Biochemical and physiological alterations induced in Diopatra neapolitana after a long-term exposure to Arsenic

Biochemical and physiological alterations induced in Diopatra neapolitana after a long-term exposure to Arsenic

Comparative Biochemistry and Physiology, Part C 189 (2016) 1–9 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Pa...

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Comparative Biochemistry and Physiology, Part C 189 (2016) 1–9

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc

Biochemical and physiological alterations induced in Diopatra neapolitana after a long-term exposure to Arsenic Francesca Coppola a, Adília Pires a, Cátia Velez a, Amadeu M.V.M. Soares a, Eduarda Pereira b, Etelvina Figueira a, Rosa Freitas a,⁎ a b

Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal Department of Chemistry & CESAM, University of Aveiro, Aveiro, Portugal

a r t i c l e

i n f o

Article history: Received 5 April 2016 Received in revised form 26 May 2016 Accepted 7 June 2016 Available online 25 June 2016 Keywords: Oxidative stress Biomarkers Arsenic Regenerative capacity Polychaete Sediment

a b s t r a c t Several authors identified polychaetes as a group of marine invertebrates that respond rapidly to anthropogenic stressors. Furthermore, several studies have demonstrated that environmental pollution lead to the impoverishment of benthic communities with species replacement and biodiversity loss, but very few studies have investigated biochemical and physiological alterations that species undergo in response to Arsenic (As) exposure. Therefore, the present study assessed the toxicity induced in the polychaete Diopatra neapolitana after a longterm (28 days) exposure to different As concentrations (0.0, 0.05, 0.25 and 1.25 mg/L). For this biochemical and physiological alterations were evaluated. Biochemical analysis included the measurement of different biomarkers such as glutathione S-transferase (GST), lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and oxidized glutathione (GSSG) were assessed in order to evaluate oxidative stress. Physiological analyzes included the observation of polychaetes regenerative capacity and the quantification of organisms total protein (PROT) and glycogen (GLY) content. The results obtained allowed to confirm the suitability of these biomarkers to identify the toxicity caused by As and moreover revealed that D. neapolitana is a good bioindicator of As pollution. © 2016 Elsevier Inc. All rights reserved.

1. Introduction All over the world coastal ecosystems have been focus of study as these areas are particularly productive but also exposed to the influence of both anthropogenic (including fisheries and industrial waste) and natural (namely erosion, volcanic eruptions, floods) contamination sources (Fossi et al., 2012; Suriya et al., 2012). In fact, over the last decades, numerous studies have been devoted to identify contamination levels, namely from inorganic contaminants in sediments and water from coastal areas, including lagoons and estuaries (Karadede and Ünlü, 2000; Mamindy-Pajany et al., 2013; Pereira et al., 2009; Seixas et al., 2005; Velez et al., 2015). Arsenic (As) is among the most common inorganic contaminants and often occurs from natural sources in the earth crust, soil, sediment, water, air and living organisms (Mandal and Suzuki, 2002). Arsenic is incorporated in N200 different minerals, in 60% of which in the form of arsenates, 20% as sulphides and sulphosalts and the remaining 20% in the form of arsenides, arsenites, oxides, silicates and elemental As (Onishi, 1969). However, as a result of human activities, such as mining, metal smelting, fossil fuel ⁎ Corresponding author at: Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. E-mail address: [email protected] (R. Freitas).

http://dx.doi.org/10.1016/j.cbpc.2016.06.006 1532-0456/© 2016 Elsevier Inc. All rights reserved.

combustion and pesticides use, As may occur in high concentrations in certain areas (Fan et al., 2008; Ko et al., 2009; Ventura-Lima et al., 2011; Xu et al., 1991). Smedley and Kinniburgh (2001) presented an overview of As concentration range in the worlds natural waters, showing that As concentrations may be found from b0.5 μg/L to N5000 μg/L. Different studies showed that As concentrations in sediments of contaminated areas may vary from 25 mg/kg to 50 mg/kg in French Mediterranean ports (Mamindy-Pajany et al., 2013) to N 200 mg/L in Indian rivers (McArthur et al., 2001). In a low contaminated system (Ria de Aveiro, Portugal) As was present in concentrations between 1.06 and 6.14 mg/kg (Freitas et al., 2012a). Besides the concern on sediments contamination levels, the interest on the relationship between environmental pollution and the impacts induced on aquatic organisms has increased in the last decades, including studies assessing the effects of contaminants on benthic macrofauna. In fact, to evaluate the environmental conditions and the effects of pollutants, especially in sediments, different benthic species have been used as sentinel and/or bioindicators (Blaise et al., 2013; Carregosa et al., 2014a; Freitas et al., 2014a, 2014b; 2012a,b; Sizmur et al., 2013; Velez et al., 2015). Among benthic communities polychaetes are an important component due to their high species richness, high biomass and density, important role in trophic chains, and high level of tolerance to adverse conditions (pollution and natural disturbance) (In-Young

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et al., 1995; Rodrigues et al., 2011). Several studies, both conducted in the environment and under laboratory conditions, have demonstrated the ability of this group of organisms to accumulate and respond to different contamination types and levels, and biochemical markers have been commonly used as early warning indicators of stressful conditions (e.g., Durou et al., 2007; Fattorini et al., 2005; Freitas et al., 2012a,b; 2015a,b; Maranho et al., 2014; Solé et al., 2009; Ventura-Lima et al., 2007). Among others, environmental studies conducted in the Mediterranean region by Bocchetti et al. (2004) demonstrated that contamination by metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) negatively affected metabolism and detoxification mechanisms of the polychaete Sabella spallanzanii. Also Pook et al. (2009) established that a metabolic cost is associated with the resistance to metals (Zn and Cu) toxicity, exhibited by Hediste diversicolor inhabiting Restronguet Creek (UK). Freitas et al. (2012a, 2012b) studied the accumulation of various metals (Cu, Pb, Cr, Hg, Cd, Mg, Mo and Ni) in Diopatra neapolitana, along the Ria de Aveiro (Portugal), demonstrating the capacity of this species to accumulate high concentrations of these elements, leading to cellular damage and increased antioxidant and biotransformation enzymes activity. Under laboratory conditions Yuan et al. (2010) exposed Perinereis aibuhitensis to sublethal Cd concentrations under a shortterm experiment (1–8 d) and suggested that Cd interfered with the antioxidant defense system of this polychaete species. Recently Bouraoui et al. (2015) showed the induction of oxidative stress biomarkers in different body regions of the polychaete Hediste diversicolor exposed to Cu. In parallel to biochemical alterations, the exposure of polychaetes to pollutants may affect their physiological performance, including their regenerative capacity (Freitas et al., 2015a, 2015c). Almost all polychaetes can regenerate parts of their body (Bely, 2006; Pires et al., 2012a), a capacity that has been recently used to evaluate the impacts of different stressors on polychaetes (Carregosa et al., 2014a; Freitas et al., 2015a, 2015b, 2015c; Nusetti et al., 2005). In this way, the present study aimed to assess the toxicity induced in the polychaete Diopatra neapolitana after a long-term (28 days) exposure to different As concentrations. For this, biochemical alterations caused by this metalloid were evaluated by measure of oxidative stress (Lipid peroxidation, Reduced and Oxidized Glutathione, Catalase, Superoxide dismutase, Glutathione-S-Transferases), and energy related (Protein and Glycogen content) biomarkers. Physiological impacts were also evaluated by studying alterations induced on the regenerative capacity of this species. 2. Material and methods 2.1. Experimental setup Diopatra neapolitana specimens were collected in the Mira channel, Ria de Aveiro (Portugal), an area considered as non-contaminated (Freitas et al., 2014b). Sampling was done in November to avoid the reproductive period of the species (Pires et al., 2012b). Organisms that were regenerating in the field were discarded and not used in this study. In order to reduce individuals contamination levels, specimens were maintained under laboratory controlled conditions during two months in aquaria, with artificial seawater (salinity 28) and a mixture of sediment from the sampling site (3:1, water:sediment), continuous aeration, temperature of 20 ± 1 °C and photoperiod of 12:12 h (light/dark). During this period water was renewed every week. After acclimation organisms were pushed out from their tubes and anaesthetized with a solution of 4% MgCl2·6H2O during 15 min. Under a stereomicroscope these individuals were amputated at segment 60 (Pires et al., 2012a). For each individual, the weight and width of the 10th chaetiger was recorded. Individuals were exposed during 28 days to 4 conditions: a) 0 mg/L As (control); b) 0.05 mg/L As; c) 0.25 mg/L As and d) 1.25 mg/L As. For each condition 3 containers were used, each one with 3 individuals. Since most of environmental As is present in its pentavalent form

(Domingo, 1995) in the present study we used sodium arsenate (Na2HAs4) as source of As. Because of its electrochemical charge, Arsenate has a stronger capacity to sorb to surfaces and is therefore less mobile than the reduced form (arsenite). For this reason arsenite has been considered to be more toxic when compared with arsenate (Singh et al., 2011). The exposure procedure was based on an adapted version of the ASTM E1562–00 (2013) - Standard Guide for Conducting Acute, Chronic, and Life-Cycle Aquatic Toxicity Tests with Polychaetes, Annelids. During exposure aeration was maintained, salinity was kept at 28, temperature at 20 ± 1 °C and photoperiod of 12:12 h (light/dark). During exposure, to assess the regenerative ability of D. neapolitana under different As concentrations, 3 organisms per condition were analyzed every week to measure the percentage of the regenerated body part (width) and the number of new chaetigers. For this, polychaetes were removed from their tubes and anaesthetized with 4% MgCl2·6H2O solution during 15 min, and the state of regeneration was observed and photographed under a stereomicroscope. The size of the regenerated body part was measured and the number of regenerating segments was counted which were identified by lighter color and narrower regenerating chaetigers compared to the rest of the body. During exposure dead organisms were removed from the containers. After exposure surviving individuals were frozen at −80 °C to evaluate biochemical alterations induced by each condition. Every week during exposure, the water was changed and As concentrations re-established. Organisms were fed ad libitum with frozen cockles every 2–3 days both during the acclimation and exposure periods. 2.2. Laboratory analysis In order to perform all biochemical analysis, each organisms frozen tissues were individually pulverized in a mill, with liquid nitrogen. The pulverized tissue was distributed into 0.2 g aliquots, which were used for As quantification and biochemical analysis. 2.2.1. As quantification in D. neapolitana Arsenic was quantified in D. neapolitana soluble and insoluble fractions of each individual. For this, samples were extracted with Tris 20 mM buffer (pH 7.6), sonicated at 4 °C during 15 s and centrifuged at 1450 g, for 15 min at 4 °C. This process originates two distinct subcellular fractions: the supernatant represents the soluble fraction and the pellet represents the insoluble fraction. The As concentration obtained in the soluble fraction can be defined as the element concentration in its free form or bound to proteins present in the cytosol, whereas As concentrations contained in the insoluble fraction can be defined as the unavailable element concentration, precipitated in insoluble metal-rich granules and cellular debris (Wallace et al., 2003; Wallace and Luoma, 2003). These fractions were separated and transferred to Teflon bombs. Samples were digested overnight at 115 °C with 2 mL of HCl:HNO3 (3v:1v). The concentration of As was quantified by ICPMS (Inductively Coupled Plasma-Mass Spectrometry) by a certified laboratory. Calibration curve was obtained through IV-ICPMS 71 A standard and verified with standard reference material (National Institute of Standards and Technology, NIST SRM 1643e), calculated measurements trueness over 90%. The results were expressed in μg per g of fresh weight (FW). To evaluate the ability of D. neapolitana to bioaccumulate As, under experimental conditions, the bioconcentration factor (BCF) was calculated for each condition corresponding to the ratio between total As concentration in the organisms and As nominal exposure concentration (McGeer et al., 2003). 2.2.2. Biochemical parameters For each biochemical analysis and from each individual 0.2 g of soft tissue was used. Extractions were performed with specific buffers for each parameter. For this, samples were sonicated for 15 s at 4 °C and

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centrifuged (10,000 g) for 15 min at 4 °C. Supernatants were stored at −80 °C or directly used to measure: i) non-enzymatic markers of oxidative stress: Lipid peroxidation, reduced and oxidized Glutathione ratio, GSH/GSSG; ii) enzymatic markers of oxidative stress: Superoxide dismutase, Catalase, Glutathione S-transferases; iii) energy related markers: glycogen and total protein content. 2.2.2.1. Non-enzymatic markers of oxidative stress. For Lipid Peroxidation (LPO) supernatants were extracted using 20% (v/v) trichloroacetic acid (TCA). LPO was measured by the quantification of malondialdehyde (MDA) equivalents, a by-product of lipid peroxidation, according to the method described by Ohkawa et al. (1979) and the modifications referred by Carregosa et al. (2014b). Absorbance was read at 532 nm (ε = 156 mM−1·cm−1). LPO levels were expressed in nmol of MDA formed per g of FW. Reduced (GSH) and oxidized (GSSG) Glutathione were determined using 0.6% sulfosalicylic acid in potassium phosphate buffer (0.1 M dipotassium phosphate; 0.1 M potassium dihydrogen phosphate; 5 mM EDTA; 0.1% (v/v) Triton X-100; pH 7.5). The glutathione content was determined according to Rahman et al. (2006), with some modifications (Carregosa et al., 2014b). The procedure was adapted to a microplate method. For GSH the absorbance was read immediately at 412 nm and expressed in μmol per g of FW. For GSSG the absorbance was read at 412 nm and expressed in μmol per g of FW. The ratio GSH/GSSG was obtained for each condition by dividing the GSH content by 2*GSSG content. 2.2.2.2. Enzymatic markers of oxidative stress. For Superoxide dismutase (SOD), Catalase (CAT), and Glutathione S-transferases (GSTs) the extraction was done with potassium phosphate buffer (50 mM sodium dihydrogen phosphate monohydrate; 50 mM disodium hydrogen phosphate dihydrate; 1 mM ethylenediamine tetraacetic acid disodium salt dihydrate (EDTA); 1% (v/v) Triton X-100; 1% (v/v) polyvinylpyrrolidone (PVP); 1 mM dithiothreitol (DTT)). SOD activity was determined based on the method of Beauchamp and Fridovich (1971). The standard curve was performed with SOD standards (0.25–60 U/mL). SOD activity was measured at 560 nm in a microplate reader. Absorbance values were read after 20 min of incubation at room temperature. SOD was expressed in U per g of FW where U represents the quantity of the enzyme that catalyzes the conversion of 1 μmol of substrate per min. The activity of CAT was quantified according to Johansson and Borg (1988). The standard curve was determined using formaldehyde standards (0–150 μM). The absorbance was read at 540 nm in a microplate reader. CAT activity was expressed in U per g of FW. One unit (U) is defined as the amount of enzyme that caused the formation of 1.0 nmol of formaldehyde per min. The activity of GSTs was determined following an adaptation of the method described by Habig et al. (1974). GSTs activity was measured spectrophotometrically at 340 nm (ε = 9.6 mM−1·cm−1) in a microplate reader. Absorbance values were read in intervals of 10 s during 5 min and this time interval was selected to calculate the enzyme activity. GST activity was expressed in U per g of FW. One unit of enzyme is defined as the amount of enzyme that causes the formation of 1 μmol of thioether per min. 2.2.2.3. Energy related parameters. For glycogen (GLY) and protein (PROT) content, the extraction was done with sodium phosphate buffer. Total PROT content was determined according to the Biuret spectrophotometric method (Robinson and Hogden, 1940), using bovine serum albumin (BSA) as standards (0–40 mg·mL−1). Absorbance was read at 540 nm. Results were expressed in mg per g of FW. Following the procedure described by Dubois et al. (1956), GLY was quantified by the phenol-sulphuric acid method. Absorbance was measured at 492 nm and results were expressed in mg per g of FW.

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2.3. Data analysis Data obtained from biochemical analyses and regenerative capacity were submitted to hypothesis testing using permutational multivariate analysis of variance with the PERMANOVA+ add-on in PRIMER v6. The pseudo-F values in the PERMANOVA main tests were evaluated in terms of significance. When the main test revealed statistical significant differences (p ≤ 0.05), pairwise comparisons were performed. The t-statistics in the pair-wise comparisons were evaluated in terms of significance. Biochemical and regeneration descriptors were analyzed following a one-way hierarchical design, with As concentration as the main fixed factor. The null hypotheses tested were: a) for As (total, soluble and insoluble) quantification, no significant differences existed among experimental conditions; b) for each biochemical parameter (PROT, GLY, LPO, CAT, SOD, GSTs, GSH, GSSG and GSH/GSSG), no significant differences existed among experimental conditions. Significance levels (p ≤ 0.05) among conditions were presented with different letters. The matrix gathering all descriptors, for each condition, was used to calculate the Euclidean distance similarity matrix. This similarity matrix was simplified through the calculation of the distance among centroids matrix based on As concentration which was then submitted to ordination analysis, performed by Principal Coordinates Analysis (PCO). Pearson correlation vectors of biochemical and regeneration descriptors (correlation N 0.75) were provided as supplementary variables and superimposed on the PCO graph. 3. Results 3.1. As accumulation in D. neapolitana The results obtained showed that total As accumulation in organisms was proportional to As exposure concentration, with significant differences among conditions (Table 1). However, the bioconcentration factor (BCF) was higher at the lowest As exposure concentration (0.05 mg/L), indicating that polychaetes presented higher accumulation rate at this condition (cf. Table 1). Moreover, in all tested conditions, the results showed that polychaetes accumulated most of the As in the soluble fraction in comparison with the concentration found in the insoluble fraction, and concentrations found at each condition for both fractions increased with the increasing exposure concentration (cf. Table 1). 3.2. D. neapolitana physiological responses 3.2.1. Mortality Results showed that at the highest As concentration (1.25 mg/L) specimens were not able to survive (100% mortality), whereby day eleven 33.3% of individuals were dead, and the remaining individuals died at days 15, 18, 27 and 28 (16.7% of organisms at each referred day). Individuals exposed to 0.05 and 0.25 mg/L As presented 33.3% mortality. Dead individuals were observed by days 15 and 27 (16.7% at each mentioned day) when exposed to 0.05 mg/L As. Individuals exposed to 0.25 mg/L of As presented 16.7% of mortality at days 11 and 18. Under control conditions (0 mg/L As) no mortality was observed. 3.2.2. Regenerative capacity Table 2 presents the regenerative capacity of D. neapolitana, as the percentage of body width regenerated, and the number of chaetigers regenerated 11, 18 and 28 days after amputation at each exposure concentration. Fig. 1 presents a photographic record of the regenerative process in the same days. Eleven days after amputation all organisms under control conditions presented a small reddish differentiated blastema with anal cirri, which are 25.04% ( ± 5.10) of the width of the remaining body part (cf. Table 1; Fig. 1A). At 0.05 mg/L As, one individual was still healing the amputated region, while the remaining specimens were regenerating

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Table 1 Total As, soluble and insoluble As fractions (μg/g FW), and bioconcentration factor (BCF) in Diopatra neapolitana. BCF was calculated as the ratio between total As concentration in the organism, under experimental conditions, and the As exposure concentration. For each parameter, different letters (a–c) represent significant differences (p ≤ 0.05) among conditions. 100% mortality was recorded at the highest As concentration (1.25 mg/L). Condition CTL (0 mg/L As) 0.05 mg/L As 0.25 mg/L As

As soluble fraction

As Insoluble fraction

a

Total As

a

0.003 ± 0.002 0.005 ± 0.002a 0.019 ± 0.011b

0.006 ± 0.002 0.018 ± 0.004b 0.054 ± 0.011c

BCF

0.009 ± +0.004 0.023 ± 0.005b 0.073 ± 0.010c

a

– 0.451 ± 0.109a 0.290 ± 0.038b

a small differentiated blastema (cf. Table 1; Fig. 1B). In the 0.25 mg/L As exposures, one individual did not regenerate the amputated region and the remaining individuals were in the healing phase (cf. Table 1; Fig. 1C). By days 18 and 28, differences among individuals at different conditions were more pronounced, with specimens under control (0.00 mg/L As) presenting a significantly higher number of new segments and a higher regenerated portion compared to individuals exposed to As (0.05 and 0.25 mg/L), where the lowest number of new chaetigers and the lowest body width was observed in polychaetes exposed to 0.25 mg/L As (cf. Table 2 and Fig. 1D-I).

to As (0.05 and 0.25 mg/L). No significant differences were observed in SOD activity between organisms exposed to both As concentrations (0.05 and 0.25 mg/L). D. neapolitana showed significantly higher CAT activity when exposed to the highest As concentration (Fig. 4B) but no significant differences were noticed between individuals under control (0.00 mg/L) and individuals exposed to the lowest As concentration (0.05 mg/L). D. neapolitana individuals showed increased GSTs activity along the increasing exposure gradient (Fig. 4C), with significant differences among all tested conditions (0.00, 0.05 and 0.25 mg/L).

3.3. D. neapolitana biochemical responses

3.3.3. Energy related markers Total protein (PROT) content in polychaetes increased along the increasing As exposure gradient (Fig. 5A), with significant differences among individuals exposed to different conditions (0.00, 0.05 and 0.25 mg/L As): D. neapolitana presented the lowest content at control, while the highest values were observed at the highest tolerated As concentration (0.25 mg/L) (cf. Fig. 5A). As for PROT, glycogen (GLY) content increased in polychaetes along the increasing As exposure gradient (Fig. 5B), with significant differences among D. neapolitana specimens exposed to different conditions (0.00, 0.05, 0.25 mg/L of As). The highest GLY content was observed in organisms exposed to the highest concentration, while the lowest content was recorded in polychaetes under control conditions (cf. Fig. 5B). Results from the PCO analysis are presented in Fig. 6. The first principal component axis (PCO 1), which accounted for 85.5% of the variability, was clearly associated with As exposure concentrations, allowing for a clear distinction between individuals exposed to the highest As concentration (positive side) and individuals exposed to control and the lowest (negative side) As concentration. PCO2 axis explained 14.5% of the total variation, separating organisms exposed to control and to the highest As concentration (positive side) from organisms exposed to the lowest As concentration (negative side). The physiological and biochemical descriptors superimposed on the PCO showed that organisms exposed to the highest As concentration (0.25 mg/L) were correlated with the increase of antioxidant and biotransformation enzymes activity, corresponding to the highest levels of GLY, PROT, LPO and oxidized glutathione (GSSG) observed. On the other hand individuals under control were associated to high GSH and GSH/GSSG values.

3.3.1. Non-enzymatic markers of oxidative stress The results obtained showed that along the increasing As gradient D. neapolitana individuals presented increased LPO levels, with significant differences between the individuals under the highest tolerated concentration (0.25 mg/L) and organisms under the remaining conditions (0.00 and 0.05 mg/L) (Fig. 2). No significant differences were observed in terms of LPO levels between individuals exposed to control and individuals under the lowest As concentration tested (cf. Fig. 2). GSH content (Fig. 3A) was significantly higher in individuals under control (0.00 mg/L) compared to individuals exposed to As (0.05 and 0.25 mg/L), with no significant differences between individuals exposed to each As concentrations (0.05 and 0.25 mg/L). An opposite trend was observed for GSSG (Fig. 3B). In this case GSSG levels were significantly higher in individuals exposed to the highest As concentration (0.25 mg/L). No significant differences were found between organisms under control (0.00 mg/L) and specimens exposed to 0.05 mg/L As. A significant decrease in GSH/GSSG ratio was observed along the increasing As exposure gradient (Fig. 3C), with significant differences among all conditions. The highest GSH/GSSG values were observed in individuals under control conditions (0.00 mg/L) while the lowest values were observed in individuals exposed to the highest tolerated As concentration (0.25 mg/L). 3.3.2. Enzymatic markers of oxidative stress Regarding SOD activity (Fig. 4A), a significant increase was noticed between individuals under control (0.00 mg/L) and individuals exposed

Table 2 Biometric and regeneration data for Diopatra neapolitana: weight (g) was measured at 60th chaetiger, immediately after amputation (day 0); width (mm) was obtained at the 10th chaetiger, immediately after amputation (day 0); % body width was obtained at the 60th chaetiger at days 11, 18 and 28 after amputation; # chaetigers was obtained at days 11, 18 and 28 after amputation. Significant differences (p ≤ 0.05) among exposure concentrations are presented with different letters. CTL Biometric data Regenerative capacity

Weight (g) Width (mm) 11 days % body width 18 days % body width # chaetigers 28 days % body width # chaetigers

[0.05]

[0.25]

[1.25]

2.01 ± 1.02a 7.83 ± 0.65a

2.03 ± 0.78a 7.87 ± 0.67a –

1.90 ± 0.87a 6.86 ± 0.22a

2.01 ± 0.61a 7.67 ± 0.47a

25.04 ± 5.10a

16.50 ± 14.59a

Healing

45.07 ± 16.06a 32.00 ± 4.36a

35.69 ± 1.65b 27.50 ± 7.77b

30.59 ± 7.10c 14.50 ± 17.67c

– –

69.19 ± 3.33a 54.01 ± 7.01a

46.38 ± 24.75b 40.00 ± 10.00b

33.06 ± 10.89c 31.50 ± 16.26c

– –

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Fig. 1. Diopatra neapolitana posterior regeneration. Photographic record of the regenerative process at 11 (A, B and C), 18 (D, E and F) and 28 days (G, H and I) after amputation for individuals exposed to a concentration range of As: Ctl-0.00 (left column), 0.05 (middle column) and 0.25 mg/L (right column).

4. Discussion Our results showed that D. neapolitana allocated most of accumulated As to the soluble fraction. These results suggested that As may be in the free form in the cytosol bound to metallothioneins, leading to high tolerance to metalloids and increased resistance by organisms. Recently Velez et al. (2016) demonstrated that the majority of As accumulated by invertebrates (clams) was bound to metallothioneins fraction when exposed to different As concentrations (0 to 4 mg/L of As). However, these detoxifying mechanisms were not enough to reduce As toxicity As in clams exposed to the highest As concentration (25 mg/L As) and mortality occurred. This may explain why in the present study D. neapolitana presented the highest mortality at the highest As exposure concentration (1.25 mg/L). Studies conducted by Freitas et al. (2012b) under environmental conditions (Ria de Aveiro, Portugal) also demonstrated that D. neapolitana accumulated As mostly in the soluble fraction. Results obtained with other species are in agreement with our findings. According to Geiszinger et al. (2002), that analyzed the accumulated As in the polychaete Arenicola marina from Odense Fjord (Denmark), N70% of total As in the organisms was found in the soluble fraction. Casado-

Fig. 2. Lipid peroxidation (LPO) in D. neapolitana exposed to different As concentrations during 28 days. Significant differences (p ≤ 0.05) among exposure concentrations are presented with different letters.

Martinez et al. (2012) evaluated the accumulation and subcellular partitioning of As in the polychaete A. marina under different laboratory exposure conditions, and revealed that As bioaccumulation in this species was mostly associated to heat stable proteins in the cytosol (~50%) (soluble fraction). Also under laboratory conditions Fattorini and Regoli (2004) reported that the polychaete Sabella spallanzanii accumulated 90% of total As in the soluble form. Berthet et al. (2003) also demonstrated that under laboratory conditions Hediste diversicolor accumulated higher concentrations of metals (Zn, Ag, Cd, Cu and Pb) in the cytosol. The present study further revealed that the highest BCF was found in polychaetes exposed to the lowest As concentration (0.05 mg/L). These findings are in agreement with studies conducted by Williams et al. (2006), where a declining BCF pattern was observed in freshwater fish as exposure to As increased. These authors suggested that organisms responses may be related to controlling mechanisms, including a change from normally functioning homeostatic and regulatory processes, since the tissue concentrations are relatively constant or at least are not increasing in proportion to increasing aqueous exposure. Similar results were obtained by Morales-Caselles et al. (2008) showing that the polychaete A. marina showed decreased BCF values with the increase of polycyclic aromatic hydrocarbons (PAHs) concentration. These authors suggested that the decrease of BCF could be due to the fact that after 21 days the organisms had been able to metabolize the PAHs with lower molecular weight or, on the other hand, during long-term contact between PAHs and sediment particles, PAHs become tightly bound to organic phases in the sediment, reducing their bioavailability. Idardare et al. (2008) further revealed higher BCFs for Zn, Ag, Cu, Cd and Pb in H. diversicolor in non-contaminated versus contaminated sites. Therefore, our results, as well as the above mentioned studies, indicate that polychaetes may control accumulation of pollutants by maintaining or decreasing concentrations in their tissues (increasing the metabolization of the contaminant and activating biotransformation mechanisms namely by the increase of GSTs activity) or may prevent contaminants accumulation by keeping their bodies for longer periods of time inside their tubes (D. neapolitana) or burrowing deeper in the sediment (H. diversicolor, A. marina). After 28 days of As exposure, polychaetes under the highest exposure concentration (1.25 mg/L) showed 100% mortality while at lower As concentrations (0.05 and 0.25 mg/L) only 17% individuals did not

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Fig. 3. A- Reduced glutathione (GSH); B- Oxidized glutathione (GSSG); and C– ratio between reduced and oxidized glutathione (GSH/GSSG), in D. neapolitana exposed to different As concentrations during 28 days. Significant differences (p ≤ 0.05) among exposure concentrations are presented with different letters.

survive. At control conditions no mortality was recorded. After exposure to As organisms showed a close correlation between As concentration and the regenerative capacity. In fact, the results obtained revealed that As negatively affected the regenerative ability of D. neapolitana organisms, which presented less new chaetigers with the increase of As concentration. Previous works also revealed that the regenerative ability of some polychaete species was affected when exposed to different stressors. The study of the impacts of pharmaceuticals paracetamol by Freitas et al. (2015a) and carbamazepine by Pires et al. (2016) revealed that D. neapolitana exposed to ecologically relevant concentrations of paracetamol (25 μg/L) and carbamazepine (0.3 to 9.0 μg/L) exhibited significantly lower capacity to regenerate their body in comparison to control organisms. Carregosa et al. (2014a) demonstrated that the regenerative capacity of D. neapolitana can also be affected by organic matter enrichment, with specimens from areas with highest concentration of organic matter presenting a longer period of time to complete regeneration. Similar findings were presented by Fattorini and Regoli (2004) demonstrating that Sabella spallazanii exposed to As showed growth retardation when exposed to As. Nusetti et al. (2005) also demonstrated a significant reduction in the number of new segments in the polychaete Eurythoe complanata after 21 days of exposure to crankcase

Fig. 4. A- Superoxide dismutase (SOD); B- Catalase (CAT) and C– Glutathione Stransferases (GSTs), in D. neapolitana exposed to different As concentrations during 28 days. Significant differences (p ≤ 0.05) among exposure concentrations are presented with different letters.

oil. In addition, studies conducted with D. neapolitana exposed to different climate change related factors (pH decrease and salinity shifts) showed that under the most stressful conditions (low pH values and salinities out of D. neapolitana optimum) polychaetes took longer to regenerate and regenerated fewer new chaetigers (Freitas et al., 2015b; Pires et al., 2015). In organisms, under non-stressful conditions, reactive oxygen species (ROS) are constantly generated during normal cell metabolism (Monserrat et al., 2007; Regoli and Giuliani, 2014). However, when organisms are exposed to stressful situations ROS are produced in excess which may damage cell macromolecules, including lipids from cell membranes causing lipid peroxidation (LPO) (Regoli and Giuliani, 2014). In the present study the cellular damage (measured by LPO) and redox status (GSH/GSSG) were evaluated to understand the biochemical impacts caused in D. neapolitana due to As exposure. The results obtained revealed that along the increasing As exposure gradient D. neapolitana individuals presented increased LPO levels. Accompanying this cellular injury, organisms diminished their redox status since the ratio GSH/GSSG decreased with the increase of As concentration. Similar findings were already obtained when D. neapolitana was exposed to organic matter, metals contamination and salinity changes, revealing higher LPO and GSSG values in polychaetes under the most

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Fig. 5. A- Total protein (PROT); and B- Glycogen (GLY) content, in D. neapolitana exposed to different As concentrations during 28 days. Significant differences (p ≤ 0.05) among exposure concentrations are presented with different letters.

stressful conditions (Carregosa et al., 2014a; Freitas et al., 2015a, 2015c; Freitas et al., 2012a). Ventura-Lima et al. (2007) showed that exposing the polychaete Laeonereis acuta to As for 2 days significantly increased the LPO and decreased the GSH/GSSG levels. Studying Avicennia marina Caregnato et al. (2008) also demonstrated a positive relationship between the increase of Zn, Cu and Pb concentrations and the increase of LPO levels. Recently Maranho et al.

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(2014) showed that H. diversicolor individuals presented increased LPO levels when exposed to different pharmaceutical drugs. When ROS are generated in excess, organisms may develop mechanisms of defense that may prevent cells from LPO, namely through the activation of antioxidant and biotransformation enzymes (Alves de Almeida et al., 2007). In the present study although D. neapolitana presented increased activity of antioxidant enzymes (SOD and CAT) with the increase of As exposure, this defense mechanism was not sufficient to prevent cells from LPO. Similar results were obtained for biotransformation enzymes, GSTs, that also increased along the increasing As exposure gradient. The present findings are in agreement with results from other studies conducted with the same polychaete species that showed higher activity of these enzymes in individuals under stressful conditions such as the increase of organic matter and alterations of salinity (Carregosa et al., 2014a; Freitas et al., 2015c). Several studies using other polychaete species also demonstrated a close relationship between the increase of contamination levels and the enhancement of antioxidant and biotransformation defense mechanisms. Geracitano et al. (2002) demonstrated higher activity of SOD and CAT in Laeonereis acuta exposed to high Cu concentration and a similar response was observed by Bocchetti et al. (2004) in the polychaete Sabella spallanzanii exposed to Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn. Solé et al. (2009) and Maranho et al. (2014) observed, respectively, an increase in antioxidant enzymes activity in H. diversicolor exposed to different metals (Cu, Zn, Ni, Co, Fe, Cd, Pb and Mn) and different drugs (ibuprofen, fluoxetine, carbamazepine, propanol). To fight against oxidative stress situations organisms may reduce their metabolism to preserve their energy reserves (Sokolova, 2013). This was the case of D. neapolitana that showed the highest glycogen and protein contents when exposed to the highest As concentration. Recently, Fossi et al. (2012) demonstrated that in the same species the energy reserves (such as glycogen) were high in organisms moderately contaminated with As, Cu, Pb, Zn, Hg, Cr and Cd than in organisms nonexposed to these contaminants. Carregosa et al. (2014a) showed the increase in glycogen and protein content in D. neapolitana in response to organic matter enrichment. Similarly, a study comparing clean and multi-contaminated sites with polychlorinated biphenyls, polycyclic aromatic hydrocarbons and metals, showed that the polychaetes

Fig. 6. Biochemical responses of D. neapolitana exposed to different As concentrations, plotted on axes 1 and 2 of a Principal Coordinates (PCO) graph. Biochemical responses are superimposed on the PCO (r N 0.75). The control (CTL) and concentrations of exposure (0.05 and 0.25 mg/L) are indicated. The biomarkers presented are: GLYC, glycogen content; PROT, protein content; GSH, reduced glutathione; GSSG, oxidized glutathione; GSH/GSSG ratio; GSTs, glutathione S-tansferases; LPO, lipid peroxidation; CAT, catalase; and SOD, superoxide dismutase.

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Abarenicola pacifica and Neanthes virens exposed to contaminated sediments presented increased the glycogen content compared to organisms from clean sediment (Durou et al., 2007). Freitas et al. (2012a) studied D. neapolitana from the Ria de Aveiro (Portugal) and showed a significant increase in the protein content in these organisms when exposed to metals (Al, Cr, Ni, Cu, Zn, Cd, Pb and Hg) and As. Studies conducted with H. diversicolor showed that when organisms were under high concentrations of metals and metalloids (Cu, Cr, Cd, Pb, Zn, Hg and metalloid such as As) protein content increased (Moreira et al., 2006). Overall, the present study demonstrated that when D. neapolitana was exposed to As the number of new regenerated chaetigers for whole body repair was diminished, evidencing a close dose-response relationship. The reduction of the ability to regenerate a new body portion could result from the oxidative stress recorded in worms exposed to As, as free radicals may cause damaging effects on the biochemical and cell functions that underly the regenerative process. The increased activity of antioxidant enzymes observed in organisms under As exposure may indicate that this defense system was induced as a consequence of As contamination. Also, the increase of GSTs activity in D. neapolitana exposed to As may be related to detoxification mechanisms developed by polychaetes exposed to the metalloid. Nevertheless, the antioxidant and biotransformation mechanisms developed by polychaetes as well as the increase of energy reserves (which may result from a lower metabolic rate) were strategies developed by D. neapolitana which did not prevent organisms from cellular damages caused by As. At high As concentrations these damages were too high and death overcame. Acknowledgments This study was supported by the Portuguese Science Foundation (FCT/MEC), co-funded by FEDER within PT2020 Partnership Agreement and Compete 2020, and through CESAM: UID/AMB/50017. Cátia Velez benefited from a PhD grant (SFRH/BD/86356/2012) and Rosa Freitas benefited from a post-doc grant (SFRH/BPD/92258/2013), both financed by National Funds through the Portuguese Science Foundation (FCT), supported by FSE and Programa Operacional Capital Humano (POCH) e da União Europeia. Adília Pires benefited from a post-doc grant (BPD/CESAM/RP/BENTONICAS/2013) funded by CESAM research own funds. References Alves de Almeida, E., Bainy, A.C.D., de Melo Loureiro, A.P., Martinez, G.R., Miyamoto, S., Onuki, J., Barbosa, L.F., Garcia, C.C.M., Prado, F.M., Ronsein, G.E., Sigolo, C.A., Brochini, C.B., Martins, A.M.G., Gennari de Medeiros, M.H., Di Mascio, P., 2007. Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: antioxidants, lipid peroxidation and DNA damage. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 146, 588–600. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Bely, A.E., 2006. Distribution of segment regeneration ability in the Annelida. Integr. Comp. Biol. 46, 508–518. Berthet, B., Mouneyrac, C., Amiard, J.C., Amiard, C., Triquet, Y., Berthelot, A., Hen, L., Mastain, O., Rainbow, P.S., Smith, B.D., 2003. Accumulation and soluble binding of cadmium, copper, and zinc in the polychaete Hediste diversicolor from coastal sites with different trace metal bioavailabilities. Arch. Environ. Contam. Toxicol. 45, 468–478. Blaise, C., Gagné, F., Gillis, P.L., Eullaffroy, P., 2013. Polychaetes as bioindicators of water quality in the Saguenay Fjord (Quebec, Canada): a preliminary investigation. J. Xenobiotics 3, 1–2. Bocchetti, R., Fattorini, D., Gambi, M.C., Regoli, F., 2004. Trace metal concentrations and susceptibility to oxidative stress in the polychaete Sabella spallanzanii (Gmelin) (Sabellidae): potential role of antioxidants in revealing stressful environmental conditions in the Mediterranean. Arch. Environ. Contam. Toxicol. 46, 244–263. Bouraoui, Z., Banni, M., Ghedira, J., Boussetta, H., 2015. Biomarkers responses in different body regions of the Polychaeta Hediste diversicolor (Nereidae, polychaete) exposed to copper. J. Integr. Coast. Zone Manage. 15 (3), 371–376. Caregnato, F.F., Koller, C.E., Geoff, R., Mac, F., Moreira, J.C.F., 2008. The glutathione antioxidant system as a biomarker suite for the assessment of heavy metal exposure and effect in the grey mangrove, Avicennia marina (Forsk.) Vierh. Mar. Pollut. Bull. 56, 1119–1127.

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