Cyanotoxins and Pollutants Interact in Surprising Ways in Freshwater Algae

A laboratory study of the freshwater microalga Chlorella vulgaris has revealed that the combined toxicity of cyanobacterial toxins and human-made contaminants cannot be predicted from the toxicity of each chemical alone. Researchers exposed the green alga to the cyanotoxins microcystin-LR and cylindrospermopsin, the herbicide terbuthylazine and the metal cadmium, both individually and in five binary mixtures, over seven days. The findings, published in the journal Ecotoxicology, show that interactions between the compounds shifted over time, with antagonism dominating after four days of exposure and more complex dose-dependent patterns, including synergism at low concentrations in three of the five mixtures, emerging after seven days. According to the authors, this time-dependent change in toxicity patterns may represent a concern for aquatic organisms living in habitats contaminated by both algal blooms and industrial or agricultural runoff.

Harmful cyanobacterial blooms are increasing worldwide as eutrophication intensifies and climate change raises water temperatures and alters precipitation patterns. Many bloom-forming cyanobacterial species produce potent toxins, and the hepatotoxic microcystins and the cytotoxin cylindrospermopsin are the most frequently detected in brackish and freshwater environments. During bloom development, microcystins are mostly retained within the cyanobacterial cells, whereas cylindrospermopsin is continuously released into the water because of its hydrophilic properties and apparent membrane permeability. Cell senescence and lysis can then release high concentrations of both toxins into the surrounding water. Microcystin levels may exceed the World Health Organization guideline values of 1 microgram per litre in drinking water and 10 micrograms per litre in recreational waters, and although surface water concentrations are generally below 100 micrograms per litre, they can exceed 1000 micrograms per litre under certain environmental conditions such as elevated pH, temperature and nutrient levels. Concentrations ranging from 4 to 6500 micrograms per litre have been detected in irrigation waters in Finland, Turkey, India and Morocco, while cylindrospermopsin in water can reach values up to 126 micrograms per litre.

Both cyanotoxins can disrupt fundamental cellular processes in aquatic organisms. Microcystins inhibit protein phosphatases 1 and 2A in mammals and higher plants, and oxidative stress appears to play a significant role in their toxicity in aquatic animals, plants and algae. In plants, microcystins also induce histone H3 hyperphosphorylation, leading to incomplete chromatid segregation and the formation of micronuclei. Cylindrospermopsin inhibits glutathione and protein synthesis and potentially interferes with DNA structure in mammals, with cytochrome P450-generated metabolites likely mediating its toxic mechanism. In plants, the toxin is recognised by alterations in microtubule formation and spindle-phragmoplast disruption. At the same time, agriculture and industry continue to release persistent anthropogenic contaminants such as metals and pesticides into aquatic systems. Although combined effects of cyanotoxins and anthropogenic contaminants have been reported for invertebrates, fish and plants, data for freshwater microalgae remained scarce, which motivated the new investigation.

The research team selected four chemicals with distinct mechanisms of action. Microcystin-LR was chosen as the most common and toxic variant of the microcystins, and cylindrospermopsin for its worldwide occurrence. Terbuthylazine, a triazine-ring herbicide frequently detected in surface waters, acts at photosystem II of photosynthetic organisms by inhibiting photosynthetic electron transport. Cadmium, a non-essential metal that enters aquatic environments from both natural and anthropogenic sources, induces oxidative stress and competes with the essential calcium binding site in photosystem II during photoactivation. The test organism, Chlorella vulgaris, is a widespread freshwater primary producer that co-occurs with cyanobacterial blooms and contaminants and is a well-established model organism. The researchers hypothesised that the alga’s growth would be adversely affected by each compound individually and that combined effects could deviate from predictions based on single-compound exposures, producing either synergistic or antagonistic interactions.

The experiments were conducted in 96-well microplates as an adaptation of the OECD 201 growth inhibition guideline. Log-phase-growing algae at approximately 5 × 10⁵ cells per millilitre were exposed for seven days, and growth was measured indirectly by optical density at 750 nanometres, with readings converted to cell concentrations using a calibration equation. Cyanotoxin concentrations were analytically verified by HPLC-PDA, and because measured values deviated by more than 20 percent from nominal concentrations, all calculations were based on measured concentrations. Stability analyses confirmed that neither microcystin-LR nor cylindrospermopsin degraded during the seven-day exposure period. Cadmium and terbuthylazine concentrations were nominal, which the authors considered appropriate because cadmium does not degrade during exposure and terbuthylazine is reported to be stable in aqueous solution with negligible hydrolysis. The toxins themselves were extracted and purified from laboratory cultures of Microcystis aeruginosa, which produces primarily microcystin-LR, and Aphanizomenon ovalisporum, which produces only cylindrospermopsin.

In single-chemical exposures, terbuthylazine proved the most toxic compound to C. vulgaris, followed by cadmium, cylindrospermopsin and microcystin-LR. After four days, terbuthylazine was about five times more toxic than cadmium, which in turn was approximately 28 times more toxic than cylindrospermopsin. After seven days the gap between terbuthylazine and cadmium narrowed, with terbuthylazine only 2.5 times more toxic, while cadmium was roughly 46 times more toxic than cylindrospermopsin. The herbicide reduced algal growth rate by 50 percent at concentrations of 0.35 to 0.39 milligrams per litre after four days, with slightly higher values after seven days, indicating some growth recovery. Cadmium showed EC50 values ranging from 0.92 to 4.15 milligrams per litre after four days and 0.76 to 1.80 milligrams per litre after seven days, becoming more toxic over time. The cyanotoxins were only toxic at high concentrations: cylindrospermopsin halved growth rates at roughly 47 to 56 milligrams per litre, while microcystin-LR produced maximum growth rate inhibitions of 23 percent and 44 percent at its lowest observed effect concentrations of 40.4 and 78.9 milligrams per litre after four and seven days respectively.

To analyse the mixtures, the team used the concentration addition model, a conservative conceptual framework that assumes mixture components have similar modes of action, implemented through the MIXTOX tool. Deviations from additivity were tested for synergism or antagonism, dose ratio dependency and dose level dependency. Because microcystin-LR and, in some experiments, terbuthylazine did not achieve 50 percent growth inhibition on their own, estimated parameters and synergistic ratios were used to characterise the interactions. All five binary mixtures deviated from the non-interactive concentration addition model, indicating genuine chemical interactions rather than simple additive or independent responses. Such deviations can occur when compounds affect one another’s bioavailability, modes of action or behaviour after uptake.

The interaction patterns depended strongly on both mixture composition and exposure duration. After four days, antagonism prevailed across all tested mixtures. In the microcystin-LR and terbuthylazine combination, the herbicide’s toxicity was reduced when microcystin-LR was present, with synergistic ratios of 0.46 at 8.7 milligrams per litre and 0.33 at 19.7 milligrams per litre of the toxin. One proposed explanation involves photosynthesis: terbuthylazine reversibly binds the D1 protein of photosystem II, but microcystin-LR at high concentrations induces reactive oxygen species that can oxidise and degrade the D1 protein, potentially leaving fewer binding sites for the herbicide. For the microcystin-LR and cadmium mixture, antagonism after four days may reflect the induction of metallothioneins and phytochelatins, detoxification proteins in algae, or the formation of microcystin-cadmium complexes that reduce the bioavailability of both compounds. The cylindrospermopsin and terbuthylazine mixture showed dose ratio-dependent antagonism, particularly when the toxin dominated the mixture, possibly because cylindrospermopsin activates glutathione-based detoxification pathways that also metabolise triazine herbicides.

After seven days, the picture changed markedly. Dose level dependency was the only deviation observed at that time point, and synergism emerged at low concentrations of each chemical in three of the five tested binary mixtures. The most striking example was the microcystin-LR and cadmium mixture, which shifted from antagonism after four days to synergism at low concentrations after seven days, with the switch occurring at concentrations below the cadmium EC50 level, at approximately 0.81 times that value. The authors suggest that the combined production of reactive oxygen species by both compounds at low concentrations could enhance oxidative stress and impair cellular detoxification mechanisms, while antagonism at high concentrations may reflect interactions in the exposure medium or reduced levels of extra detoxification proteins. For the cylindrospermopsin and terbuthylazine mixture, the model predicted that synergism would occur only at concentrations well above those tested, leaving antagonism dominant within the experimental range. The terbuthylazine and cadmium mixture remained antagonistic at both time points.

The authors emphasise that the concentrations used in the mixture experiments, particularly for the cyanotoxins, were higher than those typically found in the environment, although exceptionally high microcystin levels of up to 124.46 milligrams per litre have been measured in water samples in southern Africa. They note that the laboratory approach can be applied to real scenarios by incorporating safety factors to convert non-ecologically relevant chemical levels into more ecologically relevant ones. The antagonism observed at shorter exposures suggests that simultaneous presence of cyanotoxins and anthropogenic compounds may result in lower-than-expected toxicity under the tested conditions, whereas the synergism at low concentrations after longer exposure, especially for microcystin-LR with cadmium, points to the possibility of more severe effects than single-chemical assessments would predict. Given the expected increase in the frequency and magnitude of harmful algal blooms, the team argues that ecotoxicological assessments need to account for mixture effects and that further research is needed to elucidate the mechanisms underlying these interactions and to determine their ecological relevance under environmentally realistic exposure conditions. The data supporting the study are publicly available in the Zenodo repository.

Subject of Research: Combined toxicity of cyanotoxins, terbuthylazine and cadmium to the freshwater alga Chlorella vulgaris in single and binary mixture exposures

Article Title: Combined toxicity of cyanotoxins and anthropogenic contaminants on Chlorella vulgaris: insights from single and binary mixture exposures

Article References: Pinheiro, C., Azevedo, J., Campos, A., Silva, P. V., Vasconcelos, V., & Loureiro, S. (2026). Combined toxicity of cyanotoxins and anthropogenic contaminants on Chlorella vulgaris: insights from single and binary mixture exposures. Ecotoxicology, 35(9), Article 196. https://doi.org/10.1007/s10646-026-03171-z

Image Credits: AI Generated

DOI: 10.1007/s10646-026-03171-z

Keywords: cyanotoxins, microcystin-LR, cylindrospermopsin, terbuthylazine, cadmium, Chlorella vulgaris, binary mixtures, concentration addition, synergism, antagonism, MIXTOX, ecotoxicology