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Assessment of catalase activity, lipid peroxidation, chlorophyll-a, and growth rate in the freshwater green algae Pseudokirchneriella subcapitata exposed to copper and zinc/Evaluacion de la actividad de la catalasa, peroxidacion lipidica, clorofila-a y tasa de crecimiento en la alga verde de agua dulce Pseudokirchneriella subcapitata expuesta a cobre y zinc.

INTRODUCTION

Metals such as copper and zinc are released into the aquatic ecosystems by anthropogenic activities such as agricultural, industrial and mining (Yruela, 2005). In this ecosystems the primary producers play an important role in the structure and functioning of aquatic ecosystems. Any negative effect on them will affect upper trophic levels (Li et al., 2006). Copper is an essential redox-active transition metal, which acts as a structural element in regulatory proteins, and participates in electron transport in photosynthesis, mitochondrial respiration, oxidative stress responses, cell wall metabolism and hormone signaling (Moreno, 2003; Srivastava et al., 2006). Zinc is essential for the stability of cell membranes in the activation of over 300 enzymes in the metabolism of proteins and nucleic acids (Cakmak & Marschner, 1988). However, in high concentrations these metals can be toxic to aquatic organisms (Srivastava et al., 2006).

The metals, as other environmental factors (ultraviolet light, extreme temperatures, radiation, environmental pollution, etc.) induce ROS (reactive oxygen species) formation inside the cell (Marshall & Newman, 2002). These ROS are formed under physiological conditions in manageable proportions, while maintaining a balance due to the defensive cellular enzymes (Droge, 2003). The breakdown of this balance is known as oxidative stress, which results in alterations of the structure-function of organs, systems, or specialized transport (Ames et al., 1993).

The damage caused by oxidative stress in fatty acid rich structures, such as cell membranes, results in lose rigidity, integrity and permeability. This process known as lipidperoxidation, produces carbonylic compounds such as malondialdehyde. Aerobic organisms have developed antioxidant defence mechanisms to prevent cell damage by ROS (Finkel & Holbrook, 2000; Sohal et al., 2002). The enzyme catalase has an active participation in the reactions that reduce the intracellular levels of [H.sub.2][O.sub.2] formed from the superoxide radical (De Zwart et al., 1999).

Although copper can act at many different levels in the cell, one of the most important mechanisms of copper action in green plants is believed to be the inhibition of electron transfer in the chloroplasts, the destruction of the chloroplast membrane, and inhibition of the photosynthetic pigments formation (Yan & Pan, 2002; Charles et al., 2006).

The microalga of freshwater Pseudokirchneriella subcapitata has been used in toxicity bioassays due to their sensitivity to chemicals agents. In these tests, the exposed population rate growth is the response variable determinated (NCh2706, 2002). At present, it is known that the molecular alterations are the first detectable, quantifiable responses, allowing the evaluation of exposure to agents at sublethal level. The biomarker concept is based in that the first level of interaction of pollutants with organisms is the molecular-cellular (Huschek & Hansen, 2005).

The studies on oxidative stress caused by metals in microalgae are few, the most are in green plants (Okamoto et al., 1996; Lee & Shin, 2003; Elisabetta & Gioacchino, 2004; Mallick, 2004; Li et al., 2006). The lipidperoxidations products determination (TBARS), together with the evaluation of the catalase activity are effective methods used to assess oxidative stress (Scandalios, 1997; Nordberg & Arne, 2001). In this research is proposed that the molecular response is more sensitive than the response at the population level in P. subcapitata exposed to copper and zinc. The goal of this study is to evaluate the effect of copper and zinc on P. subcapitata, through the activity of catalase, lipidperoxidation by TBARS assay, growth rate and concentration of chlorophyll-a.

MATERIALS AND METHODS

The green alga species Pseudokirchneriella subcapitata, belonging to the order Chlorococcales, class Chlorophyceae, was acquired in the Laboratory of Phycology of University of Concepcion, Chile.

Bioassays

The bioassays were carried out according to the Chilean standard NCh2706 (2002). Prior to the bioassay a nutrient solution consisting of five stock solutions, which contain: micronutrients, macronutrients, Fe-EDTA, trace elements and NaHC[O.sub.3] was prepared. The algae was inoculated into 500 mL Erlenmeyer flasks in an equal volume for all samples, from culture stock during the exponential phase of growth. Then, they were enriched with the nutrient concentration to avoid false negatives, and volumes of copper sulphate pentahydrate (CuS[O.sub.4].5[H.sub.2]O) and zinc sulphate heptahydrate (ZnS[O.sub.4]. 7[H.sub.2]O) were added until a concentration of 0.025, 0.05, 0.075 and 0.1 mg [L.sup.-1] of Cu and Zn respectively. Finally, the flask was adjusted to 300 mL with distilled water. Additionally, a blank was prepared containing only the inoculum of P. subcapitata, nutrients and water. Each treatment and the blank, in triplicate, was exposed to continuous cool white light with a low intensity of 90 [+ or -] 10 [micro]mol [m.sup.-2] [s.sup.-1], at 23[degrees]C [+ or -] 2[degrees]C and shaken by hand every day. To have a biomass of microalgae that would make the determinations, the exposure time was 15 days. The cell density (n) was determined at the beginning and the end of the bioassay counting directly in a microscope using a Neubauer Bright Line hemacytometer.

The growth rate (k) expressed as number of cell duplications was determined from:

k = [3,3211 x log [N.sub.n] - log [N.sub.0]]/tn

Where tn is the time elapsed between the start and end of the trial (in days), [N.sub.0] is the nominal initial cell density and [N.sub.n] is the density at the end of the test.

Chlorophyll-a

To determine the chlorophyll-a concentration, 100 mL of the sample was filtered and immersed in acetone (90% V/V) for 20 h in darkness and cold. The absorbance of the solvent was determined at 630, 645 and 665 nm in a spectrophotometer (Cecil CE 2000 model 2041). The chlorophyll-a concentration was calculated through the Richards & Thompson (1952):

mg chl-a [m.sup.3] = [[(15, 6 [E.sub.665] - 2 [E.sub.645] - 0,8 [E.sub.630])]/V] x 10

Where [E.sub.665] is the absorbance at 665 nm, [E.sub.645] the absorbance at 645 nm, [E.sub.630] at 630 nm and V the volumen sample leaked.

Oxidative stress parameters

A volumen of 200 mL of each treatment was centrifuged and frozen with liquid nitrogen before grinding it with a mortar. Two mL of buffer 50 mM sodium phosphate (pH 7.0) were added to obtain an approximate volume of 3 mL, 1 mL of which was used to measure thiobarbituric acid reactive species (TBARS) and the remaining 2 mL to determine the enzymatic activity of catalase (CAT).

Determination of thiobarbituric acid reactive species (TBARS)

Lipidperoxidation was determined using the TBARS assay: samples were treated with trichloroacetic acid and then with thiobarbituric acid, the complex formed absorbed at 540 nm, the results are expressed in nanomoles of MDA/[10.sup.6] cells. MDA content was calculated using the calibration curve of MDA bis dimethylacethal.

Catalase activity (CAT)

The enzymatic activity of catalase was determined, by spectrophotometry following the hydrogen peroxide degradation rates for 1 min every 15 sec, at 240 nm which is accelerated in the presence of enzyme (Ratkevicius et al., 2003; Contreras et al., 2005; Cargnelutti et al., 2006; Hidalgo et al., 2006). The sample (2 mL) was concentrated by centrifugation at 3500 rpm for 15 min. A blank (2.9 mL sodium phosphate buffer pH 7.0 and 100 mL of supernatant) was used. The results are expressed as U/[10.sup.6] cells (Li et al., 2006). The results were analyzed through the analysis of variance Anova and correlation determined with the program Systat 5.0. For each treatment three replicates were considered.

RESULTS

The growth rate of P. subcapitata exposed to copper decreased at 0.075 mg [L.sup.-1], while in the presence of zinc no statistically significant differences to the control was found in the range of concentration tested (Fig.1), however correlations with metal concentrations (Table 1) were observed. The concentration of chlorophyll-a decreased significantly at copper concentration 0.05 mg L-1 and zinc concentration 0.075 mg [L.sup.-1]. However an increase of chlorophyll-a at 0.025 mg [L.sup.-1] of copper was observed (Fig. 2). Only the correlation with zinc was significant (Table 1).

[FIGURE 1 OMITTED]

The catalase activity increased in comparison to the control in 0.075 mg [L.sup.-1] and 0.05 mg [L.sup.-1] of copper and zinc, respectively (Fig. 3). Zinc induced the activation of the antioxidant enzyme catalase at lower concentration than copper. The correlation between metal concentration and catalase activity was significant only for zinc (Table 1). There was significant correlation between lipid damage expressed as malondialdehyde content and catalase activity (Table 1). The lipid damage in the cell membrane was higher with copper than zinc. The damage was observed in exposure to 0.025 mg [L.sup.-1] copper, while for zinc it was observed at 0.1 mg [L.sup.-1] (Fig. 4). In this case, the correlation between damage to the cell membrane expressed as nmols/106cell of malondialdehyde was significant to copper and zinc (Table 1).

DISCUSSION

Copper was more toxic than zinc in relationship at the rate growth, chlorophyll-a content and, lipoperoxidation endpoints, wich is similar to what was reported by Utgikar et al. (2004) in Vibrio fischeri. However, copper toxicity was lower than what was found by Kaneko et al. (2004); these autors reported an IC50 at 0.11 mg [L.sup.-1] of copper in Selenastrum capricornutum, similar values were reported by Bossuyt & Janseen (2004) in the same species, our study show that at copper concentration similar the inhibition on the rate growth was less to twenty percent. The differences may be due to the chemical characteristics of test water, such as pH, hardness, time exposure, concentration and type of chelating agent in the nutrients used in the these studies. On the other hand, the differences in the effects of the respective metals could be attributed to the redox potential of the metals. Copper has a greater redox potential that zinc (Cu E[omicron] = 0.16 and Zn E[omicron] = -0.76) which would lead to an increased oxidant effect at lower concentrations.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

A manifestation of the hormesis effect was expressed under the Cu exposure. The stimulation of chlorophyll-a at the most low concentration of copper was similar to that founded by Knauer et al. (1997) and Janssen & Heijrick (2003). Whitton (1970) reported that the algae growth may be stimulated by low metal concentrations and inhibited at high metal concentrations. The parameter chlorophyll-a concentration was more sensitive than growth rate. The lowest content of chlorophyll-a in the higher concentrations of the metals can be due to the peroxidation of chloroplast membranes. Metals can destabilize the membrane by oxidation due to free radicals formation. Copper alters membrane tilacoides and causes changes in its composition (De Vos et al., 1991), interference with the photosynthetic system and the a decline in the rate of photosynthesis (Cook et al., 1997; Yruela, 2005). Our results differ from those reported by Bossuyt & Janssen (2004), who found a significant increase in chlorophyll-a at higher concentrations of metals in P. subcapitata. However, these authors used a different medium to that used in the present study and their cultures were acclimated previously to 60 and 100 [micro]g [L.sup.-1], which would explain the differences with our results.

The catalase activity was higher in the highest concentrations tested. Similar results are reported by Li et al. (2006) when Pavlova viridis was exposed to copper and zinc. Contrarily, Srivastava et al. (2006) found that the catalase enzyme is dependant on concentration-time, their results show an increase in catalase activity until the 7 day of exposure with 108% induction. However, catalase activity declines in plants exposed to up 5 and 25 [micro]M copper independently of the exposure duration, indicating a maximum decline of 42% on day 7 at 25 [micro]M, probably due to enzyme degradation at higher free radicals concentration. Tripathi et al. (2006) reported less catalase activity at lower concentrations in Scenedesmus sp. when exposed to copper and zinc, than the present study. The results in our work suggest that the antioxidant enzyme catalase plays an important role at higher toxicant concentrations in P. subcapitata. The antioxidant catalase in P. subcapitata was protective at lower concentrations in P. subcapitata exposed to zinc compared to copper exposure.

Both metals provoked damage to lipid. MDA content increased significantly from 0.025 mg [L.sup.-1] and 1.0 mg [L.sup.-1] in copper and zinc respectively, indicating rise in lipid peroxidation in P. subcapitata. This is similar to that found by Li et al. (2006) with the microalga Pavlova viridis (Prymnesiophyceae), whose results show that the MDA content is expressed significantly from 0.5 mg [L.sup.-1] of copper and 3, 25 mg [L.sup.-1] of zinc. The difference with our work can be explained by the different sensibility between species. Similarly, Srivastava et al. (2006) reported a gradual increase in the content MDA and significant differences to the control in Hydrilla verticillata at the highest concentration of copper tested.

In conclusion, both metals induce the antioxidant activity of catalase. Copper induces lipidperoxidation at lower concentrations than zinc. Copper and zinc cause adverse effects on the physiology of P. subcapitata determined through the content of chlorophyll-a. The growth rate was less sensitive to the metals that catalase activity, lipidperoxidation and chlorophyl-a content. The molecular parameters can be more adecuate than growth rate to monitoring the effect in microalgae aquatic ecosystems.

DOI: 10.3856/vol39-issue2-fulltext-9

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Received: 2 August 2010; Accepted: 11 May 2011

Paulina Soto (1), Hernan Gaete (1,3) & Maria Eliana Hidalgo (2)

(1) Departamento de Biologia y Ciencias Ambientales

(2) Departamento de Quimica y Bioquimica

(3)Centro de Investigacion y Gestion de Recursos Naturales (CIGREN), Facultad de Ciencias Universidad de Valparaiso, Valparaiso, Chile

Corresponding author: Hernan Gaete (hernan.gaete@uv.cl)
Table 1. Correlation between measured parameters.

Tabla 1. Correlacion entre los parametros medidos.

          Concentration    CatCu     CatZn    ClofCu    ClofZn

CatCu         0.71
CatZn         0.98 *         -
ClofCu        0.74         -0.53        -
ClofZn       -0.93 *         -       -0.96 *
MdaCu         0.92 *        0.93 *      -      -0.69
MdaZn         0.81           -        0.78       -      -0.88 *
[mu]Cu       -0.88 *       -0.92 *      -      0.74        -
[mu]Zn       -0.90 *         -       -0.95 *     -       0.90 *

           MdaCu    MdaZn

CatCu
CatZn
ClofCu
ClofZn
MdaCu
MdaZn
[mu]Cu    -0.98 *
[mu]Zn       -      -0.78

Concentration : metal concentrations; CatCu: catalase activity
exposured to copper; CatZn: catalase activity exposured to zinc;
ClofCu: Chlorophyll-a exposured to copper; ClofZn: chlorophyll-a
exposure to zinc; MdaCu: MDA production exposured to copper ; MdaZn :
MDA production exposured to zinc; [mu]Cu: growth rate exposured to
copper;  [mu]Zn: growth rate exposured to zinc. *: indicate significant
correlation  (P < 0.05).
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Title Annotation:Research Article
Author:Soto, Paulina; Gaete, Hernan; Hidalgo, Maria Eliana
Publication:Latin American Journal of Aquatic Research
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Date:Jul 1, 2011
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