Effects of Cadmium and Nickel Mixtures on Multiple Endpoints of the Microalga Raphidocelis subcapitata.

It is crucial to investigate the effects of mixtures of contaminants on aquatic organisms, because they reflect what occurs in the environment. Cadmium (Cd) and nickel (Ni) are metals that co-occur in aquatic ecosystems, and information is scarce on their joint toxicity to Chlorophyceae using multiple endpoints. We evaluated the effects of isolated and combined Cd and Ni metals on multiple endpoints of the chlorophycean Raphidocelis subcapitata. The results showed that Cd inhibited cell density, increased reactive oxygen species (ROS) production (up to 308% at 0.075 mg L-1 of Cd), chlorophyll a (Chl a) fluorescence (0.050-0.100 mg L-1 of Cd), cell size (0.025-0.100 mg L-1 of Cd), and cell complexity in all concentrations evaluated. Nickel exposure decreased ROS production by up to 25% at 0.25 mg L-1 of Ni and Chl a fluorescence in all concentrations assessed. Cell density and oxygen-evolving complex (initial fluorescence/variable fluorescence [F0/Fv]) were only affected at 0.5 mg L-1 of Ni. In terms of algal growth, mixture toxicity showed antagonism at low doses and synergism at high doses, with a dose level change greater than the median inhibitory concentration. The independent action model and dose-level-dependent deviation best fit our data. Cadmium and Ni mixtures resulted in a significant increase in cell size and cell complexity, as well as changes in ROS production and Chl a fluorescence, and they did not affect the photosynthetic parameters. Environ Toxicol Chem 2024;43:1855-1869. © 2024 SETAC.

Ecotoxicity of losartan potassium in aquatic organisms of different trophic levels.

The intensive use of the antihypertensive losartan potassium (LOS) has culminated in its high occurrence in aquatic environments. However, insufficient studies had investigated its effects in non-target organisms. In this study, ecotoxicity of LOS was assessed in aquatic organisms from distinct trophic levels (Desmodesmus subspicatus, Daphnia magna, and Astyanax altiparanae). Genotoxicity was assessed by the comet assay in D. magna and A. altiparanae, and biochemical biomarkers for the fish. LOS was more toxic to D. subspicatus (EC50(72h) = 27.93 mg L-1) than D. magna (EC50 = 303.69 mg L-1). Subsequently, this drug showed to induce more DNA damage in D. magna than A. altiparanae, when exposed to 2.5 mg L-1. No significant stress responses were observed by the fish biomarkers, suggesting that higher trophic levels organisms are more tolerant to LOS toxicity. LOS showed relatively low toxic potential for a short period of exposure, but with different patterns of toxicity for the organisms from distinct trophic levels, contributing to further risk assessment of LOS.

Exposure to the azo dye Direct blue 15 produces toxic effects on microalgae, cladocerans, and zebrafish embryos.

Aquatic pollution caused by dyes has increased together with the growth of activities using colorants such as the textile, leather, food, and agrochemicals industries. Because most popular azo dyes are synthesized from benzidine, a carcinogenic compound, a threat to aquatic biota could be expected. The use of single species for toxicity assessment provides limited data, so a battery of test organisms, including representatives of different trophic levels such as algae, zooplankters, and fish, could undoubtedly provide more information. Therefore, our study was aimed at evaluating the toxic effect of the azo dye Direct blue 15 (DB15) on a battery of bioassays using a primary producer (Pseudokirchneriella subcapitata), a primary consumer (Ceriodaphnia dubia), and a secondary consumer (Danio rerio). P. subcapitata was more sensitive to DB15 (IC50 = 15.99 mg L-1) than C. dubia (LC50: 450 mg L-1). In the algae exposed to DB15, chlorophyll-a and -b were significantly increased, and carotenoids were reduced. The concentrations of protein, carbohydrates, and lipids per cell in P. subcapitata exposed to all DB15 concentrations were significantly higher than that measured in control. At 25 mg L-1 of DB15, survival, total progeny, and the number of released clutches were significantly decreased, and the start of reproduction was delayed in C. dubia. DB15 did not induce lethal or sublethal effects in D. rerio embryos at any of the tested concentrations from 24 to 72 h post-fertilization (hpf), but from 96 to 144 hpf, the larvae exposed to 100 and 500 mg L-1 developed yolk sac edema, curved tail, and skeletal deformations. After 144 hpf, DB15 produced a significant increase in embryos without a heartbeat, as the concentration of dye raised. The textile-used, azo dye DB15, caused toxic effects of different magnitude on microalgae, cladocerans, and zebrafish embryos; for this reason, the discharge of this colorant into waterbodies should be regulated to prevent environmental impacts.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.