Raquel Prado

Comparison of the sensitivity of different toxicity test endpoints in a microalga exposed to the herbicide paraquat.

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. Toxic effects of these pollutants on microalgae are generally evaluated using phytotoxicity tests based on growth inhibition, a population-based parameter. However, physiological cellular endpoints could allow early detection of cell stress and elucidate underlying toxicity mechanisms. Effects of the herbicide paraquat on the freshwater microalga Chlamydomonas moewusii were studied to evaluate growth rate and cellular parameters such as cellular viability and metabolic activity assayed by flow cytometry and DNA damage assayed by the comet assay. Sensitivity of growth and parameters assayed by flow cytometry were similar, showing a significant effect in cultures exposed to a paraquat concentration of 0.1 microM or higher, although in cultures exposed during 48 h to 0.05 microM, a significant stimulation of cellular fluorescein fluorescence was observed, related to cellular metabolic activity. After only 24 h of herbicide exposure significant DNA damage was observed in microalgal cells exposed to all paraquat concentrations assayed, with a 23.67% of comets in cultures exposed to 0.05 microM, revealing the genotoxicity of this herbicide. Taking into account the results obtained, comet assay provides a sensitive and rapid system for measuring primary DNA damage in Chlamydomonas moewusii, which could be an important aspect of environmental genotoxicity monitoring in surface waters.

Cell proliferation alterations in Chlorella cells under stress conditions

Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and reproduction at microalgal cell level.

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

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 microM. 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.

Ring test for whole-sediment toxicity assay with a benthic marine diatom

This work presents the results of an interlaboratory proficiency exercise for whole-sediment toxicity assays with the benthic marine diatom Cylindrotheca closterium. An assay protocol was established and followed by all participating laboratories. Cell growth after 72 h exposure was the endpoint used. Four sediment samples of unknown toxicity were assayed. The main problem encountered during this exercise was the differences in the cell growth of algae exposed to reference sediment. Those differences may be associated with changes in the physiological status of the initial culture due to temperature changes during transport to the other laboratories. In general, the method proposed presented good replicability (precision between replicates) and reproducibility (interlaboratory precision). Around 80% (17 out of 21) of results obtained were classified as satisfactory (Z-scores <2). The whole-sediment assay with C. closterium presented here can be considered sufficiently successful for possible use as a standard toxicity test. The assay is simple to perform, the proposed species is ecologically relevant as an integral component of microphytobenthos, and is widely distributed around the world. These positive factors suggest that the whole-sediment assay with the benthic marine diatom C. closterium can be used as a reliable tool in marine sediment quality assessment.

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 1h 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.

Suitability of cytotoxicity endpoints and test microalgal species to disclose the toxic effect of common aquatic pollutants

Pulse discharges of chemicals to aquatic environments may lead to high concentrations of them in surface waters for short periods of time, but enough to induce toxic effects on aquatic organisms; however, no many methods allow an early warning of toxicity of these agents. Acute effects of one representative chemical from each of three of the main groups of aquatic pollutants (pesticides, metals and pharmaceuticals) are studied on two green microalgal species (Chlamydomonas moewusii and Chlorella vulgaris). Flow cytometry protocols were used to detect the potential application of chlorophyll a fluorescent emission, cell viability, metabolic activity and membrane potential as cytotoxicity endpoints, besides an epifluorescence microscopy protocol for comet assay to detect genotoxicity level of cells. Obtained results confirm the suitability of them for the prospective assessment of the potential cytotoxicity of these aquatic pollutants. The two microalgal species analysed could be used as indicators in toxicity bioassays, being C. moewusii more sensitive than C. vulgaris. Among cell parameters assayed, the metabolic activity and the primary DNA damage stood out as sensitive cytotoxicity endpoints.