Characterization of cell response in Chlamydomonas moewusiicultures exposed to the herbicide paraquat: Induction of chlorosis

[Abstract] The use of herbicides constitutes the principal method of weed control, but the introduction of these compounds into the aquatic environment can provoke severe consequences for non-target organisms such as microalgae. Effects of the widely used herbicide paraquat were assessed on the green freshwater microalga Chlamydomonas moewusii by means of the analysis of its photosynthetic pigment content, using a traditional spectrophotometric technique that provides population bulk measurements, and by means of flow cytometry, which allowed characterizing the microalgal response at a single-cell level. Results obtained reveal that paraquat concentrations above 50 nM induce chlorosis in a percentage of microalgal cells depending on herbicide concentration and exposure time, as reflected by a reduced cell chlorophyll autofluorescence and pigment content of the biomass. Cell viability in these cultures was also reduced in a concentration dependent way. The possibility of analysing chlorotic and non-chlorotic subpopulations separately allowed the study of morphological properties and physiological status of both cell types, leading to the conclusion that chlorotic cells are non-viable cells, based on their reduced size and complexity and their inability to be stained in the fluorescein diacetate assay. In the case of non-chlorotic cells, cell viability was reduced with the increase of paraquat concentration. Non-chlorotic cells in these cultures showed an increased size and complexity in comparison with control cells, probably due to a growth inhibition. Chlorophyll fluorescence was the most sensitive parameter since even cells exposed to the lowest concentration assayed, 50 nM, although not chlorotic, showed a significantly reduced chlorophyll fluorescence with respect to control cells, reflected also by a reduced chlorophyll content of the biomass.Galicia: Consellería de Innovación, Industria e Comercio; 08MDS020103PRMinisterio de Educación y Ciencia; CGL2004-02037

Screening acute cytotoxicity biomarkers using a microalga as test organism

The present study checked the suitability of the integration of flow cytometry (FCM) as technique and a freshwater microalga (Chlamydomonas moewusii) as cell system model for ecotoxicological studies, looking for sensitive biomarkers of acute cytotoxicity of potential contaminants in aquatic systems. The detection of the potential acute toxicity of a pollutant is of interest because pulse discharges of contaminants to natural waters could lead to high concentrations of these substances that are only present for short periods of time but can affect aquatic organisms such as microalgae. Physiological alterations in C. moewusii cells were analysed after 1 h of exposure to different concentrations of the herbicide paraquat. Cell viability was not affected, but the acute toxicity of paraquat was evident at other levels of cell physiology. Herbicide-treated cells showed lower autofluorescence and higher size and internal complexity, lower esterase activity and lower mitochondrial membrane potential. Paraquat induced the depolarisation of the plasma membrane and the increase of intracellular free calcium level and cytosolic pH in a concentration-dependent percentage of cells. All these effects can be related to the oxidative stress induced by the herbicide, as revealed the significantly increased intracellular levels of reactive oxygen species in cultures exposed to paraquat concentrations which induced the physiological alterations mentioned above. Excluding cell viability and mitochondrial membrane potential, these cytotoxicity endpoints could be considered sensitive biomarkers for the short-term exposure to pollutants such as herbicides. Highlights: • This study examine the acute cytotoxicity of paraquat on microalgal physiology. • Flow cytometric protocols assayed allow the screening of different toxicity cellular endpoints. ► Cell viability is not a sensitive biomarker. • Short-term paraquat exposure induces alteration in the overall cellular ionic homeostasis. • Most alterations observed could be related with the overproduction of ROS.Xunta de Galicia; 08MDS020103P

The herbicide paraquat induces alterations in the elemental and biochemical composition of non-target microalgal species

[Abstract]Huge quantities of pesticides are dispersed in the environment, affecting non-target organisms. Since paraquat affects the photosynthetic process, the biochemical composition of affected species should be altered. The effect of paraquat on Chlamydomonas moewusii, a freshwater non-target species, was studied. After 48 h of herbicide exposure, growth rate, dry weight, and chlorophyll a and protein content were affected by paraquat concentrations above 0.05 μM. C/N ratio was also affected due to a decrease in nitrogen content in the dry biomass, while the carbon content remained constant for all paraquat concentrations assayed. Enzymes involved in nitrogen assimilation were affected by paraquat, being nitrate reductase activity more sensitive to paraquat than nitrite reductase. Based on the results obtained in the present study, paraquat exerts adverse effects upon a common freshwater green microalga, thus the application of this herbicide for weed control must be carried out very carefully, so that any disturbance affecting algae will have severe repercussions on higher trophic levels and on the elemental biogeochemical cycles.Ministerio de Educación y Ciencia; CGL 2004/02037 BO

Cytotoxic effects of pesticides on microalgae determined by flow cytometry

As much as ca. 99.7% of the applied load of pesticides is dispersed in the environment, not reaching the target pests. In this way, pesticides enter into aquatic ecosystems from agricultural runoff or leaching and, as a consequence, have become some of the most frequently ocurring organic pollutants in aquatic ecosystems. Most phytotoxicological research with herbicides has been conducted on target plants. The sensitivity of algae to many herbicides is very high, and a better understanding of their environmental effects is probably acquired by using test species representing non-target groups. Because of their short generation times, microalgae respond rapidly to environmental changes. Furthermore, microalgal tests are generally sensitive, rapid and low-cost effective. For these reasons, the use of microalgal toxicity tests is increasing, and today these tests are frequently required by authorities for notifications of chemicals and are also increasingly being used to manage chemical discharges. Cytotoxic effects of aquatic pollutants on microalgae are very heterogeneous, and they are influenced by the environmental conditions and the test species. Growth, photosynthesis, chlorophyll fluorescence and others parameters reflect the toxic effects of pollutants on microalgae; however, other relevant endpoints are less known because experimental difficulties, especially under in vivo conditions. Flow cytometry offers multiparametric analysis of cells on a cell-by-cell basis in near in vivo conditions. Applied in the study of the aquatic environments since the 80's, this technique has achieved extensive use for the study of microalgae and has been introduced as an alternative to more traditional techniques of analyzing cells in culture and from natural populations. Several parameters determined by flow cytometry, such as cell volume and granularity, chlorophyll a fluorescence, cell viability, cell proliferation, oxidative stress, membrane potential or intracellular calcium level, were employed to assess changes in the physiological status of different microalgae as a consequence of the toxic action of herbicides. The variety of results obtained in the present study reveals that flow cytometry is a useful tool in the toxicity tests with microalgae.Xunta de Galicia; PGIDIT04RFO103946PRMinisterio de Educación y Ciencia CGL2004-02037BO

Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints

Since recent evidence has demonstrated that many types of chemicals exhibit oxidative and/or genotoxic potential on living organisms, reactive oxygen species (ROS) formation and DNA damage are currently the best accepted paradigms to assess the potential hazardous biological effects of a wide range of contaminants. The goal of this study was to evaluate the sensitivity of different cytotoxicity and genotoxicity responses on the model microalga Chlamydomonas reinhardtii exposed to the prooxidant herbicide paraquat. In addition to the growth endpoint, cell viability, mitochondrial membrane potential and presence of reactive oxygen species (ROS) were assayed as potential markers of cytotoxicity using flow cytometry (FCM). To study the effects of paraquat on C. reinhardtii DNA, several genotoxicity approaches were implemented for the first time in an ecotoxicological study on microalgae. Oxidative DNA base damage was analysed by measuring the oxidative DNA lesion 8-OHdG by FCM. DNA fragmentation was analysed by different methods: comet assay, and cell cycle analysis by FCM, with a particular focus on the presence of subG1-nuclei. Finally, effects on morphology of nuclei were monitored through DAPI staining. The evaluation of these endpoints showed that several physiological and biochemical parameters reacted to oxidative stress disturbances with greater sensitivity than integrative parameters such as growth rates or cell viability. The experiments revealed concentration-dependent cytotoxicity (ROS formation, depolarization of mitochondrial membrane), genotoxicity (oxidative DNA damage, DNA strand breakage, alterations in nuclear morphology), and cell cycle disturbances (subG1-nuclei, decrease of 4N population) in paraquat-treated cells. Overall, the genotoxicity results indicate that the production of ROS caused by exposure to paraquat induces oxidative DNA damage followed by DNA single- and double-strand breaks and cell cycle alterations, possibly leading to apoptosis in C. reinhardtii cells. This is supported by the observation of typical hallmarks of apoptosis, such as mitochondrial membrane depolarization, alterations in nuclear morphology and subG1 nuclei in cells exposed to the highest assayed concentrations. To our knowledge, this is the first study that provides a comprehensive analysis of oxidative DNA base damage in unicellular algal cells exposed to a prooxidant pollutant, as well as of its possible relation with other physiological effects. These results reinforce the need for additional studies on the genotoxicity of environmental pollutants on ecologically relevant organisms such as microalgae that can provide a promising basis for the characterization of potential pollutant hazards in the aquatic environment

Population growth study of the rotifer Brachionus sp. fed with triazine-exposed microalgae

[Abstract]Few data exist on potential toxic effects that pollutants may have on zooplankton fed microalgae exposed to pesticides. For that reason, microalgal cultures were exposed to different concentrations of the triazine herbicide terbutryn, and used as exclusive food source to Brachionus sp. females, with the aim to evaluate potential deleterious effects upon population growth, survival, reproduction and feeding of the rotifer.Chlorella vulgaris cells were able to accumulate terbutryn, removing more than 90% of the total amount of herbicide in all the exposed cultures. Growth curves of Brachionus sp. showed that population density decreased as terbutryn concentration increased in the microalgal cells. In fact, this species of rotifer did not survive beyond four days when fed with microalgae exposed to 500 nM terbutryn. Percentage of reproductive females in rotifer populations fed with terbutryn-exposed microalgae decreased significantly as herbicide concentration increased. In control cultures, reproductive females laid a maximum of three eggs per individual; whereas in 100 nM cultures, reproductive females laid only one egg per individual throughout the treatment period. Terbutryn accumulated in C. vulgaris provoked a decrease in the feeding rate ofBrachionus sp. cultures fed with these microalgae with respect to control cultures. After this treatment period, all the rotifer populations, except those fed with 500 nM terbutryn-exposed microalgae, showed recovery patterns when they were returned to fresh medium containing herbicide-free microalga. Taking into account the results obtained, uptake of pesticides by phytoplankton can directly affect higher trophic levels.Xunta de Galicia; PGIDIT04RFO103946P

Toxicity induced by three antibiotics commonly used in aquaculture on the marine microalga Tetraselmis suecica (Kylin) Butch

Aquaculture facilities are a potential source of antibiotics to the aquatic ecosystems. The presence of these compounds in the environment may have deleterious effects on non-target aquatic organisms such as microalgae, which are often used as biological indicators of pollution. Therefore, the toxicity induced by chloramphenicol (CHL), florphenicol (FLO) and oxytetracycline (OTC), three antibiotics widely used in aquaculture, on the marine microalga Tetraselmis suecica was evaluated. Growth inhibition and physiological and biochemical parameters were analysed. All three antibiotics inhibited growth of T. suecica with 96 h IC50 values of 11.16, 9.03 and 17.25 mg L−1 for CHL, FLO and OTC, respectively. After 24 h of exposure no effects on growth were observed and cell viability was also unaffected, whereas a decrease in esterase activity, related with cell vitality, was observed at the higher concentrations assayed. Photosynthesis related parameters such as chlorophyll a cellular content and autofluorescence were also altered after 24 h of antibiotics addition. It can be concluded that T. suecica was sensitive to the three antibiotics tested

Physiological response of freshwater microalga (Chlorella vulgaris) to triazine and phenylurea herbicides

[Abstract]The effects of two herbicides used wide-spread, isoproturon (phenylurea) and terbutryn (triazine), on growth, dry weight, elemental composition, photosynthetic pigments and protein content, and cell volume assayed by flow cytometry in the freshwater microalgae Chlorella vulgaris were studied. Different parameters for algal activity show widely different sensitivities to these aquatic pollutants. After 96 h of herbicide exposure, terbutryn was the strongest inhibitor of growth, giving an EC50 value for growth twice lower than that for isoproturon cultures (EC50 terbutryn=0.097 μM; EC50 isoproturon=0.199 μM). However, lower concentrations of the triazine herbicide provoked an increase in the cellular density and growth rate of this microalga, not observed in the phenylurea-treated cultures. Cellular volume and dry weight of C. vulgaris cells were increased strongly in the presence of isoproturon and terbutryn. Other cellular parameters, such as pigment and protein content, were stimulated with both herbicides at higher concentrations.Xunta de Galicia; PGIDT01MAM10302P

Atrazine-Induced Chlorosis in Synechococcus elongatus Cells

The effects of a widely used herbicide, atrazine, on the freshwater cyanobacterium Synechococcus elongatus were studied. The cyanobacteria were exposed to varying concentrations of atrazine (0.025, 0.05, 0.1, 0.25, and 0.75 µM) for 96 h. Different parameters such as growth, autofluorescence of chlorophyll a, pigment content, volume, and internal granularityof the cells were determined daily. Differences were detected between cultures with and cultures without atrazine for the parameters analyzed. Atrazine exposure induced the process of chlorosis in cyanobacterial cells, given that this herbicide has an effect on photosynthesis, chlorotic subpopulations having low values of chlorophyll a autofluorescence. More unpigmented subpopulations (chlorotic) appeared as the atrazine concentration increased and better growth rates resulted. Atrazine also induced changes in cell volume and internal granularity, these being most apparent after 48 h of exposure and in cultures with higher atrazine concentrations (0.25 and 0.75 µM).Xunta de Galicia; PGIDT01MAM10302P

Flow cytometric analysis of the encystment process induced by paraquat exposure in Haematococcus pluvialis (Chlorophyceae)

The freshwater microalga Haematococcus pluvialis exhibits a unique morphological response to environmental stress, accumulating carotenoid pigment during encystment. The complexity of characterizing the different cell stages and monitoring the pigment cell content during the life cycle of this microalga is one of the main problems reported when assessing astaxanthin accumulation and degradation. Therefore, with the aim of studying the potential encystment response in this microalga by means of flow cytometry (FCM), we induced oxidative stress in cultures of vegetative growing cells by treating them with paraquat, a known generator of superoxide anion radicals. Two flow cytometric approaches were successfully used to monitor the effect of oxidative stress on morphological changes and genesis of carotenoids in H. pluvialis: (1) a cytometric characterization of different cell types based on analysis of the fluorescence of chlorophyll a vs the fluorescence of astaxanthin, and (2) staining with the fluorochromes hydroethidium (HE) and dihydrorhodamine 123 (DHR), in order to measure the in vivo intracellular levels of reactive oxygen species (ROS). FCM data showed that astaxanthin accumulation during encystment hampers the production of ROS. Furthermore, the cell content of astaxanthin seems to be a good indicator of the extent to which H. pluvialis cells undergo oxidative stress, and also of how the cells defend themselves under stress conditions.Xunta de Galicia; INCITE08ENA103032E