Behavioural effects on marine amphipods exposed to silver ions and silver nanoparticles☆
Graphical abstract
Introduction
Behaviour can be defined as the outcome of a sequence of neurophysiological events, which include the stimulation of sensory and motor neurons, muscular contractions and release of chemical messages (Lagadic et al., 1994). Additionally, behavioural responses integrate many cellular processes vital to an organism's survival and reproduction, reflecting biochemical and ecological consequences of toxic impact (Gerhardt, 1995). Several studies have used behavioural responses as tools for ecotoxicity testing and water quality monitoring (e.g. locomotor activity, response to light, ventilatory activity, feeding rate) because they are sensitive, fast, simple and cost-effective to perform (Bakker et al., 1997; Wallace and Estephan, 2004). The organism's mobility and response to light are also behavioural markers with ecological relevance, as locomotion is essential to find food, escape predators and obtain mating success; whereas the response to a light stimulus is associated with predator avoidance (Bakker et al., 1997). Any pollutant that interferes with the mobility of an organism can, therefore, result in reduced fitness and ‘ecological death’ (Arce Funck et al., 2013; Scott and Sloman, 2004; Vellinger et al., 2012).
Silver (Ag) has been widely applied in nanomaterials (Fabrega et al., 2011; Musee, 2011; Purcell and Peters, 1998), mainly because of its broad-spectrum antimicrobial properties. As a consequence of its use, Ag can be released into the environment in a variety of compounds and forms (Morgan et al., 1997; Purcell and Peters, 1998), making it an element of environmental concern. Silver released into surface waters can reach toxic concentrations to the aquatic life - from picograms per litre to micrograms per litre (Purcell and Peters, 1998; Wood et al., 1999). Free silver ion (Ag+) is the main species responsible for Ag toxicity in the aqueous phase, belonging to the highest toxicity class together with Cd, Cr(VI) and Hg (Ratte, 1999 and references therein). However, for AgNP, it is still unclear if toxicity is due only to the dissolution of Ag+ or whether the nanoparticles size, shape and defects in surface crystals contribute to their high toxicity (George et al., 2012; Vannuci-Silva et al., 2019). Once in the marine ecosystem, silver and its nanoparticles tend to agglomerate and precipitate to the sediment bottom (Forstner, 1983), leading to elevated exposure of benthic organisms such as amphipods.
Amphipods have been used in ecotoxicology studies for decades, being considered an excellent model for monitoring the health of aquatic biotopes and the effects of anthropogenic contaminants (Arce Funck et al., 2013; Felten et al., 2008; Gerhardt, 1995; Vellinger et al., 2012). They are ubiquitous to almost all water systems, abundant and ecologically relevant. These marine crustaceans play a fundamental role in the ecosystem dynamics (Melo and Nipper, 2007) and have a short life and reproductive cycle. Furthermore, amphipods require little space and resources in the laboratory for husbandry and testing (Artal et al., 2017).
Several studies on the behavioural effects of metal exposure on invertebrates can be found in the literature, including freshwater amphipods, particularly Gammarus species. Altered behaviour responses after metal exposure on Gammarus pulex were reported by Gerhardt (1995), Felten et al. (2008) and Vellinger et al. (2012). Arce Funck et al. (2013) studied behavioural responses of the male freshwater amphipod Gammarus fossarum exposed to Ag (at 0, 0.5, 1, 2, and 4 μg L−1) and found that locomotor and ventilatory activities were significantly reduced after 96 h. However, studies on the behaviour of marine amphipods are still scarce. To the best of our knowledge, there is no information on Ag and AgNP effects on swimming behaviour in these organisms.
Echinogammarus marinus is a marine amphipod which inhabits a wide range of coastline in northwestern Europe, stretching from Norway to southern Portugal. Bossus et al. (2014) and Guler & Ford (2010) studied the behavioural effects of anti-depressants on this species and found significant alterations in swimming velocity, as well as photo and geotaxis responses, which could impact population-levels.
In this work, we investigated the behaviour effects on E. marinus after exposure to AgNO3 via water and to AgNP or AgCl amended food. Both routes were evaluated because silver ions (Ag+) are promptly absorbed via water, which implies full-time interaction between contaminants and organisms - whereas food exposure is intermittent – the reason why silver salts in solution are positive controls of silver exposure (Andreï et al., 2016; Pokhrel and Dubey, 2012). Complementarily, dietary assessment of Ag and AgNP exposure is especially relevant, given that contaminated food intake is one of the primary routes for silver and metallic nanomaterials incorporation (Petersen and Henry, 2012). The aims of this study were to understand behavioural alterations and develop an assay to evaluate sub-lethal effects of silver in the marine environment. We hypothesized that silver absorbed via contaminated water and food would lead to disruption on locomotion and light response in E. marinus.
Section snippets
Reagents, material and equipment
The reagents used in this work were: reverse osmosis water, concentrated nitric acid (HNO3 - Fischer Scientific), sea salt (Red Sea®) and silver nitrate (AgNO3 - Fischer Scientific). Experimental materials included plastic pots, plastic Petri dishes, plastic pipettes, tweezers, glassware and other materials generally used in biology laboratories. All glassware was decontaminated with 10% HNO3 overnight prior to use. The equipment used in this work were: salinity meter, pH meter, analytical
Experiment A1
The mean swimming velocity peaks just after lights on and it gradually comes back to lower values. The velocity peak was inversely proportional to Ag concentration in water (Fig. 1). The faster velocity peaks were noticeable for the control organisms and the lowest concentration (5 μg L−1), which showed no significant differences between their responses to light (p = 0.973). However, velocity after lights on for the 100 and 25 μg L−1 treatments were significantly different than control
Discussion
Movement is a highly ecologically relevant behavioural marker since locomotion is required to find food, escape predation and obtain mates (Arce Funck et al., 2013). Therefore, if metals interfere with locomotor activity, they will likely reduce fitness and could lead to “ecological death” of an organism (Scott and Sloman, 2004). There are some studies reporting the possible physiological and biochemical mechanisms behind the metal effects on behaviour (Felten et al., 2008; J. Lawrence and
Conclusions
Silver affected swimming and response to light behaviour on E. marinus exposed to AgNO3 via water. Animals swam slower and responded less to light proportionally to the increase of Ag in the water. There was no difference in response to light between treatments for exposure to AgCl or AgNP incorporated in the food after 28 days. Perhaps, longer exposure times would be required for the observation of effects.
This was the first work to assess behaviour effects of silver and silver nanoparticles
Funding
This work was supported by the São Paulo Research Foundation (FAPESP) and the Coordination for the Improvement of Higher Education Personnel (CAPES) [grant numbers 2013/26301-7 and 2016/19635-4].
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank FAPESP and CAPES (grants 2013/26301-7 and 2016/19635-4, São Paulo Research Foundation - FAPESP) for the financial support and the UK Environment Agency for providing the water quality data of Langstone Harbour.
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This paper has been recommended for acceptance by Dr. Sarah Harmon.