Microalgae Raphidocelis Subcapitata Research Articles

Ecotoxic effects of the vehicle solvent dimethyl sulfoxide on Raphidocelis subcapitata, Daphnia magna and Brachionus calyciflorus

ABSTRACT Dimethyl sulfoxide (DMSO) is widely used as a vehicle solvent in ecotoxicity bioassays.Despite its frequent use, it could be toxic for organisms at some concentrations. Hence, this study aims to investigate the effects of DMSO on the model organisms: microalgae Raphidocelis subcapitata, microcrustacean Daphnia magna, and rotifer Brachionus calyciflorus. DMSO was applied to the organisms in concentrations ranging from 0.031% to 4%. For R. subcapitata the lowest observed effect concentration (LOEC) after 72 h of exposure was 0.125% DMSO. The 50% effective concentration (EC50) was 2.138 ± 0.372%. In D. magna, significant differences in the mobility after 24 h or 48 h of exposure were observed for 1% DMSO being 1.712 ± 0.207% and 1.167 ± 0.220% DMSO the EC50 observed for 24 h and 48 h exposure, respectively. For B. calyciflorus, it was not possible to validate the tests performed, as there were insufficient animals alive in the control conditions at the end of the exposure period. Therefore, we recommended avoiding DMSO as a vehicle in assays using B. calyciflorus, and to use final DMSO concentrations in an experimental solution not exceeding 0.125% for R. subcapitata and 0.5% for D. magna. Finally, using a maximum of 0.01% DMSO is safe for the organisms tested. The effects of the vehicle solvent dimethyl sulfoxide (DMSO) were investigated in three aquatic model organisms, the microalgae Raphidocelis subcapitata, the microcrustacean Daphnia magna and the rotifer Brachionus calyciflorus. As a result, negative effects on growth rate for the algae population, immobilisation of daphnids and inhibition of rotifer reproduction was observed obtaining EC50 close to 1% of DMSO for all these cases.

Attenuation of toxicity and occurrence of degradation products of the fungicide tebuconazole after combined vacuum UV and UVC treatment of drinking water

Antifungal azoles are the most frequently used fungicides worldwide and occur as active ingredients in many antifungal pharmaceuticals, biocides, and pesticides. Azole fungicides are frequent environmental contaminants and can affect the quality of surface waters, groundwater, and drinking water. This study examined the potential of combined vacuum UV (185 nm) and UVC (254 nm) irradiation (VUV/UVC) of the azole fungicide tebuconazole and the transformation product 1,2,4-trizole on degradation and changes in ecotoxicity. In vivo ecotoxicity was examined before and after UV treatment using bioassays with test organisms from different trophic levels to integrate changes in biological effect of the parent compound and the degradation products. The test battery included the luminescent bacterium Aliivibrio fischeri, the Gram-positive bacterium Bacillus subtilis, the fungus Fusarium graminearum, the green microalga Raphidocelis subcapitata, and the crustacean Daphnia magna. The combined VUV/UVC treatment of tebuconazole in drinking water efficiently degraded the parent compound at the µg/L-mg/L level and resulted in transformation products with lower toxicity than the parent compound. A direct positive correlation was observed between the applied UV dose (fluence, J/cm2), the disappearance of tebuconazole, and the decrease in ecotoxicity. The combined VUV/UVC process does not require addition of supplementary oxidants or catalysts and our study suggests that VUV/UVC-mediated photolysis of azole fungicides in water can decrease the overall toxicity and represent a potentially environmentally friendly treatment method.

Integral Valorization of Two-Phase Olive Mill Solid Waste (OMSW) and Related Washing Waters by Anaerobic Co-digestion of OMSW and the Microalga Raphidocelis subcapitata Cultivated in These Effluents.

This study evaluates the comprehensive valorization of the byproducts derived from the two-phase olive oil elaboration process [i.e., olive washing water (OWW), olive oil washing water (OOWW), and olive mill solid waste (OMSW)] in a closed-loop process. Initially, the microalga Raphidocelis subcapitata was grown using a mixture of OWW and OOWW as the culture medium, allowing phosphate, nitrate, sugars, and soluble chemical oxygen demand removal. In a second step, the microalgal biomass grown in the mixture of washing waters was used as a co-substrate together with OMSW for an anaerobic co-digestion process. The anaerobic co-digestion of the combination of 75% OMSW–25% R. subcapitata enhanced the methane yield by 7.0 and 64.5% compared to the anaerobic digestion of the OMSW and R. subcapitata individually. This schedule of operation allowed for integration of all of the byproducts generated from the two-phase olive oil elaboration process in a full valorization system and the establishment of a circular economy concept for the olive oil industry.

Effects of cadmium and cobalt mixtures on growth and photosynthesis of Raphidocelis subcapitata (Chlorophyceae).

Metals occur simultaneously in the environment, and therefore it is important to know their toxicity and mechanism of action when associated with another metal. Furthermore, anthropogenic actions increase their concentrations in the environment where they can interact and undergo transformations that can even increase their toxicity. This study aimed to evaluate the effects of cadmium (Cd) and cobalt (Co), isolated and combined, on the microalgae Raphidocelis subcapitata. Regarding the toxicity of isolated metals, the IC5096h was 0.08mgL-1 of Cd and 0.16mgL-1 of Co. Cell density decreased at all concentrations of the Cd tested. The parameters related to cell size, cell complexity and mean cell chlorophyll a (Chl a) fluorescence were significantly affected by both metals. According to species sensitivity curves (SSD), the microalgae R. subcapitata was the second most sensitive organism to Co exposure and the tenth concerning Cd. Metal mixture data were best fitted to the concentration addition (CA) model and dose-ratio dependence (DR) deviation, showing synergism at high concentrations of Co and low concentrations of Cd. Besides that, antagonism was observed at low concentrations of Co and high concentrations of Cd. Photosynthetic performance, assessed by maximum quantum yield (ΦM) and oxygen evolving complex (OEC), presented antagonism effects for both analyzed parameters. Thus, the mixture of Cd and Co showed synergistic and antagonistic interactions for the parameters analyzed in R. Subcapitata, indicating the importance of understanding the mechanisms of toxicity of metal mixtures in phytoplankton.

Enhanced degradation of sulfamethoxazole by a modified nano zero-valent iron with a β-cyclodextrin polymer: Mechanism and toxicity evaluation

Rising concern about emerging and already persisting pollutants in water has urged the scientific community to develop novel remedial techniques. A new group of remediation methods is based on the modification of nanoscale zero-valent iron particles (nZVI), which are well known for treating volatile organic compounds and heavy metals. The properties of nZVI may be further enhanced by modifying their structure or surface using “green” polymers. Herein, nZVI was modified by a β-cyclodextrin polymer (β-CDP), which is considered an environmentally safe and inexpensive adsorbent of contaminants. This composite was used for the first time for the degradation of sulfamethoxazole (SMX). Coating by β-CDP not only enhanced the degradation of SMX (>95%, under 10 min) by the nanoparticles in a wide pH range (3–9) and enabled their efficient reusability (for three cycles) but also made the coated nZVI less toxic to the model bioindicator microalga Raphidocelis subcapitata. Moreover, degradation products of SMX were found to be less toxic to Escherichia coli bacteria and R. subcapitata microalga, contrary to the SMX antibiotic itself, indicating a simple and eco-friendly cleaning process. This research aims to further stimulate and develop novel remedial techniques based on nZVI, and provides a potential application in the degradation of antibiotics in a wide pH range. Moreover, the wealth of available cyclodextrin materials used for surface modification may open a way to discover more efficient and attractive composites for environmental applications.

Bioaccumulation but no biomagnification of silver sulfide nanoparticles in freshwater snails and planarians

Bioaccumulation studies are critical in regulatory decision making on the potential environmental risks of engineered nanoparticles (NPs). The present study evaluated the toxicokinetics of silver, taken up from sulfide nanoparticles (Ag2S NPs; simulating an aged Ag NP form) and AgNO3 (ionic counterpart), in the pulmonate snail Physa acuta and the planarian Girardia tigrina. The snails were first exposed for 7 days to Ag-spiked water, along with the microalgae Raphidocelis subcapitata upon which they fed setting up a double route exposure, and subsequently provided as pre-exposed food to the planarians. Ag toxicokinetics and bioaccumulation were assessed in planarians and snails, and potential biomagnification from snail to planarian was evaluated. Gut depuration was also explored to understand whether it constitutes a factor likely to influence Ag toxicokinetics and internal concentrations in the test species. Both species revealed Ag uptake in Ag2S NP and AgNO3 treatments, with higher uptake from the latter. Uptake by the snails was probably via a combination of water exposure and ingested algae provided as food, but ingestion of algae possibly had higher relevance for Ag uptake from the Ag2S NPs compared to AgNO3. For planarians, diet probably was the most important exposure route since no Ag uptake was observed in previous waterborne exposures to Ag2S NPs. Kinetics and internal Ag concentrations did not significantly differ between depurated and non-depurated snails or planarians. The planarians fed on snails revealed no biomagnification. To the best of our knowledge this is the first study investigating the toxicokinetics and biomagnification of NPs in planarians, and with that providing important data on the kinetics and bioaccumulation of NPs in a relevant benthic species.

Effects of virgin and weathered polystyrene and polypropylene microplastics on Raphidocelis subcapitata and embryos of Danio rerio under environmental concentrations

Microplastics are ubiquitous contaminants of freshwater ecosystems. However, few ecotoxicity assays have been conducted on freshwater organisms using environmentally relevant concentrations of virgin and weathered microplastics. This work assessed the adverse effects of virgin and artificially weathered fragments of polystyrene and polypropylene on the microalga Raphidocelis subcapitata (72 h growth inhibition assay) and on embryos of the fish Danio rerio (96 h fish embryo assay) under environmentally relevant concentrations (2000–200,000 MP L−1) and high concentrations (12.5–100 mg L−1). Sizes of microplastics were measured as tens (polystyrene) to hundreds (polypropylene) of micrometers, while aging was assessed by measuring the carbonyl index. In the microalga, the tested high concentrations promoted growth, while environmentally relevant concentration induced either growth inhibition or promotion. In zebrafish embryos, environmentally relevant concentrations decreased body length and heart rates. No relevant effects were observed in organisms exposed to high concentrations for mortality, malformations, hatching rates, and swimming bladder inflation. Virgin microplastics presented slightly higher toxicity but direct comparison was hindered by the lack of a linear dose-response curve. Despite the lack of a clear pattern, adverse effects were often observed in the lowest environmentally relevant concentrations, raising concerns over the impacts of microplastics on freshwater ecosystems.

Synthesis of Purine-Based Ionic Liquids and Their Applications.

Bio-based ionic liquids (ILs) are being increasingly sought after, as they are more sustainable and eco-friendly. Purines are the most widely distributed, naturally occurring N-heterocycles, but their low water-solubility limits their application. In this work, four purines (theobromine, theophylline, xanthine, and uric acid) were combined with the cation tetrabutylammonium to synthesize bio-based ILs. The physico–chemical properties of the purine-based ILs were characterized, including their melting and decomposition temperatures and water-solubility. The ecotoxicity against the microalgae Raphidocelis subcapitata was also determined. The ILs show good thermal stability (>457 K) and an aqueous solubility enhancement ranging from 53- to 870-fold, in comparison to their respective purine percursors, unlocking new prospects for their application where aqueous solutions are demanded. The ecotoxicity of these ILs seems to be dominated by the cation, and it is similar to chloride-based IL, emphasizing that the use of natural anions does not necessarily translate to more benign ILs. The application of the novel ILs in the formation of aqueous biphasic systems (ABS), and as solubility enhancers, was also evaluated. The ILs were able to form ABS with sodium sulfate and tripotassium citrate salts. The development of thermoresponsive ABS, using sodium sulfate as a salting-out agent, was accomplished, with the ILs having different thermosensitivities. In addition, the purine-based ILs acted as solubility enhancers of ferulic acid in aqueous solution.

A Two-Step Cultivation Strategy for High Biomass Production and Lipid Accumulation of Raphidocelis subcapitata Immobilized in Alginate Gel

This work focuses on a culture strategy that combines high biomass production and lipid accumulation in the green microalgae Raphidocelis subcapitata immobilized in alginate gel in order to obtain high lipid productivity for biodiesel production. The study of the effects of nitrogen and phosphorus deficiency on lipid accumulation and biomass production in immobilized microalgae showed that both conditions (N− and P−) promoted lipid accumulation in the microalgae. The lipid contents achieved under nitrogen (31.7% ± 3.2% (dcw)) and phosphorus (19.4% ± 1.9% (dcw)) deficiency conditions were higher than those obtained in the complete medium (control) (14.9% ± 1.5% (dcw)). The highest lipid productivity was recorded under nitrogen deficiency conditions (PL = 11.1 ± 1.1 mg/L/day). This indicated that nitrogen deficiency was more effective than phosphorus deficiency in terms of triggering lipid accumulation in the microalgae. However, the conditions for inducing lipid accumulation (N− or P−) resulted in slower growth. In order to address this issue and achieve high lipid productivity, a two-step culture strategy was used. Immobilized R. subcapitata was cultivated under optimal concentrations of nitrogen and phosphorus to achieve a high biomass concentration. Thereafter, the beads containing the microalgae were transferred to a culture medium under nitrogen deficiency conditions in order to induce lipid accumulation. The concentrations 1.5 g/L of NaNO3 and 20 mg/L of K2HPO4 were determined as being the optimal concentrations for growth, and they produced the highest biomass production rates (µm max = 0.233 ± 0.023 day−1 and µm max = 0.225 ± 0.022 day−1 for NaNO3 and K2HPO4, respectively) from all of the concentrations studied. With the two-step culture strategy, immobilized R. subcapitata accumulated 37.9 ± 3.8% of their dry weight in lipid and reached a lipid productivity value of PL = 40.3 ± 4.0 mg/L/day under nitrogen deficiency conditions. This value was approximately 3.6 times higher than that obtained in the direct culture of cells under nitrogen deficiency conditions (PL = 11.1 ± 1.1 mg/L/day).

Toxicity of α-Ag2WO4 microcrystals to freshwater microalga Raphidocelis subcapitata at cellular and population levels

Silver-based materials have microbicidal action, photocatalytic activity and electronic properties. The increase in manufacturing and consumption of these compounds, given their wide functionality and application, is a source of contamination to freshwater ecosystems and causes toxicity to aquatic biota. Therefore, for the first time, we evaluated the toxicity of the silver tungstate (α-Ag2WO4), in different morphologies (cube and rod), for the microalga Raphidocelis subcapitata. To investigate the toxicity, we evaluated the growth rate, cell complexity and size, reactive oxygen species (ROS) production and chlorophyll a (Chl a) fluorescence. The α-Ag2WO4 – R (rod) was 1.7 times more toxic than α-Ag2WO4–C (cube), with IC10 and IC50 values of, respectively, 8.68 ± 0.91 μg L−1 and 13.72 ± 1.48 μg L−1 for α-Ag2WO4 – R and 18.60 ± 1.61 μg L−1 and 23.47 ± 1.16 μg L−1 for α-Ag2WO4–C. The release of silver ions was quantified and indicated that the silver ions dissolution from the α-Ag2WO4 – R ranged from 34 to 71%, while the Ag ions from the α-Ag2WO4–C varied from 35 to 97%. The α-Ag2WO4–C induced, after 24 h exposure, the increase of ROS at the lowest concentrations (8.81 and 19.32 μg L−1), whereas the α-Ag2WO4 – R significantly induced ROS production at 96 h at the highest concentration (31.76 μg L−1). Both microcrystal shapes significantly altered the cellular complexity and decreased the Chl a fluorescence at all tested concentrations. We conclude that the different morphologies of α-Ag2WO4 negatively affect the microalga and are important sources of silver ions leading to harmful consequences to the aquatic ecosystem.

Degradation of the antifungal pharmaceutical clotrimazole by UVC and vacuum-UV irradiation: Kinetics, transformation products and attenuation of toxicity

Azole fungicides are frequently detected as water contaminants due to extensive societal use, and these broad-spectrum antifungal chemicals can cause harmful effects in non-target organisms. Vacuum ultraviolet (VUV) treatment is an efficient and green technology that produces hydroxyl radical to remove contaminants from water. This study investigated the potential of UVC (254 nm) and combined VUV/UVC (185/254 nm) treatment to remove the widely used antifungal compound clotrimazole from water. Degradation kinetics, degradation mechanisms, and toxicity mitigation were investigated using a VUV photoreactor. Toxicity of clotrimazole to aquatic organisms before and after UV treatment were investigated using the luminescent bacterium Aliivibrio fischeri, the bioluminescent yeast Saccharomyces cerevisiae BLYR, the filamentous fungus Fusarium graminearum, the freshwater microalga Raphidocelis subcapitata, and the crustacean Daphnia magna. VUV irradiation efficiently degraded the persistent pollutant clotrimazole at elevated concentrations (mg/L) and at environmental concentrations (µg/L) with > 50% abatement in 1 min and > 95% removal within 32 min. VUV photolysis produced 8 transformation products manly resulting from drug hydroxylation in the phenyl ring and/or imidazole group followed by ring opening or loss of the imidazole moiety. Substantial decrease in aquatic toxicity was observed after UV treatment suggesting that VUV irradiation of aqueous clotrimazole generated less-toxic transformation products.

Specific toxicity of azithromycin to the freshwater microalga Raphidocelis subcapitata

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.

Freshwater alga Raphidocelis subcapitata undergoes metabolomic changes in response to electrostatic adhesion by micrometer-sized nylon 6 particles

Nylon powders are a type of microplastic (MP) used in personal care products such as cosmetics and sunscreens. To determine the effects of nylon polymers on freshwater microalgae, we investigated the effects of two types of micrometer-sized nylon polymers, i.e., powdered nylon 6 (Ny6-P) and nylon 12 (Ny12), and four other micrometer-sized MPs, i.e., low-density polyethylene, polyethylene terephthalate, polystyrene, and ultra-high-molecular-weight polyethylene, on the microalga Raphidocelis subcapitata. The results showed that Ny6-P inhibited R. subcapitata growth more than the other MPs; R. subcapitata growth was inhibited by 54.2% with 6.25 mg/L Ny6-P compared with the control. Ny6-P in the culture media adhered to R. subcapitata cells electrostatically, which may have disrupted growth and photosynthetic activity. Metabolomic analysis revealed that many metabolites related to the amino acid catabolic pathway and γ-glutamyl cycle were induced, which might trigger responses to avoid starvation and oxidative stress. Our study provides important information on the effects of Ny6-P on algae in freshwater environments.

Long-term ecotoxicological effects of ciprofloxacin in combination with caffeine on the microalga Raphidocelis subcapitata.

Ciprofloxacin (CIP) is an antimicrobial "pseudo-persistent" in aquatic ecosystems. Once dispersed in the water compartments, it can also affect the microalgae. Thus, the evaluation of its long-term ecotoxicological effects is necessary. CIP interactions with other pharmaceuticals are not well known. In this study, we investigated the toxic effects of CIP alone and combined with caffeine (CAF), using the modified Gompertz model parameters and the chlorophyll-a production of the microalga Raphidocelis subcapitata as endpoints, throughout a 16-day exposure assay. The exposure to CIP alone led to significant reductions of the growth rate and the cell density of the microalgae compared to control groups. The combination with CAF lowered the adverse effects of CIP to R. subcapitata. However, as the toxicity is dynamic, our results indicated that the toxic effects in respect to the studied endpoints changed throughout the exposure period, reinforcing the need for longer-term ecotoxicity assessments.

Using multiple endpoints to assess the toxicity of cadmium and cobalt for chlorophycean Raphidocelis subcapitata

Metals may cause damage to the biota of contaminated environments. Moreover, using multiple endpoints in ecotoxicological studies is useful to better elucidate the mechanisms of toxicity of these compounds. Therefore, this study aimed to evaluate the effects of cadmium (Cd) and cobalt (Co) on growth, biochemical and photosynthetic parameters of the microalgae Raphidocelis subcapitata, through quantification of lipid classes composition, chlorophyll a (Chl a) content, maximum (ΦM) and effective (Φ’M) quantum yields and efficiency of the oxygen-evolving complex (OEC). Both metals affected the algal population growth, with an IC50–96h of 0.67 and 1.53 μM of Cd and Co, respectively. Moreover, the metals led to an increase in the total lipid content and reduced efficiency of OEC and ΦM. Cell density was the most sensitive endpoint to detect Cd toxicity after 96 h of treatment. Regarding Co, the photosynthetic parameters were the most affected and the total lipid content was the most sensitive endpoint as it was altered by the exposure to this metal in all concentrations. Cd led to increased contents of the lipid class wax esters (0.89 μM) and phospholipids (PL - at 0.89 and 1.11 μM) and decreased values of triglycerides (at 0.22 μM) and acetone-mobile polar lipids (AMPL - at 0.44 and 1.11 μM). The percentage of free fatty acids (FFA) and PL of microalgae exposed to Co increased, whereas AMPL decreased in all concentrations tested. We were able to detect differences between the toxicity mechanisms of each metal, especially how Co interferes in the microalgae at a biochemical level. Furthermore, to the best of our knowledge, this is the first study reporting Co effects in lipid classes of a freshwater Chlorophyceae. The damage caused by Cd and Co may reach higher trophic levels, causing potential damage to the aquatic communities as microalgae are primary producers and the base of the food chain.

Uptake of microalgae as sublethal biomarker reveals phototoxicity of oxytetracycline to the crustacean Daphnia magna

Antibiotics are considered emerging pollutants as their presence in the environment is increasingly common. Although their environmental concentrations are generally low, they can pose risk to organisms through bioaccumulation, causing sublethal effects. Furthermore, solar radiation can trigger reactions in certain compounds after their accumulation within organisms or in the environment. Toxicity and photoinduced toxicity of oxytetracycline (OTC, widely used antibiotic in salmon aquaculture) on Daphnia magna (Crustacea, Cladocera) and microalgae Raphidocelis subcapitata (Chlorophyceae) as its food source was assessed via aqueous exposure. Also, the impact via diet (microalga) to the crustacean was examined. In addition to lethal (immobility) effect, in vivo chlorophyll fluorescence techniques were used to determine food ingestion (gut content as a biomarker of physiological health) in D. magna and physiological status of microalgae. OTC (≤10 mg L − 1) was not acutely (24 h) toxic to R. subcapitata when measured as maximum quantum yield (Fv/Fm) in darkness. However, under short (1 h) UV exposure OTC caused irreversible decrease of Fv/Fm (50%) at ≥0.5 mg L − 1. OTC was not acutely lethal to D. magna (≤10 mg L − 1), however, sublethal effects (43% decrease in food ingestion) at 10 mg L − 1 were demonstrated. UV exposure (4.5 h) strongly exacerbated toxicity of OTC, leading to lethal (87% immobility) and sublethal (81% decrease of feeding in survived individuals) effects. Uptake of OTC (aqueous exposure) and its photosensitization in tissues of D. magna under UV exposure was confirmed. On the other hand, rapid bioadsorption of OTC on cell surface was evident in R. subcapitata. Uptake of OTC in D. magna through diet could not be confirmed at short-term. Photomodification of OTC under UV exposure was observed through changes in its absorption spectrum. The results show that short exposure to summer UV levels of southern Chile can rapidly induce phototoxicity of OTC, suggesting a potential risk to aquatic organisms.

Ecotoxicological evaluation of fungicides used in viticulture in non-target organisms.

The effect of fungicides, commonly used in vine cultures, on the health of terrestrial and aquatic ecosystems has been poorly studied. The objective of this study was to evaluate the toxicity of three viticulture fungicides (myclobutanil, cymoxanil, and azoxystrobin) on non-target organisms, the bacteria Rhodopirellula rubra, Escherichia coli, Pseudomonas putida, and Arthrobacter sp., the microalgae Raphidocelis subcapitata, and the macrophyte Lemna minor. Fungicide toxicity was performed in acute cell viability assay for bacteria; 72-h and 7-day growth inhibition tests for R. subcapitata and L. minor, respectively. Contents of photosynthetic pigments and lipid peroxidation in L. minor were evaluated. Arthrobacter sp. and P. putida showed resistance to these fungicides. Even though azoxystrobin affected R. rubra and E. coli cell viability, this effect was due to the solvent used, acetone. Cell viability decrease was obtained for R. rubra exposed to cymoxanil and E. coli exposed to myclobutanil (30 min of exposure at 10 mg/L and 240 min of exposure at 46 mg/L, respectively). R. subcapitata showed about 10-fold higher sensitivity to azoxystrobin (EC50-72h = 0.25 mg/L) and cymoxanil (EC50-72h = 0.36 mg/L) than L. minor to azoxystrobin and myclobutanil (EC50-72h = 1.53 mg/L and EC50-72h = 1.89 mg/L, respectively). No lipid peroxidation was observed in L. minor after fungicide exposure, while changes of total chlorophyll were induced by azoxystrobin and myclobutanil. Our results showed that non-target aquatic organisms of different trophic levels are affected by fungicides used in viticulture.

Caffeine reduces the toxicity of albendazole and carbamazepine to the microalgae Raphidocelis subcapitata (Sphaeropleales, Chlorophyta)

AbstractPharmaceutically active compounds (PhACs) are emerging contaminants that have been widely detected in water bodies in the last decades, with ecological effects toward aquatic biota that have not been fully elucidated. Most studies concerning their toxicity to microalgae have only considered short‐term individual PhAC exposure, rather than combined exposure to several compounds for longer time periods. In this study, we investigated the effects of albendazole (ABZ) (anthelmintic) and carbamazepine (antiepileptic), alone and in combination with caffeine, on the growth and production of chlorophyll‐a of the microalgae Raphidocelis subcapitata, during 16 days of exposure. ABZ alone had a more significant effect than carbamazepine alone on the growth rate and maximum cell density of the microalgae (p < .05; analysis of variance). These results were probably related to the effect of ABZ in inhibiting enzyme complexes and cell membrane proteins related to adenosine triphosphate synthesis, which is important for cell growth. The presence of caffeine lowered the toxicities of ABZ and carbamazepine to the microalgae, probably due to its antioxidant properties, positively affecting chlorophyll‐a production, growth rate, and maximum cell density. Thus, caffeine had an antagonistic interaction with the studied PhACs. The results reinforce the importance of ecotoxicological assays that compare individual and combined PhAC exposure conditions. Our findings highlighted that caffeine can be a relevant factor influencing such assays, considering its widespread occurrence in impacted water bodies.

Wildfire effects on two freshwater producers: Combining in-situ and laboratory bioassays.

Mediterranean forests are highly susceptible to wildfires, which can cause several impacts not only within burnt areas but also on downstream aquatic ecosystems. The ashes' washout from burnt areas by surface runoff can be a diffuse source of toxic substances, such as metals, when reaching the nearby aquatic systems, and can be noxious to aquatic organisms. The present work aimed at assessing the ecotoxicological effects of post-fire contamination on two aquatic producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor) through in-situ bioassays, validating the obtained results with the outcomes of laboratory bioassays with surface water collected simultaneously. Four distinct sites were selected in a basin partially burnt (Ceira river basin; Coimbra district, Portugal) for bioassay deployment: one site upstream the burnt area in the Ceira river (RUS); three sites located under the influence of the burnt area, one immediately downstream of the burnt area in the Ceira river (RDS) and the other two in tributary streams within the burnt area (BS1 and BS2). The in-situ bioassays lasted for 13 days and began following the first post-fire major rain events. Results showed that the microalgae growth rate was able to distinguish the three sites within and downstream of the burnt area (BS1, BS2, RDS) from the site upstream (RUS). By contrast, the macrophytes growth rate only allowed to differentiate between the sites within the burnt area (BS1 and BS2) and those up- and downstream of the burnt area (RUS and RDS). The in-situ results for both species were corroborated with the results of the laboratory experiments, supporting the use of laboratory surrogates for a screening assessment of wildfire impacts in aquatic ecosystems. Direct causal relationships between the observed ecotoxicological effects on R. subcapitata and L. minor and the physical-chemical parameters of the water samples were difficult to establish, although the results suggest (i) a role of differential major and trace metal load in explaining species growth variation; (ii) interaction between metals and/or between metals and other field parameters are likely to modulate the biological responses to the challenges deriving from wildfire runoff.

Growth and Production of Lipids in Raphidocelis subcapitata Immobilized in Sodium Alginate Beads

The growth and production of lipids in the green microalga Raphidocelis subcapitata immobilized in alginate gel are studied. The beads are made from alginate (2% w/v) and CaCl2 (1% w/v). The dry weight, the concentration of cells, and the lipid content are determined after dissolution of the beads in a sodium phosphate buffer. The results show that variations in biomass do not reflect variations in the number of cells in R. subcapitata. Cells divide more rapidly (Gc = 3.45 ± 0.3 days) than biomass is produced (Gm = 4.1 ± 0.4 days) during the exponential growth phase. Therefore, the average mass of the immobilized cells decreases until it reaches its minimum at the end of the exponential phase. Thus, during the stationary phase, cell division ceases while biomass production continues, and the average mass of the immobilized cells increases. In the present study, it is shown that this increase is due to the accumulation of lipids following the depletion of nitrates and phosphates in the culture medium. A lipid content of 24.7 ± 2.5% (dcw) and a lipid productivity of LP = 29.8 ± 3.0 mg/L/day are recorded at the end of culture. These results suggest that immobilized R. subcapitata has promising potential for biodiesel production.