# Review on Cyanobacterial Studies in Portugal: Current Impacts and Research Needs
## 2 Cyanobacterial Studies
### In Vivo Studies  
Initial in vivo research findings on the effects of cyanobacteria and associated toxins focused mainly on the aquatic environment and their organisms (Figure 2). Zooplankton, bivalves and fish are among the most studied groups. These works comprehended various in vivo measurement approaches, using mostly laboratory-exposed organisms. In the early nineties, [PERSON] [42] started by studying the impact of toxic and nontoxic cyanobacteria on some freshwater microcrustacean species. Results showed that a toxic _Microcytsi aeruginosa_ strain produced total lethality of three cladocerans (_Daphnia longispina_, _Cerio-daphnia pulchella_ and _Simoccephalus vetulus_) in two days. Furthermore, the non-toxic strain of the same cyanobacteria species caused total mortality to _Daphnia_ in four days while the other two species survived longer, suggesting that other compounds apart from MC may be involved in zooplankton dynamics [42; 43]. In the following studies with this group of organisms, [PERSON] et al. [44; 45; 46] assessed bioaccumulation and effects promoted by toxic and non-toxic cyanobacterial strains using _D. magna_ as a biological model. Results suggested that the fitness and growth potential of natural _Daphnia_ populations may be affected by cyanobacteria, especially when subjected to blooms of toxic strains. Furthermore, CYN and paralytic shellfish toxins (PSTs) were detected in the tissues of _D. magna_ after exposure to the toxic strains of _Cylindrospernopsis raciborskii_ and _Aphanizomenon issatschenkoi_, respectively, indicating that daphnids are potentially capable of passing cyanotoxins to higher trophic levels. In bivalve mollusks, the accumulation and depuration of cyanotoxins was reported in several studies where freshwater and intertidal bivalves were exposed to toxic cyanobacteria. [PERSON] et al. [47] and [PERSON] and [PERSON] [48] reported the accumulation of high levels of MC by _Mytillus galloprovincialis_ when fed with toxic _M. aeruginosa_ for several days. The majority of the toxins were contained in digestive tract, whereas the other organs were barely contaminated. Results also confirmed that mussels could retain detectable toxins after transfer to a non-toxic phytoplankton food source. Later, [PERSON] et al. [49] and [PERSON] et al. [50] reported the accumulation of high levels of PSTs and CYN by _Anodonta cygnea_ after 2 weeks exposure to the toxic cyanobacteria _A. issatschenkoi_ and _C. raciborskii_, respectively. The pattern of PSTs accumulation in the various mussel organs was similar to the ones obtained for MC in _M. galloprovincialis_. In its turn, [PERSON] et al. [50] were the first to report the concentrations of cyanobacterial toxins present in the haemolymph and found that this fluid contained the highest concentrations of CYN in _A. cygnea_, followed by viscera. Following a 2-week depuration period, trace to undetectable levels of PST were found in _A. cygnea_, whereas approximately 50% of the CYN remained in the tissues. Finally, [PERSON] et al. [51] reported no bioaccumulation of anatoxin-a by _M. galloprovincialis_ fed with toxic _Anabaena_ sp. (\(5\times 10^{5}\) cell mL\({}^{-1}\)) during 2 weeks. The lack of mortality during all these experiments associated to toxin persistence in the tissues supports the idea that bivalves are very resistant to cyanotoxins and good toxin vectors. Thus, it is proposed these mollusks possess detoxification mechanisms. Changes in activity and expression of GSTs in mussels and clams frequently found in Portuguese waters have already been linked to MC-producing microalgae or their purified toxins exposure [52; 53; 54; 55]. Working with bivalves of several aquatic habitats, [PERSON] et al. [38] showed that these mollusks presented specific adaptive biotransformation responses to MCs and other cyanobacteria compounds supported by the modulation of distinct cGST classes.