Due to the growing concern about the presence of microplastics (MP) in the environment, the number of studies evaluating the toxicity of these small persistent particles on different marine species has increased in recent years. Few studies have addressed their impact on marine phytoplankton, a subject of great concern since they are primary producers of the aquatic food web. The aim of this study is to unravel the cytotoxicity of 2.5 μg mL−1 unlabelled amino-modified polystyrene beads of different sizes (0.5 and 2 μm) on the marine diatom Chaetoceros neogracile. In addition to traditional growth and photosynthesis endpoints, several physiological and biochemical parameters were monitored every 24 h in C. neogracile cells by flow cytometry during their exponential growth (72 h). Dynamic Light Scattering measurements revealed the strong aggregation and the negative charge of the beads assayed in the culture medium, which seemed to minimize particle interaction with cells and potentially associated impacts. Indeed, MP were not attached to the microalgal cell wall, as evidenced by scanning electron micrographs. Cell growth, morphology, photosynthesis, reactive oxygen species levels and membrane potential remained unaltered. However, exposure to MP significantly decreased the cellular esterase activity and the neutral lipid content. Microalgal oil bodies could serve as an energy source for maintaining a healthy cellular status. Thus, MP-exposed cells modulate their energy metabolism to properly acclimate to the stress conditions. Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site.

Chlamydomonas reinhardtii cells were exposed to a sublethal concentration of the widespread herbicide atrazine for 3 and 24h. Physiological parameters related to cellular energy status, such as cellular activity and mitochondrial and cytoplasmic membrane potentials, monitored by flow cytometry, were altered in microalgal cells exposed to 0.25μM of atrazine. Transcriptomic analyses, carried out by RNA-Seq technique, displayed 12 differentially expressed genes between control cultures and atrazine-exposed cultures at both tested times. Many cellular processes were affected, but the most significant changes were observed in genes implicated in amino acid catabolism and respiratory cellular process. Obtained results suggest that photosynthesis inhibition by atrazine leads cells to get energy through a heterotrophic metabolism to maintain their viability.

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.

Large quantities of personal care products (PCPs) are used daily and many of their chemical ingredients are subsequently released into marine environments. Cultures of the marine microalga Tetraselmis suecica were exposed for 24 h to three emerging compounds included in the main classes of PCPs: the UV filter benzophenone-3 (BP-3), the disinfectant triclosan (TCS) and the fragrance tonalide (AHTN). Concentrations tested, expressed as cellular quota (pg cell), ranged from 5 to 40 for BP-3, from 2 to 16 for TCS and from 1.2 to 2.4 for AHTN. A small cytometric panel was carried out to evaluate key cytotoxicity biomarkers including inherent cell properties, growth and metabolic activity and cytoplasmic membrane properties. BP-3 caused a significant increase in growth rate, metabolic activity and chlorophyll a fluorescence from 10 pg cell. However, growth and esterase activity decreased in cells exposed to all TCS and AHTN concentrations, except the lowest ones. Also these two compounds provoked a significant swelling of cells, more pronounced in the case of TCS-exposed cells. Although all treated cells remained viable, changes in membrane potential were observed. BP-3 and AHTN caused a significant depolarization of cells from 10 to 1.6 pg cell, respectively; however all TCS concentrations assayed caused a noticeable hyperpolarization of cells. Metabolic activity and cytoplasmic membrane potential were the most sensitive parameters. It can be concluded that the toxicological model used and the toxicological parameters evaluated are suitable to assess the toxicity of these emerging contaminants.

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