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Toxic Effects of Fungicides (Penconazole and Triadimenol) on Growth and Protein Amount of Scenedesmus acutus.

Byline: Nur Agirman, Burcin Bedil, Gokce Kendirlioglu and Ahmet. Kadri Cetin

Summary: In this work, the effects of penconazole and triadimenol to protein amount and growth of the microalgae Scenedesmus acutus were investigated. The culture of S. acutus was exposed to dissimilar concentrations of penconazole and triadimenol (1, 3, 6, 10 and 15 g/L) in the climate chamber at 23 1C and 16:8 photoperiod. Cell numbers and growth rates in liquid cultures of S. acutus were calculated for a period of four days. The results indicated toxic effects of penconazole and triadimenol on growth and protein amount of S. acutus. The protein amount and cell number of S. acutus in cultures treated with penconazole and triadimenol were rapidly decreasing at second day.

Key Words: Scenedesmus acutus, Penconazole, Triadimenol, Protein amount, Cell growth.

Introduction

Aquatic environments are generally impacted by different pollutants from various sources. On living organisms in aquatic ecosystems was conducted numerous studies examining the effects of these pollutants. [1-3]. Generally, pesticides leaved into aquatic environment from agriculture activities. There is a conspicuous risk that non-target organisms are damaged because of pesticide's toxic features, ether at the application place, or by reason of unplanned spreading, at nearby, or even faraway, areas. Chemical materials or chemical mixtures aimed to controlling and killing, any organisms is presumed as pesticides. There are many kinds of pesticides, insecticides, herbicides, defoliants, desiccants, fungicides, nematicides, avicides, and rodenticides are some of them.

Fungicides inhibiting the biosynthesis of ergosterol have been show to enhance the effect of green algae and photobacteria in aquatic environment [4]. Triadimenol is a fungicide with preventive and extirpation action. This fungicide is used for controlling of dusty mould and rusts in cereals. Triadimenol is used eliminated leaf spot disease on various plants. Penconazole is a systemic fungicide that with curative and protective effect. For controlling the pathogenic organisms as Basidiomycetes, Ascomycetes and Deuteromycetes on various fruits, vegetables and grains, this fungicide are used.

Because these fungicides are generally applied to various fields, contamination of nearby aquatic systems may occur through a variety of mechanisms, including drift, and surface runoff and leaching. Defining the opposite effects these fungicides may have on non-target algae and other organisms in aquatic ecosystems is important. One of the significant components of aquatic ecological systems is algae. Algae produce dissolved oxygen and organic material with photosynthesis. Algae produce food for different organisms in aquatic environments [3]. Algae are known to be susceptible to many chemicals in some degree. Pollutants' toxic effects on algae are usually reclaimed by using phytotoxicity tests which are based on enlargement prevention. Physiological nuclear endpoints may authorize early invention of cell stress and explain fundamental toxicity mechanisms because of enlargement prevention [1].

Scenedesmus acutus, (Chlorophyta, Sphaeropleales), is an extremely tolerant green algae. S. acutus is a freshwater unicellular a green algae found many aquatic habitats. The alga has a rapid growth and can be cultured simply and economical under autotrophic conditions. The use of S. acutus as model organism for toxicity test in aquatic environment has the advantage of S. acutus widely spread in the freshwater and its frequent use for growth inhibition toxicity tests [1, 5, 6]. The aim of our study was to investigate the effects of two significant fungicides (triadimenol and penconazole) on S. acutus. Dose-effects the two fungicides on the growth and protein amount of S. acutus. We are hoped that the results of our research would provide benifical references for the usage and environmental control of triadimenol and penconazole.

Experimental

S. acutus is green algae which generate most of the phytoplankton in fresh water. S. acutus was cultured in Jaworski's medium [7]. The medum was composed of distilled water and the following chemical ingredients: Ca(NO3)2.4H2O, KH2PO4, MgSO4.7H2O, NaHCO3, EDTA FeNa, EDTA Na2, H3BO3, MnCl2.4H2O, (NH2)6Mo7O2H2O, NaNO3, Na2HPO4, cyanocobalamin, thiamine and biotin. The culture medium was sterilized at 121C temperature and 1 atmospheric pressure for 15 min. The isolates were cultured in Erlenmeyer flasks containing modified Jaworski's medium at 23 1C in 2000 lux light intensity with 16:8 h light and dark cycles.

Algal cells were counted by plankton counting chamber under a microscope. At the same time, same samples were determined daily using a spectrophotometer at 680nm. The measurements in spectrophotometer were compared with microscope counting. The algal cell was calculated by measuring the optical density at 680 nm using a visible density spectrophotometer. A standard curve relating to optical density was produced, which was subsequently used to calculate of individual number based on their optical density. The calculated individual number (as the average of three experiments) was used to construct growth curves. The relationship curve between OD680 and cell number was plotted, and the cell number was obtained by regression.

Total protein was measured by Lowry methods. Samples were performed in the dark to prevent degradation of the Folin reagent. The spectrophotometric absorbance was converted to protein concentration using a calibration curve [8].

S. acutus cultures were exposed to various concentrations of triadimenol and penconazole (1, 3, 6, 10 and 15 g/L). Algal toxicity test of 4 days of exposure was conducted following the general design of Environmental Protection Agency [9]. After the inoculation, the stock and experimental cultures of S. acutus were grown in the same liquid medium at 23 1C and a light intensity of 2000 lux on a 16 h l and 8 h d photoperiod. The inocula was prepared from these cultures to provide an primary cell density 1145000 cells/ml of S. acutus in treated and control culture. Triadimenol was added to the S. acutus culture just after inoculums. Penconazole also added separately to other cultures. Control cultures were incubated in the same liquid medium without pesticides.

The experimental sets were run in triplicate. The control and exposed cultures with fungicides were hand shaken twice daily.

Result and Discussion

Effects of Triadimenol and Penconazole on Algal Growth

The observation of effects of various concentrations of triadimenol on S. acutus indicated a different effect in population growth. Fig. 1 shows cell counts of S. acutus exposed to different triadimenol concentrations. As shown in Fig. 1, the growth of S. acutus generally declined with increasing exposure. At the first day, S. acutus in culture exposed to 1 g/L, 3 g/L, 6 g/L, 10 g/L and 15 g/L of concentrations, was counted as 1914950 cells/ml, 1704950 cells/ml, 1494950 cells/ml, 1214950 cells/ml and 794950 cells/ml, respectively. At the second day of bioassay, S. acutus was counted in decrease by 1635000 cells/ml, 1285000 cells/ml, 1215000 cells/ml, 865000, cells/ml and 585000 cells/ml at 1 g/L, 3 g/L, 6 g/L, 10 g/L and 15 g/L of concentrations. This decrease in the cell number of S. acutus continued until the fifth day.

On the end of the fourth day, cell numbers of algae decreased to 50% as compared to the first days and the highest number of cells was determined at 1 g/L concentration at the day fourth cell count (Fig. 1). Triadimenol viewed to be a powerful inhibitor of S. acutus population growth. S. acutus was exposed to various concentrations of triadimenol (from 1 to 15 g/L), cell amount was diminished during second day. At 48 h of exposure to 3 g/L triadimenol cell amount was inhibited by 50% compared to control (Fig. 1).

Penconazole affected the population growth in liquid culture of S. acutus (Fig. 2). At the first day, S. acutus was counted in no fungicide cultures as 2125000 cells/ml, and an insignificant increase in cell numbers of S. acutus in 1, 3 and 6 g/L-treated cultures could be observed, when compared to control (Fig. 2). At the second day of exposed to penconazole, there was a significant decrease in individual number with respect to the control, in the liquid medium exposed above 3 g/L at 48 hr of exposition. At 96 hr, the cultures exposed from 3 g/L and higher, showed a considerable decrease of individual number when compared with the control cultures (Fig. 2).

Effects of Triadimenol and Penconazole on Protein Amount

Protein amount was determined and these results were shown in Fig. 3. S. acutus samples analyzed for the four days and the change of the protein amounts was found to be dependent on the exposed time and concentrations of triadimenol (Fig.3). Calculated from the first day of inoculation that the amount of total protein 42.844 g/ ml was found to 1 g/L concentration of triadimenol. Until the end of this study, the amount of the protein continued to decrease. After 48 h of exposure, protein amount in S. acutus culture exposed to 1 g/L, 3 L, 6 g/L, 10 g/L and 15 g/L of triadimenol concentrations was found as 34.389 g/ml, 26.584 g/mL, 20.730 g/ml, 16.828 g/ml and 11.625 g/ml, respectively. Until end of this study, amount of protein was continuously decreased (Fig. 3).

In the tested S. acutus in cultures exposed with penconazole, protein amount were decreased regularly with increased penconazole concentrations and exposed time (Fig. 4). At the first day, protein amount in the control culture was measured as 48.698 g/ml. But in S. acutus culture treated with 1, 3, 6, 10 and 15 g/L of penconazole, was measured as 42.844, 38.291, 36.991, 30.486 and 24.633 g/ml, respectively. At the second day, protein amount in no treated culture with penconozale was measured as 51.950 g/ml. At the same day, protein amount in the treated cultures declined by 34.389, 26.584, 20.730, 16.828 and 11.625 g/ml at 1, 3, 6, 10 and 15 g/L in sequence. The toxic effects of penconazole on protein amount in S. acutus cultures were varied when protein amount of some treated cultures, decreased significantly with respect to the control at 48 h of exposition.

Especially, there was a significant reduction of protein amount with respect to the control, of the cultures exposed above 3 g /L at second day of exposition to pencenazole. At the third and fourth days decrease of protein amount in treated cultures with penconazole could be detected, when compared with the control cultures (Fig. 4).

Toxicity in algae of some pesticides was showed by different studies [4-6, 10-13].

Scenedesmus, one of most commonly tested species of algae, was among the most sensitive of aquatic plant species tested [14]. Each of pesticides significantly decreased the cell number in Scenedesmus acutus cultures. In the present study, we observed growth and protein amount high level in cultures treated by low level concentrations of triadimenol and penconazole. Our previous studies showed that, protein amount and cell counts in S. acutus cultures has decreased depend on exposure time, concentration and kind of pesticides [15- 17]. In this study, especially S. acutus growth and protein amount decreased after second day of pesticide exposure. The increase of pesticides concentrations would inhibit algae growth and protein amount in S. acutus.

In our studies, the individual number and protein amount of S. acutus were detected to be negatively related with high concentrations of triadimenol and penconazole. In a study by Shen et al. (2014), an increase in fungicide concentration resulted in a significant decrease in the individual number and total protein amount of green algae S. acutus, and the decrease in individual number was probably due to the protein metabolisms, which was caused by high concentrations of fungicide in the culture of S. acutus [3]. The growth rate and protein amount of S. acutus was decreased with high concentration of triadimenol and penconazole.

Conclusion

The consequences of this research indicate that in first days of inoculation, sensivities of S. acutus to fungicides has been started, also at the same time, the algae population has decreased in cultures exposed to triadimenol and penconazole. Commonly, the growth rates and protein amounts of S. acutus were found to be negatively correlated with high amount of triadimenol and penconazole. Toxicity of fungicidies on S. acutus decreased chlorophyll and protein synthesis. According to these results, it could be believed that the effects of triadimenol and penconazole on algal growth and protein amount are decreased with increasing fungicides concentrations.

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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Dec 31, 2015
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