Cyanobacterial Toxins and Peptides in Lake Vegoritis, Greece

Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018–2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 μg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 μg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.


Introduction
Cyanobacteria are common photosynthetic microorganisms found in lakes and surface water reservoirs, which can, under favorable conditions, grow massively to form blooms [1]. Several cyanobacteria species produce potent toxic compounds as secondary metabolites, called cyanotoxins (CTs) [2,3]. Several incidents of wild and domestic animal poisoning as well as human health effects due to toxic cyanobacterial blooms have been reported [4][5][6][7]. not designed to provide information concerning the spatial and temporal variation of CTs. Furthermore, there is complete lack of information regarding the co-occurrence of other cyanobacterial peptides such as MGs, AERs, AEGs, and APs.
In response to the above study, a 3-year monitoring program of Lake Vegoritis was initiated in 2018, with the aim to characterize the cyanobacterial species present and to assess the occurrence of various classes of CTs and CPs in the lake. Fifteen CTs (i.e., CYN, ATX, NOD and dmMC-RR, MC-RR, MC-YR, MC-HtyR, dmMC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW, and MC-LF) and ten CPs (i.e., MG FR1, MG FR3 MG T1, MG T2, AER 602/K139, AER 298A, AEG A, AP B, AP F and oscillamide (OSC Y)) were targeted. Selection of the targeted cyanobacterial metabolites was based on their frequency of detection in other Greek lakes [31,32]. To implement this monitoring program, a new method for simultaneous determination of various classes of CPs in addition to CTs was developed and validated, extending a previously validated LC-MS/MS analytical protocol [31,33] to include MGs, AERs, AEGs and APs. Using this new protocol, the detection of several classes of CTs and CPs would be possible in a single analytical run.
Results obtained by this 3-year study enable risk assessment and management of toxic cyanobacterial blooms by the lake's authorities. The study's findings also facilitate the reliable and effective communication of the risks to the general public and stakeholders, with regards to the uses of the lake, such as irrigation, fishing, and recreational activities.

Physico-Chemical Parameters of Lake Vegoritis
Lake Vegoritis is under pressure from point source and diffuse pollution. As reported by monitoring results from 2012-2015, the lake was in moderate ecological and good chemical status [34]. The concentrations of most of the physicochemical quality parameters, with the exception of nitrates and sulfates, did not fluctuate considerably during the study period (Table S1). The F − , NO 2 − , Br − and PO 4 3− ions were measured below the method's limit of quantification (LOQ) during the whole study period. The Cl − ranged between 32-40 mg/L, with a single high measurement (58 mg/L) in July 2020. The NO 3 − ions ranged from below LOQ to 1.0 mg/L, with the highest values detected in the winter of 2018 and 2020 and in August 2020. The greatest variability was noticed for SO 4 2− levels that ranged between 84 and 200 mg/L, with no seasonal or other temporal pattern observed. The cation (Na + , K + , Mg 2+ , Ca 2+ ) concentrations measured showed relative stability throughout the study period and no temporal distribution pattern.
In 2018, the highest levels of total phosphorus (TP) were measured. Concentrations ranged from 22 to 60 µg/L and displayed the highest values in February and April (60 and 50 µg/L, respectively). In June 2018, TP decreased to 43 µg/L, while in July 2018 the mean value of the TP concentration was 38 µg/L. Concentrations declined in the following months (<29 µg/L). The following years, 2019 and 2020, TP concentrations were measured at slightly lower levels, 28-50 µg/L and 16-38 µg/L, respectively.
The transparency of the water was measured using the Secchi disc and in June 2018 there was a significant reduction to 0.3 m, from 6.5 m and 6.0 m measured in February 2018 and April 2018, respectively. Such a low value was measured for the first time in the lake throughout the operation of the National Monitoring Water Network (2012-2018). Since then, the transparency of the water in the lake, including bathing area sampling points, presented a noteworthy increase (2.4 m on 4 July 2018 and 3.0 m on 12 July 2017 and 17 July 2018). The Secchi disc was visible to the bottom of the bathing area stations until the autumn. The following years 2019-2020, transparency of the water at the NMWN (National Monitoring Water Network) sampling point ranged from 1.6 m to 4.8 m.
The total suspended solids (TSS) exhibited a high value of 8.46 mg/L in July 2018. During the summer, their concentrations in the lake decreased both at the NMWN sampling point and the bathing area (Site 1). In the next two years, TSS did not exceed 2.54 mg/L, except for two cases in June 2019 (3.75 mg/L) and in September 2020 (5.67 mg/L).

Chlorophyll α
The concentration of chlorophyll α in Lake Vegoritis in June 2018 was high (15.9 µg/L), with a decreasing trend during the following summer months at both the NMWN sampling point and the bathing area (Site 1) (3.0-8.0 µg/L). This reduction was in line with the improvement in water transparency values observed during the same period. Similar values of chlorophyll α concentration (4.2-7.7 µg/L) were measured during the following years, 2019 and 2020. In two samples collected on 10 September 2019 and 22 September 2020 much higher values (19.4 and 31.8 µg/L, respectively) were measured, but no discoloration of water was observed (Table S1).

Phytoplankton
During June 2018, the total phytoplankton biomass was estimated at 2.3 mg/L. The most abundant taxa were the green alga Sphaerocystis schroeteri (39,889,429 cells/L), cyanobacteria of the genus Dolichospermum (14,864,850 cells/L) and the species Aphanizomenon flos-aquae (3,383,107 cells/L). Chlorophyta and cyanobacteria comprised 88% of the biomass. In the subsequent samplings, no bloom, mat or scum (as defined in Directive 2006/7/EC) [35] were visually observed. Furthermore, phytoplankton biomass in samples from NMWN gradually decreased, mainly due to the decrease in the biomass of chlorophytes and dinophytes. The Dolichospermum biomass also declined sharply (from 0.75 mg/L in June 2018 to 0.02 mg/L in July 2018 and 0.04 mg/L in August 2018).
At the same period, summer 2018, in the samples from the bathing area the cyanobacteria Microcystis spp., Aphanocapsa spp., Dolichospermum spp., and Aphanizomenon flos-aquae were dominant. However, their biomass did not exceed 0.7 mg/L (Aphanizomenon flos-aquae in July 2018). The biomass values of the dominant cyanobacteria were measured at higher levels in July (1.6 mg/L), followed by sharp decline in the next months. Regarding the chlorophyte Sphaerocystis schroeteri, its biomass was measured at lower levels compared to the measurement of June 2018 at the NMWN sampling point and showed a gradual further decrease during August and September.
During the warm period of 2019 the cyanobacteria biomass (four samples from NMWN sampling point) displayed the opposite trend. Biomass gradually increased during summer until the maximum value of 5.4 mg/L (September 2019), where Aphanizomenon spp. biomass was estimated at 2.1 mg/L, Lemmermanniella spp. at 2.1 mg/L, and Raphidiopsis raciborskii at 0.5 mg/L. In all the other three samples of 2019, Aphanizomenon spp. and Dolichospermum spp. were the main representatives, though with lower biomass values (up to 0.82 mg/L).
No specific trend was observed during the warm period of 2020. In June 2020 the biomass of cyanobacteria was minimal (0.1 mg/L). Over the next three months it increased up to 1.8 mg/L, as measured in August 2020. In July a Dolichospermum species dominated (1.1 mg/L), but low biomass values were measured for Aphanocapsa cf. holsatica and Cyanodictyon species (up to 0.2 mg/L). In August and September, species of the genus Aphanizomenon prevailed (1.6 and 0.9 mg/L, respectively). Low biomass values of Microcystis species (0.1 and 0.3 mg/L) were also measured. Total phytoplankton and cyanobacteria biomass concentrations measured during the study period are given in Table S1.  Tables S2 and S3. In 2018, 14 samples were analyzed from July to November, seven from each one of the sampling sites. None of these samples were found to contain detectable amounts of CYN, ATX and NOD. The analysis of filtered water showed the presence of extracellular MCs, i.e., MC-LR, MC-RR and MC-YR, at concentrations up to 0.029, 0.023 and 0.014 µg/L, respectively. Intracellular (cell-bound) MCs were also detected, including MC-RR, MC-LR, During 2019, a total of 24 samples from the two sampling sites were analyzed from April to October. ATX and NOD were not detected in any of the samples. Contrary to findings of 2018 monitoring, CYN was detected mainly in the intracellular (cell-bound) fraction, at concentrations ranging from 0.032 to 0.685 µg/L, and at a lower level in the extracellular (dissolved) fraction. MCs were also detected, with MC-LR and dmMC-LR up to 0.233 and 0.080 µg/L (extracellular fraction) and MC-RR, dmMC-LR, MC-LR, and MC-HilR up to 0.029, 0.055, 0.241, and 0.027 µg/L, respectively (intracellular fraction).

Occurrence of Cyanotoxins (CTs) in Lake Vegoritis
In 2020, 20 samples were analyzed from May to October. Co-occurrence of CYN and MCs was again observed with CYN present during this period at concentrations reaching 0.  detected [31]. Although the findings were based on only two samples, the toxin profile (MC-RR, MC-LR) is in agreement with the present study. However, the concentrations reported in the previous study were far higher, possibly because the sampling was targeted rather than systematic, aiming at localized bloom formations.    While there are no recreational beach monitoring programs for toxins in Greece, this is the first study to investigate the presence, the concentration, and the diversity of CTs in a popular lake beach of North Greece devoted to recreational activities. In all cases, the measured concentration of CTs did not exceed the provisional guideline values proposed for recreational water by the World Health Organization (WHO) that have been recently updated and were set at 24 and 6 μg/L for MC-LR and CYN, respectively [38,39].   While there are no recreational beach monitoring programs for toxins in Greece, this is the first study to investigate the presence, the concentration, and the diversity of CTs in a popular lake beach of North Greece devoted to recreational activities. In all cases, the measured concentration of CTs did not exceed the provisional guideline values proposed for recreational water by the World Health Organization (WHO) that have been recently updated and were set at 24 and 6 μg/L for MC-LR and CYN, respectively [38,39].  Results for both sampling sites presented a similar seasonal trend in CT concentrations, with an increase during the summer followed by a second outbreak in the autumn. As expected, intracellular MCs tended to appear first, followed by extracellular MCs, due to the release of cell-bound MCs into water after the lysis of cyanobacterial cells, with the exception of June 2020 (Figure 1a,b). The maximum of total MCs, 1.02 µg/L, consisted of dmMC-RR, MC-RR, MC-YR, and MC-LR, and was measured on 7 September 2020 at Site 1, with MC-RR being the most abundant MC variant. The percentage of samples in which each CT was detected is shown in Table 1. MC-LR was detected in 79% of samples through the entire monitoring program, while MC-RR was detected in 50% of samples and was largely absent during 2019.
CYN was also detected in 71% of samples of Lake Vegoritis, while detections occurred only during 2019 and 2020, and not in samples taken in 2018. CYN was found in both extraand intracellular fractions. The maximum concentration of total CYN was 0.727 µg/L at Site 1 on 1 September 2019 ( Figure 1c). The presence of MCs could be related to the dominant cyanobacteria Dolichospermum spp. (Anabaena spp.), Aphanizomenon spp., and Microcystis spp. that were identified in Lake Vegorits during the study period [36]. The presence of CYN may also be attributed to the cyanobacteria Aphanizomenon spp. and Dolichospermum spp. as well as to Raphidiopsis raciborskii [12,37]. These results concur with a previous study where Lake Vegoritis was found to be mainly dominated by Dolichospermum spp., Aphanizomenon spp., and Microcystis spp. [31]. In the same study, CYN, MC-RR and MC-LR were identified in one sample of biomass from Lake Vegoritis (September 2008) at concentrations of <LOQ, 0.118 and 0.049 µg/L, respectively. In a water sample (July 2014), extracellular dmMC-RR, MC-RR, MC-YR, dmMC-LR, MC-LR, and MC-LY (MC-RR: 104 µg/L and MC-LR 96.3 µg/L) were also detected [31]. Although the findings were based on only two samples, the toxin profile (MC-RR, MC-LR) is in agreement with the present study. However, the concentrations reported in the previous study were far higher, possibly because the sampling was targeted rather than systematic, aiming at localized bloom formations.
While there are no recreational beach monitoring programs for toxins in Greece, this is the first study to investigate the presence, the concentration, and the diversity of CTs in a popular lake beach of North Greece devoted to recreational activities. In all cases, the measured concentration of CTs did not exceed the provisional guideline values proposed for recreational water by the World Health Organization (WHO) that have been recently updated and were set at 24 and 6 µg/L for MC-LR and CYN, respectively [38,39].

Detection, Identification and Occurrence of Cyanobacterial Peptides (CPs) in Lake Vegoritis
Although the occurrence of MCs in fresh water bodies is well documented due to the development of analytical protocols [1,31,33,40], data bases [41,42], and a number of commercially available standards, less is known regarding the presence of other CPs. Recent studies showed the presence of CPs in water bodies [17,43], but analysis was mainly done in cyanobacterial biomass, not in water samples. Analytical protocols have not been developed and validated for water samples (extracellular-intracellular fractions), to include cleanup and pre-concentration steps [22]. In this study, we present method performance and validation results for targeted LC-MS/MS analysis of water samples for CPs, based on an analytical workflow previously used for analysis of CTs [33]. The targeted CPs were MG FR1, MG FR3 MG T1, MG T2, AER 602/K139, AER 298A, AEG A, AP B, AP F and OSC Y. The validated method was then used to analyze water samples from Lake Vegoritis.

Chromatographic Separation and MS/MS Identification of CPs
A sample of cyanobacterial mass from a bloom in Lake Kastoria, Greece (September 2014) containing all target CPs was used as a reference sample. Efficient chromatographic separation of target CPs was achieved with a reversed-phase C18 column (Atlantis T3, Waters), previously applied for MCs, NODs, CYN and ATX [33].
Identification of CPs was performed using tandem mass spectrometry in a multiple reaction monitoring (MRM) mode ( Table 2). The total ion chromatogram (TIC) and MRM chromatograms of the selected quantifier transitions obtained from cyanobacterial mass extract from Lake Vegoritis (7 September 2020, Site1) are presented in Figure 4. MGs presented a characteristic fragment ion, at m/z 128.2, attributed to a part of Ahda and, at m/z 162.1, to a part of chlorinated Ahda [44,45]. The most intense common ions of MGs, which share the amino acid sequence proline (Pro)-tyrosine (Tyr)-tyrosine (Tyr) at the Cterminus in their structure (i.e., MG FR3, MG T1 and MG T2), were at m/z 233.0 [Pro-Tyr-CO + H] + and m/z 442.2 [Pro-Tyr-Tyr + H] + [45,46]. AERs were characterized by m/z 140.0 and m/z 122.0, which are the Choi immonium ion and dehydrated Choi immonium ion, respectively [47]. Additionally, m/z 221.2 was attributed to Leucine (Leu)-Choi fragment (or Isoleucine (Ile)-Choi fragment), while m/z 311.0 was indicative of the presence of the arginine derivative-argininol in the structure of AERs [45,48]. APs were characterized by m/z 84, which corresponds to the lysine (Lys) immonium ion [49]. The fragment ion m/z 201.0 is characteristic of APs that contain arginine (Arg) as a side chain [49]. Fragment ions from the loss of the side chain amino acid with the CO linkage (i.e., m/z 637.3 for AP B and m/z 651.4 for AP F) or amino acid from the ring (i.e., m/z 681.4 for OSC Y [50]) were also considered. AEGs were characterized by m/z 112.0 and was annotated in a previous study as TzlCO in case of AEG A, since it is a common ion in the fragmentation spectra of some other AEGs [20].  Figure S3.

Method Performance and Validation Results
The ability of the method for accurate quantification was assessed for AP B, for which an analytical reference standard was available. The response (quantification ion peak area) over the range 5-100 µg L −1 was linear (r 2 ≥ 0.999). Precision, expressed as relative standard deviation (%RSD), was 8.6% under repeatability (n = 3) conditions and 15.9% under reproducibility (different days, n = 15) conditions. The limit of detection (LOD) of AP B was 0.001 µg/L and the LOQ was 0.003 µg/L. The LOD was estimated from measurements (n = 8) of standard solution (5 µg/L) using the formula: LOD = t (n−1, 0.95) × SD, where t (n−1, 0.95) was the t-test value for n-1 degrees of freedom at 95% confidence level, (1.895 for n = 8) and SD was the standard deviation of measurements. Limit of quantification (LOQ) was estimated as 3 × LOD.
Quantification of APs was carried out using the class equivalent approach with concentrations expressed as AP B equivalents, while for the rest CPs concentrations were expressed as MC-LR equivalents.
Recoveries of CPs (extracellular and intracellular fractions) were evaluated by analyzing spiked samples using CP-free water and cyanobacterial biomass as matrices and the reference sample from the Lake Kastoria bloom for spiking. Results are presented in Table 3. Recovery experiments were carried out in triplicate and mean recoveries in the extracellular fraction ranged from 77.0-129.2% for all target CPs, except for AEG A which was poorly recovered (17.1%) and AER 602/K139 that showed a recovery of 163.5%. Mean recoveries in the intracellular fraction were in the range of 73.4-98.3%, except for AEG A which had a low recovery (7.5%) ( Table 3). In all recovery estimations, %RSD was <28.4%.
The validated analytical protocol can be used for detection and identification of target CTs and CPs of various chemical classes using a single analytical method. It could further serve as a basic template for analysis of cyanobacterial metabolites, expanding to more CTs and CPs in the future as they become commercially available as standards or included in mass spectral databases.   These findings consist of the first report of the occurrence of a variety of CPs, in addition to MCs and CYN, in Lake Vegoritis ( Figure S4), showing that all 10 target CPs were detected, with MGs and Aps found to be the most abundant classes of CPs compared to other classes (Figure 7). The highest concentration of total MGs (sum of intra-and extracellular), 60.0 μg/L, was measured on 7 September 2020 (Site 1), and consisted of MG FR1, MG FR3, MG T1, and MG T2, where MG T1 was the most abundant. The maximum of total APs (AP B, AP F, and OSC Y), 2.83 μg/L (Figure 2), was measured on 27 May 2019 (Site 2), with AP F presenting the highest concentration ( Figure 6).
To date, there is only one report in the literature related to the presence of MGs in cyanobacterial bloom samples collected from Greek lakes other than Vegoritis, providing only qualitative data with no information on the presence of MGs in the water phase [32]. According to that study, the most frequently detected MGs were MG FR1 (70% of samples) followed by MG T1 (52%). In another study on the occurrence of APs in the freshwater bodies of Greece, the presence of AP B and AP A was reported, however, analysis  To date, there is only one report in the literature related to the presence of MGs in cyanobacterial bloom samples collected from Greek lakes other than Vegoritis, providing only qualitative data with no information on the presence of MGs in the water phase [32]. According to that study, the most frequently detected MGs were MG FR1 (70% of samples) followed by MG T1 (52%). In another study on the occurrence of APs in the freshwater bodies of Greece, the presence of AP B and AP A was reported, however, analysis was carried out using HPLC-UV without confirmation by mass spectrometry [51].
There is still lack of information concerning the occurrence of AERs and AEG A in Greek lakes. Furthermore, there is a gap in the knowledge related to the co-existence of CPs with CTs which may cause adverse health effects to humans and animals. In the frame of the present study, it was found that in water samples of Lake Vegoritis, AP B and AP F, which were detected in almost all samples (100% and 98%, respectively), co-existed with the frequently found MC-LR, CYN, and MC-RR (79%, 71%, and 50%, respectively), (Table 1). OSC Y was also present in 68% of the samples. The frequency of occurrence of the rest of the CPs ranged from 9% to 45%, while the detected CTs ranged from 5% to 24%, respectively.
Cyanobacteria possess a great metabolic potential and are able to co-produce several peptides from different classes [15]. MGs and AEG A exhibited a similar trend in Lake Vegoritis and their production could be attributed to Microcystis spp. [21,47,52], since both CP classes were detected in 2018 and 2020, but not in 2019, similarly to Microcystis spp. Low concentrations of AERs were determined in all sampling periods and their presence in Lake Vegoritis might be related to Aphanizomenon spp. and Dolichospermum spp. [53,54] as well as Microcystis spp. [18]. Dolichospermum spp. (Anabaena spp.), the dominant cyanobacterial species during all sampling periods, are possibly related to the high frequency of APs detection, especially AP B, which was present in all samples from Lake Vegoritis [55].
Our results are in agreement with other studies investigating the presence of CPs in inland water bodies. MCs, Aps, and AERs were the main cyanobacterial metabolites identified in biomass during a bloom episode in a dam for drinking water on Lake Occhito, near the town of Foggia in Southern Italy [56]. Similarly, MCs and APs were identified in the biomass from Siemianówka Dam Reservoir (northeast Poland) during a study from 2009 to 2012 [57]. In a more recent study of six eutrophic lakes in USA by Beversdorf et al., APs were detected in all lakes together with MGs at concentrations of the same order of magnitude found in Lake Vegoritis [22]. Similarly, both MCs and CPs (APs, MGs, and cyanopeptolins) were detected in surface and raw drinking waters from the eutrophic Lake Winnebago, Wisconsin [58]. In another study on the diversity and spatial distribution of MCs, NODs, Aps, and MGs in Green Bay, Lake Michigan, an important recreational resource, the presence of MCs (mainly MC-RR and -LR) and CPs (mainly APs and MGs) was reported with the mean of total MCs and APs as 1.28 and 0.20 µg/L, respectively [23].
The occurrence of cyanobacterial metabolites was also reported for lake water samples (mostly recreational) from Canada, in the frame of a collaborative citizen-science project. CTs were present in 75% of the samples, from ng/L up to µg/L, and AP A and AP B were in 38% of the samples at concentrations up to 10 µg/L [59]. High levels of APs (µg mg/L) were also detected in water samples from the Sau-Susqueda-El Pasteral reservoir system in Spain in the autumn of 2015, although MCs were <0.3 µg/L [60].
In most of the published studies, the intracellular (cell-bound) or total concentration (sum of extracellular and intracellular fractions) of CPs was reported. There is a general lack of knowledge on whether CPs are released from cyanobacterial cells into water or remain as cell-bound in the intracellular fraction [58]. In the frame of the current study, as intra-and extracellular fractions were quantified separately, it seems that the CPs studied are mostly intracellular, with the extracellular fraction concentrations being generally lower by an order of magnitude (Tables S4, S5 and Figure 7).
From Figure 2, it was also observed that the maximum concentrations of CPs occurred at the same time period with CTs, following the same trend. Taking into consideration the co-occurrence of CTs with CPs of different classes with unknown behavior, bioactivity and environmental levels in lake water, more research and monitoring programs are urgently needed for assessing possible threats to humans and the environment. This study was the first of this kind in Greece and in Lake Vegoritis, where both recreational and fishing activities take place.

Conclusions
This study reports, for the first time, the co-occurrence of CTs and CPs in Lake Vegoritis, situated in the north of Greece. Furthermore, the study describes the variantspecific changes of CT and CP profiles over a 3-year monitoring period as well as the basic water quality parameters of the lake and phytoplankton-cyanobacteria composition.
In order to realize the study, a previously validated LC-MS/MS analytical protocol for the simultaneous determination of multi-class cyanotoxins in water was further extended and validated to be applied for the detection of multiple CPs, i.e., MGs (MG FR1, MG FR3 MG T1, and MG T2), AERs (AER 602/K139 and AER 298A), AEG A and APs (AP B, AP F, and OSC Y). Samples of Lake Vegoritis were analyzed for CTs and CPs in both extracellular and intracellular fractions.
Using the above protocol, CTs were detected in two sampling sites of Lake Vegoritis, during 2018-2020, consisting of MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, MC-HilR and CYN, with MC-LR (79%) being the most frequently detected, followed by CYN (71%). The concentrations of MCs and CYN were generally low (<1 µg/L) and they did not exceed the guideline values proposed for recreational water by WHO (24 and 6 µg/L for MC-LR and CYN, respectively). CPs belonging to the classes of APs, AERs, MGs, and AEG A were also detected, with AP B and AP F present in almost all water samples. The co-occurrence of two potent cyanotoxin classes (MCs and CYN) with multiple CPs in the lake's water supports the need for future studies on the interactions between multiple cyanobacteria metabolites with regards to possible effects on human health.

Study Area Description and Sample Collection
Lake Vegoritis, one of the largest lakes in Greece, is located in Western Macedonia region in North-Western Greece and occupies the lowest area of the Ptolemaida basin (Figure 8a). It is considered as one of the most important water resources of Western Macedonia for its multiple uses and benefits for humans [34]. The investigative monitoring of the condition of Lake Vegoritis was designed and implemented from 2018 to 2020. In compliance with the WFD provisions [35], integrated samples (from the euphotic zone, 2.5× Secchi Depth) were taken from the pelagic zone of the lake (40 •  ples (1.5 L) for toxins analysis were collected in disposable plastic bottles from the surface layer of the lake (0-30 cm) at least monthly, from July to November, April to October, and May to October during 2018, 2019 and 2020, respectively. Sampling took place at two points: Site 1, 40°43′29.5′'N, 21°45′13.6′'E from the designated bathing area of the lake, and Site 2, 40°43′12.1′'N, 21°45′07.2′'E from the pier (Figure 8b). The samples were transported to the laboratory within 24 h of collection, in dark containers and at low temperature (≈4 °C).   Lake water samples were filtered through glass fiber filters with a 47 mm diameter and 0.7 µm pore size (Millipore, Cork, Ireland) and a glass vacuum filtration device. Solid-phase extraction (SPE) was carried out using a 12-port SPE vacuum manifold with large volume samplers connected through PTFE tubes (Supelco, Bellefonte, PA, USA) and a diaphragm vacuum pump (KNF Laboport, Freiburg, Germany). SPE cartridges used for cleanup and pre-concentration purposes were Supel-Select HLB (bed wt. 200 mg, volume 6 mL, Supelco, St. Louis, MO, USA) and Supelclean ENVI-Carb (bed wt. 250 mg, volume 3 mL, Supelco, St. Louis, MO, USA).

Chemicals and Instrumentation
The analysis of target analytes was carried out using a TSQ Quantum Discovery Max triple-stage quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, PA, USA), with an electrospray ionization (ESI) source coupled to a Finnigan Surveyor LC system, equipped with a Surveyor AS autosampler (Thermo Electron Corporation, San Jose, PA, USA). Xcalibur software 2.0 was used to control the MS parameters for data acquisition and data analysis. The chromatographic column used was an Atlantis T3 (2.1 mm × 100 mm, 3 µm, Waters, Wexford, Ireland).

Physico-Chemical Parameters
Ion chromatography (IC) was applied for separation, analysis, and quantification of both anions (F − , Cl − , NO 2 − , Br − , NO 3 − , PO 4 3− , and SO 4 2− ) and cations (Na + , K + , Mg 2+ , and Ca 2+ ) in water samples. Subsamples were filtered through 0.45 µm glass fiber filters within 48 h of sampling and kept at 2-6 • C for up to 4 days. For cation analysis, samples were acidified to a pH of 3 ± 0.5 with nitric acid. Dissolved anions and cations were determined using a Dionex Aquion IC System (Thermo Fisher Scientific, Waltham, MA, USA) according to EN ISO 10,304 and 14,911 guidelines, respectively [61,62].
Determination of total phosphorus (TP) was carried out in unfiltered water subsamples (about 250 mL, stored at −18 • C for up to six months), using the ascorbic acid method following persulfate digestion [63]. Orthophosphates were determined based on spectrophotometry (Hitachi U-5100 UV/VIS, Hitachi, Tokyo, Japan).
For determination of total suspended solids (TSS), a measured volume of a water subsample (about 500 mL) was filtered through a pre-weighed glass microfiber filter (Whatman Grade 934-AH ® RTU circles). The filter was heated at 103-105 • C for an hour and then weighed. The TSS were calculated as the mass increase divided by the water volume filtered [63]. Analysis was conducted within seven days from sampling and the subsamples were kept at 4 • C in the dark.

Chlorophyll α Analysis
Subsamples of 1 L were filtered through Whatman GF/F glass fiber filters within 48 h of sampling. Chlorophyll α was measured using 90% acetone and application of the trichromatic equation [63,64]. Absorbance was measured using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).

Microscopic Analysis
For phytoplankton analysis, subsamples (about 500 mL) were transferred to plastic bottles and preserved with acid Lugol's solution [65]. Additional samples were obtained by vertical plankton net hauls (20 µm mesh, Hydro-Bios, Altenholz, Germany) through the euphotic zone, and preserved with formaldehyde. Phytoplankton identification and enumeration were based on the Utermöhl settling technique [66] as described in EN 15,204 guidelines [65]. The analysis was carried out using sedimentation chambers (Hydro-Bios) and an inverted microscope Zeiss AxioObserver.A1 equipped with an AxioCam (Carl Zeiss, Oberkochen, Germany). The phytoplankton biomass was estimated following EN 16,695 guidelines [67].  [31]. Filtered water samples were pre-treated using the dual cartridge (HLB and Envi-Carb) SPE process [33]. Briefly, water samples, after adjustment to pH 11, were passed through a dual cartridge assembly of HLB and ENVI-Carb. Recovery of extracellular CTs and CPs was achieved by reversing the cartridges and eluting with a mixture of 10 mL DCM:MeOH (40:60, v/v), containing 0.5% FA. The extract was dried and the residue was re-dissolved with 400 µL MeOH: H 2 O (5:95, v/v) prior to LC-MS/MS analysis.

LC-MS/MS Analysis
A gradient elution program was applied for chromatographic separation with solvents (A) ACN and (B) water, both containing 0.5% FA. The gradient started at 5% A (held for 3 min), which increased to 20% A in 1 min (held for 2 min), further to 35% A in 1 min (held for 7 min), 70% A in 14 min, and finally 90% in 1 min (held for 3 min). An equilibration time of 10 min was kept after each sample run. The flow rate was set at 0.2 mL/min with 20 µL injection volume and the column temperature was set at 30 • C.
Electrospray ionization (ESI) in positive mode was used and the three most intense and characteristic precursor-product ion transitions were selected for detection and identification of each compound in MRM mode. LC-MS/MS detection parameters for the target CTs was set according to Zervou et al. [33]. Parameters for the CPs detection are given in Table 2. The selection of MRM transitions was based on fragmentation spectra from previous studies [46,49,50,57,68] or in the frame of this study. In all cases, single protonated [M+H] + ions were set as the precursor ions. The most intense product ion was chosen to be the quantifier ion, while quantification was performed using external standards at concentrations of 5, 20, and 100 µg/L. For APs, since only AP B was commercially available as a standard, quantification was carried out using the class equivalent approach with AP F and OSC Y concentrations expressed as AP B equivalents. All other CPs for which no class equivalent standard was available, were expressed as MC-LR equivalents [69].

Validation of Methods for the Determination of CPs
The analytical work flow used for the target CTs [31] was also validated for its performance for CPs. Recovery studies were carried out by spiking samples with an extract of cyanobacterial mass (Lake Kastoria, September 2014) containing all target CPs. The TIC and MRM chromatograms of the selected quantification ions obtained from cyanobacterial mass extract from Lake Kastoria (September 2014) are shown in Figure S5.
To obtain the extract of cyanobacterial mass used for spiking, 10 mg of lyophilized biomass (Lake Kastoria, September 2014) was extracted two times with 1.5 mL of 75% MeOH: 25% H 2 O and a third time with 1.5 mL n-butanol. Each time the mixture was vortexed, sonicated for 15 min in a sonication bath (Bandelin Sonorex Super RK106) and then centrifuged at 4000 rpm for 10 min at room temperature (DuPont RMC-14 Refrigerated Micro-Centrifuge, Sorvall Instruments, Newtown, CT, USA) and the supernatant was separated from the pellet. All supernatants were pooled together. One milliliter of the extract was evaporated to dryness, the residue was re-dissolved in 400 µL of MeOH: H 2 O (5:95 v/v) and analyzed by LC-MS/MS. The rest of the extract was used for spiking CPfree biomass (retained on filter after passing 200 mL of lake water) and filtered water samples (400 mL) to carry out recovery experiments. Additionally, recovery experiments were carried out by spiking filtered CP-free water samples (400 mL) with AP B at the concentration level of 100 ng/L.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/toxins13060394/s1, Figure S1: structures of several classes of cyanotoxins. For the classes of Microcystins and Nodularins, R x stands for variable L-amino acids, Figure S2: structures of several classes of cyanopeptides. For the classes of Anabaenopeptins and Microginins, A x stands for variable L-amino acids, Figure S3: fragmentation pathways of AEG A involving the m/z 154.2 and 86.0 fragment ions. Fragmentation pathways predicted by Mass Frontier 8.0 software, Figure S4: heat maps showing all CP and CT concentrations detected in Lake Vegoritis throughout the two sampling points (Site 1 and Site 2), Figure S5: TIC and MRM chromatograms of quantifier ions for the cyanopeptides obtained from the biomass extract of Lake Kastoria (sampling September 2014), and Table S1: physico-chemical parameters, total phytoplankton biomass and cyanobacteria biomass concentrations measured during the study period. Table S2: concentrations of extracellular CTs from Lake Vegoritis (µg/L), Table S3: concentrations of intracellular CTs from Lake Vegoritis (µg/L), Table S4: concentrations of extracellular CPs from Lake Vegoritis (µg/L), and Table S5: concentrations of intracellular CPs from Lake Vegoritis (µg/L).