10 6 to 14×10 6 cells) during first 48 h after application of 25 µg/g captan to field soils. Agnihotri (1971) reported a significant reduction (from 2.3×10 5 to 0.  (Lipthay et al., 2003;Tuxen et al., 2006), phenoxy acids (Chilton et al., 2005), 2,4-D (Broholm et al., 2001), 2-(2,4-dichlorophenoxy)propionic acid (Lipthay et al., 2003).
The four phases normally encountered in a batch operation (lag, exponential growth, stationary growth and death phases) were observed with the batch bioreactor. After the initial lag period, the microbial population in the batch bioreactor started to increase with time reaching a maximum of 113.9×10 6 after 120 h (5 days from the start), remained relatively constant till the 240 h (for 5 days) and then started to decline. The microbial population of the continuous bioreactor also increased but at slower rate than that of the batch bioreactor due to the continuous loss of microbes from the bioreactor with the effluent.

Fig. 2. Microbial growth
It reached a steady state condition after 288 h (12 d from the start) when the rate of microbial growth in the bioreactor was equal to the rate of microbial loss from the bioreactor with the effluent. The microbial population in the continuous bioreactor at the steady state was 86.5×10 6 cells/mL (75.94% of that in the batch bioreactor).
The lag period and specific growth rate were determined from the batch operation data according to the procedure described by Ghaly et al. (1989) as shown in Fig. 3. The lag period and the specific growth rate were 22 h and 0.096 h −1 , respectively. Radianingtyas et al. (2003) reported 18 h lag period and 0.014 h −1 specific growth rate while degrading 4-choloroaniline (1 mM) in a batch reactor with a bacterial consortium comprising four different species isolated from an Indonesian agricultural soil. Lappin et al. (1985) reported 18 h lag period and 0.09 h −1 specific growth rate while degrading mecoprop (1 gm L −1 ) in a batch reactor with a microbial community isolated from wheat root systems. Rhee et al. (1997) were able to demonstrate pyridine degradation (3 mM) in a batch reactor and reported 13 h lag period and 0.08 h −1 specific growth rate using denitrifying bacteria isolated from industrial wastewater.
The lag period and net specific growth rate (µ net ) was also determined for the continuous bioreactor during the initial nonsteady state period using the same procedure as shown in Fig. 4. The lag period and net specific growth rate for the continuous bioreactor were 26 h and 0.045 h −1 , respectively. Krishna and Philip (2009) conducted studies on biodegradation of carbofuran at a concentration of 150 mg L −1 in a continuous reactor and reported 48 h lag period and 0.3928 d −1 specific growth rate using carbofuran enriched cultures.   The results indicated that the rate of microbial loss with the effluent (k r ) was 0.051 h −1 .

Pesticide Degradation
The pesticide concentrations in the effluent samples taken from the batch and continuous biorearctors overtime are shown in Fig. 5. The pesticide concentration in the batch bioreactor started to decline with time reaching zero value after 120 h (5 d) from the start. Also, the pesticide concentration in the effluent of the continuous bioreactor declined with time reaching a constant value of 15 mg L −1 after 288 h (10 d). Thus, a removal efficiency of 89.6% was achieved after 10 days with the continuous bioreactor compared to a removal efficiency of 100% after 5 d with the batch bioreactor. Megadi et al. (2010) reported a complete degradation of fungicide captan after 6 days during growth of Bacillus circulans in the Mineral Salt Medium (MSM) containing 0.1% captan. Buyanovsky et al. (1988) reported 33% degradation of captan (with an initial concentration of 50 mg L −1 ) after 2 weeks (including lag phase of 2 days) of incubation with soil bacteria, no further degradation of captan was observed after the 2 weeks period. The maximum permissible value for captan and metabolites in livestock water is set 13 µg L −1 (HC, 2010). However,

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AJES a Canadian drinking water quality guideline for captan has not been developed (HC, 2010). The batch bioreactor used in the study achieved 100% removal of captan while the effluent from continuous bioreactor contained 15 mg L −1 which is not acceptable for livestock water.
Biological degradation of pesticides refers to the use of microorganisms to destroy those chemicals in solid or liquid wastes into harmless by products. The aerobic biological treatment relies on microbial activity and aeration efficiency. Microbes that naturally occur in soil increased significantly in number and began to biodegrade pesticide in this study. The microbes utilized the pesticide as bioavailable carbon source for energy (respiration) and synthesis (growth) of microbial cells according to the following equations:

Synthesis
Organic matter + NH4 Microbes  → more microbes (3) The proposed pathway for the degradation of captan is shown in Fig. 6. The soil microbial population used in this study contained microorganisms capable of utilizing the carbon, chloride, nitrogen and sulfur found in captan (C 9 H 8 Cl 3 NO 2 S) under aerobic condition into carbon dioxide (CO 2 ), water (H 2 O), chloride (Cl), nitrate (NO 3 ) and sulphate (SO 4 ) and obtain the energy required for growth according to the following equation (Swanner and Templeton, 2011;Megadi et al., 2010;Munch et al., 1996): Soil microbes  → 9CO2 + H2O + 3HCl + NH3 + H2S+ ∆E (4) The captan degradation process takes place in several steps. In the first step, captan is converted into cis-1,2,3,6-tetrahydro phthalimide, thiocarbonyl chloride and hydrochloric acid. In the second, step cis-1,2,3,6-tetrahydro pthalimide is converted into cis-1,2,3,6-tetrahydro pthalimidic acid and thiocarbonyl chloride is converted into H 2 S, CO 2 and H 2 O. In the third step, the cis-1,2,3,6-tetrahydro pthalimidic acid is converted into O-phthalic acid and ammonia. In the forth step, the O-phthalic acid is converted into protocatechuic acid. In the fifth step, the protocatechuic acid is converted into 3carboxy-cis,cis muconic acid which is oxidized to CO 2 and H 2 O. The NH 3 is converted into NO 3 by the nitrifying bacteria while the H 2 S is converted into SO 4 by the hydrogen sulphide reducing bacteria as follows: The biodegradation of organic substrates such as pesticides in a batch system can be described by the following equation: where: P t = The concentration of pesticide at the time t (mg L −1 ) P o = The initial concentration of pesticide (mg L −1 ) k = The rate constant (h -1 ) t = The time (h) A plotting of ln (P t /P 0 ) versus time (t) yields straight line with a slope equal k. However, plotting the data obtained from the batch bioreactor did not fit a straight line for the entire period. The results (Fig. 7) showed different degradation rates for the lag period (0.0025 h -1 ) and exponential growth period (0.71 h -1 ). It appears from the results that the microorganisms are able to utilize captan as a source of carbon and energy for maintenance during the lag period. Karpouzas et al. (2005) reported 25% degradation of cadusafos (at an initial concentration of 12 mg L −1 ) during the lag period of 30 h after inoculation of Flavobacterium sp. and Sphingomonas sp. (isolated from cadusafos contaminated soil) which was followed by gradual increases in bacterial populations. (reaching 3×10 6 and 8×10 6 cells/mL for the Flavobacterium and the Sphingomonas sp. in 72 h, respectively) that result in complete degradation of cdusafos by both isolates in 78 h. Karpouzas and Walker (2000) reported a 30% degradation of ethoprophos (initial concentration of 100 mg L −1 ) after inoculation of Pseudomonas putida (isolated from ethoprophos contaminated soil) with a mineral salts Medium Supplemented with Nitrogen (MSMN) in the first 33 h and observed complete degradation after 50 h. In this study, 8.2% (12 mg L −1 ) of the captan in the batch bioreactor was degraded during the lag period of 22 h and complete degradation was achieved in 120 h. Leoni et al. (1992) reported a captan half life of 3.6 days in an activated sludge system, Hermanutz et al. (1973) reported captan half lives of 7 h at 12°C and 1 h at 25°C in Lake Superior. Ghaly et al. (2007) reported primiphos-methyle half life of 25 h in composting system (at 50-60°C). In this study, a captan half life of 52 h was observed for the batch bioreactor.

CONCLUSION
The initial cell number (30.1×10 6 cells/mL) in the soil water mixture first declined with time during the first 24 h reaching 15.6×10 6 and 11.1×10 6 cells/mL in the batch and continuous bioreactors, respectively. This was due to the inhibitory effect of pesticide on some of the soil microbial species that had less tolerance to captan at the initial concentration of 144 mg L −1 . The results indicated that microbial population reached its maximum after 5 and 12 days from the start in batch and continuous bioreactors, respectively. The lag period and the specific growth rate for the batch bioreactor were 22 h and 0.096 h −1 , respectively. Captan degradation efficiency of 89.6% was achieved after 10 days in the continuous bioreactor compared to a degradation efficiency of 100% after 5 d in the batch bioreactor. A half life of 52 h was observed in the batch bioreactor. This study showed that the batch mode of operation completely removed captan while the effluent from the continuous bioreactor had a captan concentration of 12 mg L −1 which is not acceptable for livestock water according to Health Canada Guidelines.