The obtained results revealed the presence of microcystins in farmed mussels Mytilus galloprovincialis, in Thermaikos gulf for first time, while concentrations above 1 ng/g were found in digestive gland. Their detection was linked to the presence of potentially toxic cyanobacteria. Microcystins in mussels and other seafood have been detected globally . Microcystins -LR and -RR are considered the most common , while MC-LR has been described as the toxin most detected in mussels, with MC-RR and MC-YR following [13, 34]. In a survey in freshwater bivalves Sinanodonta woodiana, Sinanodonta arcaeformis, and Unio douglasiae from a wetland in South Korea , it was found that the concentrations of MC-RR, MC-LR and MC-ΥR, were 11.2 to 70.1 µg/g dry weight in the muscles and 168.9 to 869 ng/g dry weight in the digestive gland. The same microcystins were detected in a similar study in freshwater mussels (Cristaria plicata, Hyriopsis cumingii and Lamprotula leai) in lake Taihu in China , at high concentrations mostly in the digestive gland, reaching up to 38,478.1 ng/g dry weight. Also, MC-RR, MC-LR and MC-YR were detected for the first time in lake Dau Tieng in Vietnam, in mussels Corbicula sp. and Ensidens sp. at concentrations 1.54 ± 0.21 and 3.15 ± 0.65 µg MC/g dry weight, respectively . Another study in North Baltic Sea, showed that the green mussels Mytilus edulis examined by ELISA, accumulated microcystins up to 21.50 ± 60 ng/g, expressed as equivalents of microcystins and nodularins. At the same study it was reported that these toxins were detected in the liver of the fish Platichthys flesus, at concentrations up to 99 ± 5 ng/g; hence no toxins were detected in the muscle . A similar study  in mussels harvested in the North-East Pacific Ocean of British Columbia, reports the presence of MC-LR at levels up to 600 ng/g, as measured by LC/MS, and also in mussels imported from Canada and the Netherlands.
Microcystins in Greece have been detected mostly in freshwater mussels linked to the occurrence of cyanobacterial blooms [5, 17]. In the freshwater mussel Anotonda sp. in Lake Kastoria, microcystins have been detected at the level of 3.271 ng/g equivalents of MC-LR dry weight, by the protein phosphatase 1 inhibition assay test (PP1IA) . Regarding the Mediterranean mussels Mytilus galloprovincialis, there is only one report in Amvarkikos gulf, in Western Greece, where microcystins were detected after a Synechococcus sp. and Synechocystis sp. bloom . The concentrations of the toxins were 45 ± 2 to 141.5 ± 13.5 ng/g equivalents of MC-LR (ELISA), values that according to the researchers were above the Tolerable Daily Intake (TDI), as it is set by the World Health Organization (WHO).
In Greece, microcystins have been also detected in other aquatic animals’ tissues. A research in fish from the lakes Kastoria, Iliki, Kerkini and Pamvotis, and Gallikos river, showed that microcystins were accumulated in the fish Cyprinus carpio, Carassius gibelio, Silurus aristotelis and Perca fluviatilis and in the amphibian Rana epirotica. The samples were analyzed by ELISA and the toxins’ concentrations were 20 to 1,440 ng/g dry weight in the muscles and 25 to 5,400 ng/g dry weight in visceral tissue . Microcystins were also detected in fish Carassius gibelio in lake Pamvotis. It was reported that the toxins were accumulated mostly in liver samples (124.4 ng/g) and less in muscle samples (7.1 ± 2.5 ng/g), analyzed by ELISA . Similar study was contacted in Cyprinus carpio tissues in lake Karla in Thessaly. It was observed that the highest concentrations were accumulated in liver (732 ± 350 ng/g), while kidneys (362 ± 207 ng/g) and muscles (362 ± 207 ng/g) were following .
Our study indicates that microcystins can be bioaccumulated in mussels, a finding that is in accordance with the literature [42, 43, 44, 45, 46]. The fact that mussels are filter feeders, enables them to bioaccumulate environmental pollutants , like microcystins . A study in the brackish waters of Curonian lagoon in Lithuania, revealed that mussels Dreissena polymorpha (zebra mussels), accumulated microcystins at high concentrations, up to 139 ng/g dry weight analyzed by ELISA and 284 ng/g dry weight by PPIA. The toxins were detected even at low abundances of potentially toxic cyanobacteria periods. It was assumed that the bioaccumulation in mussels could be explained by a secondary contamination by resuspended microcystins’ residues in sediment particles . Moreover, cyanobacteria are considered food for mussels, leading to the uptake of intracellular cyanotoxins. Although the detection of hepatotoxins in mussels at high levels due to the uptake of intracellular and free toxins, should be more clarified.
According to our study, the target organ of microcystins bioaccumulation seems to be the digestive gland. Several studies have reported the bioaccumulation of microcystins in the digestive gland of freshwater, brackish and marine mussels, at concentrations higher than of other organs [35, 36, 42, 43]. Moreover, during an experimental contamination, pearl mussels (Hyriopsis cumingii) from an aquaculture of Yueshan village, Ezhou city, in China, were exposed to Microcystis aeruginosa 905 for 15 days. After the analysis by HPLC-UV, it was found that MC-LR was accumulated in hepatopancreas at the highest level of 55.78 ± 6.73 µg/g dry weight, while the concentrations in other organs were 27.88 ± 2.22 µg/g dry weight in gonads, 5.66 ± 0.55 µg/g dry weight in gills and 5.17 ± 0.87 µg/g dry weight in muscles .
Except from bioaccumulation, the biomagnification of microcystins in aquatic animals has been a field of study for many researchers, leading to different opinions. According to some studies, no biomagnification is observed, or it is not documented sufficiently [36, 37, 51, 52]. Moreover, biomagnification has been observed more in lipophilic marine biotoxins rather than in hydrophilic ones, as microcystins . On the other hand, biomagnification was reported in farmed and wild bivalves (mussels, oysters and clams), where concentrations of microcystins up to 107 times higher than the ones in water were reported . Also, it has been reported that biomagnification of cyanotoxins in aquatic animals could be explained by their evolution in relation to cyanobacteria , leading to their ability to develop defense mechanisms against the cyanotoxins . Through this the aquatic animals could bioaccumulate toxins at high levels and thus be carriers in the food chain, contributing to biomagnification. In a recent meta-analysis of field studies, biomagnification and biodilution of microcystins in aquatic foodwebs (zooplankton, mollusks, fish, decapods, turtles and birds) was assessed . The biomagnification factor (BMF) for microcystins was calculated by the researchers as the ratio between their concentration detected in those organisms and their diet. Biodilution was sufficient. Zooplankton showed potential for microcystins’ biomagnification, influencing their concentrations in the liver of zooplanktivorous fishes and carnivorous jellyfish, where high values of BMF were observed. There seemed to be a proportional increase between the duration of consumers’ exposure to diet, having high microcystins’ concentrations. It was concluded that the exposure to high populations of potential toxic cyanobacteria of aquatic animals, especially those that are part of the human food chain, could be related to biomagnification.
In our study, results above 1 ng/g were observed mostly during warm seasons and especially in May. This could be explained by the increase of sunlight, the high temperatures in the water and the enrichment of Thermaikos gulf with nutrients from the adjacent rivers. A survey in Greek internal waters showed that the highest levels of MC-LR were observed during the warm months of the year . Microcystin -RR, and microcystin -LR have also been detected at 79.4% and 73.5% respectively in lakes in Greece during warm seasons . In another study by ELISA in lake Koronia, microcystins were detected at higher levels during spring . Moreover, mussels Mytilus galloprovincialis in Amvrakikos gulf accumulated the highest concentrations of microcystins in the same season . Same findings have been reported in other Mediterranean countries. In Italy, a survey in lakes Garda, Como, Iseo, Lugano and Maggiore revealed that the highest concentrations of microcystins and anatoxin-a were observed during warm seasons and mostly in May and September . Additionally, in South-East Adriatic, in mussels Mytilus galloprovincialis analyzed by ELISA, microcystins were detected up to 256 ng/g. Moreover, they were detected up to 2.3 ng/g in clams Chamelea gallina . A similar survey in Portuguese recreational waters showed that the highest levels of microcystins were recorded during spring and summer, and mostly from April to September . Moreover, it has been reported that the global climate change and the increase of the temperature in water environments induce potentially toxic cyanobacterial blooms and accumulation of microcystins in aquatic animals [1, 7].
According to our results a spatial distribution of microcystins in the same area, in different seasons was detected. This could be explained by the different levels of enrichment in the coastal zones by the runoffs of the rivers, carrying nutrients. Axios is near Kavoura in Chalastra, Aliakmonas is near Makrigialos in Pieria and Loudias is near Klidi in Imathia. Their annual runoffs are 158 up to 279 m3/s, 73 up to 137 m3/sand 5 to 10 m3/s, respectively. Large agriculture activity is located in the adjacent areas with the transferred sediments, which are rich in nitrates and phosphates, are up to 500 t/km2 . The highest amounts of runoffs in Kavoura, Imathia and the estuaries areobserved during spring , while in Makrigialos the lowest are observed during summer .
Regarding the comparison between ELISA and LC–Orbitrap HRMS, our results indicate an overestimation by ELISA. In particular, during spring the samples above 1 ng/g were 24 by ELISA and 7 by LC–Orbitrap HRMS, in summer 12 and 4 respectively and in autumn 16 and 4, respectively. Moreover, the concentrations obtained by ELISA were 6 to even 22 times higher. According to the manufacturer’s instructions the microcystins’ standards in ELISA were MC-LR, MC-RR, MC-YR, MC-LF, MC-LW, and the desmethylated [D-Asp3] MC-RR and [Dha7] MC-LR. On the other hand, the LC–Orbitrap HRMS analysis focused on the most common microcystins MC-LR, MC-RR and MC-YR. Although ELISA is considered a suitable screening method due to its high sensitivity, detection of multi analogues of microcystins, rapid results, low cost and lack of ethical issues [26, 27, 28, 29], false positive results due to matrix effects cannot be excluded. Similar observations have also been reported in other studies. In a study in bovine’s drinking water, Microcystis aeruginosa cells were added at the abundance of 105 cells/mL for 28 days. The microcystins’ concentrations in the liver were 0.92 µg MC-LR equivalents/g fresh weight measured by ELISA, although no toxins were detected by HPLC/GC-MS . It was concluded that the levels were 1000 times higher due to matrix effect. In another study during an expansion of a cyanobacterial bloom in farms of Mytilus galloprovincialis in Adriatic Sea,  the MC-LR equivalents were 256 ng/g (ELISA) in mussels’ tissues. Although only the desmethylated derivative desMe-MC-RR was confirmed by LC/ESI-Q-ToF-MS/MS at the level of 39 ng/g. In other mussel samples an overestimation was also observed (1.5 to 6.5 times higher). It was mentioned that ELISA is a useful screening tool, but results should be confirmed by chromatographic analytical methods tandem to mass spectrometry. In another study in McMurdo Ice Shelf station in Antarctica, researchers from New Zealand examined water samples and cyanobacterial mats . The concentrations of microcystins were up to 8 times higher by ELISA than LC-MS. This overestimation could lead to unnecessary bans in drinking water, so the results should be confirmed by mass spectrometry. A similar study evaluated the results obtained by ELISA and LC-MS/MS . It was proposed that ELISA could be used for screening, since it provides rapid results. Still, it was reported that the nonlinear calibration curve could lead to false positive results, since small differences in absorbances might be translated into big concentration differences. The same authors report that LC-MS/MS provides higher specificity and sensitivity and concluded that in any case the abundance of cyanobacteria and the water treatment should be considered. Also, microcystins’ concentrations have been assessed in nontoxic cyanobacterial food supplements, peals and capsules . It was reported that no statistically significant differences were observed in capsules, since the concentrations were 43 to 410 ng/capsule and 40 to 425 ng/capsule by ELISA and LC/MS, respectively. In peals the levels by ELISA were lower, 200 to 960 ng/peal, when by LC/MS they were 280 to 1,310 ng/peal. So, an underestimation at the level of 27% was observed by ELISA. This was explained by the different sensitivity of the LC-MS method in some microcystins’ analogues. Finally, in a recent study  water samples from 31 water ecosystems in Michigan, U.S.A., were examined by both methods, ELISA and LC-MS for microcystins, during July, August, September and October, when cyanobacterial blooms often occur. It was reported that no statistically significant differences were observed in July and August. On the other hand, an overestimation by ELISA was noticed in September and October. This was attributed to cross-reactivity with microcystins’ degradation products and to the quantification using nonlinear calibration curve.