Butachlor, Pretilachlor, 2,4-D and Oxyfluorfen in Two Soil Types, Viz

Indian Journal of Weed Science 47(2): 193–196, 2015

Leaching behaviour of four herbicides in two soils of Kerala

K.M. Durga Devi*, C.T. Abraham and C.N. Upasana College of Horticulture, Kerala Agricultural University, Thrissur, Kerala 680 656

Received: 7 April 2015; Revised: 23 May 2015

ABSTRACT The present study was conducted to find out the extent of leaching of butachlor, pretilachlor, 2,4-D and oxyfluorfen in two soil types, viz. Type I [coarse textured low organic matter soil (Mannuthy–Ultisol] and Type II [fine textured high organic matter soil (Alappad-Inceptisol]. Intact soil columns were collected from the paddy fields after the harvest of second crop. Butachlor, pretilachlor, 2,4-D and oxyfluorfen were applied in moist soil columns at the recommended rate of application. Soil samples from different depths up to 10 cm (top 5 segments of 2 cm each) and the leachate at 30 and 60 cm depths were analyzed for herbicide residues using gas chromatography. Among the four herbicides tested, 2,4-D registered highest level of residue in the leachate (0.20 ppm at 60 cm depth). Pretilachlor and butachlor followed the same trend in the pattern of movement of residue through the soil columns. However, the leachate of pretilachlor registered much lower quantity of residue (0.006 ppm). Fine textured organic matter rich soil recorded lower residue levels compared to the soil with coarse texture and poor organic matter. It could be attributed to the high adsorptive power of the soil, especially at the top layers with high organic matter content. Oxyfluorfen residues could not be detected in the leachate, because of its poor water solubility. Key words: 2,4-D, Butachlor, Leaching, Oxyfluorfen, Pretilachlor

Among the different pre-emergence herbicides MATERIALS AND METHODS in rice, butachlor, pretilachlor and oxyfluorfen are more popular in the paddy fields of Kerala. Sodium Intact soil columns were collected from two soil salt of 2, 4-D is the most common post-emergence types, viz. Type I (sandy loam low organic matter soil herbicide in the major rice bowls of the state, viz. of Ultisol order at Rice field of Agricultural Research Kole and Kuttanad. Herbicide movement in soils is of station, Mannuthy) and Type II (high organic matter major concern in tropical soils with heavy rainfall. clayey soil of Inceptisol order, Kole Lands, Alappad) Since these herbicides are applied to the soil surface after the harvest of second crop. Long PVC tubes (60 without incorporation, heavy rainfall soon after cm) of 16 cm diameter were taken for Type I soil. application may cause excessive leaching of the Tubes of only 30 cm and 16 cm diameter were taken herbicide from the surface zone, resulting in poor for Type II soil because the area is below mean sea weed control. In the case of herbicides with high soil level and hence collection of intact soil columns mobility, leaching may lead to injury of deeper-rooted below 30 cm was not possible. The tubes were cut desirable species. In addition, herbicide leaching may vertically and the two halves were pressed onto the result in contamination of ground and surface water lateral sides of the soil pit of 60 cm deep, dug in the (Anderson 1983). Movement of herbicide within the rice field. The soil corresponding to the different soil profile is influenced by many factors such as depths were transferred as such to the two halves of chemical nature of herbicide, the adsorptive capacity the PVC column and they were joined together using of soil and the amount of water available for adhesive tape. The tube was kept on an iron stand downward movement through the soil. Butachlor, after tying the lower end with a muslin cloth. After pretilahlor, oxyfluorfen and 2,4-D differ much in their adding water continuously to attain constant water solubility (RSC 1987). Considering these percolation rate, the herbicides were added at the factors, studies on leaching pattern of three pre- recommended rate of application, viz.1.25 , 0.75, 1.0 emergence herbicides and one post-emergence and 0.2 kg/ha for butachlor (Machete®), pretilachlor herbicide were conducted in two soil types of Kerala, (Rifit®), 2,4-D (Fernoxone®) and oxyfluorfen under All India Coordinated Research Programme on (Goal®), respectively using a spray volume of 500 L/ Weed Control during the period from 2007-08 to ha. The quantity of solution required for spraying was 2010-11. calculated based on the surface area. *Corresponding author: [email protected]

193 Leaching behaviour of four herbicides in two soils of Kerala

Water was added frequently to the top of the 30% dichloromethane). It was then eluted with 100 column at one day after spraying. A total of 1000 mL mL of (70% dichloromethane/30% hexane) solvent water was added through the column (200 mL x 5 mixture. First 20 mL was discarded and next 80 mL times) so as to simulate the normal rainfall receiving was collected. The solvent was then evaporated off in the area. Methods were standardized for estimation and the residue was dissolved in 1 mL n-hexane. residues in soil and water samples using the gas One micro litre of n- hexane extract was injected chromatograph. Soil samples from different layers in to the GC 2010 fitted with a 63Ni electron capture (top 5 segments of 2 cm each) leachate were detector, a DB-17 capillary column and a split analysed for herbicide residues. Average values of injector. The temperature of the injector, column and residues from three replications were worked out for detector were 180, 210 and 3000 C, respectively with comparison of data. a split ratio of 3:1. The residue content was calculated Extraction and estimation of 2,4-D residues from the standard curve obtained with the reference standard. Water samples were filtered out of any particulate matter. A 50 mL portion of the sample was Air dried soil sample (15 g, 2 mm sieved) was taken and saturated with sodium chloride. The pH thoroughly mixed with 10g of anhydrous sodium was adjusted to <2 with HCl. It was then extracted sulphate, 2 g of florisil (60-100 mesh size) and 0.3g with (5 x 25 mL) portions of acetonitrile. The pooled of activated charcoal. A glass column of 30 cm length extract was then concentrated to 15 mL. Fifteen mL and 2 cm internal diameter was taken. Anhydrous 10% NaOH was added and the pH was adjusted to > sodium sulphate of 3 cm layer was put on the non 13. The organic phase was evaporated off in adsorbent cotton kept at the lower end of the column. presence of the alkali. The aqueous alkaline solution Then the soil sample mixture (prepared as above) was was refluxed for 20 min. It was then cooled and added to the column and another layer of anhydrous extracted with equal volume of hexane (x3). The sodium sulphate of 1 cm was put over this layer. The hexane fraction was discarded and the aqueous herbicide was extracted with 100 mL of hexanes: portion was acidified with HCl to pH<2. It was then acetone mixture (9:1) and excess solvent was extracted with equal volumes of diethyl ether (x3) evaporated under vacuum to one mL. The evaporated sample was made up to 5 mL with n-hexane. One Twenty five gram of wet soil sample after micro litre portion of the n- hexane extract was draining excess water by spreading over a filter paper injected in to the GC 2010 fitted with a 63 Ni electron was shaken on a shaker with 80 m. extracting capture detector, a BPX-5 capillary column and a split mixture (acetonitrile: water: glacial acetic acid in the injector. The temperature conditions for butachlor ratio 80:20:25) for a period of 30 minutes at 220 rpm and petilachlor were the same (250, 220 and 3000C and filtered through Whatman No.1 filter paper. The for injector, column and the detector were filtrate was acidified with concentrated HCl (15 mL) respectively). For oxyfluorfen, optimum temperature and separated by extracting thrice each with 50 mL conditions were 220, 210 and 2400C for injector, diethyl ether. column and the detector respectively). The residue From the combined diethyl ether extract, the content was calculated from the standard curve organic phase was evaporated off and the residue obtained with the reference material (97% pure was dissolved in 3 mL of methanol. Added a 3ml reference standards obtained from Dr. Ehrenstorfer, portion of boron tri fluoride methanol reagent and GmbH, Germany) refluxed for 10 minutes on a water bath. After Gas chromatographic technique (KAU 2008) reaction, excess alcohol was evaporated off and was used for estimation of butachlor, pretilachlor and 20mL water was added, shaken vigorously for 5 min. oxyfluorfen residues in the leachate. and extracted with hexane (3x10mL). The hexane portions were combined, the organic phase RESULTS AND DISCUSSION evaporated off and the residue was concentrated Physicochemical characteristics of the soil A 5 cm bed of activated silica gel Pyrex glass column packed at the two ends each with 1g of The major physico-chemical characteristics of anhydrous sodium sulfate was used. The column was soil, viz. soil texture, pH, cation exchange capacity washed with 25 mL hexane. The extract obtained (CEC), anion exchange capacity (AEC) and organic after derivatisation as given in step II was placed on carbon content of the soil sample taken from the rice the column. The residue in the column was washed field before conducting experiment are presented in with 100 mL solvent system containing (70% hexane/ (Table 1).

194 K.M. Durga Devi, C.T. Abraham and C.N. Upasana

Table 1. Major physico chemical characteristics of the (+)/kg and 9.38 to 12.4 C mol (-)/kg respectively). soil columns collected for the study Sesquioxide content varied from 3.0 to 4.60 per cent Soil type (average values) between the soil types. Type I Type II Herbicide residues at different soil depths (Mannuthy) (Alappad) coarse textured fine textured All the herbicides registered maximum quantity Characteristics low organic high organic of residue in the upper 2 cm of the soil column. matter soil matter soil- sandy loam- clay- inceptisol Maximum value was registered by butachlor ultisol treatment (2.43 µg/g) in Type II soil and oxyfluorfen registered lowest value in both the soil types (0.55 Clay % 26.0 64.0 Organic carbon, % 0.90 1.87 and 0.61µg/g for Type I and Type II, respectively). pH 5.09 5.1 This could be attributed to the differences in the levels C.E.C, C mol(+)/kg 9.25 15.7 of application of herbicides to the columns. The A.E.C, C mol(-)/kg 12.4 9.38 recommended level of application of butachlor Sesquioxide, % 4.6 3.0 was1.25 kg/ha and that of oxyfluorfen was 0.20 kg/ ha (KAU 2011). Higher proportion of applied The soils showed wide differences in their pretilachlor compared to butachlor observed in the textural characteristics. The mean clay content varied upper layer of soil column could be attributed to from 25.98% (Type I) to 64.00% (Type II). The higher Kd (distribution coefficient) values for organic carbon content of Type I soil (Mannuthy) pretilachlor as reported by Hasna (2011). was 0.90% and that of Type II (Alappad) was 1.87%. There was considerable decrease in the residue The soils were uniformly acidic in nature (5.1). Cation and anion exchange capacities showed with increasing depth of the soil. Fine textured organic matter rich soil recorded lower residue levels variations between the soil types (9.25 to 15.75. mol

Table 2. Leaching pattern of butachlor, pretilachlor oxyfluorfen and 2,4-D, in different soil types

Herbicide and level of Depth of Concentration of herbicide (µg/g or µg/ mL) application (kg/ha) soil (cm) Coarse textured low organic matter soil Fine textured high organic matter soil Butachlor (1.25) 0-2 1.47 (35.28) 2.43 (58.32) 2-4 0.25 (6.0) 0.77 (18.48) 4-6 0.15 (3.6) 0.14 (3.36) 6-8 0.05 (1.2) 0.02 (0.48) 8-10 0.05 (1.2) 0.02 (0.48) Leachate 0.16 (3.84) (>60 cm depth) 0.04 (0.96) (>30 cm depth) Pretilachlor (0.75) 0-2 1.56 (62.4) 1.85 (74.0) 2-4 0.19 (7.6) 0.21 (8.4) 4-6 0.09 (3.6) 0.09 (3.6) 6-8 0.06 (2.4) 0.03 (1.2) 8-10 0.03 (1.2) 0.01 (0.4) Leachate 0.006 (0.24) (>60 cm depth) 0.004 (0.16) (>30 cm depth) Oxyfluorfen (0.20) 0-2 0.55 (82.5) 0.61 (91.5) 2-4 0.005 (0.75) 0.004 (0.6) 4-6 0.005(0.75) 0.002 (0.3) 6-8 0.003 (0.45) BDL 8-10 0.003(0.45) BDL

Leachate BDL(>60 cm depth) BDL(>30 cm depth) 2,4-D (1.0) 0-2 1.44 (43.2) 1.83(54.9) 2-4 0.17(5.1) 0.19 (5.7) 4-6 0.10 (3.0) 0.16(4.8) 6-8 0.08 (2.4) 0.10 (3.0) 8-10 0.05(1.5) 0.09(2.7) Leachate 0.20 (6.0) (>60 cm depth) 0.05 (1.5) (>30 cm depth) Percentage of the applied herbicide remaining at different depths is given in parentheses; BDL: Below detectable level

195 Leaching behaviour of four herbicides in two soils of Kerala

compared to the soil with coarse texture and poor low in both the soil types and may not contaminate organic matter. It could be attributed to the high groundwater under recommended rate of application adsorptive power of the soil, especially at the top of the herbicide. Similar findings were reported by layers with high organic matter content. Yen et al. (2003) after evaluating the possible Based on the quantity sprayed over the soil contamination of oxyfluorfen using the behavior column, percentage of applied herbicide remaining in assessment model and the groundwater pollution- each layer of soil was calculated (Table 2). It could be potential (GWP) model. The present study also noticed that oxyfluorfen is more strongly adsorbed in revealed that the solubility of herbicide in water is the the first soil layer of 0-2 cm (82.5% and 91.5% of the major factor determining the movement of herbicides applied herbicide in Type I and Type II soil, in the soils of Kerala. The extent of leaching followed respectively) followed by pretilachlor (62.4 and the order: 2,4-D (620 mg/L) >butachlor (50 mg/L) > 74.0%, respectively), 2,4-D (43.2% in Type I soil) pretilachlor(20 mg/L) > oxyfluorfen (0.114mg/L). and butachlor (35.28% in Type I soil). In Type II soil, REFERENCES butachlor (58.32%) adsorption was higher than that of 2,4-D (54.9%). As reported by Hasna (2011), Anderson WP. 1983. Weed Science. Principles and Practices. higher quantity of organic matter resulted in Second edition. West Publishing Co., NY. considerable increase in the adsorption of butachlor in Hasna K. 2011. Influence of Organic Matter and Soil Moisture the lateritic soil of Kerala. on the Adsorption of Chloroacetanilide Herbicides, viz., Butachlor and Pretilachlor, in Laterite Soil. M.Sc. thesis, Herbicide residues in the leachate Kerala Agricultural University, Kerala, India, 91p. KAU [Kerala Agricultural University]. 2008. Annual Report Among the four herbicides tested, 2,4-D residue 2008. All India Coordinated Research Programme on Weed was the maximum in the leachate (Table 2) followed Control. Kerala Agricultural University, Kerala, India, 80p. by butachlor, pretilachlor and oxyfluorfen. The KAU [Kerala Agricultural University]. 2011. Package of leachate collected at 30 cm depth in Type II soil Practices Recommendations: Crops, 14th edition. Crops. registered 2,4-D residues to an extent of 0.05 µg/mL Kerala Agricultural University, Thrissur, 337 p. (1.50% of the applied herbicide). In the case of Type RSC, 1987. Agro Chemical Hand book (Ed. Hartley D). Second I soil 2,4 D residues registered in the leachate at 60 edition. The Royal Society of Chemistry, Notingham, UK. cm depth was 0.2 µg/mL (6.0%). Oxyfluorfen Yen JH, Sheu WS and Wang YS. 2003. Dissipation of the residues in the leachate collected from both the soil herbicide oxyfluorfen in subtropical soils and its potential types were below the detectable level. The results to contaminate groundwater. Ecotoxicology and indicated that the mobility of oxyfluorfen was very Environmental Safety 54(2):151-6.

196 Indian Journal of Weed Science 47(2): 193–196, 2015

Leaching behaviour of four herbicides in two soils of Kerala

K.M. Durga Devi*, C.T. Abraham and C.N. Upasana College of Horticulture, Kerala Agricultural University, Thrissur, Kerala 680 656

Received: 7 April 2015; Revised: 23 May 2015

193 Leaching behaviour of four herbicides in two soils of Kerala

194 K.M. Durga Devi, C.T. Abraham and C.N. Upasana

Table 2. Leaching pattern of butachlor, pretilachlor oxyfluorfen and 2,4-D, in different soil types

195 Leaching behaviour of four herbicides in two soils of Kerala

196

International Journal of Science, Environment ISSN 2278-3687 (O) and Technology, Vol. 5, No 5, 2016, 3106 – 3114 2277-663X (P)

IMPACT OF VARIOUS DOSES OF BUTACHLOR ON WEED GROWTH, CROP YIELD OF RICE, MICROBIAL POPULATION AND RESIDUAL EFFECT ON WHEAT CROP V. Pratap Singh, Neeshu Joshi, Neema Bisht, A. Kumar, Kavita Satyawali and R.P. Singh Department of Agronomy, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar E-mail: [email protected]

Abstract: A field investigation was carried to test the bioefficacy of Butachlor 50 EC at various doses (Sponsor Vs market sample) to control the weeds in transplanted rice. Butachlor was applied at various doses as pre emergence and its market sample used at two doses, almix also applied as post emergence. All weed control treatments significantly reduced the density as well as dry matter accumulation of weeds over weedy check during both the years. The maximum suppression of density as well as dry matter accumulation of weeds and highest WCE were obtained with application of Butachlor 50% EC at higher doses (2000 and 4000 g/ha) as compared to its lower dose 1250 g/ha. Among herbicidal treatments maximum grain (4479 and 5056 kg/ha) and straw (7370 and 6963 kg/ha) yield was achieved by the application of Butachlor 50 EC (SS) at 2000 g/ha applied as pre emergence during both the years. Keywords: Bio-efficacy, Butachlor, microbial, residual and transplanted rice.

Introduction

Rice (Oryza sativa L.) is a staple food for more than 60% of the world’s population. Feeding the 9 billion people expected to inhabit our planet by 2050 will be an unprecedented challenge for the mankind. In India, rice is cultivated in an area of 44.07 m ha annually with a production of 103.40 mt, with an average productivity only 2.3 t/ha (FAO, 2012). There are several reasons for low productivity, however it has been estimated that without weed control, the yield loss can be as high as upto 90%. Weeds have caused yield reduction of 28 to 45% in transplanted rice (Singh et al., 2007; Manhas et al., 2012). High density of grassy weeds competing with crop and resulting into heavy yield losses up to 40-50% (Singh et al., 2004). Furthermore, any delay in weeding will lead to increased weed biomass which has a negative correlation with yield. The reduction in weed growth was observed with intensive puddling and shallow depth submergence in transplanted rice (Reddy and Reddy, 1999). Transplanted rice faces diverse type of weed flora, consisting of grasses, broad-leaf weeds Received Aug 24, 2016 * Published Oct 2, 2016 * www.ijset.net 3107 V. Pratap Singh, Neeshu Joshi, Neema Bisht, A. Kumar, Kavita Satyawali and R.P. Singh

and sedges. Weed competition brings reduction in yield of transplanted rice by about 50 per cent ( Mukherjee and Singh, 2005). Manual weeding is although effective and most common method, however, scarcity and high wages of labor particularly during peak period of critical competition between weed and crop period make this method uneconomical. The goal of herbicide use is to kill or stunt weed infestation allowing the rice to grow and gain a competitive advantage. Weeds are the most important biological constraint to decrease the yield of rice. Now chemical weed control method is becoming popular among the farmers because it is the most efficient means of reducing weeds competition with minimum labor cost. Butachlor, anilofos, oxadiargyl, etc. are the herbicides presently use for weed control in transplanted rice. Weed density per unit area is an important and key parameter in figuring out the impact of treatments on weed growth. Moreover, the use of herbicides, though discouraged worldwide these days because of environmental and health hazards, is inevitable due to many reasons particularly in the terms of economics and the immediate effect. Bio-efficacy of herbicides is also depends on herbicide doses and affect the crop physiology, soil health and residue effect on succeeding crop. With this thought keeping in background, the present research work on bio-efficacy of butachlor 50% EC of sponsor Vs market samples tested in transplanted rice to find out the herbicide efficacy at various doses and its impact on crop and soil health along with carryover of residues in succeeding wheat crop. Materials and Methods The field experiment was conducted at N.E. Borlaug Crop Research Centre of G.B. Pant University of Agriculture and Technology, Pantagar during two consecutive year of 2010-11 and 2011-12. The Crop Research Centre where the experiment was conducted is located at 29 o N latitude, 79.3 o E longitude, and at an altitude of 283.84 metres above the mean sea level. The soil was loamy, medium in organic matter (0.67%), available phosphorus (17.5 kg/ha) and potassium (181.2 kg/ha) with pH 7.5. The experiment consisted of eight treatments including untreated (control) and laid out in randomized block design (RBD) with three replications. The treatments were as follows of Butachlor 50% EC (sponsor sample) at 1250, 2000, 4000 g/ha and two doses of market sample at 1250 and 2000 g/ha applied as pre emergence, Almix 20%WP at 4 g/ha applied as post emergence to required volume of water 500 l/ha. Weed free and weedy check also included in experiment to compare the efficacy of herbicidal treatments. Rice variety “Sarjoo 52” seedling was planted on June 2 of kharif 2010 and June 10 of kharif 2011. The other agricultural practices used for rice transplanting in the Impact of Various Doses of Butachlor on Weed …. 3108

region were followed. Knap sack sprayer fitted with boom along with flat fan nozzle was used to apply the herbicidal solution. The basal dose was applied at the rate of 60:60:40:25 kg/ha of N:P:K and Zn, respectively. Weed were recorded species wise in each plot at 30 and 60 days after transplanting (DAT) with the help of quadrate of 0.25m 2 for the area marked for observation. The weed after uprooting are cleaned and dried in oven at 72 0C temperature and weed control efficiency was calculated by using the formula WCE = (weed biomass in unwedded control– weed biomass in managed treatment)/ weed biomass in unweeded control x 100. Besides observations for plant height, tillers (number/row), panicles/m 2, grains/panicle, 1000 grain weight, grain and straw yield were taken. Data recorded were statistically analyzed according to Gomez and Gomez (1984). Means were compared at 5% levels of significance by the least significant difference (LSD) test. Result and Discussion Weed flora The dominant weed flora of the experimental site at 30 and 60 DAT was similar during both years and comprised of grasses; Echinochloa colona, Echinochloa crusgalli, broad leaf weeds; Ammania baccifera, Caesulia auxillaris, Alternanthera sessilis and Cyperus spp . was the only species among the sedge. Effect on weeds Weed density and weed dry matter varied significantly under different herbicidal treatments (Table 1&2). Data presented in Table 1&2 indicated that all the herbicidal treatments significantly reduced the density and dry matter accumulation of weeds with increasing the dose of Butachlor 50% EC (Sponsor sample) as compared to market sample of Butachlor 50% EC at both stages during both the years except at 30 DAA in 2010 over the weedy check and thus ultimately enhanced weed control efficiency. Prakash et al ., 2013 also observed that Butachlor 1500 g/ha significantly reduced the density and dry weight of weeds over weedy check (control). Maximum reduction in weed density and dry weight of weeds was obtained with application of Butachlor 50% EC (SS) 4000 g/ha followed by its lower dose 2000 g/ha. Application of Butachlor 50% EC at 1250 and 2000 g/ha (SS) was found more effective in reducing the population of grassy weeds over the market sample of butachlor at same doses. Weed dry matter is a better parameter to measure the competition than the weed number (Bhanumurthy and Subramanian, 1989). The total dry matter accumulation of weeds reduced is an indication of the overall utilization of resources and better light interception. Application of Butachlor either SS or MS both significantly reduced weed population as well 3109 V. Pratap Singh, Neeshu Joshi, Neema Bisht, A. Kumar, Kavita Satyawali and R.P. Singh

as weeds dry matter accumulation found in rice crop over weedy check during both the years of experimentation. The lower weed dry weight in weed control treatments may be ascribed to lesser number of weeds, rapid depletion of carbohydrates reserves of weeds through rapid respiration (Hill and Santlemann, 1969). Among various herbicides, Butachlor 50% EC (SS) at 4000 g/ha recorded the lowest weed dry matter accumulation followed by its lower dose at 2000 g/ha at both the stages of crop growth due to elimination of both grassy and non-grassy weeds resulting in maximum weed control efficiency. While Almix 20% WP 4 g/ha was least effective with lowest weed control efficiency as it was not effectively control the grassy weeds.

3110

Table 1: Effect of treatments on density and total dry weight of weeds at 30 DAA during 2010 and 2011 Treatments Dose 2010 2011 (g/ha) Weed density (no./m 2) at 30 DAA Weed density (no./m 2) at 30 DAA ) ) 2 2

pp. A. A. E. E. A. A. E. E. s colona colona colona colona sessalis sessalis sessalis Cyperus weight (g/m Weight (g/m A. basifera basifera A. A. basifera basifera A. I. rugosum I.rugosum axillaris C. I. rugosum I.rugosum axillaris C. E. crusgalli crusgalli E. E. crusgalli crusgalli E. Total weed dry Total weed dry Butachlor50% EC(SS) 1250 1.1(2.7) 2.1(8.0) 0.0(0.0) 0.0(0.0) 1.5(5.3) 2.7(13.3) 0.0(0.0) 14.8 1.0(2.7) 0.5(1.3) 0.0(0.0) 1.0(2.7) 2.3(9.3) 0.0(0.0) 27.1 Butachlor 50% EC (SS) 2000 0.0(0.0) 1.3(4.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 2.4(10.7) 0.0(0.0) 13.4 0.5(1.3) 0.0(0.0) 0.0(0.0) 1.0(2.7) 2.1(8.0) 0.0 (0.0) 22.7 Butachlor 50% EC (SS) 4000 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.7(2.7) 2.3(9.3) 0.0(0.0) 7.6 0.5(1.3) 0.0(0.0) 0.0(0.0) 0.5(1.3) 2.3(9.3) 0.0(0.0) 19.4 Butachlor 50% EC (MS) 1250 0.0(0.0) 2.9(17.3) 0.0(0.0) 0.0(0.0) 0.0(0.0) 2.6(12.0) 0.0(0.0) 14.8 1.0(2.7) 1.6(4.0) 0.0(0.0) 1.0(2.7) 2.1(8.0) 1.0(2.7) 27.6 Butachlor 50% EC (MS) 2000 0.0(0.0) 1.3(4.0) 0.0(0.0) 0.0(0.0) 0.5(1.3) 2.7(13.3) 0.5(1.3) 12.7 0.5(1.3) 0.5(1.3) 0.0(0.0) 1.0(2.7) 2.3(9.3) 0.0(0.0) 22.9 Almix 20% WP 4 3.1(21.3) 4.0(52.0) 2.9(17.3) 0.0(0.0) 2.2(8.0) 2.1(8.0) 2.5(12.0) 35.0 1.8(5.3) 2.8(16.0) 0.0(0.0) 0.0(0.0) 1.4(5.3) 0.0(0.0) 52.8 Weed free - 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0 Weedy - 4.5(88.0) 4.4(82.7) 1.5(5.3) 1.5(5.3) 0.0(0.0) 0.7(2.7) 3.4(29.3) 84.3 2.1(7.3) 3.0(21.3) 1.7(4.7) 2.4(10.7) 2.7(14.7) 3.1(21.3) 93.2 LSD(0.05) 0.5 1.1 1.1 0.8 1.3 0.9 0.6 12.3 1.1 0.8 0.1 1.2 0.8 0.5 6.9 SS - Sponsor sample, MS – Market sample, Values in parentheses are original which were transformed to log (X+1) for analysis, DAA- days after herbicide application. Table 2: Effect of treatments on density and dry weight of weeds at 60 DAA during 2010 and 2011 2010 2011

2 2 )

Treatments Dose Weed density (no./m ) at 60 DAA 2 Weed density (no./m ) at 60 DAA

(g/ha)

) 2

weed dry I. pp. E. E. E. A. C. A. E. E. E. A. C. A. (g/m s colona colona colona colona dryweight sessalis sessalis sessalis Total weed basifera basifera basifera basifera axillaris axillaris axillaris axillaris Cyperus crusgalli crusgalli crusgalli crusgalli rugosum rugosum Weight (g/m Total Butachlor50% EC(SS) 1250 0.0 (0.0) 1.3 (4.0) 0.0 (0.0) 0.0 (0.0) 1.1 (2.7) 2.5 (10.7) 41.1 0.7(1.3) 2.1(7.3) 0.7(1.3) 1.3(2.7) 1.6(4.0) 3.1(21.3) 45.5 Butachlor 50% EC (SS) 2000 0.0 (0.0) 1.1 (2.7) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 2.7 (14.7) 12.9 0.0(0.0) 1.7(4.7) 0.0(0.0) 0.0(0.0) 0.5(1.3) 3.2(25.3) 34.0 Butachlor 50% EC (SS) 4000 0.5(1.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 2..4(10.7) 12.5 0.0(0.0) 1.0(2.7) 0.0(0.0) 0.0(0.0) 0.0(0.0) 3.2(24.0) 24.9 Butachlor 50% EC (MS) 1250 2.0 (6.7) 2.7(13.3) 0.0 (0.0) 1.1 (2.7) 1.1 (2.7) 2.7 (13.3) 54.1 1.2(2.7) 2.1(8.0) 0.7(1.3) 1.3(2.7) 1.6(4.0) 3.1(22.7) 46.1 Butachlor 50% EC (MS) 2000 1.1 (2.7) 0.7 (2.7) 0.0 (0.0) 0.0 (0.0) 2.0 (6.7) 3.0 (20.0) 42.7 0.7(1.3) 0.0(0.0) 0.0(0.0) 0.0(0.0 0.0(0.0) 3.0(20.0) 36.8 Almix 20% WP 4 2.3 (9.3) 3.9(49.3) 2.0 (6.7) 0.0 (0.0) 0.0 (0.0) 1.1 (2.7) 211.3 0.0(0.0) 2.1(8.0) 1.8(5.3) 0.0(0.0 0.0(0.0) 0.0(0.0) 60.9 Weed free - 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0 0.0(0.0) 0.0(0.0) 0.0 Weedy - 2.5(10.7) 3.9(50.7) 2.0 (6.7) 2.7(14.7) 2.0 (6.7) 2.1 (8.0) 256.5 1.8(5.3) 3.1(21.3) 2.0(6.7) 1.8(5.3) 1.8(5.3) 2.6(17.3) 158.8 LSD(0.05) 0.8 1.3 0.3 0.6 0.8 0.7 29.0 0.5 0.5 0.6 0.3 0.6 0.6 21.6 SS - Sponsor sample, MS – Market sample, Values in parentheses are original which were transformed to log (X+1) for analysis, DAA- days after herbicide application. 3111

Fig 1: Effect of different treatments on

WCE at 60 DAA during 2010 and 2011 2010 2011 100

Weed Control Efficiency ControlWeed Efficiency (%) SS - Sponsor sample, MS – Market sample, DAA- days after herbicide application.

Treatments

All the treatments of weed control increase the weed control efficiency in respect to increase the doses of herbicide over weedy check (Fig 1). The results revealed that maximum weed control efficiency other than weed free treatment was obtained with application of Butachlor 50 EC (SS) at higher dose 4000 g/ha followed by its lower dose 2000 g/ha during both the years. Table 3 . Effect of herbicidal treatments on yield an d yield attributes of rice crop . Treatments Dose Panicles /m 2 Grains/ 1000 grain Grain yield Straw yield (g/ha) panicle weight(g) (kg/ha) (kg/ha) 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 Butachlor 50% EC 1250 147 212 161 130 21.6 23.7 4139 4650 6771 6663 (SS) Butachlor 50% EC 2000 163 237 185 140 22.9 24.1 4479 5056 7370 6963 (SS) Butachlor 50% EC 4000 162 213 146 135 23.0 23.5 4089 4812 6589 6760 (SS) Butachlor50% EC 1250 143 211 155 127 23.7 23.7 4167 4583 6406 6500 (MS) Butachlor50% EC 2000 157 215 159 131 24.7 24.0 4427 4928 7084 6917 (MS) Almix 20% WP 4 140 194 153 118 23.4 24.1 3281 4349 6271 6322 Weed free - 172 259 166 143 25.4 23.7 4505 4974 7451 6988 Weedy - 134 172 158 97 23.9 22.7 3015 4096 6302 5390 LSD 16 30 14 18 NS NS 350 273 525 352 Effect of herbicides on yield attributes and yield of rice All the yield attributing characters except 1000 grain weight were influenced significantly due to various herbicidal treat ments during both years (Table 3). Among the herbicidal Impact of Various Doses of Butachlor on Weed …. 3112

treatments, the highest value of yield attributing characters viz., panicles/m 2 and grains /panicle was recorded with application of Butachlor 50% EC (SS) at 2000 g/ha during both years. Grain yield is the principal and primary parameter for assessment of any weed control treatments applied in experimentations. Data on grain yield revealed that the all executed treatments out-weighted over the weedy check. Among the herbicidal application, Butachlor 50 EC (SS) 2000 g/ha performed the best by giving the highest grain yield of 4479 and 5056 kg/ha during 2010 and 2011, respectively which was comparable to weed free as well as butachlor (MS) at 2000 g/ha. During 2010 highest straw yield (7370 kg/ha) was achieved with the application of butachlor (SS) at 2000g/ha which was found at par with butachlor (MS) applied at 2000g/ha and weed free while in 2011, it was found maximum (6988 kg/ha) with weed free situation which was significantly superior to butachlor (MS) applied at 1250 g/ha, almix at 4g/ha and weedy check. The effective control of weeds starting from the early crop growth stage might have resulted in better growth and yield of rice. The variation in grain and straw yield under different treatments was the result of variation in weed density and weed control efficiency. Due to maximum infestation of weeds, the lowest grain yield of rice was recorded in the untreated control plots. Effect on microbial activity of soil A healthy population of soil microorganisms can stabilize the ecological system in soil (Chauhan et al ., 2006) due to their ability to regenerate nutrients to support plant growth. Any change in their population and activity may affect nutrient cycling as well as availability of nutrients, which indirectly affect productivity and other soil functions (Wang et al ., 2008). Before herbicidal application the population of bacteria, actinomycetes and fungi was not significantly affected by the treatments during 2011 while at harvest weed control measures had no significant effect on the population of bacteria during 2010 as well as on the population of actinomycetes and fungi during 2011. Maximum bacterial population was recorded with almix 20% WP at 4 g/ha before application and with butachlor 50% EC (MS) at the time of harvest which was found superior to all other herbicidal treatments except butachlor 50% EC (SS) applied at 4000 g/ha. During the year 2010, among herbicidal treatments, application of butachlor 50% EC (MS) at 1250 g/ha before application and application of butachlor 50% EC (SS) at 1250 g/ha at harvest achieved highest population of actinomycetes as well as fungi (Table 4).

3113 V. Pratap Singh, Neeshu Joshi, Neema Bisht, A. Kumar, Kavita Satyawali and R.P. Singh

Table 4: Effect of different treatments on microbial population at different stages Treatment Doses Bacteria 10 -7 Actinomycetes 10 -5 Fungi 10 -4 (g/ha) BA AH BA AH BA AH 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 Butachlor 50% EC 14.3 22.5 17.2 11.4 1250 12.7 16.0 12.0 12.7 21.00 3.3 3.7 22.03 (SS) 8 3 2 3 Butachlor 50% EC 15.5 21.4 18.7 14.6 2000 10.7 13.7 10.0 12.0 19.03 2.3 2.3 19.36 (SS) 1 0 8 9 Butachlor 50% EC 14.8 17.7 17.6 12.7 4000 10.3 14.7 8.7 9.4 16.40 1.7 2.0 17.63 (SS) 3 3 9 7 Butachlor 50% EC 15.6 23.2 18.8 13.9 1250 12.0 14.3 12.3 12.4 21.66 4.0 3.3 22.43 (MS) 7 0 1 8 Butachlor 50% EC 14.4 21.9 19.2 15.3 2000 13.0 14.7 9.3 11.7 19.30 2.3 2.7 20.73 (MS) 9 0 6 5 Almix 20% WP 13.2 20.2 19.3 16.4 4 13.3 14.7 10.7 12.0 20.46 4.0 3.0 21.63 5 6 6 0 13.9 20.5 18.7 12.7 Weed free - 15.3 18.3 12.7 13.4 22.93 4.3 4.0 21.80 1 3 4 9 14.3 23.1 16.6 16.7 Weedy - 14.0 15.7 14.7 15.0 20.46 3.7 4.7 22.76 7 0 2 2 Sem± - 0.9 1.1 1.5 1.0 0.7 1.0 0.9 1.0 0.5 1.0 0.4 1.1 CD at 5% - 2.9 NS NS 3.2 2.4 NS 2.7 NS 1.5 NS 1.3 NS BA- before application, AH- at harvest Effect on succeeding wheat crop All yield and yield attributing characters were not influenced significantly due to weed control treatments (Table 5) and they were statistically similar and at par to each other, including weedy check plots. Pre emergence application of Butachlor (sponsor sample as well as market sample) against weeds in rice crop during kharif season was safe for growing wheat crop. Table 5: Residual effect of herbicides on succeeding crop (wheat) Plant No. Grains/ 1000 grain Grain yield Straw yield Dose height -2 Treatments Spikes (m ) spike weight (g) (kg/ha) (kg/ha) g/ha (cm) 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 Butachlor 50% 92.3 116 37.0 39.8 4083 5708 1250 91.5 255 57.4 45.80 3198 6302 EC (SS) Butachlor 50% 91.0 108 39.5 43.7 4150 7458 2000 90.6 287 48.1 48.75 3283 7344 EC (SS) Butachlor 50% 93.5 128 40.5 45.1 4250 7208 4000 92.5 259 42.8 45.30 3229 6854 EC (SS) Butachlor 50% 93.0 111 35.2 40.3 3875 7500 1250 92.6 271 48.3 44.95 2948 7427 EC (MS) Butachlor 50% 89.1 128 38.6 42.2 4000 6750 2000 89.1 300 41.1 45.68 3271 7146 EC (MS) Almix 20% WP 4 91.6 92.0 100 298 38.1 49.5 43.6 47.06 3542 3000 7250 7167 Weed free - 92.0 92.0 99 276 33.3 52.6 43.1 46.26 4167 3448 7625 7510 Weedy - 95.2 95.5 109 284 40.7 45.4 41.7 46.90 3667 3062 7458 7105 CD at 5 % NS NS NS NS NS NS NS NS NS NS NS NS

Impact of Various Doses of Butachlor on Weed …. 3114

References [1]Bhanumurthy, V.B. and Subramanian, N.S. 1989. Adaptaion of new parameters, grain yield competition for weed control study. Indian J. Agri. Sci. 59:800-801. [2] Chauhan, A.K., Das, A., Kharkwal, H., Kharkwal, A.C. and Varma, A. 2006. Impact of microorganisms on environment and health, in A.K. Chauhan and A. Varma ed., Microbes: Health and environment. [3] FAO. 2012. Rice scenario. Directorate. Rice Research News Lett. 10(3):2. Hill, L.V. and Santlemann, P.V. 1969. Comparative effect of annual weeds on Spanish peanut. Weed Sci. 17:1&2. [4] Manhas, S.S., Singh, G., Singh, D. and Khajuria, V. 2012. Effect of tank-mixed herbicides on weeds and transplanted rice (Oryza sativa L.). Ann. Agri. Res. News Series. 33(1&2):25-31. [5] Mukherjee, D. and Singh, R.P. 2005. Effect of microherbicide on weed dynamics, yield and economics of transplanted rice (Oryza sativa). Indian J. of Agro. 50(4): 292-295. Prakash, C., Shivran, R.K. and Koli, N.R. 2013. Bioefficacy of new herbicides in transplanted rice. Indian J. Weed Sci. 45(4): 282-284. [6] Q.K. Wang, S.L. Wang, Y.X. Liu. 2008. Responses to N and P fertilization in a young Eucalyptus dunnii plantation; microbial properties, enzyme activities, and dissolved organic carbon. Appl. Soil Ecol. 40:484-490. [7] Reddy, B. S., and S. R. Reddy. 1999. Effect of soil and water management on weed dynamics in lowland rice. Indian J. Weed Sci. 31: 179-182. [8] Singh, G., Singh, V.P. and Singh, M. 2004. Effect of almix and butachlor alone or in combination on transplanted rice and associated weeds. Indian J. Weed Sci. 36(1&2):64-67. [9] Singh, P., Singh, R. and Singh, K.N. 2007. Efficacy of new herbicides in transplanted rice under temperate conditions of Kashmir. Indian J. Weed Sci. 39(3&4):167-171.

Full Length Research Paper

Acute toxicity of the chloroacetanilide herbicide butachlor and its effects on the behavior of the freshwater fish Tilapia zillii

Christopher Didigwu Nwani1*, Udu Ibiam Ama2, Florence Okoh3, Uche Obasi Oji2, Rebecca Chima Ogbonyealu2, Arua Agha, Ibiam4 and Onyinyechi Udu-Ibiam2

1Department of Zoology and Environmental Biology, University of Nigeria, Nsukka, Nigeria. 2Department of Biochemistry, Ebonyi State University PMB 053 Abakaliki, Nigeria. 3Department of Applied Biology, Ebonyi State University, PMB 053, Abakaliki, Nigeria. 4Department of Industrial Mathematics, Ebonyi State University, PMB 053, Abakaliki, Nigeria.

Accepted 5 December, 2012

Acute toxicity of butachlor, a chloroacetanilide herbicide (2-chloro-N-[2, 6-diethylphenyl] acetamide) to Tilapia zillii, was studied in a semi static bioassay. The 24, 48, 72 and 96 h LC50 values (with 95% confidence limits) estimated by probit analysis were 3.13 (2.88 to 4.61), 1.93 (0.63 to 4.41), 1.27 (0.59 to -1 1.92) and 1.25 (0.60 to1.85) mgl , respectively. There were significant differences (p<0.05) in the LC50 values obtained at different exposure times and the safe levels estimated by different methods varied from 1.25×10-1 to 1.25×10-5 mgl-1. There were dose and time dependent increase in mortality rate due to exposure to the herbicide. Stress signs in form of hyperactivity, erratic swimming, skin discoloration, -1 vigorous jerks of the body followed by exhaustion and death were observed. The 96 h LC50 of 1.25 mgl obtained indicate that the herbicide was toxic to T. zillii. Agricultural use of butachlor in the environment, especially, near water bodies, must be restricted to avoid the severe risk associated with the use of the pesticide.

Key words: Tilapia zillii, butachlor, toxicity, mortality, behavioral changes, safe level.

INTRODUCTION

Butachlor, (2-chloro-N-[2, 6-diethylphenyl] acetamide) is lected from tube wells adjacent rice fields in Philippines one of the most widely used chloroacetanilide herbicide (Natarajan, 1993). The pesticide can degrade rapidly but for the control of annual grasses in rice fields and many under conditions of low temperature, low moisture, high broadleaf weeds. It can also be used in seed beds and alkalinity and lack of suitable microbial degraders, it may seed transplant fields as well as in some crop fields such remain biologically active and persist in soils for a long as wheat, barley, cotton, vegetables and peanuts. It reac- time. Lin et al. (2000) reported that increase in sunlight hes aquatic environment due to the proximities of the enhanced photo degradation of butachlor in water and agricultural country sides to water places. The repeated that the half life of the herbicide in non-filtered river water and indiscriminate use of the herbicide, careless hand- was shorter than filtered samples. Lin et al. (1999) also ling, accidental spillage or discharge of untreated efflu- reported that surface waters with high concentrations of ents into natural water ways has harmful effects on the organic and suspended solid reduced the toxicity of fish and other aquatic organisms. Butachlor contamina- pesticides possibly through photo degradation. tion of 0.163 ppb has been recorded in ground water col- The contamination of aquatic ecosystems by xenobio- tics has gained increased attention and several recent studies have demonstrated the toxicity of butachlor to fish

*Corresponding author. E-mail: [email protected]: and other aquatic organisms (Geng et al., 2005; 2010; +2348037509910. Ateeq et al., 2006; Geng et al., 2005; Peebua et al., 500 Afr. J. Biotechnol.

Table 1. Data on fish survival at different test concentrations and sampling time intervals in T. zillii.

Exposed concentration Number Number of fish alive at different time intervals (hours) Mortality (mgl-1) exposed 24 48 72 96 Survival (%) (%) 0.00 30 30 30 30 30 100 00 0.60 30 30 30 30 24 80 20 1.20 30 28 24 20 18 60 40 1.80 30 26 21 14 12 40 60 2.40 30 24 12 10 06 20 80 3.00 30 18 10 4 00 00 100

2007; Geng et al., 2010, Chang et al., 2011). Butachlor Acute toxicity bioassay has also been reported to be carcinogenic (Ou et al., Acute toxicity assay to determine the 96 h LC50 values of butachlor 2000) and can adversely disrupt the reproductive process was conducted with definitive test in a semi-static system in the and affect the thyroid and sex steroid hormones in Zebra laboratory as per the standard methods (APHA, AWWA, WPCE, fish (Chang et al., 2011). The fish Tilapia zillii is com- 2005). The range finding test was carried out prior to determine the monly found in all African countries and some other tropi- concentrations of the test solution for definitive test. The experiment cal countries of the world. It is an aquaculture candidate was conducted in glass aquaria (60 × 30 × 30 cm size) containing that can narrow the gap between demand and supply of 40 L of de-chlorinated and aerated water. The test solution was changed on every alternate day to counter-balance the decreasing animal protein in developing countries. Furthermore, pesticide concentrations. During the treatment, fish behavior was Tilapia zillii is an attractive model species for toxicity observed daily. In definitive test, a set of 10 fish specimen were studies because of its availability throughout the year, randomly exposed to nominal butachlor (0.60, 1.20, 1.80, 2.40, 3.00 ease of culture, prolific reproduction, omnivorous nature mgl-1) concentrations. Another set of 10 fish were simultaneously and their general hardiness in culture environment. maintained in tap water, without test chemical, and considered as In spite of the wide application of butachlor to farm- control. The experiment was set in triplicate and mortality of the fish due to butachlor exposure was recorded up to 96 h at every 24 h lands by our farmers and the possible ecotoxicological interval (Table 1) to obtain LC50 values of the test pesticides. The impact attached to its use, there is paucity of information LC50 of butachlor was determined following the probit analysis on its effects on many local fish species like Tilapia zillii. method described by Finney (1971). The safe level of the test The present study thus aims at the determination of the pesticides was estimated by multiplying the 96 h LC50 with different 96 h LC of butachlor to the freshwater fish Tilapia zillii, application factors (AF) and was based on Hart et al. (1948), 50 Sprague et al. (1971), Committee on water Quality Criteria (CWQC, their behavioral responses and the possible safe levels of 1972), National academy of Sciences/National Academy of Engi- the pesticide during the exposure period. neering (NAS/NAE, 1973), Canadian Council of Resources and Environmental Ministry (CCREM, 1991) and the International Joint Commission (IJC, 1977). The physicochemical properties of test MATERIALS AND METHODS water, namely temperature, dissolved oxygen; pH, total hardness, conductivity and total alkalinity were analyzed using standard Experimental fish specimen and chemicals methods (APHA, AWWA, WPCE, 2005).

The juveniles of freshwater fish Tilapia zillii (Family: Cichlidae, Data analysis Order: Perciformes) were caught from nearby rivers, ponds and lakes with the help of local fishermen. The fish specimens had an The data obtained were statistically analyzed by statistical package average (±S.D) wet weight and length of 8.304 ± 0.13g and 6.17 ± SPSS (Version 16). The data were subjected to one way analysis of 0.15cm, respectively. To avoid bias, fish of similar length and variance (ANOVA) and Duncan’s multiple range test to determine weight range were selected and used. Specimens were subjected the significance difference at 5 % probability level. to a prophylactic treatment by bathing twice in 0.05% potassium permanganate (KMnO4) for 2 min to avoid any dermal infections. The specimens were then acclimatized for 2 weeks under RESULTS laboratory conditions in semi-static systems. They were fed with commercial trout pellets daily at 2% body weight (BW) during Physico-chemical parameters of the test water acclimatization. The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water. The fish The physico-chemical characteristics of the test water are were treated in accordance with the rules conforming to principles presented in Table 2. The water temperature varied from of Laboratory Animal Care as set by set by the Institutional Animal 23.50 to 25.50°C, pH ranged from 7.15 to 7.80 while Ethics Committee (IAEC) of the University. For the present study, dissolved oxygen varied from 6.30 to 6.80 mgl-1. The commercial formulation of butachlor (50% EC) with trade name conductivity value ranged from 252 to 300 µM cm-1 “butaforce” manufactured by Anhui Futian Agrochemical Ltd -1 Shanghai, China, with CAS NO A5-0268 was purchased from the whereas total hardness varied from 174 to 180 mgl as local market and used. CaCO3 during the experimental period. Nwani et al. 501

Table 2. Physico-chemical properties of the test water.

Characteristic Mean Range Air temperature (°C) 25.5 25.40-26.42 Water temperature (°C) 24.66 23.50-25.50 PH 7.20 7.15-7.80 Dissolved oxygen (mgl-1) 6.55 6.30-6.80 Conductivity (µMcm-1) 254 252-300 Total hardness (mgl-1) 176 174-180

Table 3. Lethal concentration of butachlor (mgl-1) (95% confidence intervals) depending on exposure time for T. zillii (n=10 in three replicates).

Lethal Exposure time (h) concentration 24 48 72 96 a a b b LC10 2.14 (1.61-2.40) 1.17 (0.01-1.65) 0.53 (0.04-0.90) 0.52 (0.04-0.87) a a b b LC20 2.47 (2.13-2.77) 1.40 (0.01-1.89) 0.72 (0.10-1.10) 0.69 (0.11-1.07) a a b b LC30 2.75 (2.47-3.25) 1.57 (0.03-2.17) 0.89 (0.20-1.31) 0.87 (0.21-1.27) a ab b b LC40 3.00 (2.70-3.86) 1.75 (0.18-2.67) 1.07 (0.36-1.55) 1.05 (0.37-1.51) a ab b b LC50 3.26 (2.88-4.61) 1.93 (0.67-4.41) 1.27 (0.59-1.92) 1.25 (0.60-1.85) a a a a LC60 3.54 (3.07-5.53) 2.12 (1.30-14.23) 1.50 (0.89-2.59) 1.48 (0.89-2.46) a ab ab b LC70 3.87 (3.27-6.74) 2.36 (1.69-18.06) 1.81 (1.21-4.05) 1.79 (1.20-3.75) a ab ab b LC80 4.30 (3.51-8.53) 2.67 (1.96-65.67) 2.24 (1.54-7.69) 2.22 (1.54-6.87) a a a a LC90 4.96 (3.86-11.84) 3.16 (2.26-95.11) 3.02 (1.98-20.48) 3.00 (1.98-17.40)

Values with different alphabetic superscripts differ significantly (p<0.05) between exposure time within lethal concentration.

Behavioral response of fishes to test concentrations values (with 95% confidence limits) of different concentrations of butachlor in T. zillii were found to be 3.13 (2.88 Fish exposed to different concentrations of the pesticide to 4.61), 1.93 (0.63 to 4.41), 1.27 (0.59 to 1.92) and 1.25 -1 displayed behavioral abnormalities in response to the test (0.60 to 1.85) mgl respectively for 24, 48, 72 and 96 h chemical. At the initial exposure, fish that were alert exposure time (Table 3). A time and dose-dependent stopped swimming and remained static in position in res- increase in mortality rate was observed; thus, as the ponse to the sudden changes in the surrounding environ- exposure time increased from 24 to 96 h, the median ment. After some time, fish in the experimental groups lethal concentration required to kill the fish was reduced. tried to avoid the test water by swimming very fast, jump- There were significant differences (p < 0.05) in LC10-90 ing and displaying other random movements. In tanks values obtained for different times of exposure. During with higher concentrations of the test pesticide swimming the experimental period no mortality was recorded in the of the fish was erratic with vigorous jerky movements control experiment. The estimated safe level of butachlor along with hyper excitation. Faster opercula movement, as calculated by multiplying the 96 h LC50 with different surfacing and gulping of air were observed. Body pig- application factors are given in Table 4. The values of mentation was decreased while copious mucus which safe level of butachlor in the Tilapia zillii varied from -1 -5 -1 covered the buccal cavity, body and gills were secreted. 1.25×10 to 1.25×10 mgl . Later, the fish lost their balance, consciousness, engaged in rolling movement and became exhausted and lethargic owing to respiratory incumbency. Soon, they settled DISCUSSION down passively at the bottom of the tank with the opercu- lum wide open and ultimately died. Acute and chronic toxicity tests are widely used to eval- uate the toxicity of chemicals on non-target organisms -1 (Santos et al., 2010). The 96 h LC50 of 1.25 mgl ob- Median lethal concentration and application factor tained indicate that the herbicide is toxic to T. zillii. The toxicity is both concentration and time dependent thus, Median lethal concentration (LC50) is the concentration of accounting for the differences in the values obtained at a test chemical, which kill 50% of the test organism in a different concentrations and exposure times. When T. -1 particular length of exposure, usually 96 h. The LC50 zillii juveniles were exposed to 0.60 mgl butachlor con- 502 Afr. J. Biotechnol.

Table 4. Estimate of safe levels of butachlor at 96 h exposure time.

-1 -1 Chemical 96 h LC50 (mgl ) Method AF Safe level (mgl ) Butachlor 1.25 Hart et al. (1948)* - 2.30×10-2 Sprague (1971) 0.1 1.25×10-1

CWQC (1972) 0.01 1.25×10-2

NAS/NAE (1973) 0.01-0.00001 1.25×10-1-1.25×10-5

CCREM (1991) 0.05 6.25×10-2 -2 IJC (1977) 5% LC50 6.25×10

2 *C = 48 h LC50×0.03/S , where C is the presumable harmless concentration and S = 24 h LC50/48 h LC50.

centrations, only 20 % died after 96 h, whereas all the Our results from the present study on butachlor toxicity fish (100 %) died after 96 h expose at 3.00 mgl-1 support the conclusion that T. zillii is sensitive to the butachlor concentration. Previously reported LC50 values pesticide and their mortality rate is dose and time depen- of butachlor in Oreochromis niloticus (Wang et al., 1992), dent. This study also shows the significance of behavioral Heteropneustus fossilis (Ateeq et al., 2005) and Channa parameters in assessing the hazards of the pesticide to punctatus (Tilak et al., 2007) were 0.880 mgL-1, 2.34 ppm fish. Agricultural use of butachlor in the environment and 247.46 ppb, respectively. Geng et al. (2005) reported especially near water bodies must be restricted to avoid -1 96 h LC50 of 1.40 mgl when Rana japonica was exposed the sever risk associated with the use of the pesticide. to butachlor while Gobic and Gunasekaran (2010) obtained 96 h LC50 of 0.515 mg/kg for Eisenia fietida. From the observed results, 96 h LC50 value for butachlor ACKNOWLEDGEMENTS -1 in T. zilli was higher than the 10.20 and 381.9 µgl obtained when similar fish, Nile tilapia (Oreochromis niloti- We acknowledge Tertiary Education Trust Fund Nigeria cus) was exposed to endosulfan (Werimo and Willen, (TETFUND), and the DERIC, Ebonyi State University, 2010) and alachlor (Peebua et al., 2007), respectively Abakaliki for providing the fund and the conducive thus indicating that endosulfan and alachlor are more to- environment, respectively, to carry out the research. xic to tilapia than butachlor. The previous literature clearly indicates that the toxicity of butachlor varies from one species to another and even in strains of the same REFERENCES species. Toxicity of chemicals to aquatic organisms has been Altinok I, Capkin E, Boran H (2012). Mutagenic, genotoxic and inhibitory reported to be affected by temperature, pH, dissolved effects of carbosulfan in rainbow trout Oncorhynchus mykiss. Pest. Biochem. Physiol. 102:61-67. oxygen, size and age, type of species, water quality, APHA, AWWA, WPCF (2005). Standard methods for the examination of concentration and formulation of test chemicals (Gupta et water and waste water, 21st 401 ed. American Public Health al., 1981; Young, 2000; Nwani et al., 2010). The safe Association, Washington, DC. level obtained for butachlor in the present study varied Ateeq B, Farah M, Ahmad W (2006). Evidence of apoptotic effects of -1 -5 -1 2,4-Dand butachlor on walking catfish Clarias batrachus by from 1.25×10 to 1.25×10 mgl . However, the large transmission electron microscopy and DNA degradation studies. Life variation in safe levels determined by various methods Sci, 78:977-986. has resulted in controversy over its acceptability Banaee M, Sureda A, Mirvaghefi AR, Ahmadi K (2011). Effects of (Buikema et al., 1982; Pandey et al., 2005). Kennega diazinon on biochemical parameters of blood in rainbow trout (Oncorhynchus mykiss). Pest. Biochem. Physiol. 99:1-6. (1979) emphasized that the major weakness in calcula- Bekeh AF, Ndome CB, Bayin PRB (2011). Some hematological tion of application factor (AF) is its dependence on LC50 changes in Oreochromis niloticus juveniles exposed to butachlor. J. values. Behavioral changes are the most sensitive indica- Agric. Food Tech. 1 (6):73-80. tors of potential toxic effects (Banaee et al., 2011). The Buikema JR, Naider-Lehner AL, Cairns JR (1982). Biological monitoring: Part IV. Toxicology Testing. Environ. Mol. Mutagen. behavioral and swimming patterns of the fish in the con- 33:239-262. trol group were normal but for the experimental groups CCREM (1991). Canadian Water Quality Guidelines; Canadian Council abnormal swimming behavior increased with increasing of Resources and Environmental Ministry, Inland Waters Directorate, concentration and exposure time. Bekeh et al. (2011) Environment Canada: Ottawa, ON, Canada. Chang J, Liu S, Zhou S, Wang M, Zhu G (2011). Effects of butachlor on reported similar behavioral responses in Tilapia (Oreo- reproduction and hormone levels in adult zebrafish (Danio rerio). Exp. chromis niloticus) exposed to butachlor. The behavioral Toxicol. Pathol. doi:10.1016/j.etp.2011.08.007. symptoms observed during butachlor exposure to T. zillii Committee on Water Quality Criteria (CWQC) (1972). A Report of the also agree with the observations reported by other Committee on Water Quality Research Series, EPA-R3-73-003, US Environmental Protection Agency Report; CWQC: Cincinnati, OH, authors reporting toxicity of pesticides in fish (Kumar et USA. al., 2010, Pandey et al., 2011; Banaee et al., 2011, Finney DT (1971). Probit Analysis. Cambridge University press, Chang et al., 2011; Altinok et al., 2012). Cambridge, p.333. Nwani et al. 503

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Comparative and combined acute toxicity of butachlor, imidacloprid and chlorpyrifos on earthworm, Eiseniafetida.

Chen C1, Wang Y2, Zhao X2, Wang Q2, Qian Y1

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Chemosphere, 27 Dec 2013, 100:111-115

DOI: 10.1016/j.chemosphere.2013.12.023 PMID: 24377448

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Abstract

Various pesticides have become widespread contaminants of soils due to their large applications in agriculture and homes. An earthworm assay was used to assess the acute toxicity of butachlor, imidacloprid and chlorpyrifos with different modes of action. Ecotoxicities of these pesticides were compared for earthworm Eiseniafetida separately and in combination in artificial soil and contact filter paper tests. Imidacloprid was the most toxic for E. fetida with LC₅₀ (lethal concentration 50) values three orders magnitude lower than that of butachlor and chlorpyrifos in both tests. The toxicity of the mixtures was compared to that predicted by the concentration addition (CA) model. According to the CA model, the observed toxicities of all binary mixtures were less than additive. However, for all the mixtures in 14 d artificial soil test, and mixtures of butachlor plus chlorpyrifos and imidacloprid plus chlorpyrifos in 48 h contact filter paper test, the difference in toxicity was less than 30%, hence it was concluded that the mixtures conformed to CA. The combined effects of the pesticides in contact filter paper tests were not consistent with the results in artificial soil toxicity tests, which may be associated with the interaction of soil salts with the pesticides. The CA model provides estimates of mixture toxicity that did not markedly underestimate the measured toxicity, and therefore the CA model is the most suitable to use in ecological risk assessments of the pesticides.

Bull Environ ContamToxicol

. 2018 Feb;100(2):208-215. doi: 10.1007/s00128-017-2245-9. Epub 2017 Dec 13.

Oxidative Stress Response Induced by Butachlor in Zebrafish Embryo/Larvae: The Protective Effect of Vitamin C

Qingqing Xiang 1, Bofan Xu 1, Yilun Ding 2, Xiaoyi Liu 1, Ying Zhou 3 4, Farooq Ahmad 5

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PMID: 29236155 DOI: 10.1007/s00128-017-2245-9

Abstract

The widespread contamination and persistence of the herbicide butachlor in the environment resulted in the exposure of non-target organisms. The present study investigated the toxicity effect of butachlor (1-15 µmol/L) and the protective effect of vitamin C (VC) against butachlor-induced toxicity in zebrafish. It was found that butachlor significantly increased the mortality and malformation rates in a dose-dependent manner, which caused elevation in reactive oxygen species (ROS) and malondialdehyde (MDA) after 72 h exposure. Compared with butachlor treatment group, the protective effect of VC against butachlor-induced toxicity were observed after adding 40, 80 mg/L VC respectively. VC significantly decreased the mortality, malformation rates, ROS, MDA, and normalized antioxidant enzymes activities of zebrafish after 72 h exposure. The result shows VC has mitigative effect on butachlor-induced toxicity and it can be used as an effective antioxidant in aquaculture.

Keywords: Butachlor; Oxidative stress; Protective effect; Vitamin C; Zebrafish.