Can mixtures of cyanotoxins represent a risk to the zooplankton? The case study of Daphnia magna Straus exposed to hepatotoxic and neurotoxic cyanobacterial extracts Emanuela Cristina Freitas a, *, Carlos Pinheiro b , Odete Rocha a , Susana Loureiro b a Department of Ecology and Evolutionary Biology, Federal University of Sa ˜o Carlos, Rodovia Washington Luis, km 235, CEP 13565-905 Sa˜o Carlos, SP, Brazil b Department of Biology and Centre for Environmental and Marine Studies, University of Aveiro, 3810-193 Aveiro, Portugal 1. Introduction As a result of agricultural, industrial and urban development over the last two centuries, aquatic environments, especially freshwater bodies, have been subject to severe impacts from nutrient inputs. One of these impacts is the proliferation of cyanobacterial blooms (Azevedo et al., 2002; Barros et al., 2004; Becker et al., 2010). Cyanobacteria can produce secondary metabolites with toxic properties, known as cyanotoxins (Carmichael, 1992). Around the world, these toxins have exerted harmful effects on aquatic communities, besides inducing the death of wild and domestic animals, as well as some human fatalities (Ressom et al., 1994; Jochiminsen et al., 1998; Carmichael et al., 2001). According to their toxic effects on humans, cyanotoxins can be grouped into hepatotoxins, neurotoxins, cytotoxins, dermatotoxins and irritant toxins (lipopolysaccharides) (Jochiminsen et al., 1998; Wiegand and Pflugmacher, 2005), the types most commonly found in freshwater habitats being the hepatotoxins, followed by neuro- toxins (Chorus and Bartram, 1999). Most studies on the acute and chronic effects of cyanotoxins on aquatic organisms consist of observing the effects of exposures to single toxins in laboratory assays (Wiegand et al., 1999; LeBlanc et al., 2005; Babica et al., 2007; Ferra ˜o-Filho et al., 2008; Sotero- Santos et al., 2008). However, it is observed that in the natural environment organisms are constantly exposed to complex mixtures of cyanotoxins, such as hepatotoxins and neurotoxins. For instance, in Brazilian freshwater reservoirs, it is common to find the species Microcystis aeruginosa and Anabaena spiroides coexisting, with the potential to produce, respectively, hepatotox- ins and neurotoxins (Matsumura-Tundisi and Tundisi, 2005; Dellamano-Oliveira et al., 2007; Moschini-Carlos et al., 2009). Since cyanotoxins are rarely found alone in freshwater bodies, but commonly in combination with other stressors, there are some Harmful Algae 31 (2014) 143–152 A R T I C L E I N F O Article history: Received 11 February 2013 Received in revised form 12 November 2013 Accepted 13 November 2013 Available online Keywords: Anatoxin-a(s) Binary mixtures Cyanobacterial extracts Daphnia magna Microcystin-LR Synergism A B S T R A C T Worldwide, cyanobacterial blooms have been increasing in intensity and frequency, with toxic cyanobacteria sometimes dominant throughout the year in many freshwater bodies. Since the coexistence of more than one type of cyanotoxins in freshwater environments is a common phenomenon, studies on the joint effects of these toxins would be very useful. In this study, the single and combined effects of two cyanotoxins with different modes of action (hepatotoxic and neurotoxic) on the survival (lethal exposure) and feeding (sublethal exposure) of the cladoceran Daphnia magna were investigated. With the single exposures, it was observed that both the survival and feeding activity of the daphnids were impaired by the hepatotoxic and neurotoxic extracts at environmentally relevant concentrations. In the combined exposures, both survival and feeding rate endpoints showed a good fit to the independent action model. For the acute assay and 24 h exposure period in the feeding inhibition test, there was no interaction between components of the hepatotoxic and neurotoxic extracts, although a slight tendency to a synergistic deviation could be seen in the feeding rates. On the other hand, for the 4 h post-exposure period, a synergistic deviation was found in feeding rates at all mixture concentrations tested. Hence, the combined exposure of hepatotoxins and neurotoxins should also be taken into account in risk assessments of freshwater bodies, since the mixture of these toxins can result in more severe post-exposure effects on the feeding of daphnids than the sum of those expected for single exposures. ß 2013 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +55 16 33518319; fax: +55 16 33518322. E-mail addresses: emanuelacfreitas@gmail.com (E.C. Freitas), carlos.pinheiro@ua.pt (C. Pinheiro), doro@ufscar.br (O. Rocha), sloureiro@ua.pt (S. Loureiro). Contents lists available at ScienceDirect Harmful Algae jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/hal 1568-9883/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2013.11.004