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Microalgal toxicity tests using integrative endpoints as algal growth are regularly required to analyse the toxicity of potentially hazardous substances in the aquatic environment. However, these do not provide mechanistic information on the toxic mode of action by which contaminants may affect algae. Bottled waters can be used as a substitute for culturing media and should not impose any stress to the cultured organisms. However, certain chemical components can interfere with specific cell targets which are not revealed by general toxicity assays. The present study investigated the sensitivity of flow cytometry (FCM) to analyse sub-lethal effects of different bottled waters to the freshwater microalgae Raphidocelis subcapitata. Several endpoints were analysed including growth rate, natural pigments content, cell size, complexity, viability and cycle, Reactive Oxygen Species (ROS) formation, mitochondrial membrane potential and Lipid Peroxidation (LPO). Additionally, photosystem II (PSII) performance was analysed by PAM fluorometry, to provide further information on the absorption, distribution and use of energy in photosynthesis. Results indicated that the most sensitive endpoints were the oxidative stress related endpoints ROS formation and LPO, pigment content, morphological endpoints as cell size, complexity and cycle, with growth rate being one of the least sensitive. Although being essential macronutrients for algal growth, the chemical elements Ca, Na, Mg, and NH 4 were identified as being primarily responsible for the observed toxicological effects to exposed algae. The applied methodology proved to be of high throughput, simultaneously assembling information on morphological, biochemical, and physiological status of algal cells. FCM also showed potential to reveal mechanistic information on the toxic mode of action of the bottled waters before any effects on algal growth was observed. The used approach demonstrated its potential for being integrated into future microalgal toxicity bioassays for testing chemicals to improve the hazard information obtained from currently approved internationally accepted test guidelines. Unlabelled Image • Sub-lethal toxicity of bottled waters to Raphidocelis subcapitata • Reactive oxygen species formation, lipid peroxidation, effects in pigments, cell size, complexity and cell cycle observed • Ca, Na, Mg, and NH 4 primarily responsible for the observed effects • The methodological approach effectively revealed the observed mechanistic effects. [ABSTRACT FROM AUTHOR]

Pharmaceuticals are produced to inflict a specific physiological response in organisms. However, as only partially metabolized after administration, these types of compounds can also originate harmful side effects to non-target organisms. Additionally, there is still a lack of knowledge on the toxicological effects of legacy pharmaceuticals such as the antibiotic azithromycin. This macrolide occurs at high concentrations in the aquatic environment and can constitute a threat to aquatic organisms that are at the basis of the aquatic food chain, namely microalgae. This study established a high‐throughput methodology to study the toxicity of azithromycin to the freshwater microalga Raphidocelis subcapitata. Flow cytometry and pulse amplitude modulated (PAM) fluorometry were used as screening tools. General toxicity was shown by effects in growth rate, cell size, cell complexity, cell viability and cell cycle. More specific outcomes were indicated by the analysis of mitochondrial and cytoplasmatic membrane potentials, DNA content, formation of ROS and LPO, natural pigments content and photosystem II performance. The specific mode of action (MoA) of azithromycin to crucial components of microalgae cells was revealed. Azithromycin had a negative impact on the regulation of energy dissipation at the PSII centers, along with an insufficient protection by the regulatory mechanisms leading to photodamage. The blockage of photosynthetic electrons led to ROS formation and consequent oxidative damage, affecting membranes and DNA. Overall, the used methodology exhibited its high potential for detecting the toxic MoA of compounds in microalgae and should be considered for future risk assessment of pharmaceuticals. [Display omitted] • Flow cytometry and PAM fluorometry detected toxicity of azithromycin to microalgae. • Azithromycin negatively impacted regulation of energy dissipation at Photosystem II. • Formation of ROS and consequent oxidative damage affected membranes and DNA. • Methodology revealed mechanistic effects of azithromycin to Raphidocelis subcapitata. [ABSTRACT FROM AUTHOR]

Due to their small size, nanoplastics (NPLs) possess specific properties which can potentiate their toxicity towards aquatic organisms. As primary producers, microalgae are at the base of aquatic food chains, thus negative impacts of NPLs will likely lead to disturbances in ecosystem productivity. The majority of data available on the toxicity of NPLs is limited to polystyrene and green microalgae, leaving a significant lack of knowledge on impacts of other polymer types across different taxonomic groups. So, the main objective of this study was to evaluate the cell responses of the red microalgae Rhodomonas baltica to plain and carboxylated poly(methyl methacrylate) NPLs (PMMA and PMMA-COOH, 50 nm). Results showed different NPL behaviour in media over time, with PMMA forming micro-scale aggregates and PMMA-COOH maintaining its nominal size range. PMMA caused a higher impact in cellular and physiological parameters than PMMA-COOH, even though a decrease in algal growth was only seen for the later. Overall, PMMA caused a significant decrease in cell viability followed by an increase in cell size and complexity, overproduction of pigments, loss of membrane integrity, hyperpolarization of the mitochondrial membrane, increased production of ROS and LPO, decrease in DNA content and reduced photosynthetic capacity. Conversely, a decrease in algal growth for PMMA-COOH was connected to an impairment in cell cycle and consequent decrease in cell viability, metabolic activity and photosynthetic performance, with negligible effects in ROS formation and pigments content. This study provided a first insight into the mechanistic understanding of the toxic impacts of PMMA and PMMA-COOH NPLs in red microalgae. Results obtained suggest an interaction between both NPLs and R. baltica cell surface that is dependent on particle behaviour and surface chemistry. Future experiments focusing on the in-depth characterization of the mode of action of these particles are recommended. Unlabelled Image • Cell responses of Rhodomonas baltica to nanosized PMMA and PMMA-COOH was evaluated. • Interaction between nanoplastics and microalgae was dependent on particle behaviour. • Exposure to PMMA caused a higher impact in cellular and physiological parameters. • Growth inhibition by PMMA-COOH linked to effects in cell cycle and cell viability • First mechanistic insight on the toxicity of PMMA nanoplastics in marine microalgae [ABSTRACT FROM AUTHOR]