International Journal of Environmental Research University of Tehran ISSN: 1735-6865 EISSN: 2008-2304 Vol. 4, Num. 2, 2010, pp. 347-352

International Journal of Environmental Research, Vol. 4, No. 2, 2010, pp. 347-352

Ma, J.^1,2, Tong, S.², Wang, P.² and Chen, J.^1*

¹College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, People’s Republic of China
²School of Forestry and Bio-technology, Zhejiang Forestry College, Lin-An 311300, People’s Republic of China

*Corresponding author E-mail: mjyzjhgy@yahoo.com

Received 16 Aug. 2009; Revised 2 Dec. 2009; Accepted 12 Dec. 2009

Code Number: er10038

ABSTRACT

The toxicity of 7 herbicides to the three cyanobacteria was tested in this work. The results indicated that: (1) There was a highly significant relationship between dried weight or chlorophyll-a and OD680nm for tested cyanobacteria; (2) the toxicity of the tested herbicides with the order from high to low was: photosynthesis-inhibiting > ACCase inhibitor > protox inhibiting herbicides; (3) the sensitivity of various species exposed to cyanazine, diclofop, prometryn, simazine and simetryn varied by over one order of magnitude. The decreasing order of sensitivity of cyanobacteria to the selected herbicides was: M. Flosaquae > M. Aeruginosa > A. flos-aquae. Cyanobacteria can produce toxins including hepatotoxins e.g. microcystins and endotoxins e.g. lipopolysaccharides. Therefore, the research on comparing the differential sensitivity of cyanobacteria and green algae is of important scientific significance and realistic value

Key words: Acute Toxicity, Berbicides, Cyanobacteria, Sensitivity

The pollution of aquatic systems by pesticides has attracted public concerns, especially in herbicides. A few studies have been conducted to determine the harm of these pollutants to living organisms in the aquatic systems (Kasai, et al., 1993; Ma, 2005). Herbicides may enter freshwater ecosystems by spray drift, leaching, run-off, or accidental spills and present potential risks for aquatic flora. However, little is known about the toxicity of these herbicides against the aquatic flora, despite the importance of aquatic plants in the functioning of ecosystems (Ma, et al., 2004; Naito, et al., 2003; Wong, 2000). Alterations of the species composition of an aquatic community as a result of toxic stress may affect the structure and function of the whole aquatic ecosystem. Cyanobacteria (blue-green algae) are known to be comparatively sensitive to many chemicals and the inclusion of these organisms in test batteries has been shown to improve the capacity of battery to predict the most sensitive responses of ecosystem (Ma, et al., 2006; Real, et al., 2003). Their ecological position at the base of most aquatic food webs and the essential roles in the nutrient cycling and oxygen production are critical to all ecosystems (Breitholtz, et al., 2006). A great deal of information on toxicological aspects of pesticides on green algae, especially on Chlorella, Scenedesmus and Selenastrum is available (Ma, et al., 2007; Sabater & Carrasco, 2001). However, little is known on the toxicological aspects of pesticides on cyanobacteria (Abou-waly, et al., 1991; Ma & Chen, 2005). Cyanobacteria can produce algal toxins, but also can fixate atmospheric nitrogen, which has important application for humans and aquatic organism (An & Kampbell, 2003; Saker & Neilan, 2001). In order to compare the differential sensitivity of herbicides to cyanobacteria, the toxicity test has been devised. In the present study, 7 herbicides were tested to examine their effects on the cyanobacteria Anabaena flos-aquae, Microcystis flos-aquae and Mirocystis aeruginosa and then compare their different sensitivity.

Tested herbicides were purchased from People’s Republic of China and their chemical classes and mode of actions (Retzinger & Smith, 1997) are shown in Table 1. The tested herbicides were dissolved in a small volume of 99.5% acetone. The concentration of acetone in the medium was kept minimizing in response to the solubility of the tested herbicides.The concentration of the acetone in the medium was less than 0.05%. The US Environmental Protection Agency recommends the allowable maximal limits of 0.05% solvent for acute tests and 0.01% for chronic tests, this level was not significant with regard to toxicity (Jay, 1996).

The toxicity tests were carried out with the freshwater cyanobacteria Anabaena flos-aquae, Microcystis flos-aquae and Mirocystis aeruginosa obtained from the Wuhan Institute of Hydrobiology, the Chinese Academy of Science. The medium for cyanobacterial growth inhibition test was HGZ medium which is composed of distilled water and the following chemical ingredients (mg/L): NaNO₃ 1500, K₂HPO₄ 39, MgSO₄·7H₂O 75, Na₂CO₃ 20, CaCl₂ 27, Na₂SiO₃·9H₂O 58, EDTA1, Citricacid 6, Fe-Citric 6, and A₅ liquid 1 mL/L (ingredients of A₅ liquid are H₃BO₃ 2860, MnSO₄ 2060, ZnSO₄·7H₂O 222, Na₂MO₄·2H₂O 391 and CuSO₄·5H₂O 79). The medium was sterilized at 121^oc, 1.05 kg /cm² for 30 min.

Cells of cyanobacteria were propagated in a 250 mL Erlenmeyer flask containing 100 mL HGZ medium and kept on a rotator shaker (100rpm) at 24^oc, and illuminated with cool-white fluorescent lights at a continuous light intensity of 5000 Lx (Verdisson, et al., 2001). 20 mL HGZ medium containing cyanobacterial cells (initial concentration OD_680nm=0.008) were distributed to sterile 50 mL Erlenmeyer flasks. The medium was then treated with a variety of herbicide concentrations, and incubated at the same temperature and light intensity (Verdisson, et al., 2001). Cyanobacterial medium was scanned at 400-800 nm wavelength after 96 hours incubated with a Shimadzu UV-2401PC spectrophotometer. The most suitable wavelength for monitoring medium was 680 nm. Strong linear relationships between dry weight concentration (DWC) or Chlorophyll-a (Chl-a) concentration of the cyanobacteria cultures and OD680nm were approved in the work (see result part). The growth of cyanobacterial biomass was calculated indirectly using OD680nm data. Appropriate control systems containing no herbicide were included in each experiment. Three replicates were made for every herbicide concentration and control. And the percent inhibition values, relative to the growth in the control systems, were also calculated in each experiment by using OD680nm data. Chl-a analysis comes after the filtration of 20 ml medium samples through the filtration (0.45-µm-pore-size Whatman GF/C membranes) and extraction with cold 90% acetone. Chl-a content in samples was estimated using spectrophotometer as a trichromatic method. Dry weight of cyanobacteria was determined with a digital balance after cells filtered on a 0.45µm membrane and dried at 105! for 8h. The EC₅₀ values were calculated by using linearregression analysis of transformed herbicide concentration as natural logarithm data versus percent inhibition (Ma, et al., 2006). All raw data was analyzed under SPSS version 11.0.

The medium of the three cyanobacteria was scanned respectively by using a Shimadzu UV2401PC spectrophotometer. The most suitable wavelength for monitoring culture growth was 680 nm. There was a highly significant relationship between dried weight or chlorophyll-a and OD_680nm for three tested cyanobacteria. Their linear regression equations were shown in Table 2. All coefficient of correlation R>0.97 and significance level P<0.001.Therefore, growth of cyanobacterial biomass was calculated indirectly using OD_680nm data in this work.

The acute toxicity of 7 herbicides to the three cyanobacteria A. flos-aquae, M. flos-aquae and M. aeruginosa, was shown in Table 3. The 96 h EC₅₀ values ofACCase inhibitor diclofop-p varied around 9-311mg/L. Synthetic auxins triclopyr varied around 32-109 mg/L. The 96 h EC₅₀ values of inhibition of photosynthesis at PSII such as ametryne, simazine, prometryne, cyanazine and simetryn varied around 0.0002-1.2425 mg/L. Comparing the acute toxicity of 7 herbicides with various primary modes of action to cyanobacteria, the acute toxicity of photosynthesis-inhibiting herbicides was the highest among the tested herbicides with the order from high to low as follows: photosynthesis-inhibiting herbicides > ACCase inhibitor > protox inhibiting herbicides. Similar results also have been obtained when green algae were used as tested organism (Ma, 2005).

Wide variations occurred in response to the tested herbicides among three individual species of cyanobacteria (Table 4). Compared with M. aeruginosa, M. flos-aquae was moresensitive to 6 herbicides— cyanazine, diclofop, prometryn, simazine, ametryn, and was less sensitive to one —triclopyr. Sensitivity of various species of cyanobacteria exposed to cyanazine, diclofop, simetryn varied over one order of magnitude. However, A. flos-aquae was less sensitive to the tested 7 herbicides while compared with M. aeruginosa. The sensitivity of various species of cyanobacteria exposed to simetryn varied over one order of magnitude. In contrast with A. flosaquae, M. flos-aquae was more sensitive to the tested 7 herbicides. Whereas the sensitivity of various species of cyanobacteria exposed to cyanazine, diclofop, prometryn, simazine and simetryn varied by over one order of magnitude. The decreasing order of average sensitivity of 3 dissimilar cyanobacteria to the selected herbicides was: M. flos-aquae> M. aeruginosa> A. flosaquae.

Chlorophyll-a content or dry weight was usually used as an indicator of green algal biomass. However, the assessment method for chlorophyll-a content or dry weight was too complicated to assess quickly. With respect to green algae, a great many works have been published revealing the correlation between biomass and absorbance (Cetin & Mert, 2006) while few concerning cyanobacteria. In the present work, we think that cyanobacterial suspension absorbance could take the place of the biomass. Therefore, the absorbance data instead of Chl-a or dried weight could be used indirectly to express the biomass of cyanobacterial suspension. It could be a quick, simple and accurate method to assess toxicity of tested contamination to cyanobacteria.

Compared with our previous works, wide variation occurred in response to the tested 5 herbicides among individual species of green algae and cyanobacteria. Contrasting with the green alga C. pyrenoidosa, Cyanobacteria A. flos-aquae was less sensitive to diclofop, prometryn, simazine and ametryn. The sensitivity of various species of cyanobacteria and green alga C. pyrenoidosa that were exposed to prometryn or simazine varied by over one order of magnitude and that exposed to diclofop or ametryn varied by over two orders of magnitude (see Table 4). It may also be important for sustaining cyanobcterial bloom during special period in the aquatic ecosystem. Cyanobacteria can produce toxins including hepatotoxins e.g. microcystins and endotoxins e.g. lipopolysaccharides. Therefore, the research on comparing the differential sensitivity of cyanobacteria and green algae is of important scientific significance and realistic value.

This project was supported by the National and Zhejiang Provincial Natural Science Foundations of China (No. 20476099 & 202111).

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