Unraveling the Toxicity of a Non-Microcystin-Producing Strain (CCIBt3106) of Microcystis aeruginosa: Ecotoxicological Effects on Aquatic Invertebrates
Abstract
1. Introduction
2. Results and Discussion
2.1. Overview of Biomass and Extraction Yields
2.2. Extracts’ Toxicity Assessment in Aquatic Microcrustaceans
2.3. Fraction Toxicity and Chemical Polarity
2.4. Metabolomic Analysis
2.4.1. Molecular Networking and Class-Level Diversity
2.4.2. Toxicity-Driven Feature Distribution
2.4.3. Putative Metabolite Annotations and Diagnostic Fragmentation Patterns
2.4.4. Environmental Implications and Chemical Risk
3. Conclusions
4. Materials and Methods
4.1. Microcystis aeruginosa (CCIBt 3106 Strain) Cultivation
4.2. Cell Counting
4.3. Extraction of Cyanobacteria Secondary Metabolites
4.4. Fractioning of the Toxic Extracts
4.5. Acute Toxicity Test with Daphnia similis (Crustacea: Cladocera)
4.6. Acute Toxicity Test with Parhyale hawaiensis (Crustacea: Amphipoda)
4.7. Cytotoxicity Test with Artemia salina (Crustacea: Anostraca)
4.8. Toxicity Statistical Analysis
4.9. LC-MS and Metabolomics Analysis
4.10. Metabolite Identification
4.10.1. Processing of Raw Data
4.10.2. Molecular Networking
4.10.3. Metabolite Annotation
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ASW | Artificial seawater |
BOD | Biological oxygen demand |
CCIBt | Algae, Cyanobacteria, and Fungi Culture Collection of the Botany Institute |
CI | Confidence interval |
DMSO | Dimethyl sulfoxide |
EC50/LC50 | Median effective/lethal concentration (50%) |
EPA | Environmental Protection Agency |
F1–F6 | Fraction numbers (0–100% IPA gradient) |
GNPS | Global Natural Products Social Molecular Networking |
HR-QTOF-MS/MS | High-resolution quadrupole time-of-flight tandem mass spectrometry |
IPA | Isopropyl alcohol |
LC-MS/MS | Liquid chromatography coupled with tandem mass spectrometry |
MeOH | Methanol |
MCs | Microcystins |
NMR | Nuclear magnetic resonance |
RDA | Redundancy analysis |
RT | Retention time |
SD | Standard deviation |
TDI | Tolerable daily intake |
WHO | World Health Organization |
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Extracts | Artemia salina | Daphnia similis | Parhyale hawaiensis |
---|---|---|---|
EC50/LC50 (95% CI) (µg mL−1) | |||
Aqueous extract | >1000 | 740 (490–790) | 800 (770–850) |
MeOH 50% extract | >1000 | 940 (540–1380) | 660 (610–720) |
MeOH 100% extract | >1000 | >1000 | 600 (490–880) |
DCM: MeOH 1:1 extract | >1000 | >1000 | >1000 |
Samples | Mean m/z [M + H]+ (Measured) | Mean RT (min) | Putative Formula | Putative Compound | m/z Error (ppm) | Annotation Level * | Reference |
---|---|---|---|---|---|---|---|
All extracts and fractions | 269.0889 | 1.48 | C10H12N4O5 | Inosine | 1.152037 | 2a | Srinivasan et al. [28]. |
MeOH 100%, MeOH 50%, DCM:MeOH 1:1, F1–F4 (H2O and MeOH) | 414.1622 | 3.1 | C16H23N5O8 | Microcystbiopterin B or E | −0.700208 | 2b | Lifshits et al. [29]. |
All extracts and fractions | 591.3587 | 5.02 | C31H50N4O5S | New microginin? | 1.149896 | 2b | Welker et al. [30]. |
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Almeida, É.C.; Jacinavicius, F.R.; Médice, R.V.; Menezes, R.B.; Passos, L.S.; Anderson, D.; Yoon, J.; Faria, E.D.; Crnkovic, C.M.; Fonseca, A.L.; et al. Unraveling the Toxicity of a Non-Microcystin-Producing Strain (CCIBt3106) of Microcystis aeruginosa: Ecotoxicological Effects on Aquatic Invertebrates. Toxins 2025, 17, 321. https://doi.org/10.3390/toxins17070321
Almeida ÉC, Jacinavicius FR, Médice RV, Menezes RB, Passos LS, Anderson D, Yoon J, Faria ED, Crnkovic CM, Fonseca AL, et al. Unraveling the Toxicity of a Non-Microcystin-Producing Strain (CCIBt3106) of Microcystis aeruginosa: Ecotoxicological Effects on Aquatic Invertebrates. Toxins. 2025; 17(7):321. https://doi.org/10.3390/toxins17070321
Chicago/Turabian StyleAlmeida, Éryka Costa, Fernanda Rios Jacinavicius, Rhuana Valdetário Médice, Rafaella Bizo Menezes, Larissa Souza Passos, Dominique Anderson, Jaewon Yoon, Elaine Dias Faria, Camila Manoel Crnkovic, Ana Lúcia Fonseca, and et al. 2025. "Unraveling the Toxicity of a Non-Microcystin-Producing Strain (CCIBt3106) of Microcystis aeruginosa: Ecotoxicological Effects on Aquatic Invertebrates" Toxins 17, no. 7: 321. https://doi.org/10.3390/toxins17070321
APA StyleAlmeida, É. C., Jacinavicius, F. R., Médice, R. V., Menezes, R. B., Passos, L. S., Anderson, D., Yoon, J., Faria, E. D., Crnkovic, C. M., Fonseca, A. L., Henry, T., & Pinto, E. (2025). Unraveling the Toxicity of a Non-Microcystin-Producing Strain (CCIBt3106) of Microcystis aeruginosa: Ecotoxicological Effects on Aquatic Invertebrates. Toxins, 17(7), 321. https://doi.org/10.3390/toxins17070321