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Jaya et al., 1:11http://dx.doi.org/10.4172/scientificreports.535

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Volume 1 • Issue 11 • 2012

Keywords: Paraquat; Herbicide; Residue

IntroductionParaquat (1,1-dimetil,4,4-bipiridilium) is a commonly used

herbicide which has been extensively employed to regenerate grasslands. Considered as most toxic herbicide as indicated by its low LD50 of only 93.4 to 113.5 mg/kg, paraquat ion will cause poisoning in mammals (European Commission, 2003: 11). Due to the nature of paraquat residues in soils, the major part (almost 99.99%) of a paraquat application that reaches the soil within the typical Good Agricultural Practice (GAP) is strongly adsorbed to soils of a wide variety of textures.

Due to the extremely slow degradation of chemical residue in natural environments, enhancement of degradation or mineralization process of paraquat by microorganisms has been gaining popularity in the last decades. Therefore, it is important to study and understand the degradation capability of each bacterial species within a microcosm in order to predict the biodegradation of various contaminants in the environment. Thus, in this study we aim to investigate the profile of paraquat residue and screen indigenous soil bacteria with abilities to grow in paraquat contaminated land in Tanah Laut Regency, South Kalimantan Province.

Materials and Methods Chemical samples and equipments

The chemicals used in this study were paraquat dichloride 297 g/L (Noxone®), sodium dithionite (Brataco®, NaOH (Brataco®), methanol (Brataco®), nutrient agar (Brataco®), nutrient broth (Brataco®), ammonium nitrate (Brataco®), MgSO4.7H2O (Brataco®), NH4NO3 (Brataco®), K2HPO4 (Brataco®), Ca(NO3)2 (Brataco®), yeast extract (Brataco®) and distilled water. All soil samples were collected from maize lands in Tanah Laut regency with history of continued farming activity for several years. All lands were previously treated using some herbicides containing paraquat.

UV-Vis spectrophotometer unit was used to determine concentration of paraquat at λmax=345 nm. Other equipments used were microscope, haemocytometer, shaker, autoclave, centrifuge, analytical balance, and standard glassware.

*Corresponding author: Jaka Darma Jaya, Department of Agro-Industrial Technology, Tanah Laut Higher Vocational Education Institute (Politeknik Tanah Laut), Indonesia, Tel: +62512 21357; E-mail: jakadj2010@gmail.com

Received November 10, 2012; Published November 30, 2012

Citation: Jaya JD, Sandri D, Fatimah (2012) Paraquat Residue in Maize Lands: Case in Tanah Laut Regency, Indonesia. 1:535 doi:10.4172/scientificreports.535

Copyright: © 2012 Jaya JD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractParaquat (n,n'-dimethyl bipyridylium dichloride) is a commonly used herbicide which has been extensively

employed for maize farm in Tanah Laut regency. The residue of paraquat was considered as a high toxic and persistent herbicide which resulted in accumulation either in environment or in organism. This study aimed to verify the profile of paraquat residue collected from maize farm land in Panyiapatan district, Kurau district and Batu Ampar district, Tanah Laut regency. All three samples collected from Panyipatan district have shown to contain 0,178 ± 0,0421 mg/kg, 0,208 ± 0,023 mg/kg and 0,204 ± 0, 0038 mg/kg paraquat. Samples collected from Kurau district have also shown to containe 0,088 ± 0,0853 mg/kg, 0,356 ± 0,0252 mg/kg and 0,236 ± 0,0226 mg/kg. The approximately similar results were also reported for samples in Batu Ampar district with 0,179 ± 0,0150 mg/kg, 1,080 ± 0,1035 mg/kg, 0,350 ± 0,0141 mg/kg. Elevent morphologically distinct isolates and 6 morphologically distinct isolates were found when they were isolated on NA and MS+paraquat0,5, respectively. All isolates are potentially used in further study of biodegradation of paraquat residue in agricultural land.

Paraquat Residue in Maize Lands: Case in Tanah Laut Regency, IndonesiaJaka Darma Jaya*, Dwi Sandri and FatimahDepartment of Agro-Industrial Technology, Tanah Laut Higher Vocational Education Institute (Politeknik Tanah Laut), Indonesia

Procedure Soil sampling

All the soil samples were collected from maize farms in (three) districts in Tanah Laut regency i.e. Batu Ampar district, Kurau district and Panyipatan district (Figure 1). Three maize farm land of each district was selected to be investigated. 3-4 samples were collected from each land. Samples were collected from the top 15 cm of soil layer in order to find aerobic or facultative anaerobic bacteria which reported to be effective in degrading the chemical residue. Soil samples from each field were collected and mixed homogeneously before it is used for determination of pesticide residue testing and isolation-identification of bacteria.

Supporting data collection

Some supporting data that is considered related to the profile of pesticide residues in each soil is collected from both, field observations and interviews, as well as statistical data from the Office of Food Crops and Plantation, Tanah Laut regency.

Quantitative measurement of paraquat

Extraction of paraquat from soil samples: Fifty grams of soil samples were collected from multiple maize fields in District Panyipatan. One hundred and fifty milliliters of distilled water (or 150 mL of methanol) were added to each soil samples and vigorously shaked at 150 rpm for 4 hours. The filtrate were evaporated to be an approximately 30 ml. Concentrated filtrate was prepared for the measurement of paraquat in soil samples by UV-Vis spectrophotometer. The solvents with greater extraction capacity would be further used for the extraction of paraquat on the other soil samples.

Citation: Jaya JD, Sandri D, Fatimah (2012) Paraquat Residue in Maize Lands: Case in Tanah Laut Regency, Indonesia. 1:535 doi:10.4172/scientificreports.535

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Determination of paraquat concentration

A total of 0.5 ml of liquid samples of soil was supplemented with 1 ml of 1% sodium dithionite (sodium hydroxide 0.1 N) and then diluted with distilled water to 10 ml, and then measured at λ=345 nm. The results were plotted on a standard curve of paraquat.

Cultivation media

The medium used for either isolation and cultivation was MS medium (mineral salts medium) consisted of g MgSO4.7H2O 0:20, 0:25 NH4NO3 g, 0,675 g K2HPO4, 0:10 g Ca (NO3)2 and 0.01% peptone (per 1 liter of distilled water). Supplemented 0.1 N paraquat was added to the medium and adjusted to pH 7.0. Sterilization was then carried out by autoclaving the sample at 121°C for 15 minutes.

Paraquat was prepared in stock solution of distilled water in a concentration of 2.5 × 103 mg/l (ppm). Media for culture starter (MS+Paraquat0,5) was prepared by adding 0.02 ml stock solution in to 100 ml MS medium in order to obtain a final concentration of 0.5 ppm.

Isolation of bacteria degrading pesticides

Isolation was done by two different media, i.e. MS medium and Nutrient medium (Nutrient Broth and Nutrient Agar- OXOID) in order to compare the result between these 2 different media. Soil sample which was proven to contain highest paraquat residues was prepared as the sample source of isolation. Ten gram of soil sample was added in to MS media and Nutrient Broth and shake for 24 hours at 150 rpm. One milliliter of diluted culture broth (10-1-10-2) was then poured on MS medium+Paraquat0,5 agar, incubated at room temperature for 24-36

hours. Obtained isolates was identified based on their single cell/colony morphology and gram staining properties.

Results and DiscussionTanah Laut regency is the largest corn producer in South Kalimantan

province. In 2011, Tanah Laut has 12.476 Ha planted maize area with high production of up to 62.467 tons/years (Office of Agriculture, Crop and Plantation, 2011). The growth of maize agriculture was supported by easy access to the construction industry as marketing fodder PT. JAFPA Confeed Indonesia in Bati-Bati district which is the main raw material of corn (Figure 2).

The samples used in this study were classified into (three) classification: humus soil, sandy soil and clay. Humus soil with high organic content was reported to contain higher microbial activity than that of other soil [2]. Sandy soil has a large cavity which allowed the residue pass through the soil. On the other hand, degradation activity of the residue in sandy soil is quite low attributed to its low organic content [2]. In contrast to the above types of soil, a clay soil with a high density mineral containing (iron and aluminum) degrading microbial activity in the soil was also not great.

On the other hand, rainfall significantly affected the movement of residues from one place to another, i.e. enter to river and/or penetrate into the lower soil layers. In addition, rainfall also affects the bioavailability of pesticides against the bacteria. High rainfall will cause the greater residual mobility and bioavailability. In 2011, the average rainfall in Batu Ampar district 224,97 mm, Kurau District 196,92 mm and Panyipatan district 221,67 mm (Office of Agriculture, Crops and Plantation, 2011).

Chemical residues on farming land were addressed to the type of pesticide/herbicide used. Based on the data obtained, Noxone®

KURAUBATI - BATI

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Figure 1: Place of soil samples collection in Tanah Laut regency.

Year

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Figure 2: The growth of maize comudity in Tanah Laut regency.

District Location means ± standard deviation (mg/kg soil dry weight)

Panyipatan1 0,178 ± 0,04212 0,208 ± 0,02333 0,204 ± 0,0038

Kurau1 0,088 ± 0,08532 0,356 ± 0,02523 0,236 ± 0,0226

Batu Ampar1 0,179 ± 0,01502 1,080 ± 0,10353 0,350 ± 0,0141

Tabel 1: Concentration of paraquat residue from soil samples collected from various Maize farm in Tanah Laut regency.

Determination of maximum wavelength: A total of 0.5 mL of paraquat 20 ppm was added with 1 mL of 1% sodium dithionite (sodium hydroxide 0.1 N). The solution was then diluted with distilled water up to 10 ml. The absorbance was measured at 300-650 nm in order to find maximum wavelength attributed to its maximum absorbances [1].

Preparation of standard curve paraquat by spectrophotometer

Standard solutions of 0, 0.5, 1, 2, 3, 4, and 5 ppm were prepared by adding 0, 1.25, 2.5, 5, 7.5, 10, and 12.5 mL of stock solution of paraquat 20 ppm to 5 mL of sodium dithionite 1% (in NaOH 0.1 N) and then diluted with distilled water up to 50 mL [1]. The solution is then measured based on UV-Vis spectrophotometer absorption at λ=345 nm.

Citation: Jaya JD, Sandri D, Fatimah (2012) Paraquat Residue in Maize Lands: Case in Tanah Laut Regency, Indonesia. 1:535 doi:10.4172/scientificreports.535

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is the commonly used herbicides in all districts where the samples were collected; in addition there are Roundup®, Rambo®, Bestok® and Gramoxone®. The main chemical of all previously mentioned herbicides were paraquat. Thus, we used paraquat as an object of research. Wide use of these chemicals resulted in the accumulation of residues in soil. This comes to be a serious problem, since the residue tends to be persistent in the soil [3].

All samples contained paraquat residues with varied concentration ranging from 0,080 mg/kg up to 0,356 mg/kg soil dry weight (Table 1). The low concentration paraquat residue may be attributed to high rainfall in Tanah Laut regency (up to 224,97 mm) during the sampling, thus the residue will flow to the river along with the rain water. Paraquat can also be degraded by sunlight or as it is called photodegradation [4]. Moreover, it can also be attributed to the microbial activity in microcosm.

The results obtained from Batu Ampar district was the highest of up to 1.080 mg/kg soil dry weight. Based on observation data, the most common used land using herbicides were Noxone® and Gramoxone®. Those two herbicides contained paraquat as the main active compounds. The sample collected in Kurau district 1 displayed lowest paraquat residue of 0.088 mg/kg soil dry weight. This is attributed to that the land did not use any pesticide before cultivation, while all lands in Panyipatan district use several herbicides which all contained paraquat.

All the samples containing paraquat residue are further identified for their microbial profile. Not all bacteria can grow on the paraquat containing medium. Six isolates were proven to grow on the medium MS+Paraquat0,5 after 2-3 days incubation. Determining a value of 0.5 ppm as supplemented paraquat was based on the previous observation which offered that the bacteria began to grow when the concentration was reduced up to 0,05 ppm. On the other hand, it was shown that more bacteria can grow on Nutrient Agar (11 isolates; Tabel 2). These fact attributed to that nutrient agar contained more simple nutrient in comparison with that of mineral salt medium supplemented with 0,5 ppm of paraquat.

Now a day, herbicides are widely used in agriculture lands which

are intended to improve yields of agriculture products by suppressing grasslands [5-7]. However, significant amounts of herbicides are applied off-target, which may cause serious impact on other non-target organisms [8-10]. Utilizing microorganisms as the agent of degradation is now a promising alternative way to convert harmful compounds to the less toxic residue. In nature, bacteria have several mechanisms to be tolerant or resistant to the toxic compounds by eliminating toxic compounds by the cell [11], or by producing enzymes degrading compounds [12].

ConclusionAll nine samples collected from three districts in Pelaihari Regency

contained 0,088 up to 1,080 mg/kg soil dry weight. The value worried us since paraquat is one of the persistent chemical residue. Eleven morphologically distinct isolates and 6 morphologically distinct isolates were found when they were isolated on NA and MS+paraquat0,5, respectively. All obtained isolates are potentially used in further study of biodegradation of paraquat residue in agricultural land attributed to their capabilies to gwow on paraquat supplemented medium.

Acknowledgements

This research was financially supported by Ministry of National Education of Republic of Indonesia via Competitive Grant, while the official support was offered by Polytechnic Tanah Laut, South Borneo Province.

References

Medium District Land No IsolateColony Morphology Cell Morphology

Colour Shape Edge Elevation Gramstaining Shape

Nutrient Agar

Kurau 21 KUR 1A Yellow Pinpoint Ontire Raised + Rod2 KUR 2A White Pinpoint Ontire Raised + Cocci3 KUR 3A Pink Pinpoint Ontire Raised + Rod

Batu Ampar 2

1 BTM 1A White Circular Entire Raised - Rod2 BTM 2A Yellowish white Irregular Undulate Convex + Cocci3 BTM 3A Yellow Spreading Undulate Raised - Rod4 BTM 4A Yellow Molar-Tooth Entire Convex - Rod5 BTM 5A Yellowish white Circular Entire Convex - Rod6 BTM 6A Yellowish white Circular Undulate Raised + Cocci7 BTM 7A Yellow Circular Crose Convex + Rod

Panyipatan 21 PAN 1A White Irregular Ontire Raised - Cocci2 PAN 2A White Pin Point Ontire Raied - Rod

MS+ Para-quat0,5

Kurau 2 1 KUR 1B White Pinpoint Ontire Raise + Cocci

Batu Ampar 2

1 BTM 1 B White Pinpoint Crose Convex + Cocci2 BTM 2 B White Pinpoint Crose Convex - Rod3 BTM 3 B White Pinpoint Entire Raised + Rod4 BTM 4 B White Molar-Tooth Lobate Raised - Cocci

Panyipatan 2 1 PAN 1B White Pinpoint Ontire Raised - Cocci

Table 2: Observed cell and colony morphologies of bacterial isolates grown in NA and MS Paraquat0,5.

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Citation: Jaya JD, Sandri D, Fatimah (2012) Paraquat Residue in Maize Lands: Case in Tanah Laut Regency, Indonesia. 1:535 doi:10.4172/scientificreports.535

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