Effects of low arsenic concentration exposure on freshwater fish in the presence of fluvial biofilms
Graphical abstract
Introduction
Arsenic (As) is a highly toxic element and its carcinogenic effect on living organisms is well known (Ng et al., 2003). Elevated concentrations of metals and metalloids in water and aquatic sediments deposited from mining or industrial waste waters are a global problem (Schaller et al., 2011). There are many examples of rivers and lakes being contaminated with arsenicals and other polluters from current and old mining activities (Casiot et al., 2005, Inam et al., 2011, Wong et al., 1999). Predicting real effects in the environment requires an ecological approach since toxicity is influenced by many environmental factors (e.g. substrate type, nutrient contents, redox status) and biological ones (e.g. biotransformation) and differs among aquatic organisms. Contaminants are accumulated on organic and inorganic sediment particles and their associated microorganism communities (Schaller et al., 2011). Microorganisms play a key role in the biogeochemical cycle of arsenic. They bioaccumulate inorganic arsenicals (iAs), biotransform to methylarsenicals and complex organoarsenicals inside their cells, and then release back to the water and mineralize methyl and organoarsenicals producing iAs species (Rahman et al., 2012). In natural waters, arsenic is mostly found in inorganic form as oxyanions of trivalent arsenite (As(III)) or pentavalent arsenate (As(V)) (Hasegawa et al., 2010, Smedley and Kinniburgh, 2002). Arsenite is assumed to be the most toxic form to most organisms, including humans, whereas As(V) is more toxic than As(III) to algae (Knauer et al., 1999). In well oxygenated aquatic systems iAs should be mostly as As(V).
While arsenic toxicity to invertebrates and algae has been described at low concentration: 20 μg/L (LOEC for Daphnia magna growth in a 21-day flow-through chronic bioassay) and 50 μg/L (14-day EC50 of growth inhibition for the green alga Scenedesmus obliquus), respectively, freshwater fish has lower sensitivity. The lowest chronic LC50 for fish reported in the literature was 550 μg/L for rainbow trout, Oncorhynchus mykiss, after 28 days' exposure (CCME, 2001). Numerous data are available on the effect of arsenic on fish reproduction (Boyle et al., 2008), growth, development and survival (D'Amico et al., 2014, Erickson et al., 2011, Gonzalez et al., 2010, Gonzalez et al., 2006, Li et al., 2009).
Existing data regarding the biochemical basis and mechanisms of arsenic toxicity support the use of antioxidant enzymes as early warning signallers of arsenic toxicity in freshwater fish. One of the earliest responses to arsenic toxicity is an increase in reactive oxygen species (ROS) (Flora, 2011), during their redox cycling and metabolic activation processes that cause lipid peroxidation and DNA damage (Ratnaike, 2003). Normally, cells defend themselves against ROS damage with several enzymes including superoxide dismutase, catalases, and glutathione peroxidases (Bansal and Kaushal, 2014). The tripeptide glutathione (GSH) directly or indirectly regulates the scavenging of ROS both as an important component of antioxidant defence system in fish and also as a molecule containing a thiol group, in which arsenic has affinity. So glutathione and its dependent enzymes such as glutathione reductase and glutathione S transferase are expected to respond to arsenic exposure in fish (Srikanth et al., 2013). There are few studies measuring antioxidant enzyme activities of freshwater fish exposed to low concentrations of arsenic. Kim and Kang (2015) observed increases in SOD and GST activities in liver and gill of juvenile rockfish (Sebastes schlegelii) exposed to 200 μg/L sodium arsenite for 20 days, whereas Sarkar et al. (2014) found a triphasic alteration in CAT activities in the brain of zebrafish exposed to 50 μg/L arsenic trioxide for 90 days.
Given the intricacies of the feedback and cycling interactions contributing to arsenic toxicity in fish, in previous studies a simplified fluvial system was used to examine the interacting effects of naturally occurring periphytic biofilms, thus incorporating some of the complexity of natural systems in a laboratory experiment and allowing some specific processes from whole-ecosystem effects to be disentangled (Barral-Fraga et al., 2015, Magellan et al., 2014). Using this experimental setup, Magellan et al. (2014) found that mosquitofish exposed to arsenate (As(V), 130 μg/L over 13 days) experienced an increase in the amount of weight gained and a higher level of aggressive behaviour; effects which were aggravated by the presence of periphytic biofilms, while periphytic biofilms suffered a reduction in algal species richness, a marked inhibition of algal growth and a strong reduction in diatom cell biovolume and these effects were reported by Barral-Fraga et al. (2015).
In this study, we investigated the effects of a longer exposure (56 days) of arsenic on mosquitofish (Gambusia holbrooki) under the influence of periphytic biofilms (growing on illuminated glass substrata) and epipsammic biofilms (growing on sediment grains), thus increasing the complexity, and hence realism of experimental conditions, by including sediments. By including periphytic and epipsammic biofilms, the influence that adsorption and/or biotic activity may have on arsenic toxicity was also evaluated.
Biofilms — communities embedded within a polysaccharide matrix — play a key role in the functioning of aquatic ecosystems. Biologically, biofilm changes chemical exposure in stream ecosystems by influencing solubility of minerals, sorption of metals onto particle surfaces, transformations between oxidized and reduced species, and metabolism of aquatic biota (Balistrieri et al. 2012). Eventually, these changes will affect bioavailability and hence toxicity in fish.
The purpose of this paper was to evaluate the effects of environmentally-realistic arsenic exposure on fish under the interactive influence active biofilm communities. Effects caused to periphytic and epipsammic biofilms of this experiment were previously evaluated and reported in detail in Tuulaikhuu et al. (2015). Periphytic and epipsammic biofilms were grown under conditions of phosphorus limitation since effects of arsenic on organisms increase under lower phosphorus availability (Rodriguez Castro et al., 2014, Wang et al., 2013) and under a well-oxygenated environment to ensure that AsV was the dominant arsenic species. Arsenic exposure influenced the quality and quantity of the biofilm and its ability to purify and oxygenate the aquatic environment. Sediments played a double and antagonistic role in arsenic toxicity to biofilm by removing arsenic from the water column but enhancing its toxicity by retaining nutrients (Tuulaikhuu et al., 2015). Arsenate (As(V)) toxicity to periphyton photosynthesis and phosphate uptake under P-limiting conditions has been demonstrated at low concentration (15 μg As/L), highlighting the role of phosphate on As(V) toxicity to these aquatic communities (Rodriguez Castro et al., 2014). In this periphytic community, P-uptake capacity was already affected and algal growth was inhibited up to 61% in P-starved conditions, but not when P-availability was higher.
Generally short-term exposure or low arsenic concentrations results in an increase of the activity of these enzymes, while higher exposure may lead to a reduction of these activities if the antioxidant defences are overwhelmed (Flora, 2011). Given the low arsenic concentration used, exposure effects were expected to be chronic, thus exposure lasted for eight weeks and the effects based on antioxidant enzyme activities (AEAs) which are expected to respond to low-dose exposure. We evaluated five different AEAs (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione-S-transferase (GST), and glutathione reductase (GR)), in liver and gill proteins of eastern mosquitofish (Gambusia holbrooki) to assess the arsenic effect on this fish. Although enzymes are early warning signallers of toxicity, they are highly sensitive to changes in environmental conditions. Thus, it is essential to provide the greatest possible ecological realism in toxicity testing to better understand real toxicity. Since sediments removed a large part of arsenic from the water column (Tuulaikhuu et al., 2015), we also expected that the remaining concentration may not affect the AEAs of the fish, but the already stressed biofilms might have some effects.
Section snippets
Experimental set up
Twelve independent experimental units with three replicates of 4 different treatments each were: control (C) (with neither As (V) nor biofilm), biofilm (B), arsenic (+ As) and biofilm with arsenic (B + As). Each experimental unit had three main components: a large tank (90 L), in which fish were not in contact with the periphytic biofilm, a channel (90 × 8.5 × 7.5 cm) containing sand blasted glass tiles to provide substrate for the periphytic biofilms and a smaller tank (8 L) (S. Fig. 1). We filled the
Physical and chemical conditions
Physicochemical parameters in water were similar among the treatments at the beginning of the experiment and changed during biofilm colonisation. Water temperature was 20.9 ± 1.0 °C on average and slightly increased at the end of the experiment but was similar between treatments. Water pH was higher in the B treatment than in C at most times, except the first day when it was lower than C (Table 1). Comparing the + As and B + As treatments, pH was higher with the presence of periphytic biofilm in the
Discussion
In our experiment, arsenic exposure changed over time. It was mainly retained in the sediment with less contribution of periphyton as described in a previous publication focused on the fate and effects of arsenic on periphytic and epipsammic biofilms that included only the B and B + As treatments, the two treatments that had periphytic biofilms (Tuulaikhuu et al., 2015). The average exposure concentrations to arsenic were relatively low: 34.4 ± 1.4 μg/L for B + As and 40.5 ± 7.5 μg/L for + As treatment,
Conclusions
We can therefore conclude that chronic exposure of mosquitofish to 34–40 μg As/L will cause effects, but the presence of periphytic biofilm may influence the response. The average exposure concentrations to arsenic for fish were between 3.75 and 4.5 times lower than the criterion continuous concentration (CCC) (150 μg/L), which is an estimate of the highest concentration of a substance in surface water to which an aquatic community can be exposed indefinitely without resulting in an unacceptable
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
Financial support was provided by the Spanish Science and Education Ministry (Project CTM2009-14111-CO2-01) and the Spanish Economy and Competitiveness Ministry (Project CGL2013-43822-R). Baigal-Amar Tuulaikhuu benefited from a doctoral fellowship from the Techno 2 Program of the European Union Erasmus Mundus partnership. Thanks to Kit Magellan for providing the fish and giving recommendations for maintaining and measuring them. Thanks also to Laura Barral for helping with the lab work, and
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