Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki

STOTEN-21650; No of Pages 9 Science of the Total Environment xxx (2017) xxx–xxx

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki Bruno Nunes b, Vera Silva a,b, Isabel Campos a,c, Joana Luísa Pereira b, Patrícia Pereira b,c, Jan Jacob Keizer a, Fernando Gonçalves b, Nelson Abrantes a,⁎ a b c

Department of Environment and Planning, CESAM, University of Aveiro, Portugal Department of Biology, CESAM, University of Aveiro, Portugal The Portuguese Sea and Atmosphere Institute, Portugal

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Toxic chemicals from wildfires can induce clear toxicity towards aquatic species. • Pro-oxidative modifications are the most relevant toxic effects. • Triggered biological responses are of defensive nature. • No neurotoxicity effects were perceived from the exposure to ashes. • Biomarkers are adequate to depict exposure of organisms to ashes from wildfires.

a r t i c l e

i n f o

Article history: Received 21 October 2016 Received in revised form 15 December 2016 Accepted 17 December 2016 Available online xxxx Editor: D. Barcelo Keywords: Wildfires Metals PAHs Oxidative stress Mosquitofish Short-term exposure

a b s t r a c t The number of wildfires has markedly increased in Mediterranean Europe, including in Portugal. Wildfires are environmentally concerning, not only due to the loss of biodiversity and forest area, but also as a consequence of environmental contamination by specific compounds including metals and polycyclic aromatic compounds (PAHs). These contaminants, mostly bound to ashes, can reach downstream water bodies, namely through surface runoff, being ultimately dispersed by vast areas and contacting with aquatic biota. Being toxicologically noteworthy, the potential toxic outcomes of the input of such chemicals across the aquatic compartment must be characterized. In this context, the present study used a biomarker-based approach to find early-warning signals of toxicity triggered by the exposure of the mosquitofish, Gambusia holbrooki, to affected aqueous runoff and stream water samples collected from a forest burnt area. The chemical analysis revealed concerning levels of metals and polycyclic aromatic hydrocarbons in both runoff and stream water samples. Biological responses elicited by the collected samples showed the occurrence of pro-oxidative modifications, specifically driven by enzymatic forms involved in the metabolism of glutathione. Despite these effects, no further signs of involvement of metals and PAHs were elicited in terms of neurotoxicity. The overall set of data implicates chemicals resulting from wildfires in clear deleterious effects in exposed fish. © 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author. E-mail address: [email protected] (N. Abrantes).

http://dx.doi.org/10.1016/j.scitotenv.2016.12.129 0048-9697/© 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129

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B. Nunes et al. / Science of the Total Environment xxx (2017) xxx–xxx

1. Introduction Wildfires are becoming a major environmental problem in Mediterranean countries of southern-Europe, since they occur with a high frequency and affect large areas (European Commision, 2015). In Portugal, this scenario is likely to become increasingly frequent and severe, not just because of the foreseen increase in fire-propitious meteorological conditions related to climate change (IPCC, 2013) but also due to other causes, in part of a societal nature, reflecting rural depopulation, land abandonment and afforestation with fire-prone species (Carmo et al., 2011; Moreira et al., 2011, 2009). The environmental impacts of wildfires constitute a major concern because of their adverse and diverse effects on different compartments (Lavorel et al., 1998; Moreira et al., 2010). Among direct and indirect impacts promoted by wildfires, their role on the production, mobilization and dispersion of contaminants has been increasingly receiving research attention, in particular in what concerns polycyclic aromatic hydrocarbons (PAHs) and metals (Campos et al., 2016, 2015, 2012; Olivella et al., 2006; Schäfer et al., 2010; Smith et al., 2011; Vila-Escalé et al., 2007). Both PAHs and metals are contaminants of major biological concern, due to their high toxicity, environmental persistence, moderate to high water solubility, and tendency to bioaccumulate. Hence, their input into aquatic systems, especially by surface runoff, could lead to serious and complex impacts on water quality, with ultimate effects on aquatic biota. Several studies reported damaging effects of wildfires on aquatic communities, including periphyton and phytoplankton (Charette and Prepas, 2003; Cowell et al., 2006; Earl and Blinn, 2003; Planas et al., 2000), macroinvertebrates (Earl and Blinn, 2003; Hall and Lombardozzi, 2008; Mellon et al., 2008; Minshall, 2003; Scrimgeour et al., 2001), amphibians (Pilliod et al., 2003), and fish (Rinne and Neary, 1996; Spencer et al., 2003). Although almost all of these studies evaluated the impact of wildfires on communities' structure or functioning, direct cause-effect relationships putatively involved were hardly established. Thus, the potential ecotoxicological effects derived from the input of PAHs and metals from burnt areas has been somewhat disregarded. First steps to overcome this research gap were given by Campos et al. (2012) and Silva et al. (2015), whose studies demonstrated that compounds released or mobilized by wildfires were capable of exerting significant toxicity to standard aquatic organisms, such as the bacteria Vibrio fischeri (Beijerinck 1889) Lehmann and Neumann 1896, the microalgae Raphidocelis subcapitata (Korshikov) Nygaard, Komárek, J. Kristiansen & O.M. Skulberg 1987, and the macrophyte Lemna minor Linnaeus 1753. By contrast, no significant effects were found towards the cladoceran Daphnia magna Straus 1820. These apparently paradoxical results illustrate the difficulty of analyzing ecotoxicological effects of wildfires; in fact, and according to Campos et al. (2012), the composition of surface runoffs from wildfires (and the consequent toxic effects elicited by PAHs) is highly variable along time. Despite these efforts, a significant number of questions still remain unaddressed. Considering that some of these compounds entering aquatic ecosystems are bioavailable and can bioaccumulate in aquatic organisms (e.g. Musa Bandowe et al., 2014; Zhao et al., 2014; Srogi, 2007), their effects on organisms from higher trophic levels, such as fish, should not be neglected. In this respect, since planktivorous fish can be found everywhere and play a key role in the aquatic food web, allowing the transference of energy from lower to high trophic levels, they are considered of high ecological relevance (e.g., Hurlbert and Mulla, 1981; Van der Oost et al., 2003). Consequently, such fish species have been the object of numerous studies assessing the harmful responses to environmental toxicants (Van der Oost et al., 2003). Among several sub-lethal parameters, the study of biochemical responses are useful tools to assess potential impairments driven by toxicants on aquatic species; in particular, biomarkers of oxidative stress are important, by providing early-warning signals, related to the imbalance between pro-oxidant effects and the antioxidant intracellular

defense mechanisms of most organisms. In regard to the chemicals produced or mobilized due to wildfires (e.g. metals and PAHs) they are known to produce cytotoxic effects in rodents, by increasing oxidative stress in macrophages (Franzi et al., 2011; Wegesser et al., 2010; Williams et al., 2013), but also in humans (Nakayama Wong et al., 2011). Oxidative stress is a common outcome of metal exposure since the uptake and accumulation of metals causes an increase in highly reactive oxygen species (ROS), such as the superoxide radical, hydrogen peroxide, and the hydroxyl radical, responsible for triggering oxidative stress in fish (Atli et al., 2006; Dautremepuits et al., 2002; Radi and Matkovics, 1988; Roche and Boge, 1993). Moreover, metals are known to exert potential deleterious effects in terms of neurotoxicity. In fact, some metals possess anticholinesterasic activity (Nunes, 2011), consequently compromising neurotransmission in exposed organisms. Likewise, PAHs are also capable of interfering with these enzymes, contributing for their inhibition (Kang and Fang, 1997). Bearing this in mind, the present study aimed to evaluate the effects of exposure to surface runoff and stream water from forest burnt areas, through the quantification of biochemical responses, such as oxidative stress, but also neurotoxicity, on the freshwater fish Gambusia holbrooki Girard 1859. The selected biochemical parameters were the activities of the antioxidant enzymes glutathione-S-transferases (GSTs), catalase (CAT), glutathione reductase (GRed), and glutathione peroxidase (GPx). The extent of lipoperoxidative damage was also monitored by quantifying the amount of malondialdehyde (MDA) by the TBARS assay. Neurotoxicity was assessed through the quantification of acetylcholinesterase (AChE) activity. 2. Material and methods 2.1. Study area and samples collection Runoff and stream water samples were collected from a recently burnt catchment located in north-central Portugal, near the parish of Talhadas, Aveiro District (N 40° 39′ 54″, W 8° 21′ 47″). The selected area was burnt by a wildfire in July 2013, affecting an area of 815 ha predominantly covered by eucalypt (Eucalyptus globulus) and maritime pine (Pinus pinaster) forest plantations. According to the available wildfire history database, there were no occurrences between 2001 and 2013 in the selected catchment. Two distinct water samples were collected for analysis and testing. One, hereinafter designated StrW, corresponded to surface water collected in a permanent stream within the burnt area. This stream was equipped with a hydrometric station and an automatic sampler triggered by a water level sensor allowing the collection of water samples into 24 1-L bottles as the water level rises following a rainfall event. The water collected in the bottles was pooled to yield the composite sample used throughout the present study. A second sample, hereinafter designated RunEH, corresponded to surface runoff collected through slope-scale plots from an area of eucalypt plantation, where the wildfire was considered to be of high severity according to the methodology described in Shakesby and Doerr (2006), Keizer et al. (2008) and Keeley (2009). Briefly, the surface runoff was collected into three 500-L tanks installed at the bottom of the burnt slope following the same rainfall event affecting the surface water stream (see Machado et al. (2015) for more details on the slope-scale set-up). The runoff from all tanks was pooled into a composite sample representing the RunEH sample. All sampling efforts were conducted in October 2013 following the first post-fire rainfall events. 2.2. Chemical determinations PAHs and metals contents were analyzed in both water samples (StrW and RunEH). The samples were filtered in order to analyze the dissolved and particulate fractions of PAHs and metals separately. Each sample was filtered in triplicate.

Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129

B. Nunes et al. / Science of the Total Environment xxx (2017) xxx–xxx

The PAHs included in this study were restricted to fifteen compounds that were identified as priority contaminants by the United States Environmental Protection Agency (USEPA; ATSDR, 1995): acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), pyrene (PYR), benzo(a)anthracene (BaA), chrysene (CHR), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), dibenzo(a,h)anthracene (DBA), indeno(1,2,3-cd)pyrene (IND) and benzo(g,h,i)perylene (BGP). The samples were filtered (1000 mL) using glass fiber filters (1.2 μm) to analyze the dissolved (eluate) and particulate (filter) PAHs levels. The dissolved PAHs levels of the filtered samples were analyzed using solid phase extraction (SPE), which was conducted in an SPE vacuum six-port SPE manifold using Bakerbond speedisk H2O-Phobic DV B extraction disk from J. T. Baker (Avantor Performance Materials, USA), according to the established procedures (Munch et al., 2012). The PAHs extracts were analyzed by using a gas chromatography–mass spectrometry (GC–MS) system (Thermo® DSQ) following the conditions described by Martins et al. (2008). The particulate PAHs levels were analyzed using pressurized liquid extraction (PLE). Before the extraction, the glass fiber filters (1.2 μm) were dried at 40 °C until constant weight. The PLE was performed using an ASE (Accelerated Solvent Extraction) 200 system (Dionex, USA) as described by Martins et al. (2012). The concentrations of PAHs were determined using the same analytical procedure as described previously. Total PAHs concentrations represent the sum of the dissolved and the particulate PAHs for each sample. The analyzed metals (V, Mn, Co, Ni, Cu, As, Cd and Pb) were chosen as target analytes. Cd, Ni and Pb are included in the EU Directive, 2008/ 105/EC as priority substances or substances of concern in the field of water policy, and along with As, they are concerning given that they may exert high environmental toxicity; on the other hand, Co, Cu, Mn and V are essential trace metals trace metals (Walker et al., 2001). The rationale for metals selection also considers previous studies denoting their relevance as surface water contaminants originating in wildfires (e.g. Campos et al., 2012; Silva et al., 2015). For the particulate metal levels, the sample was filtered through a 0.45 μm polycarbonate filter. The suspended particulate matter retained in the filter (dried at 40 °C) was totally digested with an acid mixture (Aqua regia and HF) following the procedure described by Caetano et al. (2007). After the acid digestion phase, metals were analyzed by ICP-MS (Inductively Coupled Plasma–Mass spectrometry; Thermo Elemental, X-series). The aqueous filtered samples, which correspond to the dissolved metal fraction, were preserved with double-distilled HNO3 to pH b 1.5 and were analyzed directly by the ICP-MS, as previously described. Total concentrations for each individual metal represent the sum of the dissolved and the particulate phase.

2.3. Test organisms G. holbrooki, commonly designated as mosquitofish, is a euryhaline organism widely distributed in both freshwater systems and estuaries of temperate regions. G. holbrooki possesses high fecundity and holds an intermediate position as a secondary consumer in the aquatic food web. Furthermore, this species is abundant, easy to catch and also easy to maintain in the laboratory. These characteristics suggest this organism as a suitable animal model in ecotoxicology (Nunes et al., 2004; Nunes et al., 2008). Since it is not native in Portugal, ethical issues related to the capture of these animals are negligible. Nevertheless, guidelines for the care and use of animals were followed and the experiments were previously approved and authorized by the institutional and national ethical commissions. Animals were handled and the experiments were carried out in compliance with ethical provisions on the welfare of experimental animals enforced by the European Union, by B Nunes, and JL Pereira, who hold appropriate FELASA certification. All

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efforts were made to avoid animal suffering and to minimize the number of specimens sacrificed and used in the experiments. G. holbrooki specimens were collected in Pateira de Fermentelos, a natural lake located in the center of Portugal. This lake is characterized by low levels of anthropogenic pollution (Ferreira et al., 2003). Fish were captured using hand nets and immediately transported to the laboratory. Only males and immature females, with 2.0 to 2.5 cm in length, were used in tests. Prior to being sent to the laboratory, unwanted organisms were immediately released where they were originally captured. Before to the toxicity tests, specimens were kept in plastic tanks for no b15 days, for acclimation. The tanks were supplied with dechlorinated tap water, and kept under continuous aeration and constant temperature (20 ± 1 °C). Inspections were conducted twice a day in order to discard diseased and dead specimens. After this period of quarantine, fish were maintained in tanks under laboratory-controlled conditions before being exposed to the samples collected in the burnt area. The specimens were fed daily with commercially available fish food (Sera Vipan® flakes) and the water was renewed once a week. 2.4. Exposure The experiment was performed according to the OECD guideline 203 for fish acute tests (OECD, 1992). Fish specimens were exposed in plastic bottles (polyethylene terephthalate previously rinsed with distilled water) to each original non diluted sample (StrW and RunEH; corresponding to 100%), to each sample diluted 50% in dechlorinated municipal water, and to a control treatment consisting of dechlorinated municipal water. This allowed covering both a low and high load regimes that are likely to occur under real environmental scenarios. The test containers were filled with 200 mL test solution, each holding 1 fish. Each treatment had 5 replicates, each composed by 10 fish randomly distributed from the maintenance aquaria into test containers at the beginning of the experiments. Fish were exposed to the test solutions for 96 h, in conditions (temperature, photoperiod and aeration) similar to those described above for the maintenance routine. Food was not provided during the exposure. Parameters such as mortality, pH, temperature, oxygen, and conductivity were measured daily during the experimental period; all measurements were within the validation ranges of the adopted testing guideline (pH 6–8.5; temperature 20 ± 2 °C; oxygen ≥ 60% of saturation levels). 2.5. Biochemical assays After the 96 h of exposure, fish were euthanized by rapid cooling by immersion in a tank containing ice-cold phosphate buffer (≤4 °C) until animals lost the ability to swim and lost the righting reflex and opercular movements. This procedure proved to be a rapid, effective, less distressful method, and showed no evidence of producing alterations in biomarkers (Wilson et al., 2009). The head, gills and liver were then removed, and homogenized in ice-cold phosphate buffer (50 mM, pH 7.0, with 0.1% Triton X-100) using a rotary tissue homogenizer at 14,000 rpm, for the ulterior determination of oxidative stress biomarkers. The homogenized tissues were centrifuged at 15,000g for ten minutes at 4 °C. Supernatants were divided into aliquots, which were used for the different biomarker determinations. Each homogenate sample was composed by 10 livers or 10 gills, accounting to one out of 5 replicates used within each treatment. Heads were processed similarly but homogenized individually in icecold phosphate buffer (0.1 M, pH 7.2); two heads were randomly collected out of the 10 available within each exposure replicate for analysis, this resulting in 10 replicated head records per treatment. Head homogenates were subsequently centrifuged at 6,000 rpm for 3 min at 4 °C. Each sample was aliquoted, and aliquots were stored at − 80 °C until the corresponding biochemical determinations.

Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129

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B. Nunes et al. / Science of the Total Environment xxx (2017) xxx–xxx

Enzymatic activity was determined spectrophotometrically (Thermo Scientific Multiskan EX spectrophotometer coupled to the dedicated Ascent Software 2.6) for glutathione-S-transferases (GSTs), glutathione peroxidase (GPx), catalase (CAT), and glutathione reductase (GRed) in liver and gills; acetylcholinesterase (AChE) activity in head tissues was also measured. GSTs catalyze the conjugation of the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione, forming a thioether that was followed based on the increment of absorbance at 340 nm, according to Habig et al. (1974). GSTs activity was expressed as nanomoles of thioether produced per minute, per milligram of protein. The activity of GRed was determined according to Carlberg and Mannervik (1985) by monitoring NADPH oxidation through the decrease in absorbance values at 340 nm, and was expressed as micromoles of NADPH oxidized per minute and per milligram of protein. CAT activity was assayed following Aebi (1984), based on the degradation rate of the substrate H2O2, monitored at 240 nm for 30 s. The results were expressed by considering that one unit of activity equals the number of moles of H2O2 degraded per minute, per milligram of protein. GPx may occur as selenium-dependent GPx (SeGPx), in which the selenium at the active site occurs in the form of selenocysteine. SeGPx have been recognized by its protective role against metal oxidative and genotoxic damage by catalyzing the reduction of both organic and inorganic peroxides like hydrogen peroxide (H2O2) (Almar et al., 1998; Chatziargyriou and Dailianis, 2010). Hence, besides the total GPx (tGPx), SeGPx was also measured. Both forms were quantified following Flohé and Günzler (1984), by measuring the consumption of NADPH at 340 nm, using cumene hydroperoxide or hydrogen peroxide as substrates; results were expressed as millimoles of NADPH per minute per milligram of protein. The extent of lipid peroxidation was measured through the quantification of thiobarbituric acid reactive substances (TBARS), according to Buege and Aust (1978). The reaction of malondialdehyde (MDA) - which is formed by degradation of initial products of free radical attack - with 2-thiobarbituric acid (TBA) was monitored spectrophotometrically at 535 nm. TBARS concentrations were expressed as MDA equivalents. AChE activity was determined according to Ellman et al. (1961), by following spectrophotometrically (412 nm) the conjugation of 5,5dithio-bis-(2-nitrobenzoic acid) (DTNB) with thiocholine resulting from the hydrolytic activity of AChE on the substrate acetylthiocholine. The activity of the enzyme was expressed as nanomoles of the complex formed per minute per milligram protein. Protein quantification of samples was determined according to the Bradford method (Bradford, 1976), adapted to microplate, in order to

express enzymatic activity or TBARS concentration relatively to the protein content of the analyzed tissues. 2.5.1. Statistical analysis One-way ANOVA followed by the Dunnett multi-comparisons test were performed to discriminate significant changes in the biochemical parameters following exposure to the samples (50% and 100% of StrW or RunEH) compared to the corresponding control. All statistical analyses were run in Minitab® 17 (Minitab Inc.), using a significance level set at 0.05. 3. Results 3.1. PAH and metal composition of stream water and surface runoff The total (∑15 PAHs) and individual PAH contents identified in the StrW and RunEH samples are presented in Table 1. Total PAHs concentration in StrW was twofold lower than in RunEH. Two compounds clearly dominated in both samples, PHE (45% and 54%, respectively) and FLU (25% and 30%, respectively). It is worth mentioning that PYR and ACY accounted for 7% of total PAHs in RunEH, and a similar relative amount was found for PYR in StrW. Four out of the fifteen PAHs, BkF, DBA, IND and BGP, were not detected. Regarding metal composition, lower concentrations of all studied metals were consistently found in StrW (Table 2). In StrW, Mn (55 μg l−1) and V (17 μg l−1) were present in the highest concentrations, followed by Pb and Ni that presented similar levels. In RunEH, the metals that attained the highest levels were Mn, V, Pb and Cu, with concentration values ranging from 109 μg l−1 (Cu) to 324 μg l−1 (Mn). Co and Cd presented the lowest concentrations. As and Co were found at similar levels (4 and 6 μ l−1, respectively) in StrW, while in RunEH the concentration of As was approximately fourfold higher than the concentration of Co. For both samples, Cd was the metal found at the lowest levels. 3.2. Biomarker responses of G. holbrooki Although toxic compounds were found in StrW and RunEH, only residual mortality was observed (with a maximum of 8% recorded in the 100% concentration of both samples), confirming these matrices as sublethal. Fig. 1 shows the effects of StrW and RunEH in the oxidative stress biomarkers assessed following exposure of G. holbrooki. As supplementary

Table 1 Mean PAHs concentrations ± standard deviation (n = 3; ng l−1) in surface water (StrW) and surface runoff (RunEH) samples collected in the burnt catchment. Environmental quality guidelines were added for comparison with the samples: Annual Average Concentration defined in the European Union Environmental Quality Standards (EU AA-EQS) under the Directive 2013/39/EU regarding priority substances in the field of water policy; United States Environmental Protection Agency Water Quality Standards (USEPA WQS), where the benchmarks available are either specific to freshwater continuous concentrations (Freshwater Chronic Values, FWC) or more general to Human Health, Water and Organisms (HHWO). Records marked bold within the samples denote levels above at least one of the guidelines. PAHs (ng l−1)

StrW

RunEH

EU AA-EQS

USEPA WQS

Acenaphthylene (ACY) Acenaphthene (ACE) Fluorene (FLU) Phenanthrene (PHE) Anthracene (ANT) Fluoranthene (FLT) Pyrene (PYR) Benzo(a)anthracene (BaA) Chrysene (CHR) Benzo(b)fluoranthene (BbF) Benzo(k)fluoranthene (BkF) Benzo(a)pyrene (BaP) Dibenzo(a,h)anthracene (DBA) Indeno(1,2,3-cd)pyrene (IND) Benzo(g,h,i)perylene (BGP) ∑15 PAHs

1.9 ± 0.35 1.9 ± 0.42 23.9 ± 8.31 42.8 ± 9.59 2.5 ± 0.82 1.4 ± 0.32 2.8 ± 0.09 0.39 ± 0.05 0.25 ± 0.02 0.45 ± 0.03 bDL 0.61 ± 0.01 bDL bDL bDL 78.9

10.4 ± 0.65 1.9 ± 0.21 38.2 ± 7.30 70.0 ± 20.00 7.1 ± 2.23 8.4 ± 3.33 10.9 ± 1.65 1.5 ± 0.32 0.38 ± 0.04 3.8 ± 0.54 bDL 1.7 ± 0.09 bDL bDL bDL 154

– – – – 100 6.3 – – – 0.17 0.17 0.17 – 0.17 0.17 n.a.

n. av. 520,000 FWC n. av. 6300 FWC 8,300,000 HHWO 130,000 HHWO 830,000 HHWO 38 HHWO 38 HHWO n. av. 38 HHWO 38 HHWO 38 HHWO 38 HHWO n. av. n.a.

bDL - below detection limit; n.a. – not applicable; n. av. – adequate benchmark is not available, although the PAH belongs to the USEPA list of priority pollutants.

Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129

B. Nunes et al. / Science of the Total Environment xxx (2017) xxx–xxx Table 2 Mean metal concentrations ± standard deviation (n = 3; μg l−1) in surface water (StrW) and surface runoff (RunEH) samples collected in in the burnt catchment. Environmental quality guidelines were added for comparison with the samples: Annual Average Concentration defined in the European Union Environmental Quality Standards (EU MAC-EQS) under the Directive 2013/39/EU regarding priority substances in the field of water policy; United States Environmental Protection Agency Aquatic Life Criteria (USEPA ALC) for surface freshwater bodies, where the criterion based on chronic exposure (Criterion Continuous Concentration) was privileged for presentation over the criterion based on acute exposure (Criterion Maximum Concentration) whenever available. Records marked bold within the samples denote levels above at least one of the guidelines. Metal (μg l−1)

StrW

RunEH

EU AA-EQS

USEPA ALC

Mn V Pb Cu Ni As Co Cd

55 ± 3.3 17 ± 1.5 11 ± 1.3 9 ± 0.5 12 ± 0.8 4 ± 0.5 6 ± 0.3 0.2 ± 0.01

324 ± 31.1 180 ± 5.5 128 ± 8.6 109 ± 9.0 48 ± 5.6 36 ± 2.3 10 ± 0.6 0.9 ± 0.04

– – 1.2 – 4 – – 0.08–1.5a

– – 2.5 – 52 360 – 3.9

a

Range dependent on water hardness.

information, the summary of the statistics used to confirm the significance of the effects can be found in Table S1. Catalase activity remained unchanged after exposure to both the StrW and the RunEH samples, for both liver and gill tissues. Effects of both samples in GSTs activity were negligible, except in gills following exposure to StrW. Neither StrW nor RunEH caused alterations in liver GRed activity, but gills had their GRed activity significantly decreased in a dose-dependent manner following exposure to both samples. Total GPx in liver and gills was kept unchanged after exposure to both samples, while Se-dependent GPx was significantly depressed in liver after exposure to both dilutions of StrW and RunEH, and in gills following exposure to full-strength (100%) StrW. Lipoperoxidation was largely unaffected by StrW and RunEH, as interpreted from the low changes in TBARs levels compared to the control. StrW and RunEH seem to induce no neurotoxicity provided that no significant modification of cholinesterasic activity was found following exposure (Fig. 2).

4. Discussion Results concerning the chemical analysis clearly demonstrated that surface runoff (RunEH) was much richer in terms of PAHs and metals than stream water (StrW) samples. In fact, this trend was observed for all PAHs, except acenaphthene, and for all metals. Although PAH burden allowed distinguishing the samples, only FLT, BbF and BaP leveled above environmental quality guidelines used by the EU (see Table 1). Still, it is worth noticing the scarcity of specific benchmarks targeted at the protection of freshwater ecosystems. Annual average benchmark concentrations are provided by the EU only for 7 out of the 15 quantified PAHs, and safety benchmarks specifically regarding the freshwater biota are provided by USEPA for 2 out of the 15 quantified PAHs. Although this scarcity in dedicated benchmarks also applies to metals, which is likely due to the restriction of the classification as priority pollutants of Cd, Ni and Pb (added As by the USEPA), metal levels in the samples were markedly above the available safety benchmarks (Table 2). Some metallic species are particularly noteworthy. Waterborne metals such as As, Cd, Pb, Hg, Cr, Ni, Mn and Fe can increase the production of reactive oxygen species (Jadhav et al., 2007), with some of these chemicals being present in high levels in StrW, but specially in RunEH. This supports the oxidative-related response observed in the present study, specifically reflected by decreased gills GRed activity elicited by exposure to both samples. Other relevant oxidative alterations in this sense include significant reductions of hepatic SeGPx activity, noticed after exposure to both samples.

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The observed changes in enzyme activities suggest overproduction of ROS and the consequent establishment of pro-oxidative conditions. However, the effects of complex mixtures of metals in fish are not always straightforward to interpret. For example, some metals tend to favor an increase of catalase activity (Hansen et al., 2007; Radwan et al., 2010) while others, including Cu and Zn, cause significant inhibitions of this enzyme's activity (Ahmad et al., 2005; Atli et al., 2006; Loro et al., 2012). Also, the oxidative stress response can be binary, with low metal levels inducing enzymatic activity, while higher metal levels not interfering at all or driving the opposite effect (Zheng et al., 2016). Despite the apparent inconsistencies, a large set of data from the literature reinforces the involvement of a pro-oxidative pathway of metal intoxication, as suggested by our results, namely through the involvement of both forms of glutathione peroxidase (total and selenium-dependent). The role of antioxidant enzymes, including glutathione peroxidase, in metallic intoxication of fish is not new; the review by Srikanth et al. (2013) shows the central activity of glutathione metabolism (and enzymes involved in such pathway) in the prevention of metal-driven toxic effects. The study by Eroglu et al. (2015) evidenced the effects of several metallic species (namely copper, cadmium, chromium, lead, and zinc) on fish glutathione metabolism. It was made clear that only specific metals were capable of altering particular enzymes; exposure to cadmium, for instance, resulted in significant increases in glutathione peroxidase activity. Furthermore, exposure to metallic contaminants was responsible for a significant decrease of glutathione peroxidase activity in the fish species Notopterus notopterus, as demonstrated by Mohanty and Samanta (2016), a result that corroborates our findings. Considering the overall complexity of the StrW and RunEH matrices as to metal composition, it is very difficult to pinpoint an individual metal that can be held responsible for the observed prooxidative effects. However, its occurrence is undisputable, and the involvement of liver (where the stronger modifications were reported) in this response clearly indicates that oxidative-based alterations took place. Being the main organ of xenobiotic metabolism in fish, the liver is likely to be more impacted by oxidative effects, thus reflecting adaptive responses including effects on enzymes involved in antioxidant defenses. Biotransformation is a common requisite for the transformation of hydrocarbon compounds into more toxic metabolites, including reactive intermediates with oxidant nature. The metabolism of PAHs is usually undertaken by the activity of cytochrome P450, often resulting in the production of ROS, including the superoxide radical (Stegeman and Hahn, 1994; Livingstone, 2003; Schlezinger et al., 2006). This highly reactive and unstable intermediate contributes also for the establishment for pro-oxidative conditions, triggering the liberation of other intermediates (Borg and Schaich, 1984; Hermes-Lima, 2004), with putative consequences at the level of the enzymatic antioxidant system. By increasing the in vivo activation of PAHs into more toxic intermediates, it is likely that aquatic organisms may need to cope with oxidative aggression, by enhancing the efficacy of its anti-oxidant defenses (Balk et al., 2011). Nevertheless, definitive trends in the response of antioxidant systems to PAH exposure have been difficult to establish. Some antioxidant enzymes may suffer initial inductions during the time course of the intoxication, while others remain at low levels and only respond after long periods of exposure. Sun et al. (2006) exposed the goldfish Carassius auratus (Linnaeus, 1758) to phenanthrene, and found increased activities of antioxidant enzymes, showing their involvement in the defense against the pro-oxidative effect of the PAHs. On the opposite, fish from extremely polluted areas (by metals and PAHs) can show lower levels of antioxidant enzymes, compared to fish from less polluted sites, while specimens collected in intermediately polluted areas can show higher levels of antioxidant enzymes compared to those from reference sites (Dupuy et al., 2015). Gene expression patterns under exposure can also confound the interpretation of the antioxidant response to PAHs. As shown by Koenig et al. (2013), genes involved in the

Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129

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Fig. 1. Effects of short-term exposure of Gambusia holbrooki to surface water (StrW) and surface runoff (RunEH) in oxidative stress biomarkers (activity of catalase; glutathione-Stransferases, GSTs; glutathione reductase, GRed; total and selenium-dependent glutathione peroxidase, tGPx and Se-GPx) and in TBARS concentration as an oxidative damage biomarker, measured in gills (black and dark grey marks) and liver (white and light grey marks) tissue. The marks represent the mean of five replicates and error bars represent the standard error. The dotted line was added to facilitate trends interpretation.* denotes significant differences relative to the control (p b 0.05).

Fig. 2. Effects of short-term exposure of Gambusia holbrooki to surface water (StrW) and surface runoff (RunEH) in soluble acetylcholinesterase (AChE) activity measured in head tissues. The marks represent the mean of ten replicates and error bars represent the standard error. The dotted line was added to facilitate trends interpretation.

Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129

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biosynthesis of antioxidant enzymes can be up-regulated following PAH exposure, with no concomitant increase in corresponding enzymatic activities. The absence of a common trend relating PAH contamination with enzymatic effects act as a serious confounding factor when interpreting effects of highly complex matrices, such as the one here tested. Moreover, the combination of oxidative stress effects elicited by metals and PAHs has not been commonly assessed. Vega-López et al. (2013) could not establish clear-cut patterns for the oxidative response of freshwater phytoplankton to PAHs, which is consistent with the outcome of the present study. Despite the establishment of significant oxidative-related alterations, no oxidative damage occurred. Exposure to common aquatic environmental pollutants (including metals and PAHs) often leads to oxidative stress and consequent peroxidative alterations (Van der Oost et al., 2003), translating into noteworthy cellular damage. In the present study, no modifications were registered in TBARS levels, suggesting the implementation of compensatory mechanisms for successfully coping with the chemical insult. Given the observed alteration in enzymes involved in the homeostasis of glutathione (namely, GSTs, GRed, and GPx), it is reasonable to hypothesize that oxidative damage was prevented with the direct or indirect involvement of glutathione. The absence of anticholinesterasic effects after exposure to both samples suggests that metal and PAH exposure levels were not high enough to trigger neurotoxic effects. Previous studies showed that exposure of fish to a combination of metals and PAHs can result in cholinesterasic inhibition, mainly due to the contribution of metals such as Pb, Cd and Cu (Oliva et al., 2012), whose levels were especially high in RunEH. Increased hydrolytic activity of fish cholinesterases following exposure to Cu was also described by Romani et al. (2003). Despite the evidences involving some metals in cholinesterasic inhibition, this relationship is still matter of debate, with the characteristics of exposure (duration, levels) and the test organism used being important sources of variation. For example, Brandão et al. (2013) showed no inhibitory role of Cu, Zn, Pb and Cd towards cholinesterases of G. holbrooki. In fact, the mechanisms ruling the interaction between different metals and acetylcholinesterase is still not entirely known (Frasco et al., 2005), which may lead to ambiguous or erroneous interpretation (Nunes, 2011). A final note is worth making on the significant increase of GSTs activity observed in gills of fish exposed to StrW; a similar pattern was apparent for fish exposed to RunEH, but it was not statistically confirmed. In fact, all pollutants quantified in RunEH were also present in StrW, but in markedly lower amounts. This may have constrained the interplay between larger oxidative alterations (as observed for fish exposed to RunEH) and increased efficacy of conjugation with glutathione reflected by higher GSTs activity (as observed for fish exposed to StrW). This particular GSTs response following exposure to the least pollutant burdened StrW sample can thus reflect increased detoxification capacity through toxicant conjugation with glutathione, rather than representing a pro-oxidative deleterious effect. Aromatic hydrocarbons metabolism is often complemented by inductions of GSTs activities in aquatic organisms (Van der Oost et al., 2003; Martínez-Gómez et al., 2009), and a similar trend has already been reported for fish inhabiting metal-contaminated areas (Lenartova et al., 1997; Nunes et al., 2014). 5. Conclusions This study highlighted that the mobilization of toxic chemicals produced by wildfires is not innocuous to aquatic species. Pro-oxidative modifications were the most relevant toxic effects observed. Additionally, metabolic alterations related with toxicant conjugation with glutathione were affected by the exposure to contaminated surface water and runoff, evidencing the defensive nature of triggered biological responses. Contrasting, no neurotoxicity effects were noticed. The responsiveness of G. holbrooki as a test species is also noteworthy, since realistic conditions of exposure resulted in significant modifications in

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several oxidative stress biomarkers, namely in the activity of GSTs, GRed and GPx. This study further indicates that the use of a biomarker-based approach may provide early-warning signals depicting exposure of aquatic species to toxicants originating in wildfires.

Acknowledgements This study was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) under the scope of the FIRETOX project (PTDC/AAG-GLO/4176/2012). Thanks are also due, for the financial support to CESAM (UID/AMB/ 50017), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. Bruno Nunes and Nelson Abrantes were hired through the Investigator FCT program (IF/01744/2013 and IF/01198/2014, respectively). Isabel Campos, Joana Luísa Pereira and Patrícia Pereira were recipients of grants from the FCT (SFRH/BD/62327/2009, SFRH/BPD/101971/2014 and SFRH/BPD/69563/2010, respectively). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.12.129.

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Please cite this article as: Nunes, B., et al., Off-site impacts of wildfires on aquatic systems — Biomarker responses of the mosquitofish Gambusia holbrooki, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2016.12.129