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.

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.

Photosynthetic, morphological and biochemical biomarkers as tools to investigate copper oxide nanoparticle toxicity to a freshwater chlorophyceae.

Copper oxide nanoparticles (CuO NP) have been produced on a large scale due to their economically interesting thermophysical properties. This heightens the concern about risks they may pose on their release into the environment, possibly affecting non-target organisms. Microalga are important organisms in ecotoxicological studies as they are at the base of the aquatic food chain, but information about their biochemical and photosynthetic changes in response CuO NP are still scarce. We studied the effects of CuO NP in Raphidocelis subcapitata using morphological, photosynthetic and biochemical biomarkers. Our results showed that the NP affected microalgal population growth with 0.70 mg Cu L-1 IC50-96 h (inhibition concentration). Based on predicted environmental concentrations of Cu NPs in aquatic environments, our results indicate potential risks of the NP to microalgae. Algal cell size, granularity and photosynthetic efficiencies were affected by the CuO NP at 0.97 and 11.74 mg Cu L-1. Furthermore, lipid metabolism was affected mostly at the highest NP concentration, but at environmentally relevant values (0.012 and 0.065 mg Cu L-1) the production of sterols (structural lipids) and triacylglycerols (reserve lipid) increased. Moreover, we found evidence of cell membrane impairment at the highest CuO NP concentration, and, as a photosynthetic response, the oxygen evolving complex was its main site of action. To the best of our knowledge, this is the first study to date to investigate microalgal lipid composition during CuO NP exposure, showing that it is a sensitive diagnostic tool. This research demonstrated that CuO NP may affect the physiology of R. subcapitata, and because they were observed in a primary producer, we foresee consequences to higher trophic levels in aquatic communities.

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 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.