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Uptake and the effects of cyanazine on Scenedesmus obliquus and Anabaena flos-aquae
Desalination 249 (2009) 1294–1297
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Desalination j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l
Uptake and the effects of cyanazine on Scenedesmus obliquus and Anabaena flos-aquae Azza M. AbdEl-Aty ⁎, M.A. El-Dib Water Pollution Research Department, National Research Center, Dokki-12311, Cairo, Egypt
a r t i c l e
i n f o
Article history: Accepted 4 August 2008 Available online 13 October 2009 Keywords: Algae Toxicity Cyanazine herbicide Uptake
a b s t r a c t Microalgal species vary in their sensitivity to the triazine herbicides, including cyanazine. The current investigation was carried out to examine the uptake and the effects of cyanazine on Chl (a) content, growth rate, EC50, and protein and amino acid contents of two different algal species; namely Scenedesmus obliquus (green alga) and Anabaena flos-aquae (blue green alga). With regard to the differential sensitivity, the present findings revealed that S. obliquus is more sensitive to the inhibitory effects of cyanazine compared to A. flos-aquae (EC50 = 26 vs. 113 µg/L, respectively). Additionally, the lowest applied concentrations of the tested herbicide increased the Chl (a) content of both algal cultures as well as its rate of uptake. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Intensive applications of herbicides, which are used in agriculture to control weed, have attracted great concerns from the public. It was reported that herbicides reached the surface water bodies either through leaching from treated lands, discharge of polluted drainage water, or via direct application to water bodies [1]. Pollution with herbicides must be considered as a potential risk for aquatic systems as well as for the quality of drinking water [2]. Cyanazine has been used as an aquatic herbicide to control submerged and floating weeds [3] and hence, could affect many non target organisms [4]. Algae are among the essential components of aquatic ecosystems, which produce oxygen and organic substances on which most organisms, including fishes and invertebrates are dependant upon [5]. Chemical effects on algae can directly affect the structure and the function of an ecosystem resulting in oxygen depletion and decreased primary productivity [6]. As triazine herbicides are toxic to algae by disrupting photosynthesis, this study was designed to investigate the differences in algal sensitivity to cyanazine that might be attributed to several factors, including variation in cyanazine uptake, algal cell size, pigment profile, and amino acid and protein N syntheses. The present study was, therefore, carried out to assess the uptake of cyanazine by two algal species; Scenedesmus obliquus (green alga) and Anabaena flos-aquae (blue green alga); and evaluating its effect on chlorophyll (a) content, growth rate (μ/d), effective concentration (EC50), and nitrogen metabolism (synthesis) represented by amino acids and protein N. In the present study, protein and amino acids were evaluated because they represent the important fractions of the cell structure liable to affect the growth rate as well as the cell wall.
The triazine herbicide, cyanazine 2-{(4-chloro-6-(ethylamino)-1,3,5triazin-2-yl)amino} 2-methylpropanenitrile (99.9% purity) was used in this study. Cyanazine has the following physico-chemical properties: Mol. Wt 240.7, Mol. formula of C9H13ClN6, Kow log P=2.1, solubility in water 171 mg/L, and stable to heat, light, and solution between pH 5 and 9 [7]. The degree of hydrophobicity is relatively low as indicated by log Kow. In the air/water system, the chemical tends to partition in water. Such data are in agreement with our finding that the concentration of cyanazine solutions was chemically stable and was not adsorbed to the walls of the containers, nor subjected to biodegradation within the tested period. The residual levels were determined by gas chromatograph HP6890 equipped with an electron-capture detector (Ni63) (GC-ECD). Chromatographic separation was performed on a 30 m×0.032 mm ID HP0.5 capillary column coated with 5% PHME syloxane with a film thickness of 0.25 μm in splitless injector mode. The column, injector, and detector temperatures were maintained at 180, 220, and 260 °C, respectively. Nitrogen was used as a carrier gas (2 mL/min). Two species of algae, both common in the aquatic ecosystem, were used as test organisms. Scenedesmus obliquus (S. obliquus) of green algae and Anabaena flos-aquae (A. flos-aquae) of blue green algae were isolated from the Nile River water and recultivated under controlled conditions. S. obliquus was grown in a modified BG11 with the following macroelements: K2HPO4, MgSO4, CaCl2, NaNO3, Na2EDTA, citric acid, and ferric ammonium citrate [8]. In refer to Ali [9] a minor modification was made by diluting the concentration of NaNO3 to 1/5th of the original concentration. On the other hand, a modified Watanabe media [10] was used for cultivation of A. flos-aquae. The test organisms were in the logarithmic phase of growth, when introduced into the algal culture media. One liter Erlenmeyer™ bioassay flasks with 500 mL of algal suspension media
⁎ Corresponding author. Tel.: +20 2 25164019. E-mail address: [email protected] (A.M. AbdEl-Aty). 0011-9164/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2009.09.003
A.M. AbdEl-Aty, M.A. El-Dib / Desalination 249 (2009) 1294–1297
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were covered with aluminum foil, and four holes were punched in the foil for gas exchange. Under continuous illumination (2500lx), the cultures were incubated at 23±2 and 30±2 °C, the optimal temperatures for S. obliquus and A. flos-aquae, respectively. The cultures were swirled once a day, to prevent clumping and adherence of algal cells to the containers. The cyanazine solution was prepared in ethanol and the applied concentrations ranged from 7.5 to 120 μg/L in the case of S. obliquus and from 30 to 480 μg/L in the case of A. flos-aquae. Each concentration was represented by three replicates for each run. As a control (blank), three culture media free from cyanazine represented the normal growth order of the algae. The blank sample contained ethanol, which was considered as a proper solvent in toxicity test [11]. Another blank, free from ethanol was used, however a control having the herbicide solutions and free from algae was run for the investigated period (10 days) to show that the cyanazine maintained its concentrations. Algal growth was determined by measuring the Chl (a) content according to the American Public Health Association [12]. Growth rate was determined in respects to the following equation: μ = d = ðln ChlðaÞT2 − ln Chl ðaÞ at T1 Þ = T2 −T1 where T is time in day. The effective concentration (EC50 which causes 50% growth inhibition based on Chl (a) content) was determined according to the method previously published by Finney [13] at 96 h. After 8 days of incubation, the algal mass was collected and was used for the determination of protein according to the method previously described [14] and modified by Quian and Wang [15] and amino acids according to the colorimetric method described by Lee and Takahashi [16]. The rate of cyanazine uptake by algal cells was determined by the classic equation stated by several authors [17,18]. Assuming a first order rate reaction, the equation is: log C− log C0 = kt = 2:303 where C is the concentration at time t, C0 is the initial concentration at t = 0, and k is the specific reaction rate constant. A plot of the log C vs. time gives a straight line with a negative slope. Time required for 50% of the reaction to proceed is given by the equation: t0.5 = 0.693 / k where t0.5 is the half-time of reaction. 3. Results and discussion Changes in the Chl (a) content of S. obliquus and A. flos-aquae treated with different concentrations of cyanazine are presented in Fig. 1. In the case of Scenedesmus, the maximum Chl (a) content of the control culture was attained at the 8th day of incubation. Chl (a) content of both controls was almost the same indicating that the ethanol has no adverse effect on algal growth as mentioned previously. Cyanazine at a concentration rate of 7.5 µg/L, exerted a slight inhibition of Scenedesmus cells up to the 6th day of incubation. Thereafter, the algal cells began to recover and its Chl (a) content exceeded that of the control. However, as the concentration of cyanazine in the algal culture increased, a corresponding decrease in Chl (a) content was attained. Control culture of Anabaena showed maximum Chl (a) content by the end of the 9th day of incubation. Treatment of Anabaena culture by 30 µg/L cyanazine showed an increase of Chl (a) content starting from the 6th day of incubation and its concentration exceeded that of the control (Fig. 1). However, the higher levels of cyanazine resulted in a sharp decrease in the Chl (a) content of algal cells. Previous studies also revealed that several herbicides, when present at low concentrations promoted the algal growth as indicated by the increase in Ch1 (a) content [19,20]. Probit analysis of Chl (a) content of algal cultures revealed that the EC50 values of cyanazine at 96 h were 26 and 113 µg/L for Scenedesmus and Anabaena, respectively. Previous studies indicated that EC50 (96 h) for cyanazine was subjected to variation according to the test algal
Fig. 1. Effect of cyanazine on the chlorophyll (a) contents of the two different algal species.
species. According to Ma et al. [4] the EC50 of green alga, Raphidocelis subcapitata, treated with cyanazine was 0.0596 mg/L, while in the case of Chlorella vulgaris the EC50 was 0.128 mg/L [21]. The current findings revealed that green algae, S. obliquus may be more sensitive to cyanazine than the blue green algae, A. flos-aquae. This result was in agreement with other studies, which stated that chlorophytes were the most sensitive followed by cyanophytes [20,22,23]. Such finding indicates that blue green algae may prevail in the natural surface waters and exert their undesirable toxic effects on drinking water resources [24]. The growth rates of S. obliquus and A. flos-aquae treated with cyanazine were derived from the Chl (a) content at short time (Tables 1 and 2). In the control culture, the maximum growth rate was achieved by the end of the 1st day of incubation. However, the growth rates of the algal cultures in the presence of cyanazine progressively decreased compared to the control. In the case of Scenedesmus, the cells showed a tendency to recover by the end of the 2nd day especially in the presence of the low cyanazine concentration and a significant relation exists between the growth rates and the concentration up to the 5th day. In case of Anabaena, the growth rates of the algal cultures showed a similar trend as that of Scenedesmus where the growth rate relatively increased by the end of the 2nd and 3rd days of incubation, and a significant relation was attained between the
Table 1 Growth rate of S. obliquus treated with cyanazine at short time intervals. Concentration of cyanazine (μg/L) 0 7.5 15 30 60 120 r2
Growth rates over time (days) 0–1
1–2
2–3
3–4
4–5
1.05 0.969 0.877 0.823 0.407 − 0.166 0.992
0.854 0.873 0.894 0.849 0.62 0.382 0.947
1.049 1.014 0.885 0.402 0.197 − 0.02 0.841
0.558 0.569 0.546 0.299 0.258 − 0.22 0.962
0.466 0.569 0.232 0.052 0.014 − 0.151 0.779
1296
A.M. AbdEl-Aty, M.A. El-Dib / Desalination 249 (2009) 1294–1297
Table 2 Growth rate of A. flos-aquae treated with cyanazine at short time intervals. Concentration of cyanazine (μg/L) 0 30 60 120 240 480 r2
Growth rates over time (days) 0–1
1–2
2–3
3–4
4–5
0.823 0.698 0.371 0.266 − 0.091 − 0.631 0.952
0.633 0.579 0.766 0.611 0.636 0.284 0.67
0.694 0.744 0.593 0.695 0.454 0.268 0.894
0.625 0.582 0.646 0.459 0.267 0.115 0.925
0.417 0.579 0.415 0.557 0.428 0.036 0.706
applied cyanazine concentration and the growth rates up to the 5th day (Table 2). According to Hersh and Crumpton [25] growth rates and effects caused by toxins must be determined when nutrient and toxin concentrations are less affected by cell number. This is best accomplished early in the experiment, as soon as log phase growth is apparent in the control. Similarly, a research work noted that the growth rate depressions must be determined early in assays (usually by the 2nd day) before the toxicant fate effects [26]. Uptake of cyanazine by algal cells of both species attained high levels when its concentration in the culture media was relatively low (Fig. 2). Such a result coincides with the relatively high growth rate of algae exposed to the lower concentrations of cyanazine. Uptake of cyanazine by algal cells proceeds as a pseudo first order rate (Fig. 3). The specific rate constants progressively decreased when cyanazine concentration increased in the algal media (Table 3). Similar trends were reported by other investigators [19,20,27]. Such a trend may be attributed to the relatively high algal growth rate attained at low herbicide concentrations and the subsequent increase in cell numbers and their surface area, which enhances the process of surface adsorption followed by absorption into the cell. Walsh [28] reported that herbicide uptake by aquatic microorganisms would be favored by their relatively high surface area–volume ratio and enhanced by the selective portioning of lipophilic compound into cellular
Fig. 3. Kinetic plot for the adsorption of cyanazine by the two different algal species.
lipids, especially at cell surface. Results presented in Tables 1 and 2 also revealed that the relatively high growth rates of algae attained at low herbicide concentration coincide with the shorter half-life time to which algal cells will be exposed to the inhibitory effects of the herbicide. Results presented in Fig. 4 reveal that cyanazine treatments affected both amino acid and protein syntheses by S. obliquus and A. flos-aquae. The lowest concentrations of cyanazine namely 7.5 μg/L and 30 μg/L stimulated the synthesis of amino acids and protein for Scenedesmus and Anabaena, respectively. However, application of higher concentrations led to decrease in the level of the two parameters for both algal species. A previous study [29] indicated that sub-lethal concentrations of atrazine dosed to the green algae Chlorella sp. induced general inhibition on growth, photosynthesis, and reduction in protein synthesis on increasing the herbicide concentration. 4. Conclusion The study revealed that low concentrations of the herbicide, liable to reach surface waters, increased Chl (a), growth rate, and rate of uptake by algal cells which followed a pseudo first order rate. The impacts of the herbicide on the previous parameters coincide with effect on amino acids and protein content of the algal cells. S. obliquus was more sensitive to the inhibitory effect of the herbicide. Such a result indicates that pollution of fresh drinking water resources by the herbicide may lead to the over growth of the blue green algae which exert toxic effects on drinking water.
Table 3 Kinetic data for the uptake of cyanazine by the two different algal species.
Fig. 2. Percentage uptake of cyanazine by the two different algal species.
Cyanazine (μg/L)
Scenedesmus obliquus Rate constant (K)
t0.5 (days)
7.5 15 30 60 120
0.12 0.10 0.04 0.036 0.028
5.8 6.9 17.3 19.2 24.7
Cyanazine (μg/L)
30 60 120 240 480
Anabaena flos-aquae Rate constant (K)
t0.5 (days)
0.124 0.072 0.036 0.010 0.005
5.6 9.6 19.2 69.3 138.5
A.M. AbdEl-Aty, M.A. El-Dib / Desalination 249 (2009) 1294–1297
Fig. 4. Effect of cyanazine on protein and amino acids contents of the two different algal species.
Acknowledgment The authors wish to express their appreciation to the Institute of National Agricultural Products Quality Management Service, Gwangju, Republic of Korea for a generous gift of the herbicide. References [1] M.P. Brooker, R.W. Edward, Freshw. Biol. 3 (1973) 383–389. [2] H. Katsumata, S. Kaneco, T. Suzuki, K. Ohta, Anal. Chim. Acta 577 (2006) 214–219. [3] C. Tomlin, The Pesticide Manual, Brithish Crop Protection Council, Hampshire, UK, 2003, p. 1341. [4] J. Ma, S. Wang, P. Wang, L. Ma, X. Chen, R. Xu, Ecotoxicol. Environ. Saf. 63 (2006) 456–462.
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[5] A. Berad, Bull. Environ. Contam. Toxicol. 57 (1996) 183–190. [6] P.K. Wong, Chemosphere 41 (2000) 177–182. [7] C. Tomlin (Ed.), Pesticide Manual, 10th edition, British Crop Protection Council, Surrey, UK, 1994. [8] W.W. Carmichael, Isolation, culture and toxicity testing of toxic fresh water cyanobacteria (blue–green algae), in: V. Shilo (Ed.), Fundamental Research in Homogenous Catalysis, Gordon & Breach, New York, 1986, pp. 1249–1262. [9] G.H. Ali, Fluoride 37 (2004) 88–95. [10] A.S. Nawawy, M. Lotfi, M. Fahmy, Agri. Res. Rev. 36 (1958) 308–320. [11] H.F. Abou Waly, Int. J. Environ. Stud. 57 (2000) 411–418. [12] APHA (American Public Health Association), Standard Methods for the Examination of Water and Wastewater, 20th Ed., 1998 AWWA, WEF. [13] D. Finney, Probit Analysis, Cambridge University, Press, Cambridge, 1971 1–333. [14] O.H. Lowery, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. [15] K. Quian, S. Wang, Haiyang Yu Huzhao 20 (1989) 192–196. [16] Y.P. Lee, T. Takahashi, Anal. Biochem. 14 (1966) 71–77. [17] S. Glasstone, Textbook of Physical Chemistry and Kinetics, Macmillan, London, 1951 1050–1054. [18] I. Meites, An Introduction to Chemical Equilibrium and Kinetics, Pergamon Press, Oxford, 1981 33–48. [19] M.A. El-Dib, S.A. Shehata, H.F. Abou-Waly, Water Air Soil Pollut. 55 (1991) 295–303. [20] S.A. Shehata, M.A. El-Dib, H.F. Abou-Waly, Bull. Environ. Contam. Toxicol. 50 (1993) 369–376. [21] J. Ma, L. Xu, S. Wang, R. Zheng, S. Jin, S. Huang, Y. Huang, Ecotoxicol. Environ. Saf. 51 (2002) 128–132. [22] H.F. Abou-Waly, M.M. Abou-Setta, H.N. Nigg, L.L. Mallory, Bull. Environ. Contam. Toxicol. 46 (1991) 223–229. [23] C. Gaggi, G. Sbrilli, A.M. Hasab El Naby, M. Bucci, M. Duccini, E. Bacci, Environ. Toxicol. Chem. 14 (1995) 1065–1069. [24] W.H.O. World Health Organization, Guidelines for Drinking Water Quality, Geneva, Vol, 1993 2. [25] C.M. Hersh, W.G. Crumpton, Bull. Environ. Contam. Toxicol. 39 (1987) 1041–1048. [26] G.E. Walsh, Aquat. Toxiciol. 3 (1983) 209–213. [27] M.A. El-Dib, H.F. Abou-Waly, H. El-Tantawy, A.M. El-Naby, Egypt. J. Microbiol. 35 (2000) 569–581. [28] G.E. Walsh, Environ. Toxicol. Chem. 7 (1988) 979–987. [29] M.M. El-Sheekh, H.M. Kotkat, O.H.E. Hammouda, Ecotoxicol. Environ. Saf. 29 (1994) 349–358.
Contents lists available at ScienceDirect
Desalination j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l
Uptake and the effects of cyanazine on Scenedesmus obliquus and Anabaena flos-aquae Azza M. AbdEl-Aty ⁎, M.A. El-Dib Water Pollution Research Department, National Research Center, Dokki-12311, Cairo, Egypt
a r t i c l e
i n f o
Article history: Accepted 4 August 2008 Available online 13 October 2009 Keywords: Algae Toxicity Cyanazine herbicide Uptake
a b s t r a c t Microalgal species vary in their sensitivity to the triazine herbicides, including cyanazine. The current investigation was carried out to examine the uptake and the effects of cyanazine on Chl (a) content, growth rate, EC50, and protein and amino acid contents of two different algal species; namely Scenedesmus obliquus (green alga) and Anabaena flos-aquae (blue green alga). With regard to the differential sensitivity, the present findings revealed that S. obliquus is more sensitive to the inhibitory effects of cyanazine compared to A. flos-aquae (EC50 = 26 vs. 113 µg/L, respectively). Additionally, the lowest applied concentrations of the tested herbicide increased the Chl (a) content of both algal cultures as well as its rate of uptake. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Intensive applications of herbicides, which are used in agriculture to control weed, have attracted great concerns from the public. It was reported that herbicides reached the surface water bodies either through leaching from treated lands, discharge of polluted drainage water, or via direct application to water bodies [1]. Pollution with herbicides must be considered as a potential risk for aquatic systems as well as for the quality of drinking water [2]. Cyanazine has been used as an aquatic herbicide to control submerged and floating weeds [3] and hence, could affect many non target organisms [4]. Algae are among the essential components of aquatic ecosystems, which produce oxygen and organic substances on which most organisms, including fishes and invertebrates are dependant upon [5]. Chemical effects on algae can directly affect the structure and the function of an ecosystem resulting in oxygen depletion and decreased primary productivity [6]. As triazine herbicides are toxic to algae by disrupting photosynthesis, this study was designed to investigate the differences in algal sensitivity to cyanazine that might be attributed to several factors, including variation in cyanazine uptake, algal cell size, pigment profile, and amino acid and protein N syntheses. The present study was, therefore, carried out to assess the uptake of cyanazine by two algal species; Scenedesmus obliquus (green alga) and Anabaena flos-aquae (blue green alga); and evaluating its effect on chlorophyll (a) content, growth rate (μ/d), effective concentration (EC50), and nitrogen metabolism (synthesis) represented by amino acids and protein N. In the present study, protein and amino acids were evaluated because they represent the important fractions of the cell structure liable to affect the growth rate as well as the cell wall.
The triazine herbicide, cyanazine 2-{(4-chloro-6-(ethylamino)-1,3,5triazin-2-yl)amino} 2-methylpropanenitrile (99.9% purity) was used in this study. Cyanazine has the following physico-chemical properties: Mol. Wt 240.7, Mol. formula of C9H13ClN6, Kow log P=2.1, solubility in water 171 mg/L, and stable to heat, light, and solution between pH 5 and 9 [7]. The degree of hydrophobicity is relatively low as indicated by log Kow. In the air/water system, the chemical tends to partition in water. Such data are in agreement with our finding that the concentration of cyanazine solutions was chemically stable and was not adsorbed to the walls of the containers, nor subjected to biodegradation within the tested period. The residual levels were determined by gas chromatograph HP6890 equipped with an electron-capture detector (Ni63) (GC-ECD). Chromatographic separation was performed on a 30 m×0.032 mm ID HP0.5 capillary column coated with 5% PHME syloxane with a film thickness of 0.25 μm in splitless injector mode. The column, injector, and detector temperatures were maintained at 180, 220, and 260 °C, respectively. Nitrogen was used as a carrier gas (2 mL/min). Two species of algae, both common in the aquatic ecosystem, were used as test organisms. Scenedesmus obliquus (S. obliquus) of green algae and Anabaena flos-aquae (A. flos-aquae) of blue green algae were isolated from the Nile River water and recultivated under controlled conditions. S. obliquus was grown in a modified BG11 with the following macroelements: K2HPO4, MgSO4, CaCl2, NaNO3, Na2EDTA, citric acid, and ferric ammonium citrate [8]. In refer to Ali [9] a minor modification was made by diluting the concentration of NaNO3 to 1/5th of the original concentration. On the other hand, a modified Watanabe media [10] was used for cultivation of A. flos-aquae. The test organisms were in the logarithmic phase of growth, when introduced into the algal culture media. One liter Erlenmeyer™ bioassay flasks with 500 mL of algal suspension media
⁎ Corresponding author. Tel.: +20 2 25164019. E-mail address: [email protected] (A.M. AbdEl-Aty). 0011-9164/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2009.09.003
A.M. AbdEl-Aty, M.A. El-Dib / Desalination 249 (2009) 1294–1297
1295
were covered with aluminum foil, and four holes were punched in the foil for gas exchange. Under continuous illumination (2500lx), the cultures were incubated at 23±2 and 30±2 °C, the optimal temperatures for S. obliquus and A. flos-aquae, respectively. The cultures were swirled once a day, to prevent clumping and adherence of algal cells to the containers. The cyanazine solution was prepared in ethanol and the applied concentrations ranged from 7.5 to 120 μg/L in the case of S. obliquus and from 30 to 480 μg/L in the case of A. flos-aquae. Each concentration was represented by three replicates for each run. As a control (blank), three culture media free from cyanazine represented the normal growth order of the algae. The blank sample contained ethanol, which was considered as a proper solvent in toxicity test [11]. Another blank, free from ethanol was used, however a control having the herbicide solutions and free from algae was run for the investigated period (10 days) to show that the cyanazine maintained its concentrations. Algal growth was determined by measuring the Chl (a) content according to the American Public Health Association [12]. Growth rate was determined in respects to the following equation: μ = d = ðln ChlðaÞT2 − ln Chl ðaÞ at T1 Þ = T2 −T1 where T is time in day. The effective concentration (EC50 which causes 50% growth inhibition based on Chl (a) content) was determined according to the method previously published by Finney [13] at 96 h. After 8 days of incubation, the algal mass was collected and was used for the determination of protein according to the method previously described [14] and modified by Quian and Wang [15] and amino acids according to the colorimetric method described by Lee and Takahashi [16]. The rate of cyanazine uptake by algal cells was determined by the classic equation stated by several authors [17,18]. Assuming a first order rate reaction, the equation is: log C− log C0 = kt = 2:303 where C is the concentration at time t, C0 is the initial concentration at t = 0, and k is the specific reaction rate constant. A plot of the log C vs. time gives a straight line with a negative slope. Time required for 50% of the reaction to proceed is given by the equation: t0.5 = 0.693 / k where t0.5 is the half-time of reaction. 3. Results and discussion Changes in the Chl (a) content of S. obliquus and A. flos-aquae treated with different concentrations of cyanazine are presented in Fig. 1. In the case of Scenedesmus, the maximum Chl (a) content of the control culture was attained at the 8th day of incubation. Chl (a) content of both controls was almost the same indicating that the ethanol has no adverse effect on algal growth as mentioned previously. Cyanazine at a concentration rate of 7.5 µg/L, exerted a slight inhibition of Scenedesmus cells up to the 6th day of incubation. Thereafter, the algal cells began to recover and its Chl (a) content exceeded that of the control. However, as the concentration of cyanazine in the algal culture increased, a corresponding decrease in Chl (a) content was attained. Control culture of Anabaena showed maximum Chl (a) content by the end of the 9th day of incubation. Treatment of Anabaena culture by 30 µg/L cyanazine showed an increase of Chl (a) content starting from the 6th day of incubation and its concentration exceeded that of the control (Fig. 1). However, the higher levels of cyanazine resulted in a sharp decrease in the Chl (a) content of algal cells. Previous studies also revealed that several herbicides, when present at low concentrations promoted the algal growth as indicated by the increase in Ch1 (a) content [19,20]. Probit analysis of Chl (a) content of algal cultures revealed that the EC50 values of cyanazine at 96 h were 26 and 113 µg/L for Scenedesmus and Anabaena, respectively. Previous studies indicated that EC50 (96 h) for cyanazine was subjected to variation according to the test algal
Fig. 1. Effect of cyanazine on the chlorophyll (a) contents of the two different algal species.
species. According to Ma et al. [4] the EC50 of green alga, Raphidocelis subcapitata, treated with cyanazine was 0.0596 mg/L, while in the case of Chlorella vulgaris the EC50 was 0.128 mg/L [21]. The current findings revealed that green algae, S. obliquus may be more sensitive to cyanazine than the blue green algae, A. flos-aquae. This result was in agreement with other studies, which stated that chlorophytes were the most sensitive followed by cyanophytes [20,22,23]. Such finding indicates that blue green algae may prevail in the natural surface waters and exert their undesirable toxic effects on drinking water resources [24]. The growth rates of S. obliquus and A. flos-aquae treated with cyanazine were derived from the Chl (a) content at short time (Tables 1 and 2). In the control culture, the maximum growth rate was achieved by the end of the 1st day of incubation. However, the growth rates of the algal cultures in the presence of cyanazine progressively decreased compared to the control. In the case of Scenedesmus, the cells showed a tendency to recover by the end of the 2nd day especially in the presence of the low cyanazine concentration and a significant relation exists between the growth rates and the concentration up to the 5th day. In case of Anabaena, the growth rates of the algal cultures showed a similar trend as that of Scenedesmus where the growth rate relatively increased by the end of the 2nd and 3rd days of incubation, and a significant relation was attained between the
Table 1 Growth rate of S. obliquus treated with cyanazine at short time intervals. Concentration of cyanazine (μg/L) 0 7.5 15 30 60 120 r2
Growth rates over time (days) 0–1
1–2
2–3
3–4
4–5
1.05 0.969 0.877 0.823 0.407 − 0.166 0.992
0.854 0.873 0.894 0.849 0.62 0.382 0.947
1.049 1.014 0.885 0.402 0.197 − 0.02 0.841
0.558 0.569 0.546 0.299 0.258 − 0.22 0.962
0.466 0.569 0.232 0.052 0.014 − 0.151 0.779
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A.M. AbdEl-Aty, M.A. El-Dib / Desalination 249 (2009) 1294–1297
Table 2 Growth rate of A. flos-aquae treated with cyanazine at short time intervals. Concentration of cyanazine (μg/L) 0 30 60 120 240 480 r2
Growth rates over time (days) 0–1
1–2
2–3
3–4
4–5
0.823 0.698 0.371 0.266 − 0.091 − 0.631 0.952
0.633 0.579 0.766 0.611 0.636 0.284 0.67
0.694 0.744 0.593 0.695 0.454 0.268 0.894
0.625 0.582 0.646 0.459 0.267 0.115 0.925
0.417 0.579 0.415 0.557 0.428 0.036 0.706
applied cyanazine concentration and the growth rates up to the 5th day (Table 2). According to Hersh and Crumpton [25] growth rates and effects caused by toxins must be determined when nutrient and toxin concentrations are less affected by cell number. This is best accomplished early in the experiment, as soon as log phase growth is apparent in the control. Similarly, a research work noted that the growth rate depressions must be determined early in assays (usually by the 2nd day) before the toxicant fate effects [26]. Uptake of cyanazine by algal cells of both species attained high levels when its concentration in the culture media was relatively low (Fig. 2). Such a result coincides with the relatively high growth rate of algae exposed to the lower concentrations of cyanazine. Uptake of cyanazine by algal cells proceeds as a pseudo first order rate (Fig. 3). The specific rate constants progressively decreased when cyanazine concentration increased in the algal media (Table 3). Similar trends were reported by other investigators [19,20,27]. Such a trend may be attributed to the relatively high algal growth rate attained at low herbicide concentrations and the subsequent increase in cell numbers and their surface area, which enhances the process of surface adsorption followed by absorption into the cell. Walsh [28] reported that herbicide uptake by aquatic microorganisms would be favored by their relatively high surface area–volume ratio and enhanced by the selective portioning of lipophilic compound into cellular
Fig. 3. Kinetic plot for the adsorption of cyanazine by the two different algal species.
lipids, especially at cell surface. Results presented in Tables 1 and 2 also revealed that the relatively high growth rates of algae attained at low herbicide concentration coincide with the shorter half-life time to which algal cells will be exposed to the inhibitory effects of the herbicide. Results presented in Fig. 4 reveal that cyanazine treatments affected both amino acid and protein syntheses by S. obliquus and A. flos-aquae. The lowest concentrations of cyanazine namely 7.5 μg/L and 30 μg/L stimulated the synthesis of amino acids and protein for Scenedesmus and Anabaena, respectively. However, application of higher concentrations led to decrease in the level of the two parameters for both algal species. A previous study [29] indicated that sub-lethal concentrations of atrazine dosed to the green algae Chlorella sp. induced general inhibition on growth, photosynthesis, and reduction in protein synthesis on increasing the herbicide concentration. 4. Conclusion The study revealed that low concentrations of the herbicide, liable to reach surface waters, increased Chl (a), growth rate, and rate of uptake by algal cells which followed a pseudo first order rate. The impacts of the herbicide on the previous parameters coincide with effect on amino acids and protein content of the algal cells. S. obliquus was more sensitive to the inhibitory effect of the herbicide. Such a result indicates that pollution of fresh drinking water resources by the herbicide may lead to the over growth of the blue green algae which exert toxic effects on drinking water.
Table 3 Kinetic data for the uptake of cyanazine by the two different algal species.
Fig. 2. Percentage uptake of cyanazine by the two different algal species.
Cyanazine (μg/L)
Scenedesmus obliquus Rate constant (K)
t0.5 (days)
7.5 15 30 60 120
0.12 0.10 0.04 0.036 0.028
5.8 6.9 17.3 19.2 24.7
Cyanazine (μg/L)
30 60 120 240 480
Anabaena flos-aquae Rate constant (K)
t0.5 (days)
0.124 0.072 0.036 0.010 0.005
5.6 9.6 19.2 69.3 138.5
A.M. AbdEl-Aty, M.A. El-Dib / Desalination 249 (2009) 1294–1297
Fig. 4. Effect of cyanazine on protein and amino acids contents of the two different algal species.
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