Comparative Biochemistry and Physiology, Part C 172–173 (2015) 36–44

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The influence of zinc chloride and zinc oxide nanoparticles on air-time survival in freshwater mussels François Gagné a,⁎, Joëlle Auclair a, Caroline Peyrot b, Kevin J. Wilkinson b a b

Emerging Methods Aquatic Contaminants Research Division, Water Science and Technology, Environment Canada, 105 McGill, Montréal, QC H2Y 2E7, Canada Département de Chimie, Université de Montréal, Montréal, QC H2V 2B8, Canada

a r t i c l e

i n f o

Article history: Received 21 October 2014 Received in revised form 22 April 2015 Accepted 29 April 2015 Available online 7 May 2015 Keywords: Elliptio complanata Air-time survival inflammation Oxidative stress

a b s t r a c t The purpose of this study was to determine the cumulative effects of exposure to either dissolved zinc or nanozinc oxide (nanoZnO) and air-time survival in freshwater mussels. Mussels were exposed to each forms of zinc for 96 h then placed in air to determine survival time. A sub-group of mussels before and after 7 days of exposure to air were kept aside for the determination of the following biomarkers: arachidonate-dependent cyclooxygenase (COX) and peroxidase (inflammation and oxidative stress), lipid metabolism (total lipids, esterases activity, HO-glycerol, acetyl CoA and phospholipase A2) and lipid damage (lipid peroxidation [LPO]). The results showed that air-time survival was decreased from a mean value of 18.5 days to a mean value of 12 days in mussels exposed to 2.5 mg/L of nanoZnO although it was not lethal based on shell opening at concentrations below 50 mg/L after 96 h. In mussels exposed to zinc only, the median lethal concentration was estimated at 16 mg/L (10–25 95% CI). The air-time survival did not significantly change in mussels exposed to the same concentration of dissolved Zn. Significant weight losses were observed at 0.5 mg/L of nanoZnO and at 2.5 mg/L for dissolved zinc chloride, and were also significantly correlated with air-time survival (r = 0.53; p b 0.01). Air exposure significantly increased COX activity in control mussels and in mussels exposed to 0.5 mg/L of nanoZnO and zinc chloride. The data also suggested fatty acid breakdown and β-oxidation. Mussels exposed to contaminants are more susceptible to prolonged exposure to air during low water levels. Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.

1. Introduction Nanotechnologies are becoming a major consumer product. For example, they are found in optical devices (e.g. solar panels), microelectronic circuits, drug delivery systems, and personal care products such as textiles, creams and sunscreens (Lee et al., 2008). The commercial interests in nanoproducts reside in their high ratio of surface area to volume, which brings about new and enhanced quantum properties. Consequently, consumer products and cosmetics represent a major area of economic growth for nanotechnology in most countries (NCI, 2006). However, legitimate concerns have been raised by the public and regulatory community about nanotechnology's safety for the human population and environment. For example, in an effort to decrease the risk of developing skin cancers from sun exposure, cream-based products composed of zinc oxide show strong UV-absorbing properties compared to other metal oxides, and have been promoted as a broad spectrum sunscreen over the last decade (Mitchnick et al., 1999). Nanoparticles of zinc oxide (nanoZnO) have the additional advantage of being transparent compared to zinc oxide-based creams, which appear as a white paste. NanoZnO also has anti-microbial properties which is considered an additional ⁎ Corresponding author. E-mail address: [email protected] (F. Gagné).

http://dx.doi.org/10.1016/j.cbpc.2015.04.005 1532-0456/Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.

advantage for consumers. Hence, the increasing use of these sunscreens could result in the release of nanoZnO and dissolved zinc if they degrade to the water column during swimming or via wastewaters. Currently, the impacts of nanoparticles on aquatic biota are not well understood; the low solubility of zinc oxide suggests that it will partition in the benthic environment at the sediment/water interface. Most freshwater mussels live in the benthic environment, where, typically, they are partly buried in sediment. They are sessile, live for long periods, and feed on suspended material in the water column. The breathing/feeding behavior of mussels makes them species vulnerable to contaminants absorbed to fine particles or associated to fine suspended matter such as nanoparticles (Canesi et al., 2012). Indeed, the gills possess ciliary activity and act as a primordial mouth where trapped particles are directed in the digestive gland. In a recent study, exposure of marine mussels Mytilus galloprovincialis to copper nanoparticles led to the accumulation of copper in the digestive gland and produced toxicity such as lipid oxidation (Gomes et al., 2012). In another study, exposure of the oyster Crassostrea gigas to nanoZnO led to zinc accumulation in gills within the first 24 h followed by the digestive gland (24 to 48 h), which is consistent with the feeding behavior of mussels. Ultrastructural electron microscopy and biochemical analyses revealed signs of mitochondrial disruption and oxidative stress (decreased thiols and increased lipid peroxidation [LPO]) in both tissues (Trevisan et al., 2014). In a recent study,

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exposure to zinc nanoparticles (3 μM) to freshwater mussels Unio tumidus increased metallothionein (MT) levels and induced oxidative stress as with oysters. This response was also exacerbated by increase in water temperature at 25 °C (Falfushynska et al., 2015). Both marine and freshwater mussels have the capacity to survive for long periods in air, from days to weeks depending on conditions such as temperature and seasonal period. In stressful situations, mussels can close their shells for days or weeks, which represents an important survival mechanism for these organisms. It is also anticipated that, in the context of climate change, warmer temperatures could result in lower water levels (droughts) in some areas, increasing exposure to air and higher temperatures (Kim et al., 2014). Air-time survival is well-studied in marine organisms (Viarengo et al., 1995; Hellou and Law, 2003), but virtually no information exists for freshwater mussels. In marine organisms, exposure to air for long periods leads to anoxic conditions and restricted food intake. Moreover, long emersion times of blue mussels leads to increased gene expression involved in energetic metabolism, protein chaperoning and maintenance of the cytoskeleton in cells (Letendre et al., 2011). They also showed that the presence of some contaminants could impact the same genes, which could have cumulative deleterious effects on mussels. During prolonged exposure to air, mussels are known to adopt a gaping strategy, which is a behavioral compromise between air intake and of drying tissues. Air exposure also led to increased acid phosphatase in blue mussels' hemocytes, indicating the presence of bacterial activity and leading to the liberation of phosphates from proteins (Kuchel et al., 2010). During that time (air exposure), important physiological stresses are at play where mussels must endure for survival. Fasting will lead to depleted energy stores involving lipid mobilization, where anoxic/dry conditions could lead to loss of energy consumption and inflammation. The purpose of this study, therefore, was to examine the cumulative toxicity of nanoZnO, dissolved zinc and aerial exposure to the freshwater unionid Elliptio complanata. We first determined the acute lethal concentration of zinc oxide nanoparticles and dissolved zinc chloride for comparison purposes at 15 °C for 96 h. Secondly, we exposed mussels to low doses of zinc oxide nanoparticles and zinc chloride for 96 h at 15 °C followed by exposure in air to determine the air time survival. A group of mussels were collected at day 7 to determine the physiological stress using a test battery of effect biomarkers known to respond to either zinc- or metal-based nanoparticles and air emersion (Gagné et al., 2008; Canesi et al., 2012). Special attention was given to lipid metabolism, oxidative stress and inflammation, which readily respond to air emersion. Lipid metabolism was determined by measuring total tissue lipids and markers of lipid mobilization, by following changes in general esterase activity (lipase), phospholipase 2 (PLA2), which is involved in the hydrolysis of carbon-2 fatty acids or inflammation precursors (arachidonic acid) on the glycerol backbone of phospholipids, and levels of glycerol from the hydrolysis of phospholipids. Our attention focused on the effects of nanoZnO and dissolved zinc on the coping mechanisms during air emersion.

preparations. Control mussels were exposed to aquarium water only. Twenty mussels per 20 L container lined with polyethylene bags were used in duplicates and exposed for 96 h at 15 °C. At the end of the exposure period, mortality (shell opening) was recorded. Because mussels have the capacity to close their shells in stressful conditions for many days if not weeks, the air-time survival was used to determine the capacity of mussels to resist emersion from water for long periods (Viarengo et al., 1995). The air-time survival was therefore determined for up to 28 days in air at 20 °C under humidified atmosphere (80%) with N = 20 mussels per treatment. Mortality was determined by sustained shell opening, recorded every day for 28 days. A subsample of surviving mussels (N = 5) were also removed after exposure to zinc for 96 h and following exposure to air for 7 days (N = 5) for stress biomarker analysis. The proportions of weight loss were determined by the following formula: % weight loss = ((mussel weight at the end of exposure to air / mussel weight before exposure to air) × 100) − 100.

2. Materials and methods

The levels of total lipids were determined in soft tissue homogenate using the phosphovanillin method (Frings et al., 1972). Calibration was achieved with Triton X-100 instead of olive oil as the standard. The data were expressed as mg lipid equivalents/mg protein. Non-specific esterase (lipases) activity was determined using the carboxy-fluorescein diacetate methodology. Briefly, 50 uL of the S12 fraction of the visceral mass was mixed with 100 μL of 10 μM of carboxy-fluorescein diacetate in 50 mM of Tris–HCl, pH 7.4, containing 0.1% dimethyl sulfoxide. The release of fluorescein was measured at 485 nm excitation and 520 nm emission at 0, 10, 20 and 30 min at 30 °C. LPO was determined in the visceral mass homogenates by the thiobarbituric acid method (Wills, 1987). Thiobarbituric acid reactants (TBARS) were determined by fluorescence at 530 nm for excitation and 630 nm for emission using a fluorescent microplate reader. Standard solutions of tetramethoxypropane were used for calibration. The data were expressed as μg TBARS/mg proteins in the visceral mass.

2.1. Exposure of freshwater mussels to zinc chloride and zinc oxide nanoparticles Freshwater mussels were collected in June at a pristine Laurentian Lake located north of the city of Montreal (QC, Canada). Mussels between 6–9 cm were collected and were transported back to the laboratory in iceboxes. Mussels were then transferred in 300-L tanks supplied with charcoal filter and UV-treated tap water of the City of Montréal maintained at 15 °C under constant aeration. They were fed with commercial coral reef solution enriched with 100 million/mL of Pseudokirchneriella subcapitata algae every 2 days. For determination of the acute lethality based on shell opening, E. complanata mussels were exposed to increasing concentrations of total zinc equivalents (0.5, 5, 25 and 50 mg/L), from either zinc chloride or nanoZnO

2.2. Zinc oxide nanoparticle characterization NanoZnO stock solution was purchased from Sigma Aldrich (# 721077) at 50% w/v at pH 7.1. The nanoZnO particles were capped with cationic 3-aminopropyl triethoxysilane to ensure stability in the stock solution. The nanoparticles were then diluted at 0.5, 2.5 and 10 mg/L in aquarium water, which was charcoal- and UV-treated tap water from the City of Montréal (pH 7.8, conductivity of 270 ± 10 μS × cm− 1, and total organic carbon content of 1 mg/L). Particle size was determined by a dynamic light scattering (DLS) instrument with a gel electromobility option (Wyatt-Instrument Mobius, 532-nm laser). Zeta potential was determined from gel mobility data as described in Domingos et al., 2013. 2.3. Tissue preparation for biomarker analyses Biomarkers of stress were determined in mussels exposed to increasing concentrations of either nanoZnO or zinc chloride for 96 h before and after air emersion for 7 days. Mussel weight and shell length were determined before and after exposure to each form of zinc, and after 7 days of exposure to air. At the end of the exposure to air, the mussels were placed on ice and the soft tissues were removed and weighted. The visceral mass homogenate was prepared by adding 5 volumes of ice-cold homogenization buffer, composed of 50 mM of NaCl, 25 mM of Tris–HCl, pH 7.5, 10 μg/mL of apoprotinin and 1 mM of dithiothreitol. A portion of the homogenate was centrifuged at 12,000 ×g for 20 min at 4 °C, and the supernatant (S12 fraction) was collected and stored at −85 °C. Total proteins were determined using the Coomassie Brillant blue dye binding assay (Bradford, 1976). Standard solutions of serum bovine albumin were used for calibration. 2.4. Lipid status and metabolism

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PLA2 activity was determined using the EnzChek phospholipase A2 assay kit (Life Technologies, Burlington, Ontario, Canada). Briefly, 50 μL of S12 fraction was mixed with one volume of the substrateliposome reagent and incubated at room temperature for 10 min. Fluorescence readings were taken at 0 and 10 min at 460 nm excitation and 515 nm emission (Synergy 4, Biotek microplate reader). Data were expressed as PLA2 units/mg of proteins. The activity of arachidonicdependent cyclooxygenase (COX) activity was also determined in the visceral mass homogenates using the peroxidase dichlorofluorescein detection reagent for hydrogen peroxide (Gagné, 2014). Briefly, 50 μL of the S12 fraction of the visceral mass homogenate was mixed with 150 μL of the assay mixture composed of 50 μM of arachidonate, 2 μM of dichlorofluorescein, and 0.1 μg/mL of horseradish peroxidase in 50 mM of Tris–HCl buffer, pH 8, containing 0.05% Tween 20. The reaction mixture was incubated for 0, 10, 20 and 30 min at 30 °C, and fluorescence was measured at 485 nm for excitation and 520 nm for emission using a Bioscan (USA) microplate reader. The data were expressed as increase in relative fluorescence units/(min × mg proteins). The levels of HO-glycerol were determined in the lipid fraction of visceral mass homogenate using H+ nuclear resonance magnetic (NMR) spectroscopy. The lipid fraction of the homogenate was prepared by adding 3 volumes of dichloromethane-methanol (3:1) to mussel soft tissue homogenates and mixed for 30 min. The mixture was centrifuged at 3000 ×g for 5 min to separate the phases. The organic phase was removed and evaporated under nitrogen stream. The material was resuspended in 200 μL of deuterated chloroform containing 1% tetramethylsilane (TMS) as the internal reference. A volume of 40 μL was injected into a miniature high-resolution NMR instrument equipped with a 45 MHz magnet (PicoSpin, benchtop NMR instrument, Cole Parmer, USA). The resonance line for the hydrogen of the OHgroups in glycerol (4.7 ppm relative to TMS) was used for quantitation (peak height). The data were expressed in relative peak height/mg of proteins. The breakdown of lipids was further examined by measuring the levels of acetyl CoA using a commercial assay kit (MAK039, Sigma Aldrich, Ontario, Canada). The S12 samples were deproteinized using an Amicon-10 kDa cutoff spin column. A volume of 50 μL of the filtrate was mixed with one volume of the reaction mix. Samples were incubated for 10 min at 37 °C. Fluorescence readings were taken at 540 nm excitation and 600 nm emission using a microplate reader (Synergy 4, Biotek Instrument). Standard preparations of acetyl CoA were used for calibration. The data were expressed as nmole of acetyl CoA/mg of proteins.

3. Data analysis Freshwater mussels (N = 20 per treatment tank) were exposed to increasing concentrations of zinc chloride and nanoZnO in duplicate for 96 h at 15 °C. Mussels were kept aside and exposed for 7 days in air. The normality and homogeneity of variance were tested with the Shapiro–Wilk and Bartlett tests, respectively. Data were subjected to an analysis of variance (ANOVA) and critical differences between treatment groups and controls (zinc concentrations before and after air exposure for 7-days) were determined by the Least Significant Difference tests, to highlight changes between the zinc exposure concentrations before and after 7-day air-time exposure and differences between exposure concentration and after 7-days of air exposure. Correlation analysis was achieved using the Pearson-moment procedure. Discriminant function and factorial analysis were performed to seek out biomarkers that best discriminate between zinc exposure (96 h) and air-time exposure (= 7 days in air). The determination of the median lethal concentration that kills 50% of mussels were determined by regression analysis. Significance was set at α = .05 and the Statistica (version 8) software was used.

4. Results 4.1. Zinc oxide nanoparticle characteristics The zinc oxide nanoparticles were obtained from a stock solution in distilled water at 50% weight/volume. They were coated with 3-aminopropyl triethoxysilane, giving a net positive charge at the surface of the particle, which prevents the formation of aggregates in water. Analysis of the commercial sample in bidistilled water revealed a mean diameter of 59.7 ± 4.7 nm, which is technically close to the manufacturer's specifications (50-nm mean diameter). The physical and chemical properties of nanoZnO in the exposure water are described in Table 1. The aquarium water was obtained from dechlorinated and UV-treated tap water from the City of Montréal, with the following characteristics: pH 7.8, conductivity of 270 ± 20 μS × cm−1, and total organic carbon content of 1 ± 0.5 mg/L. The aquarium water could neutralize the surface charge of nanoZnO and initiate aggregation. Indeed, the mean size of the nanoparticles jumped to 409 ± 114 nm at the lowest concentration (0.5 mg/L), and increased with the exposure concentrations ending at 1781 ± 102 nm with 10 mg/L of nanoZnO. Neither the Zeta potential of particles nor the electrophoretic mobility was significantly affected by the nanoZnO concentration. Hence, nanoZnO forms aggregates in a dose-dependent manner in aquarium water composed of tap water. The actual concentration of Zn was also determined through ICP-mass spectrometry and corresponded to the 85–95% of the nominal Zn concentrations for either nanoZnO or ZnCl2. The data were reported as nominal total Zn concentrations.

4.2. Exposure of zinc chloride and zinc oxide nanoparticles to mussels Mussels were exposed to increasing concentrations of zinc in the form of zinc chloride (dissolved) and nanoZnO for 96 h at 18 °C. The Lethal Concentration 50 (LC50) for zinc chloride was estimated at 16 mg/L (10–25 mg/L, 95% confidence interval) where no mortality occurred at concentrations b10 mg/L. For nanoZnO, no toxicity based on shell opening of mussels were observed at concentrations of 10, 25 and 50 mg/L. Hence, for mussels, zinc chloride appeared to be more toxic than nanoZnO. The capacity of mussels to maintain closed shells was determined by the air-time survival test using sublethal zinc concentrations (Fig. 1A). Indeed, exposure of mussels to low concentrations of Zn did not elicit changes in shell opening behavior as mussels were freely breathing at 0.5 and 2.5 mg/L of zinc. The data are expressed as total zinc for zinc chloride and nanoZnO here and for the following figures. The air-time survival was significantly reduced from a mean of 18.5 days in air to a mean of 12 days in mussels exposed to 2.5 mg/L of nanoZnO. Although the air-time survival was reduced from a mean of 18.5 days to a mean of 15 days in the 2.5 mg/L of zinc chloride group, the difference was not statistically significant (p N 0.05). This suggests that although the acute lethality of zinc chloride is higher than nanoZnO for E. complanata mussels, the latter is more potent in decreasing air-time survival. We also determined the mussel weight/shell length ratio in mussels at day 7 exposure to determine whether weight loss could predict irreversible mussel shell opening (death) (Fig. 1B). In mussels exposed to zinc chloride, the weight loss was significantly lower in the 2.5 mg/L exposure group. In mussels exposed to nanoZnO, significant weight loss was observed at 0.5 and 2.5 mg/L of total zinc equivalent. We measured total lipid content normalized either with shell length or soft tissue weights and found no significant effects, indicating that weight loss was not associated by lower total lipid content in mussels. Total proteins normalized against shell length but not with soft tissues wet weight were significantly lower. The weight loss was significantly correlated with the air-time survival for dissolved zinc (r = 0.46; p b 0.001) and nanoZnO (r = 0.55; p b 0.01), suggesting that weight loss is associated with lower air-time survival. A comparison between the

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Table 1 Characterization of zinc oxide (ZnO) nanoparticles. Sample

Mean size (nm)

Zeta potential mV

Electrophoretic mobility (μm cm/s V)

0.5 mg/L (aquarium water) 2.5 mg/L (aquarium water) 10 mg/L (aquarium water)

409 ± 114 629 ± 61 1781 ± 102

−18.96 ± 2.18 −21.98 ± 0.82 −22.62 ± 0.66

−1.32 ± 0.15 −1.53 ± 0.06 −1.57 ± 0.04

Mean particle size and Zeta analyses were determined by dynamic light scattering (DLS). Electrophoretic mobility was determined on agarose gel electrophoresis as described in Materials and methods.

24

A

Mean Standard error 22

Air time survival (days)

20

18

16

* 14

12

all zinc exposure groups. Peroxidase activity was negatively correlated with COX activity (r = −0.33; p b 0.05). The effects of zinc and air-time exposure were also examined at the level of lipid metabolism. General lipid hydrolysis was determined by following the activity of non-specific esterase (Est) in the visceral mass of mussels (Fig. 4). In mussels exposed to dissolved zinc, the only significant increase in Est activity occurred in mussels exposed to 2.5 mg/L of zinc after 7 days of air exposure compared to mussels exposed to zinc before air exposure. No changes were observed in mussels exposed to nanoZnO. However, Est activity was significantly correlated with COX activity in mussels (r = 0.67; p b 0.001) in mussels, which indicates a contribution of Est activity in inflammation. The levels of acetyl CoA were determined in mussels to ascertain the metabolic breakdown of lipids though β-oxidation, given that mussels were fasting during the 96 h exposure to zinc and 7 days of air exposure, i.e., a total of 11 days without food intake. Air exposure increased acetyl CoA levels in control mussels. In mussels exposed to dissolved zinc, the levels of acetyl CoA were increased at 2.5 mg/L of zinc (Fig. 5A). In mussels exposed to nanoZnO, acetyl CoA levels were increased for all zinc exposure concentrations (Fig. 5B). The levels of acetyl CoA were increased following 7 days in air in controls, and this increase was abolished in the presence of nanoZnO. The levels of glycerol were determined in mussels by NMR spectroscopy, which determines the hydrogen from the OH group (Fig. 6). Exposure to air, and neither nanoZnO nor dissolved zinc, significantly increased HO-glycerol levels. In mussels exposed to dissolved zinc, hydroxyl levels were not significantly affected by zinc concentration (Fig. 6A). However, the levels of glycerol were significantly higher in mussels exposed to 2.5 mg/L of zinc after 7 days of air exposure compared to either zinc-treated mussels before air exposure or in control mussels after 7 days of exposure. In mussels exposed to nanoZnO, HOglycerol levels also increased in mussels exposed to both concentrations of zinc following air exposure. The activity of PLA2 was also determined in mussels treated to both forms of zinc and air exposure (Fig. 7). Exposure to air also led to increased PLA2 activity in control mussels. The

B Mussel weight to shell length ratio (g/mm)

correlation coefficients for dissolved zinc and nanoZnO did not significantly differ. After the 96 h of exposure to each form of zinc, a sub-group of mussels was kept for 7 additional days in air, to understand the cumulative effects of zinc exposure for 4 days (96 h) and of air exposure. The activity of arachidonic acid COX was determined in the visceral mass of mussels after 96 h of exposure to both forms of zinc and after air exposure for 7 days (Figs. 2A and B). In mussels exposed to dissolved zinc, the levels of COX activity did not change. After 7 days in air, COX activity was readily increased with no apparent additive effects of zinc. Indeed, COX activity was increased approximately 1.3-fold in mussels exposed to air for 7 days, regardless of the zinc exposure concentration. In mussels exposed to nanoZnO, COX activity increased with the exposure concentration of total zinc, reaching 1.4-fold induction at the highest zinc concentration (2.5 mg/L). Air exposure increased COX activity for the controls and 0.5 mg/L zinc as dissolved zinc, but this was lost for the highest exposure group. Oxidative stress was further examined by following changes in LPO and peroxidase activity in mussels (Fig. 3A to D). In mussels exposed to dissolved zinc, no change in LPO was observed. Air exposure did not change LPO in control mussels, but LPO was readily increased in mussels exposed to zinc (0.5 and 2.5 mg/L) followed by air exposure (Fig. 3A). In mussels exposed to nanoZnO, the exposure to the nanoparticles did not increase LPO in mussels or in mussels treated to air exposure (Fig. 3B). LPO actually significantly dropped in mussels exposed to 2.5 mg/L zinc and air for 7 days compared to mussels exposed to 2.5 mg/L of zinc only. LPO levels were significantly correlated with COX activity, albeit weakly (r = 0.30; p b 0.05). In mussels exposed to dissolved zinc, peroxidase activity was not affected (Fig. 3C). Peroxidase activity was significantly reduced in the controls after 7 days of air emersion compared to the controls (i.e. before air emersion). In mussels exposed to nanoZnO, neither the exposure to zinc nor the air exposure alone produced changes in peroxidase activity (Fig. 3D). However, peroxidase activity was significantly reduced after 7 days of exposure in air compared to

0,62 Mean Standard error

0,60 0,58 0,56

*

0,54

*

0,52

*

0,50 0,48 0,46 0,44 0,42 control

0.5 mg/L ZnCl2

2.5 mg/L ZnCl2

0.5 mg/L nZnO

2.5 mg/L nZnO

10 Control

0.5 mg/L ZnCl2

2.5 mg/L ZnCl2

0.5 mg/L nZnO

Exposure concentration (mg/L)

2.5 mg/L nZnO

Exposure treatment (mg/L)

Fig. 1. Decreased air-time survival in mussels exposed to zinc chloride and zinc oxide nanoparticles. The air-time survival represents the number of days required to die as evidenced by constant shell opening (A). Mussel weight to shell length was also measured after 7 days (B). The data represent the mean with standard error (n = 8). The * indicates a significant difference from the controls at α = 0.05.

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F. Gagné et al. / Comparative Biochemistry and Physiology, Part C 172–173 (2015) 36–44

B

1400 Mean

Stand. error

c

1300

b,c

1200

c

1100

1000

900

800

1400

COX activity (relative fluorescein units/min/mg proteins)

COX activity (relative fluorescein units/min/mg proteins)

A

Mean

1300

a

Stand. error

c c

1200

1100

1000

900

800

700

Control

+7-d air

0.5

+ 7-d air

2.5

700

+ 7-d air

Control

+ 7-d air

0.5

ZnCl2 (mg/L)

+ 7-d air

2.5

+ 7-d air

NanoZnO

Fig. 2. Cumulative effects of zinc and air exposure on COX activity in mussels. Mussels were exposed to zinc chloride (A) and nanoZnO (B) for 96 h at 15 °C followed by air exposure for 7 days. COX activities were determined in the visceral mass in mussels before (96 h) and after exposure to air (7 days). The data are expressed as the mean with standard error (n = 8). A significant difference between the controls and zinc concentrations is denoted by “a”; a significant difference between the controls and 96 h exposure to zinc after 7 days in air is denoted by “b”; and “c” indicates a significant difference before and after exposure to air for 7 days.

increase in PLA2 was abolished when dissolved zinc was present. In mussels treated with nanoZnO, a significant increase in PLA2 was observed for the highest concentration tested (2.5 mg/L). Following

A

2,8 Mean

B

b, c

Stand. error

treatment to air exposure, PLA2 was significantly decreased at 0.5 mg/L of zinc. However, PLA2 activity was significantly correlated with COX activity in mussels (r = 0.39; p b 0.01). Analysis of covariance

Mean

Stand. error

2,6

2,6

2,4

b,c 2,2 2,0 1,8 1,6

Lipid peroxidation (ug TBARS/mg proteins)

2,4

Lipid peroxidation (ug TBARs/mg proteins)

2,8

2,2

2,0

1,8

1,6

1,4

1,4

1,2

1,2

c

1,0

1,0

Control

+ 7-d air

0.5

+ 7-d air

2.5

Control

+ 7-d air

+ 7-d air

0.5

C

0,20 Mean

D

Stand. error

2.5

+ 7-d air

0,20

Mean

Stand. error

0,18

0,18

Peroxidase activity (relative units/min/mg proteins)

Peroxidase activity (relative units/min/mg proteins)

+ 7-d air

NanoZnO (mg/L)

ZnCl2 (mg/L)

0,16

0,14

c 0,12

0,10

0,16

c

0,14

c

c

0,12

0,10

0,08

0,08

Control

+ 7-d air

0.5

+ 7-d air

ZnCl2 (mg/L)

2.5

+ 7-d air

Control

+ 7-d air

0.5

+ 7-d air

2.5

+ 7-d air

NanoZnO (mg/L)

Fig. 3. Effects of zinc exposure followed by exposure to air on oxidative stress in mussels. Mussels were exposed to zinc chloride (A) and nanoZnO (B) for 96 h at 15 °C followed by air exposure for 7 days. The levels of LPO were determined in the visceral mass in mussels before (96 h) and after exposure to air (7 days). The levels of TBARS were determined for zinc chloride (A) and nanoZnO (B). Peroxidase activity was also determined for zinc chloride (C) and nanoZnO (D). The data are expressed as the mean with standard error (n = 8). A significant difference between the controls and zinc concentrations is denoted by “a”; a significant difference between the controls and 96 h exposure to zinc after 7 days in air is denoted by “b”; and “c” indicates a significant difference before and after exposure to air for 7 days.

F. Gagné et al. / Comparative Biochemistry and Physiology, Part C 172–173 (2015) 36–44

B

1,3

Mean

1,2

c

Stand. error

1,1

1,0

0,9

0,8

0,7

Esterase acitivity (nmol fluorescein formed/min/mg proteins)

Esterase activity (nmol fluorescein formed/min/mg proteins)

A

41

1,3 Mean

Stand. error

1,2

1,1

1,0

0,9

0,8

0,7

0,6

0,6 Control

+ 7-d to air

0.5

+ 7-d to air

2.5

Control

+ 7-d to air

+ 7-d to air

0.5

+ 7-d to air

2.5

+ 7-d to air

NanoZnO (mg/L)

ZnCl2

Fig. 4. Effects of exposure to zinc and air-time stress on general esterase activity in mussels. Mussels were exposed to zinc chloride (A) and nanoZnO (B) for 96 h at 15 °C followed by air exposure for 7 days. The activity of non-specific Est was determined in the visceral mass in mussels before (96 h) and after exposure to air (7 days). The data are expressed as the mean with standard error (n = 8). A significant difference between the controls and zinc concentrations is denoted by “a”; a significant difference between the controls and 96 h exposure to zinc after 7 days in air is denoted by “b”; and “c” indicates a significant difference before and after exposure to air for 7 days. Data for zinc chloride and nanoZnO are shown in A and B, respectively.

of COX activity against PLA2 revealed that COX activity was significantly influenced by either nanoZnO or zinc chloride concentrations as well as PLA2 activity, with the latter more significant than the former. This highlights the close relationship between PLA2 and COX activities. Canonical analysis between oxidative stress biomarkers (COX, LPO and peroxidase) and lipid metabolism (Est, acetyl CoA, PLA2 and glycerol) revealed a significant correlation between these two groups: Rc = 0.68; p b 0.001. Discriminant function analysis was performed to determine whether Zn exposure and air stress could be well discriminated with others and determine which physiological biomarkers responded more to the cumulative effects of Zn and air exposure (Fig. 8). In mussels exposed to air stress only, the main changes occurred with COX activity, peroxidase activity and acetylCoA levels. This suggests that air stress brings about inflammation and fasting of mussels under low oxygen. In mussels exposed to ZnCl2 and nanoZnO, the major changes occurred at the same axis than for mussels exposed to air only. Mussels treated to dissolved Zn and air stress produced changes on the x axis which involved LPO, EST and acetylCoA levels. In mussels treated to nanoZnO and air stress, the response pattern was more diffuse with no clear changes on either axes.

A

Mussels placed in air for long periods of time leads to anaerobia and lack of food intake. During this period, mussels expend metabolic energy under low oxygen tension, leading to increased anaerobic metabolism and loss of energy reserves from proteins, sugars and lipids. Mussels treated to air emersion only revealed increased acetyl CoA, PLA2 and COX activity. In some cases, exposure to either nanoZnO or dissolved zinc also increased these endpoints, which are involved in air exposure stress, and which forms the basis of cumulative effects of stressors acting on similar endpoints. Indeed, significant decreases in air-time survival and mussel weight were found with nanoZnO. There was no sign of oxidative stress, because peroxidase and LPO either decreased or remained constant respectively. Reduced peroxidase activity with no changes in LPO is consistent with anaerobic conditions during air emersion for mussels. The increased levels of acetyl CoA could be indicative of reduced aerobic metabolism and β-oxidation for energy expenses from lipids reserves during fasting (Abraham et al., 1984). This was consistent with increased levels of HO-glycerol, from which fatty acids were released from the glycerol backbone for energy metabolism

B

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5. Discussion

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20 Control

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Fig. 5. Effects of zinc exposure followed by exposure to air on acetyl CoA in mussels. Mussels were exposed to zinc chloride (A) and nanoZnO (B) for 96 h at 15 °C followed by air exposure for 7 days. The levels of acetyl CoA were then determined in the visceral mass in mussels before (96 h) and after exposure to air (7 days). The data are expressed as the mean ng AcetylCoA/ mg proteins with standard error (n = 8). A significant difference between the controls and zinc concentrations is denoted by “a”; a significant difference between the controls and 96 h exposure to zinc after 7 days in air is denoted by “b”; and “c” indicates a significant difference before and after exposure to air for 7 days. Data for zinc chloride and nanoZnO are shown in A and B, respectively.

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A

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NanoZnO (mg/L)

ZnCl2 (mg/L)

Fig. 6. Effects of zinc exposure followed by exposure to air on glycerol in mussels. Mussels were exposed to zinc chloride (A) and nanoZnO (B) for 96 h at 15 °C followed by air exposure for 7 days. The levels of glycerol were determined by the corresponding hydroxyl protons of glycerol by NMR. The data are expressed as the mean with standard error (n = 8). A significant difference between the controls and zinc concentrations is denoted by “a”; a significant difference between the controls and 96 h exposure to zinc after 7 days in air is denoted by “b”; and “c” indicates a significant difference before and after exposure to air for 7 days. Data for zinc chloride and nanoZnO are shown in A and B, respectively.

through β-oxidation. One drawback of this adaptive mechanism is the increase in PLA2 activity. PLA2 activity is involved in calciumdependent lysosomal membrane destabilization, which is the main organite involved in catabolism and xenobiotic removal in marine mussels M. galloprovincialis (Burlando et al., 2002). Indeed, exposure of mussels to estradiol-17β leads to the release of free calcium, destabilization of lysosomal membranes and PLA2 activation. Lysosome destabilization by estradiol-17β was prevented by either a free calcium chelator or PLA2 activity inhibitor. In another study, exposure to air alone led to decreased lysosomal membrane stability after 72 h (3 days) and was related to mortality (Brenner et al., 2012). PLA2 activity releases arachidonic acid at the carbon 2 of glycerol, which is the precursor of inflammatory mediators when oxidized by COX during the formation of prostaglandins and eicosanoids. This is consistent with the significant correlation between PLA2 and COX activities in mussels exposed to air only (r = 0.55; p b 0.05). PLA2 was also strongly associated with nonspecific Est activity (r = 0.7; p b 0.01) in mussels exposed to air only, indicating that Est was partly involved in the mobilization of lipids for energy, leading to increased acetyl CoA precursors for energy metabolism. Indeed, Ests are involved in the hydrolysis of fatty acid esters where phospholipases (an Est) hydrolyze fatty acids from the phospholipids. Hence, prolonged fasting, even during anoxia for many days, leads to lipid hydrolysis (β-oxidation) and to inflammation in mussels as well. However, inflammation (oxidative stress) did not increase

A

0,090 Mean 0,085

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c Phospholipase 2 activity (U/mg proteins)

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LPO levels in mussels under anaerobic stress (exposed to air). However, dissolved zinc pre-treatment, and to some extent nanoZnO, increased LPO after air exposure, which suggests an interaction of zinc toxicity with air exposure (anaerobic metabolisms and food deprivation). It was shown in vertebrates that zinc binding to endogenous PLA2 resulted in increased enzyme activity (Lindahl and Tagesson, 1996). Hence, the cumulative stimulatory effects of zinc and air exposure to PLA2 lead to inflammatory damage as LPO in mussels. NanoZnO did not increase LPO at significant levels (α b 0.05), but a significant decrease between exposure to the highest concentration (i.e., 2.5 mg/L) of dissolved zinc and nanoZnO and 7 days of exposure to air was found, suggesting that normoxic conditions are required for LPO in mussels exposed to nanoparticles. The lack of LPO and peroxidase responses during air exposure could be explained by the increased activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) when mussels are exposed to air during the first hours (Letendre et al., 2009). Inflammation could also be the result of bacterial growth during air exposure. However, it was shown by Brenner et al. (2012) that autophagic processes of macrophages (phagocytosis) were not involved during the early adaptive processes from which mussels cope with aerial exposures. A negative relationship between air-time survival and weight loss was found in mussels exposed to either dissolved zinc or nanoZnO. This can be explained by the cumulative effects of zinc exposure and

0,075

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Fig. 7. Effects of zinc and air exposures on PLA2 activity in mussels. Mussels were exposed to zinc chloride (A) and nanoZnO (B) for 96 h at 15 °C followed by air exposure for 7 days. PLA2 activity was then determined in the visceral mass after 96 h exposure to zinc and after 7 days in air. The data are expressed as the mean with standard error (n = 8). A significant difference between the controls and zinc concentrations is denoted by “a”; a significant difference between the controls and 96 h exposure to zinc after 7 days in air is denoted by “b”; and “c” indicates a significant difference before and after exposure to air for 7 days. Data for zinc chloride and nanoZnO are shown in A and B, respectively.

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5 ZnCl 2 and air stress (100%) ZnCl 2 (63%) Control (50%) Control and air stress (63%) nZnO and air stress (50%) nZnO (38%)

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Component 2 (COX>Perox>acetylCoA)

3 2 1 0 -1 -2 -3 -4 -5 -4

-3

-2

-1

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4

Component 1 (LPO>Est>acetylCoA) Fig. 8. Discriminant function analysis of exposure to zinc forms and air-time stress. The biomarker data on the exposure of mussels to zinc chloride and nanoZnO before and after air emersion for 7 days were analyzed using discriminant function analysis. The classification performance is provided in percentages in the legend, and the biomarkers under the x and y axes are the three most important ones as determined by factorial analysis. The control mussels are represented by the dotted ellipse. The (%) in the legend represents the classification efficiency.

the stress of anoxic (and dry) conditions without any food intake. For example, during air exposure the release of water could account, in part at least, for the weight loss. Acetyl CoA is the end product of lipid mobilization during β-oxidation and gluconeogenesis for energy production, which was consistent with increased HO-glycerol (from the breakdowns of phospholipids) based on H NMR analysis of lipid extracts of the visceral mass of mussels. However, acetyl CoA is mainly utilized by the citric cycle under aerobic metabolism, which is in keeping with air-gaping behavior of mussels maintained for long periods to air. In the presence of hypoxia, the β-oxidation of lipids would release FADH2 and NADH2 for metabolic energy. This process would result in the accumulation of acetyl CoA, because aerobic metabolism (the citric acid cycle) is limited in anoxic conditions but could be partially compensated by air-gaping. Exposure to both forms of zinc also increased the levels of acetyl CoA, whereas no evidence of cumulative effects with air exposure was found. Recent evidence suggests that intracellular free zinc inhibits aerobic energy production, such as the mitochondrial electron transport chain and components of glycolysis and the citric acid cycle (Dineley et al., 2003), which contributes to the alreadyimpaired aerobic metabolism during prolonged periods in air. Dissolved cadmium could also suppress anaerobic metabolism, which could decrease air-time survival in marine mussels, likely by lowering ATP reserves in tissues (Ivanina et al., 2010). In oysters, gill and digestive gland mitochondria were particularly sensitive to zinc oxide nanoparticles, leading to mitochondria membrane disruption, loss of cristae, and LPO at zinc concentration (4 mg/L; 24–48 h) above those tested in the present study (Trevisan et al., 2014). As with LPO, peroxidase activity was diminished after exposure to air for 7 days, as explained previously. However, this effect was abolished by exposure to dissolved zinc, but not with nanoZnO, which indicates that its toxicity was not mediated by the release of ionic zinc. This is consistent with the behaviour of the nanoparticles in aquarium water when they tended to form large aggregates. This was further corroborated by the increased LPO following air exposure in mussels exposed to dissolved zinc but not nanoZnO. It is noteworthy that the combined effects of zinc and air exposure were not always additive. For example, acetyl CoA levels and PLA2 activities were induced by both forms of zinc and exposure to air, but zinc exposures led to stronger increases in acetyl CoA than did air exposure. This

suggests that a physiological limit in this response was reached and accounted for the loss of air-time survival at 2.5 mg/L of nanoZnO and perhaps dissolved zinc. Decreased survival to air did not appear to be related to valve activity or periods of valve opening during immersion in aquarium water (Ait Ayad et al., 2011). Based on the air-time survival test, nanoZnO appeared to be more toxic than dissolved zinc in freshwater mussels, although dissolved zinc was acutely more lethal than nanoZnO. Exposure to 2.5 mg/L of zinc in the form of nanoZnO resulted in a decrease in air-time survival from approximately 18 days in controls to 12 days in exposed mussels. Decreased air-time survival was associated with a decreased ratio of mussel weight to shell length, representing a 20% drop in mussel weight. Given that mussels could close their shells for many days, this could complicate acute lethal evaluations of toxicants especially at high concentrations for short periods of time. Shell opening behavior should be examined during toxicity testing. In another study, marine mussels (Mytilus edulis) exposed to 2 mg/L of nanoZnO for 12 weeks accumulated zinc in tissues and grew 40% less than controls (Hanna et al., 2013). The gills in mussels are ciliated and behave like a primordial mouth, and bring particles (micro-organisms) to the digestive gland during feeding, thus changing the route of exposure for particles. Indeed, mussels were identified as species at risk of contamination by fine particles and nanoparticles in the aquatic environment (Gagné et al., 2008; Canesi et al., 2012). This was further supported in a previous study with E. complanata exposed to nanoZnO, leading to increased total zinc, metallothioneins and LPO in the digestive gland but not in gills (Gagné et al., 2013). Gills are considered as the first contact organ for metals, but find their way in the digestive gland when the metals are presented in the form of nanoparticles (Gagné et al., 2014). NanoZnO was found to be bioavailable in mussels (with a bioconcentration factor of 88), but was removed as zinc in the pseudofeces without any discernible nanoparticles, suggesting the breakdown of nanoZnO in mussel tissues (Montes et al., 2012). In conclusion, exposure to prolonged periods to air of freshwater mussels leads to important physiological changes in the attempt to survive outside of water. Exposure to contaminants such as dissolved zinc and nanoZnO could tamper with these adaptations. This is especially the case for nanoZnO, which significantly decreased air-time survival and produced effects in the biomarkers responding

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to air exposure. This study also showed that the observed effects of nanoZnO could not be solely explained by the dissolved zinc. Acknowledgments This work was funded by the Chemicals Management Plan of Environment Canada. Nicolas Siron provided technical assistance in tissue preparation and biomarker assessments, and Len Goldberg provided editorial assistance. References Abraham, S., Hansen, H.J., Hansen, F.N., 1984. The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla). Comp. Biochem. Physiol. B 79, 285–289. Ait Ayad, M., Ait Fdil, M., Mouabad, A., 2011. Effects of Cypermethrin (pyrethroid insecticide) on the valve activity behavior, byssal thread formation, and survival in air of the marine mussel Mytilus galloprovincialis. Arch. Contam. Toxicol. 60, 462–470. 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. Brenner, M., Broeg, K., Wilhelm, C., Buchholz, C., Koehler, A., 2012. Effect of air exposure on lysosomal tissues of Mytilus edulis L. from natural intertidal wild beds and submerged culture ropes. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 161, 327–336. Burlando, B., Marchi, B., Panfoli, I., Viarengo, A., 2002. Essential role of Ca2+-dependent phospholipase A2 in estradiol-induced lysosome activation. Am. J. Physiol. Cell Physiol. 283, 1461–1468. Canesi, L., Ciacci, C., Fabbri, R., Marcomini, A., Pojana, G., Gallo, G., 2012. Bivalve molluscs as a unique target group for nanoparticle toxicity. Mar. Environ. Res. 76, 16–21. Dineley, K.E., Votyakova, T.V., Reynolds, I.J., 2003. Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration. J. Neurochem. 85, 563–570. Domingos, R.F., Rafiei, A.C.Z., Monteiro, B.C.E., Khan, M.A.K., Wilkinson, K.J., 2013. Agglomeration and dissolution of zinc oxide nanoparticles: role of pH, ionic strength and fulvic acid. Environ. Chem. 10, 306–312. Falfushynska, H., Gnatyshyna, L., Yurchak, I., Sokolova, I., Stoliar, O., 2015. The effects of zinc nanooxide on cellular stress responses of the freshwater mussels Unio tumidus are modulated by elevated temperature and organic pollutants. Aquat. Toxicol. 162, 82–93. Frings, C.S., Fendley, T.W., Dunn, R.T., Queen, C.A., 1972. Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. Clin. Chem. 18, 673–674. Gagné, F., 2014. Biomarkers of infection and diseases. Biochemical Ecotoxicology—Principles and Methods, First Edition Elsevier Inc., USA (Chapter 11). Gagné, F., Gagnon, C., Blaise, C., 2008. Aquatic nanotoxicology: a review. Curr. Top. Toxicol. 4, 1–14. Gagné, F., Turcotte, P., Auclair, J., Gagnon, C., 2013. The effects of zinc oxide nanoparticles on the metallome in freshwater mussels. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 158, 22–28.

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