Ecotoxicology and Environmental Safety 104 (2014) 294–301 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv ROI-scavenging enzyme activities as toxicity biomarkers in three species of marine microalgae exposed to model contaminants (copper, Irgarol and atrazine) Pablo Lozano, Chiara Trombini, Elena Crespo, Julián Blasco, Ignacio Moreno-Garrido n Instituto de Ciencias Marinas de Andalucía (CSIC). Campus Río San Pedro s/n, 11510 Puerto Real, Cádiz, Spain art ic l e i nf o a b s t r a c t Article history: Received 23 August 2013 Received in revised form 18 March 2014 Accepted 19 March 2014 There is a need to develop efficient tools to prevent damage to marine ecosystems due to pollution. Since microalgae play a key role in marine ecosystems, they are considered potentially useful for quick and sensitive toxicity bioassays. In this study an integrative analysis has been carried out of the anti-oxidant enzyme activities of marine microalgae species. Three marine microalgae species (Cylindrotheca closterium, a benthic diatom; Phaeodactylum tricornutum, a diatom which has been used as model organism in toxicity bioassays; and Rhodomonas salina, a cryptophyceae which is considered to present a certain level of heterotrophy) were exposed to selected concentrations of three model pollutants: copper (5 and 10 mg L 1), atrazine (25 and 50 mg L 1) and Irgarol (0.5 and 1.0 mg L 1). These pollutant concentrations are environmentally relevant for coastal ecosystems, and have been selected for checking the efficiency of the reactive oxygen intermediate (ROI) scavenging enzyme system of these organisms. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx) and glutathione peroxidase (GPx) activities were measured at the end of 24 h exposure. The integrated biomarker response (IBR) index – in our case for oxidative stress – has been employed to evaluate the ROI-scavenging enzyme system for each species and each treatment. In general, the SOD and CAT enzyme activities measured were higher in exposed populations than in controls, whereas APx and GPx activities showed the opposite trend. These microalgae showed significant responses of oxidative stress biomarkers at environmentally relevant concentrations for the assayed pollutants and short exposure periods, conditions that most other model organisms cannot match. Therefore microalgae present clear advantages over other species for their prospective employment in an “early warning system”. & 2014 Elsevier Inc. All rights reserved. Keywords: Microalgae Toxicity Anti-oxidant enzymes Bioassay 1. Introduction Marine microalgae comprise the basis of trophic networks in marine and coastal ecosystems. They constitute the base level of the food chain and play a key role in the biogeochemical cycles of substances that are potential pollutants. These organisms can be very useful as bioindicators, given their high surface/volume ratio and their noted capacity for accumulating large amounts of several xenobiotics (Volterra and Conti, 2000; Barreiro et al., 2002; Conti et al., 2007). Bioassays with microalgae classically involved measuring parameters such as growth rate, biomass or chlorophyll content (OECD, 1998; Nyholm and Källqvist, 1989). However, new molecular tools are capable of detecting cell-stress after short exposure times, and provide rapid information about cellular status (Hook and Osborn, n Corresponding author. E-mail address: ignacio.moreno@icman.csic.es (I. Moreno-Garrido). http://dx.doi.org/10.1016/j.ecoenv.2014.03.021 0147-6513/& 2014 Elsevier Inc. All rights reserved. 2012). Algal cells are able to produce specific molecules or to increase specific enzyme activities in response to stress caused by the presence of substances that are toxic to them (Mittler, 2002). Therefore, with the appropriate tools, these organisms can be used in the design of better “early warning systems” of pollution in the environment. Most of the few publications concerning toxic stress biomarkers in microalgae have focused mainly on freshwater species: there are studies in the literature on alterations in the activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx) and glutathione reductase (GR) in Scenedesmus obliquus in the presence of copper and a fungicide (Dewez et al., 2005), and on the responses of the enzyme SOD, in populations of the same species, to the presence of pesticides (Li et al., 2005). In marine species, the few references found are focused on the variation of SOD, CAT and glutathione peroxidase (GPx) levels in Pavlova viridis populations exposed to different metals (Li et al., 2006; Liu et al., 2010; Janknegt et al., 2009; Ebenezer and Ki, 2013). Other study focused on the detection of enzyme activity levels in P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 three species of phytochelatin-producing marine microalgae in the presence of metals (Morelli et al., 2009). The model substances for the experiments conducted in this work are copper, a metal of ecological significance involved in the processes of coastal pollution, and two organic pollutants: a herbicide, atrazine, and an antifouling substance, Irgarol, whose concentrations in estuaries and coastal ecosystems can reach levels representing environmental risk to populations of microdler et al., 2013; Bérard et al., 2003; Nyströ m et al., 2002). algae (Nö Copper is used in anti-fouling compounds on ships and offshore installations due to its bacteriostatic properties, and in fungicides and wood preservatives. Copper is toxic to many organisms, and can cause alterations in the redox system of the organism's cells (Anderson and Morel, 1978; Araújo et al., 2008). Atrazine is one of the most-widely used herbicides for weed control; as a result of its widespread use, its relatively high solubility in water, and its persistence, it has been identified as one of the main pollutants in surface water (Solomon et al., 1996). Furthermore, these authors have shown that microalgae are one of the groups most sensitive to atrazine. Irgarol 1051 is a herbicide used in copper-based antifouling paints. Recently, it has been banned in Denmark, but is still approved in the United States and in most European countries. Atrazine and Irgarol belong to the triazine group of chemicals, and both produce inhibition of the electron transport chain in photosynthesis (Fuerst and Norman, 1991). The purpose of the study is to analyze the response of a battery of biomarkers related to oxidative stress in three species of microalgae, after being exposed for 24 h to sub-lethal and ecologically relevant concentrations of three pollutants. An integrated response plot is performed in order to summarize the enzymatic anti-oxidant mechanisms. 2. Material and methods 295 2.4. Lipid peroxidation Lipid peroxidation refers to the oxidative degradation of lipid membrane and is used as an indicator of cell damage caused by the Reactive Oxidative Intermediates (ROIs). The method of thiobarbituric acid reactive substances (TBARS) was employed to measure lipid peroxidation (Buege and Aust, 1978). This assay measures the malondialdehyde (MDA) in the sample; MDA is one of the final products that are formed in the decomposition of certain products of lipid peroxidation. 2.5. SOD activity Superoxide dismutase (SOD) activity was measured using a commercial kit from SIGMA (SOD Assay Kit – WST). This method uses a tetrazolium salt: WST-1 (2 – (4-iodophenyl) – 3 – (4-nitrophenyl) –5 – (2,4-disulfo-phenyl)-2H-tetrazolium). This salt is reduced in the presence of the superoxide anion (SOD substrate) and produces a formazin-type dye, allowing the colorimetric method to be used. 2.6. Catalase activity Catalase (CAT) activity was measured using a method based on spectrophotometric evaluation of the rate of disappearance of H2O2 added to the sample (Beers and Sizer, 1952). A volume of 20 mL of each sample was placed in a 96-well microplate transparent to ultraviolet light. Then 250 mL of 30 mM H2O2 in 0.05 M phosphate buffer pH 7.8 was added, and the absorbance was measured at 240 nm every 10 s for 2 min at room temperature. 2.7. Glutathione peroxidase activity Glutathione peroxidase (GPx) activity was measured by modifying the assay described by Günzler and Flohe (1985). This indirect method is based on the oxidation of GSH which is catalyzed by GPx, through the reactions of the following process: oxidized glutathione (GSSG) is reduced, catalyzed by glutathione reductase (GR); this requires NADPH, which becomes NADP þ . In practice, this technique involves measuring the decrease in absorbance at 340 nm, the wavelength at which NADPH is absorbed, and thus the GPx activity can be estimated. 2.1. Organisms and maintenance media 2.8. Ascorbate peroxidase activity The three species used in the study were obtained from the Collection of Microalgae Strains held by the Marine Sciences Institute of Andalusia (CSIC) (Lubián and Yúfera, 1989); they were routinely cultured in filtered natural seawater sterilized by autoclaving and enriched with f/2 medium (Guillard and Ryther, 1962). Silicate (50 mg L 1) was also added to the diatom cultures. Ascorbate peroxidase (APx) activity and the oxidized form of dehydroascorbate reductase (DAsA) were determined according to the protocol described by Law et al. (1983) with some modifications, and adapted to a multi-plate reader. 2.2. Experimental design 2.9. Standardization Exposures were performed in conical flasks of borosilicate glass. For each species 21 flasks were prepared each with 100 mL of culture with a cell density of 0.5  106 cells mL 1. The standard cell concentration for exposure in marine microalgae bioassays (104 cells mL 1) (OECD, 1998) was not used because it was necessary to have a final protein concentration sufficiently high to be measured by the Bradford method. Microalgae were concentrated to remove as much culture medium as possible and then diluted with seawater without artificial medium. This was to prevent interactions of the components of the f/2 medium (such as EDTA) with some of the chemical substances (Moreno-Garrido et al., 1999). Each species was exposed for 24 h at two sub-lethal concentrations of copper (5 and 10 mg L 1) (Moreno-Garrido et al., 1999), atrazine (25 and 50 mg L 1) m et al., 2002), in (Solomon et al., 1996) and Irgarol (0.5 and 1.0 mg L 1) (Nyströ triplicate. At the end of exposure period, the content of each flask was divided into two falcon-type tubes of 50 mL, and then centrifuged for 20 min at 250g and 4 1C. Supernatant was removed and the pellet was re-suspended and centrifuged again in eppendorf vials. These vials were stored at 80 1C for subsequent enzyme assays, and the supernatant water was stored at 20 1C, for measurement of pollutant concentrations after exposure. All enzyme activities were normalized to total protein concentration. The protein concentration was determined using the standard Bradford colorimetric assay, with bovine serum albumin as the standard (Bradford, 1976). 2.3. Chemical analysis of incubation water Atrazine and Irgarol concentrations were measured by high performance liquid chromatography (HPLC Agilent Technologies model 1200, EC-C18 Poroshell 100 mm  3 mm  1.7 mm). Copper concentrations were measured by inductive coupled plasma spectrophotometry (ICP-MS G3272B). 2.10. Integrated response plot A method for combining all the measured biomarker responses as a general “stress index” termed Integrated Biomarker Response (IBR; Beliaeff and Burgeot, 2002) was applied for each species. In our case we use the activities of the main ROI-scavenging enzymes in the microalgae and, therefore, we will obtain a general “oxidative stress index” which could be useful as an “early warning system”. In each treatment, for each substance and each species, we obtain a polygon where each vertex represents the measurement of an enzyme. The top vertex and the right vertex represent the activity of SOD and CAT respectively; increases of these activity levels compared with the control indicate an increase of the oxidative stress. The bottom vertex and the left vertex represent the activity of GPX and APX respectively; decreases of these activity levels compared with the control indicate an increase of the oxidative stress. In order to allow visual comparison in the plots, values for each enzyme activity (X) were standardized to obtain Y, where Y ¼(X m)/s, being m the media and s the standard deviation. Then Z was obtained by Z¼ Y or Z¼Y in function of activation or inhibition. The minimum value (Min) for each treatment was added to Z to compute the score (S), being thus S ¼Zþ |Min|. S is always 40, as |Min| is the absolute value of the minimum. Then, the “stress index” was computed following Beliaeff and Burgeot (2002). 296 P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 3. Results and discussion 3.1. Incubation water analysis Atrazine concentrations after 24 h of exposure were half of the initial concentrations; copper remained at values close to the nominal concentration; and at the end of incubation period Irgarol concentrations were below the sensitivity threshold of the analytical method. In the control treatment tank no detectable amounts of atrazine and Irgarol were found, but a residual content of copper appears (Table 1.) 3.2. Lipid peroxidation After 24 h of exposure to each of the separate pollutants, MDA concentration was just significantly higher (P o0.05) compared to control for Phaeodactylum tricornutum exposed to copper concentrations (10 mg L 1). Cylindrotheca closterium and Rhodomonas salina did not show any significant variation in MDA concentration. Background levels for P. tricornutum and R. salina were similar while in C. closterium they are almost double (Fig. 1). MDA is a final product that is formed in the decomposition of certain products of lipid peroxidation, and it serves as an indicator of the cell damage produced by ROIs (Valavanidis et al., 2006). The finding that a toxic exposure does not increase the concentration of MDA may indicate that cellular damage is not occurring, either because the substances are not producing ROIs or because enzymatic detoxification mechanisms or antioxidant molecules are working properly and prevent the substances from producing lipid peroxidation. A third possibility would be that peroxidation is occurring but, instead of producing MDA, there are other end products that are not detected by the TBARS method (Janero, 1990). Few studies have been published on lipid peroxidation by contaminants in algae, and no clear conclusions have been reported. For copper exposure, authors such as Vavilin et al. (1998) have not found significant increases (Po0.05) in MDA concentration after exposure of microalgae Chlorella pyrenoidosa at 0.1 mg L 1 for 24 h; however, other authors such as Soto et al. (2011) have observed a significant increase for Pseudokirchneriella subcapitata exposed to 0.025 mg L 1 of copper, for the same exposure period. Similar discrepancies appear in studies carried out on the effect of triazine-derivative herbicides (Hourmant et al., 2009): Chaetoceros gracilis was exposed to bentazon (an herbicide like atrazine) at different concentrations for different periods of time; significant increases in MDA concentration were reported for 0.05 mg L 1, after two days of exposure. Furthermore, in a recent study (Cima et al., 2013), the coral species Sarcophyton cf. glaucum was exposed to an antifouling substance very similar to Irgarol (Sea-Nine 211TM); this coral did not show any significant increases in the concentration of MDA after 72 h exposure at 0.1 mg L 1. The cellular mechanisms for detoxification of ROIs have Table 1 Copper and atrazine concentrations (mg L 1) after 24 h incubation. Irgarol concentrations are not noted as all samples resulted below the detection limit of the analytical technique (0.3 mg L 1). This limit is 1 mg L 1 for Atrazine and copper. Error means standard deviation (n ¼3). (mg L 1 ) Copper 0 Copper 5 Copper 10 Atrazine 0 Atrazine 25 Atrazine 50 P. tricornutum R. salina C. costerium 7.5 7 1.4 7.17 0.9 10.0 7 1.4 nd. 12.2 7 2.4 26.3 7 2.3 8.17 4.2 6.6 73.4 14.0 7 0.3 nd. 11.3 70.7 33.3 7 12 6.2 7 4.2 9.2 7 0.8 15.2 7 0.5 nd. 8.5 7 1.0 24.57 6.2 nd. – means “not detected”. Fig. 1. Estimation of lipid peroxidation in each species, after being exposed for 24 hours to two sublethal concentrations of copper (5 and 10 mg L 1), atrazine (25 and 50 mg L 1) and Irgarol (0.5 and 1.0 mg L 1). The units are mol of malondialdehyde (MDA). Data are expressed as the mean of three replicates, and standard deviation. Bars with * are significantly different from control (Po0.05). been shown to be effective for reduction cell damage in all cases, except for P. tricornutum exposed to 10 mg L 1 Cu. Assuming the TBARS method is a good indicator of lipid peroxidation, it can be concluded that, for the species, substances, doses and exposure times selected, the cellular mechanisms for the detoxification of ROIs have been sufficiently effective to prevent cell damage in all cases, except for the samples of P. tricornutum exposed to 10 mg L 1 of copper. 3.3. SOD activity After exposure for 24 h to sub-lethal concentrations of three model pollutants, SOD activity showed a significant increase (Po0.05) in two of the species selected, and for two of the pollutants: in P. tricornutum, SOD activity increased for atrazine exposure, regardless of the assayed P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 P. tricornutum 297 0.4 P. tricornutum 300 * * U/mg U/mg 0.3 200 0.2 * * * * * * 100 0.1 0 0.0 R. salina 0.4 R. salina 300 U/mg U/mg 0.3 200 0.1 0.0 0 0.4 C. closterium C. closterium 300 0.2 * 100 * * * 0.3 U/mg U/mg * 200 100 0.1 0.0 0 Copper Atrazine Irgarol Copper P. tricornutum * * * * * 100 nmol/mg U/mg 200 150 100 * 0 R. salina R. salina 200 * 200 * nmol/mg U/mg Irgarol 50 0 150 100 100 50 0 * * * * * 0 C. closterium 200 nmol/mg 300 U/mg Atrazine 200 P. tricornutum 300 300 * 0.2 200 100 C. closterium Controls Low concentration High concentration 150 100 50 * * * 0 0 Copper Atrazine Irgarol Copper Atrazine Irgarol Fig. 2. Enzymatic activities for each species, after being exposed for 24 h to two sublethal concentrations of copper (5 and 10 mg L 1), atrazine (25 and 50 mg L 1) and Irgarol (0.5 and 1.0 mg L 1). Data are expressed as the mean of three replicates and standard deviation. Bars with * are significantly different from control (Po 0.05). Fig. 2a is SOD activity per mg of protein; Fig. 2b is CAT activity per mg of protein; Fig. 2c is GPx activity per mg of protein; and Fig. 2d is APx activity into nmol per mg of protein. 298 P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 concentration. R. salina showed no significant differences between control and exposed samples, but a tendency for activity to increase for the model contaminants was seen. C. closterium showed an increase of SOD activity for copper and atrazine exposure, but it did not show any significant difference at different concentrations. Irgarol, at the tested concentrations, did not provoke any observable effect on the phytoplankton species for this biomarker. The basal levels of SOD activity were not the same in all species: C. closterium and P. tricornutum showed twice the activity level of R. salina (Fig. 2.) SOD is the first enzyme to act as an antioxidant defense system for plants; it transforms the O2 into H2O2 (Mittler, 2002). There are few previous studies on the response of SOD to contaminants in microalgae (El-Baky et al., 2004; Janknegt et al., 2007; Okamoto and Colepicolo, 1998). Most of them show activity increasing when algae are exposed to metals (Okamoto and Colepicolo, 1998) or herbicides (Bowler et al., 1992). Atrazine causes the greatest noticeable increase in SOD activity, but not in the case of R. salina which is a facultative heterotrophic cryptophyta, and this could explain the absence of measurable response to herbicides. Copper affects C. closterium, which has proven to be a very sensitive species to this substance (Araújo et al., 2010). Irgarol at the selected concentrations does not cause measurable effects in any of these species. 3.4. Catalase activity Catalase activity increased significantly (Po0.05) in comparison to controls in P. tricornutum for the tested substances, although the differences detected were dependent on concentration. For R. salina, CAT activity only increased in the samples exposed at the lower copper concentration (5 mg L 1) and C. closterium did not show any significant variation with respect to the controls. In this case again basal levels of the three species do not match. P. tricornutum has double the basal levels of R. salina, and C. closterium presents more than three times the basal activity level of R. salina (Fig. 3.) Catalase is an enzyme that transforms relatively large amounts of H2O2 into water and oxygen and, therefore, its activity relates to the detoxification of this ROI during oxidative stress (Mittler, 2002). Several studies have demonstrated the increase of catalase activity by exposing microalgae populations to copper (Morelli and Scarano, 2004; Manimaran et al. (2012)); however, those studies were performed with longer exposures and/or higher doses than those used in this study; and in the case of Morelli and Scarano (2004) high concentrations have been used (0.6 mg L 1), provoking thus an increase in CAT activity of 200 Percent. Manimaran et al. (2012) used a concentration of 0.02 mg L 1 (similar to the lower dose used in this study) and an exposure time of one week. In that study, a dramatic increase in CAT activity was observed in populations of Odontella mobilionsis. Regarding CAT response, P. tricornutum is the most sensitive species to all the treatments tested. R. salina showed a significant increase in CAT activity only when exposed to copper. CAT activity in C. closterium appears to be unaffected by the treatments. 3.5. Glutathione peroxidase activity After 24 h exposure to sub-lethal concentrations of the three pollutants, GPx activity in P. tricornutum showed a significant reduction (P o0.05) for all treatments except at the higher Irgarol concentration, which did not cause variations. R. salina showed increased GPx activity in the samples exposed to the lower doses of copper (5 mg L 1) and Irgarol (0.5 mg L 1). In C. closterium copper did not cause variations in GPx activity, while atrazine and Irgarol provoked enzyme inhibition. R. salina and C. closterium showed similar basal levels of GPx activity, whereas P. tricornutum showed five times more GPx activity (Fig. 3). As happens with SOD and CAT activity, GPx activity is increased or inhibited depending on the species, the pollutant, the concentration and the exposure time. In the case of copper, Li et al. (2006) have observed that GPx activity in the algae Pavlova viridis increased when it was exposed to copper concentrations higher than 0.5 mg L 1 for 24 h. On the other hand, Srivastava et al. (2006) observed that GPx activity was inhibited in the aquatic plant Hydrilla verticilata after being exposed to copper concentrations of 0.3 mg L 1 for 48 h. For herbicides such as atrazine and Irgarol, there are no studies in which GPx activity in algae has been measured. However, there is ample literature in higher plants, and many studies indicate the inhibition of GPx when plants are exposed to these substances (Hassan and Nemat Alla, 2005; Light et al., 2005). 3.6. Ascorbate peroxidase activity APx activity did not show significant changes (P o0.05) with respect to the control in P. tricornutum for any of the substances, except for samples exposed to the lower dose of Irgarol (0.5 mg L 1). In the case of R. salina, APx activity was significantly inhibited by exposure to the three substances; C. closterium however did not show any variation compared to control. Basal levels in P. tricornutum and R. salina were similar, while in C. closterium the basal level of activity was double. APx, GPx and CAT are enzymes responsible for the detoxification of H2O2 in plants. APx and GPx act together in the glutathione– ascorbate cycle to convert H2O2 to O2 and H2O. Since the affinity of both enzymes for hydrogen peroxide is three orders of magnitude lower than catalase, this suggests that CAT is primarily responsible for the degradation of H2O2 which produce large quantities of this ROI (Mittler, 2002). This would explain why, in this case, the APx activity did not increase compared to the control, and is even inhibited, as reported in Morelli and Scarano (2004). 3.7. Integrated response SOD is the first enzyme to act on the antioxidant defense system of plants, transforming O2 into H2O2. CAT, GPx and APx, however, are responsible subsequently for converting H2O2 to H2O and O2 (Mittler, 2002). CAT is primarily responsible for detoxification during stress, when H2O2 levels are high, while APx and GPx act together to degrade H2O2 with a lower affinity and a finer modulation (Asada and Takahashi, 1974), possibly linked to the functions that these enzymes have as secondary messengers. When the microalgae are exposed to the contaminant, GPx and APx activities decrease. This pollutant stress can provoke suppression of photosynthesis or damage to the photosynthetic apparatus, and the ascorbate–glutathione cycle reduces the energy available for ROI-scavenging. However, because CAT and SOD do not require a supply of reducing equivalents for their function, they might be insensitive to the redox status of cells and their function might not be affected during stress, unlike the other mechanisms (Mittler, 2002). In this study, we can observe clearly this behavior in the case of P. tricornutum. In all exposures, the activity of SOD and CAT increases, compared to control samples; in addition, GPx, APx or both are inhibited, also in all cases. Therefore, we can say that it could be a perfect example of a situation of oxidative stress, and this species would be a good candidate to work with as a bioindicator of oxidative stress. For R. salina, CAT activity does not increase or inhibit in any of the cases, while SOD activity increases in some cases, but not significantly (Po 0.05). In those cases in which SOD appears to increase (copper 10 μg L 1 and Irgarol 1 μg L 1), GPx increases P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 Fig. 3. SOD, CAT, GPx and APx activities are plotted in an integrated way for each species and each exposure condition. 299 300 P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 too. APx was inhibited in all exposures. This species was not affected by these contaminants at the measured doses and the exposure time. As mentioned previously, R. salina is a facultative heterotrophic, which could explain the lack of measurable response to herbicides. Basal levels of CAT in C. closterium are higher than the basal levels of the other species assayed, and this level is maintained after exposure to the selected pollutants. SOD activity increased in all cases except for Irgarol. GPx and APx do not vary in the case of copper but they are inhibited when exposed to atrazine and the higher concentration of Irgarol. The results show that C. closterium supports a high level of oxidative stress. This benthic species lives half buried in the sediment to protect itself from intense light, and to avoid oxidation. Nevertheless, C. closterium does show oxidative stress depending on concentration and exposure. This species could serve as a good “early warning” bioindicator for the benthic ecosystem. Although higher concentrations of the pollutants or longer exposure times would surely produce more evident effects on microalgal anti-oxidant enzymatic systems, in this study it was decided to keep concentrations at environmentally-realistic values. Additionally, short exposure times can improve the efficiency of potential monitoring bioassays. The aim of this study has been to confirm the basis for the design of useful enzyme activity bioassays for coastal biomonitoring capable of the rapid detection of differences in natural populations submitted to toxic stress. Further research focused on natural waters from actual locations, with natural mixed populations of microalgae, needs to be carried out in the future. 4. Conclusions Marine microalgae are proposed as excellent bioindicators of pollution due to their high sensitivity, which can give warning of the toxic effects of chemicals sooner than any other species. The microalgae used in this study showed significant responses in oxidative stress biomarkers for environmentally-relevant concentrations and short exposure periods, conditions under which most organisms used in assays of this kind do not show differences in levels of enzyme activity. An IBR index could be an useful tool to draw a general pattern of response against oxidative stress because it allow to represent the complex interaction between the different enzymes that work in the scavenging of the oxidative damage. Each species presents a different enzymatic activity under the same conditions. This means that the most appropriate measurement of the response of the population of algae in any particular location will be a combination of the responses of the species involved. Acknowledgments This research was funded by the project PHYTOBIOMARK (CTM2009-10563/MAR). References Anderson, D.M., Morel, F.M.M., 1978. Copper sensitivity of Gonyaulax tamarensis. Limnol. Oceanogr. 23 (2), 283–294. Araújo, C.V.M., Diz, F.R., Lubián, L.M., Blasco, J., Moreno Garrido, I., 2010. Sensitivity of Cylindrotheca closterium to copper: influence of three endpoints and two test methods. Sci. Total Environ. 408, 3696–3703. Araújo, C.V.M., Moreno-Garrido, I., Diz, F.R., Lubián, L.M., Blasco, J., 2008. Effects of cold-dark storage on growth of Cylindrotheca closterium and its sensitivity to copper. Chemosphere 72, 1366–1372. Asada, K., Takahashi, M., 1974. Production and scavenging of active oxygen in photosynthesis. In: Kyle, D.J., et al. (Eds.), Photoinhibition. Elsevier, pp. 227–287 Barreiro, R., Picado, L., Real, C., 2002. Biomonitoring heavy metals in estuaries: a field comparison of two brown algae species inhabiting upper estuarine reaches. Environ. Monit. Assess. 75, 121–134. Beers, R.F., Sizer, I.W.A., 1952. Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Beliaeff, B., Burgeot, T., 2002. Integrated biomarker response: a useful tool for ecological risk assessment. Environ. Toxicol. Chem. 21 (6), 1316–1322. Bérard, A., Dorigo, U., Mercier, I., Becker-van Slooten, K., Grandjean, D., Leboulanger, C., 2003. Comparison of the ecotoxicological impact of triazines Irgarol 1051 and atrazine on microalgal cultures and natural microalgal communities in Lake Geneva. Chemosphere 53, 935–944. Bowler, C., Montagu, M.V., Inze, D., 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 83–116. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation methods. Enzymology 52, 302–310. Cima, F., Ferrari, G., Ferreira, N.G.C., Rocha, R.J.M., Serôdio, J., Loureiro, S., Calado, R., 2013. Preliminary evaluation of the toxic effects of the antifouling biocide SeaNine 211TM in the soft coral Sarcophyton cf. glaucum (Octocorallia, Alcyonacea) based on PAM fluorometry and biomarkers. Marine Environ. Res. 93, 16–22. Conti, M.E., Lacobucci, M., Cecchetti, G.A., 2007. Biomonitoring study: trace metals in seagrass, algae and mollusks in a reference marine ecosystem (Southern Thyrrenian Sea). Int. J. Environ. Pollut. 29, 308–332. Dewez, D., Geoffroy, L., Vernet, G., Popovic, R., 2005. Determination of photosynthetic and enzymatic biomarkers sensitivity used to evaluate toxic effects of copper and fludioxonil in alga Scenedesmus obliquus. Aquat. Toxicol. 74, 150–159. Ebenezer, V., Ki, J.S., 2013. Physiological and biochemical responses of the marine dinoflagellate Prorocentrum minimum exposed to the oxidizing biocide chlorine. Ecotoxicol. Environ. Saf. 92, 129–134. El-Baky, H.H.A., El-Baz, F.K., El-Baroty, G.S., 2004. Production of antioxidant by the Green Alga Dunaliella salina. Int. J. Agric. Biol. 6 (1), 1560–8530. Fuerst, E.P., Norman, M.A., 1991. Interactions of herbicides with photosynthetic electron transport. Weed Sci. 39, 458–464. Guillard, R.R.L., Ryther, J.H., 1962. Studies on marine planktonic diatoms, I. Cyclotella nana Hustedt and Detonula confervaceae (Cleve) Gran. Can. J. Microbiol. 8, 229–239. Günzler, W.A., Flohe, L., 1985. Glutathione peroxidase. In: Greenwald, R.A. (Ed.), Handbook of Methods for Oxygen Research. CRC press, Boca Raton, FL, pp. 265–285 Hassan, N.M., Nemat Alla, M.M., 2005. Oxidative stress in herbicide-treated broad bean and maize plants. Acta Physiol. Plant. 27 (4), 429–438. Hook, S.E., Osborn, H.L., 2012. Comparison of toxicity and transcriptomic profiles in a diatom exposed to oil, dispersants, dispersed oil. Aquat. Toxicol., 139–151 Hourmant, A., Amara, A., Pouline, P., Durand, G., Arzul., G., Quiniou, F., 2009. Effect of Bentazon on growth and physiological responses of marine diatom: Chaetoceros gracilis. Toxicol. Mechan. Methods 19 (2), 109–115. Janero, D.R., 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic. Biol. Med. 9 (6), 515–540. Janknegt, P.J., Marco de Graaff, C., van de Poll, W.H., Visser, R.J.W., Rijstenbil, J.W., Buma, A.G.J., 2009. Short-term antioxidative responses of 15 microalgae exposed to excessive irradiance including ultraviolet radiation. Eur. J. Phycol. 44 (4), 525–539. Janknegt, P.J., Rijstenbil, J.W., van de Poll, W.H., Gechev, T.S., Buma, A.G., 2007. A Comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae. J. Photochem. Photobiol. 87, 218–226. Law, M.Y., Charles, S.A., Halliwell, B., 1983. Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplast. The effect of hydrogen peroxide and paraquat. Biochem. J. 210, 899–903. Li, M., Hu, C., Zhu, Q., Chen, L., Kong, Z., Liu, Z., 2006. Copper and zinc induction of lipid peroxidation and effects on an antioxidant enzyme activities in the microalga Pavlova viridis (Prymnesiophyceae). Chemosphere 62, 565–572. Li, X., Ping, X., Xiumei, S., Zhenbin, W., Liqiang, X., 2005. Toxicity of cypermethrin on growth, pigments, and superoxide dismutase of Scenedesmus obliquus. Ecotoxicol. Environ. Saf. 60, 188–192. Light, G.G., Mahan, J.R., Roxas, V.P., Allen, R.D., 2005. Transgenic cotton (Gossypium hirsutum L.) seedlings expressing a tobacco glutathione S-transferase fail to provide improved stress tolerance. Planta 222 (2), 346–354. Liu, Y., Guan, Y., Gao, Q., Tam, N.F.Y., Zhu, W., 2010. Cellular responses, biodegradation and bioaccumulation of endocrine disrupting chemicals in marine diatom Navicula incerta. Chemosphere 80 (5), 592–599. Lubián, L.M., Yúfera, M. 1989. Colección de Cepas de Microalgas del Instituto de Ciencias Marinas de Andalucía (CSIC). Acuicultura intermareal. M. Yúfera. Instituto de Ciencias Marinas de Andalucía, Cádiz. Manimaran, K., Karthikeyan, P., Ashokkumar, S., Prabu, V.A., Sampathkumar, P., 2012. Effect of copper on growth and enzyme activities of marine diatom, Odontella mobiliensis. Bull. Environ. Contam. Toxicol. 88, 30–37. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Morelli, E., Marangi, M.L., Fantozzi, L.A., 2009. A phytochelatin-based bioassay in marine diatoms useful for the assessment of bioavailability of heavy metals released by polluted streams. Environ. Intern. 35 (5), 532–538. P. Lozano et al. / Ecotoxicology and Environmental Safety 104 (2014) 294–301 Morelli, E., Scarano, G., 2004. Copper-induced changes of non-protein thiols and antioxidant enzymes in the marine microalga Phaeodactylum tricornutum. Plant Sci. 167, 289–296. Moreno-Garrido, I., Lubián, L., Soares, A.M.V.M., 1999. Oxygen production rate as a test for determining toxicity of copper to Rhodomonas salina Hill & Wetherbee (Cryptophyceae). Bull. Environ. Cont. Tox. 62, 776–782. Nö dler, K., Licha, T., Voutsa, D., 2013. Twenty years later – Atrazine concentrations in selected coastal waters of the Mediterranean and the Baltic Sea. Mar. Pollut. Bull. 70, 112–118. Nyholm, N., Källqvist, T., 1989. Methods for growth inhibition toxicity tests with freshwater algae. Environ. Toxicol. Chem. 8, 689–703. Nyströ m, B., Slooten, K.V., Bérard, A., Grandjean, D., Druart, J.C., Leboulanger, C., 2002. Toxic efects of Irgarol 1051 on phytoplankton and macrophytes in Lake Geneva. Water Res. 36, 2020–2028. OECD, Paris, France Okamoto, O.K., Colepicolo, P., 1998. Response of superoxide dismutase to pollutant metal stress in marine dinoflagelate, Gonyaulax polyedra. Comp. Biochem. Physiol. 119C (1), 67–73. Solomon, K.R., Baker, D.B., Richards, R.P., Dixon, K.R., Klaine, S.J., La Point, T.W., Kendall, R.J., Weisskopf, C.P., Giddings, J.M., Giesy, J.P., Hall, L.W., Williams, W.M., 301 1996. Ecological risk assessment of atrazine in North American surface waters. Environ. Toxicol. Chem. 15 (1), 31–76. Soto, P., Gaete, H., Hidalgo, M.E., 2011. Assessment of catalase activity, lipid peroxidation, chlorophyll-a, and growth rate in the freshwater green algae Pseudokirchneriella subcapitata exposed to copper and zinc. Lat. Am. J. Aquat. Res. 39 (2), 280–285. Srivastava, S., Mishra, S., Tripathi, R.D., Dwivedi, S., Gupta, D.K., 2006. Copperinduced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle. Aquat. Toxicol. 80 (4), 405–415. Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M., 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol. Environ. Saf. 64 (2), 178–189. Vavilin, D.V., Ducruet, J.M., Matorin, D.N., Venediktov, P.S., Rubin, A.B., 1998. Membrane lipid peroxidation, cell viability and Photosystem II activity in the green alga Chlorella pyrenoidosa subjected to various stress conditions. J. Photochem. Photobiol. B: Biol. 42 (3), 233–239. Volterra, L., Conti, M.E., 2000. Algae as biomarkers, bioaccumulators and toxin producers. Int. J. Environ. Pollut. 13, 92–125.