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;00:1-15. © 2024 SETAC.

Effects of ZnWO4 nanoparticles on growth, photosynthesis, and biochemical parameters of the green microalga Raphidocelis subcapitata.

Nanoparticles have applications in many sectors in the society. ZnWO4 nanoparticles (ZnWO4-NPs) have potential in the fabrication of sensors, lasers, and batteries, and in environmental remediation. Thus, these NPs may reach aquatic ecosystems. However, we still do not know their effects on aquatic biota and, to our knowledge, this is the first study that evaluates the toxicity of ZnWO4-NPs in a eukaryotic organism. We evaluated the toxicity of ZnWO4-NPs on the green microalga Raphidocelis subcapitata for 96 h, in terms of growth, cell parameters, photosynthesis, and biochemical analysis. Results show that most of Zn was presented in its particulate form, with low amounts of Zn2+, resulting in toxicity at higher levels. The growth was affected from 8.4 mg L-1, with 96h-IC50 of 23.34 mg L-1. The chlorophyll a (Chl a) content increased at 30.2 mg L-1, while the fluorescence of Chl a (FL3-H) decreased at 15.2 mg L-1. We observed increased ROS levels at 44.4 mg L-1. Regarding photosynthesis, the NPs affected the oxygen evolving complex (OEC) and the efficiency of the photosystem II at 22.9 mg L-1. At 44.4 mg L-1 the qP decreased, indicating closure of reaction centers, probably affecting carbon assimilation, which explains the decay of carbohydrates. There was a decrease of qN (non-regulated energy dissipation, not used in photosynthesis), NPQ (regulated energy dissipation) and Y(NPQ) (regulated energy dissipation via heat), indicating damage to the photoprotection system; and an increase in Y(NO), which is the non-regulated energy dissipation via heat and fluorescence. The results showed that ZnWO4-NPs can affect the growth and physiological and biochemical parameters of the chlorophycean R. subcapitata. Microalgae are the base of aquatic food chains, the toxicity of emerging contaminants on microalgae can affect entire ecosystems. Therefore, our study can provide some help for better protection of aquatic ecosystems.

Isolated and combined effects of cobalt and nickel on the microalga Raphidocelis subcapitata.

Aquatic organisms are exposed to several compounds that occur in mixtures in the environment. Thus, it is important to investigate their impacts on organisms because these combined effects can be potentiated. Cobalt (Co) and nickel (Ni) are metals that occur in the environment and are used in human activities. To the best of our knowledge, there are no studies that investigated the combined effects of these metals on a freshwater Chlorophyceae. Therefore, this study analyzed the isolated and combined effects of Co and Ni in cell density, physiological and morphological parameters, reactive oxygen species (ROS), carbohydrates and photosynthetic parameters of the microalga Raphidocelis subcapitata. Data showed that Co affected the cell density from 0.25 mg Co L-1; the fluorescence of chlorophyll a (Chl a) (0.10 mg Co L-1); ROS production (0.50 mg Co L-1), total carbohydrates and efficiency of the oxygen evolving complex (OEC) at all tested concentrations; and the maximum quantum yield (ΦM) from 0.50 mg Co L-1. Ni exposure decreased ROS and cell density (0.35 mg Ni L-1); altered Chl a fluorescence and carbohydrates at all tested concentrations; and did not alter photosynthetic parameters. Regarding the Co-Ni mixtures, our data best fitted the concentration addition (CA) model and dose-ratio dependent (DR) deviation in which synergism was observed at low doses of Co and high doses of Ni and antagonism occurred at high doses of Co and low doses of Ni. The combined metals affected ROS production, carbohydrates, ΦM, OEC and morphological and physiological parameters.

Single and combined effects of Zn and Al on photosystem II of the green microalgae Raphidocelis subcapitata assessed by pulse-amplitude modulated (PAM) fluorometry.

Increasing metal concentrations in aquatic environments are mainly due to anthropogenic actions, which is a matter of concern for the biodiversity of aquatic biota. It is known that metals coexist in environments, however environmental risk assessments do not usually take into account the effects of these mixtures. We aimed to test Zn and Al mixtures on the photosynthetic apparatus of a green microalga, for the first time, using PAM fluorometry. After 72 h exposure, single concentrations from 0.08 to 0.46 µM Zn and 22.24 to 37.06 µM Al affected the photosynthetic parameters of Raphidocelis subcapitata. Metals affected the efficiency of the oxygen-evolving complex - OEC (F0/Fv), increasing it by 25% at 0.46 µM Zn and by 82% at 37.06 µM Al - concentrations where, 57% and 78% of growth inhibition occurred, respectively. We observed that the algal growth was more sensitive to infer Zn toxicity, while F0/Fv was more affected by Al. Regarding quenching, there was an increase in passive energy dissipation ((Y(NO)) at 0.46 µM Zn, and we observed an increase in both regulated ((NPQ and Y(NPQ)) and non-regulated energy dissipation ((qN and (Y(NO)) at 37.06 µM Al. Our results showed synergism and antagonism at different concentrations in mixtures, the antagonism prevailing at higher metal concentrations and, in some cases, synergism at lower concentrations of Zn and Al. Since we observe more than additive and less than additive effects, it is of the utmost importance to take mixture toxicity tests into account when performing risk assessments on green algae.

Individual and Combined Effects of Manganese and Chromium on a Freshwater Chlorophyceae.

Manganese (Mn), an essential metal in trace amounts, and chromium (Cr), a nonessential metal to algae, are often found in effluent discharges and may co-occur in contaminated aquatic environments. Therefore, we investigated the effects of Mn and Cr, and their mixtures, on a freshwater Chlorophyceae, Raphidocelis subcapitata, using a multiple endpoint approach. Regarding the single exposure of metals, Mn was 4 times more toxic (median inhibitory concentration at 72 h [IC5072 h ] = 4.02 ± 0.45 µM Mn) than Cr (IC5072 h = 16.42 ± 4.94 µM Cr) for microalgae, considering the effects on the relative growth rate. Moreover, this species was the most sensitive to Mn, according to the species sensitivity distribution curve. Overall, the tested metals did not lead to significant changes in reactive oxygen species production, cellular complexity, and cell relative size but significantly decreased the algal growth and the mean cell chlorophyll a (Chl a) fluorescence at the highest concentrations (3.64-14.56 µM of Mn and 15.36-19.2 µM of Cr). The decreased mean cell Chl a fluorescence indicates an impact on pigment synthesis, which may be related to the observed growth inhibition. In the mixture tests, the reference models concentration addition and independent action were used to analyze the data, and the independent action model was the best fit to describe our results. Therefore, the Mn and Cr mixture was noninteractive, showing additive effects. This is the first study to address the combined toxicity of Mn and Cr regarding freshwater Chlorophyceae. Environ Toxicol Chem 2022;41:1004-1015. © 2022 SETAC.

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 IC5096 h was 0.08 mg L-1 of Cd and 0.16 mg L-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.

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.

Zinc and aluminum mixtures have synergic effects to the algae Raphidocelis subcapitata at environmental concentrations.

A large number of metals is present in aquatic ecosystems, often occurring simultaneously, however, the isolated toxicity of them are better well known than their mixtures. Based on that, for the first time we aimed to test the effects of zinc (Zn) and aluminum (Al) mixtures to the microalgae Raphidocelis subcapitata. Regarding isolated toxicity, the 96 h IC50 of Zn and Al based on specific growth rates occurred, respectively, at 0.40 and 27.40 µM, thus Zn was ≈70-fold more toxic than Al. Both Zn and Al altered the cell size and complexity of R. subcapitata at the highest concentrations, although only during Zn exposure was the chlorophyll a fluorescence significantly diminished. Microalgae exposed to Al produced more ROS than during Zn exposure. Moreover, algae produced less ROS at the highest Zn concentration than in the lower concentrations. According to species sensitivity curves (SSD), R. subcapitata was the most sensitive organism to Zn and one of the most sensitive to Al. With respect to mixture toxicity tests, there were significant deviations for both CA (concentration addition) and IA (independent action) models, although data best fitted the CA model and DL (dose level-dependence) deviation, in which metals showed synergic effects at low concentrations and antagonist effects at higher concentrations.

Effects of iron oxide nanoparticles (Fe3O4) on life history and metabolism of the Neotropical cladoceran Ceriodaphnia silvestrii.

Nanoparticles (NPs) production is increasing worldwide. These products are likely to end up in aquatic environments. However, few studies evaluated the chronic toxicity of iron-based NPs (Fe-NPs) to cladocerans and their potential ecotoxicological hazards. In this study we aimed to investigate the effects of iron oxide nanoparticles (Fe3O4-NPs) to Ceriodaphnia silvestrii Daday, 1902, assessing acute (48 h) and chronic toxicity (up to 14 d). Besides traditional endpoints (immobility and lethality), we also evaluated physiological responses (respiration rates) in a 48 h-exposure. No immobility was observed (EC50 > 100 mg L-1) after 48 h, whereas respiration rates at the highest concentration were 400% of that in control, indicating that this endpoint was more sensitive during acute toxicity. In chronic assays, Fe3O4-NPs affected body length (8.24% growth inhibition in 7 d-exposure) and number of eggs (7-d IC10: 3.53, IC20: 6.69 mg Fe L-1) and neonates (7-d IC10: 1.25, IC20: 3.75 mg Fe L-1). Based on species sensitivity distribution (SSD), C. silvestrii was a sensitive organism, suggesting Fe-NPs as a possible threat for this species. Our results also indicate that the NPs caused a physical barrier, impairing food absorption, since we observed NPs agglomerations into cladocerans' gut. We demonstrate that Fe3O4-NPs affects C. silvestrii metabolism and reproduction and our results support the use of sublethal endpoints to assess environmental safety. The release of these NPs into freshwater environments should be carefully evaluated, since disturbances on cladoceran population dynamics could cause strong impacts on the entire food web structure and ultimately on ecosystem functioning.

Effects of 2,4-D-based herbicide (DMA® 806) on sensitivity, respiration rates, energy reserves and behavior of tadpoles.

Increased use of sugarcane pesticides and their destination to non-target environments in Brazil has generated concerns related to the conservation of more vulnerable groups, such as amphibians. Besides the high skin permeability, tadpoles are constantly restricted to small and ephemeral ponds, where exposure to high concentrations of pesticides in agricultural areas is inevitable. This study evaluated chronic effects caused by sub-lethal concentrations of 2,4-dichlorophenoxyacetic acid herbicide on energy storage, development, respiration rates, swimming performance and avoidance behavior of bullfrog tadpoles (Lithobates catesbeianus). Firstly, we conducted acute toxicity test (96 h) to estipulate sub-lethal concentrations of 2,4-D and evaluate the sensitivity of three tadpoles' species to this herbicide. Results showed that Leptodactylus fuscus presented the lowest LC50 96 h, 28.81 mg/L, followed by Physalaemus nattereri (143.08 mg/L) and L. catesbeianus (574.52 mg/L). Chronic exposure to 2,4-D (125, 250 and 500 µg/L) delayed metamorphosis and inhibited the growth of tadpoles at concentrations of 125 µg/L. Effects on biochemical reserves showed that 2,4-D increased total hepatic lipids in tadpoles, although some individual lipid classes (e.g. free fatty acids and triglycerides) were reduced. Protein and carbohydrates contents were also impaired by 2,4-D, suggesting a disruption on energy metabolism of amphibians by the herbicide. In addition to biochemical changes, respiration rates and swimming speed were also decreased after chronic exposure to 2,4-D, and these responses appeared to be correlated with the changes detected in the basic energy content. Avoidance test indicated that tadpoles of L. catesbeinus avoided the presence of 2,4-D, however they were unable to detect increasing gradients of the contaminant. Our data showed that chronic exposure to 2,4-D impaired biochemical, physiological and behavioral aspects of tadpoles, which may compromise their health and make them more vulnerable to environmental stressors in natural systems.

Responses of Raphidocelis subcapitata exposed to Cd and Pb: Mechanisms of toxicity assessed by multiple endpoints.

Microalgae have been widely used in ecotoxicological studies in order to evaluate the impacts of heavy metals in aquatic ecosystems. However, there are few studies that analyze the effects of metals in an integrative way on photosynthetic apparatus of freshwater microalgae in the generation of reactive oxygen species (ROS) and biochemical composition. Therefore, this study aimed to assess cadmium (Cd) and lead (Pb) toxicity using synchronously physiological and biochemical endpoints, specially detecting lipidic classes for the very first time during Cd and Pb-exposure to Raphidocelis subcapitata. Here we show that analyzing the algae growth, the IC50-72 h for Cd was 0.04 µM and for Pb was 0.78 µM. In general, the Cd affected the biochemical parameters more, leading to an increase in total lipid content (7.2-fold), total carbohydrates (3.5-fold) and ROS production (3.7-fold). The higher production of lipids and carbohydrates during Cd-exposure probably acted as a defense mechanism, helping to reduce the extent of damage caused by the metal in the photosynthetic apparatus. For Pb, the physiological parameters were more sensitive, which resulted in changes of chlorophyll a synthesis and a reduction of both efficiency of oxygen-evolving complex and quantum yields. Besides that, we observed changes in the lipid class composition during Cd and Pb-exposure, suggesting these analyses as great biomarkers to assess metal toxicity mechanisms in ecological risk assessments. Thereby, here we demonstrate the importance of using multiple endpoints in ecotoxicological studies in order to obtain a better understanding of the mechanisms of metal toxicity to R. subcapitata.