Effect of chromium oxide (III) nanoparticles on the production of reactive oxygen species and photosystem II activity in the green alga Chlamydomonas reinhardtii.

With the growth of nanotechnology and widespread use of nanomaterials, there is an increasing risk of environmental contamination by nanomaterials. However, the potential implications of such environmental contamination are hard to evaluate since the toxicity of nanomaterials if often not well characterized. The objective of this study was to evaluate the toxicity of a chromium-based nanoparticle, Cr2O3-NP, used in a wide diversity of industrial processes and commercial products, on the unicellular green alga Chlamydomonas reinhardtii. The deleterious impacts of Cr2O3-NP were characterized using cell density measurements, production of reactive oxygen species (ROS), esterase enzymes activity, and photosystem II electron transport as indicators of toxicity. Cr2O3-NP exposure inhibited culture growth and significantly lowered cellular Chlorophyll a content. From cell density measurements, EC50 values of 2.05±0.20 and 1.35±0.06gL(-1) Cr2O3-NP were obtained after 24 and 72h of exposure, respectively. In addition, ROS levels were increased to 160.24±2.47% and 59.91±0.15% of the control value after 24 and 72h of exposition to 10gL(-1) Cr2O3-NP. At 24h of exposure, the esterase activity increased to 160.24% of control value, revealing a modification of the short-term metabolic response of algae to Cr2O3-NP exposure. In conclusion, the metabolism of C. reinhardtii was the most sensitive to Cr2O3-NP after 24h of treatment.

Different toxicity mechanisms between bare and polymer-coated copper oxide nanoparticles in Lemna gibba.

In this report, we investigated how the presence of a polymer shell (poly(styrene-co-butyl acrylate) alters the toxicity of CuO NPs in Lemna gibba. Based on total Cu concentration, core-shell CuO NPs were 10 times more toxic than CuO NPs, inducing a 50% decrease of growth rate at 0.4 g l(-1) after 48-h of exposure while a concentration of 4.5 g l(-1) was required for CuO NPs for a similar effect. Toxicity of CuO NPs was mainly due to NPs solubilization in the media. Based on the accumulated copper content in the plants, core-shell CuO NPs induced 4 times more reactive oxygen species compared to CuO NPs and copper sulfate, indicating that the presence of the polymer shell changed the toxic effect induced in L. gibba. This effect could not be attributed to the polymer alone and reveals that surface modification may change the nature of NPs toxicity.

Toxicity of PAMAM-coated gold nanoparticles in different unicellular models.

Polyamidoamine (PAMAM) dendrimers are used for many pharmaceutical and biomedical applications. However, the toxicological risks of several PAMAM-based compounds are still not fully evaluated, despite evidences of PAMAM deleterious effects on biological membranes, leading to toxicity. In this report, we investigated the toxicity of generation 0 PAMAM-coated gold nanoparticles (AuG0 NPs) in four different models to determine how different cellular systems are affected by PAMAM-coated NPs. Toxicity was evaluated in two mammalian cell lines, Neuro 2A and Vero, in the green alga Chlamydomonas reinhardtii and the bacteria Vibrio fischeri. AuG0 NP treatments reduced cell metabolic activity in algal and bacterial cells, measured by esterase enzymatic activity (C. reinhardtii) and luminescence emission (V. fischeri). EC50 value after 30 min of treatment was similar in both organisms, with 0.114 and 0.167 mg mL(-1) for C. reinhardtii and V. fischeri, respectively. On the other hand, AuG0 NPs induced no change of mitochondrial activity in mammalian cells after 24 h of treatment to up to 0.4 mg mL(-1) AuG0 NPs. Change in the absorption spectra of AuG0 NP in the mammalian cell culture media may indicate an alteration of NP properties that contributed to the low toxicity of AuG0 NPs in mammalian cells. For a safe development of PAMAM-based nanomaterials, the difference of sensitivity between mammalian and microbial cells, as well as the modulation of NPs toxicity by medium properties, should be taken into account when designing PAMAM NPs for applications that may lead to their introduction in the environment.

Evaluation of toxicity and oxidative stress induced by copper oxide nanoparticles in the green alga Chlamydomonas reinhardtii.

Copper oxide nanoparticles (CuO NP) are frequently employed for their antimicrobial properties in antifouling paints. Their extensive use can contaminate aquatic ecosystems. However, the toxicological effects of this NP in the environment are poorly known. In this study, we evaluated the toxicity and oxidative stress induced by CuO NP on Chlamydomonas reinhardtii using several toxicological assays. CuO NP was found to induce growth inhibition and a significant decrease in carotenoids levels. From data on cells density after 72 h of CuO NP exposure in light, the EC50 value was calculated to be 150.45±1.17 mg L(-1) and the NOEC≤100 mg L(-1). Evaluation of esterase activity demonstrates a decrease in cell metabolism activity with the increase of CuO NP concentration. The CuO NP induced an increase of reactive species level (190±0.45% at 1000 mg L(-1) after 72 h of exposition, compared to control) and lipid peroxidation of cellular membranes (73±2% at 1000 mg L(-1) of CuO NP in 72 h of exposition, compared to control). Investigation of CuO NP uptake showed the presence of NP into C. reinhardtii cells in different sites of the cell and, biomarkers of enzymatic antioxidants showed a change of activity after CuO NP exposition. In conclusion, C. reinhardtii was shown to be sensitive to the presence of CuO NP in their environment and CuO NP treatments induced a toxic response from 0.1 mg L(-1) after 72 h of treatment.

Alleviation of silver toxicity by calcium chloride (CaCl2) in Lemna gibba L.

The toxicity effects of silver (Ag) and the protective role of calcium chloride (CaCl2) was studied in Lemna gibba L. (L. gibba) plants. Silver speciation showed that silver toxicity in L. gibba culture medium can be attributed to free ionic Ag(+) concentration. Frond abscission, intracellular reactive oxygen species (ROS) formation and intracellular uptake of Ag(+) were investigated when L. gibba plants were exposed to AgNO3 concentrations (0.5, 1, 5, and 10 µM) supplemented or not by 10 µM CaCl2. An increase in frond abscission, intracellular ROS and intracellular uptake of Ag(+) were detected in L. gibba plants for all tested concentrations of AgNO3 after 24 h treatment. However, addition of 10 µM CaCl2 to the L. gibba culture medium reduced the toxic effects of Ag by decreasing silver uptake into the plant and intracellular ROS formation. The results suggest that Ag-induced toxicity was attributed to Ag(+) accumulation and chloride was able to protect L. gibba plants against Ag toxicity by formation of complexes with Ag and then alleviation of the metal induced oxidative stress.

Alteration of chromium effect on photosystem II activity in Chlamydomonas reinhardtii cultures under different synchronized state of the cell cycle.

The inhibitory effect of chromium (Cr) on photosystem II (PSII) activity was investigated in the green alga Chlamydomonas reinhardtii during different phases of the cell cycle. Algae were cultivated in continuous light or a light/dark cycle (16:8 h) to obtain a synchronously dividing cell culture. The cell division phases were determined with the DNA-specific fluorescent probe SYBR green using flow cytometry. The effect of Cr on PSII activity was investigated after a 24-h treatment with algal cultures having different proportions of newly divided cells (G(0)/G(1)), dividing cells at the DNA replication phase (S), and dividing cells at the mitosis phase (G(2)/M). Using chlorophyll a fluorescence parameters based on PSII electron transport capacity in dark- (Φ(M)II) and light-adapted (Φ'(M)II) equilibrium state, we found that the effect of Cr differs depending on the stage of the cell cycle. When algal cultures had a high proportion of cells actively dividing (M phase), the toxic effect of Cr on PSII activity appeared to be much higher and PSII quantum yield was decreased by 80 % compared to algal cultures mainly in the G(0)/G(1) phase. Therefore, the inhibitory effect of Cr on photosynthesis appears to be different according to the cell cycle state of the algal population.

Genotoxic effects of copper oxide nanoparticles in Neuro 2A cell cultures.

Copper oxide nanoparticles (CuO NPs) are used for their biocide potential however they were also shown to be highly toxic to mammalian cells. Therefore, the effects of CuO NPs should be carefully investigated to determine the most sensitive processes for CuO NP toxicity. In this study, the genotoxicity of CuO NPs was investigated in vitro, using the mouse neuroblastoma cell line Neuro-2A. Genotoxic effects related to DNA fragmentation, DNA methylation and chromosomal damage, as well as lipid peroxidation, were investigated and compared to cytotoxic effects, measured by the mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan. Based on mitochondrial activity, CuO NPs were found to be cytotoxic. At the highest concentration tested (400 mg l⁻¹), 63% of cell viability was found in Neuro-2A cells after 24 h of treatment to CuO NPs. CuO NPs were also found to induce DNA fragmentation, lipid peroxidation and micronucleus formation. The micronucleus assay was the most sensitive to evaluate CuO NP genotoxicity and micronucleus frequency was increased significantly at 12.5 mg l⁻¹ CuO NPs after 24h of treatment. At this concentration, no significant change of cell viability was found using the mitochondrial activity assay. These results highlight the important risk of genotoxic effects of CuO NPs and show that genotoxicity assays are a sensitive approach to evaluate the risk of CuO NP toxicity.

Induction to oxidative stress by saxitoxin investigated through lipid peroxidation in Neuro 2A cells and Chlamydomonas reinhardtii alga.

Saxitoxin (STX) is a cyanotoxin, which can cause neurotoxic effects and induce ecological changes in aquatic environments, a potential risk to public and environmental health. Many studies of cytotoxicity on animal cells and algae have been performed, although few compare the toxic effects between the two models. In this sense, we investigated the oxidative stress induced by STX (0.4-3.0 nM) in two different cellular models: Neuro-2A (N2A) cells and Chlamydomonas reinhardtii alga by quantification of malondialdehyde (MDA) levels as indicative of lipid peroxidation (LPO). Also was evaluated the antioxidant defense of these cells systems after exposure to STX by the addition of antioxidants in N2A cells culture, and by the measure of antioxidants enzymes activity in C. reinhardtii cells. The MDA levels of N2A cells increased from 15% to 113% for 0.4 and 3.0 nM of STX, respectively, as compared to control. Superoxide-dismutase and catalase did not appear to protect the cell from STX effect while, in cells treated with vitamin E, the rates of MDA production decreased significantly, except for higher concentrations of STX. No MDA productions were observed in algal cells however some effects on antioxidant enzymes activity were observed when algae were exposed to 3.0 nM STX. Our results indicate that the concentrations of STX that may induce oxidative stress through LPO are different in animal and phytoplankton communities. A combination of algal and animal bioassays should be conducted for reliable assessment of oxidative stress induced by STX.

Polymer coating of copper oxide nanoparticles increases nanoparticles uptake and toxicity in the green alga Chlamydomonas reinhardtii.

Copper oxide nanoparticles (CuO NPs) are frequently used in a polymer-coated form, to be included in paints or fabrics for antimicrobial properties. Their application in antifouling paints may lead to the contamination of aquatic ecosystems. However, the toxicological risk of NPs in the environment is hard to evaluate due to a lack of knowledge on the mechanisms of NP interaction with biological systems. In this study, we investigated the effect of polymer coating on CuO NP toxicity in the green alga Chlamydomonas reinhardtii by comparing bare and polymer-coated CuO NPs prepared from the same CuO nanopowder. Both CuO NP suspensions were toxic to C. reinhardtii after 6 h treatment to concentrations of 0.005-0.04 g L(-1). Bare and polymer-coated CuO NPs induced a decrease of Photosystem II activity and the formation of reactive oxygen species. Polymer-coated CuO NP was found to be more toxic than the uncoated CuO NP. The higher toxicity of CS-CuO NP was mainly associated with the increased capacity of polymer-coated CuO NP to penetrate the cell compared to bare CuO NPs. These results indicates that the high toxicity of polymer-coated CuO NPs in algal cells results of intracellular interactions between NPs and the cellular system.

Interactive effects of temperature and copper on photosystem II photochemistry in Chlorella vulgaris.

In natural aquatic ecosystems, temperature conditions may undergo changes depending on the depth of the water column or micro-environmental conditions. In this study, copper effect on the photosynthetic performance of Chlorella vulgaris was investigated at different temperatures by using chlorophyll a fluorescence transients and fluorescence imaging parameters. Copper as a pollutant is known to be an inhibitor of photosystem II (PSII) photochemistry; therefore it was important to know how the change of temperature may alter this effect. PSII photochemistry was investigated when C. vulgaris, affected by different copper concentrations, was exposed to 24, 28 and 31 °C. Increase of temperature induced higher alterating effects to PSII quantum yield, primary photosynthetic electron transport from water splitting system and consequently higher decrease of total photosynthetic performance if compared to copper effect alone. Additional temperature effect to copper inhibition increased energy dissipation via non-photochemical pathway. In this study we indicated that, when C. vulgaris changes temperature conditions, inhibitory effect of copper also undergoes changes. For natural aquatic system we may suppose, when algae are distributed at different depths of water column, that toxicity effect will be dependent to the temperature conditions of the site.

Temperature influence on silver nanoparticles inhibitory effect on photosystem II photochemistry in two green algae, Chlorella vulgaris and Dunaliella tertiolecta.

PURPOSE: In this study, the effect of silver nanoparticles (AgNPs) on the photosynthetic performance of two green algae, Chlorella vulgaris and Dunaliella tertiolecta, was investigated at 25°C and 31°C. METHODS: To induce AgNPs effect, algal cells were exposed for 24 h to concentrations varying from 0 to 10 mg/L. The polyphasic OJIP fluorescence transient was used to evaluate photosystem II (PSII). RESULTS: We show that growth media and temperature had different effects in AgNPs agglomerates formation and Zeta potential. When temperature conditions change, inhibitory effect of AgNPs also undergoes changes. Increase of temperature induced higher altering effects to PSII quantum yield, primary photosynthetic electron transport, and consequently higher decrease of total photosynthetic performance if compared to AgNPs effect alone. AgNPs has a negative effect on D. tertiolecta compared to C. vulgaris. CONCLUSION: We conclude that temperature tends to enhance the toxic effects on aquatic alga and these alterations might have serious consequences on ecosystem equilibrium and aquatic plant communities.

Okadaic acid inhibits cell growth and photosynthetic electron transport in the alga Dunaliella tertiolecta.

Okadaic acid (OA), which is produced by several dinoflagellate species, is a phycotoxin known to induce a decrease of biomass production in phytoplankton. However, the mechanisms of OA cytotoxicity are still unknown in microalgae. In this study, we exposed the green microalga Dunaliella tertiolecta to OA concentrations of 0.05 to 0.5 µM in order to evaluate its effects on cell division, reactive oxygen species production and photosynthetic electron transport. After 72 h of treatment under continuous illumination, OA concentrations higher than 0.10 µM decreased culture cell density, induced oxidative stress and inhibited photosystem II electron transport capacity. OA effect in D. tertiolecta was strongly light dependent since no oxidative stress was observed when D. tertiolecta was exposed to OA in the dark. In the absence of light, the effect of OA on culture cell density and photosystem II activity was also significantly reduced. Therefore, light appears to have a significant role in the toxicity of OA in microalgae. Our results indicate that the site of OA interaction on photosynthetic electron transport is likely to be at the level of the plastoquinone pool, which can lead to photo-oxidative stress when light absorbed by the light-harvesting complex of photosystem II cannot be dissipated via photochemical pathways. These findings allowed for a better understanding of the mechanisms of OA toxicity in microalgae.

Interaction of gold nanoglycodendrimers with algal cells (Chlamydomonas reinhardtii) and their effect on physiological processes.

With the rise of nanotechnologies, the risk of contamination of aquatic ecosystems with nanoparticles is increasing. Glycodendrimer-coated gold nanoparticles have been developed for biomedical applications; however, their effect on microalgae has never been studied. In this report, their interactions with algae were investigated using two strains of Chlamydomonas reinhardtii, a wild type having cell wall and a cell wall-deficient mutant. Cultures were exposed 48 h to 6 and 12 ng ml⁻¹ of gold nanoparticles coated with mannose generation 0 polyamidoamine dendrimer. Culture aggregation was found only for wild type cells, probably because of interactions between mannose and cell wall glycoproteins. Nanoparticles penetrated cytoplasm in both strains; however, inhibition of algal growth and photosynthetic activity was found only in the wild type. We conclude that nanoparticles' deteriorating effect in algae is caused by interactions with the cell wall, causing an aggregation of cell culture, and not by nanoparticle penetration inside the cytoplasm.

Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta.

Freshwater microalga Chlorella vulgaris and marine microalga Dunaliella tertiolecta were used to investigate toxic effects induced by 50 nm silver nanoparticles (AgNPs). To induce AgNPs effect, we exposed Chlorella vulgaris and Dunaliella tertiolecta for 24h to 0-10 mg/L. We showed that growth media had different effects in AgNPs agglomerates' formation. Cellular viability, reactive oxygen species (ROS) formation and lipids peroxidation were employed to assess the toxic effects of AgNPs. AgNPs were able to interact directly with the Chlorella vulgaris cells surface and large aggregates were observed. AgNPs have a negative effect on Chlorella vulgaris and Dunaliella tertiolecta, as manifested by a strong decrease in chlorophyll content, viable algal cells, increased ROS formation and lipids peroxidation. The variability in sensitivity of both algae towards AgNPs was observed. We conclude that AgNPs have a negative effect on aquatic algae and these alterations might have serious consequences on structure and function of aquatic plant communities.

Carotenogenesis up-regulation in Scenedesmus sp. using a targeted metabolomics approach by liquid chromatography-high-resolution mass spectrometry.

Carotenoids have potent antioxidant activity as well as therapeutic value, and their formation has been seen to be induced in algae by stress, including high-salt culture conditions. A differential profiling of carotenoids was conducted using a targeted metabolomics approach with accurate mass data generated by liquid chromatography-electrospray-time-of-flight (LC-ESI-TOF) mass spectrometry followed by postacquisition filtering based on isotope patterns and mass defects to detect carotenoids up-regulated in Scenedesmus sp. exposed to high-salt conditions. Algal cultures treated with high concentrations of sodium acetate or sodium chloride were found to cause an increase in various carotenoids. On the basis of differential analysis, astaxanthin and canthaxanthin increased upon salt treatment. Astaxanthin, in its free form and as fatty acid esters, was seen to increase in Scenedesmus sp. using accurate mass MS. A few other carotenoid compounds increased upon salt treatment, including echinenone and adonirubin, involved in the pathway of astaxanthin biosynthesis from ß-carotene, as well as isomers of astaxanthin and canthaxanthin. A time course study of acetate treatment was done to observe the time-dependent up-regulation of carotenogenesis.

Investigation of animal and algal bioassays for reliable saxitoxin ecotoxicity and cytotoxicity risk evaluation.

Contamination of water bodies by saxitoxin can result in various toxic effects in aquatic organisms. Saxitoxin contamination has also been shown to be a threat to human health in several reported cases, even resulting in death. In this study, we evaluated the sensitivity of animal (Neuro-2A) and algal (Chlamydomonas reinhardtii) bioassays to saxitoxin effect. Neuro-2A cells were found to be sensitive to saxitoxin, as shown by a 24 h EC50 value of 1.5 nM, which was obtained using a cell viability assay. Conversely, no saxitoxin effect was found in any of the algal biomarkers evaluated, for the concentration range tested (2-128 nM). These results indicate that saxitoxin may induce toxic effects in animal and human populations at concentrations where phytoplankton communities are not affected. Therefore, when evaluating STX risk of toxicity, algal bioassays do not appear to be reliable indicators and should always be conducted in combination with animal bioassays.

Effect of cadmium on photosystem II activity in Chlamydomonas reinhardtii: alteration of O-J-I-P fluorescence transients indicating the change of apparent activation energies within photosystem II.

In this study, we evaluated how cadmium inhibitory effect on photosystem II and I electron transport may affect light energy conversion into electron transport by photosystem II. To induce cadmium effect on the photosynthetic apparatus, we exposed Chlamydomonas reinhardtii 24 h to 0-4.62 µM Cd(2+). By evaluating the half time of fluorescence transients O-J-I-P at different temperatures (20-30°C), we were able to determine the photosystem II apparent activation energies for different reduction steps of photosystem II, indicated by the O-J-I-P fluorescence transients. The decrease of the apparent activation energies for PSII electron transport was found to be strongly related to the cadmium-induced inhibition of photosynthetic electron transport. We found a strong correlation between the photosystem II apparent activation energies and photosystem II oxygen evolution rate and photosystem I activity. Different levels of cadmium inhibition at photosystem II water-splitting system and photosystem I activity showed that photosystem II apparent activation energies are strongly dependent to photosystem II donor and acceptor sides. Therefore, the oxido-reduction state of whole photosystem II and I electron transport chain affects the conversion of light energy from antenna complex to photosystem II electron transport.

Effect of aluminum on cellular division and photosynthetic electron transport in Euglena gracilis and Chlamydomonas acidophila.

The present study investigated aluminum's effect on cellular division and the photosynthetic processes in Euglena gracilis and Chlamydomonas acidophila at pH 3.0, at which Al is present mostly as Al(3+), AlSO(4) (+), and Al(SO(4))(2) (-). These algal species were exposed to 100, 188, and 740 microM Al, and after 24 h cell-bound Al was significantly different from control only for the highest concentration tested. However, very different effects of Al on algal cellular division, biomass per cell, and photosynthetic activity were found. Aluminum stimulated cell division but decreased at some level biomass per cell in C. acidophila. Primary photochemistry of photosynthesis, as Photosystem II quantum yield, and energy dissipation via nonphotochemical activity were slightly affected. However, for E. gracilis, under the same conditions, Al did not show a stimulating effect on cellular division or photosynthetic activity. Primary photochemical activity was diminished, and energy dissipation via nonphotochemical pathways was strongly increased. Therefore, when Al is highly available in aquatic ecosystems, these effects may indicate very different response mechanisms that are dependent on algal species.

Effect of core-shell copper oxide nanoparticles on cell culture morphology and photosynthesis (photosystem II energy distribution) in the green alga, Chlamydomonas reinhardtii.

The effect of core-shell copper oxide nanoparticles with sizes smaller than 100 nm on cellular systems is still not well understood. Documenting these effects is pressing since core-shell copper oxide nanoparticles are currently components of pigments used frequently as antifouling paint protecting boats from crustacean, weed and slime fouling. However, the use of such paints may induce strong deteriorative effects on different aquatic trophic levels that are not the intended targets. Here, the toxic effect of core-shell copper oxide nanoparticles on the green alga, Chlamydomonas reinhardtii was investigated with regards to the change of algal cellular population structure, primary photochemistry of photosystem II and reactive oxygen species formation. Algal cultures were exposed to 0.004, 0.01 and 0.02 g/l of core-shell copper oxide nanoparticles for 6h and a change in algal population structure was observed, while the formation of reactive oxygen species was determined using the 2',7'-dichlorodihydrofluorescein diacetate marker measured by flow cytometry. For the study of the photosystem II primary photochemistry we investigated the change in chlorophyll a rapid rise of fluorescence. We found that core-shell copper oxide nanoparticles induced cellular aggregation processes and had a deteriorative effect on chlorophyll by inducing the photoinhibition of photosystem II. The inhibition of photosynthetic electron transport induced a strong energy dissipation process via non-photochemical pathways. The deterioration of photosynthesis was interpreted as being caused by the formation of reactive oxygen species induced by core-shell copper oxide nanoparticles. However, no formation of reactive oxygen species was observed when C. reinhardtii was exposed to the core without the shell or to the shell only.

Dichromate effect on energy dissipation of photosystem II and photosystem I in Chlamydomonas reinhardtii.

In this study, we investigated the energy dissipation processes via photosystem II and photosystem I activity in green alga Chlamydomonas reinhardtii exposed to dichromate inhibitory effect. Quantum yield of photosystem II and also photosystem I were highly decreased by dichromate effect. Such inhibition by dichromate induced strong quenching effect on rapid OJIP fluorescence transients, indicating deterioration of photosystem II electron transport via plastoquinone pool toward photosystem I. The decrease of energy dissipation dependent on electron transport of photosystem II and photosystem I by dichromate effect was associated with strong increase of non-photochemical energy dissipation processes. By showing strong effect of dichromate on acceptor side of photosystem I, we indicated that dichromate inhibitory effect was not associated only with PSII electron transport. Here, we found that energy dissipation via photosystem I was limited by its electron acceptor side. By the analysis of P700 oxido-reduction state with methylviolagen as an exogenous PSI electron transport mediator, we showed that PSI electron transport discrepancy induced by dichromate effect was also caused by inhibitory effect located beyond photosystem I. Therefore, these results demonstrated that dichromate has different sites of inhibition which are associated with photosystem II, photosystem I and electron transport sink beyond photosystems.