Toxins produced by cyanobacteria (cyanotoxins) belong to the most harmful substances in natural aquatic environments (Neumann et al. 2007; Everson et al. 2009; Weirich & Miller 2014; Kubickova et al. 2019). One of the most common cyanotoxins is the alkaloid cylindrospermopsin (CYN; Fig. 1), which is produced by several freshwater cyanobacteria, such as
Chemical structure of CYN: A – sulfate group, B – tricyclic guanidine moiety, C – uracil ring
In previous studies, we described the chemical structure of the CYN decomposition products formed under alkaline conditions combined with boiling temperature or UV-B irradiation (Adamski et al. 2016a; Adamski et al. 2016b). The objective of this research was to assess their potential toxicity based on tests with
The decomposition of CYN under alkaline conditions combined simultaneously with high temperature or UV-B irradiation was performed as described by Adamski et al. (2016a, b). In short, CYN samples dissolved in buffer at pH 10 were exposed to boiling (100°C ± 1°C) or UV-B irradiation (36 μmol m−2 s−1) until complete decomposition of the toxin. The progressive loss of CYN was monitored every half hour using high-pressure liquid chromatography (HPLC) according to the method described by Adamski et al. (2016a, b). In brief, a Waters Atlantis® dC18 column (3.9 × 100 mm, 3 μm) maintained at 35°C was used, and the following gradient elution was applied: from 99 to 88% of water/trifluoroacetic acid (0.05% v/v) for 24 min at a flow rate of 1.0 ml min−1. Eluent B was acetonitrile/trifluoroacetic acid (0.05%, v/v). CYN was quantified using its absorbance at λ = 262 nm. The CYN decomposition products were identified using an ultra-performance liquid chromatography tandem-mass spectrometer (UPLC-MS/MS) coupled with a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole) according to the method developed by Adamski et al. (2016a, b). Chromatographic separation was achieved using an Acquity UPLC BEH (bridged ethyl hybrid) C18 column (2.1×100 mm, 1.7 μm) with an Acquity UPLC BEH C18 VanGuard pre-column (2.1×5 mm, 1.7 μm) maintained at 40°C under the following conditions: 100% of eluent A for 2 min and a gradient elution from 100 to 30% of eluent A for 10 min at a flow rate of 0.3 ml min−1. Eluent A was water and eluent B was acetonitrile. Both were acidified with formic acid (0.1% v/v). The MS detection settings of the Waters TQD mass spectrometer were as follows: a source temperature of 150°C, a desolvation temperature of 350°C, a desolvation gas flow rate of 600 l h−1, a cone gas flow rate of 100 l h−1, a capillary potential of 3.00 kV, and a cone potential of 20 V. Collision activated dissociation (CAD) analyses were conducted with an energy of 30 eV, and all fragmentations were in the source. The ion spectra were obtained by scanning from 30 to 500 m/z. The concentrations of mixtures of the CYN decomposition products were expressed as the equivalent of the CYN initial concentrations.
Evaluation and comparison of the toxicity of CYN and its decomposition products were performed using a commercially available Thamnotoxkit FTM toxicity test and a Deltatox® II analyzer according to the respective standard operational procedures. The Thamnotoxkit FTM assay is based on the determination of the percentage mortality of
Samples of CYN and its decomposition products were adjusted to neutral pH by 0.1 M HCl and prepared at five concentrations, 0.2, 0.4, 0.75, 1.5 and 3 μg ml−1, by dilution in the standard medium (Thamnotoxkit FTM) or Milli-Q water (Deltatox® II). The standard medium and Milli-Q water were used as controls in the Thamnotoxkit FTM and the Deltatox® II, respectively.
The Thamnotoxkit FTM test was purchased from MicroBioTests, Inc. (Gent, Belgium). The Deltatox® II test came from Modern Water, Inc. (New Castle, DE, USA). The commercial standard of CYN was obtained from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water came from Millipore (Bedford, MA, USA). All other reagents were of MS/MS, HPLC or analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Data obtained from the chromatographic studies (not presented here) and mass spectrometry analyses revealed the presence of two and four decomposition products of CYN formed after the exposure of samples to pH 10 combined with boiling (Fig. 2) or UV-B irradiation (Fig. 3), respectively. In Figure 2, the compound with a retention time (Rt) of 1.90 min represents one of the two diastereoisomers indicated
Mass spectra of CYN decomposition products formed under alkaline conditions (pH 10) combined with boiling temperature: A – CP-2 or CP-3, B – CP-6
previously as CP-2 or CP-3, and the substance with an Rt of 3.85 min was identified as CP-6 (Table 1; Adamski et al. 2016a). In Figure 3, in turn, CYN derivatives with an Rt of 1.88 min, 3.87 min, 4.46 min and 4.55 min were labeled as CPI-1, CPI-2, CPI-4 and CPI-5, respectively (Table 2; Adamski et al. 2016b).
Mass spectra of CYN decomposition products formed under alkaline conditions (pH 10) during UV-B irradiation: A – CPI-1, B – CPI-2, C – CPI-4, D – CPI-5
Products of total CYN decomposition formed under alkaline conditions (pH 10) combined with boiling temperature
Decomposition products | Retention [min] time | [M+H]+ | Fragmentation ions | Proposed structure |
---|---|---|---|---|
CP-2 or CP-3 | 1.90 | 336.14 | 194.1, 176.1 | |
CP-6 | 3.85 | 292.01 | 212.1, 194.1, 176.1 |
Products of the total CYN decomposition formed under alkaline conditions (pH 10) during UV-B irradiation
Decomposition products | Retention [min] time | [M+H]+ | Fragmentation ions | Proposed structure |
---|---|---|---|---|
CPI-1 | 1.88 | 290.29 | 210.1, 192.1 | |
CPI-2 | 3.87 | 292.15 | 212.1, 194.1, 176.1 | |
CPI-4 | 4.46 | 338.34 | 196.1, 178.1, 137.1, 110.1 | |
CPI-5 | 4.55 | 214.13 | 196.1, 178.1, 137.1, 110.1 |
The total decomposition of the CYN molecule under both tested conditions resulted in relatively similar compounds. These are the products of separation of the uracil ring (CP-6, CPI-1, CPI-2), the sulfate group (CP-2/CP-3, CPI-4) or both (CPI-5) from the tricyclic guanidine moiety. Recent studies also confirmed the presence of similar CYN decomposition derivatives in uncooked or cooked/steamed muscles of tilapia fish (
The concentrations of CYN and its decomposition products used in this study reflect the toxin concentrations commonly found in natural water bodies (for a review, see Adamski et al. 2014) as well as its highest concentration ever detected in nature (1.5 μg ml−1; Santos et al. 2015). Moreover, the toxicity of the concentration twice as high as the highest concentration determined for CYN (3 μg ml−1) was analyzed. This allowed a satisfactory comparison of the toxicity of the toxin and the mixture of its derivatives.
The LC50 value of CYN for
Toxicity of CYN and its decomposition products assessed with Thamnotoxkit FTM and Deltatox® II tests expressed as the concentration causing death of 50% of crustacean larvae (LC50) or 50% inhibition of the bacterial bioluminescence (IC50)
Tested substances | Toxicity tests | |
---|---|---|
Thamnotoxkit FTM LC50 [μg ml−1] | Deltatox® II IC50 [μg ml−1] | |
CYN | 0.39 | < 0.2 |
CYN decomposition products formed under boiling temperature at pH 10 | > 3 | > 3 |
CYN decomposition products formed under UV-B irradiation at pH 10 | > 3 | > 3 |
The results obtained by applying the Deltatox® II test, according to the classification described by Hsieh et al. (2004), showed a high sensitivity of
Decrease in bioluminescence observed in the Deltatox® II test: A – pure CYN (aqueous solution), B – mixture of CYN decomposition products formed under alkaline conditions (pH 10) at 100°C, C – mixture of CYN decomposition products formed under alkaline conditions (pH 10) during UV-B irradiation
products did not cause any significant changes in the level of bacterial bioluminescence (Figs 4B and C; Table 3).
The presented results confirmed the less toxic character of these derivatives. As a consequence, it can be assumed that they do not pose a threat to the life and health of organisms such as crustaceans and bacteria living in water reservoirs.
Taking into account the less toxic properties of the studied decomposition compounds, our results suggest that the toxicity of CYN is determined by the presence of the tricyclic guanidine moiety, the uracil ring and the sulfate group (Fig. 1), and the structural modification of any of the above-mentioned parts may contribute to the reduction of its toxic properties. In comparison to the CYN decomposition process described in our previous studies (Adamski et al. 2016a,b), its complete breakdown was also associated with the loss of all compounds structurally similar to CYN. The obtained results clearly show that the applied conditions result in the decomposition of CYN to less toxic products. Norris et al. (1999) studied the
To date, only some limited information has been reported on the removal of CYN from drinking water in wastewater treatment plants (De la Cruz et al. 2013). The results presented in this study advance the knowledge about the toxicity of CYN decomposition products and may be useful in the design and implementation of water treatment systems.
In many cases, extensive and persistent cyanobacterial blooms contribute to the alkalization of water (Gao et al. 2012). The combination of alkaline pH and UV irradiation affects the amount of CYN in the natural environment. Further studies should focus on the process of CYN degradation in nature and the toxicity of its decomposition products formed under such conditions.
During the complete decomposition of CYN under alkaline conditions (pH 10), combined with high temperature (100ºC ± 1°C) or UV-B irradiation (36 μmol m−2 s−1), less toxic decomposition products were formed. The obtained results indicate that the modifications of the chemical structure of CYN, such as the separation of the uracil ring or the sulfate group and changes in the guanidine moiety, decrease its toxic properties. Given the limited information available on the fate and toxicology of CYN in the natural aquatic environment, further studies should focus on these issues.