Relationship between metal and polybrominated diphenyl ether ...

Marine Pollution Bulletin 100 (2015) 383–392

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Relationship between metal and polybrominated diphenyl ether (PBDE) body burden and health risks in the barnacle Balanus amphitrite Lianguo Chen a, James C.W. Lam b,c, Xiaohua Zhang b,c, Ke Pan a, Cui Guo a, Paul K.S. Lam b,c,d, Wenxiong Wang a, Hongbin Liu a, Pei-Yuan Qian a,⁎ a

Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China Research Centre for the Oceans and Human Health, Shenzhen Key Laboratory for Sustainable Use of Marine Biodiversity, City University of Hong Kong Shenzhen Research Institute Building, Shenzhen 518057, China d Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China b c

a r t i c l e

i n f o

Article history: Received 29 May 2015 Received in revised form 6 August 2015 Accepted 7 August 2015 Available online 29 August 2015 Keywords: Barnacle Balanus amphitrite Metals PBDEs Biological responses

a b s t r a c t In the present study, we employed the widespread and gregarious barnacle species Balanus amphitrite in a biomonitoring program to evaluate coastal pollution around three piers (i.e., Tso Wo Hang, Sai Kung and Hebe Haven) in Hong Kong. An integrated approach was used herein, combining both the chemical determination of contaminant concentrations, including metals and polybrominated diphenyl ethers (PBDEs), and a suite of biological responses across the entire barnacle lifecycle (i.e., adult, nauplius, cyprid and juvenile). The analytical results revealed a distinct geographical distribution of metals and PBDEs. Adult physiological processes and larval behaviors varied significantly among the three piers. Furthermore, a correlation analysis demonstrated a specific suite of biological responses towards metal and PBDE exposure, likely resulting from their distinct modes of action. Overall, the results of this study indicated that the combination of chemical and biological tests provided an integrated measure for the comprehensive assessment of marine pollution. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The use of sentinel organisms as biomonitors to indicate environmental safety is a well-established approach in the environmental risk assessment of a wide variety of anthropogenic contaminants (Durou et al., 2007; Phillips and Rainbow, 1993; Rainbow et al., 2000). During exposure to environmental pollution, biomonitors are able to accumulate trace aqueous contaminants to a much higher level in their tissues, eventually causing the emergence of physiological disturbances. Working together with chemical monitoring of geographical fluctuations of contaminants, the integrated application of a suite of biological responses can provide early warning signals of environmental disturbances and offer ecotoxicologically relevant tools for the comprehensive evaluation of the combined effects of contaminants (Cheung et al., 2002; Durou et al., 2007; Dionísio et al., 2013). Therefore, serving as an efficient supplement to chemical detection, biomonitoring has become widely applied, and various biomonitors, such as mussels, oysters and barnacles, are generally considered in an attempt to obtain a complete assessment of the risks associated with contaminants (Blackmore, 1998; Blackmore et al., 1998; Morillo et al., 2005; Páez-Osuna et al., 1999). ⁎ Corresponding author. E-mail address: [email protected] (P.-Y. Qian).

http://dx.doi.org/10.1016/j.marpolbul.2015.08.020 0025-326X/© 2015 Elsevier Ltd. All rights reserved.

The barnacle is an intertidal crustacean species and shows a global distribution, playing ecologically important roles in the structure and function of coastal ecosystems (Cheng et al., 2004; Chiang et al., 2003). The lifecycle of the barnacle comprises two stages: one planktonic stage and one sessile stage (Fig. 1). In the planktonic stage, nauplius larvae are released from adult barnacles. After feeding to deposit adequate energy, the nauplii will transform into non-feeding cyprids, which are competent for later settlement. In general, cyprids will search the habitat surface first and then attach onto the chosen site to metamorphose into sessile juvenile barnacles (He et al., 2012). During their whole lifecycle, barnacles will be subjected to environmental pollution and suffer from adverse effects. As sessile filterfeeders, barnacles tend to ingest large quantities of contaminant-rich particles from ambient water matrices, and thus, they exhibit a high uptake and bioaccumulation of environmental contaminants (Rainbow, 1995; Ramos et al., 2014). The barnacle mostly fulfills the qualifications of an ideal biomonitor and consequently is being widely used as a suitable indicator in programs designed to assess coastal pollution (Rainbow et al., 2000). For example, previous research has employed adult barnacles of various species to indicate the bioavailability of trace metals through measuring the metal concentrations in soft tissues (Blackmore, 1998; Dionísio et al., 2013; Fialkowski and Newman, 1998; Páez-Osuna et al., 1999; Rainbow et al., 2000). In addition, at Quequén Harbor in Argentina, the population recruitment of the barnacle Balanus

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(Billinghurst et al., 1998; Chiang et al., 2003; Faimali et al., 2006; Greco et al., 2006; Lam et al., 2000; Qiu et al., 2005; Wu et al., 1997a,b). Therefore, in the present study, the widespread and gregarious barnacle B. amphitrite Darwin was used as a convenient, easily accessed biomonitor to evaluate the geographical variation in coastal pollution in Hong Kong. Pollution assessment around three piers (i.e., Tso Wo Hang, Sai Kung and Hebe Haven) was facilitated first by analytical measurements of the concentrations of metals and polybrominated diphenyl ethers (PBDEs). In addition, a wide spectrum of biological responses, including physiological and behavioral assays across the whole lifecycle of B. amphitrite (i.e., adult, nauplius, cyprid and juvenile), was also determined to complement the chemical tools for the comprehensive monitoring of marine pollution. 2. Materials and methods 2.1. Study sites and adult barnacle collection

Fig. 1. The lifecycle of the barnacle B. amphitrite includes a planktonic stage and a sessile stage. After release from the adult barnacle, the feeding nauplius larvae will transform into the non-feeding cyprid larvae, which is competent for later attachment and metamorphosis into the sessile juvenile barnacle.

amphitrite Darwin is used to assess the environmental quality; recruitment failure as a result of sewage outfall leads to a steady decrease in the population density (Calcagno et al., 1998). Physiological changes involved in oxidative stress and neural signaling, as indicated by sensitive biomarkers with distinct biological meanings, are documented in barnacles as responses to exposure to marine pollution (Niyogi et al., 2001; Ramos et al., 2014; Zanette et al., 2015). Furthermore, the relatively high sensitivities of developmental and behavioral assays, including both phototaxis and settlement tests, allow the extensive incorporation of barnacle nauplii and cyprids into the ecotoxicity assessment battery

As shown in Fig. 2, adult barnacles were collected from three piers (i.e., Tso Wo Hang, Sai Kung and Hebe Haven) in the eastern Kowloon area of Hong Kong on September 14, 2014. Although the three piers under investigation are closely located geographically, their associated human activities, which are considered the primary sources of marine pollution, are much different. Around Sai Kung pier with a dense population, the heavy domestic discharge and shipping activities are the likely causes of the burden on the marine environment. Hebe Haven, which is a largely enclosed bay with limited water circulation and renewal, has the most intense shipping activities, which range from ship building, traveling and dry dock maintenance, despite not being densely populated like Sai Kung. However, compared with Sai Kung and Hebe Haven, the Tso Wo Hang area lacks a dense residential population and heavy shipping, and thus, it was considered as the reference site. The physiochemical conditions of the seawater at the three piers, including the pH, temperature, salinity and dissolved oxygen, were measured (Table 1). Despite spatial separation and distinct anthropogenic interference, the seawater around the three piers exhibited identical physiochemical properties. Barnacles were scraped from the intertidal zone of the piers, placed temporarily in polythene bags and stored at −80 °C in the laboratory

Fig. 2. Location map of the three piers used for barnacle collection in Hong Kong.


2.3. Physiological responses

Table 1 Seawater physiochemical factors in the three piers.a Piers

pH

Temperature (°C)

Salinity (‰)

Dissolved oxygen (mg/L)

Tso Wo Hang Sai Kung Hebe Haven

8.6 ± 0.01 8.5 ± 0.01 8.5 ± 0.03

30.7 ± 0.2 30.5 ± 0.1 30.7 ± 0.1

31.0 ± 0.0 30.0 ± 0.0 30.0 ± 0.0

6.6 ± 0.0 6.5 ± 0.2 6.8 ± 0.1

a

385

Data are represented as mean ± SEM of three measurements.

for subsequent analysis. The growth of barnacles from different piers (n = 30) was measured and recorded, including the basal size (rostro-carinal), the total weight comprising both the shell and soft tissue, and the wet and dry weight of the soft tissue alone. Drying at 60 °C for two days allowed the tissues to achieve constant weight. The ratio of the wet weight to the dry weight of the soft tissues was also calculated for each pier. 2.2. Contaminant concentrations in adult soft tissues 2.2.1. Concentration of metals To measure the accumulation of metals in barnacles from different piers, individual barnacles with a similar size (n = 6) were selected, and their soft tissues were dissected out. After drying at 60 °C for two days until a constant weight was achieved, the tissues were completely digested in 1 mL of concentrated nitric acid (70%) at 60 °C for another two days. After centrifuging the digests at 10,000 ×g for 10 min, the supernatant was transferred to new Falcon tubes and diluted with ultrapure water up to a volume of 10 mL to analyze the Zn, Cu, Fe, Ni, Cd, Pb, Cr, Sn and As using an inductively coupled plasma-optical emission spectrometer (ICP-OES) (Optima 7000 DV, PerkinElmer) (Wang et al., 2011). The concentrations of the metals were expressed as μg/g dry weight. 2.2.2. PBDE concentrations The PBDE content in barnacle tissues was measured following the method described in previous studies (Chen et al., 2012a; Zhu et al., 2014) with some modifications. Because of the relatively low concentrations of PBDEs in the coastal environment, approximately twenty barnacle bodies were dissected and pooled together as one replicate. Each pier contained six replicates in total. After freeze-drying, the dry weight of each replicate was recorded. Each surrogate standard (5 ng), including 13C12-labeled BDE47, 99, 100, 153, 154 and 183, was added to the samples, and then the samples were subjected to accelerated solvent extraction (Dionex ASE® 200, USA) using a mixture of dichloromethane (DCM) and hexane (4:1 v/v, 40 mL) at 1500 psi and 110 °C for 6 min. The extracts were then injected into the gel permeation chromatography column (25 mm i.d. × 500 mm; Bio-Beads S-X3, Bio-Rad Laboratories, Hercules, CA, USA) to remove lipids after elution in a mixture of DCM and hexane (1:1, v/v) at 5 mL/min for 80 min. Further cleanup of the samples was conducted using a packed column containing 1 g of anhydrous sodium sulfate and 5 g of activated silica gel (60 Å average pore size) from top to bottom. A mixed solution of DCM and hexane (2:1, v/v) was used for the elution. 13C12-labeled BDE139 was added as a recovery spike prior to the instrumental analysis. The identification and quantification of PBDEs were performed using a liquid chromatography–tandem mass spectrometer system (LC–MS/MS) consisting of an Agilent 1290 Infinity LC (Agilent Technologies, Palo Alto, CA) coupled to an AB SCIEX QTRAP® 5500 LC–MS/MS system with an atmospheric pressure chemical ionization (APCI) interface. A Zorbax SB-C18 column (2.1 mm × 100 mm, 1.8 μm; Agilent Technologies, Palo Alto, CA) was used for the chromatography separation. Data were acquired and processed using AB SCIEX Analyst software (version 1.6). The concentrations of the analytes were expressed as ng/g dry weight.

2.3.1. Energy reserves and respiration rate Energy reserves, including protein, carbohydrate and triglyceridebased lipids, were measured, respectively, in individual soft tissues (n = 12). The total protein content in the aqueous homogenate was quantified using the Bradford Coomassie brilliant blue method with an absorbance at 595 nm. Bovine serum albumin (BSA) was used as the standard protein. The content of total carbohydrates was estimated based on the anthrone-sulfuric acid reaction, which produces a blue-colored product with an optical density that can be measured at a wavelength of 620 nm (Roe, 1955). Triglyceride concentrations were determined by a colorimetric reaction of an enzyme that produces hydrogen peroxide with an absorbance at 510 nm (Fossati and Prencipe, 1982). Glycerol solution was employed as the standard for the triglyceride measurements. To provide an integrated overview of the available energy reserves, the total energy equivalent was calculated from the concentrations of protein, carbohydrate and lipid using the energy conversion factors 18.00, 17.16 and 35.24 kJ/g, respectively (Whyte et al., 1992). The respiration rate of individual barnacles (n = 12) was measured using Winkler's method (Outdot et al., 1988). Reduction of dissolved oxygen levels in the seawater was used to monitor the respiration rate. The barnacles were placed in stoppered bottles filled with 0.22-μm filtered seawater and incubated for 3 h. Next, 0.5 mL of Winkler I solution (400 g of MnCl2·4H2O in 1000 mL of distilled water) and 0.5 mL of Winkler II solution (500 g of NaOH, 1 g of NaN3 and 150 g of KI in 1000 mL of distilled water) were immediately added to the bottles, and after repeated inversion, the remaining dissolved oxygen was fixed, forming a brownish-orange cloud of precipitate. The precipitate was dissolved in 1 mL of acid mixture (350 mL of 85% H3PO4 and 350 mL of 36% HCl in 1000 mL of distilled water). A 50-mL aliquot of the extract from the bottle contents was transferred and automatically titrated using 0.01 M Na2S2O3 (716 DMS Titrino Metrohm). The consumption of dissolved oxygen by barnacle respiration was converted using the titration volume of 0.01 M Na2S2O3 when the titration end point was reached. The respiration rate was expressed as mg dissolved oxygen consumed/h/g body weight. 2.3.2. Metallothionein (MT) protein content The MT concentrations in the barnacle tissues were measured using a mercury-saturation method according to previous research (Pan and Zhang, 2006). Briefly, five barnacle bodies were pooled together as one replicate (n = 4) and homogenized on ice using a tissue tearer (BioSpec Products, Bartlesville, OK, USA) in 2 mL of Tris–HCl buffer (pH 7.2; 25 mM). After centrifugation at 10,000 ×g at 4 °C for 20 min, a 1-mL aliquot of the supernatant was spared and used to determine the MT concentrations. The homogenate was first heated at 100 °C for 10 min to enable the precipitation of non-MT protein and then centrifuged at 10,000 ×g at 4 °C for 20 min. Subsequently, 200 μL of HgCl2 solution (50 mg Hg/L) prepared in 10% trichloroacetic acid (TCA) was added to a 200-μL aliquot of the supernatant and incubated for 10 min to saturate the MT proteins. For the blank control, 200 μL of Tris–HCl lysis buffer (pH 7.2; 25 mM) was used as a replacement for the heated supernatant. The excess Hg was then removed by the addition of 400 μL of BSA solution, followed by centrifugation at 20,000 ×g at 4 °C for 20 min. The resulting sample was then digested in 3 mL of concentrated nitric acid (70%; Fisher Scientific) and analyzed after appropriate dilution using a Cold Vapor Atomic Fluorescence mercury analyzer (QuickTrace™ M-8000, USA) (Pan et al., 2011). The total protein concentration was determined using the Bradford Coomassie brilliant blue method with BSA as the standard. The MT concentrations were expressed as μmol/mg protein. 2.3.3. Acetylcholinesterase (AChE) activity The soft tissues of five barnacle individuals were pooled together as one replicate (n = 4) and homogenized on ice in 1.0 mL of normal saline

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(0.9% sodium chloride) using a tissue tearer (BioSpec Products, Bartlesville, OK, USA). After centrifugation at 3500 ×g at 4 °C for 10 min, the supernatant was transferred to a new tube to measure the enzyme activity. The AChE activity in the barnacle soft tissues was determined by quantifying the hydrolysis and conversion rate of the acetylcholine (ACh) substrates into the yellow colored product sym-trinitrobenzene after formation of the complex of thiocholine with dithiobisnitrobenzoate (Chen et al., 2014; Ramos et al., 2014). The increase in optical density was measured colorimetrically at 412 nm, and the enzyme activity was expressed as μmol acetylcholine hydrolyzed/mg protein at 37 °C within 6 min. The protein concentration was measured using the Bradford Coomassie brilliant blue method with BSA as the standard. 2.3.4. Oxidative stress The soft tissues of five barnacles were pooled together as one replicate, and each group contained four replicates. The tissues were homogenized on ice using a tissue tearer in 1.0 mL of normal saline (0.9% sodium chloride) and then centrifuged at 12,000 ×g at 4 °C for 30 min. The supernatant was transferred to a new tube and divided into several aliquots for subsequent oxidative stress measurements using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The enzymatic activities of superoxide dismutase (SOD) were measured according to the instructions provided with the kit, which was based on the ability of SOD to inhibit nitrite formation from superoxide radical-dependent hydroxylamine oxidation. The rate of nitrite formation was monitored colorimetrically at 550 nm. The glutathione peroxidase (GPx) activities were measured based on the reaction of GSH with dithionitrobenzene to generate a stable yellow product with an absorbance at 412 nm. The activities of the enzymes were expressed as the decrease in μmol GSH/min/mg protein. The enzyme activities of glutathione S-transferase (GST) were determined based on the catalysis ability of the reaction between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) with an optical density measured at 412 nm. One unit of GST activity was defined in terms of the decrease in 1 μmol/L GSH/mg protein after 1 min of incubation at 37 °C. The catalase (CAT) enzyme activities were measured colorimetrically based on the catalyzed consumption of H2O2, resulting into a decrease in the absorbance at 405 nm. The CAT activities were expressed as μmol H2O2 consumption/s/mg protein. The enzyme activities of glutathione reductase (GR) were determined by the conversion capability of GSSG towards GSH and were expressed as the increase in GSH concentrations at 420 nm. The contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined utilizing the reaction with dithionitrobenzoic acid to produce a yellow compound, which could be quantified at 420 nm. Lipid peroxidation (LPO) was determined by the formation of thiobarbituric acid reactive substances (TBARS), including malondialdehyde (MDA), in tissues. The formation of TBARS led to the production of a red color with a maximum absorbance at 532 nm. The production of reactive oxygen species (ROS) in barnacle tissues was determined using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO) (Chen et al., 2014). ROS content was expressed as the fluorescence intensity of the produced DCF/mg protein, which was measured using a microplate reader Wallac Victor 2/1420 multilabel counter (Perkin Elmer Life Sciences, Boston, MA) with an excitation at 502 nm and an emission at 530 nm. The protein concentration was measured using the Bradford Coomassie brilliant blue method with BSA as the standard.

with a commercial kit (Tiangen Biotech., Beijing, China). The DNA samples were divided into three equal portions and used for the determination of dsDNA, ssDNA and partially unwound DNA (auDNA) using the fluorescent DNA binding dye bisbenzamide. The fluorescence was measured using a microplate reader Wallac Victor 2/1420 multilabel counter (Perkin Elmer Life Sciences, Boston, MA) with an excitation wavelength at 355 nm and an emission wavelength at 460 nm. The indicator of DNA integrity, the F value, which is inversely proportional to the number of DNA strand breaks, was calculated as follows: F = (auDNA − ssDNA) / (dsDNA − ssDNA). 2.4. Status of larval and juvenile barnacles 2.4.1. Viability of larval barnacles After exposing adult barnacles to light, the newly released nauplius larvae were collected according to their phototaxis behaviors. The viability of the collected nauplii was quantitatively measured based on their phototactic responses. The nauplius larvae were then cultured in 0.22-μm filtered seawater at 28 °C and fed sufficient Chaetoceros gracilis Schutt until day 4 when most of the nauplii had metamorphosed into cyprids, which are competent to settle. After the cyprids were collected, the survival rate and metamorphosis rate during the culture and metamorphosis processes were recorded. The phototaxis and settlement behaviors of the cyprids were also measured, respectively, to indicate the larval viability associated with the different piers. The phototaxis behavior was measured according to the protocol described in previous research (Zhang et al., 2011). Briefly, twenty nauplius or cyprid larvae were distributed into 10-mL glass tubes filled with 0.22-μm filtered seawater (n = 12). After sealing the tops of the tubes with a parafilm membrane, the tubes were wrapped in aluminum foil to block light with the exception of the bottom part, along which 1/10 of the tube length was exposed to light. Next, the tubes were inverted (top–down) and kept in the dark. To ensure random distribution of the larvae, the tubes were first gently agitated and placed horizontally in the dark for 1 min prior to light exposure. After illumination for 2 min, the number of larvae at the exposed bottom was counted as an indicator of the phototactic response. The settlement percentage of cyprids was also determined according to a previous protocol (Chen et al., 2015b). Approximately twenty freshly molted cyprids were placed into each well of a 24-well plate (Nunc, Naterville, USA; n = 12). Each well contained 1 mL of 0.22-μm filtered seawater. After incubation in the dark for 24 h and 48 h, the number of settled and swimming cyprids, respectively, was counted under a stereomicroscope to calculate the settlement percentage. 2.4.2. Growth of juvenile barnacles To assess whether the growth of juvenile barnacles was affected at the different piers, PVC panels (100 mm × 75 mm) were employed to present the surface for cyprid settlement. Each pier included four panels. The surfaces of the panels were first sandblasted and washed using 0.22-μm filtered seawater prior to the addition of cyprids. After incubation for 48 h, all of the panels with juvenile barnacles were transferred to a new tank and fed a diet of C. gracilis Schutt for 30 days at 24 °C. The growth of the juvenile barnacles (twenty random individuals per panel) was then recorded, including the basal size and total wet weight. 2.5. Statistical analysis

2.3.5. Measurement of DNA integrity The DNA integrity was assessed using the alkaline unwinding assay as described previously, in which the transition rate of double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) is proportional to the breaks in the phosphodiester backbone and thus can be used as a measure of the DNA integrity (Ching et al., 2001; Siu et al., 2003). The extraction and purification of DNA from the soft tissues of each barnacle individual (n = 12) were conducted according to the manual provided

All of the data were expressed as the mean ± SEM. The normality and variance homogeneity were checked first using the Kolmogorov– Smirnov test and Levene's test, respectively. Logarithmic transformation of the data was conducted if necessary. Pearson correlation analysis was used to test the correlation between the physiological responses and the body burdens of pollutants. Significant difference among sampling sites was determined by one-way analysis of variance (ANOVA) followed by


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Table 2 Growth of adult barnacles in three piers.⁎ Piers

Basal size (cm)

Total weight (g)

Wet weight (g)

Dry weight (g)

Wet weight/dry weight

Tso Wo Hang Sai Kung Hebe Haven

1.58 ± 0.04a 1.52 ± 0.03a 1.11 ± 0.03b

1.07 ± 0.10a 0.87 ± 0.05a 0.35 ± 0.02b

0.13 ± 0.02a 0.15 ± 0.02a 0.05 ± 0.00b

0.021 ± 0.003a 0.021 ± 0.002a 0.008 ± 0.001b

7.73 ± 0.87a 7.48 ± 0.44a 5.82 ± 0.25b

⁎ Different letters (a or b) denote statistically significant difference among sampling sites and the values are represented as mean ± SEM of thirty adult barnacle individuals.

the LSD post-hoc test. A P-value b 0.05 was set as the criteria to define significant difference. All statistical analysis were performed using SPSS v13.0 software (SPSS, Chicago, IL, USA). 3. Results 3.1. Growth of adult barnacles The results of the growth parameters of the adult barnacles at the three piers showed that the growth of barnacles from Hebe Haven was significantly inhibited, as demonstrated by the decreased basal diameter and weight. In contrast, the barnacles at Tso Wo Hang and Sai Kung had similar growth statuses (Table 2). The ratio of wet weight to dry weight was also significantly decreased in barnacles from Hebe Haven (Table 2). 3.2. Contaminant concentrations in adult barnacles In the soft tissues, both metal and PBDE accumulation were examined (Fig. 3). Barnacles from Hebe Haven contained the highest levels of metals (19,610.9 ± 3171.8 μg/g dry weight) as initially

predicted compared with the other two sites, following the order Hebe Haven N Tso Wo Hang N Sai Kung (Fig. 3A). Of the detected metals, Zn, Cu and Fe accounted for the majority, among which Zn was predominant in all three sites and reached much higher concentrations compared with the other metals (Fig. 3A). The concentration of Zn was much higher in barnacles collected from Hebe Haven (18,440.4 ± 3089.4 μg/g) than those from Tso Wo Hang (12,929.4 ± 1372.8 μg/g) and Sai Kung (10,855.2 ± 913.7 μg/g). A similar trend was observed for Cu, which displayed the highest concentrations in barnacles from Hebe Haven (915.1 ± 104.5 μg/g), while Fe was most abundant in barnacles from Tso Wo Hang (273.2 ± 35.1 μg/g) relative to Sai Kung (260.1 ± 18.3 μg/g) and Hebe Haven (230.0 ± 24.4 μg/g). In addition to Zn, Cu and Fe, the other investigated metals (i.e., Ni, Cd, Pb, Cr, Sn and As) showed low and comparable levels among the three piers (Fig. 3A). In contrast to the metals, a different geographical distribution among the piers was observed for PBDEs (Fig. 3B). Unexpectedly, the highest bioaccumulation of PBDEs was detected in barnacles from Tso Wo Hang (887.5 ± 67.1 ng/g dry weight), while Sai Kung (401.5 ± 50.9 ng/g dry weight) and Hebe Haven (370.4 ± 45.8 ng/g dry weight) showed comparable levels of PBDEs in barnacle tissues. Among the six

Fig. 3. Burden of (A) metals (μg/g dry weight) and (B) PBDE congeners (ng/g dry weight) in the soft tissues of adult barnacles from different piers. All of the data are presented as the mean ± SEM of six replicates.

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Fig. 5. MT protein content (A) and AChE enzyme activities (B) in barnacles from different piers. All of the data are presented as the mean ± SEM of four replicates. Significant differences among sampling sites are indicated by different letters (a or b).

3.4. MT protein content and AChE enzyme activities

Fig. 4. Energy reserves (A), energy equivalents (B) and respiration rate (C) of adult barnacles collected from different piers. Data for energy reserves and the respiration rate are presented as the mean ± SEM of twelve individual barnacles. The numbers on top of the columns denote the sum of the energy equivalents. Significant differences among sampling sites are indicated by different letters (a or b).

congeners of interest, BDE47 was the most abundant congener at all three piers, followed by BDE99, 100, 153, 154 and 183 (Fig. 3B). 3.3. Energy reserves and respiration rates The concentrations of protein, carbohydrate and lipid were measured to evaluate the energy reserves in the barnacles from the three piers. The results showed that barnacles at Hebe Haven had increased reserves of protein and lipid in comparison with those at the other two sites (Fig. 4A). The overview of energy reserves showed that more energy equivalents were present in Hebe Haven barnacles, mainly contributed by an increase in lipid content (Fig. 4B). The metabolic rate characteristic of oxygen consumption during respiration was also significantly accelerated in adult barnacles from Hebe Haven at 43.7 ± 5.7 mg O2/h/g body weight (Fig. 4C).

The concentrations of MT protein were significantly increased to 51.6 ± 6.5 μmol Hg-binding sites/mg protein in the soft tissues of barnacles from Hebe Haven compared with 34.6 ± 3.8 μmol/mg protein in Tso Wo Hang and 26.2 ± 4.4 μmol/mg protein in Sai Kung (Fig. 5A). In addition, the enzyme activities of AChE were significantly decreased by 49.9% in Sai Kung and by 50.9% in Hebe Haven relative to those in Tso Wo Hang (Fig. 5B).

3.5. Oxidative stress and DNA integrity The enzymatic activities of SOD were markedly decreased by 71.6% in Hebe Haven relative to Tso Wo Hang (Fig. 6A). A significant increase in ROS production was observed in barnacles from both Sai Kung and Hebe Haven to 109.6 ± 1.7% and 116.3 ± 2.2%, respectively, relative to the levels at Tso Wo Hang (Fig. 6A). In addition, there were also significant increases of 35.9% and 26.1% in the enzymatic activities of GPx along with a decreased content of GSH by 15.4% and 32.3% in barnacles from Sai Kung and Hebe Haven, respectively (Fig. 6A). The other parameters related to oxidative stress, such as the enzymatic activities of CAT, GST and GR as well as the contents of GSSG and TBARS, were not significantly modified (data not shown). The F value, a measure of DNA integrity, was significantly decreased in barnacles from Hebe Haven,


Fig. 6. Oxidative stress (A) and DNA integrity (B) in the soft tissues of adult barnacles from different piers. All of the data for oxidative stress are presented as the mean ± SEM of four replicates, and the data for DNA integrity are presented as the mean ± SEM of twelve individual barnacles. Significant differences among sampling sites are indicated by different letters (a or b).

indicating an increase in breaks and damage to the DNA strands based on the inverse relationship between the F value and DNA strand breaks (Fig. 6B). 3.6. Status of larval and juvenile barnacles To determine whether the larval and juvenile barnacles were affected at the three piers, the viability of larvae (i.e., nauplius and cyprid) and the growth of juveniles were monitored. During the culture and metamorphosis periods, although the survival rates among the piers were not significantly different, the metamorphosis rate from nauplius to cyprid at 4 days was decreased in both Sai Kung and Hebe Haven (72.4% and 63.2%, respectively) compared with 88.1% in Tso Wo Hang (Table 3). Furthermore, the phototactic behaviors of both the nauplii

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Fig. 7. Changes in the viability of nauplius and cyprid larvae from different piers. (A) Variation in the phototaxis behavior of both nauplius and cyprid larvae and (B) settlement percentages of cyprids after incubation for 24 h and 48 h. Values represent the mean ± SEM of twelve replicates (each containing 20 larvae). Significant differences among the sampling sites are indicated by different letters (a, b or c).

and cyprids released from Hebe Haven adults were significantly inhibited (Fig. 7A). The settlement percentages of cyprids after a 24-h incubation were also significantly decreased to 26.6% and 42.7%, respectively, at Sai Kung and Hebe Haven compared with 53.7% at Tso Wo Hang, while incubation for 48 h allowed more cyprids to settle but eliminated the significant difference (Fig. 7B). After culturing the settled barnacles for 30 days, the growth parameters for the juvenile barnacles, including the basal diameter and total wet weight, were recorded and found to be inhibited significantly in the Sai Kung and Hebe Haven groups relative to that from the Tso Wo Hang pier (Table 3).

3.7. Correlation between contaminant burden and organism responses

Table 3 Viability of barnacle larvae from different piers.⁎ Larval viability

Tso Wo Hang

Sai Kung

Hebe Haven

Survival rate (%)d Metamorphosis rate (%)d Juvenile Basal size (mm)e barnacle Total weight (mg)e

55.0 ± 5.8a 88.1 ± 3.1a 3.3 ± 0.06a 4.7 ± 0.25a

60.1 ± 16.2a 72.4 ± 5.0b 2.7 ± 0.07b 2.9 ± 0.24b

64.2 ± 26.3a 63.2 ± 5.0b 3.1 ± 0.05c 3.8 ± 0.19c

⁎ Different letters (a, b or c) denote statistically significant difference among sampling sites; d Values represent the mean ± SEM of three replicate tanks; e Values represent the mean ± SEM of four replicate panels, each randomly collecting twenty juvenile barnacles.

The correlation analysis between the contaminant burden, including the total metals and total PBDEs, and the physiological and behavioral responses of the organisms revealed that metals and PBDEs possessed specific responsive indices (Table 4). In detail, metals showed significant correlation, either positive or negative, with MT protein content, DNA integrity, respiration rate, lipid and protein reserves and the phototaxis behavior. In contrast to metals, PBDEs showed a distinct responsive pattern and correlated significantly with AChE enzyme activity, GPx activity, GSH content, ROS level, SOD activity, settlement and phototaxis behavior (Table 4). The exception was the phototaxis behavior,

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where both metals and PBDEs correlated significantly but with an opposite direction (Table 4). For the effects significantly correlated with metals, a positive correlation was observed for MT content, DNA integrity, metabolic rate and energy reserves, while metals correlated negatively with the phototactic behavior of nauplii and cyprids (Table 4). However, for the PBDEcorrelated effects, the positive correlation included AChE, GSH, SOD, settlement and phototaxis, with the exception of GPx and ROS, which showed a negative correlation with the PBDE concentration (Table 4). 4. Discussion Since the filter-feeding barnacles can accumulate ambient trace contaminants to high levels in their tissues, barnacles were used herein as sentinel species in a biomonitoring program to assess the environmental impact at three piers in Hong Kong that are subjected to varying anthropogenic influences. A combined approach of chemical measurements and biological responses was applied, which has been shown to be an effective technique to evaluate the risks of coastal pollution (Cheung et al., 2002). The accumulation of metals and PBDEs has been detected in barnacle tissues, and our results revealed a geographical variation in the concentrations of metals and PBDEs among the three piers. Furthermore, a suite of specific biological responses appeared to be responsive to metals and PBDEs, respectively, presumably ascribed to their different modes of action. In the assessment of environmental quality, the concentration determination of toxic substances that accumulates in organisms has been suggested to be the first step in an integrated biomonitoring system (Zauke et al., 1996). In the present study, chemical analysis of metal concentrations revealed the highest accumulation in barnacles collected from Hebe Haven, particularly for Zn and Cu, compared with the other two piers. The accumulation of metals in barnacles has been well documented, in which the concentrations of Zn and Cu are generally higher than the other metals (Blackmore et al., 1998; Dionísio et al., 2013; Morillo et al., 2005; Páez-Osuna et al., 1999). For example, in B. amphitrite barnacles collected from Mission Bay in San Diego, the concentrations of Zn and Cu have been reported to be as high as 37,900 μg/g and 3750 μg/g, respectively (Fialkowski and Newman, 1998). Up to 23,300 μg/g of Zn and 1810 μg/g of Cu have also been detected in B. amphitrite sampled around the coast of Hong Kong (Rainbow and Blackmore, 2001), which is comparable to the concentrations of Zn (18,440 μg/g) and Cu (915 μg/g) that are currently measured in barnacles from Hebe Haven. The great potential of barnacles to take up Zn and Cu from an ambient seawater environment without evidence of regulation and excretion supports their consistently high bioavailability and also suggests that they are good biomonitors of Zn and Cu in coastal pollution (Powell and White, 1990; Rainbow and White, 1989). Furthermore, because there is no evidence for regulation of the accumulated Zn and Cu, it can be concluded that, in locations where barnacles contain greater amounts of metals, the seawater accordingly has a higher metal bioavailability (Páez-Osuna et al., 1999). Therefore, elevated concentrations of Zn and Cu in barnacle tissues from Hebe Haven could be directly referred to as a higher biological availability of Zn and Cu in the associated seawater. In addition, although Zn and Cu are regarded as essential elements that are involved in the maintenance of normal biological functions when kept at homeostatic levels (Apostoli, 2002), adverse effects are eventually induced under conditions in which the levels of the bioavailable metals are increased beyond the tolerance capacity of the organisms. In the present study, MT protein production was significantly induced in Hebe Haven barnacles, and a significant correlation was observed between metals and MT protein contents. The induction of MT protein is frequently used as a sensitive biomarker to indicate metal pollution in aquatic environments (Zanette et al., 2015). MT protein is involved in the metabolism and detoxification of metals, and after being induced by and binding to metals, the formation of the non-

toxic metal–MT complex protects organisms against direct exposure to metals and thus alleviates the toxic effects of metal accumulation (Viarengo et al., 1997). Therefore, it is conceivable that the induction of MT protein could be a self-adaption and self-protection strategy of barnacles in response to the increased bioavailability of metals. However, if the metal concentrations increase to levels beyond the sequestration capacity of MT protein, then other toxic effects will start to emerge, as characterized by the decrease in DNA integrity observed herein. DNA strand breaks represent another widely used biomarker of metal pollution (Regoli et al., 2004). In addition, the energy reserves (i.e., protein and lipid) and metabolic rate in barnacles from Hebe Haven were both significantly increased, showing a positive correlation with the accumulation of metals. The amount of energy available for biological functioning provides a rapid and sensitive measure of stress in organisms, which generally need to consume large quantities of energy due to the metabolic cost of xenobiotic detoxification following exposure to a suboptimal environment (Smolders et al., 2004). The increased energy demand activates the metabolic processes involved in energy production and drives organisms to produce and allocate sufficient energy for xenobiotic elimination (Chen et al., 2015a; Smolders et al., 2004). Furthermore, the impact on physiological energetics following long-term exposure to environmental stress will eventually be reflected in the indices of health (e.g., growth and reproduction) (Adams and Greeley, 2000; Mersch et al., 1996), which might provide an explanation for the decreased growth of the barnacles from Hebe Haven. In addition to metals, in the present study, we also measured the bioaccumulation of organic PBDE contaminants in the soft tissues of barnacles. Compared with the metals, a contrasting distribution pattern of PBDEs was observed, with highest concentration detected in Tso Wo Hang, which was initially chosen as the reference site. Furthermore, among the PBDE congeners detected, the concentrations followed the following order: BDE47 N BDE99 N BDE100 N BDE153 N BDE154 N BDE183. This order likely reflects the effect of the variation in bromination on the bioaccumulation potential (McDonald, 2002). Furthermore, because congeners of PBDEs with higher levels of bromination can be debrominated to congeners with lower levels (Noyes et al., 2011; Stapleton et al., 2004), the highest bioaccumulation of BDE47 might have also resulted from the debromination of PBDE congeners with higher levels of bromination. Based on the different bioaccumulation factors and intracellular metabolism, the accumulation pattern of PBDE congeners in organisms is less indicative of aqueous concentrations in coastal pollution compared with highly persistent metals. However, despite being detected at much lower concentrations than the metals, PBDE bioaccumulation and chronic exposure would likely lead to adverse effects in barnacles. There is mounting evidence that PBDEs can act as neurotoxicants to disturb neurotransmission, affect the development of the nervous system and lead to alterations in behavioral performance (Chen et al., 2012a; Costa and Giordano, 2007). In the present study, the enzymatic activity of AChE, a sensitive indicator of neurotoxicity, correlated positively with the PBDE concentrations. Previous research has also reported that AChE activity is induced with increasing PBDE concentrations (Chen et al., 2012b; Liang et al., 2010), although the initial induction changes to inhibition following an extended duration of exposure (Liang et al., 2010). The biphasic response is a recognized phenomenon in physiological parameters according to the intensity and duration of the toxic stressor (Regoli et al., 2014). Therefore, when barnacles are exposed to PBDEs within the tolerance threshold, the induction of AChE could be a positive adaption that plays a role in the regulation of physiological homeostasis. However, individual biomarkers possess different sensitivities towards chemical stress (Regoli et al., 2004). For GPx antioxidant activities, a negative correlation of GPx with PBDEs was observed, probably implying that the load of toxicant stress had overwhelmingly depressed the antioxidant capacity and abolished the supposed up-regulation of GPx enzymatic activities. Despite an initial induction, a significant decrease in GPx activities has also been reported in previous research


391

Table 4 Correlation between contaminant burden and organism responses.a Contaminants

∑Metals ∑PBDEs

AChE

− √

GPx

− ×

GSH

− √

MT

√√√ −

ROS

− ×

SOD

− √√

DNA integrity

√ −

Respiration

√√ −

Lipid

√√√ −

Protein

√√ −

Settlement

− √√√

Phototaxis Nauplius

Cyprid

× √√

× √√

a √, significantly positive correlation at P b 0.05; √√, significantly positive correlation at P b 0.01; √√√, significantly positive correlation at P b 0.001; ×, significantly negative correlation at P b 0.05; −, not significantly correlated.

investigating contaminant exposure in mussels (Cheung et al., 2004; Richardson et al., 2008). Because the antioxidant enzyme GPx is responsible for the conversion from GSH to GSSG during the removal of hydrogen peroxide (Chen et al., 2014), the depression of GPx activities in Tso Wo Hang would reduce the consumption of GSH and thus result in the higher levels of GSH detected in the barnacles at that pier. Alternatively, another critical antioxidant enzyme, SOD, was significantly induced in Tso Wo Hang to scavenge the excessive oxyradicals such that ROS production was diminished in barnacles collected from Tso Wo Hang pier. Considering the coincidence of the increased ROS levels and greater DNA damage in barnacles from Hebe Haven, the enhanced oxidative stress at that pier might also have contributed in part to the observed DNA damage (Regoli et al., 2004). In addition, although nauplius and cyprid larvae were cultured in clean seawater without direct exposure, larval behaviors, including both phototaxis and settlement, were also significantly positively correlated with the accumulation of PBDEs. It is known that parental exposure to PBDEs can maternally transfer the chemical load into the offspring (Nyholm et al., 2008; Ostrach et al., 2008; van de Merwe et al., 2011), leading to developmental neurotoxicity and disturbances in larval behavioral performance (Chen et al., 2012a). Furthermore, the behavioral hyperactivity observed with increasing toxicant stress could be termed hormesis, which is a widespread phenomenon in various organisms exposed to diverse toxicants and is regarded as an adaptive response by which the organism protects itself against the effects of the toxin (Chen et al., 2012b; Irons et al., 2010; MacPhail et al., 2009). In summary, in the present study, barnacles were used in a biomonitoring program designed to assess the coastal pollution around three piers (i.e., Tso Wo Hang, Sai Kung and Hebe Haven) in Hong Kong. An integrated approach was used, including chemical measurements of the contaminant concentrations and a wide spectrum of biological responses during the entire lifecycle of the barnacles, with the aim of providing a more complete and more ecologically relevant assessment of the environmental quality. Metal contamination was mainly observed in Hebe Haven, while PBDEs accumulated to the highest extent in Tso Wo Hang barnacles. A suite of specific responses correlated significantly with the presence of metals and PBDEs, respectively, likely indicating their distinct modes of action. Therefore, the integration of chemical and biological tests was found to be a useful tool in the comprehensive assessment of environmental risks. The varying contamination pattern and reduced environmental safety along the coast of Hong Kong highlights the need for extensive environmental surveillance for the future establishment of a regulation framework.

Acknowledgments This project was supported by grants from the China Ocean Mineral Resources Research and Development Association (DY125-15-T-02), the Research Grants Council of the Hong Kong Special Administrative Region (GRF661611 and GRF662413), the National Natural Science Foundation of China (41206080, 41276111), the Science Technology and Innovation Committee of Shenzhen Municipality (JCYJ20130401145617289), and the Hong Kong Research Grants Council (CityU 11100614).

References Adams, S.M., Greeley, M.S., 2000. Ecotoxicological indicators of water quality: using multiresponse indicators to assess the health of aquatic ecosystems. Water Air Soil Pollut. 123, 103–115. Apostoli, P., 2002. Elements in environmental and occupational medicine. J. Chromatogr. B 778, 63–97. Billinghurst, Z., Clare, A.S., Fileman, T., Mcevoy, M., Readman, J., Depledge, M.H., 1998. Inhibition of barnacle settlement by the environmental oestrogen 4-nonylphenol and the natural oestrogen 17ß-oestradiol. Mar. Pollut. Bull. 36, 833–839. Blackmore, G., 1998. An overview of trace metal pollution in the coastal waters of Hong Kong. Sci. Total Environ. 214, 21–48. Blackmore, G., Morton, B., Huang, Z.G., 1998. Heavy metals in Balanus amphitrite and Tetraclita squamosa (Crustacea: Cirripedia) collected from the coastal waters of Xiamen. Chin. Mar. Pollut. Bull. 36, 32–40. Calcagno, J.A., López Gappa, J., Tablado, A., 1998. Population dynamics of the barnacle Balanus amphitrite in an intertidal area affected by sewage pollution. J. Crustac. Biol. 18, 128–137. Chen, L., Yu, K., Huang, C., Yu, L., Zhu, B., Lam, P.K., Lam, J.C., Zhou, B., 2012a. Prenatal transfer of polybrominated diphenyl ethers (PBDEs) results in developmental neurotoxicity in zebrafish larvae. Environ. Sci. Technol. 46, 9727–9734. Chen, L., Huang, C., Hu, C., Yu, Y., Yang, L., Zhou, B., 2012b. Acute exposure to DE-71: effects on locomotor behavior and developmental neurotoxicity in zebrafish larvae. Environ. Toxicol. Chem. 31, 2338–2344. Chen, L., Ye, R., Xu, Y., Gao, Z., Au, D.W.T., Qian, P.Y., 2014. Comparative safety of the antifouling compound butenolide and 4,5-dichloro-2-n-octyl-4-isothiazolin-3one (DCOIT) to the marine medaka (Oryzias melastigma). Aquat. Toxicol. 149, 116–125. Chen, L., Sun, J., Zhang, H., Au, D.W.T., Lam, P.K.S., Zhang, W., Bajic, V.B., Qiu, J.W., Qian, P.Y., 2015a. Hepatic proteomic responses in marine medaka (Oryzias melastigma) chronically exposed to antifouling compound butenolide [5-octylfuran-2(5H)-one] or 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT). Environ. Sci. Technol. 49, 1851–1859. Chen, L., Xu, Y., Wang, W.X., Qian, P.Y., 2015b. Degradation kinetics of a potent antifouling agent, butenolide, under various environmental conditions. Chemosphere 119, 1075–1083. Cheng, S.H., Chan, K.W., Chan, P.K., So, C.H., Lam, P.K.S., Wu, R.S.S., 2004. Whole-mount in situ TUNEL method revealed ectopic pattern of apoptosis in cadmium treated naupliar larvae of barnacle (Balanus amphitrite Darwin). Chemosphere 55, 1387–1394. Cheung, C.C.C., Zheng, G.J., Lam, P.K.S., Richardson, B.J., 2002. Relationships between tissue concentrations of chlorinated hydrocarbons polychlorinated biphenyls and chlorinated pesticides and antioxidative responses of marine mussels, Perna viridis. Mar. Pollut. Bull. 45, 181–191. Cheung, C.C.C., Siu, W.H.L., Richardson, B.J., De Luca-Abbott, S.B., Lam, P.K.S., 2004. Antioxidant responses to benzo(a)pyrene and Aroclor 1254 exposure in the green-lipped mussel, Perna viridis. Environ. Pollut. 128, 393–403. Chiang, W.L., Au, D.W., Yu, P.K., Wu, R.S., 2003. UV–B damages eyes of barnacle larvae and impairs their photoresponses and settlement success. Environ. Sci. Technol. 37, 1089–1092. Ching, E.W.K., Siu, W.H.L., Lam, P.K.S., Xu, L., Zhang, Y., Richardson, B.J., Wu, R.S.S., 2001. DNA adduct formation and DNA strand breaks in green-lipped mussels (Perna viridis) exposed to benzo[a]pyrene: dose- and time-dependent relationships. Mar. Pollut. Bull. 42, 603–610. Costa, L.G., Giordano, G., 2007. Developmental neurotoxicity of polybrominated diphenyl ether (PBDE) flame retardants. Neurotoxicology 28, 1047–1067. Dionísio, M., Costa, A., Rodrigues, A., 2013. Heavy metal concentrations in edible barnacles exposed to natural contamination. Chemosphere 91, 563–570. Durou, C., Poirier, L., Amiard, J.C., Budzinski, H., Gnassia-Barelli, M., Lemenach, K., Peluhet, L., Mouneyrac, C., Roméo, M., Amiard-Triquet, C., 2007. Biomonitoring in a clean and a multi-contaminated estuary based on biomarkers and chemical analyses in the endobenthic worm Nereis diversicolor. Environ. Pollut. 148, 445–458. Faimali, M., Garaventa, F., Piazza, V., Greco, G., Corrà, C., Magillo, F., Pittore, M., Giacco, E., Gallus, L., Falugi, C., Tagliafierro, G., 2006. Swimming speed alteration of larvae of Balanus amphitrite as a behavioural end-point for laboratory toxicological bioassays. Mar. Biol. 149, 87–96. Fialkowski, W., Newman, W.A., 1998. A pilot study of heavy metal accumulation in a barnacle from the Salton Sea, Southern California. Mar. Pollut. Bull. 36, 138–143. Fossati, P., Prencipe, L., 1982. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28, 2077–2080. Greco, G., Corrà, C., Garaventa, F., Chelossi, E., Faimali, M., 2006. Standardization of laboratory bioassays with Balanus amphitrite larvae for preliminary oil dispersants toxicological characterization. Chem. Ecol. 22, S163–S172.

392


He, L.S., Xu, Y., Matsumura, K., Zhang, Y., Zhang, G., Qi, S.H., Qian, P.Y., 2012. Evidence for the involvement of p38 MAPK activation in barnacle larval settlement. PLoS One 7, e47195. Irons, T.D., MacPhail, R.C., Hunter, D.L., Padilla, S., 2010. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neurotoxicol. Teratol. 32, 84–90. Lam, K.S., Wo, K.T., Wu, R.S.S., 2000. Effects of cadmium on the development and swimming behavior of barnacle larvae Balanus amphitrite Darwin. Environ. Toxicol. 15, 8–13. Liang, S., Gao, H., Zhao, Y., Ma, X., Sun, H., 2010. Effects of repeated exposure to decabrominated diphenyl ether (BDE-209) on mice nervous system and its self repair. Environ. Toxicol. Pharmacol. 29, 297–301. MacPhail, R.C., Brooks, J., Hunter, D.L., Padnos, B., Irons, T.D., Padilla, S., 2009. Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Neurotoxicology 30, 52–58. McDonald, T.A., 2002. A perspective on the potential health risks of PBDEs. Chemosphere 46, 745–755. Mersch, J., Wagner, P., Pihan, J.C., 1996. Copper in indigenous and transplanted zebra mussels in relation to changing water concentrations and body weight. Environ. Toxicol. Chem. 15, 886–893. Morillo, J., Úsero, J., Gracia, I., 2005. Biomonitoring of trace metals in a mine-polluted estuarine system (Spain). Chemosphere 58, 1421–1430. Niyogi, S., Biswas, S., Sarker, S., Datta, A.G., 2001. Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons. Mar. Environ. Res. 52, 13–26. Noyes, P.D., Hinton, D.E., Stapleton, H.M., 2011. Accumulation and debromination of decabromodiphenyl ether (BDE-209) in juvenile fathead minnows (Pimephales promelas) induces thyroid disruption and liver alterations. Toxicol. Sci. 122, 265–274. Nyholm, J.R., Norman, A., Norrgren, L., Haglund, P., Andersson, P.L., 2008. Maternal transfer of brominated flame retardants in zebrafish (Danio rerio). Chemosphere 73, 203–208. Ostrach, D.J., Low-Marchelli, J.M., Eder, K.J., Whiteman, S.J., Zinkl, J.G., 2008. Maternal transfer of xenobiotics and effects on larval striped bass in the San Francisco Estuary. Proc. Natl. Acad. Sci. U. S. A. 105, 19354–19359. Outdot, C.R., Gerard, R., Morin, P., Gningue, I., 1988. Precise shipboard determination of dissolved oxygen (Winkler procedure) for productivity studies with a commercial system. Limnol. Oceanogr. 33, 146–150. Páez-Osuna, F., Bójorquez-Leyva, H., Ruelas-Inzunza, J., 1999. Regional variations of heavy metal concentrations in tissues of barnacles from the subtropical pacific coast of Mexico. Environ. Int. 25, 647–654. Pan, L., Zhang, H., 2006. Metallothionein, antioxidant enzymes and DNA strand breaks as biomarkers of Cd exposure in a marine crab. Charybdis japonica. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 144, 67–75. Pan, K., Lee, O.O., Qian, P.Y., Wang, W.X., 2011. Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia. Mar. Pollut. Bull. 62, 1140–1146. Phillips, D.J.H., Rainbow, P.S., 1993. Biomonitoring of Aquatic Trace Contaminants. Chapman and Hall, London. Powell, M.I., White, K.N., 1990. Heavy metal accumulation by barnacles and its implications for their use as biological monitors. Mar. Environ. Res. 30, 91–118. Qiu, J.W., Thiyagarajan, V., Cheung, S.C.K., Qian, P.Y., 2005. Toxic effects of copper on larval development of the barnacle Balanus amphitrite. Mar. Pollut. Bull. 51, 688–693. Rainbow, P.S., 1995. Biomonitoring of heavy metal availability in the marine environment. Mar. Pollut. Bull. 31, 183–192. Rainbow, P.S., Blackmore, G., 2001. Barnacles as biomonitors of trace metal availabilities in Hong Kong coastal waters: changes in space and time. Mar. Environ. Res. 51, 441–463. Rainbow, P.S., White, S.L., 1989. Comparative strategies of heavy metal accumulation by crustaceans: zinc, copper and cadmium in a decapod, an amphipod and a barnacle. Hydrobiologia 174, 245–262.

Rainbow, P.S., Wolowicz, M., Fialkowski, W., Smith, B.D., Sokolowski, A., 2000. Biomonitoring of trace metals in the Gulf of Gdansk, using mussels (Mytilus edulis) and barnacles (Balanus improvisus). Water Res. 34, 1823–1829. Ramos, A.S., Antunes, S.C., Goncalves, F., Nunes, B., 2014. The gooseneck barnacle (Pollicipes pollicipes) as a candidate sentinel species for coastal contamination. Arch. Environ. Contam. Toxicol. 66, 317–326. Regoli, F., Frenzilli, G., Bocchetti, R., Annarumma, F., Scancelli, V., Fattorini, D., Nigro, M., 2004. Time-course variations of oxyradical metabolism, DNA integrity, and lysosomal stability in mussels, Mytilus galloprovincialis, during a field translocation experiment. Aquat. Toxicol. 68, 167–178. Regoli, F., Pellegrini, D., Cicero, A.M., Nigro, N., Benedetti, M., Gorbi, S., Fattorini, D., D'Errico, G., Di Carlo, M., Nardi, A., Gaion, A., Scuderi, A., Giuliani, S., Romanelli, G., Berto, D., Trabucco, B., Guidi, P., Bernardeschi, M., Scarcella, V., Frenzilli, G., 2014. A multidisciplinary weight of evidence approach for environmental risk assessment at the Costa Concordia wreck: integrative indices from Mussel Watch. Mar. Environ. Res. 96, 92–104. Richardson, B.J., Mak, E., De Luca-Abbott, S.B., Martin, M., McClellan, K., Lam, P.K.S., 2008. Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): do mussels “integrate” biomarker responses? Mar. Pollut. Bull. 57, 503–514. Roe, J.H., 1955. The determination of sugar in blood and spinal fluid with anthrone reagent. J. Biol. Chem. 212, 335–343. Siu, W.H.L., Hung, C.L.H., Wong, H.L., Richardson, B.J., Lam, P.K.S., 2003. Exposure and time-dependent DNA strand breakage in hepatopancreas of green-lipped mussels (Perna viridis) exposed to Aroclor 1254, and mixtures of B[a]P and Aroclor 1254. Mar. Pollut. Bull. 46, 1285–1293. Smolders, R., Bervoets, L., De Coen, W., Blust, R., 2004. Cellular energy allocations in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization. Environ. Pollut. 129, 99–112. Stapleton, H., Alaee, M., Letcher, R., Baker, J., 2004. Debromination of the flame retardant decabromodiphenyl ether by juvenile carp (Cyprinus carpio) following dietary exposure. Environ. Sci. Technol. 38, 112–119. van de Merwe, J.P., Chan, A.K.Y., Lei, E.N.Y., Yau, M.S., Lam, M.H.W., Wu, R.S.S., 2011. Bioaccumulation and maternal transfer of PBDE 47 in the marine medaka (Oryzias melastigma) following dietary exposure. Aquat. Toxicol. 103, 199–204. Viarengo, A., Ponzano, E., Dondero, F., Fabbri, R., 1997. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Mar. Environ. Res. 44, 69–84. Wang, W.X., Yang, Y., Guo, X., He, M., Guo, F., Ke, C., 2011. Copper and zinc contamination in oysters: subcellular distribution and detoxification. Environ. Toxicol. Chem. 30, 1767–1774. Whyte, J.N.C., Bourne, N., Ginther, N.G., Hodgson, C.A., 1992. Compositional changes in the larva to juvenile development of the scallop Crassadoma gigantea (Gray). J. Exp. Mar. Biol. Ecol. 163, 13–29. Wu, R.S.S., Lam, P.K.S., Zhou, B., 1997a. A settlement inhibition assay with cyprid larvae of the barnacle Balanus amphitrite. Chemosphere 35, 1867–1874. Wu, R.S.S., Lam, P.K.S., Zhou, B., 1997b. Effects of two oil dispersants on phototaxis and swimming behaviour of barnacle larvae. Hydrobiologia 352, 9–16. Zanette, J., Monserrat, J.M., Bianchini, A., 2015. Biochemical biomarkers in barnacles Balanus improvisus: pollution and seasonal effects. Mar. Environ. Res. 103, 74–79. Zauke, G., Petri, G., Ritterhoff, J., Meurs, H., 1996. Theoretical background for the assessment of the quality status of ecosystems: lessons from studies of heavy metals in aquatic invertebrates. Senckenberg. Marit. 27, 207–214. Zhang, Y.F., Wang, G.C., Xu, Y., Sougrat, R., Qian, P.Y., 2011. The effect of butenolide on behavioral and morphological changes in two marine fouling species, the barnacle Balanus amphitrite and the bryozoan Bugula neritina. Biofouling 27, 467–475. Zhu, B., Lai, N.L.S., Wai, T.C., Chan, L.L., Lam, J.C.W., Lam, P.K.S., 2014. Changes of accumulation profiles from PBDEs to brominated and chlorinated alternatives in marine mammals from the South China Sea. Environ. Int. 66, 65–70.