Effect of herbicide (atrazine and paraquat) application on soil bacterial population

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1 Sky Journal of Soil Science and Environmental Management Vol. (9), pp , December, 013 Available online ISSN Sky Journals Full Length Research Paper Effect of herbicide (atrazine and paraquat) application on soil bacterial population Stanley H. O 1., Maduike E. M * and Okerentugba P. O 1. 1 Department of Microbiology, Faculty of Science, University of Port Harcourt, P.M.B. 533 Choba, Port Harcourt, Nigeria. Department of Animal and Environmental Biology, Faculty of Science, University of Port Harcourt, P.M.B. 533 Choba, Port Harcourt, Nigeria. Accepted 1 November, 013 The effect of two herbicides, - atrazine and paraquat on soil bacterial population, soil ph and percentage moisture content was assessed over a period of eight s. Soil samples from uncultivated, open field were treated with herbicides at recommended rates and half (0.5x) recommended rates. The soil ph was affected while the differences in percentage moisture content of the treated soils depended on factors other than the treatment. Each collection point was sampled at two depths, 10 and 0 cm. Bacterial populations decreased upon treatment with herbicides at both concentrations when compared with the control (untreated soil). Concentrations of herbicide treatments at recommended rates resulted in much lower bacterial count and diversity compared to soils treated with half the recommended dose. The bacterial population for all treatments dropped at the 4 th of post treatment, but increased progressively at the 6th and 8th. Herbicide treatments also resulted in the suppression of some bacterial species, e.g. Proteus sp, and Actinomycetes. Bacillus sp. and Pseudomonas sp. were the most frequently isolated bacteria from herbicide treated soils. Thus, the microorganisms which were sensitive to herbicide application, therefore, could serve as a reliable indicator of the biological value of soils while the resistant ones could be further studied for bioremediation purpose. Key words: Microorganisms, concentrations, soil ph, soil moisture content. INTRODUCTION Fertile soil is inhabited by the root systems of higher plants, many animal forms (e.g., rodents, insects and worms), and by tremendous numbers of microorganisms. Usually, the density of organisms is less in cultivated soil than uncultivated/virgin land and population decreases with soil acidity. The bacterial population of the soil exceeds the population of all other groups of microorganisms in both number and variety. In modern agricultural production, herbicide application is a regular practice. The problems caused by the increased application of herbicides call for multidisciplinary approach (Dobrovoljskiy and Grishina, 1985). *Corresponding author. meekebere@yahoo.com. Herbicides are chemical substances or preparations designed to kill plants, especially weeds, or to inhibit their growth. Herbicides become incorporated in soil directly, during plant treatment and indirectly, via water or residues of plant and animal origin. After application, herbicides can be (i) evaporate (volatilize),(ii) washed through surface run-off,(iii) leach into deep soil strata and ground water, (iv) inactivated by plants, or (v) be adsorbed in soil in which case they become subject to chemical or microbiological degradation. An ideal pesticide should have the ability to destroy target pest quickly and should be able to degrade as non-toxic substances as quickly as possible. The increased use of herbicides in agricultural soils causes the contamination of the soil with toxic chemicals. When herbicides are applied, the possibilities exist that these chemicals may exert certain effects on non-target organisms, including

2 10 Sky. J. Soil. Sci. Environ. Manage. soil microorganisms (Simon-Sylvestre and Fournier, 1979; Wardle and Parkinson, 1990). The microbial biomass plays an important role in the soil ecosystem where they fulfill a crucial role in nutrient cycling and decomposition (De-Lorenzo et al., 001). Herbicides, when applied could then accumulate to toxic levels in the soil and become harmful to microorganisms, plant, wild life and man (Amakiri, 198). There is an increasing concern that herbicides not only affect the target organisms (weeds) but also the microbial communities present in soils, and these non-target effects may reduce the performance of important soil functions (De-Lorenzo et al., 001). There is an increasing concern that herbicides not only affect the target organisms (weeds), but also the microbial communities present in soils, and these non-target effects may reduce the performance of important soil functions. Atrazine is an example of triazine herbicides with the trade name, Multrazine. The triazines were shown to inhibit photosystem II, but have no effect on photosystem I (Trebst, 008) while paraquat is an example of quaternary ammonium herbicides with the trade name, Gramoxone. Paraquat is known to act on the Phosystem I within the photosynthetic membrane. On microorganisms, they have inhibitory effects, repressing effects, reduces enzyme activity and mycelia (growth). Having known the actions of these herbicides on plants, this work is targeted to ascertain the common soil microorganisms in the study site and how herbicides affect their population and distribution. MATERIALS AND METHODS Study area The study area was located at the uncultivated field behind Ofrima building within University of Port Harcourt, Nigeria. The site (open field) was divided into smaller plots (blocks) of 9m areas. Herbicides The herbicides used were purchased from a local agricultural dealer store in Port Harcourt. The herbicides are Gramoxone (designated G in this study) which contains 76 g paraquat dichloride (00 g paraquat ion) per litre and Multrazine (designated as M ) which contains 50% atrazine as the active ingredient. Gramoxone is distributed by CG Biostadt Limited while Multrazine is marketed by Multichem Industries Limited. Soil treatments The herbicides were applied at recommended rates (G1 = paraquat, M1 = atrazine) and half (0.5x) the recommended rates (G = paraquat, M = atrazine) to know their effect on the soil bacterial population. The treatments were carried out at recommended rates of 0.6 L/ha (at ml in 10 L sprayer) for paraquat and 3 L/ha (at 150 ml in 10 L sprayer) for atrazine. Thereafter, two other preparations were made which contained half the recommended concentration of each herbicide. Soil treatments were carried out in duplicates. The preparations were made as described by Pal et al. (1994). Sample collection The soil samples were collected using the soil augar. Two depths (10 and 0 cm) of soil samples were collected from each block of field during each collection from the same point. The soil samples were made free of large stones and plant debris. The research was an eight s post-treatment sampling at two s interval. Physicochemical analyses Soil ph A soil: water ratio of 1 : was used for the determination of soil ph of herbicide treated soils using Equip-Tronics digital ph meter model EQ-610. The method described by Onyeike and Osuji (003) was used. Moisture content The thermal drying technique was used at 100 C in an air circulation oven. B & T Laboratory thermal Equipment (oven) and OHAUS Portable Scale Scout II (weighing balance) were used. The method described by Onyeike and Osuji (003) was used. Moisture (%) = Loss in weight on drying (g) x 100 Initial sample weight (g) 1 Temperature The temperature of the soil was taken using mercury-inbulb thermometer during each collection. Bacterial enumeration and identification A ten-fold serial dilution was made for each soil sample. Nutrient agar (NA) was used for the enumeration of total heterotrophic bacteria by the spread plate method in triplicate plates. Incubation was done at C for 4-48 h. Bacterial and actinomycetes isolates were characterized based on cultural characteristics, staining reactions and biochemical reactions as described by

3 Mean Stanley et al. 103 Table 1. Effect of herbicide application on soil ph. Soil treatments G 1 G M 1 M *Insignificant Weeks Mean 5.* 5.7* 5.1* 5.6* 5.3 Table. Percentage moisture content and temperature values in control and herbicide treated soils. Wks (% moisture) 10cm 0cm Paraquat (% moisture) Atrazine (% moisture) Temp. Recommended rate (G 1) x0.5 recommended rate (G ) Recommended rate (M 1) x0.5 recommended rate (M ) ( C) 10cm 0cm 10cm 0cm 10cm 0cm 10cm 0cm Cheesbrough (000). Identification was thereafter made with reference to Bergey s manual of systemic bacteriology (1984). Statistical analysis Data generated from the study were subjected to analysis of variance (ANOVA). RESULTS The statistical test of significance of the effect of herbicide treatments on soil ph showed that the differences obtained (Table 1) as a result of the treatments are not significant since the computed F value (1.4ns) is lower than the tabular F value (3.06) at 5% level of significance. However, the coefficient of variation showed a 91.65% chance in the results from the experiment occurring as a result of treatments (indicates that, soil ph was affected by the treatments). Considering the effect on percentage moisture content, at 10 cm depth, the statistical result showed; computed F value, 0.19 < tabular F value, 3.06, at 5% level of significance. At 0 cm depth, the result showed; computed F value, 0.16 < tabular F value, 3.06, at 5% level of significance. Therefore, herbicide treatments at both depths had no significant effect on percentage moisture content of the soil. Thus, indicating that changes in moisture content of soils depended on factors (e.g. temperature) other than the treatments (Table ). All the herbicides used for treatment (both the recommended and 0.5x recommended rates) in this study resulted in significant reduction in soil bacterial population, diversity and distribution (Figure 1). Discussion From the ANOVA result at 10 cm depth, the differences on the number of viable bacterial counts by the treatments are highly significant since the computed F value (14.7**) is greater than the tabular F value (4.89) at 1% level of significance. Analysis of variance at 0 cm depth also showed a high significant effect on bacterial count. At recommended rates, maximum mean bacterial counts were observed at the 8th of post treatment in the two herbicide treated soils; G 1 = 1.8 x 10 6 cfu/ml, M 1 =. x 10 6 cfu/ml. These counts were lower compared to the control soil which was.9 x10 6 cfu/ml. Treatments at x0.5 recommended rates resulted in much higher bacterial counts compared to soils treated at recommended rates; G =.6 x10 6 cfu/ml, M =.7 x 10 6 cfu/ml. The bacterial populations for all soil samples dropped at 4 of post treatment, but increased progressively at the 6th and 8th s (Figure 1). Ayansina and Oso (006) discovered that higher

4 104 Sky. J. Soil. Sci. Environ. Manage. Cfu/ml (x10 6 ) >> >> 4 ooo ooo 6 //// //// 8 0 G1 G M1 M Herbicide treated soil Figure 1. Effect of herbicides on soil bacterial populations. G 1 = paraquat treated soils (recommended conc.); G = paraquat treated soils (0.5 x recommended conc.); M 1 = atrazine treated soils (recommended conc.); M = atrazine treated soils (0.5 x recommended concentration). concentrations of herbicide treatments resulted in much lower microbial counts when compared to soils treated with recommended doses. This study agrees with the above statement because the recommended concentrations resulted in much lower bacterial counts when compared with soils treated with half the recommended doses. The high bacterial counts observed in soil treatments at two could be due to the fact that the soil microflora are able to temporarily mineralize and use the herbicides as energy sources. However, this was followed by a general decline in microbial counts. Taiwo and Oso (1997) have also suggested that this decline in microbial counts must have been due to the fact that microbial populations that were tolerant of treated pesticides (herbicides) were susceptible to the products of soil-pesticides (herbicides) interactions, which could have possibly been bactericidal. Also, Cloete et al. (006) also stated that system under stress usually cause species diversity to decrease and may result in an increase in numbers of the species capable of tolerating stress. This supports the decline in 4 (Figure 1). Conclusion Based on the results obtained from this study, it is obvious that; (i) when herbicides are applied, the chemicals exert certain effects on non-target organisms, including soil microorganisms, (ii) paraquat reduced the population and diversity of bacteria more than atrazine, (iii) in an open field, the top soil (10 cm depth) harbors

5 Stanley et al. 105 Table 3. Bacteria isolated from control and herbicide treated soils in relation to depth. Soil treatment 10cm depth Bacillus cereus, Pseudomonas sp., Actinomycetes, Bacillus subtilis, Proteus sp., Micrococcus sp. Bacterial isolates 0cm depth Bacillus cereus, Pseudomonas sp., Actinomycetes Bacillus subtilis, Proteus sp., Micrococcus sp. G 1 Bacillus cereus, Pseudomonas sp. Bacillus cereus. G Bacillus cereus, Pseudomonas sp., Bacillus cereus, Pseudomonas sp. Bacillus subtilis. M 1 Bacillus cereus, Pseudomonas sp., Bacillus cereus, Pseudomonas sp., Micrococcus sp. Bacillus subtilis. M Bacillus cereus, Pseudomonas sp., Micrococcus sp. Bacillus cereus, Pseudomonas sp. Microbiological research should be focused on isolating microbial strains which, on application, effectively degrade herbicides, irrespective of the natural microbial population in the soil. This will help to restore the soil back to its initial state before herbicide application. more microorganisms (both in population and diversity) than the sub soil (0 cm depth), (iv) The herbicides at recommended rates, G1 and M1, reduced the population and diversity of bacteria in the soil than 0.5x recommended rates, G and M. The microorganisms, Proteus sp. and Actinomycetes which were sensitive to herbicide application (Table 3), therefore, may serve as a reliable indicator of the biological value of soil. Inoculants based on microorganisms that possess the potential to degrade herbicides could be used as bio-preparations in combination with chemical preparations. Simon-Sylvestre G, Fournier JC (1979). Effects of pesticides on soil micro flora. Adv. Agron., 31: 1-9. Taiwo LB, Oso BA (1997). The Influence of some pesticides on soil microbial flora in relation to changes in nutrient level, rock phosphate solubilization and P-release under laboratory conditions. Agric. Ecosystem & Environ.,65: Trebst A (008). The mode of action of triazine herbicides, In: The triazine herbicides: 50 years revolutionizing agriculture, LeBaron, H.M., McFarland, J.E. & Burnside, O. (Eds.), , Elsevier, Oxford (UK). Wardle DA, Parkinson D (1990). Effects of three herbicides on soil microbial biomass and activity. Plant Soil, 1(1): 1-8. REFERENCES Amakiri MA (198). Microbial Degradation of soil applied herbicides. Nig. J. Microbiol., : Ayansina ADV, Oso, BA (006). Effect of two commonly used herbicides on soil micro flora at two different concentrations. Afr. J. Biotechnol., 5(): Bergey H, Krieg NR, Holt JG (1984). Bergey s manual of systemic bacteriology. Baltimore. Cheesbrough M (000). District Laboratory Practice in Tropical Countries Part. Press Syndicate of the University of Cambridge. Cloete TE, Atlas RM (006). Basic and Applied Microbiology. Van Schalk publishers, Hatfield Pretoria. pp De Lorenzo ME, Scott GI, Ross PE (001). Toxicity of pesticides to aquatic microorganisms: a review. Environ. Toxicol. Chem., 0: D o b r o v o l j s k i y GV, G r i s h i n a LA (1985). Security soil. Moscow: Kolos, p. 4. Onyeike EN, Osuji JO (003). Research Techniques in Biological and Chemical Sciences. Springfield Publishers Ltd. pp Pal Sk, Das Gupta SK (1994). Pest (Skill Development Series no. 15) Training and Fellowship program, International Crop Research Institute for Semi Arid Tropics (ICRISAT), Patancheru 5034, Andhra Pradesh, India. pp