Optimization of cell disruption in Raphidocelis subcapitata and Chlorella vulgaris for biomarker evaluation

Raphidocelis subcapitata and Chlorella vulgaris are bioassay microalgae with rigid cellulosic cell wall which can hinder the release of intracellular proteins often studied as toxicity biomarkers. Since cell disruption is necessary for recovering intracellular biomolecules in these organisms, this study investigated the efficiency of ultrasonication bath; ultrasonication probe; vortexer; and bead mill in disintegrating the microalgae for anti-oxidative enzyme extraction. The extent of cell disruption was evaluated and quantified using bright field microscopy. Disrupted algae appeared as ghosts. The greatest disintegration of the microalgae (83-99.6 %) was achieved using bead mill with 0.42-0.6 mm glass beads while the other methods induced little or no disruption. The degree of cell disruption using bead mill increased with exposure time, beads-solution ratio and agitation speed while larger beads caused less disruption. Findings revealed that bead milling, with specific parameters optimized, is one of the most effective methods of disintegrating the robust algal cells

EURO-ECOLE: Assessment of the Bioavailability and Potential Ecological Effects of Copper in European Surface Waters ; subproject 4: Evaluation and improvement of the ecological relevance of laboratory generated toxicity data

This report summarizes the acute and chronic toxicity of copper to algae, Daphnia and a few other freshwater species in standard laboratory test water and a wide range of natural surface waters (collected across Europe), with a wide range of pH, dissolved organic carbon (DOC) concentration and hardness. These data can be used for validation of bioavailability models such as the biotic ligand model (BLM)

Features of the microalga Raphidocelis subcapitata: physiology and applications

The microalga Raphidocelis subcapitata was isolated from the Nitelva River (Norway) and subsequently deposited in the collection of the Norwegian Institute of Water Research as ``Selenastrum capricornutum Printz''. This freshwater microalga, also known as Pseudokirchneriella subcapitata, acquired much of its notoriety due to its high sensitivity to different chemical species, which makes it recommended by different international organizations for the assessment of ecotoxicity. However, outside this scope, R. subcapitata continues to be little explored. This review aims to shed light on a microalga that, despite its popularity, continues to be an ``illustrious'' unknown in many ways. Therefore, R. subcapitata taxonomy, phylogeny, shape, size/biovolume, cell ultra-structure, and reproduction are reviewed. The nutritional and cultural conditions, chronological aging, and maintenance and preservation of the alga are summarized and critically discussed. Applications of R. subcapitata, such as its use in aquatic toxicology (ecotoxicity assessment and elucidation of adverse toxic outcome pathways) are presented. Furthermore, the latest advances in the use of this alga in biotechnology, namely in the bioremediation of effluents and the production of value-added biomolecules and biofuels, are highlighted. To end, a perspective regarding the future exploitation of R. subcapitata potentialities, in a modern concept of biorefinery, is outlined.info:eu-repo/semantics/publishedVersio

Oxidative stress responses and cellular energy allocation changes in microalgae following exposure to widely used human antibiotics

The individual effect of four human antibiotics on the microalgae Raphidocelis subcapitata was investigated following a 120-h exposure. The effects were assessed by analyzing growth, and biochemical parameters related with: 1) antioxidant capacity and oxidative damage by measuring superoxide dismutase (SOD) activity and lipid peroxidation (LPO) levels; and 2) cellular energy allocation (CEA) by quantifying the content in energy reserves, which represents the energy available (Ea), and the electron transport system activity that represents a measure of oxygen and cellular energy consumption (Ec). Growth yield inhibitory concentrations of sulfamethoxazole (18-30%), clarithromycin (28.7%), ciprofloxacin (28%) and erythromycin (17-39%) were found to elicit a considerable increase in Ec, thereby causing a significant decrease in the CEA. The elevated Ec can be a result of the need to respond to oxidative stress occurring under those conditions given the significant increase in SOD activity at these levels. For sulfamethoxazole, erythromycin and ciprofloxacin, the antioxidant responses do not seem to be enough to cope with the reactive oxygen species and prevent oxidative damage, given the elevated LPO levels observed. A stimulatory effect on growth yield was observed (up to 16%) at ciprofloxacin lowest concentration, which highly correlated with the increase in CEA. Based on the no observed effect concentration (NOECs) and/or effective concentration (EC10) results, Ec, SOD and CEA were more sensitive than the classical endpoint of growth rate for all the tested antibiotics. By revealing the antibiotic stress effects in R. subcapitata at the cellular level, this study suggests CEA as a more reliable indicator of the organisms' physiological status.info:eu-repo/semantics/publishedVersio

Citotoxicidade dos produtos de degradación de microcontaminantes emerxentes sobre a microalga doceacuícola Raphidocelis subcapitata

[Abstract] Emerging contaminants (EC), such as pharmaceuticals, pose a potential risk in aquatic environments since their presence in water media can induce toxic effects onto the environment’s biota. This kind of compounds, not completely degraded after their use/application, can easily reach different water bodies, like lakes or oceans, given that many water treatment systems cannot efficiently eliminate them nowadays. Thus, it is of great concern the potential negative effect these compounds can cause on representative organisms of these environments, such as microalgae, given their essential role on the trophic net as primary productors. In recent years, advance oxidation processes (AOP) have been researched as promising mechanisms of remediation and elimination of these emerging contaminants from aquatic environments. In the present study several experiments were carried out in order to test the possible toxic effect of the antibiotic and emerging contaminant sulfamethoxazole (SMX) on the freshwater microalga Raphidocelis subcapitata. For that, several parameters, like proliferation, viability, vitality, photosystem II’s quantum yield, oxidative stress, and potential of cytoplasmic and mitochondrial membrane were monitored through flow cytometry (FCM). The effective concentration of SMX that induces a 10% inhibition on the growth of the microalga was calculated (EC10). Furthermore, the toxicity of photolyzed SMX (PLSMX), obtained after exposure of SMX solutions at their EC10 to UV light, was also studied by monitoring of the same parameters. The results obtained demonstrated that exposure to both compounds, pure and photolyzed, significantly induces (p<0.05) great levels of cells with low vitality. Nevertheless, PLSMX also induces total cell growth inhibition and the decrease of daughter cells percentage. Further, the cells also show a loss in cell viability and a decrease on the quantum yield of their photosystem II. Moreover, R. subcapitata exposed to PLSMX also exhibited enhanced levels of intercellular reactive oxygen species (ROS) production and great values of cells with both cytoplasmic and mitochondrial depolarized membranes. From the results obtained it can be concluded that PLSMX showed a greater toxic effect on the studied microalga compared to pure SMX. This has great ecological relevance, since remediation of SMX by AOP could affect the aquatic environments in a more toxic manner compared to pure SMX, scenario aggravated by the fact that microalgae are the fundamental primary productors of these environments. Thus, further research on SMX remediation is needed.[Resumo] Os contaminantes emerxentes (EC), coma os medicamentos, supoñen un potencial risco nos medios acuáticos dado que a súa presencia nestes ambientes pode inducir efectos tóxicos na biota do medio. Este tipo de compostos, os cales non se degradan completamente despois do seu uso/aplicación, poden alcanzar facilmente diferentes masas de auga, coma lagos ou océanos, dado que a día de hoxe os sistemas de tratado de auga non as poden eliminar de maneira eficiente. Polo tanto, é de elevada preocupación o potencial efecto negativo que estes compostos poden causar nos organismos representativos destes medios, coma as microalgas, dado o seu papel esencial na rede trófica coma produtores primarios. En anos recentes, os procesos de oxidación avanzada (AOP) foron investigados como mecanismos prometedores de remediación e eliminación destes contaminantes emerxentes en medios acuáticos. Neste estudo varios experimentos foro levados a cabo para comprobar o posible efecto tóxico do antibiótico e contaminante emerxente sulfametoxazol (SMX) na microalga de auga doce Raphidocelis subcapitata. Para iso, varios parámetros, coma a proliferación celular, a viabilidade, a vitalidade, o rendemento cuántico do fotosistema II, o estrés oxidativo, e o potencial das membranas citoplasmática e mitocondrial foron monitorizados por citometría de fluxo (FCM). A concentración efectiva de SMX que induce unha inhibición do 10% no crecemento da microalga foi calculada (EC10). Por outra parte, a toxicidade de SMX fotolizado (PLSMX), obtido logo da exposición de solucións de SMX na súa EC10 con luz UV, tamén foi estudiada por monitorización dos mesmos parámetros. Os resultados obtidos demostraron que a exposición de R. subcapitata a ambos compostos, puro e fotolizado, induce significativamente (p<0.05) elevados niveis de células con baixa actividade metabólica. Sen embargo, PLSMX tamén induce a inhibición total do crecemento celular e a redución da porcentaxe de células fillas. Ademais, as células tamén mostran unha perda da viabilidade celular e unha redución no rendemento cuántico do fotosistema II. Adicionalmente, R. subcapitata exposta a PLSMX tamén exhibiu niveles elevados de produción intracelular de especies reactivas de osíxeno (ROS) e de células con membrana citoplasmática e mitocondrial despolarizadas. Cos resultados obtidos pódese concluír que PLSMX mostrou un maior efecto tóxico na microalga estudiada en comparación con SMX puro. Isto é de gran relevancia ecolóxica, dado que a remediación de SMX con AOP pode afectar aos medios acuáticos dunha maneira máis tóxica ca SMX puro, escenario agravado polo feito de que as microalgas son os produtores primarios fundamentais destes medios. Polo tanto, maior investigación na remediación de SMX é necesaria.[Resumen] Los contaminantes emergentes (EC), como los fármacos, suponen un potencial riesgo en los ecosistemas acuáticos dado que su presencia en estos ambientes puede inducir efectos tóxicos en la biota del medio. Este tipo de compuestos, los cuales no se degradan completamente después de su uso/aplicación, pueden alcanzar fácilmente diferentes masas de agua, como lagos u océanos, dado que a día de hoy los sistemas de tratamiento de aguas no los pueden eliminar de manera eficiente. Por lo tanto, es de elevada preocupación el potencial efecto negativo que estos compuestos pueden causar en los organismos representativos de estos medios, como las microalgas, dado su papel esencial en la red trófica como productores primarios. En años recientes, los procesos de oxidación avanzada (AOP) han sido investigados como mecanismos prometedores de remediación y eliminación de estos contaminantes emergentes en medios acuáticos. En este estudio varios experimentos fueron llevados a cabo para comprobar el posible efecto tóxico del antibiótico y contaminante emergente sulfametoxazol (SMX) en la microalga de agua dulce Raphidocelis subcapitata. Para ello, varios parámetros, como la proliferación celular, la viabilidad, la vitalidad, el rendimiento cuántico del fotosistema II, el estrés oxidativo, o el potencial de las membranas citoplasmática y mitocondrial fueron monitorizados por citometría de flujo (FCM)., La concentración efectiva de SMX que induce una inhibición del 10% en el crecimiento de la microalga fue calculada (EC10). Por otra parte, la toxicidad de SMX fotolizado (PLSMX), obtenido luego de la exposición de soluciones de SMX en su EC10 con luz UV, también fue estudiada por monitorización de los mismos parámetros. Los resultados obtenidos demostraron que la exposición de R. subcapitata a ambos compuestos, puro y fotolizado, induce significativamente (p<0.05) elevados niveles de células con baja vitalidad. Sin embargo, PLSMX también induce la inhibición total del crecimiento y la reducción del porcentaje de células hijas. Además, las células también muestran una pérdida de viabilidad celular y una reducción en su rendimiento cuántico del fotosistema II. Adicionalmente, R. subcapitata expuesta a PLSMX también exhibió niveles elevados de producción intracelular de especies reactivas de oxígeno (ROS) y de células con membrana citoplasmática y mitocondrial despolarizadas. Con los resultados obtenidos se puede concluir que PLSXM mostró un mayor efecto tóxico en la microalga estudiada en comparación con SMX puro. Esto es de gran relevancia ecológica, dado que la remediación de SMX con AOP puede afectar a los medios acuáticos de una manera más tóxica que el SMX puro, escenario agravado por el hecho de que las microalgas son los productores primarios fundamentales de estos medios. Por lo tanto, mayor investigación en la remediación de SMX es necesaria.Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2020/202

Effects of long-term exposure to colloidal gold nanorods on freshwater microalgae

Gold nanorods have shown to pose adverse effects to biota. Whether these effects may be potentiated through prolonged exposure has been rarely studied. Therefore, this work aimed at evaluating the effects of long-term exposure to sublethal levels of cetyltrimethylammonium bromide (CTAB) coated gold nanorods (Au-NR) on two freshwater microalgae: Chlorella vulgaris and Raphidocelis subcapitata. These algae were exposed to several concentrations of Au-NR for 72 h and, afterwards, to the corresponding EC5,72h, for growth, during 16 days. The sensitivity of the two algae to Au-NR was assessed at days 0, 4, 8, 12 and 16 (D0, D4, D8, D12 and D16, respectively) after a 72-h exposure to several concentrations of Au-NR. At the end of the assays, effects on yield and population growth rate were evaluated. Raphidocelis subcapitata was slightly more sensitive to Au-NR than C. vulgaris: EC50,72h,D0 for yield were 48.1 (35.3-60.9) and 70.5 (52.4-88.6) μg/L Au-NR, respectively while for population growth rate were above the highest tested concentrations (53 and 90 μg/L, respectively). For R. subcapitata the long-term exposure to Au-NR increased its sensitivity to this type of nanostructures. For C. vulgaris, a decrease on the effects caused by Au-NR occurred over time, with no significant effects being observed for yield or population growth rate at D12 and D16. The capping agent CTAB caused reductions in yield above 30% (D0) for both algae at the concentration matching the one at the highest Au-NR tested concentration. When exposed to CTAB, the highest inhibition values were 69% (D4) and 21.3% (D8) for R. subcapitata, and 64% (D12) and 21% (D16) to C. vulgaris, for yield and population growth rate, respectively. These results suggested long-term exposures should be included in ecological risk assessments since short-term standard toxicity may either under- or overestimate the risk posed by Au-NR.publishe

Ecotoxicidad del herbicida Glifosato sobre cuatro algas clorófitas dulceacuícolas

The increasing use of glyphosate in Argentina is directly related to the increase of areas cultivated with a glyphosate-tolerant transgenic variety of soybean. That has raised concern about the effects of this herbicide in the pampean aquatic ecosystems. Hence, the ecotoxicity of pure (active ingredient) and commercial grade (Roundup) of the herbicide glyphosate was evaluated towards four green freshwater algae: Scenedesmus acutus, Scenedesmus quadricauda, Chlorella vulgaris, and Raphidocelis subcapitata. Toxic effects of glyphosate were assessed on both short-term (photosynthetic rates, determined as oxygen production) and at long-term (growth of the populations, determined as number of cells). A stimulation of the photosynthetic rate was observed at low herbicide concentrations (hormesis). Pure grade short-term effects on the photosynthetic rates appeared at concentrations between 50 and 166 mg L−1, whereas long-term effects on growth appeared in the 1.55-4 mg L−1 range. The commercial grade resulted more toxic than the pure grade; its long-term effects appeared at concentrations between 0.1-3.7 mg L−1. These concentrations are clearly below the expected environmental concentrations (EEC) for this herbicide. Recovery experiments showed that both the pure grade and the commercial grade had algistatic and not algicidal effects. Possible effects and implications at algal community level regarding competitiveness are discussed.El incremento en el uso de glifosato en Argentina está directamente relacionado con el incremento de las áreas cultivadas con una variedad de soja transgénica tolerante al glifosato. Este hecho ha incrementado la preocupaci'on acerca de los efectos de este herbicida sobre los ecosistemas acuáticos de la región pampeana. Así, la ecotoxicidad del herbicida glifosato en su forma pura (ingrediente activo) y su formulado comercial (Roundup) fue evaluada hacia cuatro algas verdes dulceacuícolas: Scenedesmus acutus, Scenedesmus quadricauda, Chlorella vulgaris y Raphidocelis subcapitata. Los efectos tóxicos del glifosato fueron evaluados tanto en su toxicidad a corto plazo (tasa fotosintética, determinada como producción de oxígeno) como en su toxicidad a largo plazo (crecimento poblacional, determinado como número de células). Una estimulación de la tasa fotosintética fue observada a bajas concentraciones del herbicida (hormesis). Los efectos a corto plazo del principio activo puro sobre la tasa fotosintética aparecieron a concentraciones entre 50 y 166 mg L−1 mientras los efectos a largo plazo sobre el crecimiento aparecieron en el rango de 1.55-4 mg L−1. El formulado comercial resultó más tóxico que el ingrediente activo puro; sus efectos a largo plazo aparecieron a concentraciones entre 0.1 y 3.7 mg L−1. Estas concentraciones se encuentran claramente por debajo de la Concentración Esperada en el ambiente (CEA) para este herbicida. Las experiencias de recuperación mostraron que tanto el ingrediente activo en forma pura y el formulado comercial ejercieron efectos algistáticos y no algicidas. Se discute los posibles efectos e implicancias a nivel de la comunidad algal en relaci'on a la capacidad competitiva

The use of algae to remove copper and lead from industrial wastewater

The aim of the research was to evaluate the effectiveness of the removal of Cu and Pb ions by algae. The experiments were carried out in the presence of two algal populations: a pure culture of Raphidocelis subcapitata, and a mixed chlorophyta population. The research involved a model study, experiments in the presence of wastewater from the manufacture of batteries, and the study of process kinetics. The wastewater pH was 4.0, and the initial concentrations of metal ions in the wastewater were 95.4 mg/L for Pb and 48.3 mg/L for Cu, respectively. The maximum sorption capacity of the pure Raphidocelis subcapitata culture was 14.8 mg/g d.m. for Pb, corresponding to the removal of 72% of lead, and 6.1 mg/g d.m. for Cu, corresponding to the removal of 43% of copper from the wastewater. The best ion sorption efficiency in the case of the mixed chlorophyta population was 7.0 mg/g d.m. for Pb, i.e., 61% removal of lead, and 12.8 mg/g d.m. for Cu, i.e., 69% removal of copper ions from the wastewater. The optimum duration of the process was found to be 1 hour, since the majority of biomass samples reached the maximum saturation after that time. On the basis of the obtained results (Lagergren models), it was found that the dominant mechanism of the process was chemisorption

Allelopathic Effect of Cereal Straw Extracts on Growth of Raphidocelis subcapitata and Microcystis aeruginosa

Harmful algal blooms are increasing in size, duration, and intensity around the globe. For several decades, cereal straws have been recognized as a viable algal control method, though the mechanisms by which cereal straws inhibit algae remain a topic of research. The objectives of this study were to evaluate the effectiveness of decomposing cereal straw extract, particularly rice (Oryza sativa) and barley (Hordeum vulgare) straw extract, to inhibit the green alga Raphidocelis subcapitata or cyanobacteria Microcystis aeruginosa in bioassays and to determine if the effectiveness of decomposing rice straw extract is altered by the presence of natural organic matter or microbial communities in pond water. In rice and barley straw extracts of the same concentration, total phenolic and flavonoid concentrations were also similar; however, rice straw was more effective than barley straw at inhibiting the growth of both R. subcapitata and M. aeruginosa. Combined with a lack of significant changes in H₂O₂ between cereal straw treatments, results support the hypothesis that algal inhibition cannot be attributed to H₂O₂ alone but depends on the release of specific phenolic and polyphenolic compounds from decomposing cereal straw. In media-based assays, both M. aeruginosa and R. subcapitata were inhibited by 5.0 g/L rice straw extract (P\u3c0.05). In pond water bioassays, 10 g/L rice straw extract showed the greatest inhibitory effect in cultures with and without bacteria. M. aeruginosa showed signs of recovery 15 days following treatment with 2.5 g/L in media and 5.0 g/L rice straw extract in pond water. For reliable long-term suppression, greater concentrations of rice straw are necessary, possibly due to the instability of allelochemicals. In pond water, variability in responses between the controls (with and without bacteria) and 5.0 g/L treatments with bacteria versus without bacteria show that the microbial community within the context of abiotic conditions of natural waters (pH, nutrient concentrations, sediment, and temperature, etc.) is important in determining bloom behavior and treatment response. Though rice straw was an effective method for the control of M. aeruginosa and R. subcapitata, complex interactions between the algae, the microbial community, and abiotic factors found in natural waters make the determination the of the inhibitory mechanism a challenge

Influence of Water Hardness on Accumulation and Effects of Silver in the Green Alga, Raphidocelis subcapitata

Metal pollution from anthropogenic sources can pose a threat to aquatic systems. Silver is released into the environment from various industrial processes. In excess, silver can accumulate and cause adverse effects in aquatic organisms, particularly those in lower trophic levels, such as phytoplankton. Water chemistry parameters, such as hardness, have been shown to modify toxicity of metals because divalent cations compete with the metal for binding sites on the biological membrane. The objective of this study was to assess population growth and silver accumulation in the green alga, Raphidocelis subcapitata, after silver exposure in waters of varying hardness for 7 d. Throughout the exposure period, a decrease in algal cell density was observed with increasing silver concentrations. Silver accumulation in the algae decreased and cell density increased with increasing water hardness. Additionally, at least some degree of protection was observed against silver toxicity due to increased water hardness