Toxicological assessment of photocatalytically destroyed mixed azo dyes by chlorella vulgaris

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ГОДИШНИК НА МИННО-ГЕОЛОЖКИЯ УНИВЕРСИТЕТ “СВ. ИВАН РИЛСКИ”, Том 57, Св. II, Добив и преработка на минерални суровини, 2014

ANNUAL OF THE UNIVERSITY OF MINING AND GEOLOGY “ST. IVAN RILSKI”, Vol. 57, Part ІI, Mining and Mineral processing, 2014

Alexandre Loukanov *, Milena Georgieva, Anatoly Angelov
University of Mining and Geology “St. Ivan Rilski”, 1700 Sofia, Laboratory of Eng. NanoBiotechnology, *
ABSTRACT. The study aims to evaluate the toxic effects photocatalytically destroyed mixed azo dyes on the growth of Chlorella vulgaris and to assess its bioaccumulation capacity as a function of the dye concentration and the time of contact with the xenobiotic. The assays were accomplished in a set of miniaturized tests, which contains the bioindicator. The exponentially growing algae cultures were exposed to various concentrations of model solution before and after photooxidation treatment. The toxicological effect of the dyes was determined by measuring of the grown inhibition over a fixed period. The toxicity to Chlorella vulgaris, expressed as log(1/EC50) of xenobiotics has been examined. All data demonstrate that the growth inhibition was greater with untreated azo dyes than the destroyed reaction mixture. The final dye concentration in the supernatant was measured spectrophotometrically in the presence of microalgae. The data demonstrated the efficiency of Chlorella vulgaris to remove the dye from wastewaters.

Александър Луканов *, Милена Георгиева, Анатолий Ангелов

Минно-геоложки университет „Св. Иван Рилски”, 1700 София, *
Резюме. Изследването е насочено към изучаване на токсичния ефект на фотокаталитично разрушена смес на азобагрила върху растежа на Chlorella vulgaris и да се оцени биоакумулационния капацитет, като функция на концентрацията на багрилото и времето на контакт с ксенобиотика. Анализите са извършени в набор на миниатюризирани тестове, съдържащи биоиндикатор. Експоненциално растящи култури на микроводорасли бяха изложени на различни концетрации на моделно багрило преди и след фотоокислително третиране. Токсикологичния ефект на багрилата беше определен чрез измерване инхибирането на растежната за определен времеви период. Токсичността на ксенобиотика беше определена спрямо log(1/EC50) на Chlorella vulgaris. Получените данни показват, че нетретираното багрило инхибира в по-голяма степен растежа на микроводораслите, отколкото реакционната смес, получена в резултат на фотоокисление. Крайната концентрация на багрилото в супернатантата беше измерена спектрофотометрично в присъствие на микроалгите. Получените резултати доказват ефективността на Chlorella vulgaris да отстраняват багрилото от отпадната вода.


Large volumes of polluted effluents are discharged from textile processing which consumes large amounts of water. Effluent from the textile industry is characterized by strong colour, high salinity, high temperature, variable pH and high chemical oxygen demand (COD; Mantzavinos and Psillakis 2004). The textile dyes are hazardous and their strong colour can reduce light availability, upsetting the biological activity in aquatic life. Algae have been shown to be capable of removing colour from various dyes through mechanisms such as biosorption, bioconversion and bioagulation (Daneshwar et al., 2007). Algae are of particular importance for such studies not only because of their widespread distribution and their fundamental importance to aquatic ecosystems, but also because they can react rapidly to environmental change, not least because of a short life cycle.

Microalgae, the primary producers in the food chain, are most sensitive to contaminants than are fish and invertebrates; they are, therefore, important organisms for monitoring water quality and aquatic toxicity. Investigating the toxicity of untreated and photooxidized azo dyes to algae and explaining the mechanism clearly is, therefore, of great importance and can potentially lead to strategies to remediate the potentially adverse effects of textile wastewater on the environment (Maynard et al., 2006). Immobilized algae are also used in bioremediation of various xenobiotics. For instance, C. vulgaris in alginate beads has been shown to be useful in removing tributylin by biosorption and biodegradation (Luan et al. 2006). However, there have been very few studies on the use of algae to remove colour from dyes. One study reported that immobilized C. pyrenoidosa in calcium alginate is better than suspension cultures in degradation of a brown dye (Huang et al. 2000). The objective of the present study was to investigate the potential use of Chlorella vulgaris for the removal of colour from textile dyes and toxicological assessment of untreated and photocatalytically destroyed mixed azo dyes. The effect of the azo dye on microalgae growth and the microalgae efficiency to accumulate the organic xenobiotic is studied in order to investigate the relationships between these two processes.

Experimental procedures

The purpose of the growth inhibition test is to determine the effects of a substance (azo dye) on the growth of unicellular green algal species. Definitions: cell concentration is the number of cells per ml, growth is the increase in cell concentration over the test period, growth rate is the increase in cell concentration per unit of time, EC50 is that concentration of test substance which results in a 50 % reduction in either growth or growth rate relative to the control, NOEC (no observed effect concentration) is the highest concentration tested at which the measured parameter show no significant inhibition of growth relative to control values, reference substance may be tested as a means of detecting unsatisfactory test conditions. Potassium dichromate can be used as a reference substance. Exponentially-growing culture of selected green algae are exposed to various concentrations of the test substance (untreated and photooxidized azo dye) over several generations under defined conditions. The inhibition of growth in relation to a control culture is determined over a fixed period. The cell concentration in the control cultures should have increased by a factor of at least 16 within three days. Disappearance of the test substance from the water into the biomass does not necessarily invalidate the test.

Culture apparatus: a cabinet or chamber in which a temperature in the range 21 to 25 °C can be maintained at ± 2 °C and continuous uniform illumination provided with a quantum flux of 0.72x1020 photons/m2s ± 20% in the spectral range 400 – 700 nm. Apparatus to determine cell concentrations, e.g. electronic particle counter, microscope with counting chamber, fluorimeter, spectrophotometer, colorimeter. The following Algal medium is recommended: NH4Cl = 15 mg/L, MgCl2.6H2O = 12 mg/L, CaCl2.2H2O = 18 mg/L, MgSO4.7H2O = 15 mg/L, KH2PO4 = 1.6 mg/L, FeCl3.6H2O = 0.08 mg/L, Na2EDTA.2H2O = 0.1 mg/L, H3BO3 = 0.185 mg/L, MnCl2.4H2O = 0.415 mg/L, ZnCl2 = 3x10–3 mg/L, CoCl2.6H2O = 1.5x10–3 mg/L, CuCl2.2H2O = 10–5 mg/L, Na2MoO4.2H2O = 7x10– 3 mg/L, NaHCO3 = 50 mg/L. The pH of this medium after equilibration with air is approximately 8.

It is suggested that the species of green algae used be a fast-growing species that is convenient for culturing and testing. Chlorella vulgaris CCAP 211/11b is considered as suitable. It is recommended that the initial cell concentration in the test cultures be approximately 104 cells/ml. The concentration range in which effects are likely to occur is determining on the basis of results from range-finding tests. For the test, at least five concentrations arranged in a geometric series, should be selected. The lowest concentration tested should have no observed effect on the growth of the algae. The highest concentration tested should inhibit growth by at least 50 % relative to the control and, preferably, stop growth completely. The test design should include preferably three replicates at each test concentration and ideally twice that number of controls. If justified the test design may be altered to increase the number of concentrations and reduce the number of replicates per concentrations.

Test cultures containing the desired concentrations of test substance (mixed azo dyes) and the desired quantity of algal inoculums are prepared by diluting with filtered algal medium aliquots of stock solutions of the test substance and of algal suspension. The culture flasks are shaken and placed in the culturing apparatus. During the test it is facilitate transfer of CO2. To this end cultures should be maintained at a temperature in the range of 21 to 25 °C, controlled at ± 2 °C. The cell concentration in each flask is determined at least at 24, 48 and 72 hours after the start of the test. Filtered algal medium is used to determine the background when using particle counters or as a blank when using spectrophotometers. The pH is measured at the beginning of the test and at 72 hours. The pH of the solutions should not normally deviate by more than one unit during the test.
Result and discussion

The metabolites produced from dye degradation are in many cases, more toxic that the parent dye. Several azo dyes and the amines from their degradation have shown mutagenic responses in Salmonella and mammalian assay systems, and their toxicity depends on the nature and position of the substituents in the molecule. For example, the dyes Acid Red 18 and Acid Red 27 are non-mutagenic, whereas the structurally similar dye Acid Red 26 is carcinogenic because of the presence of a methyl group and the difference in the position of the sodium sulphonate. Therefore, in general, it becomes very important for any bioremediation technology to assess the toxicity of the pollutants and metabolities formed after dye degradation in order to study the feasibility of the method. Chlorella grew in 100 % textile water although the final biomass attained was significantly lower (p < 0.05) than in 20 – 80 % textile water. Colour removal by Chlorella decreases with the increase in initial colour, especially in medium containing > 60 % textile water. This agrees with other studies using algae such as Synechocystis and Phormidium for removing colour from reactive dyes (Karacakaya et a;., 2009). Without Chlorella (control), colour removal due to adsorption or bioconversion by the algae did not take place, hence, very low colour removal was observed.

The equilibrium experiment curves of azo dye biosorption onto Chlorella vulgaris at different temperatures (298, 308, 318, 328 K) are presented in Figure 1.

Fig. 1. Biosorption equilibrium isotherm
The biosorption isotherms were characterized by an initial step of weak attraction between Chlorella and the dye, followed by a strong increase in the biosorption capacity, and finally a plateau was observed. It can be observed that a temperature decrease caused an increase in biosorption capacity, the best result obtained at 298 K. This behavior can be explained by two main aspects: Firstly, the temperature increase causes an increase in the solubility of the dyes (Koprivanac and Kusic, 2009) so, the interaction forces between the dyes and the solvent become stronger than those between dyes and algae. Coupled to this, according to Aksu (2005), at temperature above 318 K damage of sites on the surface of biomass can occurs and, consequently, a decrease in the surface activity. Piccin et al. (2011) showed that the temperature increase caused a decrease in the adsorption capacity of FD&C red n˚40 onto chitosan. Similar behavior was obtained by Aksu and Tezer (2005) in the biosorption of remazol red and remazol golden yellow using Chlorella vulgaris as biosorbent. They obtained best results at 298 K.

The EC50 values in Reactive Black 5 azo dye (untreated and photooxidized) tests were 0.94, 1.00 and 0.45 mg/L at 24, 48, and 72 h, respectively. There was a decrease in the culture rate as the exposure time and dye concentration increased. The growth rate was significantly lower and different to the control in C4, C5 and C6 after being exposed for 48 h (p < 0.01). After 72 h, there also were significantly differences between the control and all the concentration tested (p<0.01) (Fig. 2).

Fig. 2. Growth rate of Chlorella vulgaris after exposure to six different concentrations of azo dye Reactive Black 5

The mean initial and final pH values ranged from 6.20 (± 0.05) to 6.00 (± 0.3), without significant differences at the beginning and the end of each assay (p > 0.05).


In conclusion, the present study showed that Chlorella vulgaris were able to grow in the textile wastewater contained mixed azo dyes. Of the three dyes tested, the cultures removed the highest percentage of colour from Reactive Black 5. The percentage of colour removal by algae decreases with increasing initial concentration of the dye. Initial untreated dyes possess higher toxicity effect on Chlorella than the photooxidized and destructed dyes. The temperature decrease caused an increase in the biosorption capacity.

Acknowledgements. The support of this project by the Bulgarian National Scientific Fund (Project No. DDVU 02-36/2010) is gratefully acknowledged.

Aksu, Z., Tezer, S., (2005) Biosorption of reactive dyes on the green alga Chlorella vulgaris. Process Biochemistry 40, 1347-1361.

Daneshwar, N., Ayazloo, M., Khatee, A., Pourhassan, M., (2007) Biological decolourisation of dye solution containing malachite green by microalgae Closterium sp. Bioresour Technol 98, 1176-1182.

Huang, G., Sun, H., Cong, L., (2000) Study on the physiology and degradation of dye with immobilized algae. Artif Cells Blood Substit Immobil Biotechnol 28(4), 347-363.

Karacakaya, P., Kilic, N.K., Duyugu, E., Donmez, G., (2009) Stimulation of reactive dye removal by cyanobacteria in media containing triacontrol hormone. J. Hazard. Mater. 172, 1635-1639.

Koprivanac, N., Kusic, H., (2009) Hazardous Organic Pollutants in Colored Wastewaters. New Science Publishers, New York.

Luan, T., Jin, J., Chan, S., Wong, S., Tam, N., (2006) Biosorption and biodegradation of tributyltin (TBT) by alginate immobilized Chlorella vulgaris beads in several treatment cycles. Proc Biochem 41, 1560-1565.

Mantzavinos, D., Psillakis, E., (2004) Enhancement of biodegradability of industrial wastewater by chemical oxidation pretreatment. J Chem Tech Biotechnol 79, 431-454.

Maynard, A., Aitken, R., Butz, T., Colvin, V., Donaldson, K. Et al., (2006) Safe handling of nanotechnology. Nature 444, 267-269.

Piccin, J.S., Dotto, G.L., Pinto, L.A.A., (2011) Adsorption isotherms and thermochemical data of FD&C Red n˚40 binding by chitosan. Brasilian Journal of Chemical Engineering 28, 295-304.

The article has been recommended for publication by department “Engineering Geoecology”.

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