Metal bioaccumulation, oxidative stress and antioxidant responses in oysters Crassostrea gasar transplanted to an estuary in southern Brazil

Science of the Total Environment 685 (2019) 332–344

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Metal bioaccumulation, oxidative stress and antioxidant responses in oysters Crassostrea gasar transplanted to an estuary in southern Brazil Clarissa P. Ferreira a, Daína Lima b, Raphaella Paiva b, Juliano M. Vilke a, Jacó J. Mattos c, Eduardo A. Almeida e, Suelen C. Grott e, Thiago C. Alves e, Jacyara N. Corrêa f, Marianna B. Jorge f, Mariana Uczay d, Carla I.G. Vogel d, Carlos H.A.M. Gomes g, Afonso C.D. Bainy b, Karim H. Lüchmann h,⁎ a

Fishery Engineering Department, Santa Catarina State University, Laguna 88790-000, Brazil Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, Federal University of Santa Catarina, Florianópolis 88034-257, Brazil c Aquaculture Pathology Research Center, Federal University of Santa Catarina, Florianópolis 88034-257, Brazil d Animal and Food Production Department, Santa Catarina State University, Lages 88520-000, Brazil e Department of Natural Sciences, Regional University of Blumenau, Blumenau 89012-170, Brazil f Oceanography and Limnology Department, Federal University of Maranhão, São Luís 65080-805, Brazil g Laboratory of Marine Mollusks, Federal University of Santa Catarina, Florianópolis 88034-257, Brazil h Department of Scientific and Technological Education, Santa Catarina State University, Florianópolis 88035-001, Brazil b

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

• Oysters Crassostrea gasar were transplanted to an estuary impacted by anthropogenic activities. • Oysters bioaccumulated metals, especially Al, Cd and Zn in gill and digestive gland. • A protective induction of antioxidant defense to deal with oxidative damage was revealed. • Gill and digestive gland react differently to cope with possible metal-induced stress.

a r t i c l e

i n f o

Article history: Received 22 December 2018 Received in revised form 7 May 2019 Accepted 24 May 2019 Available online 28 May 2019 Editor: Daniel Wunderlin Keywords: Oysters Metal Biochemical biomarkers Estuarine pollution

a b s t r a c t The present study assessed the spatial and temporal variations on metal bioaccumulation and biochemical biomarker responses in oysters Crassostrea gasar transplanted to two different sites (S1 and S2) at the Laguna Estuarine System (LES), southern Brazil, over a 45-days period. A multi-biomarker approach was used, including the evaluation of lipid peroxidation (MDA) levels, and antioxidant defense enzymes (CAT, GPx, GR and G6PDH) and phase II biotransformation enzyme (GST) in the gills and digestive gland of oysters in combination with the quantification of Al, Cd, Cu, Pb, Fe, Ni and Zn in both tissues. The exposed oysters bioaccumulated metals, especially Al, Cd and Zn in gills and digestive gland, with most prominent biomarker responses in the gills. Results showed that GPx, GR and G6PDH enzymes offered an increased and coordinated response possibly against metal (Zn, Ni, Cd and Cu) contamination in gills. GST was inversely correlated to Cd levels, being its activity significantly lowered over the 45-d exposure periods at S2. On contrary, in digestive gland GST was slightly positively correlated to Cd, revealing a compensatory mechanism between tissues to protect oysters' cells against oxidative damages, since MDA levels also decreased. CAT also appeared to be involved in the cellular protection against oxidative stress,

⁎ Corresponding author. E-mail address: [email protected] (K.H. Lüchmann).

https://doi.org/10.1016/j.scitotenv.2019.05.384 0048-9697/© 2019 Elsevier B.V. All rights reserved.

C.P. Ferreira et al. / Science of the Total Environment 685 (2019) 332–344

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being increased in gills. However, CAT was negatively correlated to Al levels, which might suggest a possible inhibitory effect of this metal in the gills of C. gasar. Differences between tissues were evident by the Integrative Biomarker Responses version 2 (IBRv2) indexes, which showed different pattern between tissues when studying the sites and exposure periods separately. This study provided evidence for the effectiveness of using a multibiomarker approach in oyster C. gasar to monitor estuarine metal pollution. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Estuaries are complex, dynamic and biologically rich environments where seawater and freshwater meet. Because of this, these areas are dominated by frequent variation in salinity, due to tidal oscillations and changes in freshwater input rates. These areas are continuously impacted by local and diffuse contaminants from a variety of anthropic activities (Day et al., 2012), being metal contamination one of the main concern for estuaries worldwide. In Brazil, metal pollution in estuaries has been receiving increasing attention due to the recent industry and agricultural development (Barletta et al., 2019). In the southern region, the Laguna Estuarine System (LES) represents an economically important area due to fishery, industry and tourism activities (Eichler et al., 2006). The Tubarão River, which flows into the LES, has been extensively impacted during the last 130 years by coal mining rejects and by other sources of metal contamination (Silva et al., 2013). Agriculture in adjacent areas also contribute to the high input of metals into LES (Eichler et al., 2006) due to the use of metal-enriched fertilizers (Gonçalves Jr et al., 2014) and pesticides containing metals in their formulation, such as glyphosate-based herbicides (Defarge et al., 2018). Thus, the location of rice farms adjacent to the LES makes possible direct inputs of metals to waters and/or indirect inputs from lixiviation of contaminated soils. Moreover, the largest coal burning thermoelectric complex of Latin America, the Jorge Lacerda Complex, had until recently an open cycle of ashes and a sedimentation basin connected to the Tubarão River (Rodriguez-Iruretagoiena et al., 2015), contributing to local contamination by metals with long lasting implications. In this river, As, Pb, Cr, Cu, and Zn were the major metals, with their concentrations in the sediments ranging between 0.005 and 25 mg·kg−1 (As), 0.5–57 mg·kg−1 (Pb), 0.001–15 mg·kg−1 (Cr), 0.4–23 mg·kg−1 (Cu), and 29–84 mg·kg−1 (Zn) (Silva et al., 2013). According to the Canadian Freshwater Sediment Guidelines (MacDonald et al., 2000), the threshold effect levels for As, Pb, Cr, Cu, and Zn are 5.9, 35, 37.3, 35.7 and 123 mg·kg−1, respectively. Thus, the concentrations of As and Pb found in the sediment of Tubarão River are of particular concern for aquatic organisms and their biological effects should be investigated. Although economically and ecologically important, there is a scarce number of studies assessing both metal contamination and toxic effects to biota in the LES. Nevertheless, toxic effects of metals have been demonstrated at molecular, cellular and organismic levels in numerous aquatic organisms elsewhere (Wang et al., 2018). One of the main consequences of metal toxicity in bivalves has been attributed to oxidative stress (Meng et al., 2018). Substantial data provides evidence that metal intoxication promotes the generation of reactive oxygen species (ROS), which either triggers protection mechanisms or causes oxidative cellular damages, such as lipid peroxidation (Chan and Wang, 2018). To minimize the negative effects of ROS, animals have evolved effective antioxidant defenses, which includes the enzymes catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), and also the multifunctional enzyme glutathione S-transferase (GST) (Livingstone et al., 1992; Liu and Wang, 2016a,b; Noctor and Foyer, 1998; Prakash and Rao, 1995; Wang et al., 2018). For this reason, the evaluation of antioxidant responses and lipid peroxidation levels has been extensively used to assess the effects of metal exposure in bivalves in monitoring studies (Almeida et al., 2004; Company et al., 2004).

Economically and ecologically important along the Brazilian coast, the mangrove oysters Crassostrea gasar (sin. Crassostrea brasiliana, Lamarck, 1819) is sessile and filter feeder species capable to bioaccumulate higher levels of organic contaminants, revealing them as an ideal bioindicator for monitoring organic pollutants (Lima et al., 2018; Lüchmann et al., 2011, 2014, 2015; Pessatti et al., 2016; Zacchi et al., 2018). In contrast, very few studies have addressed the capacity of metal bioaccumulation in different organs of C. gasar (KuranchieMensah et al., 2016). With this background in mind, in the present study we transplanted oysters C. gasar to two different sites at the LES over a period of 45 days in order to identify possible metal sources and the time evolution of metal bioaccumulation and biomarker responses. After 7-, 15-, 30and 45-days oysters were collected among sites and biological variations were assessed upon the application of multi-biomarker approach, including the measurement of CAT, GPx, GR, G6PDH, and GST activities, and the levels of lipid peroxidation in the gills and digestive gland. In addition, the concentrations of metals (Al, Cd, Cu, Pb, Fe, Ni and Zn) were measured in the gills and digestive gland to investigate the possible association between biomarker responses and pollutant levels. Finally, to summarize the biomarkers responses in each site of the LES, Integrated Biomarker Response version 2 (IBRv2) indexes were used for all biological parameters measured in the gills and digestive gland. 2. Materials and methods 2.1. Study area The study was done at the Laguna Estuarine System (LES) during a period of 45 days (October–December 2017). The estuary is located in the state of Santa Catarina, southern Brazil (28°12′S - 48°38′W) and is composed of three lagoons: Mirim, Imaruí and Santo Antônio dos Anjos, which is connected to the Atlantic Ocean (Fig. 1). The two sites at the LES selected for the study were either located near industrial, urban or agriculture areas, which substantially contributes to pollutants input to the estuary: site 1 (S1) is located near the Laguna city and receives effluents from untreated sanitary discharges and from the Tubarão River, which is highly impacted by discharges from coal mining, urban areas and industries (Silva et al., 2013); and site 2 (S2) is mainly impacted by rice culture and fishery activities (RodriguezIruretagoiena et al., 2015). Due to difficulties to find a location in the LES less impacted by anthropic activities and with similar physicochemical characteristics, the reference site (Ref) was selected in Florianópolis Island, in a bay free of contaminant sources, where oysters are farmed for human consumption (Fig. 1). 2.2. Experimental animals and transplants Adult oysters of similar genetic stock and shell length (6.31 ± 0.31 cm) were supplied by an oyster farm located in Florianópolis, Santa Catarina, southern Brazil. The oysters were placed in mini floating rafts (100 × 50 cm) and transplanted to S1 and S2 (1 raft per site). Before the in situ exposure, ten oysters were collected to represent the initial time (0 d), and then other 80 animals were kept in each site for 7, 15, 30 and 45 days. For each sampling time, 16 oysters from each site (Ref, S1 and S2) were collected and transported to the laboratory, where they

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Fig. 1. Sites of in situ transplantation in the Laguna Estuarine System, southern Brazil. Oysters Crassostrea gasar were transplanted from the reference site – Ref, in Florianópolis, to S1 and S2.

were immediately dissected for biochemical and metal analyses. The raft of site S1 disappeared before 45 d, and oysters could not be sampled at this time. Salinity and temperature were measured at each oyster collection without remarkably variation between different sites at same exposure time. For each sampling site and exposure time, six oysters were killed and collected for the analysis of metal concentration. Gills and digestive gland samples were rinsed with artificial salt water, dried at 60 °C up to reach the dry weight and stored at room temperature until metal analysis. For biochemical assays, ten oysters from each exposure time and site were killed, and had the gills and digestive gland immediately excised, frozen in liquid nitrogen and stored at −80 °C until biomarkers analyses.

analyses were performed by inductively coupled plasma optical emission spectrometry (ICP-OES) (Shimadzu, ICPE-9800) to measure aluminum (Al), cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), lead (Pb) and zinc (Zn). All metals measurements were performed in triplicate. Results are expressed as ug·g−1 dry tissue. The accuracy of the methodology, reported as recovery, was evaluated throughout by analysis of standard reference material (TORT-3: Lobster Hepatopancreas and ERM®-CE278k: Mussel, NRC). The recoveries were ranging from 66.2% to 106.4%. The following instrumental parameters for analytes determination by ICP-OES were: Radiofrequency (kW) – 1.2; Plasma Gas (L/ min) – 10.0; Auxiliary Gas (L/min) – 0.6; Carrier Gas (L/min) – 0.7; Exposure time (sec) – 30; Sensitivity – Wide range; View direction – Axial; Wavelength (nm) – Al (396.15), Cd (214.43), Cu (324.75), Pb (220.35), Fe (238.20), Ni (231.60) and Zn (202.54).

2.3. Metal concentration in oyster tissues For determination of metal concentrations in gills and digestive gland, the tissues were dried at ~60 °C to a constant weight (approximately one week). The dried tissues were weighed and homogenized with a mortar and a pestle into a fine powder. Approximately 0.1 g of the dried and ground material was accurately weighed and transferred into the Teflon extraction vessel with 2 mL of HNO3 (65% v/v). The extraction vessel was placed in the microwave oven (One Touch Technology, Mars 6) and the digestion was run as follow: ramp time – 1100 W, reaching 200 °C for 59 min; hold time – 1100 W, 200 °C for 55 min; cooling for 15 min. All the acid extracts were respectively moved to a new tube and diluted to 30 mL with Milli-Q water. The chemical

2.4. Biochemical biomarkers 2.4.1. Antioxidants and biotransformation enzymes The gills and digestive gland (approximately 150 mg each) were individually homogenized for the measurements of enzymatic activities in 1:4 (w:v) chilled buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M sucrose, 0.15 M KCl, 1 mM DTT, and 0.1 mM PMSF) using a tissue homogenizer Tissue-Tearor (BioSpec Products, USA). Samples were maintained on ice throughout homogenization. Homogenates were centrifuged at 9000g for 30 min at 4 °C, and the resulting supernatants were used for enzymatic assays.

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Catalase (CAT) activity was measured by the decrease in absorbance at 240 nm by H2O2 decomposition, according to Aebi (1984). Glutathione peroxidase (GPx) activity was quantified indirectly by monitoring the NADPH oxidation rate at 340 nm using cumene hydroperoxide (C6H5C(CH3)2OOH) as substrate (Wendel, 1981). Glutathione reductase (GR) activity was determined by the NADPH oxidation rate at 340 nm, due to the conversion of GSSG into GSH (Carlberg and Mannervik, 1985). Glucose-6-phosphate dehydrogenase (G6PDH) activity was determined following the method of Glock and McLean (1953), which evaluates the increase in absorbance at 340 nm, caused by the reduction of NADP to NADPH. Glutathione S-transferase (GST) activity was assayed by increasing absorbance at 340 nm, using 1-chloro-2.4 dinitrobenzene (CDNB) as substrate (Keen et al., 1976). Total protein levels were quantified according to Bradford (1976), using bovine serum albumin as standard. All assays were performed in duplicate in a 96-well plate reader (SpectraMax 250, Molecular Devices, Sunnyvale, CA, USA). 2.4.2. Lipid peroxidation levels Levels of lipid peroxidation in gills and digestive gland were assessed by the quantification of product formed by the reaction of malondialdehyde (MDA) and thiobarbituric acid (TBA) according to Almeida et al. (2004). Briefly, 100 mg of each tissue were homogenized in 0.3 mL of 0.1 M Tris-HCl buffer containing 0.05% butylated hydroxytoluene (BHT), pH 8.0, and added 0.3 mL of the TBA solution (0.4% in 0.2 M HCl) to each sample. The mixture was heated at 90 °C for 40 min and colorimetric derivative was extracted with 1 mL of nbutanol. Samples were then subjected to a centrifugation at 5000g for 3 min at 4 °C, and analysis was performed using High Performance Liquid Chromatography (HPLC) with Diodes Array Detection (DAD). The supernatant fraction was collected and inserted into a HPLC system and monitored at 532 nm. The results were evaluated using the software Chromeleon 7 and MDA estimation was based on a standard calibration curve obtained by the hydrolysis of tetramethoxypropane. The components of the HPLC system were two pumps with automatic sampler, a mobile phase 50 mM monobasic potassium phosphate buffer (pH 7.0) with 40% methanol, flow of 0.5 mL. min-1 and a LC-18 (250 × 4.6 mm, 5 μm pore diameter) column (Dionex Ultimate 3000, Thermo Fisher Scientific, CA, USA). 2.5. Integrated biomarker response The biomarkers results obtained in this study were applied to the Integrated Biomarker Response Index version 2 (IBRv2), described by Beliaeff and Burgeot (2002) and modified by Sanchez et al. (2013). This version of IBR is based on the principle of reference deviation between a disturbed and undisturbed state (Sanchez et al., 2013). In the present study, the deviation between biomarkers measured in the gills and digestive glands of oysters maintained for 7, 15 and 30-d at S1 and S2 were compared to those measured in oysters from the Ref site. The IBRv2 was not calculated for 45-d due to the disappearance of the raft kept at S1 after 30 days of exposure. For each individual biomarker, the ratio between the mean value obtained at the experimental sites (Xi) and the Ref site mean value (X0) was log-transformed [Yi = log (Xi/X0)]. A general mean (μ) and standard deviation (s) was calculated, considering Yi values of a given biomarker measured at each site. Then, Yi values were standardized by the formula: Zi = (Yi − μ)/s and the difference between Zi and Z0 (reference site) were used to define the biomarker deviation index (A = Zi − Z0). To find the integrated multiple biomarkers response (IBRv2), the absolute value of A parameters calculated for each biomarker in each experimental site were summed (IBRv2 = ∑|A|). For each experimental site (S1 and S2), A parameters were reported in a star plot to represent the reference deviation of each investigated biomarker for each tissue. The area above zero (0) reflects biomarker induction, and the area below zero (0) indicates a biomarker inhibition.

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2.6. Statistical analyses All statistical analyses were carried out using the open source R software (3.0.5 version). Results are presented as mean ± standard deviation unless otherwise stated. Linear regression or Gamma Log-linear regression by package contrast were conducted to compare intra- and intergroups contrasts by the interaction between time and site (time*site) for each biomarker, metal, as well as each tissue (McCullagh and Nelder, 1989). Spearman's correlation was chosen, since it does not require the assumption that the relationship between the variables is linear (Spearman, 2010). The correlation analyses were performed between metals and biochemical data by functions rcorr and the package corrplot. A correlation analysis was also performed in each tissue to verify the potential relationship between the levels of metals, as previously stated by Jaminska et al. (2011). According to the authors, some metals have similar pathways of metabolism and storage, and the information on their joint bioaccumulation is of extreme importance to predict the putative toxic effects of such exposure at the biota. Statistical significance of data was set up at p b 0.05, and the critical value of Spearman coefficient was set up at ±1. According to Mukaka (2012), the stronger the correlation, the closer the correlation coefficient comes to ±1.

3. Results 3.1. Metal bioaccumulation in oyster tissues The concentration of metals (Al, Cd, Cu, Fe, Ni, Pb and Zn) in the gills and digestive gland of Crassostrea gasar collected in farming area before transplantation (0 d), and over the sampling periods at Ref, S1 and S2 sites are shown in Table 1. In general, the concentrations of Cd and Zn were higher in the gills, while Al, Cu, Fe, Pb and Ni levels were similar in gills and digestive gland. Furthermore, metal concentrations fluctuated during the sampling sites and periods according to the tissue (Tables 1 and 2). Out of the seven metals, only Ni and Pb concentrations remained unchanged in both the gills and digestive gland of oysters from S1 compared to Ref (Tables 1 and 2). The gills concentrations of Cd and Zn were higher in animals sampled at S1 after 7-, 15- and 30-d, whereas Cu levels were higher after 7- and 15-d, compared to the same periods in Ref. Over the 30-d period, the levels of Fe were significantly lower, while the metal concentrations increased after 15-d in digestive gland. In addition, the digestive gland of oysters from S1 presented higher levels of Al following 15- and 30-d of in situ exposure, and also higher Cd levels after 15-d. Significant spatial variations of metal concentrations were also documented for S2 (Table 2). C. gasar presented higher levels of Cd and Zn in gills after 7-, 15- and 30-d when compared to Ref. Cd levels were also significantly higher after 45-d in the gills from S2 oysters compared to oysters from Ref. In the gills of animals kept at S2 for 45-d, the concentrations of Al, Fe and Ni were lower than those from the Ref group. In the digestive gland, the concentrations of Al, Pb and Fe were increased after 7-d in oysters from S2, while Ni levels were decreased in the same group. Similar to the results on gills, Cd levels in the digestive gland were also increased after 15-, 30- and 45- in the oysters from the S2 group, compared to the Ref group. Unlike previous metals, Cu remained unchanged in oysters from S2 in both the gills and digestive gland (Tables 1 and 2). At the end of the in situ exposure, oysters from S1 and S2 had the highest concentrations of Cd and Zn in gills and digestive gland. No obvious temporal trend was documented for most metals, except for Cd and Zn, which temporal fluctuations were consistent at different tissues (Table 2). However, changes on Cd concentrations were more dramatic in the gills at S2 during the whole experimental period (Tables 1 and 2). Curiously, for both tissues, Cd levels were positively correlated to Cu and

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Table 1 Concentration of metal (mean ± standard deviation) in gills and digestive gland of oysters Crassostrea gasar from reference site (Ref), in Florianópolis, and transplanted to sites S1 and S2 at the Laguna Estuarine System for 7, 15, 30 and 45 days. Data are presented as ug·g−1 dry tissue. Site Gills

Digestive gland

0 d* Mean

7d SD

Mean

15 d SD

Mean

30 d SD

Mean

45 d SD

Mean

Aluminum (Al) Ref 140.14 86.37 120.42 44.03 148.51 49.96 129.68 16.09 169.70 S1 126.41 12.74 126.72 9.22 112.11 16.85 – S2 171.34 30.51 133.00 58.94 226.95 181.43 89.87 Cadmium (Cd) Ref 5.03 1.17 S1 S2

4.87 6.69 11.83

0.96 0.72 3.01

3.55 7.76 11.47

0.48 1.19 2.89

5.03 7.70 12.72

0.88 1.36 2.34

5.90 – 13.57

Copper (Cu) Ref 16.09 S1 S2

13.28 18.53 20.54

3.22 3.78 5.22

13.70 19.37 19.12

3.27 2.12 6.30

16.21 17.39 17.08

5.18 3.55 14.12

21.91 – 21.29

0.57 0.62 0.66

0.31 0.24 0.29

0.64 0.66 0.57

0.12 0.29 0.19

0.59 0.51 0.62

0.06 0.16 0.30

0.64 – 0.42

Lead (Pb) Ref 0.64 S1 S2

3.01

0.28

Iron (Fe) Ref 96.98 10.32 101.43 14.59 84.16 7.20 103.10 S1 104.78 7.11 96.46 6.86 80.95 S2 133.26 25.64 106.99 22.83 102.41 Nickel (Ni) Ref 3.19 S1 S2

0.76

3.12 3.16 2.98

0.29 0.15 0.27

2.77 3.24 3.01

0.20 0.13 0.20

3.93 3.11 3.05

21.94 108.52 15.65 – 50.15 63.16

1.74 0.33 0.18

0d SD

Mean

7d SD

Mean

SD

Mean

30 d SD

Mean

45 d SD

Mean

92.18 106.33 27.31 133.86 41.57 111.79 34.60 83.90 22.63 139.84 187.75 81.59 217.43 132.01 145.75 63.20 – 38.69 294.92 298.32 154.03 64.81 137.05 49.52 154.20

0.92

2.38

0.31

4.79

6.73

11.42

4.58

9.48

0.36

0.77

0.13

0.14

12.78

79.06

6.90

13.85

4.03 – 2.45

1.05 0.70

2.69

0.20

2.81 3.31 3.64

0.97 0.25 0.98

2.47 3.70 3.69

0.23 0.62 0.87

2.49 2.58 4.95

0.50 1.15 1.15

3.59 – 5.81

15.25 14.66 14.56

5.97 3.61 1.88

12.96 13.41 16.10

2.43 4.38 4.87

9.87 11.45 11.75

2.63 5.39 7.39

16.48 – 19.89

0.87 0.78 4.19

0.25 0.27 8.13

0.74 1.12 0.67

0.23 1.42 0.17

0.78 0.74 0.85

0.09 0.41 0.09

1.01 – 0.94

24.00 81.38 37.84 132.21 52.40 94.49

16.10 46.40 24.40

84.94 123.17 134.42

2.81 2.85 6.38

Zinc (Zn) Ref 283.11 45.48 247.36 39.48 216.58 33.34 284.28 51.55 401.74 47.93 141.74 34.89 168.92 S1 305.77 42.06 357.76 60.49 448.26 47.42 – 164.14 S2 340.58 48.18 384.82 80.81 404.26 172.76 399.00 149.13 192.11 *

15 d

0.19 0.59 9.07

2.62 4.41 2.62

52.54 124.29 30.85 170.55 64.28 181.05

0.18 3.92 0.07

65.33 10.44 111.05 95.88 43.75 – 85.48 33.46 92.39

2.61 2.58 2.64

0.50 1.40 0.26

2.89 – 3.25

SD 55.83 39.63

0.31 1.68

5.19 13.52

0.47 0.45

12.34 20.71

0.50 0.85

26.20 132.67 43.90 173.87 40.63 14.58 166.27 77.36 – 38.43 160.50 77.89 237.73 128.05

0 d represents the period when oysters were sampled before transplantation to LES.

Zn (gills: r = 0.80; r = 0.83, respectively; digestive gland: r = 0.71; r = 0.76, respectively; p b 0.05). 3.2. Biochemical biomarkers The activities of antioxidant enzymes and lipid peroxidation levels in gills and digestive gland are presented in Fig. 2. In general, biomarker responses fluctuated between sites and sampling periods, which revealed some tissue-dependent patterns. With respect to antioxidant enzymes, no alterations were observed in the activities of CAT, GPx, GR and G6PDH in the gills of oysters from S1, compared to the Ref group (Table 3), and only GST activity changed. In the S1 group, the GST activity was significantly lower in the gills after 15- and 30-d, compared to oysters from the Ref group. In the digestive gland of oysters from S1 group, only G6PDH activity was altered, with the oysters presenting higher activity after 15-d when compared to the activity in the digestive gland of Ref oysters. Similar to the S1 group, GST was also lowered in the gills of oysters from S2 compared to those from the Ref group, after 15-, 30- and 45-d (Fig. 2). Oysters from S2 presented higher CAT but lower GR activities in the gills compared to the Ref group, after 45-d. In the digestive gland of S2 oysters, the activities of GR and GST were higher than Ref values after 7-d, while G6PDH was higher following 15-d of exposure. After 45-d, the activities of GPx, GR, G6PDH and GST in the digestive gland of S2 oysters were lower than the activities in same organ of oysters from the Ref group (Fig. 2). Regarding lipid peroxidation, oysters from the S1 presented higher MDA levels than Ref oysters after 7- and 15 days in the gills, and after 7-days in the digestive gland (Fig. 2 and Table 3). Oysters from S2

presented higher lipid peroxidation levels than Ref oysters after 15 days in the gills, but lower lipid peroxidation after 45-d in same organ. In the digestive gland, oysters kept in S2 for 15-d also showed lower lipid peroxidation levels than Ref oysters. Most of biomarker responses showed some degree of temporal variation. Comparing with the initial activities (0 d), CAT showed a significant temporal increase in gills at S1 after 15-d, and at S2 following 30and 45-d exposure, whereas such effect was observed in digestive gland only for 7-d at oysters from S2. A similar pattern of increase was observed for GPx, with temporal changes at Ref, S1 and S2 in all exposure times, notably in gills, while digestive gland showed increased activity after 15-, 30- and 45-d at S1, S2 and Ref, respectively. Regarding the ancillary enzymes, G6PDH was the most affected by temporal fluctuation, with time-increase responses for most sites and tissues. Interestingly, GST activity also varied across exposure periods, but the trend varied according to the tissue: while gills GST diminished along time, activity in digestive gland was temporally higher after 15-d at S1 and 30-d at S2, when compared to the initial activity. Moreover, GST activity in digestive gland was positively correlated to GPx and G6PDH activities (r = 0.72 and r = 0.58, respectively, p b 0.05), the last being negatively correlated to MDA levels (r = −0.62, p b 0.05). Regarding the lipid peroxidation levels, the digestive gland presented significant lower contents at the end of whole experiment in oysters from S2, whereas oysters from Ref presented a significant increase at the end of 45-d period, when compared to the initial time. Interestingly, the temporal increases of CAT and GPx activities in digestive gland and gills, respectively, were positively correlated to decreased MDA levels (digestive gland: r = 0.72, p b 0.05; gills: r = 0.57, p b 0.05).

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Table 2 Summarized significant differences (p value) among sampling time (0 d × 7 d, 15 d, 30 d and 45 d) and sites (Ref × S1, Ref × S2, and S1 × S2) for metals analyzed in gills and digestive gland of oysters Crassostrea gasar from Ref, in Florianópolis, and sites S1 and S2 at the Laguna Estuarine System. Values marked in bold indicate statistical significance (p b 0.05). Metal

Time

Gills

Digestive gland

Site

Al

Cd

Cu

Pb

Fe

Ni

Zn

0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d

Ref x

Ref x

Site

Ref x

Ref x

Ref

S1

S2

S1

S2

Ref

S1

S2

S1

S2

– 0.551 0.818 0.759 0.454 – 0.770 0.004 0.998 0.143 – 0.292 0.368 0.964 0.035 – 0.637 0.998 0.737 0.978 – 0.585 0.094 0.458 0.179 – 0.883 0.362 0.178 0.130 – 0.149 0.007 0.964 0.001

– 0.588 0.596 0.250 – – 0.011 0.000 0.001 – – 0.199 0.089 0.489 – – 0.898 0.886 0.367 – – 0.281 0.939 0.018 – – 0.914 0.842 0.765 – – 0.370 0.012 0.000 –

– 0.555 0.870 0.157 0.187 – 0.000 0.000 0.000 0.000 – 0.405 0.551 0.844 0.309 – 0.891 0.660 0.890 0.133 – 0.082 0.549 0.739 0.017 – 0.534 0.578 0.673 0.019 – 0.313 0.086 0.050 0.058

– 0.710 0.231 0.271 – – 0.002 0.000 0.000 – – 0.018 0.011 0.580 – – 0.685 0.867 0.515 – – 0.694 0.107 0.007 – – 0.904 0.178 0.048 – – 0.024 0.000 0.000 –

– 0.225 0.677 0.051 0.030 – 0.000 0.000 0.000 0.000 – 0.120 0.221 0.843 0.887 – 0.542 0.654 0.834 0.115 – 0.076 0.095 0.962 0.001 – 0.729 0.527 0.055 0.001 – 0.046 0.001 0.021 0.962

– 0.220 0.784 0.207 0.148 – 0.159 0.745 0.704 0.002 – 0.144 0.550 0.546 0.057 – 0.467 0.847 0.909 0.131 – 0.506 0.792 0.129 0.001 – 0.541 0.730 0.715 0.321 – 0.257 0.464 0.702 0.183

– 0.053 0.021 0.249 – – 0.036 0.008 0.591 – – 0.231 0.456 0.991 – – 0.972 0.392 0.932 – – 0.053 0.023 0.443 – – 0.851 0.140 0.901 – – 0.406 0.288 0.364 –

– 0.012 0.259 0.430 0.258 – 0.006 0.005 0.000 0.000 – 0.478 0.294 0.941 0.064 – 0.039 0.795 0.839 0.702 – 0.005 0.403 0.726 0.469 – 0.055 0.950 0.961 0.619 – 0.265 0.382 0.675 0.040

– 0.173 0.015 0.036 – – 0.278 0.014 0.813 – – 0.813 0.855 0.527 – – 0.754 0.273 0.876 – – 0.052 0.012 0.117 – – 0.960 0.063 0.964 – – 0.857 0.090 0.212 –

– 0.015 0.261 0.097 0.725 – 0.066 0.006 0.000 0.002 – 0.856 0.409 0.621 0.370 – 0.015 0.811 0.841 0.877 – 0.003 0.411 0.209 0.244 – 0.021 0.995 0.978 0.704 – 0.548 0.146 0.471 0.103

3.3. Relationship between metal and biochemical biomarkers The possible relationships between metals bioaccumulated either in gills or in digestive gland, and the biochemical parameters measured in both tissues of C. gasar, were assessed by Spearman correlation analysis. The matrix correlations for both tissues are presented in Fig. 3. In gills, MDA levels and GPx activity presented a slight, but significant positive association with Zn concentration (r = 0.58 and r = 0.72, respectively, p b 0.05), while CAT activity was negatively correlated with Al concentrations (r = −0.56, p b 0.05). In addition, the ancillary enzyme GR was positively correlated with Ni levels (r = 0.54, p b 0.05), whereas G6PDH activity was positively correlated with Cd, Cu and Zn contents (r = 0.65, r = 0.62 and r = 0.56, respectively, p b 0.05). On the other hand, a putative inverse relationship between GST activity and Cd concentrations was observed (r = −0.56, p b 0.05) in the gills, while in digestive gland the GST activity presented a slight but significant positive correlation with Al, Cd, Pb and Fe levels (r = 0.58, r = 0.68, r = 0.55 and r = 0.69, respectively, p b 0.05). 3.4. Integrated biomarker responses Results of general IBRv2 for each experimental site and time are presented in Fig. 4. The IBRv2 values were similar between S1 and S2, with the highest ∑IBRv2 total value (44.80) observed for S2, followed by S1, which presented a slightly lower value (43.85). The star plots clearly showed the difference between studied areas at the LES for each tissue. In the gills of oysters kept for 7-d at the LES, the IBRv2 at S1 was slightly higher (7.4) than that of S2 (5.9). However, while the S1

IBRv2 value was due to increased CAT and GPx activities and MDA levels, with simultaneous decreases in further biomarkers, the value of S2 IBRv2 was mainly due to increased GPx and G6PDH activities. In the digestive gland, the IBRv2 value for S1 was lower (5.9) than that for S2 (9.8). At S1, the IBRv2 alteration was mainly due to increased CAT, GPx, GST and MDA, while at S2 the IBRv2 change resulted from increased responses of all biomarkers analyzed. After 15-d transplantation, the IBRv2 in the gills of oysters at S1 was higher (8.4) than that of S2 oysters (7.7). The main variation was due to induction of GR and G6PDH activities at S1, while the GST remained with decreased activity. On the other hand, the star plot for S2 indicates an increased MDA levels along with all antioxidant enzymes activity. In digestive gland, the IBRv2 of S1 was slightly higher (7.6) than that of S2 oysters (7.4). However, while the S1 IBRv2 value was due to increased activity of all protective biomarkers and decreased MDA levels, the value of S2 IBRv2 was mainly due to increased G6PDH and GST activities, with parallel decrease in further biomarker. After 30 days of exposure, the IBRv2 in gills was higher (8.4) at S1 than the value observed at S2 (6.0). Curiously, the outcome pointed for an inhibition of all biomarkers at S1, while an increase in CAT and GR activities and MDA levels, together with an inhibition of further biomarkers, were observed for oysters maintained at S2. Finally, the digestive gland IBRv2 value was higher at S2 (8.1) compared to S1 (5.9), mainly because of the increased CAT activity and MDA levels at S1. Interestingly, GST and the other biomarkers presented lowered activities. At S2, the IBRv2 change resulted from increased GPx and GST, and decreased CAT, GR, G6PDH and MDA (Fig. 4).

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Fig. 2. Biochemical biomarkers measured in gills and digestive gland of oysters Crassostrea gasar from Ref, in Florianópolis, and sites S1 and S2, in the Laguna Estuarine System. Box-plots illustrate the median, 25% and 75% percentiles, minimum and maximum, and the horizontal line represents the median of the initial value (0 d).

4. Discussion 4.1. Metal bioaccumulation in oysters Our study focused on the relationships between metal contamination and biomarker responses in the gills and digestive gland of mangrove oyster Crassostrea gasar transplanted to an estuary impacted by rejects from rice farming, the thermoelectric power plant and untreated urban and sanitary effluents. The concentrations of most metals (Al, Cu, Fe, Ni, Pb and Zn) in both gills and digestive gland determined in this study over a 45-day period were comparable to those measured in shellfish from unpolluted locations (Capolupo et al., 2017; Lu et al., 2017; Wang and Lu, 2017). However, the levels of Zn bioaccumulated in C. gasar exceeded the maximum acceptable limit of 50 mg·kg−1 wet weight for human consumption (Congresso Nacional, 1965; Ministério da Saúde, 1998). Moreover, Cd concentrations, especially in oysters from S2, ranged from 7.5 μg·g−1 in digestive gland to 15 μg·g−1 in the gills of C. gasar, which were very close to the concentrations found in the tissues of C. virginica from Jamaica Bay, New York, a site historically polluted by metals (Rodney et al., 2007). Indeed, Cd concentrations observed in oysters from LES were in line with the concentrations reported for C. hongkongensis and C. angulata transplanted to a metal-polluted estuary in China over a two-month period (Liu and Wang, 2016a). This result indicates that the status of metal pollution at the LES should be considered of particular concern, since Cd ranks seventh place in the

priority list of top 20 hazardous substances of the Agency for Toxic Substances and Disease Registry (ATSDR) of the US, which prioritizes the substances that pose the most significant potential threat to human health. Indeed, Cd environmental exposure has been associated with numerous conditions and human diseases (Glenn, 2002). Furthermore, it is also important to mention that the concentrations of Cd in transplanted oysters exceeded the food safety standards of 2 mg·kg−1 wet weight (Congresso Nacional, 1965; Ministério da Saúde, 1998). Interestingly, some studies have reported that Cd bioaccumulation can be facilitated by Zn and Cu presence (Liu and Wang, 2013, 2014), which might explain the slight but significant positive correlation found between Cd and concentrations of Cu and Zn in C. gasar. One feasible explanation points to the ability of Cd to displace Zn and/or Cu from cysteine-rich proteins, such as metallothionein (MT), facilitating Cd accumulation (Wang and Lu, 2017). This free Zn, in turn, is a key element for the activation of transcription factors involved in metal homeostasis and thereby the released Zn induces synthesis of more MT (Huang et al., 2004). Moreover, the highest Cd levels in oyster's gills, when compared to the values reported for the digestive gland, were expected since the gills are in constant contact with water from external environment, which is favored by the large surface area of gills epithelium (Fernández et al., 2010). Thus, oysters assimilate metals by adsorption onto this tissue and membrane surfaces, and by ion exchange of dissolved metals across lipophilic membranes of the gills. Indeed, considering that gills are rich on Ca2 channels for ion exchange, along

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Table 3 Summarized significant differences (p value) among sampling time (0 d × 7 d, 15 d, 30 d and 45 d) and sites (Ref × S1, Ref × S2 and S1 × S2) for biochemical biomarkers in gills and digestive gland of oysters Crassostrea gasar from Ref, in Florianópolis, and sites S1 and S2 at the Laguna Estuarine System. Values marked in bold indicate statistical significance (p b 0.05). Biomarker

Time

Gills

Digestive gland

Site

CAT GPx

GR G6PDH

GST

MDA

0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d 0d 7d 15 d 30 d 45 d

Ref x

Ref x

Site

Ref x

Ref x

Ref

S1

S2

S1

S2

Ref

S1

S2

S1

S2

– 0.846 0.830 0.255 0.438 – 0.810 0.014 0.000 0.001 – 0.784 0.205 0.499 0.257 – 0.529 0.179 0.033 0.077 – 0.254 0.084 0.082 0.420 – 0.550 0.090 0.162 0.040

– 0.106 0.046 0.627 – – 0.555 0.014 0.001 – – 0.283 0.144 0.395 – – 0.424 0.008 0.172 – – 0.007 0.022 0.003 – – 0.052 0.523 0.156 –

– 0.912 0.448 0.008 0.002 – 0.052 0.003 0.000 0.005 – 0.608 0.766 0.189 0.206 – 0.013 0.004 0.002 0.000 – 0.012 0.924 0.331 0.015 – 0.124 0.182 0.103 0.633

– 0.204 0.108 0.554 – – 0.729 0.910 0.870 – – 0.507 0.969 0.147 – – 0.986 0.371 0.287 – – 0.406 0.000 0.000 – – 0.008 0.009 0.838 –

– 0.871 0.801 0.641 0.002 – 0.113 0.817 0.658 0.302 – 0.904 0.252 0.731 0.025 – 0.320 0.664 0.687 0.553 – 0.610 0.049 0.007 0.008 – 0.363 0.005 0.789 0.013

– 0.556 0.139 0.505 0.965 – 0.654 0.431 0.325 0.008 – 0.749 0.761 0.512 0.708 – 0.779 0.641 0.000 0.000 – 0.803 0.441 0.097 0.000 – 0.193 0.122 0.694 0.060

– 0.279 0.090 0.442 – – 0.674 0.039 0.278 – – 0.521 0.506 0.283 – – 0.753 0.002 0.028 – – 0.027 0.009 0.099 – – 0.003 0.911 0.381 –

– 0.014 0.470 0.503 0.577 – 0.300 0.921 0.028 0.695 – 0.057 0.327 0.043 0.057 – 0.412 0.010 0.033 0.091 – 0.000 0.057 0.000 0.258 – 0.005 0.273 0.325 0.012

– 0.582 0.780 0.900 – – 0.355 0.268 0.902 – – 0.662 0.316 0.526 – – 0.961 0.000 0.151 – – 0.068 0.080 0.906 – – 0.003 0.156 0.163 –

– 0.059 0.324 0.933 0.603 – 0.215 0.317 0.697 0.001 – 0.018 0.477 0.141 0.016 – 0.292 0.003 0.121 0.009 – 0.008 0.518 0.101 0.000 – 0.072 0.002 0.523 0.535

with the similar chemical properties shared between Cd and calcium ions, we can suggest a higher uptake of estuarine Cd by the oyster's gills. In our study, Al, Ni and Pb did not vary significantly among sites, but their levels ranged greatly from 45 to 476 μg·g−1, 0.33 to 4.72 μg·g−1, and from 0.06 to 1.94 μg·g−1, respectively, in the oyster's tissues. Similar values have been detected in mollusks exposed to metals under

controlled laboratory conditions, such as the mussel Mytilus galloprovincialis, that accumulated up to 0.28 and 8.53 μg·g−1 of Ni following 24-h and 8-d exposure, respectively (Attig et al., 2010). It is important to highlight that interactions among metals, even at low concentrations, can yield synergistic or antagonistic effects in the exposed organisms. In fact, Benavides et al. (2016) reported increased

Fig. 3. Spearman correlation matrix between metal concentration and biochemical biomarkers in gills and digestive gland of oysters Crassostrea gasar from Ref, in Florianópolis, and sites S1 and S2, at the Laguna Estuarine System. Positive correlations are displayed in blue and negative correlations in red. Color intensity and the size of the circle are proportional to the correlation coefficients. Significance indicated through * = p b 0.05, and ** = p b 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Integrated biomarker response index (IBRv2) values and associated star plots for gills and digestive gland of oysters (Crassostrea gasar) maintained for 7-, 15- and 30-d at sampling sites at the Laguna Estuarine System. Abbreviations: CAT – catalase activity; GPx – glutathione peroxidase activity; GR – glutathione reductase activity; G6PDH – glucose 6-posphate dehydrogenase activity; GST – glutathione S-transferase activity; MDA content; Ref – reference site; S1 – site 1; S2 – site 2. The dotted circle represents the Ref group and the black line represents the exposed group.

toxicity of combined Al2O3 and ZnO nanoparticles in the fish Carassius auratus. Likewise, Hu et al. (2018) observed higher toxicity in the marine microalga, Isochrysis galbana, exposed to mixtures of Pb and Al2O3, when compared to free lead exposure. Interestingly, our results revealed a slight but significant positive relationship between oyster's uptake of Al and Cd, Cu, Fe, Ni and Zn in the gills of C. gasar, as well as a possible positive correlation between Al and Pb concentrations in the digestive gland. Taken together these findings indicate the influence of Al on the uptake of other metals, especially in the gills, which could be

explained by the putative anionic unbalance on the distribution and number of chloride cells in the this tissue caused by the presence of Al, as previously observed in fish gills (Camargo et al., 2009). Still with regard to the metal bioaccumulation in C. gasar, a temporal trend of increasing levels was observed for Cd and Zn at the end of the exposure period. However, no obvious temporal variation was observed for further elements, which highlights the occurrence of anthropogenic activities responsible for Cd and Zn releases into aquatic environments of LES. This outcome also suggests enhanced releases of Cd and Zn rich

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wastewater during the months of November and December, which might be related to the increased fertilizer use in the rice culture in this period (Silva et al., 2011). However, whether any industrial activities in the area present temporal variations in the release of metalcontaminated effluents needs to be investigated further. 4.2. Biomarker responses in oysters Metal intoxication can accelerate the production of cellular ROS, which is expected to instigate the response of antioxidant system (Wang et al., 2012). The response to oxidative stress involves key antioxidant and ancillary enzymes, such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). However, when ROS generation exceeds the protective defense, oxidative stress occurs resulting in oxidative damage to macromolecules. The evaluation of lipid peroxidation levels has been used as a measure of oxidative stress induced by aquatic pollutants including metals, as previously reported in the mussel M. galloprovincialis after exposure to sublethal concentrations of Cu, Cd, Fe and Pb (Vlahogianni and Valavanidis, 2007), and in the brown mussel Perna perna exposed to Zn (Trevisan et al., 2014), and to Fe, Cu, and Cd (Almeida et al., 2004). Liu and Wang (2016b) also found higher lipid peroxidation levels in C. hongkongensis transplanted to a Chinese estuary polluted by metals. Interestingly, Meng et al. (2017) observed that Cd plays an important role as an indirect inducer of ROS, leading to oxidative stress. Cd is a bivalent cationic metal unable to generate ROS directly, however they can be produced by the replacement of Fenton metals from cysteine-rich proteins by Cd (Casalino et al., 1997). In accordance, Dorta et al. (2003) reported that Cd interacts with specific protein thiols resulting in membrane permeability transition, which is followed by ROS generation along with Fe mobilization leading to lipid peroxidation of the mitochondrial membrane (Cuypers et al., 2010; Ercal et al., 2001). Likely, in our study, oysters bioaccumulated significant concentrations of Cd in both gills and digestive gland, with simultaneous increases of MDA levels in the first two weeks of in situ exposure at the LES. However, the MDA levels significantly decreased at the end of 45-d exposure period, possibly due to the coordinated induction in CAT, and possibly other antioxidant enzymes, resulting in reduced oxidative stress. This finding is further corroborated by the increased CAT activity in the gills, together with the positive correlation found between CAT and MDA contents in the digestive gland. CAT converts hydrogen peroxide (H2O2) to water and oxygen, acting in the first line of the enzymatic defense against oxidative stress induced by environmental metals (Borković-Mitić et al., 2013). Thus, the increased CAT activity detected in the gills of C. gasar indicates higher rates of H2O2 production in oysters over the period of 45-d transplantation at S2. Our results agree with other field studies that have demonstrated significant correlations between metal accumulation and CAT activity in C. angulata (Funes et al., 2006) and M. galloprovincialis (Vidal-Liñán et al., 2010), and an increase in CAT in C. gigas exposed to Cd under laboratory conditions (Jo et al., 2008). Interestingly, a slight, but significant negative correlation between CAT activity and Al levels was observed in the gills of C. gasar. Al has been attributed to controversially alter CAT activity by inhibiting it in human erythrocytes (Zatta et al., 2002), as well as in rats liver incubated with AlCl3 (Chainy et al., 1996), or activating its enzyme activity as reported in the diencephalon of neonatal rats exposed to high levels (35 μg·g−1) of AlCl3 (Yuan et al., 2012). Nevertheless, gills of C. gasar from the LES bioaccumulated even higher levels of Al, ranging from 45 to 476 μg·g−1, which herein possibly resulted in decreased CAT activity. In contrast to the response of CAT, GPx activity did not vary among sites at the LES, but presented strong temporal variations, especially in the gills. GPx catalyzes not only the reduction of hydrogen peroxide into water, but also the degradation of lipid hydroperoxides into corresponding alcohols (Lesser, 2006). In accordance to this, a positively

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correlation was found between GPx and lipid peroxidation in the gills of oysters transplanted to LES compared to Ref site, which indicates a probable increase in ROS production that has led to increased MDA levels. Furthermore, GPx was positively correlated with Zn, which has been known as an element providing protection against oxidative stress (Galazyn-Sidorczuk et al., 2012). Thus, the temporal increase in GPx activity concomitant to higher Zn concentrations might indicate that, to some extent, the enhancing of Zn uptake by the gills possibly enhanced the antioxidant capacity of C. gasar. Indeed, oysters from Ref site presented increased GPx activity, enhanced MDA levels and higher Zn concentrations at the end of the exposure period. Our results agree with the increased GPx activity reported in the gills of C. gigas exposed to Zn under laboratory conditions (Cong et al., 2012). Nevertheless, in digestive gland the temporal increases in GPx activity seemed to likely avoid the formation of lipid hydroperoxides, protecting the cells from oxidative damage, as the MDA levels decreased at 45-d in C. gasar maintained at S2. However, no correlation was observed among metals, GPx and MDA in digestive gland. This outcome indicates either: 1) the gills is a more susceptible organ to metal-induced oxidative stress, as indicated by the substantial alterations in the IBRv2 pattern of oysters from S2; and 2) oysters may have been exposed to some other substances, not included in the scope of this study, capable to induce such response in the digestive gland, in particular at S2. With respect to the ancillary enzymes, GR presented disperse responses, without any clear spatial-temporal relationship. GR is a NADPH-dependent enzyme essential to maintain GPx and GST functions through the regeneration of reduced glutathione (GSH) from its oxidized form (GSSG) (Deponte, 2013). GSH is one of the most important non-enzymatic antioxidant in the cell and increased GR activity is likely an important strategy by organisms to counter oxidative stress. In the present study, GR activity increased after 7-d in digestive gland of oysters kept at S2, possibly as a response to increase the cellular GSH supply, but it decreased at the end of exposure time in both tissues. In addition, the GR activity in gills was slightly positively correlated to Ni concentrations, which suggests the putative role of this element on inducing cellular protection, since GSH is also considered the first line of antioxidant defense. In accordance, Palermo et al. (2015) observed an increase of GR activity along with higher GSH levels in the gills of fish exposed to Ni in laboratory; though, the authors did not find any GR response in the liver. Thus, we can hypothesize that the GR induction in C. gasar exhibited a pivotal role during the putative stress elicited by Ni, which also might indicate an effort to maintain the redox elements to deal with ROS production due to metal occurrence at the LES. G6PDH is the key enzyme responsible for the generation of NADPH, used by GR to maintain GSH levels within the cells (Bainy et al., 1996). Our results showed an increased G6PDH activity in the gills of oysters kept at S1 after 15 and 30 d- exposure, with no significant increases in digestive gland. In addition, G6PDH activity was positively correlated with CAT in gills, and GPx in both gills and digestive gland, which corroborates its role as an ancillary enzyme for the antioxidant response. Our finding is also consistent with those observed by Zacchi et al. (2018) in C. gasar collected in aquaculture areas, suggesting major effects of pollutants in GR and G6PDH in oysters from the most contaminated area. Moreover, increases in GR and G6PDH activities were observed in C. gigas exposed to domestic sewage (Flores-Nunes et al., 2015), an important pollutant source at the LES. Interestingly, the gills G6PDH activity was positively correlated with Cd, Cu and Zn levels, which suggests a potential coordinate role of these metals on such antioxidant response. GST activity exhibited spatial-temporal variations in both gills and digestive gland. GSTs play an essential cellular role by removing ROS products, such as lipid peroxidation by-products, as well as other harmful substances, converting them into non-reactive water-soluble substances to further excretion from the cell (Doyen et al., 2008; Fukuda et al., 1997). Indeed, numerous studies have reported induction of GST activity in clams and mussels exposed to metals (Bao et al., 2016;

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Bonnail et al., 2018; Bouzahouane et al., 2018; Cantú-Medellín et al., 2009) or collected in metal-polluted areas (Fernández et al., 2010; Vidal-Liñán et al., 2014). Contrariwise, a decrease in GST activity in C. hongkongensis exposed to a multi-metal contaminated estuary has been previously associated with increased levels of Cu and Cd (Liu and Wang, 2016b). Similarly, our study also showed a decrease in GST activity in gills of oysters from the LES compared to Ref oysters, which was also slightly inversely correlated to Cd concentrations. We can hypothesize that these changes might be explained by the possible inhibitory effects of metal ions on GST enzyme activity, as was recently reported for fish Chalcalburnus tarichii Pallas exposed in vitro to Cd, Cu, Zn and Ag (Özaslan et al., 2017). Decreased GST activity could increase oyster's susceptibility to negative impacts of pollutants, because it turns the oysters less efficient in biotransformation process, in particular organic pollutants, feasible to occur at the LES. Moreover, an enhanced GST activity was observed in digestive gland compared to gills, which in turn reveals a compensatory mechanism between tissues to protect oysters' cells against oxidative damages. Further, GST activity was positively correlated to four out of seven metals analyzed, reinforcing its potential role in protecting cells against harmful effects of metal exposure. In order to better understand the overall biomarker responses in C. gasar reported for each site at the LES, integrated biomarker response (IBRv2) indexes were generated in this study for each tissue. According to Sanchez et al. (2013), the IBRv2 values ranging from 1.1 to 3.7 are designated for low contaminated sites, whereas those heavily contaminated vary between 5.8 and 10.3. In the present study, IBRv2 was not able to clearly discriminate a pollutant site from another, since S1 and S2 presented very similar scores. However, these values highlight the existence of important biological responses in the oysters transplanted to the LES, which we can postulate to the increased bioaccumulation of Zn and Cd in the oysters' tissues. Although the biochemical analyses and the general IBRv2 values were not able to distinguish which of the two studied sites from the LES was more or less impacted, when compared the IBRv2 values from gills and digestive gland separately, it was clear that oyster's responses were quite different between S1 and S2. These discrepancies point to differences in the characteristics of the contaminants present at each site. It should be mentioned that, despite this study has focused on the relationships between metal bioaccumulation and biomarker responses, possible effects due to other contaminants present in the LES should be not disregarded. As commented previously, main sources of contaminants in LES are derived from agriculture fields (especially rice cultivation), coal mining activity and urban rejects, which could also contribute with numerous other classes of pollutants, such as polycyclic aromatic hydrocarbons, pesticides and numerous other compounds from sanitary or industrial sewages. These other contaminants could be substantially contributing for the alterations seen in the biomarkers analyzed in the oysters, and evidenced by the different IBRv2 patterns between tissues. However, any relationships between other classes of contaminants and biochemical biomarker responses in oysters need to be further investigated. 5. Conclusions Our study revealed the bioaccumulation of metals in transplanted oysters to the Laguna Estuarine System, southern Brazil, and related biological responses of metal pollution on oysters. This study also provided the first evidence of Cd, Zn, Ni, Cu and Al effects on biochemical biomarkers in the gills and digestive gland of C. gasar. The potential toxic effects lead to an activation of the antioxidant system to protect cells against oxidative damages possibly induced by metals. Different responses were observed for gills and digestive gland, as they displayed some variations in the sensitivity of some biomarkers possibly to cope with metal stress. Furthermore, this study provided evidence for the effectiveness of using a multi-biomarker approach in the oyster C. gasar to monitor metal pollution in Brazilian estuarine areas.

Acknowledgments The authors acknowledge the financial support of the National Council for Scientific and Technological Development (CNPq, grant number 428997/2016-3), and the Program to Support Research - Foundation for Research and the State of Santa Catarina Innovation (PAPFAPESC, grant number 2017 TR744) during the present study to K.H. Lüchmann and CAPES/FAPESC for Master's Scholarship to C.P. Ferreira. E.A. de Almeida and A.C.D. Bainy are recipients of CNPq productivity fellowship. They also thank Juliana R. Moser, Flávia L. Zacchi, Bárbara P.H. Righetti and Isis M. Martins for their contributions to the success of this work. E.A. de Almeida and A.C.D. Bainy are recipients of CNPq productivity fellowship. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. https://doi.org/10.1016/ S0076-6879(84)05016-3. Almeida, E.A., Miyamoto, S., Bainy, A.C.D., De Medeiros, M.H.G., Di Mascio, P., 2004. Protective effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals. Mar. Pollut. Bull. 49, 386–392. https://doi.org/10.1016/j.marpolbul.2004.02.020. Attig, H., Dagnino, A., Negri, A., Jebali, J., Boussetta, H., Viarengo, A., Dondero, F., Banni, M., 2010. Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations. Ecotoxicol. Environ. Saf. 73, 1712–1719. https:// doi.org/10.1016/j.ecoenv.2010.08.007. Bainy, A.C.D., Saito, E., Carvalho, P.S.M., Junqueira, V.B.C., 1996. Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site. Aquat. Toxicol. 34, 151–162. https://doi.org/10.1016/0166-445X(95)00036-4. Bao, Y., Liu, X., Zhang, W., Cao, J., Li, W., Li, C., Lin, Z., 2016. Identification of a regulation network in response to cadmium toxicity using blood clam Tegillarca granosa as model. Sci. Rep. 6, 35704. https://doi.org/10.1038/srep35704. Barletta, M., Lima, A.R.A., Costa, M.F., 2019. Distribution, sources and consequences of nutrients, persistent organic pollutants, metals and microplastics in South American estuaries. Sci. Total Environ. 651, 1199–1218. https://doi.org/10.1016/J. SCITOTENV.2018.09.276. Beliaeff, B., Burgeot, T., 2002. Integrated biomarker response: a useful tool for ecological risk assessment. Environ. Toxicol. Chem. 21, 1316–1322. Benavides, M., Fernandez-Lodeiro, P.Coelho, Lodeiro, C., Diniz, M.S., 2016. Single and combined effects of aluminum (Al2O3) and zinc (ZnO) oxide nanoparticles in a freshwater fish, Carassius auratus. Environ. Sci. Pollut. Res. 23 (24), 24578–24591. https://doi. org/10.1007/s11356-016-7915-3. Bonnail, E., Cunha Lima, R., Bautista–Chamizo, E., Salamanca, M.J., Cruz-Hernández, P., 2018. Biomarker responses of the freshwater clam Corbicula fluminea in acid mine drainage polluted systems. Environ. Pollut. 242, 1659–1668. https://doi.org/ 10.1016/j.envpol.2018.07.111. Borković-Mitić, S., Pavlović, S., Perendija, B., Despotović, S., Gavrić, J., Gačić, Z., Saičić, Z., 2013. Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River. Ecol. Indic. 32, 212–221. https://doi.org/10.1016/J.ECOLIND.2013.03.024. Bouzahouane, H., Barour, C., Sleimi, N., Ouali, K., 2018. Multi-biomarkers approach to the assessment of the southeastern Mediterranean Sea health status: preliminary study on Stramonita haemastoma used as a bioindicator for metal contamination. Chemosphere 207, 725–741. https://doi.org/10.1016/j.chemosphere.2018.05.118. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. Camargo, M.M.P., Fernandes, M.N., Martinez, C.B.R., 2009. How aluminium exposure promotes osmoregulatory disturbances in the neotropical freshwater fish Prochilus lineatus. Aquat. Toxicol. 94, 40–46. https://doi.org/10.1016/j.aquatox.2009.05.017. Cantú-Medellín, N., Olguín-Monroy, N.O., Méndez-Rodríguez, L.C., Zenteno-Savín, T., 2009. Antioxidant enzymes and heavy metal levels in tissues of the black chocolate clam Megapitaria squalida in Bahía de La Paz, Mexico. Arch. Environ. Contam. Toxicol. 56, 60–66. https://doi.org/10.1007/s00244-008-9156-z. Capolupo, M., Franzellitti, S., Kiwan, A., Valbonesi, P., Dinelli, E., Pignotti, E., Birke, M., Fabbri, E., 2017. A comprehensive evaluation of the environmental quality of a coastal lagoon (Ravenna, Italy): integrating chemical and physiological analyses in mussels as a biomonitoring strategy. Sci. Total Environ. 598, 146–159. https://doi.org/ 10.1016/J.SCITOTENV.2017.04.119. Carlberg, I., Mannervik, B., 1985. Glutathione reductase. Methods Enzymol. 113, 484–490. https://doi.org/10.1016/S0076-6879(85)13062-4. Casalino, E., Sblano, C., Landriscina, C., 1997. Enzyme activity alteration by cadmium administration to rats: the possibility of iron involvement in lipid peroxidation. Arch. Biochem. Biophys. 346, 171–179. https://doi.org/10.1006/abbi.1997.0197. Chainy, G.B., Samanata, L., Rout, M., 1996. Effect of aluminium on superoxide dismutase, catalase and lipid peroxidation of rat liver. Res. Commun. Mol. Pathol. Pharmacol. 94, 217–220. Chan, C.Y., Wang, W.-X., 2018. A lipidomic approach to understand copper resilience in oyster Crassostrea hongkongensis. Aquat. Toxicol. 204, 160–170. https://doi.org/ 10.1016/j.aquatox.2018.09.011.

C.P. Ferreira et al. / Science of the Total Environment 685 (2019) 332–344 Company, R., Serafim, A., Bebianno, M.J., Cosson, R., Shillito, B., Fiala-Médioni, A., 2004. Effect of cadmium, copper and mercury on antioxidant enzyme activities and lipid peroxidation in the gills of the hydrothermal vent mussel Bathymodiolus azoricus. Mar. Environ. Res. 58, 377–381. https://doi.org/10.1016/j.marenvres.2004.03.083. Cong, M., Wu, H., Liu, X., Zhao, J., Wang, X., Lv, J., Hou, L., 2012. Effects of heavy metals on the expression of a zinc-inducible metallothionein-III gene and antioxidant enzyme activities in Crassostrea gigas. Ecotoxicology 21, 1928–1936. https://doi.org/10.1007/ s10646-012-0926-z. Congresso Nacional, 1965. Decreto no 55871, de 26 de março de 1965, Modifica o Decreto no 50.040, de 24 de janeiro de 1961, referente a normas reguladoras do emprêgo de aditivos para alimentos, alterado pelo Decreto no 691, de 13 de março de 1962. Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., Opdenakker, K., Nair, A.R., Munters, E., Artois, T.J., Nawrot, T., Vangronsveld, J., Smeets, K., 2010. Cadmium stress: an oxidative challenge. BioMetals 23, 927–940. https://doi.org/10.1007/ s10534-010-932. Day, J.W., Yáñez-Arancibia, A., Kemp, W.M., Crump, B.C., 2012. Introduction to estuarine ecology. Estuarine Ecology. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 1–18 https://doi.org/10.1002/9781118412787.ch1. Defarge, N., Spiroux de Vendômois, J., Séralini, G.E., 2018. Toxicity of formulants and heavy metals in glyphosate-based herbicides and other pesticides. Toxicol. Rep. 5, 156–163. https://doi.org/10.1016/j.toxrep.2017.12.025. Deponte, M., 2013. Glutathione catalysis and the reaction mechanisms of glutathionedependent enzymes. Biochim. Biophys. Acta Gen. Subj. 1830, 3217–3266. https:// doi.org/10.1016/J.BBAGEN.2012.09.018. Dorta, D.J., Leite, S., DeMarco, K.C., Prado, I.M.R., Rodrigues, T., Mingatto, F.E., Uyemura, S.A., Santos, A.C., Curti, C., 2003. A proposed sequence of events for cadmiuminduced mitochondrial impairment. J. Inorg. Biochem. 97, 251–257. https://doi.org/ 10.1016/S0162-0134(03)00314-3. Doyen, P., Bigot, A., Vasseur, P., Rodius, F., 2008. Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium-dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 147, 69–77. https://doi.org/10.1016/J. CBPC.2007.08.002. Eichler, P., Castelão, G., Pimenta, F., Eichler, B., Miranda, L., Rodrigues, A., Pereira, E., 2006. Foraminifera and thecamoebians as indicator of hydrodynamic process in a choked coastal lagoon, Laguna estuarine system, SC, Brazil. J. Coast. Res. 1144–1148. Ercal, N., Gurer-Orhan, H., Aykin-Burns, N., 2001. Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539. Fernández, B., Campillo, J.A., Martínez-Gómez, C., Benedicto, J., 2010. Antioxidant responses in gills of mussel (Mytilus galloprovincialis) as biomarkers of environmental stress along the Spanish Mediterranean coast. Aquat. Toxicol. 99, 186–197. https:// doi.org/10.1016/j.aquatox.2010.04.013. Flores-Nunes, F., Mattos, J.J., Zacchi, F.L., Serrano, M.A.S., Piazza, C.E., Sasaki, S.T., Taniguchi, S., Bicego, M.C., Melo, C.M.R., Bainy, A.C.D., 2015. Effect of linear alkylbenzene mixtures and sanitary sewage in biochemical and molecular responses in pacific oyster Crassostrea gigas. Environ. Sci. Pollut. Res. 22, 17386–17396. https://doi.org/ 10.1007/s11356-015-4486-7. Fukuda, A., Nakamura, Y., Ohigashi, H., Osawa, T., Uchida, K., 1997. Cellular response to the redox active lipid peroxidation products: induction of glutathione S-transferase P by 4-hydroxy-2-nonenal. Biochem. Biophys. Res. Commun. 236, 505–509. https://doi. org/10.1006/bbrc.1997.6585. Funes, V., Alhama, J., Navas, J.I., López-Barea, J., Peinado, J., 2006. Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic littoral. Environ. Pollut. 139, 214–223. https://doi.org/10.1016/J.ENVPOL.2005.05.016. Galazyn-Sidorczuk, M., Brzóska, M.M., Rogalska, J., Roszczenko, A., Jurczuk, M., 2012. Effect of zinc supplementation on glutathione peroxidase activity and selenium concentration in the serum, liver and kidney of rats chronically exposed to cadmium. J. Trace Elem. Med. Biol. 26, 46–52. https://doi.org/10.1016/j. jtemb.2011.10.002. Glenn, V.D., 2002. Top 20 hazardous substances from the CERCLA priority list of hazardous substances for 2001. Ref. Rev. 16, 29–30. Glock, G.E., McLean, P., 1953. Further studies on the properties and assay of glucose 6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. J. 55, 400–408. Gonçalves Jr., A.C., Nacke, H., Schwantes, D., Coelho, G.F., 2014. Heavy metal contamination in Brazilian agricultural soils due to application of fertilizers. In: HernandezSoriano, M.C. (Ed.), Environmental Risk Assessment of Soil Contamination. Intech Open. Hu, J., Zhang, Z., Zhang, C., Liu, S., Zhang, H., Li, D., Zhao, J., Han, Z., Liu, X., Pan, J., Huang, W., Zheng, M., 2018. Al2O3 nanoparticle impact on the toxic effect of Pb on the marine microalga Isochrysis galbana. Ecotoxicol. Environ. Saf. 161, 92–98. https://doi.org/ 10.1016/j.ecoenv.2018.05.090. Huang, M., Shaw, I.F., Petering, D.H., 2004. Interprotein metal exchange between transcription factor IIIa and apo-metallothionein. J. Inorg. Biochem. 98, 639–648. https://doi.org/10.1016/j.jinorgbio.2004.02.004. Jaminska, A., Konieczka, P., Skóra, K., Namieśnik, J., 2011. Bioaccumulation of metals in tissues of marine animals, part I: the role and impact of heavy metals on organisms. Pol. J. Environ. Stud. 20 (5), 1117–1125. Jo, P.G., Choi, Y.K., Choi, C.Y., 2008. Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 147, 460–469. https://doi.org/10.1016/ j.cbpc.2008.02.001. Keen, J.H., Habig, W.H., Jakoby, W.B., 1976. Mechanism for the several activities of the glutathione S-transferases. J. Biol. Chem. 251, 6183–6188.

343

Kuranchie-Mensah, H., Teyssié, J.-L., Oberhänsli, F., Tumnoi, Y., Pouil, S., Warnau, M., Metian, M., 2016. Bioconcentration of Ag, Cd, Co, Mn and Zn in the mangrove oyster (Crassostrea gasar) and preliminary human health risk assessment: a radiotracer study. Bull. Environ. Contam. Toxicol. 97, 413–417. https://doi.org/10.1007/s00128016-1825-4. Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiological ecology. Annu. Rev. Physiol. 68, 253–278. https://doi.org/10.1146/annurev. physiol.68.040104.110001. Lima, D., Zacchi, F.L., Mattos, J.J., Flores-Nunes, F., Gomes, C.H.A. de M., de Mello, Á.C.P., Siebert, M.N., Piazza, C.E., Taniguchi, S., Sasaki, S.T., Bícego, M.C., Bebianno, M.J., de Almeida, E.A., Bainy, A.C.D., 2018. Molecular and cellular effects of temperature in oysters Crassostrea brasiliana exposed to phenanthrene. Chemosphere 209, 307–318. https://doi.org/10.1016/j.chemosphere.2018.06.094. Liu, F., Wang, W.X., 2013. Facilitated bioaccumulation of cadmium and copper in the oyster Crassostrea hongkongensis solely exposed to zinc. Environ. Sci. Technol. 47, 1670–1677. https://doi.org/10.1021/es304198h. Liu, F., Wang, W.-X., 2014. Differential influences of Cu and Zn chronic exposure on Cd and Hg bioaccumulation in an estuarine oyster. Aquat. Toxicol. 148, 204–210. https://doi.org/10.1016/j.aquatox.2014.01.014. Liu, X., Wang, W.X., 2016a. Time changes in biomarker responses in two species of oyster transplanted into a metal contaminated estuary. Sci. Total Environ. 544, 281–290. https://doi.org/10.1016/j.scitotenv.2015.11.120. Liu, X., Wang, W.X., 2016b. Antioxidant and detoxification responses of oysters Crassostrea hongkongensis in a multimetal-contaminated estuary. Environ. Toxicol. Chem. 35, 2798–2805. https://doi.org/10.1002/etc.3455. Livingstone, D.R., Lips, F., Martinez, P.G., Pipe, R.K., 1992. Antioxidant enzymes in the digestive gland of the common mussel Mytilus edulis. Mar. Biol. 112, 265–276. Lu, G.Y., Ke, C.H., Zhu, A., Wang, W.X., 2017. Oyster-based national mapping of trace metals pollution in the Chinese coastal waters. Environ. Pollut. 224, 658–669. https://doi.org/10.1016/j.envpol.2017.02.049. Lüchmann, K.H., Mattos, J.J., Siebert, M.N., Granucci, N., Dorrington, T.S., Bícego, M.C., Taniguchi, S., Sasaki, S.T., Daura-Jorge, F.G., Bainy, A.C.D., 2011. Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction. Aquat. Toxicol. 105, 652–660. https://doi.org/10.1016/j. aquatox.2011.09.003. Lüchmann, K.H., Dafre, A.L., Trevisan, R., Craft, J.A., Meng, X., Mattos, J.J., Zacchi, F.L., Dorrington, T.S., Schroeder, D.C., Bainy, A.C.D., 2014. A light in the darkness: new biotransformation genes, antioxidant parameters and tissue-specific responses in oysters exposed to phenanthrene. Aquat. Toxicol. 152, 324–334. https://doi.org/ 10.1016/j.aquatox.2014.04.021. Lüchmann, K.H., Clark, M.S., Bainy, A.C.D., Gilbert, J.A., Craft, J.A., Chipman, J.K., Thorne, M.A.S., Mattos, J.J., Siebert, M.N., Schroeder, D.C., 2015. Key metabolic pathways involved in xenobiotic biotransformation and stress responses revealed by transcriptomics of the mangrove oyster Crassostrea brasiliana. Aquat. Toxicol. 166, 10–20. https://doi.org/10.1016/j.aquatox.2015.06.012. MacDonald, D.D., Ingersoll, C.G., Berger, T.A., 2000. Development and evaluation of consensus-based sediment quality guidelines for freshwater ecosystems. Arch. Environ. Contam. Toxicol. 39, 20–31. https://doi.org/10.1007/s002440010075. McCullagh, P. (Peter), Nelder, J.A., 1989. Generalized Linear Models. Chapman and Hall. Meng, J., Wang, W., Li, L., Yin, Q., Zhang, G., 2017. Cadmium effects on DNA and protein metabolism in oyster (Crassostrea gigas) revealed by proteomic analyses. Sci. Rep. 7, 11716. https://doi.org/10.1038/s41598-017-11894-7. Meng, J., Wang, W.-X., Li, L., Zhang, G., 2018. Tissue-specific molecular and cellular toxicity of Pb in the oyster (Crassostrea gigas): mRNA expression and physiological studies. Aquat. Toxicol. 198, 257–268. https://doi.org/10.1016/j.aquatox.2018.03.010. Ministério da Saúde, 1998. Portaria no 685, de 27 de agosto de 1998. Mukaka, M.M., 2012. Statistics corner: a guide to appropriate use of correlation coefficient in medical research. Malawi Med. J. 24, 69–71. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. https://doi.org/ 10.1146/annurev.arplant.49.1.249. Özaslan, M.S., Demir, Y., Küfrevioğlu, O.I., Çiftci, M., 2017. Some metals inhibit the glutathione S-transferase from Van Lake fish gills. J. Biochem. Mol. Toxicol. 31, e21967. https://doi.org/10.1002/jbt.21967. Palermo, F.F., Risso, W.E., Simonato, J.D., Martinez, C.B.R., 2015. Bioaccumulation of nickel and its biochemical and genotoxic effects on juveniles of the neotropical fish Prochilodus lineatus. Ecotoxicol. Environ. Saf. 116, 19–28. https://doi.org/10.1016/j. ecoenv.2015.02.032. Pessatti, T.B., Lüchmann, K.H., Flores-Nunes, F., Mattos, J.J., Sasaki, S.T., Taniguchi, S., Bícego, M.C., Dias Bainy, A.C., 2016. Upregulation of biotransformation genes in gills of oyster Crassostrea brasiliana exposed in situ to urban effluents, Florianópolis Bay, Southern Brazil. Ecotoxicol. Environ. Saf. 131, 172–180. https://doi.org/10.1016/j. ecoenv.2016.04.003. Prakash, N.T., Rao, K.S., 1995. Modulations in antioxidant enzymes in different tissues of marine bivalve Perna viridis during heavy metal exposure. Mol. Cell. Biochem. 146, 107–113. https://doi.org/10.1007/BF00944602. Rodney, E., Herrera, P., Luxama, J., Boykin, M., Crawford, A., Carroll, M.A., Catapane, E.J., 2007. Bioaccumulation and tissue distribution of arsenic, cadmium, copper and zinc in Crassostrea virginica grown at two different depths in Jamaica Bay, New York. In Vivo 29, 16–27. Rodriguez-Iruretagoiena, A., Fdez-Ortiz de Vallejuelo, S., Gredilla, A., Ramos, C.G., Oliveira, M.L.S., Arana, G., de Diego, A., Madariaga, J.M., Silva, L.F.O., 2015. Fate of hazardous elements in agricultural soils surrounding a coal power plant complex from Santa Catarina (Brazil). Sci. Total Environ. 508, 374–382. https://doi.org/10.1016/j. scitotenv.2014.12.015.

344

C.P. Ferreira et al. / Science of the Total Environment 685 (2019) 332–344

Sanchez, W., Burgeot, T., Porcher, J.-M., 2013. A novel “Integrated Biomarker Response” calculation based on reference deviation concept. Environ. Sci. Pollut. Res. 20, 2721–2725. https://doi.org/10.1007/s11356-012-1359-1. Silva, A.P., Santos, A.C., Dreyer, J.P., Prudêncio, J.M., Ferrarini, M.A.Z., Ferreira, V.C., Fonseca, A., 2011. Efeito da rizicultura sobre as características físico-químicas e dos nutrientes inorgânicos dissolvidos na coluna d'água ao longo do Rio da Madre (SC, Brasil). Conference: XXIII Semana Nacional de Oceanografia. Silva, L.F.O., Fdez-Ortiz de Vallejuelo, S., Martinez-Arkarazo, I., Castro, K., Oliveira, M.L.S., Sampaio, C.H., de Brum, I.A.S., de Leão, F.B., Taffarel, S.R., Madariaga, J.M., 2013. Study of environmental pollution and mineralogical characterization of sediment rivers from Brazilian coal mining acid drainage. Sci. Total Environ. 447, 169–178. https:// doi.org/10.1016/j.scitotenv.2012.12.013. Spearman, C., 2010. The proof and measurement of association between two things. Int. J. Epidemiol. 39, 1137–1150. https://doi.org/10.1093/ije/dyq191. Trevisan, R., Flesch, S., Mattos, J.J., Milani, M.R., Bainy, A.C.D., Dafre, A.L., 2014. Zinc causes acute impairment of glutathione metabolism followed by coordinated antioxidant defenses amplification in gills of brown mussels Perna perna. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 159, 22–30. https://doi.org/10.1016/j. cbpc.2013.09.007. Vidal-Liñán, L., Bellas, J., Campillo, J.A., Beiras, R., 2010. Integrated use of antioxidant enzymes in mussel. Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain). Chemosphere 78, 265–272. Vidal-Liñán, L., Bellas, J., Etxebarria, N., Nieto, O., Beiras, R., 2014. Glutathione Stransferase, glutathione peroxidase and acetylcholinesterase activities in mussels transplanted to harbour areas. Sci. Total Environ. 470–471, 107–116. https://doi. org/10.1016/j.scitotenv.2013.09.073.

Vlahogianni, T.H., Valavanidis, A., 2007. Heavy-metal effects on lipid peroxidation and antioxidant defence enzymes in mussels Mytilus galloprovincialis. Chem. Ecol. 23, 361–371. https://doi.org/10.1080/02757540701653285. Wang, W.X., Lu, G., 2017. Heavy metals in bivalve mollusks. Chemical Contaminants and Residues in Food, Second edition Elsevier, pp. 553–594 https://doi.org/10.1016/B9780-08-100674-0.00021-7. Wang, Z., Yan, C., Vulpe, C.D., Yan, Y., Chi, Q., 2012. Incorporation of in situ exposure and biomarkers response in clams Ruditapes philippinarum for assessment of metal pollution in coastal areas from the Maluan Bay of China. Mar. Pollut. Bull. 64, 90–98. https://doi.org/10.1016/j.marpolbul.2011.10.017. Wang, W.-X., Meng, J., Weng, N., 2018. Trace metals in oysters: molecular and cellular mechanisms and ecotoxicological impacts. Environ. Sci. Processes Impacts 20, 892–912. https://doi.org/10.1039/C8EM00069G. Wendel, A., 1981. Glutathione peroxidase. Methods Enzymol. 77, 325–333. https://doi. org/10.1016/S0076-6879(81)77046-0. Yuan, C.Y., Lee, Y.J., Hsu, G.S., 2012. Aluminum overload increases oxidative stress in four functional brain areas of neonatal rats. J. Biomed. Sci. 21, 19–51. https://doi.org/ 10.1186/1423-0127-19-51. Zacchi, F.L., Flores-Nunes, F., Mattos, J.J., Lima, D., Lüchmann, K.H., Sasaki, S.T., Bícego, M.C., Taniguchi, S., Montone, R.C., de Almeida, E.A., Bainy, A.C.D., 2018. Biochemical and molecular responses in oysters Crassostrea brasiliana collected from estuarine aquaculture areas in Southern Brazil. Mar. Pollut. Bull. 135, 110–118. https://doi.org/ 10.1016/J.MARPOLBUL.2018.07.018. Zatta, P., Kiss, T., Suwalsky, M., Berthon, G., 2002. Aluminum (III) as promoter of cellular oxidation. Coord. Chem. Rev. 228, 271–284. https://doi.org/10.1016/S0010-8545 (02)00074-7.