Marine Pollution Bulletin xxx (2014) xxx–xxx

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

Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete Paul K.S. Shin a,b,⇑, Singaram Gopalakrishnan c, Alice K.Y. Chan a, P.Y. Qian d, Rudolf S.S. Wu c,⇑ a

Department of Biology and Chemistry, City University of Hong Kong, Hong Kong State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong c School of Biological Sciences, The University of Hong Kong, Hong Kong d Division of Life Sciences, Hong Kong University of Science and Technology, Hong Kong b

a r t i c l e

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Keywords: Hypoxia PBDE Interactive effect Polychaete Capitella Settlement

a b s t r a c t Marine benthic polychaete Capitella sp. I is widely known to adapt to polluted habitats; however, its response to xenobiotics under hypoxic conditions has been rarely studied. This research aimed to test the hypothesis that interactive effects of hypoxia and congener BDE-47 of polybrominated diphenyl ethers (PBDE), which is ubiquitous in marine sediments, may alter the settlement of Capitella sp. I. Our results revealed that under hypoxic condition, settlement success and growth in body length of Capitella larvae were significantly reduced compared to those under normoxia of similar BDE-47 concentration. While no significant changes in morphology of settled larvae were noted in both exposure conditions, the presence of BDE-47 could enhance polychaete growth. The present findings demonstrated that the interactive effects of hypoxia and environmentally realistic concentrations of BDE-47 in sediments could affect polychaete settlement, which, in turn, reduce its recruitment and subsequent population size in the marine benthic ecosystem. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Hypoxia is a major environmental problem occurring in a wide range of coastal ecosystems and is a growing global concern because of the increasing extent of anthropogenic eutrophication (Wu, 2002; Levin et al., 2009). In the marine environment, hypoxia occurs when the level of dissolved oxygen (DO) falls below 2 mg L1, at which point most of benthic fauna could show aberrant behaviour (Diaz and Rosenberg, 2008). In recent years, the responses of marine organisms to hypoxia have been reported at the individual and/or population level, in terms of settlement (Baker and Mann, 1992), growth (Zhou et al., 2001), reproduction (Thomas et al., 2006; Thomas and Rahman, 2009), mortality (Diaz and Rosenberg, 1995), spatial distribution (Pihl et al., 1991) and species interactions (Breitburg et al., 1997). The effect of hypoxia can be greatly influenced by direct and indirect inputs of xenobiotics, which are harmful to the biota. Earlier studies have been focused either on the direct effects of hypoxia or contaminants that may impair physiological functions and settlement in ⇑ Corresponding authors. Addresses: Department of Biology and Chemistry, City University of Hong Kong, Hong Kong. Tel.: +852 34427720; fax: +852 34420522 (P.K.S. Shin), School of Biological Sciences, The University of Hong Kong, Hong Kong. Tel.: +852 22990837; fax: +852 25599114 (R.S.S. Wu). E-mail addresses: [email protected] (P.K.S. Shin), [email protected] (R.S.S. Wu).

benthic species (Borowsky et al., 1997; Sundelin and Eriksson, 1998; Schiedek et al., 2006; Lam et al., 2010). However, very few studies focused on the combined effect such as hypoxia and/or anoxia as co-varying stressors which could cause biological damage that is often more dramatic than the direct effects of contaminants alone (Stickle et al., 1989). Polybrominated diphenyl ethers (PBDE) are man-made chemicals designed and extensively used as flame retardants in consumer products and are hazardous contaminants which can accumulate in the environment (Alaee et al., 2003). Among various congeners of PBDE, BDE-47 has aroused great concern due to its persistence, bioaccumulation potential and possible adverse effects on the organisms (Lema et al., 2007). Important sources of PBDE to the coastal environment may include atmospheric deposition, discharge of domestic sewage, surface runoff and river outflow through releases during its manufacture and use as well as treatment and recycling of the end products containing PBDE. Though most of the developed countries have banned PBDE, it has been revealed that these xenobiotics can still be released from products incorporating these compounds (Watanabe and Sakai, 2003). This could further lead to the possibility for the persistence of these compounds in the environment and their interactions with other ubiquitous contaminants or environmental stressors such as hypoxia. Aquatic organisms could concentrate PBDE from water or their diet through trophic transfer (Bartrons et al., 2012). Previous

http://dx.doi.org/10.1016/j.marpolbul.2014.04.037 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Shin, P.K.S., et al. Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.04.037

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P.K.S. Shin et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

studies revealed that BDE-47 also affected the survival rate and physiological biomarkers in grass shrimp (Key et al., 2008), caused morphological abnormalities during embryogenesis in zebra fish (Lema et al., 2007), and delayed hatching and reduced fecundity in the Japanese medaka and fathead minnows (Muirhead et al., 2006). Although most existing studies were focused on the response of vertebrates to PBDE, their effects on invertebrates, especially on marine polychaetes, are poorly known. It is well established that polychaetes have long been used as a representative animal group to reflect the health of benthic communities, as they are habitually the most abundant invertebrate macrofauna occupying in sediments, both in terms of number of species and abundance (Dean, 2008). In addition, polychaetes are a pivotal part of the marine food web and occupy in different trophic levels, with their diverse feeding strategies (Fauchald and Jumars, 1979; Pagliosa, 2005). They also serve as an important descriptor of the state of the benthic environment (Gambi and Giangrande, 1986; Inglis and Kross, 2000; Samuelson, 2001). The larvae of most sedentary polychaetes are free swimming but they need to settle on a suitable substratum for metamorphosis into juveniles. Hence, the relative immobility of sedentary polychaetes poses the threat that they are chronically exposed to xenobiotics in the marine environment, leading to reduction in recruitment success and fitness of these species for their survival in the benthic ecosystem. The release of accumulated contaminants from the marine sediments and their subsequent uptake by the organisms are becoming an increasingly important issue, although the input of primary sources of many xenobiotics has been substantially reduced (Wu, 1999; Schiedek et al., 2007). While the surface layer of the sediment showed a decrease in the contaminants of certain banned products, the deeper layer still maintained relatively higher levels (Jonsson et al., 2000; Isosaari et al., 2002) and could exert adverse effect particularly on burrowing organisms. Moreover, the impact of burial depth of pollutants on their uptake in infauna has received little attention in the literature. In our previous study, we reported that BDE-47 affected polychaete settlement in a species-specific manner and that the settlement of Capitella sp. I was enhanced in the BDE-47 contaminated sediments in Hong Kong (Lam et al., 2010). In marine sediments of Hong Kong, the concentrations of PBDE reported were amongst the highest in the world (Liu et al., 2005). Some of the locations where these sediment samples were collected are also prone to hypoxic events, especially in the summer, as revealed in the monthly monitoring programme undertaken by the Hong Kong Environmental Protection Department (http://epic.epd.gov.hk/ca/uid/marinehistorical/p/1). The presence of contaminants could cause an additional stress on marine organisms which are already impacted due to low level of oxygen in some coastal waters. It has also been reported that hypoxia could increase the toxicity of a number of pollutants to aquatic fish (Alabaster et al., 1983). Here, we build on our previous work by testing the hypothesis that exposure to contaminated sediments with PBDE under hypoxic conditions would adversely affect the settlement of benthic animals. Specifically, the present study aimed to ascertain the interactive effect of BDE-47 doses and hypoxia on larval settlement and subsequent growth of a marine polychaete Capitella sp. I and investigate whether hypoxia can modulate the bioaccumulation of BDE-47 in its body tissue.

2005). The conditions of the aquaria used to culture Capitella sp. I were as follows: sediment at 4 cm depth, temperature 24 ± 1 °C, pH 7.9 ± 0.1 and salinity 32 ± 1‰. Adult Capitella were fed twice a week with commercial fish food Tetramin flakes (Melle, Germany). The sediment samples used in the present study were collected from Tung Lung Chau, east of Victoria Harbour, Hong Kong (22°190 N, 114°160 E) using a van Veen grab (0.1 m2) as described in our earlier study (Lam et al., 2010). This site, located offshore, is free from PBDE contamination (Liu et al., 2005). Freshly collected sediment with total organic carbon of about 6.00 ± 0.80% (referred to as ‘‘natural sediment’’ hereafter) was homogenized and sieved through a 250 lm mesh, rinsed with natural seawater (NSW) and stored at 80 °C for subsequent experiments. To kill the benthic animals/larvae in the natural sediments, samples were frozen at 80 °C for 2 weeks, and then later thawed for 6 h at 4 °C before being used in subsequent treatments. All sediment samples were used quickly for experimentation within two weeks after thawing. The life cycle of Capitella sp. I has been described elsewhere (Grassle and Grassle, 1976; Tsutsumi et al., 2001), in which larval settlement involves the trochophore larvae burrowing into sediment and metamorphose into juveniles with building of mucoid tubes within a few hours. 2.2. Sediment preparation for laboratory tests The BDE-47 (99% purity; Wellington Laboratories, USA) stock solution was prepared by dissolving 0.5 mg of BDE-47 in 10 mL of hexane. Natural sediment was spiked with BDE-47 stock solution as treatments of low and high BDE-47 levels, viz. 0.5 ng g1 (ppb) and 3.0 ng g1 (ppb) dw, which were within the range reported in marine sediments in Hong Kong (Liu et al., 2005). Two controls were also employed, including the untreated, natural sediment (control) and sediment spiked with an equal volume of hexane as that of treatment with high concentration of BDE-47 (solvent control). All treatments were vortexed vigorously after the addition of hexane and/or BDE-47 and kept at 4 °C overnight to prevent microbial degradation. Hexane was first evaporated under fume hood and all sediment samples were centrifuged at 2000 rpm for 5 min to remove excessive seawater, if any, prior to use in the subsequent experiments. 2.3. Experimental system The experimental system was mainly composed of a glass tank (10 L) with dimension of 27  15  20 cm, a digital DO controller (model No. 01972-00, Cole Parmer, Illinois, USA), and a cylinder of compressed nitrogen, regulator and an air pump. The level of oxygen in the experimental tank was monitored automatically by the oxygen probe of the controller. When the desired oxygen of level deviated from the fixed value, the controller would send a signal to the valves connecting to the nitrogen gas tank and/or air pump so as to restore the desired oxygen level by delivering either nitrogen and/or air into the experimental tank. For hypoxic exposure, the DO concentration was regulated at 1.5 mg L1 O2 (Brown-Peterson et al., 2008) and for normoxic exposure, 6.5 mg L1 O2 was used (Lam et al. 2010). Other physical parameters were maintained constantly throughout the experimental exposure similar to that in culture of adult Capitella sp. I at temperature 24 ± 1 °C, pH 7.9 ± 0.1 and salinity 32 ± 1‰.

2. Materials and methods

2.4. Larval multiple-choice settlement and growth experiments

2.1. Culture of polychaete larvae and sediment sample collection

A total of 16 small petri dishes, each with a diameter of 3 cm and height of 1.5 cm, were placed in a glass chamber, following the modified method of Grassle et al. (1992). Four different types of sediment: (a) natural sediment (control), (b) sediment spiked

All experiments were performed with laboratory-cultured Capitella sp. I adopted from previous studies (Thiyagarajan et al.,

Please cite this article in press as: Shin, P.K.S., et al. Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.04.037

P.K.S. Shin et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

with hexane (solvent control), (c) sediment spiked with 0.5 ng g1 dw of BDE-47 (designated as ‘‘low concentration’’ hereafter), (d) sediment spiked with 3.0 ng g1 dw BDE-47 (designated as ‘‘high concentration’’ hereafter), were assigned to the petri dishes following a 4  4 Latin-square design with four replicates per treatment or control. Each petri dish received 10 g (w/w) sediment and the whole chamber was filled up with 2 L of filtered seawater (FSW). The sediments were exposed to the experimental conditions two days before starting the exposure. Short (24-h) and long-term (28-d) studies were conducted separately. For testing larval settlement, 300 competent larvae were added into each chamber with aeration in normoxic treatment and for hypoxic condition, the oxygen level was slowly reduced to 1.5 mg L1 in a period of 3–4 h and the whole experimental setup was kept in darkness at room temperature either for 24 h or 28 d. For counting the number of settled larvae after 24-h exposure, the water in the experimental chamber was reduced slowly and the petri dishes retrieved. The sediment in the petri dishes was gently sieved with filtered seawater to separate the larvae settled in the sediment and counted under a dissecting microscope. For 28-d exposure, the settled larvae were fed with Tetramin flakes once every two days. After the exposure period, the metamorphosed larvae (juveniles) were carefully retrieved from the petri dishes using a pair of fine forceps and counted. The body length of 30 juveniles were randomly sampled from each treatment and measured using an image analysis software analySIS v3.2 (Soft Imaging System, Olympus, Germany). Each experiment was repeated thrice (n = 3) using larvae from different batches of Capitella cultures.

ion monitoring mode using a GC (Agilent 7890) equipped with a mass-selective detector (Agilent 5975) and DB-5HT fused silica capillaries (J & W Scientific Inc.; 0.25 mm i.d.  30 m  0.25 lm film thickness). A total of three replicate sediment and polychaete samples were analyzed. 2.7. Statistical analysis All the data were normalized by log (x + 1) transformation followed by Levene’s test for analysis of homogeneity. For both 24-h and 28-d exposure experiments, numbers of settled larvae or juveniles were analyzed using a replicated Latin-Square ANOVA, followed by Tukey’s HSD tests if significant differences were found (Lam et al., 2010). Under each exposure, effects of row, column, sediment treatment (fixed factor), and replicate (random factor) in each experiment were analyzed. To study the effect of DO and BDE-47 levels on larval settlement and growth, two-way factorial ANOVA was performed. For each treatment, the number of settled larvae in the four replicate wells in each experimental chamber was summed and treated as one sample. In case that significant interaction between the main factors (DO and BDE-47) was found, individual t-tests were used to compare difference between the two DO levels (normoxia and hypoxia) under each BDE-47 treatment and one-way ANOVA, followed by Tukey’s HSD tests, to compare BDE-47 treatments under each DO level. If there was no significant interactive effect between the two factors, one-way ANOVA, followed by Tukey’s HSD tests, was further performed on each factor to identify difference among treatments.

2.5. Scanning electron microscopy

3. Results

After 28-d exposure, 10 specimens of Capitella were randomly selected and fixed in 2.5% glutaraldehyde, 0.1 M sodium cacodylate, 2% paraformaldehyde and 0.4 M sucrose for 2 h at room temperature, and were further rinsed several times in buffer (0.25 M sucrose in 0.1 M sodium cacodylate) and in distilled water. The specimens were then transferred into a secondary fixation process (2% OsO4 in 0.1 M cacodylate buffer at pH 8 in dark) for 2 h, followed by dehydration in an ethanol series (30, 50, 70, 80, 95 and 100%). They were further dried using hexamethyldisilazane for 3 times with 5 min each and then dried completely in a fume hood chamber for 30 min. Finally the specimens were mounted on aluminum stub with carbon tape followed by sputter coating with gold or gold–palladium (BAL-TEC SCD 005 Sputter Coater). All specimens were viewed at an appropriate magnification and digital photographs were taken using FEI/Philips XL30 ESEM FEG scanning electron microscope (SEM).

3.1. Settlement of larvae after 24-h exposure

2.6. Quantification of BDE-47 in sediment and polychaete samples Concentrations of BDE-47 in both sediment samples and settled Capitella juveniles were measured after 28-d exposure experiment. About 3 g of the sediment samples and 10 specimens of metamorphosed Capitella from normoxic and hypoxic treatments were separately freeze-dried, ground with anhydrous sodium sulphate (Sigma, USA), and extracted at 100 °C with a mixture of dichloromethane/hexane (3:1) for 10 min twice in an accelerated solvent extractor (Dionex 350, USA). The extract was evaporated to dryness and weighed. An appropriate amount of 13C-labeled BDE-47 (Wellington Laboratories, USA) was added to the samples as surrogate before extraction. Activated copper was added to desulphurize the sediment samples and cleaned up by silica gel (Sigma 391484, USA) and 13C-labeled BDE-47 (Wellington Laboratories, USA) was added as an internal standard. BDE-47 was quantified either by negative chemical impact or electron impact with single

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The total numbers of larvae settled in control and treatment groups under different exposure conditions in multiple-choice settlement experiment are shown in Fig. 1. The results in larval settlement were consistent in all replicates with uniform sediment treatment response by the competent larvae, with no significant row and column effects (Latin-Square ANOVA, normoxic exposure: p = 0.907 for row effect, p = 0.896 for column effect; hypoxic exposure: p = 0.217 for row effect, p = 0.140 for column effect). On average, about 83% of larvae settled after 24-h exposure under normoxic condition. Settlement of larvae in sediments spiked with high and low concentrations of BDE-47 was significantly greater than that in the control groups. Further, the number of larvae settled in the sediment spiked with high concentration of BDE-47 was significantly more than the other groups. However, on average only 26.8% of the larvae were settled under hypoxic condition after 24 h of exposure. Between different exposures the number of settled larvae under hypoxic condition was significantly less than that of the respective group under normoxic condition (Fig. 1). The results of two-way factorial ANOVA further revealed the significant interactive effect of DO and BDE-47 levels to inhibit larval settlement after 24-h exposure (p < 0.001, Table 1). 3.2. Settlement of larvae after 28-d exposure The total numbers of larvae settled and metamorphosed into juveniles under normoxic and hypoxic conditions are shown in Fig. 2. The results in larval settlement were also consistent in all replicates, without significant row and column effects (LatinSquare ANOVA, normoxic exposure: p = 0.088 for row effect, p = 0.101 for column effect; hypoxic exposure: p = 0.098 for row effect, p = 0.662 for column effect). 72% of the larvae of Capitella settled in sediment samples and metamorphosed into juveniles

Please cite this article in press as: Shin, P.K.S., et al. Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.04.037

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P.K.S. Shin et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

Fig. 1. 24-h exposure experiment: Number of Capitella sp. I larvae settled on control and BDE-47 dosed sediments exposed under normoxia (6.5 mg O2 L1) and hypoxia (1.5 mg O2 L1). Data are mean + SE of three experimental chambers. Two separate one-way ANOVA tests, followed by Tukey’s HSD tests, were carried out to examine the dose effect on (1) normoxic exposure, annotated by lowercase letters and (2) hypoxic exposure, annotated by uppercase letters. Different letters in each test group denote statistical significant difference at p < 0.05.  Denotes significant difference between DO effect under each sediment treatment (t-test, p < 0.05).

Table 1 Results of two-way ANOVA showing the main and interactive effect of DO and BDE-47 during the settlement after 24-h and settlement and growth after 28-d exposure for Capitella sp. I. Significant p value

Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete.

Marine benthic polychaete Capitella sp. I is widely known to adapt to polluted habitats; however, its response to xenobiotics under hypoxic conditions...
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