The combined effect of polystyrene microplastics (mPS) and dibutyl phthalate (DBP), a common plastic additive, on the microalgae Chlorella pyrenoidosa was investigated in the present study. The 96 h-IC₅₀ value of DBP was 2.41 mg L⁻¹. Polystyrene microplastics exhibited size-dependent inhibitory effect to C. pyrenoidosa, with the 96 h-IC₅₀ at 6.90 and 7.19 mg L⁻¹ for 0.1 and 0.55 μm mPS respectively, but little toxicity was observed for 5 μm mPS. The interaction parameter ρ based on the response additive response surface (RARS) model varied from −0.309 to 5.845, indicating the interaction pattern varying with exposure concentrations of chemical mixtures. A modified RARS model (taking ρ as a function of exposure concentration) was constructed and could well predict the combined toxicity of mPS and DBP. More than 20% reduction of DBP was observed at 20 mg L⁻¹ mPS, while 1 mg L⁻¹ mPS had no significant effect on the bioavailability of DBP at different sampling time points. Volume, morphological complexity and chlorophyll fluorescence intensity of microalgal cells were disturbed by both DBP and mPS. The antagonistic effect of high concentrations of mPS might be partially attributed to the combination of hetero- and homo-aggregation and the reduced bioavailability of DBP. The overall findings of the present study profiled the combined toxic effects of mPS and DBP on marine phytoplankton species which will be helpful for further evaluation of ecological risks of mPS and DBP in marine environment.

As an important part of extracellular secondary metabolites, extracellular polymeric substances (EPS) can play a significant role in protecting cells from the threat of exogenous substances, including nanoparticles (NPs). However, the regulation mechanisms of EPS secretion under NPs exposure remain largely unknown. This study investigated the signaling pathways and molecular responses related to EPS secretion of algae (Chlorella pyrenoidosa) upon the exposures to anatase and rutile TiO₂ NPs (nTiO₂-A and nTiO₂-R, respectively) at two similar toxic (20% and 50% of algal growth inhibition) concentrations. The results showed that EPS responded to nTiO₂ stress via excess secretion and compositional variation, and nTiO₂-A induced more EPS secretion than nTiO₂-R at similar toxicity concentrations. The up-regulation of the Ca²⁺ signaling pathway might play a greater role in promoting EPS secretion under nTiO₂-R exposure compared with nTiO₂-A exposure, while the significantly increased intracellular ROS could mainly account for the increased EPS secretion under nTiO₂-A exposure. The up-regulated genes related to biological synthesis and protein metabolism and the enhanced biosynthetic metabolism might be the direct causes of the increased EPS secretion. The increased ROS could have a greater effect on the amino acid metabolism and related genes upon the exposure to nTiO₂-A than nTiO₂-R to induce more EPS secretion. More serious membrane damage caused by nTiO₂-R than nTiO₂-A would affect the intracellular inositol phospholipid metabolism more severely, while the inositol phospholipid pathway and Ca²⁺ signaling pathway might agree and communicate with each other inherently to regulate EPS secretion upon nTiO₂-R exposure. The findings address the regulation mechanisms of algal EPS secretion under nTiO₂ exposure and provide new insights into algal bio-responses to nTiO₂ exposure.

The top-selling strobilurin, azoxystrobin (AZ), is a broad-spectrum fungicide that protects against many kinds of pathogenic fungi by preventing their ATP production. The extensive use of AZ can have negative consequences on non-target species and its effects and toxic mechanisms on algae are still poorly understood. In this work, Chlorella pyrenoidosa that had been grown in BG-11 medium was exposed to AZ (0.5–10 mg L⁻¹) for 10 d. The physiological and molecular responses of the algae to AZ treatment, including photosynthetic efficiency, lipid peroxidation level, antioxidant enzyme activities, as well as transcriptome-based analysis of gene expression, were examined to investigate the potential toxic mechanism. Results shows that the photosynthetic pigment (per cell) increased slightly after AZ treatments, indicating that the photosystem of C. pyrenoidosa may have been strengthened. Glutathione and ascorbate contents were increased, and antioxidant enzyme activities were induced to relieve oxidative damage (e.g., from lipid peroxidation) in algae after AZ treatment. Transcriptome-based analysis of gene expression combined with physiological verification suggested that the 5 mg L⁻¹ AZ treatment did not inhibit ATP generation in C. pyrenoidosa, but did significantly alter amino acid metabolism, especially in aspartate- and glutamine-related reactions. Moreover, perturbation of ascorbate synthesis, fat acid metabolism, and RNA translation was also observed, suggesting that AZ inhibits algal cell growth through multiple pathways. The identification of AZ-responsive genes in the eukaryotic alga C. pyrenoidosa provides new insight into AZ stress responses in a non-target organism.

In this study, we investigated the bioaccumulation and elimination of 14C-labeled BPA by the green alga Chlorella pyrenoidosa and the subsequent transfer of 14C-BPA residues from the contaminated alga to the rotifer Brachionus calyciflorus. After 10 days of BPA exposure, the algal cells accumulated 15% of the initial radioactivity from the medium, with 71% of the accumulated radioactivity occurring in the form of non-extractable bound residues. An approximate steady state of the accumulation of the 14C-BPA residues in the algae was reached after about 4 days of exposure. The bioconcentration factor of total radioactivity in the algae was 106 mL (g dry weight)−1 at steady state. During the elimination phase, only the extractable residues were released from the algae into the water whereas the bound residues, following their ingestion by the rotifers, were converted to extractable forms and then also released. Furthermore, our results demonstrated the biomagnification of BPA-related residues in the food chain between algae and rotifers. The trophic transfer of these BPA-derived residues from the algae to rotifers and thus the environmental hazard may posed by this pathway, because of subsequent effects on the food chain.

Phosphorus (P) plays a critical role in algal growth; therefore, a better understanding of P availability is essential to control harmful algal blooms. Three algae species, Microcystis aeruginosa, Chlorella pyrenoidosa, and Pseudokirchneriella subcapitata, were mono-cultured and co-cultured on three types of P substrates, dissolved inorganic P (DIP), phosphomonoesters glucose-6-phosphate (G-6-P) and β-glycerol phosphate (β-glycerol-P), and phosphonate (glyphosate), to explore their growth and P utilization. All three species could utilize dissolved organic P (DOP) to sustain their growth, whereas DIP was their preferred P substrate in both culture types. Algae could regulate the P uptake capacity under different P conditions, and the added P could be rapidly accumulated at the beginning of the culture and slowly utilized during the subsequent life cycle. M. aeruginosa exhibited wider P selectivity and could utilize all three P substrates, whereas the other two species could only use phosphomonoester (G-6-P and β-glycerol-P) in the mono-cultures. However, in the co-cultures, the relative bioavailability of DOP for M. aeruginosa and C. pyrenoidosa was enhanced, and M. aeruginosa might contribute to the growth of C. pyrenoidosa and P. subcapitata when fed with glyphosate. The three species showed an intrinsic ability to produce alkaline phosphatase (AP), and AP activity (APA) was regulated by Pi stress. However, high APA did not necessarily lead to high Pi release and algal growth on unfavorable substrates. Although M. aeruginosa was not superior in growth rate in the mono-cultures, it showed a better P accumulation ability and maintained stable growth on different P substrates. Moreover, it was a good competitor, suppressing the thriving growth of the other species in co-cultures. Overall, the findings indicated the strategic flexibility of P utilization by algae and the strong competitive ability of M. aeruginosa in Pi-limited and DOP-enriched natural waters.

With the increasing environmental application and discharge of iron-based nanoparticles (NPs), a comprehensive understanding of their fate and ecotoxicological effect in the aquatic environment is very urgent. In this study, toxicities of 4 zero-valent iron NPs (nZVI) of different sizes, 2 Fe2O3 NPs of different crystal phases, and 1 type of Fe3O4 NPs to a green alga (Chlorella pyrenoidosa) were investigated, with a focus on the effects of particle size, crystal phase, oxidation state, and environmental aging. Results show that the algal growth inhibition of nZVI increased significantly with decreasing particle size; with similar particle sizes (20–30 nm), the algal growth inhibition decreased with oxidation of the NPs with an order of nZVI > Fe3O4 NPs > Fe2O3 NPs, and α-Fe2O3 NPs presented significantly higher toxicity than γ-Fe2O3 NPs. The NP-induced oxidative stress was the main toxic mechanism, which could explain the difference in algal toxicity of the NPs. The NP-cell heteroagglomeration and physical interactions also contributed to the nanotoxicity, whereas the effect of NP dissolution was negligible. The aging in distilled water and 3 surface water samples for 3 months increased surface oxidation of the iron-based NPs especially nZVI, which decreased the toxicity to algae. These findings will be helpful for the understanding of the fate and toxicity of iron-based NPs in the aquatic environment.

Butyl Xanthate (BX) is a typical flotation reagent used to extract non-ferrous nickel ores, discharged into the surrounding environment of mining areas in large quantities. However, few studies have focused on the toxicity of combined pollution of BX and nickel (Ni) on aquatic plants, especially phytoplankton, the main producer of aquatic ecosystems. The toxicity and potential mechanism of single and combined pollution of BX and Ni at different concentrations (0–20 mg L⁻¹) on typical freshwater algae (Chlorella pyrenoidosa) were studied. BX slightly stimulated the growth of C. pyrenoidosa on the first day, but Ni and Ni/BX mixture significantly inhibited it during incubation. Results showed that the inhibition rate (I) of the pollutants on the growth of C. pyrenoidosa followed the order: Ni/BX mixture > Ni > BX. The 96-h 20% effective inhibitory concentrations (96h-EC₂₀) of Ni and BX on C. pyrenoidosa growth were 3.86 mg L⁻¹ and 19.25 mg L⁻¹, respectively, indicating C. pyrenoidosa was sensitive to pollutants. The content of total soluble protein (TSP) and chlorophyll a (Chl-a) changed significantly, which may be caused by the damage of pollutants to cell structures (cell membranes and chloroplasts). In addition, the I of pollutants on C. pyrenoidosa growth was related to dose, superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA). The increasement of reactive oxygen species (ROS), antioxidant enzymes (SOD and CAT), and MDA content, suggested C. pyrenoidosa suffered from oxidative stress, leading to lipid oxidation. These results will help to understand the toxicity mechanism of pollutants in typical mining areas and assess the environmental risks of pollutants to primary producers in aquatic ecosystems.

Plastic pollution has become a pressing issue due to its persistence in the environment. Smaller plastics are more easily ingested, potentially exerting greater influences on organisms. In this study, the effects of polystyrene nanoplastics (NP) on the toxic effects, bioaccumulation, biodegradation and enantioselectivity of ibuprofen (IBU) in algae Chlorella pyrenoidosa were explored. The influences on the growth rate, chlorophyll a, total antioxidant capacity (T-AOC), reactive oxygen species (ROS) and lipid peroxidation (MDA) were evaluated after 96 h of exposure to a combination of polystryene NP (1 mg L⁻¹) and IBU (5–100 mg L⁻¹). The results indicated that the inhibitory effect of IBU on C. pyrenoidosa growth was alleviated in the presence of NP. For instance, the 96 h-IC₅₀ value for rac-IBU in the treatment lacking NP was 45.7 mg L⁻¹, and the corresponding value in the treatment containing NP was 63.9 mg L⁻¹. The co-exposure of NP led to a significant enhancement of T-AOC and slight reduction of ROS and MDA compared with the individual exposure (IBU) group, suggesting a decreased oxidative stress. In addition, treatment with NP led to a decreased bioaccumulation and accelerated biodegradation of IBU in C. pyrenoidosa and enhanced removal in the medium. The enantioselective toxicity, bioaccumulation and biodegradation of IBU were observed both in the absence and presence of NP. S-IBU exhibited a greater toxicity, and R-IBU was preferentially accumulated and degraded in C. pyrenoidosa. No interconversion of the two enantiomers occurred regardless of the presence of NP. This consequence implied that the influence of coexistent NP should be considered in the environmental risk assessment of pharmaceuticals and personal care products in aquatic environments.

Polyacrylonitrile polymer (PAN), a common representative textile material and a microplastic, has significant influence on phytoplankton algae, especially with co-exposure with other pollutants, e.g. Cu²⁺. In the present study, we carried out experiments to reveal the population size variation trends of Chlorella pyrenoidosa over time (during a whole growth cycle of 6 days) under PAN and/or Cu²⁺. The levels of pigments (chlorophyll a, b, total chlorophyll and carotenoids), chlorophyll a fluorescence parameters, and other physiological and biochemical indices, containing total protein measurements of H₂O₂, catalase (CAT), and malondialdehyde (MDA) under different treatment groups were measured to explain the physio-ecological mechanism of the effect of PAN and/or Cu²⁺ on the growth of C. pyrenoidosa. The results showed that PAN, Cu²⁺ and the combination of PAN and Cu²⁺ inhibited the growth of C. pyrenoidosa. Chlorophyll a and b decreased significantly with increasing levels of pollutants (PAN and/or Cu²⁺); however, the carotenoid levels increased with increasing levels of pollutants (PAN and/or Cu²⁺) for the first three cultivation days. The oxygen-evolving complexes (OECs) of C. pyrenoidosa had been damaged under Cu²⁺ pollution. The results also showed that CAT activity, MDA content and H₂O₂ activity of C. pyrenoidosa increased with increasing levels of pollutants (PAN and/or Cu²⁺); however, total protein content decreased with increasing levels of pollutants (PAN and/or Cu²⁺) at the first cultivation day. These results indicate that pollutants (PAN and/or Cu²⁺) are harmful to the growth of the C. pyrenoidosa population and negatively affect the levels and function of the pigments in C. pyrenoidosa by decreasing chlorophyll a and b levels, increasing carotenoid levels, and increasing antioxidant enzyme activity.

Insecticide ethiprole, the alternative of fipronil which has been restricted in many countries, may contaminant water bodies through surface runoff after agricultural application, however, the aquatic toxicity and environmental behavior of ethiprole is still unknown. In this study, five metabolites of ethiprole (ethiprole sulfone, ethiprole sulfide, ethiprole amide, desethylsulfinyl ethiprole and ethiprole sulfone amide) were synthesized and their toxic effects on photosynthetic pigment and antioxidase in aquatic plant Chlorella pyrenoidosa (C. pyrenoidosa) were evaluated on an enantiomeric level. Besides, the accumulation and metabolism of rac-ethiprole and its enantiomers in algae suspension and algae were studied. Ethiprole sulfide was found to be more toxic than ethiprole, with the 96h EC₅₀ value seven times lower than ethiprole. Enantioselective toxicity was observed with R-ethiprole more toxic than S-ethiprole. The contents of chlorophyll were significantly reduced by all the chemicals at higher concentrations, and the levels of protein, malondialdehyde (MDA) and the activity of antioxidant defense enzymes were dose-dependent. The half-life of rac-ethiprole in algae suspension was 13.6 days and ethiprole amide was the major metabolite. However, ethiprole sulfide was the main metabolite in algae, suggesting different metabolic pathways in algae suspension and algae. Enantioselective metabolism in algae suspension was found with S-ethiprole metabolized faster than R-ethiprole. The preferentially accumulated and metabolized of R-ethiprole in algae was observed and C. pyrenoidosa had limited capacity to convert one enantiomer into the other. These findings indicated the toxicity of ethiprole to C. pyrenoidosa is lower than fipronil. The individual enantiomers of chiral pollutants and their metabolites should be considered in risk assessments.