Ecotoxicology and Environmental Safety 108 (2014) 72–77

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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: Extensive use of synthetic dyes in many industrial applications releases large volumes of wastewater.
Received 28 February 2014 Wastewaters from dying industries are considered hazardous and require careful treatment prior to
Received in revised form discharge into receiving water bodies. Dyes can affect photosynthetic activities of aquatic flora and
27 May 2014
decrease dissolved oxygen in water. The aim of this study was to evaluate the effect of Congo red on
Accepted 28 May 2014
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
Keywords: emission showed that the donor side of photosystem II was affected at high concentrations of Congo red.
Chlorella vulgaris The quantum yield for electron transport (φEo), the electron transport rate (ETR) and the performance
Chlorophyll a fluorescence
index (PI) also decreased. The reduction in the ability to absorb and use the quantum energy increased
Congo red
non-photochemical (NPQ) mechanisms for thermal dissipation. Overall, Congo red affects growth and
JIP-test
Photosynthesis metabolic activity in photosynthetic organisms in aquatic environments.
Respiration & 2014 Elsevier Inc. All rights reserved.

1. Introduction photosynthetic process (Annuar et al., 2009). The resulting

changes in the concentration of dissolved O2 damage the aquatic
Azo compounds constitute the largest and most diverse group biota present and increase the biochemical oxygen demand of the
of synthetic dyes and are widely used by the textile, food, surrounding water (Ali, 2010). In addition, some azo dyes, such as
cosmetic, and pulp and paper industries (El-Sheekh et al., 2009). Congo red, Direct Blue 15 and Direct Red 2, have been reported to
At the global level, 280,000 t of textile dyes are discharged into be toxic, mutagenic, and carcinogenic to aquatic life (Bafana et al.,
industrial effluent each year (Jin et al., 2007). It is estimated that 2008; Golka et al., 2004; Saratale et al., 2011).
the quantity of dye that does not fix to textile fibers depends on In aquatic ecosystems the green algae are the primary produ-
the application method, varying from 2 percent when basic dyes cers and are key indicator organisms (Wen et al., 2011) when
are used to 50 percent when reactive dyes are used (Hai et al., used to evaluate water quality and the ecotoxicity of contami-
2007; Pearce et al., 2003). The discharge of azo dyes into the nants (Xu et al., 2013), such as metals, herbicides, insecticides,
environment is a serious problem and should be avoided not only and other xenobiotic compounds (Jena et al., 2012; Levy et al.,
for esthetic reasons but also because of the threat it poses to public 2007; Qian et al., 2008). On the other hand, the chlorophyll a
health and ecosystems (Golka et al., 2004). In aquatic environ- fluorescence kinetic has been used to indicate alterations of the
ments, the colors produced by azo dyes impede plant develop- photosynthetic capacity when damage is induced by pollutants
ment by decreasing the passage of light, thereby affecting the 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). Further-
n
Corresponding author. Fax: þ52 55 50613313. more, studies have shown that changes in photosystem II (PSII)
E-mail addresses: [email protected] (M. Hernández-Zamora),
can be quantified using the JIP-test (Jena et al., 2012; Strasser et
[email protected] (H.V. Perales-Vela),
cmfl[email protected] (C.M. Flores-Ortíz), al., 2000). However, as of yet, no information is available regard-
[email protected] (R.O. Cañizares-Villanueva). ing the impact of azo dyes on the photosynthetic apparatus of

http://dx.doi.org/10.1016/j.ecoenv.2014.05.030
0147-6513/& 2014 Elsevier Inc. All rights reserved.
 M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77 73

algae. Such information is important because it would permit us 2. The probability that a trapped exciton moves an electron into the electron
transport chain beyond QA ; Ψo ¼ 1  Vj
to determine the toxicity to the aquatic environment caused by
3. The quantum yield of electron transport; φEo ¼ [1  (Fo/Fm) (1  Vj)]¼ (φPo) (Ψo)
this type of dye. For this reason, the objective of the current study 4. Reaction centers per excited cross section; (RC/ABS) ¼ [(Mo/Vj) (1  (Fo/Fm))]
is to evaluate the toxic effects of the azo dye Congo red on the 5. The performance index; PIABS ¼ (RC/ABS) [φPo/(1  φPo)] [Ψo/(1  Ψo)]
growth and photosynthetic metabolism of the microalga Chlorella
vulgaris. 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).
2. Materials and methods 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
2.1. Dye used
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
Congo red (Sigma-Aldrich), also called Direct Red 28, was used for the optical-density value (0.3 680 nm) and these were washed using Bold´s Basal mineral
experiments described herein. Its molecular form is C32H22N6O6S2Na2, and its medium to remove adsorbed dye completely.
molecular weight is 696.7 g mol  1. All experiments employed a 100 mg L  1 stock Each cellular suspension was vacuum-filtered using Millipores membranes of
solution of dye, which, prior to its addition in each experimental unit, was sterilized 5.0-μm pore diameter and were later adapted to darkness for 5 min. Signatures for
by filtration using Millipores membranes of 0.22 μm pore diameter. Chl-a fluorescence were detected directly from the filter surface (Perales-Vela et al.,
2007).
2.2. Experimental units 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
Experiments were conducted under sterile conditions with axenic cultures of
10,000 μmol m  2 s  1 for 0.8 s, to close all reaction centers and obtain the
the green microalga C. vulgaris donated by the Laboratorio de Hidrobiología
maximum fluorescence value of Fm in darkness. Immediately after the saturating
Experimental of the Escuela Nacional de Ciencias Biológicas of the Instituto
pulse, the samples were irradiated with a non-saturating actinic white light
Politécnico Nacional. The strain was collected and isolated from temporary ponds
200 μmol m  2 s  1 for 5 min to achieve a steady state and then these were
in the State of Mexico (Atlacomulco). The registration number is LHE-Chl 01. The
superimposed a saturating white light pulse of 10,000 μmol m  2 s  1 for 0.8 s to
experimental culture units were glass bottles with flat surfaces, each of which had
obtain the value of Fm0 .
a total capacity of 0.5 L and a working volume of 0.25 L, that were inoculated with
Finally the values were calculated as follows:
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 1. Relative electron transport rate (rETR) ¼ [((Fm0  Fs)/Fm0 )] (0.5)(PAR). The value of
the culture medium. The culture conditions were as follows: temperature, 0.5 corresponds to the proportion of light that is transferred to each of the two
257 3 1C; illumination, 120 μmoles photons m  2 s  1; photoperiod, 12/12 h photosystems (PSII and PSI) and the photosynthetically active radiation (PAR)
(light/darkness); and air supply, 200 ml min  1. 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 2.5. Measurement of photosynthesis and respiration
(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, Measurements were made using an oximeter (Oxylab-Hansatech, UK) at a
pre-treated for a constant weight. Later, the samples were dried at 80 1C for 48 h. controlled temperature of 30 1C in exponentially growing cultures. Previously, the
Growth rate of algal culture is a measure of the increase in biomass over time cells were washed using Bold´s Basal mineral medium to remove adsorbed dye
and it was determined by the following equation: 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.
K ¼ In ðN 2 =N 1 Þ=ðT 2  T 1 Þ
The oxygen release rate (photosynthesis) was obtained by illuminating each of the
where, K is growth rate, N1 and N2, biomass content at time (T1) and the time (T2), samples with an actinic red light of 400 μmol m-2 s-1 for 1 min. Immediately after
respectively chlorophyll a and b and total carotenoids were determined using 3 ml turning off the light source, the oxygen consumption rate (respiration) was
of each sample, after the sample was centrifuged for 10 min. Subsequently, the recorded for 2 min.
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 2.6. Statistical analysis
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 All experiments were performed in triplicate, and the results obtained were
sample was centrifuged at room temperature. The supernatant was separated and statistically analyzed via one-way analysis of variance (P o 0.05) and Tukey's test
the concentrations of Chl-a, Chl-b, and total carotenoids were determined with a for comparison of means using the statistical package Sigma-Plot (version 11.0).
UV–Vis spectrophotometer (Genesys 10UV Thermo Electron Corporation) accord-
ing to the equations of Wellburn (1994).
3. Results and discussion
2.4. Chlorophyll fluorescence
3.1. Effect of Congo red on growth
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
The microalga C. vulgaris was affected after 96 h of exposure to
chamber for liquid phase HPEA/LPA (Hansatech, UK). For each of the experimental Congo red. A decrease in growth rate (μ) was observed in every
units, samples were corrected to the same optical density (0.3 680 nm) and these experimental unit after increasing the concentration of dye in the
were washed using Bold´s Basal mineral medium to remove adsorbed dye culture medium; this decrease was significant (Po 0.05) for
completely. After, 2 ml samples of each treatment (n ¼4) were taken and incubated
concentration over 15 mg L  1 compared to the control (Fig. 1).
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 μ value was 0.657 day  1 for cells with no dye, while the
The equipment automatically recorded the following values: (i) minimal values of 0.538 and 0.391 day  1 were obtained when cells were
fluorescence (Fo) at 50 μs, (ii) maximum fluorescence (Fm) between 200 and exposed to 5 and 10 mg L  1 of dye, respectively. However, the μ
500 ms, (iii) variable fluorescence at 2 ms (Vj ¼ Fj  Fo/Fm  Fo) and (iv) the slope at values remained at 0.201 day  1 when cells were exposed to dye
the beginning of fluorescence Mo ¼ 4 (FK  Fo)/(FM  Fo). These data were used to
calculate the following parameters (Strasser et al., 2004):
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
1. The maximum quantum yield of primary photochemistry; φPo ¼ [1  (Fo/Fm)] ¼ separately to 30 mg L  1 of Supranol Red 3BW, Lanaset Red 2GA,
Fv/Fm 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 red dye on content of photosynthetic pigments. It can be seen that
dye (control). the total chlorophyll (aþ b) content decreases significantly with
Lim et al. (2010) obtained μ values of 0.34 day  1 in C. vulgaris respect to the control as the dye concentration is increased.
cells exposed to Supranol Red 3BM and 0.05 day  1 when residual Acuner and Dilek (2004) have reported similar results with
textile water was used. These values were lower than the control C. vulgaris and the dye Tectilon Yellow G. This reduction in
(μ 0.40 day  1). chlorophyll content can affect the conversion of light energy and,
There have been very few attempts to explain what causes thus, photosynthetic electron transport (Strasser et al., 2004).
cellular growth inhibition resulting from exposure to azo dyes; Furthermore, as seen in Fig. 2, the total carotenoid content was
however there are reports of high tolerance and susceptibility in not significantly altered (P o0.05), which can indicate that the
algae. For example, Acuner and Dilek (2004) showed that C. dye affects each photosynthetic pigment differently. Carotenoids
vulgaris can grow without damage in the presence of 400 mg L  1 are important because they are involved in the removal of the
of Tectilon Yellow 2G. Furthermore, other algae like Pseudokirch- oxygen free radicals which are generated when the metabolism
neriella subcapitata (Selenastrum capricornutum) could only grow is inhibited, as well as being important in the regulation of the
in 0.5 mg L  1 of Disperse Blue 3 (Novotny et al., 2006). light excitation energy in the reaction center of photosystem II
The inhibitory effect of textile dyes was reported by Ogawa (Choudhury and Behera, 2001; Czerpak and Piotrowska, 2006).
et al. (1989). They found that inhibition was caused by intercala-
tion of dye compounds between DNA base pairs, so preventing 3.2. Chlorophyll-a fluorescence
enzymatic activity and cell replication (Ogawa et al., 1988).
Ganesh et al. (1994) noted inhibition of biomass in a waste- An increase in the yield of chlorophyll fluorescence from the
water system treating with a reactive dye and suggested that this dark into the light over a time period of around 1 s, it has been
was caused by the products of dye degradation rather the dye explained as a consequence of reduction of electron acceptors in
itself. Other studies have demonstrated that one of the possible the photosynthetic pathway, downstream of PSII, notably plasto-
causes of the observed growth inhibition is related to the altera- quinone and in particular, QA. Once PSII absorbs light and QA has
tion of photosynthesis, especially at the PSII level (Perron and accepted an electron, it is not able to accept another until it has
Juneau, 2011). 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
3.1.1. Photosynthetic pigment content to an overall reduction in the efficiency of photochemistry and so
In plants and algae, photosynthetic pigments (Chl-a, Chl-b, and to corresponding increase in the yield of fluorescence (Maxwell
carotenoids) are important as an indirect measure of photosyn- and Johnson, 2000). The polyphasic chlorophyll a fluorescence
thetic function (Marr et al., 1995). Fig. 2 shows the effect of Congo 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 0.7087 0.004a 0.563 7 0.016a 0.3777 0.011a 0.3327 0.005a 0.996 7 0.002a 0.378 70.018a
5 0.7157 0.003a 0.5687 0.018a 0.384 7 0.013a 0.343 7 0.008a 0.9977 0.003a 0.404 70.022b
10 0.626 7 0.005b 0.542 7 0.006b 0.3177 0.004b 0.281 7 0.011b 1.0007 0.002a 0.215 70.005c
15 0.4917 0.013c 0.508 7 0.015c 0.234 7 0.012c 0.1927 0.008c 0.993 7 0.014a 0.083 70.007d
20 0.4997 0.006c 0.5447 0.007b 0.2517 0.003d 0.2167 0.011d 1.0007 0.001a 0.101 70.004e
25 0.5167 0.007d 0.540 7 0.014b 0.256 7 0.009d 0.2247 0.023d 1.0007 0.001a 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). electron flow inhibition beyond QA in PSII, the quantum yield of
Some authors have attributed the decrease in the maximal electron transport (φEo) should decrease markedly. The current study
fluorescence (inflection P) to deactivation or reduced fluorescence shows that increasing the Congo red dye concentration in the culture
emissions, due to the presence in the “pool” of plastoquinones medium results in decreased (φEo) values. The former indicates that
(PQ) that are oxidized (Haldimann and Tsimilli-Michael, 2005). On the toxic effect of Congo red on electron transport in C. vulgaris is
the other hand, Govindjee (1995) and Perales-Vela et al. (2007) predominantly related to the reduction of activity in the donor side
have suggested that a decrease in the phases J and I illuminates of photosystem II or to the light phase, represented by the φPo values,
how electron-transport inhibition on the donor side of PSII results in and to a lesser extent by the acceptor side represented by the
the accumulation of P680 þ and can reduce fluorescence emission. reduction of carriers beyond QA (Ψo).
The results in the photochemical activity determined by the Table 1 shows that the Mo values did not increase significantly
fluorescence emission, shows that the dye decreased significantly with respect to the control, this confirms that there is no inhibition
(P o0.05) the maximum quantum yield of primary photochemis- in electron transport between QA and QB (Ψo), and that the
try (φPo), the probability that a trapped exciton moves an electron decrease in electron transport (φEo) is the result of the decrease
into the electron transport chain beyond (Ψo), and the quantum in the maximum quantum efficiency φPo. It can also be seen that
yield of the electron transport (φEo). Corresponding values are the values of RC/ABS decreased significantly (P o0.05) at a con-
shown in Table 1. At the maximum dye concentration (25 mg L  1) centration of 10 mg L  1 of dye with respect to control. Reducing
the values of φPo, Ψo, and φEo decrease by 27.11, 4.08, and 32.09 the number in active reaction centers means that less energy is
percent, respectively, compared to the control. used to drive electron transport, therefore, this energy must be
The maximum quantum yield of primary photochemistry (φPo) dissipated by non-photochemical mechanisms (Perron et al., 2012).
uniquely reflects the efficiency of light-phase reactions (Strasser et Furthermore the performance index (Table 1) decreased sig-
al., 2004). It has been reported that when a decrease in φPo occurs, nificantly (P o0.05). At the maximum dye concentration
it is due to destruction or modification of the photosystem, which (25 mg L  1), the value of PIABS was reduced by 71.2 percent.
is to say that the reaction center of PSII does not have its normal Strasser et al. (2004) demonstrated that the performance index
quantum efficiency. The reduction in φPo found in this investiga- (PIABS) is the parameter that is most sensitive to the JIP-test, given
tion is the result of the decrease in the values of the minimum (Fo) that this parameter incorporates several steps in electron trans-
and maximum (FM) fluorescence. port and comprises three independent components, RC/ABS, φPo,
The decrease in the values of parameters Ψo indicate that the and Ψo. A decrease in PIABS values can be attributed to a change in
yield of the electron transport per a trapped exciton in treated one, two, or three parameters (Xiang et al., 2013). The assayed
cells with Congo red (10–25 mg L  1) is lower than in the control concentrations of Congo red dye affected the three parameters
and that the energy needed to close all reaction centers decreases (φPo, Ψo, and RC/ABS) that are involved in the PIABS. As shown by
(Strasser et al., 1995). Strasser et al. (2000), a decline in the performance index results
Studies conducted by Jena et al. (2012) have associated decreased from a disruption of energy absorption, trapping or transfer
values of φPo, Ψo, and φEo with a reduction of the electron flow from beyond the QA quinone. Aksmann and Tukaj (2008) found that
PSII to PSI. Moreover, Xiang et al. (2013) have mentioned that upon 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 assayed and that this inhibition increased as the dye concentration
(φPo, Ψo, and φEo). Until now, no results have been reported that in the medium was increased. At the minimum and maximum
explain the PIABS effect of cellular exposure of microalgae to azo concentrations of Congo red tested (5 and 25 mg L  1), the photo-
dyes. However, the current study shows that the parameter most synthetic process was inhibited by 84 and 98 percent and the
sensitive to dye was RC/ABS, followed by φPo and Ψo. respiratory process by 76 and 96 percent, respectively, with
The electron transport rate (ETR) in vivo showed decreases up respect to the control (P o0.05). Even when the photosynthetic
to 67.4 percent at the highest concentration tested (Fig. 4). In and respiratory rate decreases drastically, the cells continue to
contrast, the decrease in the electron transport rate (ETR), resulted grow at a relatively slow speed compared to the control (Fig. 1).
in an increase of non-photochemical quenching (NPQ) up to 75 C. vulgaris is a photosynthetic microalga, and its growth is
percent for the maximum dye concentration (25 mg L  1) with highly dependent on photosynthetic performance. Any change in
respect to the control (Fig. 4). Müller et al. (2001) showed that this environmental conditions requires the development of acclima-
behavior is due to partial inhibition of the primary photosynthetic tion mechanisms that adjust the energy flow from the light-
process, which is necessary to regulate the excitation energy harvesting complex to the Calvin cycle (Wilson et al., 2006), and
captured by the antenna complex so that it can be balanced with the decline photosynthesis will ultimately influence growth, as
electron transport and ATP and NADPH synthesis to reduce been by the results in Figs. 1 and 5. Consistent with the results
potential damage to PSII by a non-channeled excitation-energy mentioned above, Xu et al. (2013) have suggested that photo-
excess, which can cause photo-oxidation. On the other hand, synthesis and cell division are key factors in algae growth and that
White et al. (2011) mentioned that the NPQ value is generally any interference or damage to photosynthesis could negatively
interpreted as the increase in thermal energy release may be affect growth. Other studies have shown that a malfunctioning
caused by the operation of the xanthophylls cycle or slow photosynthetic apparatus inevitably diverts the light energy
reversible damages to the photosynthetic apparatus. absorbed to other processes within the electron-transport chain
Until now, no reports have demonstrated the effects of azo dyes and subsequently if the excited states of chlorophyll cannot be
on NPQ and ETR. However, we can infer that the behavior of the used, the energy is dissipated as fluorescence or heat by the non-
parameters, as demonstrated by our results, is similar to those photochemical quenching mechanisms (Rocchetta and Küpper
reported by other authors for microalgae exposed to different toxic 2009), as seen by the results in Figs. 4 and 5.
compounds, such as heavy metals and herbicides (Perales-Vela et
al., 2007; Deblois et al., 2013).
4. Conclusions
3.3. Photosynthesis and respiration
Exposure of C. vulgaris to Congo red causes a reduction in
Fig. 5 shows that the metabolic rate (photosynthesis and growth rate. This reduction is associated with a decrease in
respiration) was inhibited at all concentrations of Congo red metabolic activity. In particularly, photosynthetic activity is
 M. Hernández-Zamora et al. / Ecotoxicology and Environmental Safety 108 (2014) 72–77 77

affected at the donor side of photosystem II, represented as the Maxwell, K., Johnson, G.N., 2000. Chlophyll fluorescence. A practical guide. J. Exp.
light phase (φPo). This reduction in the ability to absorb and use Bot. 51, 659–668.
Müller, P., Xiao-Ping, L.i., Niyogi, K.K., 2001. Non-photochemical quenching: a
the quantum energy causes an increase in non-photochemical response to excess light energy. Plant Physiol. 125, 1158–1566.
mechanisms for thermal dissipation. 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
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Nielsen, H.D., Nielsen, S.L., 2005. Photosynthetic responses to Cu2 þ exposure are
Acknowledgments
independent of light acclimation and uncoupled from growth inhibition in
Fucus serratus (Phaeophyceae). Mar. Pollut. Bull. 51, 715–721.
Hernández-Zamora M., received a post-graduate scholarship 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
from Consejo Nacional de Ciencia y Tecnología (Grant no. 204491)
toxicity of azo- and anthraquinone dyes. Chemosphere 63, 1436–1442.
for her doctoral studies. 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.
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