Antioxidant responses in estuarine invertebrates exposed to repeated oil spills: Effects of frequency and dosage in a field manipulative experiment
Introduction
The production of reactive oxygen species (ROS) occurs naturally during cellular aerobic respiration processes (Vidal-Liñán and Bellas, 2013), but can also be highly affected by environmental factors, such as salinity and temperature (Lushchak, 2011), or exposure to contaminants (Monserrat et al., 2007, Lüchmann et al., 2011, Marques et al., 2014). Increased ROS levels may induce lipid, protein and DNA oxidation, leading to several deleterious effects at cellular level (Monserrat et al., 2007, Vidal-Liñán and Bellas, 2013). Cells are protected against the deleterious effects of oxyradical generation by maintaining ROS at low levels through several antioxidant defenses, which include both enzymatic and non-enzymatic antioxidants (Kaloyianni et al., 2009 Lüchmann et al., 2011, Turja et al., 2013, Zanette et al., 2015).
Changes in antioxidant defenses can be used as indicators of contaminant exposure. The antioxidant system involves enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Among the non-enzymatic defenses, glutathione (GSH) participates in many important biological processes including protection against toxic compounds (Lüchmann et al., 2011). Moreover, enzymes involved in the elimination of ROS byproducts, such as glutathione S-transferase (GST), play an important role as indirect antioxidant (Boutet et al., 2004, Lüchmann et al., 2011, Zanette et al., 2015). Eventually, deficiency in the antioxidant system of cells can increase the lipid peroxide levels (LPO), a major mechanism by which oxyradicals can damage the cellular membrane lipids (Turja et al., 2013, Zanette et al., 2015).
Polycyclic aromatic hydrocarbons (PAHs) are a common source of contamination in the aquatic environment, mostly as a result of petroleum-related activities (Lüchmann et al., 2014). PAHs are primarily associated with anthropogenic sources, particularly fossil fuels and their derivatives. The process of partial combustion, accidental oils spills and the disposal of domestic and industrial effluents are the major sources of PAHs to coastal systems (Martins et al., 2011, Abreu-Mota et al., 2014). PAHs may affect aquatic organisms in many ways and the oxidative stress is one of the key elements of their toxicity (Lushchak, 2011). PAHs are primarily metabolized via hydroxylation (phase-I reactions) and detoxified by enzymes in the cytochrome P450 system (Lushchak, 2011, Lüchmann et al., 2014).
Many studies have reported changes in oxidative stress biomarkers as a response to PAHs exposure in marine invertebrates, particularly in bivalves (Turja et al., 2013, Marques et al., 2014, Lüchmann et al., 2014, Turja et al., 2014, Vidal-Liñán et al., 2014, Won et al., 2013), but also in polychaetes (Nesto et al., 2010 Ramos-Gómez et al., 2011, Won et al., 2013). Filter-feeding mollusks are often used as sentinels in pollution monitoring due to their significant ability to bioaccumulate pollutants as well as to respond to their presence (Solé et al., 2009, Lüchmann et al., 2011). Polychaete worms are also good sentinels because they can adapt to stressful environmental conditions, are distributed worldwide and present a sedentary lifestyle (Solé et al., 2009, Díaz-Jaramillo et al., 2011). However, few studies have investigated the effects of PAHs on oxidative stress biomarkers in other marine invertebrates, such as crabs (Martín-Díaz et al., 2008, Morales-Caselles et al., 2008, Ricciardi et al., 2010) and gastropod mollusks (Reid and MacFarlane, 2003, Sarkar et al., 2006, Tim-Tim et al., 2009).
Experiments evaluating biomarker responses have often been done under laboratory conditions (Silva et al., 2005, Lüchmann et al., 2011, Luna-Acosta et al., 2011) to isolate the putative effects of PAH exposure from other factors. Such experiments, however, do not include the full set of naturally occurring abiotic and biotic variables (Goodsell et al., 2009), which can affect the persistence of contaminants and, ultimately, the response of selected biomarkers. Thus, results from laboratory studies should be compared to robust field experiments in order to generate ecologically relevant information (Reid and MacFarlane, 2003, Nesto et al., 2010, Díaz-Jaramillo et al., 2013, Marques et al., 2014). Field experiments can be conducted whether by transplanting organisms to polluted areas (e.g. Díaz-Jaramillo et al., 2013, Turja et al., 2014) or by experimentally adding contaminants to natural sites (e.g. Marques et al., 2014).
Particularly in coastal and estuarine habitats, the intense traffic of small and mid size ships, together with fishing and recreational boats are often responsible for the release of petroleum products at a range of frequencies and intensities. Most of these vessels use marine diesel oil as fuel, which is less persistent than crude oil although it is highly toxic (Lytle and Peckarsky, 2001). Nonetheless, biomarker responses to PAH exposure in marine invertebrates are often evaluated from acute, non-cumulative, single-dosage oil spills. Impact assessments are commonly carried out after accidents through descriptive approaches (Tim-Tim et al., 2009, Morales-Caselles et al., 2008, Sureda et al., 2011), but also by the use of field manipulative experiments (Marques et al., 2014). Consequently, little is known of how repeated oil spills at varying frequencies and intensities can affect biomarkers responses, especially in the field.
In this study, we examined the effects of the frequency and intensity of experimental diesel spills on enzyme activities (SOD, CAT, GST and GPx), levels of reduced glutathione (GSH) and lipid peroxidation (LPO) in three macrofaunal species: the bivalve Anomalocardia brasiliana, the gastropod Neritina virginea and the polychaete Laeonereis culveri (formerly identified as Laeonereis acuta). These species were chosen because they are adapted to stressful environmental conditions, relatively sessile, widely distributed and occupy different trophic levels. A. brasiliana is a filter feeder that feeds mostly on plankton; N. virginea is a grazer that feeds mainly on epiphytic algae, and L. culveri is a deposit feeder that forages within the sediment column.
By comparing the effects of three frequencies of exposure events against two dosages of oil in a factorial experiment with asymmetrical controls, we tested the following hypotheses: 1) if selected biomarkers are affected by repeated oil spill events, then biomarker responses in organisms exposed to frequent spills will be significantly different from those in the control treatment; 2) if different exposure regimes are determinant causes of variability, then biomarker responses in organisms exposed to frequent low-dosage spills will be significantly different from those exposed to infrequent high-dosage spills; 3) if the time elapsed since the last oil spill is determinant, then biomarker responses in organisms exposed to the same dosage of oil under the same frequency, but for which the timing of exposure differed, will vary significantly.
Section snippets
Study area
Experimental oil spills were conducted on an intertidal flat at Papagaios Island in the polyhaline Cotinga sub-estuary (Fig. 1), a 20-km channel located in Paranaguá Bay (southern Brazil). Local tidal flats are mainly composed by moderately to well-sorted very fine sands (Souza et al., 2013) and are often covered with seaweeds such as Ulva and Enteromorpha (Ulvaceae, Chlorophyta) or diatom biofilms. The tidal regime is mainly semidiurnal, with diurnal inequalities, and may reach up to 1.7 m in
Biomarker responses in the bivalve A. brasiliana
The activities of SOD, GST and levels of LPO were significantly increased by frequent high-dosage oil spills (1d500) compared to the control treatment. Moreover, GSH concentration was significantly reduced in bivalves exposed to frequent high-dosage spills. However, CAT and GPx activities were not affected by this exposure regime (Control vs. 1d500 comparison in Table 2; Fig. 2).
The major differences in biomarker responses between treatments were caused by the frequency of oil spills (Table 2).
PAHs
The concentration of PAHs in the sediment indicated that oil-exposed plots were effectively contaminated by diesel, reaching levels close to those reported for polluted regions (Venturini et al., 2008, Martins et al., 2011). According to Notar et al. (2001) ΣPAH concentrations higher than 500 ng g−1 are indicative of highly contaminated sediments. This threshold value was exceeded in most of the impacted plots, particularly those exposed to high dosages of diesel. Moreover, none of the control
Conclusions
Experimental in situ simulations of oil exposure events with different frequencies and intensities provide a useful tool for detecting and quantifying environmental impacts. We have shown that non-enzymatic antioxidants such as glutathione, together with enzymatic antioxidants, biotransformation enzymes and lipid peroxidation in the bivalve A. brasiliana and the polychaete L. culveri are suitable biomarkers of petroleum pollution. When exposed to the same overall diesel release, but at distinct
Acknowledgements
Our special thanks to many friends and colleagues for their assistance in fieldwork. We are also grateful to Adriana Sardi, Kristine Hopland, Júlia Bilibiu and Manuela Santana for their valuable help with tissue dissection. This research was funded by the Brazilian National Council for Scientific and Technological Development − CNPq (Proc. 475592/2012-3). L. Sandrini-Neto acknowledges a PhD fellowship from CNPq. L. Camus is funded by the Latin America program of the Norwegian Research Council
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