The measurement of induced oxidative stress and associated cellular damage in aquatic microorganisms has become, in the last years, a recognized end point and an accepted paradigm to assess and compare the toxicity of different micropollutants on microalgae . Consequently, the measurement of oxidative stress and associated cellular damage, together with standard ecotoxicological endpoints such as growth and photosynthesis inhibition are of high relevance for understanding the mode of micropollutant action. Toxic metals, nanoparticles, and organic compounds [2-5], and also some physical stressors like UV radiation  have the potential to cause oxidative damage in microalgae. The methods currently used to assess oxidative stress are numerous and range from direct quantification of reactive oxygen species (ROS) to the indirect evaluation of oxidative stress through measurement of the induced cellular effects . In studies focused on the evaluation of oxidative stress in algae, these methods are currently applied and are either based on the evaluation of cellular damage (e.g., lipid [8, 9] and protein oxidation ) or on the quantification of antioxidant enzymes and radical scavengers [11-13]. In recent years, the use of fluorescent probes specifically designed to detect ROS in vivo or to label ROS-induced cellular damage has notably increased since these methods couple high sensitivity with simple use . Among the fluorescent probes available for the investigation of oxidative stress in living cells, probes based on dihydrofluoresceine diacetate have been applied with microalgal cells [15, 16]. However, their use is limited by their high photosensitivity, low dye penetration in the cells and the necessity to add substances to enhance membrane permeability [17, 18]. To our knowledge there are no dyes that are systematically applied for the detection of lipid oxidation in microalgal cells, which is why in this work we propose a lipophilic fluorescent dye 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) to probe oxyl-radical induced lipid oxidation by flow cytometry (FCM). C11-BODIPY581/591 is a fatty acid analog with specific fluorescent properties . When excited with blue light at 488 nm wavelength, the molecule has a constitutive fluorescence emission with a maximum at 595 nm. Following oxyl-radical induced oxidation, the fluorescence emission shifts to shorter wavelengths with a maximum emission at 520 nm. Due to its lipophilic properties, the molecules easily enter the lipid bilayer and once inside the cellular membrane are subject to oxidation by oxyl-radicals together with the endogenous fatty acids . C11-BODIPY581/591 is a good candidate for routine analyses and ecotoxicity tests; it has a high quantum yield of fluorescence emission that assures a good signal measurement even at low concentration, and it has high photostability and its fluorescence emission is not prone to pH changes and solvent polarity. Moreover, its oxidation induced fluorescence shift can be triggered only by oxy-radicals and not by superoxides, nitric oxides, transition metal ions and hydroperoxides . The last feature is particularly important in ecotoxicity testing since the specificity of the dye for radical species reduces the occurrence of false positives generated by the reaction of the probe with other chemicals present in the medium. C11-BODIPY581/591 has been already proposed to determine lipid peroxidation  and has been used as a lipid peroxidation probe in mammalian cells [21-25]. Furthermore, comparison of lipid peroxidation obtained by using C11-BODIPY581/591 with mass spectrometry quantification of an oxidized lipid, the 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine, revealed the tendency of C11-BODIPY581/591 to overestimate lipid peroxidation in the cells but confirmed its capability to highlight oxidative stress conditions at the membrane level that have the potential to cause lipid peroxidation . Dyes from the BODIPY-family have been also applied in studies on microalgae, notably in algal biotechnology for the evaluation of intracellular lipids  and the estimation of the antioxidant activity of algal pigments . Recently, the potential of this dye was explored to study lipid peroxidation induced by silver nanoparticles on the microalgae Chlorella vulgaris and Dunaliella tertiolecta . In this study, the results obtained with C11-BODIPY581/591 positively correlated with the measurement of cellular oxidative stress determined by a fluorescein diacetate probe. However, no information was reported concerning the staining procedure. To our knowledge, no other studies with microalgae were published using this dye in an ecotoxicological context. Therefore, there is a lack of systematic knowledge about the influence of different staining conditions on the dye performance in microalgal species, as well as no established procedures.
FCM is a single cell technique that provides a multi-parameter assessment of the physiological state of individual cells of a population. In studies concerning algae, this technique has been extensively applied to investigate the distribution of phytoplankton in natural environments  and to discriminate, identify and isolate algal species [27, 28]. Because of its capability to discriminate different morphological features in cells of the same population, this technique is increasingly applied in the optimization of biotechnological microalgal processes  and in ecotoxicological studies [17, 30-34]. A combination of FCM and appropriate fluorescent dyes is used in ecotoxicology to investigate the effects induced either by metallic or organic micropollutants. Examples include testing altered membrane permeability with the propidium iodide staining [30, 31], esterase activity by FDA staining [32-34] and oxidative stress using different probes like 5-(and-6)-carboxy-2′,7′-dihydrodifluorofluorescein di-acetate (H2DFFDA) , 2′,7′-dichlorodihydro-fluorescein diacetate (H2DCFDA)  or dihydrorhodamine 123 (DHR123) [31, 33]. Nonetheless, the potential of FCM in the field of ecotoxicology is still underexploited.
The aim of the present proof-of-concept study is therefore to examine the suitability of the dye C11-BODIPY581/591 in combination with FCM for the assessment of lipid oxidation in the microalga Chlamydomonas reinhardtii under a variety of exposure conditions to different micropollutants. Microalgae were chosen as model organisms because of their important ecological functions being the base of the food chain as well as because of their wide use in ecotoxicity testing. In the adopted experimental strategy, the dye was first tested treating C. reinhardtii cells with different H2O2 concentrations and exposure durations to verify its ability to reveal oxyl-radical induced lipid oxidation and to set optimal staining conditions. Then, the lipid oxidation potential of different environmental micropollutants such as copper and mercury, copper oxide nanoparticles (CuO-NPs), and two pesticides, a generic antimicrobial agent diethyldithiocarbamate (DDC) and the herbicide diuron were quantified in a long- and short-term exposure using the optimized staining conditions.