Department Environmental Chemistry

Cyanobacterial blooms require monitoring, as they pose a threat to ecosystems and human health, especially by the release of toxins. Along with widely reported microcystins, cyanobacteria coproduce other bioactive metabolites; however, information about their dynamics in surface waters is sparse. We investigated dynamics across full bloom successions throughout a five-year lake monitoring campaign (Greifensee, Switzerland) spanning 150 sampling dates. We conducted extensive suspect screening of cyanobacterial metabolites using the database CyanoMetDB. Across all 850 samples, 35 metabolites regularly co-occurred. Microcystins were present in 70% of samples, with [d-Asp³,(E)-Dhb⁷]MC-RR reaching concentrations of 70 ng/L. Anabaenopeptins, meanwhile, were detected in 95% of all samples with concentrations of Oscillamide Y up to 100-fold higher than microcystins. Based on LC-MS response and frequency, we identified indicator metabolites exclusively produced by one of three cyanobacteria isolated from the lake, these being [d-Asp³,(E)-Dhb⁷]MC-RR from Planktothrix sp. G2020, Microginin 761B from Microcystis sp. G2011, and Ferintoic acid B from Microcystis sp. G2020. These indicators showed distinct temporal trends and peaking seasons that reflect the variance in either the abundance of the producing cyanobacteria or their toxin production dynamics. Our approach demonstrates that selecting high LC-MS response and frequent and species-specific indicator metabolites can be advantageous for cyanobacterial monitoring.

The UV–vis absorbance spectra, molar extinction coefficients and circular dichroism spectra, as well as NMR and high resolution tandem mass spectrometry spectra were determined for two prominent secondary metabolites from cyanobacteria, namely anabaenopeptin A and anabaenopeptin B. The compounds were extracted from the cyanobacterium Planktothrix rubescens CBT929 and purified by flash chromatography and HPLC. Exact amounts of isolated compounds were assessed by quantitative ¹H-NMR with internal calibrant ethyl 4-(dimethylamino)benzoate in DMSO‑d₆ at 298 K with a recycle delay (d1) of 120 s. UV–vis absorbance spectra were recorded in methanol at room temperature. Molar extinction coefficients were determined at 278 nm as 4190 M⁻¹ cm⁻¹ and 2300 M⁻¹ cm⁻¹ in methanol for anabaenopeptin A and anabaenopeptin B, respectively. Circular dichroism spectra and secondary fragmentation mass spectra are also reported.

The mass proliferation of cyanobacteria, episodes known as blooms, is a concern worldwide. One of the most critical aspects during these blooms is the production of toxic secondary metabolites that are not limited to the four cyanotoxins recognized by the World Health Organization. These metabolites comprise a wide range of structurally diverse compounds that possess bioactive functions. Potential human and ecosystem health risks posed by these metabolites and co-produced mixtures remain largely unknown. We studied acute lethal and sublethal effects measured as impaired mobility on the freshwater microcrustaceans Thamnocephalus platyurus for metabolite mixtures from two cyanobacterial strains, a microcystin (MC) producer and a non-MC producer. Both cyanobacterial extracts, from the MC-producer and non-MC-producer, caused acute toxicity with LC₅₀ (24 h) values of 0.50 and 2.55 mg_{dw_biomass}/mL, respectively, and decreased locomotor activity. Evaluating the contribution of different cyanopeptides revealed that the Micropeptin-K139-dominated fraction from the MC-producer extract contributed significantly to mortality and locomotor impairment of the microcrustaceans, with potential mixture effect with other cyanopeptolins present in this fraction. In the non-MC-producer extract, compounds present in the apolar fraction contributed mainly to mortality, locomotor impairment, and morphological changes in the antennae of the microcrustacean. No lethal or sublethal effects were observed in the fractions dominated by other cyanopetides (Cyanopeptolin 959, Nostoginin BN741). Our findings contribute to the growing body of research indicating that cyanobacterial metabolites beyond traditional cyanotoxins cause detrimental effects. This underscores the importance of toxicological assessments of such compounds, also at sublethal levels.

Cyanobacterial blooms occur at increasing frequency and intensity, notably in freshwater. This leads to the introduction of complex mixtures of their products, i.e., cyano-metabolites, to drinking water treatment plants. To assess the fate of cyano-metabolite mixtures during ozonation, a novel multicompound ozone (O₃) competition kinetics method was developed. Sixteen competitors with known second-order rate constants for their reaction with O₃ ranging between 1 and 10⁸ M^–1 s^–1 were applied to cover a wide range of the O₃ reactivity. The apparent second-order rate constants (k_app,O3) at pH 7 were simultaneously determined for 31 cyano-metabolites. k_app,O3 for olefin- and phenol-containing cyano-metabolites were consistent with their expected reactivity (0.4–1.7 × 10⁶ M^–1 s^–1) while k_app,O3 for tryptophan- and thioether-containing cyano-metabolites were significantly higher than expected (3.4–7.3 × 10⁷ M^–1 s^–1). Cyano-metabolites containing these moieties are predicted to be well abated during ozonation. For cyano-metabolites containing heterocycles, k_app,O3 varied from <10² to 5.0 × 10³ M^–1 s^–1, giving first insights into the O₃ reactivity of this class of compounds. Due to lower O₃ reactivities, heterocycle- and aliphatic amine-containing cyano-metabolites may be only partially degraded by a direct O₃ reaction near circumneutral pH. Hydroxyl radicals, which are formed during ozonation, may be more important for their abatement. This novel multicompound kinetic method allows a high-throughput screening of ozonation kinetics.

Cyanobacterial blooms affect aquatic ecosystems across the globe and one major concern relates to their toxins such as microcystins (MC). Yet, the ecotoxicological risks, particularly non-lethal effects, associated with other co-produced secondary metabolites remain mostly unknown. Here, we assessed survival, morphological alterations, swimming behaviour and cardiovascular functions of zebrafish (Danio rerio) upon exposure to cyanobacterial extracts of two Brazilian Microcystis strains. We verified that only MIRS-04 produced MCs and identified other co-produced cyanopeptides also for the MC non-producer NPCD-01 by LC-HRMS/MS analysis. Both cyanobacterial extracts, from the MC-producer and non-producer, caused acute toxicity in zebrafish with LC₅₀ values of 0.49 and 0.98 mg_{dw_biomass}/mL, respectively. After exposure to MC-producer extract, additional decreased locomotor activity was observed. The cyanopeptolin (micropeptin K139) contributed 52% of the overall mortality and caused oedemas of the pericardial region. Oedemas of the pericardial area and prevented hatching were also observed upon exposure to the fraction with high abundance of a microginin (Nostoginin BN741) in the extract of the MC non-producer. Our results further add to the yet sparse understanding of lethal and sublethal effects caused by cyanobacterial metabolites other than MCs and the need to better understand the underlying mechanisms of the toxicity. We emphasize the importance of considering mixture toxicity of co-produced metabolites in the ecotoxicological risk assessment of cyanobacterial bloom events, given the importance for predicting adverse outcomes in fish and other organisms.

The frequency and intensity of cyanobacterial blooms continue to increase in freshwater systems across the globe. Cyanobacteria can release toxins and several bioactive secondary metabolites and analytical methods are needed to effectively assess their concentrations in surface waters. Since booms can evolve rapidly in parts of a lake, high resolution of spatial and temporal sampling increases the complexity of monitoring efforts. Here, we present the validation of an automated, online-solid phase extraction (SPE) high performance liquid chromatography (HPLC)-high resolution tandem mass spectrometry (HRMS/MS) method. This online-SPE HPLC-HRMS/MS methods enables quantitative monitoring of surface waters for 17 cyanobacterial peptides (cyanopeptides), spanning 5 distinct cyanopeptide classes, including: microcystins, anabaenopeptins, nodularins, cyclamides and cyanopeptolins. The method can quantify these cyanopeptides in the low ng/L-range with high accuracy (85-116%) and low relative matrix effects (<25%). We demonstrated its application to Swiss lake waters (Zürichsee, Hallwilersee, Greifensee), which also highlighted the value of adding cyanopeptides beyond common microcystins when monitoring surface waters for cyanobacteria.

Cyanobacteria, also known as blue-green algae, are photosynthetic bacteria that can colonize different habitats, including extreme ones. They are of great interest to the scientific community, especially because of their ability to produce cyanotoxins: toxic secondary metabolites potentially harmful to organisms especially when released to surface waters.

Within the natural products field there is an increasing emphasis on the study of compounds from microbial sources. This has been fuelled by interest in the central role that microorganisms play in mediating both interspecies interactions and host-microbe relationships. To support the study of natural products chemistry produced by microorganisms we released the Natural Products Atlas, a database of known microbial natural products structures, in 2019. This paper reports the release of a new version of the database which includes a full RESTful application programming interface (API), a new website framework, and an expanded database that includes 8128 new compounds, bringing the total to 32 552. In addition to these structural and content changes we have added full taxonomic descriptions for all microbial taxa and have added chemical ontology terms from both NP Classifier and ClassyFire. We have also performed manual curation to review all entries with incomplete configurational assignments and have integrated data from external resources, including CyanoMetDB. Finally, we have improved the user experience by updating the Overview dashboard and creating a dashboard for taxonomic origin. The database can be accessed via the new interactive website at www.npatlas.org.

To assess the risks associated with cyanobacterial blooms, the persistence and fate processes of cyanotoxins and other bioactive cyanobacterial metabolites need to be evaluated. Here, we investigated the reaction with photochemically produced singlet oxygen (¹O₂) for 30 cyanopeptides synthesized by Dolichospermum flos aquae, including 9 anabaenopeptins, 18 microcystins, 2 cyanopeptolins, and 1 cyclamide. All compounds were stable in UVA light alone but in the presence of a photosensitizer we observed compound-specific degradation. A strong pH effect on the decay was observed for 18 cyanopeptides that all contained tyrosine or structurally related moieties. We can attribute this effect to the reaction with ¹O₂ and triplet sensitizer that preferentially react with the deprotonated form of tyrosine moieties. The contribution of ¹O₂ to indirect phototransformation ranged from 12 to 39% and second-order rate constants for 9 tyrosine-containing cyanopeptides were assessed. Including the pH dependence of the reaction and system-independent second-order rate constants with ¹O₂ will improve the estimation of half-lives for multiclass cyanopeptide in surface waters. Our data further indicates that naturally occurring triplet sensitizers are likely to oxidize deprotonated tyrosine moieties of cyanopeptides and the specific reactivity and its pH dependence needs to be investigated in future studies.

Background: Bloom-forming cyanobacteria occur globally in aquatic environments. They produce diverse bioactive metabolites, some of which are known to be toxic. The most studied cyanobacterial toxins are microcystins, anatoxin, and cylindrospermopsin, yet more than 2000 bioactive metabolites have been identified to date. Data on the occurrence of cyanopeptides other than microcystins in surface waters are sparse.
Results: We used a high-performance liquid chromatography–high-resolution tandem mass spectrometry/tandem mass spectrometry (HPLC–HRMS/MS) method to analyse cyanotoxin and cyanopeptide profiles in raw drinking water collected from three freshwater reservoirs in the United Kingdom. A total of 8 cyanopeptides were identified and quantified using reference standards. A further 20 cyanopeptides were identified based on a suspect-screening procedure, with class-equivalent quantification. Samples from Ingbirchworth reservoir showed the highest total cyanopeptide concentrations, reaching 5.8, 61, and 0.8 µg/L in August, September, and October, respectively. Several classes of cyanopeptides were identified with anabaenopeptins, cyanopeptolins, and microcystins dominating in September with 37%, 36%, and 26%, respectively. Samples from Tophill Low reservoir reached 2.4 µg/L in September, but remained below 0.2 µg/L in other months. Samples from Embsay reservoir did not exceed 0.1 µg/L. At Ingbirchworth and Tophill Low, the maximum chlorophyll-a concentrations of 37 µg/L and 22 µg/L, respectively, and cyanobacterial count of 6 × 10⁴ cells/mL were observed at, or a few days after, peak cyanopeptide concentrations. These values exceed the World Health Organization’s guideline levels for relatively low probability of adverse health effects, which are defined as 10 µg/L chlorophyll-a and 2 × 10⁴ cells/mL.
Conclusions: This data is the first to present concentrations of anabaenopeptins, cyanopeptolins, aeruginosins, and microginins, along  with microcystins, in U.K. reservoirs. A better understanding of those cyanopeptides that are abundant in drinking water reservoirs can inform future monitoring and studies on abatement efficiency during water treatment.

Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.

Background: Cyanobacteria and their toxins occur in high concentrations during the so-called bloom events in surface waters. To be able to assess the risks associated with cyanobacterial blooms, we need to understand the persistence and fate processes of these toxins and other bioactive metabolites. In this study, we investigated the photochemical fate of 54 cyanopeptides extracted from two strains of Microcystis aeruginosa (PCC7806 and UV006), Planktothrix rubescens, and Dolichospermum flos aquae. We determined half-lives during sunlight exposure in lake water and inspected the effect of pH on transformation kinetics for 27 microcystins, 8 anabaenopeptins, 14 cyanopeptolins, 2 cyclamides, and 3 aeruginosins.
Results: For cyanopeptides from D. flos aquae and P. rubescens, we observed the highest removal of 28 and 26%, respectively, after 3-h sunlight exposure. Most cyanopeptides produced by the two M. aeruginosa strains were rather persistent with only up to 3% removal. The more reactive cyanopeptides contained amino acids known to undergo phototransformation, including methionine and tyrosine moieties or their derivatives. Photochemical half-lives of 14 tyrosine-containing cyanopeptides decreased by one order of magnitude from nearly persistent conditions at pH 7 (half-life > 70 h) to shorter half-lives at pH 10 (< 10 h).
Conclusions: More work is needed to distinguish the contribution of different photochemical reaction pathways including the contributions to the pH effect. To the best of our knowledge, this is the first assessment of transformation kinetics of such a wide range of cyanopeptides. The abundant and persistent cyanopeptides that have not been studied in detail yet should be prioritized for the evaluation of their ecosystem and human health risks and for their abatement during drinking water treatment.

Intensified cyanobacterial bloom events are of increasing global concern because of adverse effects associated with the release of bioactive compounds, including toxic cyanopeptides. Cyanobacteria can produce a variety of cyanopeptides, yet our knowledge about their abundance and co-production remains limited. We applied a suspect-screening approach, including 700 structurally known cyanopeptides, and identified 11 cyanopeptides in Microcystis aeruginosa and 17 in Dolichospermum flos-aquae. Total cyanopeptide concentrations ranged from high nmol to μmol g_dry^-1 with slightly higher cell quotas in the mid-exponential growth phase. Relative cyanopeptide profiles were unchanged throughout the growth cycle. We demonstrate that quantification based on microcystin-LR equivalents can introduce an error of up to 6-fold and recommend a class-equivalent approach instead. In M. aeruginosa, rarely studied cyclamides dominated (>80%) over cyanopeptolins and microcystins. While all nutrient reductions caused less growth, only lowering phosphorous and micronutrients reduced cyanopeptide production by M. aeruginosa. Similar trends were observed for D. flos-aquae and only lowering nitrogen decreased cyanopeptide production while the relative abundance of individual cyanopeptides remained stable. The synchronized production of other cyanopeptides along with microcystins emphasizes the need to make them available as reference standards to encourage more studies on their occurrence in blooms, persistence, and potential toxicity.

Harmful cyanobacterial blooms in freshwater ecosystems produce bioactive secondary metabolites including cyanopeptides that pose ecological and human health risks. Only adverse effects of one class of cyanopeptides, microcystins, have been studied extensively and have consequently been included in water quality assessments. Inhibition is a commonly observed effect for enzymes exposed to cyanopeptides and has mostly been investigated for human biologically relevant model enzymes. Here, we investigated the inhibition of ubiquitous aquatic enzymes by cyanobacterial metabolites. Hydrolytic enzymes are utilized in the metabolism of aquatic organisms and extracellularly by heterotrophic bacteria to obtain assimilable substrates. The ubiquitous occurrence of hydrolytic enzymes leads to the co-occurrence with cyanopeptides especially during cyanobacterial blooms. Bacterial leucine aminopeptidase and alkaline phosphatase were exposed to cyanopeptide extracts of different cyanobacterial strains (Microcystis aeruginosa wild type and microcystin-free mutant, Planktothrix rubescens) and purified cyanopeptides. We observed inhibition of aminopeptidase and phosphatase upon exposure, especially to the apolar fractions of the cyanobacterial extracts. Exposure to the dominant cyanopeptides in these extracts confirmed that purified microcystins, aerucyclamide A and cyanopeptolin A inhibit the aminopeptidase in the low mg L^-1 range while the phosphatase was less affected. Inhibition of aquatic enzymes can reduce the turnover of nutrients and carbon substrates and may also impair metabolic functions of grazing organisms.

Cyanobacterial bloom events that produce natural toxins occur in freshwaters across the globe, yet the potential risk of many cyanobacterial metabolites remains mostly unknown. Only microcystins, one class of cyanopeptides, have been studied intensively and the wealth of evidence regarding exposure concentrations and toxicity led to their inclusion in risk management frameworks for water quality. However, cyanobacteria produce an incredible diversity of hundreds of cyanopeptides beyond the class of microcystins. The question arises, whether the other cyanopeptides are in fact of no human and ecological concern or whether these compounds merely received (too) little attention thus far. Current observations suggest that an assessment of their (eco)toxicological risk is indeed relevant: First, other cyanopeptides, including cyanopeptolins and anabaenopeptins, can occur just as frequently and at similar nanomolar concentrations as microcystins in surface waters. Second, cyanopeptolins, anabaenopeptins, aeruginosins and microginins inhibit proteases in the nanomolar range, in contrast to protein phosphatase inhibition by microcystins. Cyanopeptolins, aeruginosins, and aerucyclamide also show toxicity against grazers in the micromolar range comparable to microcystins. The key challenge for a comprehensive risk assessment of cyanopeptides remains their large structural diversity, lack of reference standards, and high analytical requirements for identification and quantification. One way forward would be a prevalence study to identify the priority candidates of tentatively abundant, persistent, and toxic cyanopeptides to make comprehensive risk assessments more manageable.