- Email: [email protected]
Physiological and biochemical responses of Chlorella vulgaris to Congo red
Ecotoxicology and Environmental Safety 108 (2014) 72–77
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Physiological and biochemical responses of Chlorella vulgaris to Congo red Miriam Hernández-Zamora a, Hugo Virgilio Perales-Vela b, César Mateo Flores-Ortíz c, Rosa Olivia Cañizares-Villanueva a,n a Laboratorio de Biotecnología de Microalgas, Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. IPN 2508, San Pedro Zacatenco, C.P. 07360 México DF, México b Laboratorio de Bioquímica, Unidad de Morfología y Función, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala, Av. de los Barrios #1, Estado de México, México c Laboratorio de Biogeoquímica, Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala, Av. de los Barrios #1, Estado de México, México
art ic l e i nf o
a b s t r a c t
Article history: Received 28 February 2014 Received in revised form 27 May 2014 Accepted 28 May 2014
Extensive use of synthetic dyes in many industrial applications releases large volumes of wastewater. Wastewaters from dying industries are considered hazardous and require careful treatment prior to discharge into receiving water bodies. Dyes can affect photosynthetic activities of aquatic flora and decrease dissolved oxygen in water. The aim of this study was to evaluate the effect of Congo red on growth and metabolic activity of Chlorella vulgaris after 96 h exposure. Exposure of the microalga to Congo red reduced growth rate, photosynthesis and respiration. Analysis of chlorophyll a fluorescence emission showed that the donor side of photosystem II was affected at high concentrations of Congo red. The quantum yield for electron transport (φEo), the electron transport rate (ETR) and the performance index (PI) also decreased. The reduction in the ability to absorb and use the quantum energy increased non-photochemical (NPQ) mechanisms for thermal dissipation. Overall, Congo red affects growth and metabolic activity in photosynthetic organisms in aquatic environments. & 2014 Elsevier Inc. All rights reserved.
Keywords: Chlorella vulgaris Chlorophyll a fluorescence Congo red JIP-test Photosynthesis Respiration
1. Introduction Azo compounds constitute the largest and most diverse group of synthetic dyes and are widely used by the textile, food, cosmetic, and pulp and paper industries (El-Sheekh et al., 2009). At the global level, 280,000 t of textile dyes are discharged into industrial effluent each year (Jin et al., 2007). It is estimated that the quantity of dye that does not fix to textile fibers depends on the application method, varying from 2 percent when basic dyes are used to 50 percent when reactive dyes are used (Hai et al., 2007; Pearce et al., 2003). The discharge of azo dyes into the environment is a serious problem and should be avoided not only for esthetic reasons but also because of the threat it poses to public health and ecosystems (Golka et al., 2004). In aquatic environments, the colors produced by azo dyes impede plant development by decreasing the passage of light, thereby affecting the
n
Corresponding author. Fax: þ52 55 50613313. E-mail addresses: [email protected] (M. Hernández-Zamora), [email protected] (H.V. Perales-Vela), cmfl[email protected] (C.M. Flores-Ortíz), [email protected] (R.O. Cañizares-Villanueva). http://dx.doi.org/10.1016/j.ecoenv.2014.05.030 0147-6513/& 2014 Elsevier Inc. All rights reserved.
photosynthetic process (Annuar et al., 2009). The resulting changes in the concentration of dissolved O2 damage the aquatic biota present and increase the biochemical oxygen demand of the surrounding water (Ali, 2010). In addition, some azo dyes, such as Congo red, Direct Blue 15 and Direct Red 2, have been reported to be toxic, mutagenic, and carcinogenic to aquatic life (Bafana et al., 2008; Golka et al., 2004; Saratale et al., 2011). In aquatic ecosystems the green algae are the primary producers and are key indicator organisms (Wen et al., 2011) when used to evaluate water quality and the ecotoxicity of contaminants (Xu et al., 2013), such as metals, herbicides, insecticides, and other xenobiotic compounds (Jena et al., 2012; Levy et al., 2007; Qian et al., 2008). On the other hand, the chlorophyll a fluorescence kinetic has been used to indicate alterations of the photosynthetic capacity when damage is induced by pollutants or by environmental conditions (Jena et al., 2012; Kalaji et al., 2012). The advantage of such a method is that it is non-invasive and reliable (Muller et al., 2008; Xia and Tian 2009). Furthermore, studies have shown that changes in photosystem II (PSII) can be quantified using the JIP-test (Jena et al., 2012; Strasser et al., 2000). However, as of yet, no information is available regarding the impact of azo dyes on the photosynthetic apparatus of
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
algae. Such information is important because it would permit us to determine the toxicity to the aquatic environment caused by this type of dye. For this reason, the objective of the current study is to evaluate the toxic effects of the azo dye Congo red on the growth and photosynthetic metabolism of the microalga Chlorella vulgaris.
2. Materials and methods 2.1. Dye used Congo red (Sigma-Aldrich), also called Direct Red 28, was used for the experiments described herein. Its molecular form is C32H22N6O6S2Na2, and its molecular weight is 696.7 g mol 1. All experiments employed a 100 mg L 1 stock solution of dye, which, prior to its addition in each experimental unit, was sterilized by filtration using Millipores membranes of 0.22 μm pore diameter. 2.2. Experimental units Experiments were conducted under sterile conditions with axenic cultures of the green microalga C. vulgaris donated by the Laboratorio de Hidrobiología Experimental of the Escuela Nacional de Ciencias Biológicas of the Instituto Politécnico Nacional. The strain was collected and isolated from temporary ponds in the State of Mexico (Atlacomulco). The registration number is LHE-Chl 01. The experimental culture units were glass bottles with flat surfaces, each of which had a total capacity of 0.5 L and a working volume of 0.25 L, that were inoculated with 15 ml of C. vulgaris culture in the exponential phase (11 mg L 1 dry biomass) Bold's Basal mineral medium (Stein, 1973) and different concentrations of dye (5, 10, 15, 20, and 25 mg L 1 of Congo red), the control used contained only the inoculum and the culture medium. The culture conditions were as follows: temperature, 257 3 1C; illumination, 120 μmoles photons m 2 s 1; photoperiod, 12/12 h (light/darkness); and air supply, 200 ml min 1.
73
2. The probability that a trapped exciton moves an electron into the electron transport chain beyond QA ; Ψo ¼ 1 Vj 3. The quantum yield of electron transport; φEo ¼ [1 (Fo/Fm) (1 Vj)]¼ (φPo) (Ψo) 4. Reaction centers per excited cross section; (RC/ABS) ¼ [(Mo/Vj) (1 (Fo/Fm))] 5. The performance index; PIABS ¼ (RC/ABS) [φPo/(1 φPo)] [Ψo/(1 Ψo)] Data were interpreted using the Handy-PEA software developed by Hansatech Instruments Ltd., U.K., and Biolyzer-HP3 software designed at the Bioenergetics Laboratory of the University of Geneva, Switzerland (van Heerden et al., 2003). Modulated fluorescence was measured using the fluorescence modulated system (FMS 2 Hansatech Instruments Ltd., Norfolk, UK) in accordance with Nielsen and Nielsen (2005) and Masojídek et al. (1999). All measurements were obtained at room temperature (25 1C) and in dark conditions. Prior to examining Chl-a fluorescence, samples of each experimental unit were adjusted to the same optical-density value (0.3 680 nm) and these were washed using Bold´s Basal mineral medium to remove adsorbed dye completely. Each cellular suspension was vacuum-filtered using Millipores membranes of 5.0-μm pore diameter and were later adapted to darkness for 5 min. Signatures for Chl-a fluorescence were detected directly from the filter surface (Perales-Vela et al., 2007). The value of Fo at room temperature was obtained by irradiating the sample with a modulated light of low intensity (0.1 μmol m 2 s 1) and after 10 s the sample was superimposed with one saturating pulse of white light of 10,000 μmol m 2 s 1 for 0.8 s, to close all reaction centers and obtain the maximum fluorescence value of Fm in darkness. Immediately after the saturating pulse, the samples were irradiated with a non-saturating actinic white light 200 μmol m 2 s 1 for 5 min to achieve a steady state and then these were superimposed a saturating white light pulse of 10,000 μmol m 2 s 1 for 0.8 s to obtain the value of Fm0 . Finally the values were calculated as follows: 1. Relative electron transport rate (rETR) ¼ [((Fm0 Fs)/Fm0 )] (0.5)(PAR). The value of 0.5 corresponds to the proportion of light that is transferred to each of the two photosystems (PSII and PSI) and the photosynthetically active radiation (PAR) utilized was of 162 μmol m 2 s 1. 2. Non-photochemical quenching (NPQ)¼ [(Fm Fm0 )/Fm0 ].
2.3. Growth and photosynthetic pigments Growth was determined using the dry-weight values obtained at the beginning (0 h) and at the end (96 h) of the experiment. Each treatment used 5-ml aliquots that were vacuum-filtered using Millipores membranes of 5.0-μm pore diameter, pre-treated for a constant weight. Later, the samples were dried at 80 1C for 48 h. Growth rate of algal culture is a measure of the increase in biomass over time and it was determined by the following equation: K ¼ In ðN 2 =N 1 Þ=ðT 2 T 1 Þ where, K is growth rate, N1 and N2, biomass content at time (T1) and the time (T2), respectively chlorophyll a and b and total carotenoids were determined using 3 ml of each sample, after the sample was centrifuged for 10 min. Subsequently, the pellet was added 2 ml of methanol and stirred for 1 min. Previously the pellet was washed using Bold´s Basal mineral medium to remove adsorbed dye completely. This procedure was done to prevent the absorption spectrum of Congo red which interferes with the absorption spectrum of photosynthetic pigments, especially carotenoids. The sample was kept in water bath at 60 1C for 10 min. Again, each sample was centrifuged at room temperature. The supernatant was separated and the concentrations of Chl-a, Chl-b, and total carotenoids were determined with a UV–Vis spectrophotometer (Genesys 10UV Thermo Electron Corporation) according to the equations of Wellburn (1994).
2.5. Measurement of photosynthesis and respiration Measurements were made using an oximeter (Oxylab-Hansatech, UK) at a controlled temperature of 30 1C in exponentially growing cultures. Previously, the cells were washed using Bold´s Basal mineral medium to remove adsorbed dye completely. The samples of each experimental unit were adjusted to the same optical-density value (0.3 680 nm) before the photosynthesis and respiration study. The oxygen release rate (photosynthesis) was obtained by illuminating each of the samples with an actinic red light of 400 μmol m-2 s-1 for 1 min. Immediately after turning off the light source, the oxygen consumption rate (respiration) was recorded for 2 min. 2.6. Statistical analysis All experiments were performed in triplicate, and the results obtained were statistically analyzed via one-way analysis of variance (P o 0.05) and Tukey's test for comparison of means using the statistical package Sigma-Plot (version 11.0).
3. Results and discussion 2.4. Chlorophyll fluorescence For all treatments, the Chl-a fluorescence emitted via photosystem II was measured at 96 h in samples conditioned in the dark using a portable fluorometer, model HANDY-PEA (Hansatech Instruments Ltd., Norfolk, UK) coupled to the chamber for liquid phase HPEA/LPA (Hansatech, UK). For each of the experimental units, samples were corrected to the same optical density (0.3 680 nm) and these were washed using Bold´s Basal mineral medium to remove adsorbed dye completely. After, 2 ml samples of each treatment (n ¼4) were taken and incubated at dark for 20 min at room temperature (25 1C), then the samples were irradiated for 1 s with red light (660 nm) saturating (3000 μmol photons m 2 s 1). The equipment automatically recorded the following values: (i) minimal fluorescence (Fo) at 50 μs, (ii) maximum fluorescence (Fm) between 200 and 500 ms, (iii) variable fluorescence at 2 ms (Vj ¼ Fj Fo/Fm Fo) and (iv) the slope at the beginning of fluorescence Mo ¼ 4 (FK Fo)/(FM Fo). These data were used to calculate the following parameters (Strasser et al., 2004): 1. The maximum quantum yield of primary photochemistry; Fv/Fm
φPo ¼ [1 (Fo/Fm)] ¼
3.1. Effect of Congo red on growth The microalga C. vulgaris was affected after 96 h of exposure to Congo red. A decrease in growth rate (μ) was observed in every experimental unit after increasing the concentration of dye in the culture medium; this decrease was significant (Po 0.05) for concentration over 15 mg L 1 compared to the control (Fig. 1). The μ value was 0.657 day 1 for cells with no dye, while the values of 0.538 and 0.391 day 1 were obtained when cells were exposed to 5 and 10 mg L 1 of dye, respectively. However, the μ values remained at 0.201 day 1 when cells were exposed to dye concentrations between 15 and 25 mg L 1. Chu et al. (2009) reported μ values of 0.06–0.35 day 1 in cells of C. vulgaris exposed separately to 30 mg L 1 of Supranol Red 3BW, Lanaset Red 2GA, and Levafix Navy Blue EBNA. The cultures grown in textile dyes
74
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
Fig. 1. Effect of Congo red on growth rate μ (day 1) in Chlorella vulgaris following dye exposure for 96 h. The mean of the bars with different letters at each treatment indicated that they were significantly different at P o0.05 according to one-way ANOVA test.
Fig. 2. Effect of Congo red on chlorophyll (aþ b) and carotenoids content in Chlorella vulgaris after 96 h of dye treatment. The mean of the bars with different letters at each treatment indicated that they were significantly different at Po 0.05 according to one-way ANOVA test.
attained significantly lower μ (Po 0.05) than those grown without dye (control). Lim et al. (2010) obtained μ values of 0.34 day 1 in C. vulgaris cells exposed to Supranol Red 3BM and 0.05 day 1 when residual textile water was used. These values were lower than the control (μ 0.40 day 1). There have been very few attempts to explain what causes cellular growth inhibition resulting from exposure to azo dyes; however there are reports of high tolerance and susceptibility in algae. For example, Acuner and Dilek (2004) showed that C. vulgaris can grow without damage in the presence of 400 mg L 1 of Tectilon Yellow 2G. Furthermore, other algae like Pseudokirchneriella subcapitata (Selenastrum capricornutum) could only grow in 0.5 mg L 1 of Disperse Blue 3 (Novotny et al., 2006). The inhibitory effect of textile dyes was reported by Ogawa et al. (1989). They found that inhibition was caused by intercalation of dye compounds between DNA base pairs, so preventing enzymatic activity and cell replication (Ogawa et al., 1988). Ganesh et al. (1994) noted inhibition of biomass in a wastewater system treating with a reactive dye and suggested that this was caused by the products of dye degradation rather the dye itself. Other studies have demonstrated that one of the possible causes of the observed growth inhibition is related to the alteration of photosynthesis, especially at the PSII level (Perron and Juneau, 2011).
3.1.1. Photosynthetic pigment content In plants and algae, photosynthetic pigments (Chl-a, Chl-b, and carotenoids) are important as an indirect measure of photosynthetic function (Marr et al., 1995). Fig. 2 shows the effect of Congo
red dye on content of photosynthetic pigments. It can be seen that the total chlorophyll (aþ b) content decreases significantly with respect to the control as the dye concentration is increased. Acuner and Dilek (2004) have reported similar results with C. vulgaris and the dye Tectilon Yellow G. This reduction in chlorophyll content can affect the conversion of light energy and, thus, photosynthetic electron transport (Strasser et al., 2004). Furthermore, as seen in Fig. 2, the total carotenoid content was not significantly altered (P o0.05), which can indicate that the dye affects each photosynthetic pigment differently. Carotenoids are important because they are involved in the removal of the oxygen free radicals which are generated when the metabolism is inhibited, as well as being important in the regulation of the light excitation energy in the reaction center of photosystem II (Choudhury and Behera, 2001; Czerpak and Piotrowska, 2006). 3.2. Chlorophyll-a fluorescence An increase in the yield of chlorophyll fluorescence from the dark into the light over a time period of around 1 s, it has been explained as a consequence of reduction of electron acceptors in the photosynthetic pathway, downstream of PSII, notably plastoquinone and in particular, QA. Once PSII absorbs light and QA has accepted an electron, it is not able to accept another until it has passed the first onto a subsequent electron carrier (QB). During this period, the reaction center is said to be “closed”. At any point in time, the presence of a proportion of closed reaction centers leads to an overall reduction in the efficiency of photochemistry and so to corresponding increase in the yield of fluorescence (Maxwell and Johnson, 2000). The polyphasic chlorophyll a fluorescence kinetics was modified as the dye increased in the culture medium,
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
75
Fig. 3. Effect of Congo red on chlorophyll a fluorescence transients in Chlorella vulgaris after 96 h of dye treatment.
Table 1 Experimental expressions of the JIP-test and their calculated values obtained for Chlorella vulgaris cell subjected for 96 h to Congo red. The maximum quantum yield of primary photochemistry (φPo); the probability that a trapped exciton moves an electron into the electron transport chain beyond QA (Ψo); the quantum yield of electron transport (φEo); the initial slope at the beginning of the variable fluorescence (Mo); the total number of active reaction center per absorption (RC/ABS) and performance index (PIABS) in Chlorella vulgaris after 96 h of dye treatment. Congo red (mg L 1)
φPo
Ψo
φEo
RC/ABS
Mo
PI
0 5 10 15 20 25
0.7087 0.004a 0.7157 0.003a 0.626 7 0.005b 0.4917 0.013c 0.4997 0.006c 0.5167 0.007d
0.563 7 0.016a 0.5687 0.018a 0.542 7 0.006b 0.508 7 0.015c 0.5447 0.007b 0.540 7 0.014b
0.3777 0.011a 0.384 7 0.013a 0.3177 0.004b 0.234 7 0.012c 0.2517 0.003d 0.256 7 0.009d
0.3327 0.005a 0.343 7 0.008a 0.281 7 0.011b 0.1927 0.008c 0.2167 0.011d 0.2247 0.023d
0.996 7 0.002a 0.9977 0.003a 1.0007 0.002a 0.993 7 0.014a 1.0007 0.001a 1.0007 0.001a
0.378 70.018a 0.404 70.022b 0.215 70.005c 0.083 70.007d 0.101 70.004e 0.109 70.008f
The parameters with different letters at each treatment indicated that they were significantly different at Po 0.05 according to one-way ANOVA test.
the maximal fluorescence particularly (FM) was reduced (Fig. 3). Some authors have attributed the decrease in the maximal fluorescence (inflection P) to deactivation or reduced fluorescence emissions, due to the presence in the “pool” of plastoquinones (PQ) that are oxidized (Haldimann and Tsimilli-Michael, 2005). On the other hand, Govindjee (1995) and Perales-Vela et al. (2007) have suggested that a decrease in the phases J and I illuminates how electron-transport inhibition on the donor side of PSII results in the accumulation of P680 þ and can reduce fluorescence emission. The results in the photochemical activity determined by the fluorescence emission, shows that the dye decreased significantly (P o0.05) the maximum quantum yield of primary photochemistry (φPo), the probability that a trapped exciton moves an electron into the electron transport chain beyond (Ψo), and the quantum yield of the electron transport (φEo). Corresponding values are shown in Table 1. At the maximum dye concentration (25 mg L 1) the values of φPo, Ψo, and φEo decrease by 27.11, 4.08, and 32.09 percent, respectively, compared to the control. The maximum quantum yield of primary photochemistry (φPo) uniquely reflects the efficiency of light-phase reactions (Strasser et al., 2004). It has been reported that when a decrease in φPo occurs, it is due to destruction or modification of the photosystem, which is to say that the reaction center of PSII does not have its normal quantum efficiency. The reduction in φPo found in this investigation is the result of the decrease in the values of the minimum (Fo) and maximum (FM) fluorescence. The decrease in the values of parameters Ψo indicate that the yield of the electron transport per a trapped exciton in treated cells with Congo red (10–25 mg L 1) is lower than in the control and that the energy needed to close all reaction centers decreases (Strasser et al., 1995). Studies conducted by Jena et al. (2012) have associated decreased values of φPo, Ψo, and φEo with a reduction of the electron flow from PSII to PSI. Moreover, Xiang et al. (2013) have mentioned that upon
electron flow inhibition beyond QA in PSII, the quantum yield of electron transport (φEo) should decrease markedly. The current study shows that increasing the Congo red dye concentration in the culture medium results in decreased (φEo) values. The former indicates that the toxic effect of Congo red on electron transport in C. vulgaris is predominantly related to the reduction of activity in the donor side of photosystem II or to the light phase, represented by the φPo values, and to a lesser extent by the acceptor side represented by the reduction of carriers beyond QA (Ψo). Table 1 shows that the Mo values did not increase significantly with respect to the control, this confirms that there is no inhibition in electron transport between QA and QB (Ψo), and that the decrease in electron transport (φEo) is the result of the decrease in the maximum quantum efficiency φPo. It can also be seen that the values of RC/ABS decreased significantly (P o0.05) at a concentration of 10 mg L 1 of dye with respect to control. Reducing the number in active reaction centers means that less energy is used to drive electron transport, therefore, this energy must be dissipated by non-photochemical mechanisms (Perron et al., 2012). Furthermore the performance index (Table 1) decreased significantly (P o0.05). At the maximum dye concentration (25 mg L 1), the value of PIABS was reduced by 71.2 percent. Strasser et al. (2004) demonstrated that the performance index (PIABS) is the parameter that is most sensitive to the JIP-test, given that this parameter incorporates several steps in electron transport and comprises three independent components, RC/ABS, φPo, and Ψo. A decrease in PIABS values can be attributed to a change in one, two, or three parameters (Xiang et al., 2013). The assayed concentrations of Congo red dye affected the three parameters (φPo, Ψo, and RC/ABS) that are involved in the PIABS. As shown by Strasser et al. (2000), a decline in the performance index results from a disruption of energy absorption, trapping or transfer beyond the QA quinone. Aksmann and Tukaj (2008) found that PIABS reduction in cells of Chlamydomonas reinhardtii cw92 was
76
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
Fig. 4. Effect of Congo red on the electron transport rate (ETR) and non-photochemical quenching (NPQ) in Chlorella vulgaris after 96 h of dye treatment. The mean of the bars with different letters at each treatment indicated that they were significantly different at P o 0.05 according to one-way ANOVA test.
Fig. 5. Effect of Congo red on the photosynthesis (O2 evolution) and respiration (O2 consumption) in Chlorella vulgaris after 96 h of dye treatment. The mean of the bars with different letters at each treatment indicates that they were significantly different at Po 0.05 according to one-way ANOVA test.
due to low quantum efficiency of electron trapping and transport (φPo, Ψo, and φEo). Until now, no results have been reported that explain the PIABS effect of cellular exposure of microalgae to azo dyes. However, the current study shows that the parameter most sensitive to dye was RC/ABS, followed by φPo and Ψo. The electron transport rate (ETR) in vivo showed decreases up to 67.4 percent at the highest concentration tested (Fig. 4). In contrast, the decrease in the electron transport rate (ETR), resulted in an increase of non-photochemical quenching (NPQ) up to 75 percent for the maximum dye concentration (25 mg L 1) with respect to the control (Fig. 4). Müller et al. (2001) showed that this behavior is due to partial inhibition of the primary photosynthetic process, which is necessary to regulate the excitation energy captured by the antenna complex so that it can be balanced with electron transport and ATP and NADPH synthesis to reduce potential damage to PSII by a non-channeled excitation-energy excess, which can cause photo-oxidation. On the other hand, White et al. (2011) mentioned that the NPQ value is generally interpreted as the increase in thermal energy release may be caused by the operation of the xanthophylls cycle or slow reversible damages to the photosynthetic apparatus. Until now, no reports have demonstrated the effects of azo dyes on NPQ and ETR. However, we can infer that the behavior of the parameters, as demonstrated by our results, is similar to those reported by other authors for microalgae exposed to different toxic compounds, such as heavy metals and herbicides (Perales-Vela et al., 2007; Deblois et al., 2013).
assayed and that this inhibition increased as the dye concentration in the medium was increased. At the minimum and maximum concentrations of Congo red tested (5 and 25 mg L 1), the photosynthetic process was inhibited by 84 and 98 percent and the respiratory process by 76 and 96 percent, respectively, with respect to the control (P o0.05). Even when the photosynthetic and respiratory rate decreases drastically, the cells continue to grow at a relatively slow speed compared to the control (Fig. 1). C. vulgaris is a photosynthetic microalga, and its growth is highly dependent on photosynthetic performance. Any change in environmental conditions requires the development of acclimation mechanisms that adjust the energy flow from the lightharvesting complex to the Calvin cycle (Wilson et al., 2006), and the decline photosynthesis will ultimately influence growth, as been by the results in Figs. 1 and 5. Consistent with the results mentioned above, Xu et al. (2013) have suggested that photosynthesis and cell division are key factors in algae growth and that any interference or damage to photosynthesis could negatively affect growth. Other studies have shown that a malfunctioning photosynthetic apparatus inevitably diverts the light energy absorbed to other processes within the electron-transport chain and subsequently if the excited states of chlorophyll cannot be used, the energy is dissipated as fluorescence or heat by the nonphotochemical quenching mechanisms (Rocchetta and Küpper 2009), as seen by the results in Figs. 4 and 5.
4. Conclusions 3.3. Photosynthesis and respiration Fig. 5 shows that the metabolic rate (photosynthesis and respiration) was inhibited at all concentrations of Congo red
Exposure of C. vulgaris to Congo red causes a reduction in growth rate. This reduction is associated with a decrease in metabolic activity. In particularly, photosynthetic activity is
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
affected at the donor side of photosystem II, represented as the light phase (φPo). This reduction in the ability to absorb and use the quantum energy causes an increase in non-photochemical mechanisms for thermal dissipation. Acknowledgments Hernández-Zamora M., received a post-graduate scholarship from Consejo Nacional de Ciencia y Tecnología (Grant no. 204491) for her doctoral studies. References Acuner, A., Dilek, F.B., 2004. Treatment of tectilon yellow 2G by Chlorella vulgaris. Process. Biochem. 39, 623–631. Aksmann, A., Tukaj, Z., 2008. Intact anthracene inhibits photosynthesis in algal cells: a fluorescence induction study on Chlamydomonas reinhardtii cw92 strain. Chemosphere 74, 26–32. Ali, H., 2010. Biodegradation of synthetic dyes – a review. Water Air Soil Pollut. 213, 251–273. Annuar, M.S.M., Adnan, S., Vikineswary, S., Chisti, Y., 2009. Kinetics and energetics of azo dye decolorization by Pycnoporus sanguineus. Water Air Soil Pollut. 202, 179–188. Bafana, A., Krishnamurthi, K., Devi, S., Chakrabarti, T., 2008. Biological decolourization of C. I. Direct Black 38 by E. gallinarum. J. Hazard. Mater. 157 (1), 187–193. Chu, W.L., See, Y.C., Phang, S.M., 2009. Use of immobilized Chlorella vulgaris for the removal of colour from textile dyes. J. Appl. Phycol. 21, 641–648. Choudhury, N.K., Behera, R.K., 2001. Photoinhibition of photosynthesis: role of carotenoids in photoprotection of chloroplasts. Photosynthetica 39, 481–488. Czerpak, R., Piotrowska, A., 2006. Jasmonic acid affects changes in the growth and some components content in Chlorella vulgaris. Acta Physiol. Plant. 28, 195–203. Deblois, C.P., Dufresne, K., Juneau, P., 2013. Response to variable light intensity in photoacclimated algae and cyanobacteria exposed to atrazine. Aquat. Toxicol. 126, 77–84. El-Sheekh, M.M., Gharieb, M.M., Abou-El-Souod, G.W., 2009. Biodegradation of dyes by some green algae and cyanobacteria. Int. Biodeterior. Biodegrad. 63, 699–704. Ganesh, R., Boardman, G.D., Michelson, D., 1994. Fate of azo dyes in sludges. Water Res. 28, 1367–1376. Golka, K., Kopps, S., Myslak, W.Z., 2004. Carcinogenicity of azo colorants: influence of solubility and bioavailability. Toxicol. Lett. 15, 203–210. Govindjee, 1995. Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust. J. Plant Physiol. 34, 1073–1079. Hai, F.I., Yamamoto, K., Fukushi, K., 2007. Hybrid treatment systems for dye wastewater. Crit. Rev. Env. Sci. Technol. 37, 315–377. Haldimann, P., Tsimilli-Michael, M., 2005. Non photochemical quenching of chlorophyll a fluorescence by oxidized plastoquinone pool in broken spinach chloroplasts. Biochim. Biophys. Acta 1706, 239–249. Jena, S., Acharya, S., Mohapatra, P.K., 2012. Variation in effects of four OP insecticides on photosynthetic pigment fluorescence of Chlorella Vulgaris Beij. Ecotoxicol. Environ. Saf. 80, 111–117. Jin, X.C., Liu, G.Q., Xu, Z.H., Tao, W.Y., 2007. Decolorization of a dye industry effluent by Aspergillus fumigatus XC6. Appl. Microbiol. Biotechnol. 74, 239–243. Kalaji, M.H., Carpentier, R., Allakhverdiev, S.I., Bosa, Karolina, 2012. Fluorescence parameters as early indicators of light stress in Barley. J. Photochem. Photobiol. B 112, 1–6. Levy, J.L., Stauber, J.L., Jolley, D.F., 2007. Sensitivity of marine microalgae to copper: the effect of biotic factors on copper adsorption and toxicity. Sci. Total Environ. 387, 141–154. Lim, S.L., Chu, W.L., Phang, S.M., 2010. Use of Chlorella vulgaris for bioremediation of textile wastewater. Bioresour. Technol. 101 (19), 7314–7322. Marr, I.L., Suryana, N., Lukulay, P., Marr, M., 1995. Determination of chlorophyll a and b by simultaneous multi-component spectrophotometry. Fressenius J. Anal. Chem. 352, 456–460. Masojídek, J., Torzillo, G., Koblízek, M., Kopecký, J., Bernardini, P., Sacchi, A., Komenda, J., 1999. Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and xanthophylls cycle. Planta 209 (1), 126–135.
77
Maxwell, K., Johnson, G.N., 2000. Chlophyll fluorescence. A practical guide. J. Exp. Bot. 51, 659–668. Müller, P., Xiao-Ping, L.i., Niyogi, K.K., 2001. Non-photochemical quenching: a response to excess light energy. Plant Physiol. 125, 1158–1566. Muller, R., Schreiber, U., Escher, B.I., Quayle, P., Bengtson Nash, S.M., Mueller, J.F., 2008. Rapid exposure assessment of PSII herbicides in surface water using a novel chlorophyll a fluorescence imaging assay. Sci. Total Environ. 401, 51–59. Nielsen, H.D., Nielsen, S.L., 2005. Photosynthetic responses to Cu2 þ exposure are independent of light acclimation and uncoupled from growth inhibition in Fucus serratus (Phaeophyceae). Mar. Pollut. Bull. 51, 715–721. Novotny, C., Dias, N., Kapanen, A., Malachova, K., Vandrovcova, M., Itavaara, M., Lima, N., 2006. Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes. Chemosphere 63, 1436–1442. Ogawa, T., Fuj, H., Kawai, K., Yatome, C., Idaka, E., 1989. Growth inhibition of Bacillus subtilis upon interaction between basic dyes and DNA. Bull. Environ. Contam. Toxicol. 42, 402–408. Ogawa, T., Shibata, M., Yatome, C., Idaka, E., 1988. Growth inhibition of Bacillus subtilis by basic dyes. Bull. Environ. Contam. Toxicol. 40, 545–552. Pearce, C.I., Lloyd, J.R., Guthrie, J.T., 2003. The removal of color from textile wastewater using whole bacterial cells: a review. Dye Pigment 58, 179–196. Perales-Vela, H.V., González-Moreno, S., Montes-Horcasitas, C., CañizaresVillanueva, R.O., 2007. Growth, photosynthetic and respiratory responses to sub-lethal copper concentrations in Scenedesmus incrassatulus (Chlorophyceae). Chemosphere 67, 2274–2281. Perron, M.C., Juneau, P., 2011. Effect of endocrine disrupters on photosystem II energy fluxes of green algae and cyanobacteria. Environ. Res. 111, 520–529. Perron, M.C., Qiu, B., Boucher, N., Bellemare, F., Juneau, P., 2012. Use of chloropyll a fluoresecence to detect the effect of microcystins on photosynthesis and photosystem II energy fluxes of green algae. Toxicon 59, 567–577. Qian, H.F., Chen, W., Sheng, G.D., Xu, X.Y., Liu, W.P., Fu, Z.W., 2008. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris. Aquat. Toxicol. 88, 301–307. Rocchetta, I., Küpper, H., 2009. Chromium- and copper-induced inhibition of photosynthesis in Euglena gracilis analyzed on the single-cell level by fluorescence kinetic microscopy. New Phytol. 182, 405–420. Saratale, R.G., Saratale, G.D., Chang, J.S., Govindwar, S.P., 2011. Bacterial decolorization and degradation of azo dyes: a review. J. Taiwan Inst. Chem. Eng. 42, 138–157. Stein, J.R., 1973. Handbook of Phycological Methods. Culture Methods and Growth Measurements. Cambridge University Press, London, pp. 7–24. Strasser, R.J., Srivastava, A., Govindjee, A., 1995. Polyphasic chlorophyll a fluorescence transient in plant and cyanobacteria. J. Photochem. Photobiol. 61, 32–42. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty, P. (Eds.), Probing Photosynthesis Mechanisms Regulation and Adaptation. Taylor and Francis, London–New York, pp. 445–483. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2004. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll Fluorescence a Signature of Photosynthesis. Advances in Photosynthesis and Respiration Series. Kluwer Academic Publisher, Dordrecht, pp. 321–362. van Heerden, P.D.R., Tsimilli-Michael, M., Krüger, G.H.J., Strasser, R.J., 2003. Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiol. Plant. 117, 476–491. Wellburn, A.R., 1994. The spectral determination of chlorophyll a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144, 307–313. Wen, Y.Z., Chen, H., Shen, C.S., Zhao, M.R., Liu, W.P., 2011. Enantioselectivity tuning of chiral herbicide dichlorprop by copper: roles of reactive oxygen species. Environ. Sci. Technol. 45, 4778–4784. White, S., Anandraj, A., Bux, F., 2011. PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids. Bioresour. Technol. 102, 1675–1682. Wilson, K.E., Ivanov, A.G., Oquist, G., Grodzinski, B., Sarhan, F., Huner, N.P.A., 2006. Energy balance, organellar redox status and acclimation to environmental stress. Can. J. Bot. 84, 1335–1370. Xia, J., Tian, Q., 2009. Early stage of excess copper to photosystem II of Chlorella pyrenoidosa-OJIP chlorophyll a fluorescence analysis. J. Environ. Sci. 21 (11), 1569–1574. Xiang, M., Chen, S., Wang, L., Dong, Z., Huang, J., Zhang, Y., 2013. Effect of vulculic acid produced by Nimbya alternantherae on the photosynthetic apparatus of Alternanthera philoxeroides. Plant Physiol. Biochem. 65, 81–88. Xu, D., Li, C., Chen, H., Shao, B., 2013. Cellular response of fresh water green algae to perfluorooctanoic acid toxicity. Ecotoxicol. Environ. Saf. 88, 103–107.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Physiological and biochemical responses of Chlorella vulgaris to Congo red Miriam Hernández-Zamora a, Hugo Virgilio Perales-Vela b, César Mateo Flores-Ortíz c, Rosa Olivia Cañizares-Villanueva a,n a Laboratorio de Biotecnología de Microalgas, Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. IPN 2508, San Pedro Zacatenco, C.P. 07360 México DF, México b Laboratorio de Bioquímica, Unidad de Morfología y Función, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala, Av. de los Barrios #1, Estado de México, México c Laboratorio de Biogeoquímica, Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala, Av. de los Barrios #1, Estado de México, México
art ic l e i nf o
a b s t r a c t
Article history: Received 28 February 2014 Received in revised form 27 May 2014 Accepted 28 May 2014
Extensive use of synthetic dyes in many industrial applications releases large volumes of wastewater. Wastewaters from dying industries are considered hazardous and require careful treatment prior to discharge into receiving water bodies. Dyes can affect photosynthetic activities of aquatic flora and decrease dissolved oxygen in water. The aim of this study was to evaluate the effect of Congo red on growth and metabolic activity of Chlorella vulgaris after 96 h exposure. Exposure of the microalga to Congo red reduced growth rate, photosynthesis and respiration. Analysis of chlorophyll a fluorescence emission showed that the donor side of photosystem II was affected at high concentrations of Congo red. The quantum yield for electron transport (φEo), the electron transport rate (ETR) and the performance index (PI) also decreased. The reduction in the ability to absorb and use the quantum energy increased non-photochemical (NPQ) mechanisms for thermal dissipation. Overall, Congo red affects growth and metabolic activity in photosynthetic organisms in aquatic environments. & 2014 Elsevier Inc. All rights reserved.
Keywords: Chlorella vulgaris Chlorophyll a fluorescence Congo red JIP-test Photosynthesis Respiration
1. Introduction Azo compounds constitute the largest and most diverse group of synthetic dyes and are widely used by the textile, food, cosmetic, and pulp and paper industries (El-Sheekh et al., 2009). At the global level, 280,000 t of textile dyes are discharged into industrial effluent each year (Jin et al., 2007). It is estimated that the quantity of dye that does not fix to textile fibers depends on the application method, varying from 2 percent when basic dyes are used to 50 percent when reactive dyes are used (Hai et al., 2007; Pearce et al., 2003). The discharge of azo dyes into the environment is a serious problem and should be avoided not only for esthetic reasons but also because of the threat it poses to public health and ecosystems (Golka et al., 2004). In aquatic environments, the colors produced by azo dyes impede plant development by decreasing the passage of light, thereby affecting the
n
Corresponding author. Fax: þ52 55 50613313. E-mail addresses: [email protected] (M. Hernández-Zamora), [email protected] (H.V. Perales-Vela), cmfl[email protected] (C.M. Flores-Ortíz), [email protected] (R.O. Cañizares-Villanueva). http://dx.doi.org/10.1016/j.ecoenv.2014.05.030 0147-6513/& 2014 Elsevier Inc. All rights reserved.
photosynthetic process (Annuar et al., 2009). The resulting changes in the concentration of dissolved O2 damage the aquatic biota present and increase the biochemical oxygen demand of the surrounding water (Ali, 2010). In addition, some azo dyes, such as Congo red, Direct Blue 15 and Direct Red 2, have been reported to be toxic, mutagenic, and carcinogenic to aquatic life (Bafana et al., 2008; Golka et al., 2004; Saratale et al., 2011). In aquatic ecosystems the green algae are the primary producers and are key indicator organisms (Wen et al., 2011) when used to evaluate water quality and the ecotoxicity of contaminants (Xu et al., 2013), such as metals, herbicides, insecticides, and other xenobiotic compounds (Jena et al., 2012; Levy et al., 2007; Qian et al., 2008). On the other hand, the chlorophyll a fluorescence kinetic has been used to indicate alterations of the photosynthetic capacity when damage is induced by pollutants or by environmental conditions (Jena et al., 2012; Kalaji et al., 2012). The advantage of such a method is that it is non-invasive and reliable (Muller et al., 2008; Xia and Tian 2009). Furthermore, studies have shown that changes in photosystem II (PSII) can be quantified using the JIP-test (Jena et al., 2012; Strasser et al., 2000). However, as of yet, no information is available regarding the impact of azo dyes on the photosynthetic apparatus of
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
algae. Such information is important because it would permit us to determine the toxicity to the aquatic environment caused by this type of dye. For this reason, the objective of the current study is to evaluate the toxic effects of the azo dye Congo red on the growth and photosynthetic metabolism of the microalga Chlorella vulgaris.
2. Materials and methods 2.1. Dye used Congo red (Sigma-Aldrich), also called Direct Red 28, was used for the experiments described herein. Its molecular form is C32H22N6O6S2Na2, and its molecular weight is 696.7 g mol 1. All experiments employed a 100 mg L 1 stock solution of dye, which, prior to its addition in each experimental unit, was sterilized by filtration using Millipores membranes of 0.22 μm pore diameter. 2.2. Experimental units Experiments were conducted under sterile conditions with axenic cultures of the green microalga C. vulgaris donated by the Laboratorio de Hidrobiología Experimental of the Escuela Nacional de Ciencias Biológicas of the Instituto Politécnico Nacional. The strain was collected and isolated from temporary ponds in the State of Mexico (Atlacomulco). The registration number is LHE-Chl 01. The experimental culture units were glass bottles with flat surfaces, each of which had a total capacity of 0.5 L and a working volume of 0.25 L, that were inoculated with 15 ml of C. vulgaris culture in the exponential phase (11 mg L 1 dry biomass) Bold's Basal mineral medium (Stein, 1973) and different concentrations of dye (5, 10, 15, 20, and 25 mg L 1 of Congo red), the control used contained only the inoculum and the culture medium. The culture conditions were as follows: temperature, 257 3 1C; illumination, 120 μmoles photons m 2 s 1; photoperiod, 12/12 h (light/darkness); and air supply, 200 ml min 1.
73
2. The probability that a trapped exciton moves an electron into the electron transport chain beyond QA ; Ψo ¼ 1 Vj 3. The quantum yield of electron transport; φEo ¼ [1 (Fo/Fm) (1 Vj)]¼ (φPo) (Ψo) 4. Reaction centers per excited cross section; (RC/ABS) ¼ [(Mo/Vj) (1 (Fo/Fm))] 5. The performance index; PIABS ¼ (RC/ABS) [φPo/(1 φPo)] [Ψo/(1 Ψo)] Data were interpreted using the Handy-PEA software developed by Hansatech Instruments Ltd., U.K., and Biolyzer-HP3 software designed at the Bioenergetics Laboratory of the University of Geneva, Switzerland (van Heerden et al., 2003). Modulated fluorescence was measured using the fluorescence modulated system (FMS 2 Hansatech Instruments Ltd., Norfolk, UK) in accordance with Nielsen and Nielsen (2005) and Masojídek et al. (1999). All measurements were obtained at room temperature (25 1C) and in dark conditions. Prior to examining Chl-a fluorescence, samples of each experimental unit were adjusted to the same optical-density value (0.3 680 nm) and these were washed using Bold´s Basal mineral medium to remove adsorbed dye completely. Each cellular suspension was vacuum-filtered using Millipores membranes of 5.0-μm pore diameter and were later adapted to darkness for 5 min. Signatures for Chl-a fluorescence were detected directly from the filter surface (Perales-Vela et al., 2007). The value of Fo at room temperature was obtained by irradiating the sample with a modulated light of low intensity (0.1 μmol m 2 s 1) and after 10 s the sample was superimposed with one saturating pulse of white light of 10,000 μmol m 2 s 1 for 0.8 s, to close all reaction centers and obtain the maximum fluorescence value of Fm in darkness. Immediately after the saturating pulse, the samples were irradiated with a non-saturating actinic white light 200 μmol m 2 s 1 for 5 min to achieve a steady state and then these were superimposed a saturating white light pulse of 10,000 μmol m 2 s 1 for 0.8 s to obtain the value of Fm0 . Finally the values were calculated as follows: 1. Relative electron transport rate (rETR) ¼ [((Fm0 Fs)/Fm0 )] (0.5)(PAR). The value of 0.5 corresponds to the proportion of light that is transferred to each of the two photosystems (PSII and PSI) and the photosynthetically active radiation (PAR) utilized was of 162 μmol m 2 s 1. 2. Non-photochemical quenching (NPQ)¼ [(Fm Fm0 )/Fm0 ].
2.3. Growth and photosynthetic pigments Growth was determined using the dry-weight values obtained at the beginning (0 h) and at the end (96 h) of the experiment. Each treatment used 5-ml aliquots that were vacuum-filtered using Millipores membranes of 5.0-μm pore diameter, pre-treated for a constant weight. Later, the samples were dried at 80 1C for 48 h. Growth rate of algal culture is a measure of the increase in biomass over time and it was determined by the following equation: K ¼ In ðN 2 =N 1 Þ=ðT 2 T 1 Þ where, K is growth rate, N1 and N2, biomass content at time (T1) and the time (T2), respectively chlorophyll a and b and total carotenoids were determined using 3 ml of each sample, after the sample was centrifuged for 10 min. Subsequently, the pellet was added 2 ml of methanol and stirred for 1 min. Previously the pellet was washed using Bold´s Basal mineral medium to remove adsorbed dye completely. This procedure was done to prevent the absorption spectrum of Congo red which interferes with the absorption spectrum of photosynthetic pigments, especially carotenoids. The sample was kept in water bath at 60 1C for 10 min. Again, each sample was centrifuged at room temperature. The supernatant was separated and the concentrations of Chl-a, Chl-b, and total carotenoids were determined with a UV–Vis spectrophotometer (Genesys 10UV Thermo Electron Corporation) according to the equations of Wellburn (1994).
2.5. Measurement of photosynthesis and respiration Measurements were made using an oximeter (Oxylab-Hansatech, UK) at a controlled temperature of 30 1C in exponentially growing cultures. Previously, the cells were washed using Bold´s Basal mineral medium to remove adsorbed dye completely. The samples of each experimental unit were adjusted to the same optical-density value (0.3 680 nm) before the photosynthesis and respiration study. The oxygen release rate (photosynthesis) was obtained by illuminating each of the samples with an actinic red light of 400 μmol m-2 s-1 for 1 min. Immediately after turning off the light source, the oxygen consumption rate (respiration) was recorded for 2 min. 2.6. Statistical analysis All experiments were performed in triplicate, and the results obtained were statistically analyzed via one-way analysis of variance (P o 0.05) and Tukey's test for comparison of means using the statistical package Sigma-Plot (version 11.0).
3. Results and discussion 2.4. Chlorophyll fluorescence For all treatments, the Chl-a fluorescence emitted via photosystem II was measured at 96 h in samples conditioned in the dark using a portable fluorometer, model HANDY-PEA (Hansatech Instruments Ltd., Norfolk, UK) coupled to the chamber for liquid phase HPEA/LPA (Hansatech, UK). For each of the experimental units, samples were corrected to the same optical density (0.3 680 nm) and these were washed using Bold´s Basal mineral medium to remove adsorbed dye completely. After, 2 ml samples of each treatment (n ¼4) were taken and incubated at dark for 20 min at room temperature (25 1C), then the samples were irradiated for 1 s with red light (660 nm) saturating (3000 μmol photons m 2 s 1). The equipment automatically recorded the following values: (i) minimal fluorescence (Fo) at 50 μs, (ii) maximum fluorescence (Fm) between 200 and 500 ms, (iii) variable fluorescence at 2 ms (Vj ¼ Fj Fo/Fm Fo) and (iv) the slope at the beginning of fluorescence Mo ¼ 4 (FK Fo)/(FM Fo). These data were used to calculate the following parameters (Strasser et al., 2004): 1. The maximum quantum yield of primary photochemistry; Fv/Fm
φPo ¼ [1 (Fo/Fm)] ¼
3.1. Effect of Congo red on growth The microalga C. vulgaris was affected after 96 h of exposure to Congo red. A decrease in growth rate (μ) was observed in every experimental unit after increasing the concentration of dye in the culture medium; this decrease was significant (Po 0.05) for concentration over 15 mg L 1 compared to the control (Fig. 1). The μ value was 0.657 day 1 for cells with no dye, while the values of 0.538 and 0.391 day 1 were obtained when cells were exposed to 5 and 10 mg L 1 of dye, respectively. However, the μ values remained at 0.201 day 1 when cells were exposed to dye concentrations between 15 and 25 mg L 1. Chu et al. (2009) reported μ values of 0.06–0.35 day 1 in cells of C. vulgaris exposed separately to 30 mg L 1 of Supranol Red 3BW, Lanaset Red 2GA, and Levafix Navy Blue EBNA. The cultures grown in textile dyes
74
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
Fig. 1. Effect of Congo red on growth rate μ (day 1) in Chlorella vulgaris following dye exposure for 96 h. The mean of the bars with different letters at each treatment indicated that they were significantly different at P o0.05 according to one-way ANOVA test.
Fig. 2. Effect of Congo red on chlorophyll (aþ b) and carotenoids content in Chlorella vulgaris after 96 h of dye treatment. The mean of the bars with different letters at each treatment indicated that they were significantly different at Po 0.05 according to one-way ANOVA test.
attained significantly lower μ (Po 0.05) than those grown without dye (control). Lim et al. (2010) obtained μ values of 0.34 day 1 in C. vulgaris cells exposed to Supranol Red 3BM and 0.05 day 1 when residual textile water was used. These values were lower than the control (μ 0.40 day 1). There have been very few attempts to explain what causes cellular growth inhibition resulting from exposure to azo dyes; however there are reports of high tolerance and susceptibility in algae. For example, Acuner and Dilek (2004) showed that C. vulgaris can grow without damage in the presence of 400 mg L 1 of Tectilon Yellow 2G. Furthermore, other algae like Pseudokirchneriella subcapitata (Selenastrum capricornutum) could only grow in 0.5 mg L 1 of Disperse Blue 3 (Novotny et al., 2006). The inhibitory effect of textile dyes was reported by Ogawa et al. (1989). They found that inhibition was caused by intercalation of dye compounds between DNA base pairs, so preventing enzymatic activity and cell replication (Ogawa et al., 1988). Ganesh et al. (1994) noted inhibition of biomass in a wastewater system treating with a reactive dye and suggested that this was caused by the products of dye degradation rather the dye itself. Other studies have demonstrated that one of the possible causes of the observed growth inhibition is related to the alteration of photosynthesis, especially at the PSII level (Perron and Juneau, 2011).
3.1.1. Photosynthetic pigment content In plants and algae, photosynthetic pigments (Chl-a, Chl-b, and carotenoids) are important as an indirect measure of photosynthetic function (Marr et al., 1995). Fig. 2 shows the effect of Congo
red dye on content of photosynthetic pigments. It can be seen that the total chlorophyll (aþ b) content decreases significantly with respect to the control as the dye concentration is increased. Acuner and Dilek (2004) have reported similar results with C. vulgaris and the dye Tectilon Yellow G. This reduction in chlorophyll content can affect the conversion of light energy and, thus, photosynthetic electron transport (Strasser et al., 2004). Furthermore, as seen in Fig. 2, the total carotenoid content was not significantly altered (P o0.05), which can indicate that the dye affects each photosynthetic pigment differently. Carotenoids are important because they are involved in the removal of the oxygen free radicals which are generated when the metabolism is inhibited, as well as being important in the regulation of the light excitation energy in the reaction center of photosystem II (Choudhury and Behera, 2001; Czerpak and Piotrowska, 2006). 3.2. Chlorophyll-a fluorescence An increase in the yield of chlorophyll fluorescence from the dark into the light over a time period of around 1 s, it has been explained as a consequence of reduction of electron acceptors in the photosynthetic pathway, downstream of PSII, notably plastoquinone and in particular, QA. Once PSII absorbs light and QA has accepted an electron, it is not able to accept another until it has passed the first onto a subsequent electron carrier (QB). During this period, the reaction center is said to be “closed”. At any point in time, the presence of a proportion of closed reaction centers leads to an overall reduction in the efficiency of photochemistry and so to corresponding increase in the yield of fluorescence (Maxwell and Johnson, 2000). The polyphasic chlorophyll a fluorescence kinetics was modified as the dye increased in the culture medium,
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
75
Fig. 3. Effect of Congo red on chlorophyll a fluorescence transients in Chlorella vulgaris after 96 h of dye treatment.
Table 1 Experimental expressions of the JIP-test and their calculated values obtained for Chlorella vulgaris cell subjected for 96 h to Congo red. The maximum quantum yield of primary photochemistry (φPo); the probability that a trapped exciton moves an electron into the electron transport chain beyond QA (Ψo); the quantum yield of electron transport (φEo); the initial slope at the beginning of the variable fluorescence (Mo); the total number of active reaction center per absorption (RC/ABS) and performance index (PIABS) in Chlorella vulgaris after 96 h of dye treatment. Congo red (mg L 1)
φPo
Ψo
φEo
RC/ABS
Mo
PI
0 5 10 15 20 25
0.7087 0.004a 0.7157 0.003a 0.626 7 0.005b 0.4917 0.013c 0.4997 0.006c 0.5167 0.007d
0.563 7 0.016a 0.5687 0.018a 0.542 7 0.006b 0.508 7 0.015c 0.5447 0.007b 0.540 7 0.014b
0.3777 0.011a 0.384 7 0.013a 0.3177 0.004b 0.234 7 0.012c 0.2517 0.003d 0.256 7 0.009d
0.3327 0.005a 0.343 7 0.008a 0.281 7 0.011b 0.1927 0.008c 0.2167 0.011d 0.2247 0.023d
0.996 7 0.002a 0.9977 0.003a 1.0007 0.002a 0.993 7 0.014a 1.0007 0.001a 1.0007 0.001a
0.378 70.018a 0.404 70.022b 0.215 70.005c 0.083 70.007d 0.101 70.004e 0.109 70.008f
The parameters with different letters at each treatment indicated that they were significantly different at Po 0.05 according to one-way ANOVA test.
the maximal fluorescence particularly (FM) was reduced (Fig. 3). Some authors have attributed the decrease in the maximal fluorescence (inflection P) to deactivation or reduced fluorescence emissions, due to the presence in the “pool” of plastoquinones (PQ) that are oxidized (Haldimann and Tsimilli-Michael, 2005). On the other hand, Govindjee (1995) and Perales-Vela et al. (2007) have suggested that a decrease in the phases J and I illuminates how electron-transport inhibition on the donor side of PSII results in the accumulation of P680 þ and can reduce fluorescence emission. The results in the photochemical activity determined by the fluorescence emission, shows that the dye decreased significantly (P o0.05) the maximum quantum yield of primary photochemistry (φPo), the probability that a trapped exciton moves an electron into the electron transport chain beyond (Ψo), and the quantum yield of the electron transport (φEo). Corresponding values are shown in Table 1. At the maximum dye concentration (25 mg L 1) the values of φPo, Ψo, and φEo decrease by 27.11, 4.08, and 32.09 percent, respectively, compared to the control. The maximum quantum yield of primary photochemistry (φPo) uniquely reflects the efficiency of light-phase reactions (Strasser et al., 2004). It has been reported that when a decrease in φPo occurs, it is due to destruction or modification of the photosystem, which is to say that the reaction center of PSII does not have its normal quantum efficiency. The reduction in φPo found in this investigation is the result of the decrease in the values of the minimum (Fo) and maximum (FM) fluorescence. The decrease in the values of parameters Ψo indicate that the yield of the electron transport per a trapped exciton in treated cells with Congo red (10–25 mg L 1) is lower than in the control and that the energy needed to close all reaction centers decreases (Strasser et al., 1995). Studies conducted by Jena et al. (2012) have associated decreased values of φPo, Ψo, and φEo with a reduction of the electron flow from PSII to PSI. Moreover, Xiang et al. (2013) have mentioned that upon
electron flow inhibition beyond QA in PSII, the quantum yield of electron transport (φEo) should decrease markedly. The current study shows that increasing the Congo red dye concentration in the culture medium results in decreased (φEo) values. The former indicates that the toxic effect of Congo red on electron transport in C. vulgaris is predominantly related to the reduction of activity in the donor side of photosystem II or to the light phase, represented by the φPo values, and to a lesser extent by the acceptor side represented by the reduction of carriers beyond QA (Ψo). Table 1 shows that the Mo values did not increase significantly with respect to the control, this confirms that there is no inhibition in electron transport between QA and QB (Ψo), and that the decrease in electron transport (φEo) is the result of the decrease in the maximum quantum efficiency φPo. It can also be seen that the values of RC/ABS decreased significantly (P o0.05) at a concentration of 10 mg L 1 of dye with respect to control. Reducing the number in active reaction centers means that less energy is used to drive electron transport, therefore, this energy must be dissipated by non-photochemical mechanisms (Perron et al., 2012). Furthermore the performance index (Table 1) decreased significantly (P o0.05). At the maximum dye concentration (25 mg L 1), the value of PIABS was reduced by 71.2 percent. Strasser et al. (2004) demonstrated that the performance index (PIABS) is the parameter that is most sensitive to the JIP-test, given that this parameter incorporates several steps in electron transport and comprises three independent components, RC/ABS, φPo, and Ψo. A decrease in PIABS values can be attributed to a change in one, two, or three parameters (Xiang et al., 2013). The assayed concentrations of Congo red dye affected the three parameters (φPo, Ψo, and RC/ABS) that are involved in the PIABS. As shown by Strasser et al. (2000), a decline in the performance index results from a disruption of energy absorption, trapping or transfer beyond the QA quinone. Aksmann and Tukaj (2008) found that PIABS reduction in cells of Chlamydomonas reinhardtii cw92 was
76
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
Fig. 4. Effect of Congo red on the electron transport rate (ETR) and non-photochemical quenching (NPQ) in Chlorella vulgaris after 96 h of dye treatment. The mean of the bars with different letters at each treatment indicated that they were significantly different at P o 0.05 according to one-way ANOVA test.
Fig. 5. Effect of Congo red on the photosynthesis (O2 evolution) and respiration (O2 consumption) in Chlorella vulgaris after 96 h of dye treatment. The mean of the bars with different letters at each treatment indicates that they were significantly different at Po 0.05 according to one-way ANOVA test.
due to low quantum efficiency of electron trapping and transport (φPo, Ψo, and φEo). Until now, no results have been reported that explain the PIABS effect of cellular exposure of microalgae to azo dyes. However, the current study shows that the parameter most sensitive to dye was RC/ABS, followed by φPo and Ψo. The electron transport rate (ETR) in vivo showed decreases up to 67.4 percent at the highest concentration tested (Fig. 4). In contrast, the decrease in the electron transport rate (ETR), resulted in an increase of non-photochemical quenching (NPQ) up to 75 percent for the maximum dye concentration (25 mg L 1) with respect to the control (Fig. 4). Müller et al. (2001) showed that this behavior is due to partial inhibition of the primary photosynthetic process, which is necessary to regulate the excitation energy captured by the antenna complex so that it can be balanced with electron transport and ATP and NADPH synthesis to reduce potential damage to PSII by a non-channeled excitation-energy excess, which can cause photo-oxidation. On the other hand, White et al. (2011) mentioned that the NPQ value is generally interpreted as the increase in thermal energy release may be caused by the operation of the xanthophylls cycle or slow reversible damages to the photosynthetic apparatus. Until now, no reports have demonstrated the effects of azo dyes on NPQ and ETR. However, we can infer that the behavior of the parameters, as demonstrated by our results, is similar to those reported by other authors for microalgae exposed to different toxic compounds, such as heavy metals and herbicides (Perales-Vela et al., 2007; Deblois et al., 2013).
assayed and that this inhibition increased as the dye concentration in the medium was increased. At the minimum and maximum concentrations of Congo red tested (5 and 25 mg L 1), the photosynthetic process was inhibited by 84 and 98 percent and the respiratory process by 76 and 96 percent, respectively, with respect to the control (P o0.05). Even when the photosynthetic and respiratory rate decreases drastically, the cells continue to grow at a relatively slow speed compared to the control (Fig. 1). C. vulgaris is a photosynthetic microalga, and its growth is highly dependent on photosynthetic performance. Any change in environmental conditions requires the development of acclimation mechanisms that adjust the energy flow from the lightharvesting complex to the Calvin cycle (Wilson et al., 2006), and the decline photosynthesis will ultimately influence growth, as been by the results in Figs. 1 and 5. Consistent with the results mentioned above, Xu et al. (2013) have suggested that photosynthesis and cell division are key factors in algae growth and that any interference or damage to photosynthesis could negatively affect growth. Other studies have shown that a malfunctioning photosynthetic apparatus inevitably diverts the light energy absorbed to other processes within the electron-transport chain and subsequently if the excited states of chlorophyll cannot be used, the energy is dissipated as fluorescence or heat by the nonphotochemical quenching mechanisms (Rocchetta and Küpper 2009), as seen by the results in Figs. 4 and 5.
4. Conclusions 3.3. Photosynthesis and respiration Fig. 5 shows that the metabolic rate (photosynthesis and respiration) was inhibited at all concentrations of Congo red
Exposure of C. vulgaris to Congo red causes a reduction in growth rate. This reduction is associated with a decrease in metabolic activity. In particularly, photosynthetic activity is
M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77
affected at the donor side of photosystem II, represented as the light phase (φPo). This reduction in the ability to absorb and use the quantum energy causes an increase in non-photochemical mechanisms for thermal dissipation. Acknowledgments Hernández-Zamora M., received a post-graduate scholarship from Consejo Nacional de Ciencia y Tecnología (Grant no. 204491) for her doctoral studies. References Acuner, A., Dilek, F.B., 2004. Treatment of tectilon yellow 2G by Chlorella vulgaris. Process. Biochem. 39, 623–631. Aksmann, A., Tukaj, Z., 2008. Intact anthracene inhibits photosynthesis in algal cells: a fluorescence induction study on Chlamydomonas reinhardtii cw92 strain. Chemosphere 74, 26–32. Ali, H., 2010. Biodegradation of synthetic dyes – a review. Water Air Soil Pollut. 213, 251–273. Annuar, M.S.M., Adnan, S., Vikineswary, S., Chisti, Y., 2009. Kinetics and energetics of azo dye decolorization by Pycnoporus sanguineus. Water Air Soil Pollut. 202, 179–188. Bafana, A., Krishnamurthi, K., Devi, S., Chakrabarti, T., 2008. Biological decolourization of C. I. Direct Black 38 by E. gallinarum. J. Hazard. Mater. 157 (1), 187–193. Chu, W.L., See, Y.C., Phang, S.M., 2009. Use of immobilized Chlorella vulgaris for the removal of colour from textile dyes. J. Appl. Phycol. 21, 641–648. Choudhury, N.K., Behera, R.K., 2001. Photoinhibition of photosynthesis: role of carotenoids in photoprotection of chloroplasts. Photosynthetica 39, 481–488. Czerpak, R., Piotrowska, A., 2006. Jasmonic acid affects changes in the growth and some components content in Chlorella vulgaris. Acta Physiol. Plant. 28, 195–203. Deblois, C.P., Dufresne, K., Juneau, P., 2013. Response to variable light intensity in photoacclimated algae and cyanobacteria exposed to atrazine. Aquat. Toxicol. 126, 77–84. El-Sheekh, M.M., Gharieb, M.M., Abou-El-Souod, G.W., 2009. Biodegradation of dyes by some green algae and cyanobacteria. Int. Biodeterior. Biodegrad. 63, 699–704. Ganesh, R., Boardman, G.D., Michelson, D., 1994. Fate of azo dyes in sludges. Water Res. 28, 1367–1376. Golka, K., Kopps, S., Myslak, W.Z., 2004. Carcinogenicity of azo colorants: influence of solubility and bioavailability. Toxicol. Lett. 15, 203–210. Govindjee, 1995. Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust. J. Plant Physiol. 34, 1073–1079. Hai, F.I., Yamamoto, K., Fukushi, K., 2007. Hybrid treatment systems for dye wastewater. Crit. Rev. Env. Sci. Technol. 37, 315–377. Haldimann, P., Tsimilli-Michael, M., 2005. Non photochemical quenching of chlorophyll a fluorescence by oxidized plastoquinone pool in broken spinach chloroplasts. Biochim. Biophys. Acta 1706, 239–249. Jena, S., Acharya, S., Mohapatra, P.K., 2012. Variation in effects of four OP insecticides on photosynthetic pigment fluorescence of Chlorella Vulgaris Beij. Ecotoxicol. Environ. Saf. 80, 111–117. Jin, X.C., Liu, G.Q., Xu, Z.H., Tao, W.Y., 2007. Decolorization of a dye industry effluent by Aspergillus fumigatus XC6. Appl. Microbiol. Biotechnol. 74, 239–243. Kalaji, M.H., Carpentier, R., Allakhverdiev, S.I., Bosa, Karolina, 2012. Fluorescence parameters as early indicators of light stress in Barley. J. Photochem. Photobiol. B 112, 1–6. Levy, J.L., Stauber, J.L., Jolley, D.F., 2007. Sensitivity of marine microalgae to copper: the effect of biotic factors on copper adsorption and toxicity. Sci. Total Environ. 387, 141–154. Lim, S.L., Chu, W.L., Phang, S.M., 2010. Use of Chlorella vulgaris for bioremediation of textile wastewater. Bioresour. Technol. 101 (19), 7314–7322. Marr, I.L., Suryana, N., Lukulay, P., Marr, M., 1995. Determination of chlorophyll a and b by simultaneous multi-component spectrophotometry. Fressenius J. Anal. Chem. 352, 456–460. Masojídek, J., Torzillo, G., Koblízek, M., Kopecký, J., Bernardini, P., Sacchi, A., Komenda, J., 1999. Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and xanthophylls cycle. Planta 209 (1), 126–135.
77
Maxwell, K., Johnson, G.N., 2000. Chlophyll fluorescence. A practical guide. J. Exp. Bot. 51, 659–668. Müller, P., Xiao-Ping, L.i., Niyogi, K.K., 2001. Non-photochemical quenching: a response to excess light energy. Plant Physiol. 125, 1158–1566. Muller, R., Schreiber, U., Escher, B.I., Quayle, P., Bengtson Nash, S.M., Mueller, J.F., 2008. Rapid exposure assessment of PSII herbicides in surface water using a novel chlorophyll a fluorescence imaging assay. Sci. Total Environ. 401, 51–59. Nielsen, H.D., Nielsen, S.L., 2005. Photosynthetic responses to Cu2 þ exposure are independent of light acclimation and uncoupled from growth inhibition in Fucus serratus (Phaeophyceae). Mar. Pollut. Bull. 51, 715–721. Novotny, C., Dias, N., Kapanen, A., Malachova, K., Vandrovcova, M., Itavaara, M., Lima, N., 2006. Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes. Chemosphere 63, 1436–1442. Ogawa, T., Fuj, H., Kawai, K., Yatome, C., Idaka, E., 1989. Growth inhibition of Bacillus subtilis upon interaction between basic dyes and DNA. Bull. Environ. Contam. Toxicol. 42, 402–408. Ogawa, T., Shibata, M., Yatome, C., Idaka, E., 1988. Growth inhibition of Bacillus subtilis by basic dyes. Bull. Environ. Contam. Toxicol. 40, 545–552. Pearce, C.I., Lloyd, J.R., Guthrie, J.T., 2003. The removal of color from textile wastewater using whole bacterial cells: a review. Dye Pigment 58, 179–196. Perales-Vela, H.V., González-Moreno, S., Montes-Horcasitas, C., CañizaresVillanueva, R.O., 2007. Growth, photosynthetic and respiratory responses to sub-lethal copper concentrations in Scenedesmus incrassatulus (Chlorophyceae). Chemosphere 67, 2274–2281. Perron, M.C., Juneau, P., 2011. Effect of endocrine disrupters on photosystem II energy fluxes of green algae and cyanobacteria. Environ. Res. 111, 520–529. Perron, M.C., Qiu, B., Boucher, N., Bellemare, F., Juneau, P., 2012. Use of chloropyll a fluoresecence to detect the effect of microcystins on photosynthesis and photosystem II energy fluxes of green algae. Toxicon 59, 567–577. Qian, H.F., Chen, W., Sheng, G.D., Xu, X.Y., Liu, W.P., Fu, Z.W., 2008. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris. Aquat. Toxicol. 88, 301–307. Rocchetta, I., Küpper, H., 2009. Chromium- and copper-induced inhibition of photosynthesis in Euglena gracilis analyzed on the single-cell level by fluorescence kinetic microscopy. New Phytol. 182, 405–420. Saratale, R.G., Saratale, G.D., Chang, J.S., Govindwar, S.P., 2011. Bacterial decolorization and degradation of azo dyes: a review. J. Taiwan Inst. Chem. Eng. 42, 138–157. Stein, J.R., 1973. Handbook of Phycological Methods. Culture Methods and Growth Measurements. Cambridge University Press, London, pp. 7–24. Strasser, R.J., Srivastava, A., Govindjee, A., 1995. Polyphasic chlorophyll a fluorescence transient in plant and cyanobacteria. J. Photochem. Photobiol. 61, 32–42. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty, P. (Eds.), Probing Photosynthesis Mechanisms Regulation and Adaptation. Taylor and Francis, London–New York, pp. 445–483. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2004. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll Fluorescence a Signature of Photosynthesis. Advances in Photosynthesis and Respiration Series. Kluwer Academic Publisher, Dordrecht, pp. 321–362. van Heerden, P.D.R., Tsimilli-Michael, M., Krüger, G.H.J., Strasser, R.J., 2003. Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiol. Plant. 117, 476–491. Wellburn, A.R., 1994. The spectral determination of chlorophyll a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144, 307–313. Wen, Y.Z., Chen, H., Shen, C.S., Zhao, M.R., Liu, W.P., 2011. Enantioselectivity tuning of chiral herbicide dichlorprop by copper: roles of reactive oxygen species. Environ. Sci. Technol. 45, 4778–4784. White, S., Anandraj, A., Bux, F., 2011. PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids. Bioresour. Technol. 102, 1675–1682. Wilson, K.E., Ivanov, A.G., Oquist, G., Grodzinski, B., Sarhan, F., Huner, N.P.A., 2006. Energy balance, organellar redox status and acclimation to environmental stress. Can. J. Bot. 84, 1335–1370. Xia, J., Tian, Q., 2009. Early stage of excess copper to photosystem II of Chlorella pyrenoidosa-OJIP chlorophyll a fluorescence analysis. J. Environ. Sci. 21 (11), 1569–1574. Xiang, M., Chen, S., Wang, L., Dong, Z., Huang, J., Zhang, Y., 2013. Effect of vulculic acid produced by Nimbya alternantherae on the photosynthetic apparatus of Alternanthera philoxeroides. Plant Physiol. Biochem. 65, 81–88. Xu, D., Li, C., Chen, H., Shao, B., 2013. Cellular response of fresh water green algae to perfluorooctanoic acid toxicity. Ecotoxicol. Environ. Saf. 88, 103–107.