Environ. Sci. Technol. 2010, 44, 7130–7137
Polybrominated Diphenylethers (PBDEs) Alter Larval Settlement of Marine Benthic Polychaetes CINDY LAM,† REBECCA NEUMANN,† PAUL K. S. SHIN,† DORIS W. T. AU,† P . Y . Q I A N , ‡ A N D R U D O L F S . S . W U * ,†,§ Centre for Marine Environmental Research and Innovative Technology, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, China, Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China, and School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
Received April 21, 2010. Revised manuscript received August 3, 2010. Accepted August 3, 2010.
Polybrominated diphenylethers (PBDEs) are found ubiquitously in marine environments worldwide. Sediment is the major sink of PBDEs, with the congener BDE 47 being most abundant. In this study, laboratory experiments were carried out to test the hypothesis that contamination of BDE 47 at environmentally realistic sediment concentrations can alter polychaete larval settlement. Using multiple-choice experiment, settlement of three polychaete species (Pseudopolydora vexillosa, Polydora cornuta, and Capitella sp. I) on four types of spiked sediment was studied and compared: (i) low BDE 47 concentration (0.5 ng g-1 dry weight); (ii) high BDE 47 concentration (3.0 ng g-1 dry weight), (iii) hexane (solvent control), and (iv) natural sediment (control). Our results showed that settlement of P. vexillosa and Capitella sp. I larvae was significantly promoted, while settlement of P. cornuta reduced, at high BDE 47 concentration in sediment compared with the respective controls under both short- (24 h) and long-term (4 week) exposures. After 4 weeks, body burden of BDE 47 in all polychaete species was directly related to the spike concentration, and body length of settled juveniles of P. vexillosa and Capitella sp. I at the high-concentration treatment was significantly longer compared with that of other treatments and controls. For the first time, we demonstrated that environmentally realistic concentrations of BDE 47 in sediment can affect polychaete settlement in species-specific and dose-dependent manners. Given the global contamination of PBDE in marine sediment, BDE 47 may potentially alter the settlement pattern of marine polychaetes and hence their benthic composition over large areas.
Introduction Polybrominated diphenylethers (PBDEs) have been used extensively as flame retardants in a wide range of products such as plastics, textiles, and electric circuitry (1). PBDEs are generally added into consumer products as commercial mixtures containing different congeners of penta-BDE, octa* Corresponding author e-mail:
[email protected]. † City University of Hong Kong. ‡ Hong Kong University of Science and Technology. § The University of Hong Kong. 7130
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 18, 2010
BDE, or deca-BDE (2). Among various congeners of PBDEs, BDE 47 has aroused great concern due to its persistence, bioaccumulation potential, and possible adverse effects on humans and wildlife (3). Serious health disorders such as thyroidogenic, estrogenic, carcinogenic, and hepatic effects, and impairment of neuro-development have been reported from humans exposed to BDE 47 (4, 5). PBDEs can be introduced into the coastal environment by various processes, such as discharge of domestic sewage and industrial wastewater, agricultural inputs, runoff from nonpoint sources and atmospheric deposition (2). In Southern China, including the Pearl River Delta, extremely high levels of PBDEs have been found in sediment (0.04-94.7 ng g-1 dry weight, (6) and Hong Kong (0.96-58.5 ng g-1 dry weight, (7)). Marine organisms in higher trophic levels (e.g., fish) can concentrate PBDEs from water or their diet. Oral exposure of BDE 47 delayed hatching and reduced fecundity in the Japanese medaka and fathead minnows (9). Developmental disorders such as reduced growth, abnormal morphology, irregular cardiac function, and altered cerebrospinal fluid flow were found in zebrafish upon exposure to high concentration of PBDEs (100-5000 µg L-1) (3). Brief exposure to BDE 47 causes morphological abnormalities during embryogenesis in zebrafish, implying that PBDEs can be teratogenic (3). Chronic exposure to BDE 47 can disrupt thyroid hormones and affect various key enzymes regulating steroidogenesis and receptors in fish gonads, subsequently altering levels of gonadotropins and thereby impairing fish reproduction (9). While the vast majority of the existing studies were on vertebrates, effects of PBDEs on invertebrates remained very poorly known. The life cycle of many marine invertebrates (e.g., polychaetes, barnacles, and bivalves) involves a planktonic larval stage followed by a sessile juvenile/adult stage after metamorphosis. It has been well established that larval settlement of marine invertebrates on both sediment and hard substratum is influenced by both positive and negative settlement cues produced by bioorganic films (10). When settling on hard substrate, exploring larvae respond to both physical (i.e., water current, light intensity) factors and chemical cues produced by micro-organisms on the substrate surface, while settlement of larvae on soft-bottom is guided by factors such as organic contents (11), sediment disturbance by water flow (12, 13), grain sizes (14), presence of conspecific juveniles or adults (15, 16), and haloaromatic metabolites of sympatric organisms (17). Polychaetes are not only an important food source for consumers at higher trophic levels (18), but also among the most dominant taxa in marine benthic communities worldwide. Disruption in polychaete settlement will therefore affect recruitment and fitness of this species, and hence its population dynamics in a habitat. Previous investigations have shown that polychaetes (Streblospio and Capitella) larvae prefer to settle on natural sediment than on polyaromatic hydrocarbons (PAHs)-contaminated sediment, and pollutants can affect their settling behavior as well as reduce growth of juveniles after settlement (19-21). Despite sediment being the major sink for PBDEs and well documented bioaccumulation of PBDEs by marine benthic fauna (22, 23), the potential impact of PBDEs on settlement of marine benthic polychaetes remains largely unknown. Such information is clearly important in identifying the hazards of PBDEs contamination to the marine environment. Of the 209 congeners of PBDEs, congener 47 is ubiquitously found in sediment and biota (3). Although polychaetes are abundant in sediment, limited studies related to toxi10.1021/es1012615
2010 American Chemical Society
Published on Web 08/20/2010
cological effects of such congener on settlement have been addressed. We hypothesize that BDE 47 in sediment will affect polychaete settlement in a species-specific manner. Experiments were carried out to investigate both short- and longterm effects of BDE 47 on settlement of three different species of polychaetes (i.e., two marine infaunal spionid polychaetes Pseduopolydora vexillosa and Polydora cornuta, and one Capitellidae, Capitella sp. I), using a multiple-choice experiment. Metamorphosis and growth (in terms of body length) of juveniles settled in contaminated and natural sediment were also studied and compared after exposure to sediment PBDEs for 24 h and 4 weeks. Body burden of BDE 47 in settled juveniles after 4 weeks was also determined and compared.
Materials and Methods Study Organisms. Two tube-building, infaunal spionid polychaetes, Pseudopolydora vexillosa and Polydora cornuta, and one deposit-feeding polychaete Capitella sp. I, were used for experiments. These species were selected because they are globally abundant in marine sediment. The life cycles of Capitella sp. I (24, 25), P. vexillosa (26), and P. cornuta (27) have been described elsewhere. Larval settlement of Spionidae and Capitellidae involves burrowing into sediment and metamorphosis into juveniles with building of mucoid tubes within a few hours. Collection and Treatment of Sediment Samples. Sediment was collected from Tung Lung Chau (TLC), east of Victoria Harbor, Hong Kong (22°19′ N, 114°16′ E) using a van Veen grab (0.1 m2). This site was selected because it is relatively clean and has low levels of pollutants (28, 29). Newly collected sediment with total organic carbon of 6.00 ( 0.80% (referred to as “natural sediment” hereafter), was homogenized and sieved through a 250-µm mesh, rinsed with natural seawater (NSW) and stored at -80 °C (designated as natural sediment). Sediment was frozen at -80 °C for 2 weeks to kill off the benthic animals/larvae therein, and thawed for 6 h at 4 °C before being used for experiment. In any case, sediment samples would not be kept for more than 2 weeks after thawing. Laboratory Maintenance of Polychaetes. Culture techniques of P. cornuta, P. vexillosa, and Capitella sp. I were adopted from refs 30, 26, and 28, respectively, with some minor modifications. Briefly, adult worms seeded in small culture containers (Ø 8 cm, height 6 cm) were maintained in plastic aquaria with fully aerated seawater under a photoperiod of 12 h light:12 h dark at constant temperature (24 °C) and salinity (32‰). Biweekly, adult P. vexillosa were fed with unicellular algae Chaetoceros gracilis, Capitella sp. I were fed with ground fish food (Tetra Marin, Germany), and P. cornuta were fed with a mixture of C. gracilis, Isochrysis galbana, and Dunaliella tertiolecta. Larvae of P. cornuta and P. vexillosa were collected and sieved through a 38- and 74µm mesh, respectively, and rinsed into a 1-L culture vessel for cultivation. Larval cultures were maintained under the same conditions as adults. Algae were cultured in f1/2 medium in filtered seawater (FSW) according to ref 31. Larvae were cultured until they become competent at 12-14 chaetigers stage in 7-9 days (P. vexillosa) and at 14-15 setigers in 11-12 days (P. cornuta), whereas Capitella sp. I larvae were harvested from brooding females and used directly for experiment. Competent larvae were defined according to morphology and number of chaetigers (32) and setigers (33). Sediment Preparations. Due to its high octanol-water partitioning coefficient (Kow), BDE 47 is lipophilic and shows a strong affinity to organic carbons in the sediment. Natural sediment was spiked with low (0.5 ng g-1 dry weight (d.w.) or high (3.0 ng g-1 d.w.) concentrations of BDE 47 (99% purity; Wellington Laboratories, USA), freeze-dried overnight, and
then homogenized. A stock solution of BDE 47 was prepared by dissolving 0.5 mg of BDE 47 in 10 mL of hexane before being added into the sediment. Both testing concentrations are within the reported range in marine sediment in the Pearl River Delta region (7). Natural sediment with and without hexane served as solvent control and control, respectively. An equal volume of solvent was used in all spiked sediments. All treatments were vigorously vortexed after the addition of BDE 47 or hexane and kept at 4 °C overnight to prevent microbial degradation. Hexane was first evaporated under fumehood and all sediment samples were centrifuged at 2000 rpm for 5 min to remove excessive seawater prior to multiple-choice settlement assays. Multiple-Choice Settlement Assays. A replicated 4 × 4 Latin-square design was used for the larval choice experiment. Multiple-choice chambers (L: 39 cm, W: 32 cm, H: 20 cm), each containing 16 wells (Ø 5 cm; depth 0.8 cm; Falcon 1006, USA), were set up for the multiple-choice experiment, following the method of 11. Four different types of sediment: (a) natural sediment (control), (b) sediment spiked with hexane (solvent controls), (c) sediment spiked with 3.0 ng g-1 d.w. BDE 47 (designated as “high concentration” hereafter), and (d) sediment spiked with 0.5 ng g-1 d.w. of BDE 47 (designated as “low concentration” hereafter), were assigned to the wells following a Latin-square design with four replicates per treatment/control. Each well received 1.0 g (w.w.) sediment and the whole chamber was filled up with 2 L of filtered seawater (FSW). For each of the three species tested, around 500 competent larvae were added into each individual multiple-choice chamber with aeration, and the whole experimental setup was kept in darkness at room temperature for 24 h. Another identical set of multiple-choice chambers was prepared and kept under the same conditions for 4 weeks, before larval settlement and growth were examined. Five hundred mL of FSW in the container was added weekly to adjust the salinity and feed the settled juveniles with the aforementioned algae (at 10-50 total chlorophyll mL-1). Total number of settled juveniles in each well within the same multiple-choice chamber was counted under stereomicroscope at the conclusion of the exposure period (i.e., after 24 h and after 4 weeks). Swimming larvae with no metamorphosis were excluded from the analysis. Each experiment was repeated thrice (n ) 3) for each species using larvae from different batches of larval cultures. Quantification of BDE 47 in Sediment and Polychaetes after Exposure. An identical set of multiple-choice settlement assays without larvae was established together with the ones with larvae under the same experimental condition stated above. Concentration of BDE 47 in sediment after settlement assays was measured after 24 h and after 4 weeks, while BDE 47 concentration in each polychaete species was measured after 4 weeks. Both sediment and polychaete samples from each treatment were freeze-dried, ground with anhydrous sodium sulfate (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, weighed, and its lipid content was determined. An appropriate amount of 13C12labeled 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), while polychaete samples was cleaned up by acidified silica gel. 13 C12-labeled BDE 77 (Wellington Laboratories) was added as an internal standard. BDE 47 was quantified either by NCI or EI with SIM mode using a GC (Agilent 7890) equipped with a mass-selective detector (Agilent 5975) with DB-5HT fused silica capillaries (J & W Scientific Inc.; 0.25 mm i.d. × 30 m × 0.25 µm film thickness). VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7131
FIGURE 1. Twenty-four h exposure experiment: Number of juvenile Pseudopolydora vexillosa (A-C, replicates 1-3), Polydora cornuta (D-F, replicates 1-3), and Capitella sp. I (G-I, replicates 1-3) settled on the various types of sediment. Data are mean ( standard deviation of four replicate wells in each multiple-choice chamber. Different lower-case letters denote significant differences compared with the natural sediment control (C) (Tukey’s test, G < 0.05) and solvent control (SC).
Statistical Analysis. Prior to statistical analysis, larval settlement data were normalized by log (x + 1) transformation. Levene’s test was used for homogeneity check. A lower significance level was used (F ) 0.01) when significant heterogeneity of variance could not be removed (34). For both 24 h and 4 weeks exposure, numbers of settled larvae were analyzed using a replicated Latin-Square ANOVA (11). In each experiment, effects of row, column, sediment treatment (fixed factor), and replicate (random factor) were analyzed. If the effects of row, column, and sediment treatment were inconsistent among replicates, the number of settled larvae per treatment in each assay was analyzed separately using one way Latin-Square ANOVA. If there was a significant difference among sediment treatments, Tukey’s multiple comparison tests were used to identify significant difference between different treatments/controls. 7132
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 18, 2010
Results Experiment 1: Larval Settlement after 24 h Exposure. About 90% of Pseudopolydora vexillosa, Polydora cornuta, and Capitella sp. I larvae settled in sediment samples and metamorphosed into juveniles after 24 h exposure. Total numbers of larvae settled in different treatments and controls in multiple-choice settlement assays are shown in Figure 1. For P. vexillosa and Capitella sp. I, larval settlement in sediment with high concentration of BDE 47 (3.0 ng g-1 d.w.) was significantly higher than that in sediment with low concentration, as well as those in both natural sediment and solvent controls (Tukey’s test, F < 0.05 for P. vexillosa; F < 0.001 for Capitella). In contrast, settlement of P. cornuta larvae was significantly lower in sediment with high BDE 47 concentration than that in low concentration and both controls (Tukey’s test, F < 0.05). The results in larval settlement of three tested species were consistent in all replicates. Neither
FIGURE 2. Four weeks exposure experiment: Number of juvenile Pseudopolydora vexillosa (A-C, replicates 1-3), Polydora cornuta (D-F, replicates 1-3), and Capitella sp. I (G-I, replicates 1-3) settled on various types of sediment. Data are mean ( standard deviation of four replicate wells in a multiple-choice chamber. Different lower-case letters denote significant differences compared with the natural sediment control (C) (Tukey’s test, G < 0.05) and solvent control (SC). row nor column effects were significant in all experiments (Latin Square ANOVA of P. vexillosa: F ) 0.27 for row effect, F ) 0.11 for column effect; P. cornuta: F ) 0.71 for row effect, F ) 0.33 for column effect; Capitella: F ) 0.83 for row effect; F ) 0.21 for column effect), suggesting a good experimental precision and uniform sediment treatment response by competent larvae. Experiment 2: Larval Settlement after 4 Weeks Exposure. Over 90% of P. vexillosa, P. cornuta, and Capitella sp. I larvae settled in sediment samples and metamorphosed into juveniles/adult worms exhibiting distinct morphology (i.e., palps and gonads) after 4 weeks. Total numbers of juveniles settled in different treatments and controls are shown in Figure 2. For P. vexillosa and Capitella sp. I, juvenile settlement in sediment with high concentration was significantly higher than that spiked with low concentration, as well as in both natural sediment and solvent
control (Tukey’s test, F < 0.01 for P. vexillosa; F < 0.001 for Capitella sp. I). Juvenile settlement of P. cornuta was reduced in the high-concentration treatment when compared with that of the controls (Tukey’s test, F ) 0.009). Results of juvenile settlement in all three tested species were consistent in all replicates. Neither row nor column effect were significant in all experiments (Latin Square ANOVA of P. vexillosa: F ) 0.29 for row effect, F ) 0.23 for column effect; P. cornuta: F ) 0.32 for row effect, F ) 0.10 for column effect; Capitella sp. I: F ) 0.11 for row effect, F ) 0.063 for column effect). Growth of Juveniles after Exposure to Sediment BDE 47 for 24 h and 4 Weeks. Body length of each juvenile (from head to tail without palps) of each tested species exposed to high and low BDE 47 concentrations was measured and compared with their respective untreated and solvent controls after 24 h and 4 weeks. VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7133
FIGURE 3. Body length of juveniles of Pseduopolydora vexillosa (A), Polydora cornuta (B), and Capitella sp. I (C) after 24 h and 4 weeks exposure to BDE 47 in the multiple-choice experiment. Data are mean ( standard deviation of 50 worms randomly sampled from each treatment. Different lower-case letters denote significant differences compared with the respective natural sediment control (C) (Tukey’s test, G < 0.05) and solvent control (SC) within the same exposure period. After 24 h exposure, body length of settled juveniles in both treatments and controls ranged from 0.89 ( 0.21 to 1.00 ( 0.29 mm for P. vexillosa; from 1.11 ( 0.56 to 1.31 ( 0.54 mm for P. cornuta; and from 0.58 ( 0.14 to 0.63 ( 0.08 mm for Capitella sp. I. No significant difference was observed between treatments and controls in all tested species (Tukey’s test, F ) 0.99 for P. vexillosa and P. cornuta; F ) 1.0 for Capitella sp. I; Figure 3). Body lengths of three polychaete species in both treatments and controls under 4 weeks exposure are shown in Figure 3. An increase in body length was clearly observed in settled juveniles of P. vexillosa and Capitella sp. I at the high concentration treatment (5.27 ( 1.46 and 3.47 ( 1.29 mm, respectively) compared to those settled at the low concentration (3.85 ( 1.05 µm for P. vexillosa; 2.18 ( 0.70 mm for Capitella sp. I) and natural sediment (3.03 ( 0.73 mm for P. vexillosa; 2.23 ( 0.80 mm for Capitella sp. I) and solvent controls (3.41 ( 0.89 mm for P. vexillosa; 2.39 ( 1.11 mm for Capitella sp. I; Tukey’s test, F < 0.001). In contrast, body length of juvenile P. cornuta settled in the high-concentration treatment was 1.41 ( 0.52 mm, which was significantly shorter than those settled in the low concentration treatment (1.95 ( 0.78 mm), as well as the natural sediment (1.86 ( 0.95 mm) and solvent control (1.72 ( 0.89 mm; Tukey’s test, F ) 0.029). 7134
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 18, 2010
BDE 47 Concentration in Spiked Sediments and Polychaetes. Mean concentration of BDE 47 in freshly collected natural sediment was 0.26 ( 0.00080 ng g-1 d.w. (n ) 2). To study the stability of BDE 47 in spiked sediment, concentration of BDE 47 in all controls and treatments was measured under different exposure times. For 24 h exposure, concentrations of BDE 47 in untreated and solvent controls were 0.21 ( 0.017 and 0.16 ( 0.078 ng g-1 d.w., respectively; whereas BDE 47 concentrations in sediment spiked with 0.5 and 3.0 ng g-1 d.w. were 0.83 ( 0.13 and 3.88 ( 0.28, showing that the measured values were within the range of spiking concentrations. Levels of BDE 47 in untreated and solvent controls after 4 weeks exposure were 0.20 ( 0.049 and 0.22 ( 0.029 ng g-1 d.w.; whereas levels in sediment spiked with BDE 47 at high and low concentrations were 0.46 ( 0.039 and 2.05 ( 0.038 ng g-1 d.w., respectively. The recoveries of BDE 47 in sediment after 24 h and 4 weeks were over 80% and 70%, respectively (Table 1). Method recovery of 13C12labeled BDE 47 ranged from 66.20 to 81.30% in the 24 h exposure experiment, and 61.10 to 98.90% for the 4 weeks exposure experiment. Concentrations of BDE 47 in each of the three polychaete species after 4 weeks exposure are shown in Table 2 (n ) 2). The highest body burden of BDE 47 was found in sediment spiked with high concentration of BDE 47 (694.37 ( 95.09 Fg mg-1 lipid for P. vexillosa; 261.43 ( 28.35 Fg mg-1 lipid for P. cornuta; and 453.09 ( 108.09 Fg mg-1 lipid for Capitella sp. I), followed by sediment spiked with low concentration of BDE 47 (64.94 ( 26.42 Fg mg-1 lipid for P. vexillosa; 104.06 ( 16.62 Fg mg-1 lipid for P. cornuta; and 87.08 ( 11.17 Fg mg-1 lipid for Capitella sp. I), which were significantly higher than those in the solvent control (34.11 ( 9.55 Fg mg-1 lipid for P. vexillosa; 82.52 ( 9.39 Fg mg-1 lipid for P. cornuta; and 46.72 ( 3.54 Fg mg-1 lipid for Capitella sp. I) and natural sediment (6.25 ( 5.03 Fg mg-1 lipid for P. vexillosa; 35.29 ( 13.31 Fg mg-1 lipid for P. cornuta; and 22.99 ( 0.66 Fg mg-1 lipid for Capitella sp. I). Recovery of 13C12-labeled BDE 47 ranged from 63.06 to 107.51%.
Discussion BDE 47 had no effect on larval metamorphosis, and all settled larvae metamorphosed and developed into juveniles and adults in sediment spiked with BDE 47, regardless of concentration and exposure time. Invertebrate larvae are sensitive to pollutants in sediment (35) and, therefore, have often been used as bioindicators for various organic pollutants (24, 36). Polychaete settlement can be either stimulated or inhibited by sediment-bound organic matters (11). For example, Capitella sp. I (28, 29) and P. vexillosa (26) larvae preferentially settle and metamorphose into juveniles in organically enriched sediment; whereas high concentrations of polyaromatic hydrocarbons in sediment moderately inhibit settlement of another polychaete Streblospio benedicti (21). For the first time, we demonstrate that polychaetes settlement can be affected in a species-specific manner and also a dosedependent manner following both short-term and long-term exposure to BDE 47. Sediment-larvae-juveniles interaction is a complicated process, probably guided by multiple settlement cues (36, 37). Sediment characteristics such as organic carbon content and grain size have been identified as positive settlement cues of many polychaetes (11, 14). For instance, larval settlement of P. cornuta, Capitella sp. I, and Streblospio benedicti was lower in ashed sediment than in natural sediment, indicating the importance of organic carbon in inducing larval settlement (33, 38). Such sediment choice made by polychaete larvae is directly related to the nutrition quality of a habitat that supports higher postsettlement performance and growth of juveniles
TABLE 1. Concentrations of BDE 47 in Sediment Samples Before and After 24 h and 4 Weeks Exposure in the Multiple-Choice Assays treatments before settlement assays control for settlement assays control solvent control 0.5 ppb 3.0 ppb
spiked concentration (ng g-1 d.w.)
measured concentration (ng g-1 d.w.)
NA
0.20 0.20 0.50 3.00
blank recovery (%)
after 24 h exposure 0.21 ( 0.017 0.16 ( 0.078 0.83 ( 0.13 3.88 ( 0.28 0.11 66.20-81.28
TABLE 2. Tissue Concentration of BDE 47 (Gg mg-1 lipid) in Three Polychaetes Species after 4 Weeks of Exposures
control solvent control 0.5 ppb 3.0 ppb
P. vexillosa
P. cornuta
Capitella sp.
6.25 ( 5.03 34.11 ( 9.55 64.94 ( 26.42 694.37 ( 95.09
35.29 ( 13.31 82.52 ( 9.39 104.06 ( 16.62 261.43 ( 28.35
22.99 ( 0.66 46.72 ( 3.54 87.08 ( 11.17 453.09 ( 108.09
in the preferred sediment (40). In the natural environment, however, PBDEs are strongly adsorbed to sediment with high organic matters due to their lipophilic nature (41). Thus, a higher level of PBDEs would be expected in marine sediment with a high % of organic matters in the natural habitat, and this may therefore further augment larval settlement of P. vexillosa and Capitella sp. I. Organic matters in sediment is not a contributing factor to the observed difference in larval settlement in the present study, because percent of organic matter is the same in all treatments and controls (6.00 ( 0.80%). Although BDE 47 does not have any discernible effect on growth of all three polychaete species after 24 h, long-term (4 weeks) exposure to high concentrations of BDE 47 significantly promoted growth of settled juveniles of both P. vexillosa and Capitella sp. I (Figure 3). On the contrary, reductions in settlement and body length of P. cornuta may affect the recruitment and fitness of this species, and hence the population dynamics and species interactions in contaminated area over a long period of time. It is not known whether abnormalities would be observed in the F1 generation produced from adults dwelling in sediment contaminated with BDE 47, and study is now underway in our laboratory to further test this. Molecules that interfere with signal transduction pathways involved in settlement and metamorphosis of many marine invertebrates have received considerable attention recently. A variety of chemicals, including juvenile hormones (42), polysaccharides (43), free fatty acids (44, 45), protein and small peptides (46), and neurotransmitters other than amino acids (46), have been suggested as possible settlement cues. Sediment enriched in proteins and lipids have previously served as effective settlement cues to promote settlement of some polychaete species (29, 46). Numerous studies have shown that chemicals produced by microbes on sediment surfaces serve as important cues for induction of larval settlement of marine invertebrates (12, 33, 36, 37, 45). Future work should be carried out to study whether and how PBDEs affect bacteria-derived settlement cues and receptors of competent larvae. Toxicity of PBDEs is related to degree and position of bromination, exposure concentration, and body burden
13
C BDE 47 recovery (%) 105.01 80.98 166.23 129.44
0.26 ( 0.00080 after 4 weeks exposure 0.20 ( 0.049 0.22 ( 0.029 0.46 ( 0.039 2.05 ( 0.038
13 C BDE 47 recovery (%) 100.01 110.98 92.43 68.76
0.42 61.07-98.93
in an organism. Body burdens in fish and marine benthic polychaetes were also compared. Oral exposure to highdose BDE 47 (ppm level) for 2 weeks has been shown to lower the fecundity in medaka and fathead minnow (9), and accumulation of BDE 47 was found within a few hours and equilibrium was reached after 3-10 days. In this study, body burden of BDE 47 showed a significant increase in all three species after 4 weeks (Table 2), and bioaccumulation was directly related to the spike concentration in the sediment. Although gonads were developed in polychaetes after 4 weeks of exposure, no larvae were produced in both treatments and controls. It would be interesting to study maternal transfer of BDE 47 in polychaetes and whether the fitness of the F1 generation would be affected. Stability and environmental fate of PBDEs in sediment is highly dependent on its chemical properties. Results from our chemical analysis showed that levels of BDE 47 in sediment retained over 70% of the original nominal concentrations after 4 weeks (Table 1), suggesting that BDE 47 is relatively stable and degradation is not significant within 4 weeks under laboratory conditions. Recent study has demonstrated that PBDEs could be effectively degraded by microorganisms under anaerobic conditions (47). Thus, the effects of sediment on polychaete settlement under hypoxic conditions might be very different and warrant further study, as hypoxia occurs over large coastal areas worldwide (48). In summary, this study clearly demonstrates that environmentally realistic concentrations of BDE 47 in sediment can affect polychaete settlement in speciesspecific and dose-dependent manners. Because sustainability of marine benthic polychaete populations is highly dependent upon larval recruitment, alteration in settlement patterns of different species may change the normal recruitment patterns and eventually species composition of the benthic community. Given the ubiquitous contamination of PBDEs in marine sediment worldwide, PBDEs may potentially alter polychaetes settlements and hence structure of benthic communities over large areas. It would be interesting to carry out experiments to test whether PBDEs have a similar effect on settlement of other important benthic invertebrate species of other taxa (especially keystone species which are important in maintaining community structure). Effects of PBDEs on settlement behaviors of polychaetes in the natural environment is a complicated process and may not necessarily be the same as revealed in the present laboratory study. To verify the present laboratory findings, field deployment of sediment trays with spiked BDE 47 should be carried out to study larval settlement pattern and benthic comVOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7135
munity composition in treated and untreated sediments under field conditions.
Acknowledgments The work described in this paper was fully supported by a grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (AoE/P-04/04). Special thanks to A. Chan and E. Lei for technical support for GC-MS analysis and W. Tse for helping with the multiplechoice settlement assays.
Literature Cited (1) WHO. Environmental Health Criteria 162: Brominated Diphenyl Ethers; World Health Organization: Geneva, 1994; pp 31-34. (2) Alaee, M.; Arias, P.; Sjo¨din, A.; Bergman, Å. An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release. Environ. Int. 2003, 29, 683–689. (3) Lema, S. C.; Schultz, I. R.; Scholz, N. L.; Incardona, J. P.; Swanson, P. Neural defects and cardiac arrhythmis in fish larvae following embryonic exposure to 2,2′,4,4′-tetrabromodiphenyl ether (PBDE 47). Aquat. Toxicol. 2007, 82, 296–307. (4) Darnerud, P. O.; Eriksen, G. S.; Jo´hannesson, T.; Larsen, P. B.; Viluksela, M. Polybrominated diphenyl ethers: occurrence, dietary exposure, and toxicology. Environ. Health Perspect. 2001, 109 (Suppl. 1), 49–68. (5) McDonald, T. A. A perspective on the potential health risks of PBDEs. Chemosphere 2002, 46, 745–755. (6) Xiang, C. H.; Luo, X. J.; Chen, S. J.; Yu, M.; Mai, B. X.; Zheng, E. Y. Polybrominated diphenyl ethers in biota and sediments of the Pearl River Estuary, South China. Environ. Toxicol. Chem. 2006, 26, 616–623. (7) Liu, Y.; Zheng, G. J.; Yu, H.; Martin, M.; Richardson, B. J.; Lam, M. H. W.; Lam, P. K. S. Polybrominated diphenyl ethers (PBDEs) in sediment and muscle tissues from Hong Kong marine waters. Mar. Pollut. Bull. 2005, 50, 1173–1184. (8) Timme-Laragy, A. R.; Levin, E. D.; Di Giulio, R. T. Developmental and behavioral effects of embryonic exposure to the polybrominated diphenylether mixture DE-71 in the killfish (Fundulus heteroclitus). Chemosphere 2006, 62, 1097–1104. (9) Muirhead, E. K.; Skillman, A. D.; Hook, S. E.; Schultz, I. R. Oral exposure of PBDE 47 in fish: toxicokinetics and reproductive effects in Japanese Medaka (Oryzias latipes) and fathead minnows (Pimephales promelas). Environ. Sci. Technol. 2006, 40, 523–528. (10) Woodin, S. A. Recruitment of infauna - positive and negative cues. Am. Zool. 1991, 31, 797–807. (11) Grassle, J. P.; Butman, C. A.; Mills, S. W. Active habitat selection by Capitella sp. I larvae. 2. Multiple-choice experiments in still water and flume flows. J. Mar. Res. 1992, 50, 717–743. (12) Woodin, S. A.; Todd, C. D. Inhibition and facilitation of settlement of epifaunal marine invertebrate larvae by microbial biofilm cues. Biofouling 1998, 12, 81–118. (13) Marinelli, R. L.; Woodin, S. A. Disturbance and recruitment: a test of solute and substrate specificity using Mercenaria mercenaria and Capitella sp. I. Mar. Ecol.: Prog. Ser. 2004, 269, 209–221. (14) Pinedo, S.; Sarda, R.; Rey, C.; Bhaud, M. Effect of sediment particle size on recruitment of Owenia fusiformis in the Bay of Blanes (NW Mediterranean Sea): an experimental approach to explain field distribution. Mar. Ecol.: Prog. Ser. 2000, 203, 205– 213. (15) Olivier, F.; Desroy, N.; Retiere, C. Habitat selection and adult recruit interactions in Pectinaria koreni (Malmgren) (Annelida: Polychaeta) post-larval populations: results of flume experiments. J. Sea Res. 1996, 36, 217–226. (16) Hardege, J. D.; Bentley, M. G.; Snape, L. Sediment selection by juvenile Arenicola marina. Mar. Ecol.: Prog. Ser. 1998, 166, 187– 195. (17) Woodin, S. A.; Marinelli, R. L.; Lincoln, D. E. Allelochemical inhibition of recruitment in a sedimentary assemblage. J. Chem. Ecol. 1993, 19, 517–530. (18) Commito, J. A.; Ambrose, W. G., Jr. Multiple trophic levels in soft-bottom communities. Mar. Ecol.: Prog. Ser. 1985, 26, 289– 293. (19) Chandler, G. T.; Scott, G. I. Effects of sediment-bound endosulfan on survival, reproduction and larval settlement of meiobenthic polychaetes and copepods. Environ. Toxicol. Chem. 1991, 10, 375–382. 7136
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 18, 2010
(20) Bridges, T. S.; Levin, L. A.; Cabrera, D.; Plaia, G. Effects of sediment amended with sewage, algae or hydrocarbons on growth and reproduction in two opportunistic polychaetes. J. Exp. Mar. Biol. Ecol. 1994, 177, 99–119. (21) Chandler, G. T.; Shipp, M. R.; Donelan, T. L. Bioaccumulation, growth and larval settlement effects of sediment-associated polynuclear aromatic hydrocarbons on the estuarine polychaete, Streblospio benedicti (Webster). J. Exp. Mar. Biol. Ecol. 1997, 213, 95–110. (22) Voorspoels, S.; Covaci, A.; Maervoet, J.; De Meester, I.; Schepens, P. Distribution of PCBs/OCPs in benthic organisms and fish from the North Sea Continental Shelf and Scheldt estuary. Mar. Pollut. Bull. 2004, 49, 393–404. (23) Yang, R. Q.; Lv, A. H.; Shi, J. B.; Jiang, G. B. The levels and distribution of organochlorine pesticides (OCPs) in sediments from the Haihe River, China. Chemosphere 2005, 61, 347354. (24) Grassle, J. P.; Grassle, J. F. Sibling species in the marine pollution indicator Capitella (Polychaeta). Science 1976, 192, 567–569. (25) Tsutsumi, H.; Wainright, S.; Montani, S.; Saga, M.; Ichihara, S.; Kogure, K. Exploitation of a chemosynthetic food resources by the polychaete Capitella sp. I. Mar. Ecol.: Prog. Ser. 2001, 216, 119–127. (26) Mok, F.; Thiyagarajan, V.; Qian, P. Y. Larval development and metamorphic behavior of the subtropical spionid polychaete Pseudopolydora vexillosa. J. Exp. Mar. Biol. Ecol. 2008, 357, 99– 108. (27) Blake, J. Reproduction and larval development of Polydora cornuta from northern New England (Polychaeta: Spionidae). Ophelia 1969, 7, 1–63. (28) Thiyagarajan, V.; Soo, L.; Qian, P. Y. The role of sediment organic matter composition in larval habitat selection by the polychaete Capitella sp. I. J. Exp. Mar. Biol. Ecol. 2005, 323, 70–83. (29) Thiyagarajan, V.; Soo, L.; Shin, P. K. S.; Qian, P. Y. Spatio-temporal variation in sediment biochemistry alters larval habitat selection and juvenile performance in the polychaete Capitella sp. I. Mar. Ecol.: Prog. Ser. 2006, 327, 207–222. (30) Irvine, S. Q.; Martindale, M. Q. Laboratory culture of the larvae of Spionidan polychaetes. Mar. Models Electron. Rec. 1999, 29. (31) Guillard, R. R.; Ryther, J. H. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve). Gran. Can. J. Microbiol. 1962, 8, 229–239. (32) Hsieh, H. L. Larval development and substrate preference at settlement in Pseudopolydora diopatra (Polychaeta: Spionidae). Invertebr. Reprod. Dev. 1999, 25, 205–214. (33) Sebesvari, Z.; Esser, F.; Harder, T. Sediment-associated cues for larval settlement of the infaunal spionid polychaetes Polydora cornuta and Streblospio benedicti. J. Exp. Mar. Biol. Ecol. 2006, 337, 109–120. (34) Underwood, A. J. Experiments in Ecology: Their Logical Design and Interpretation Using Analysis of Variance; Cambridge University Press: Cambridge, 1997. (35) Bock, M. J.; Miller, D. C. Fluid flow and suspended particulates as determinants of polychaete feeding behavior. J. Mar. Res. 1996, 54, 565–588. (36) Pawlik, J. R. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar. Biol. Annu. Rev. 1992, 30, 273– 335. (37) Hadfield, M. J.; Paul, V. J. Natural chemical cues for the settlement and metamorphosis of marine invertebrate larvae. In McClintock, J. G., Baker, B. J., Eds.; Marine Chemical Ecology; CRC Press: Boca Raton, FL, 2001; pp 431-461. (38) Butman, C. A.; Grassle, J. P.; Buskey, E. J. Horizontal swimming and gravitational sinking of Capitella sp. I (Annelida: Polychaeta) larvae: implications for settlement. Ophelia 1998, 29, 43–57. (39) Cohen, R. A.; Pechenik, J. A. Relationship between sediment organic content, metamorphosis, and postlarval performance in the deposit-feeding polychaete Capitella sp. I. J. Exp. Mar. Biol. Ecol. 1999, 240, 1–18. (40) Moon, H. B.; Kannan, K.; Lee, S. J.; Choi, M. Polybrominated diphenyl ethers (PBDEs) in sediment and bivalves from Korean coastal waters. Chemosphere 2007, 66, 243–251. (41) Pawlik, J. R.; Faulkner, D. J. Specific free fatty acids induce larval settlement and metamorphosis of the reef-building tube worm Phragmatopoma californica (Fewkes). J. Exp. Mar. Biol. Ecol. 1986, 102, 301–310.
(42) Biggers, W. J.; Laufer, H. Detection of juvenile hormone-active compounds by larvae of the marine annelid Capitella sp. I. Arch. Insect. Biochem. Physiol. 1996, 32, 475–484. (43) Kirchman, D.; Graham, S.; Reish, D.; Mitchell, R. Lectins may mediate in the settlement and metamorphosis of Janua (Dexiospira) brasiliensis Grube (Polychaeta: Spirorbidae). Mar. Biol. Lett. 1982, 3, 131–142. (44) Pawlik, J. R. Chemical induction of larval settlement and metamorphosis in the reef-building tube worm Phragmatopoma california (Sabellariidae: Polychaeta). Mar. Biol. 1986, 91, 59–68. (45) Jensen, R. A.; Morse, D. E. Chemically induced metamorphosis of polychaete larvae in both the laboratory and the ocean environment. J. Chem. Ecol. 1990, 16, 911–930.
(46) Jin, T.; Qian, P. Y. Effect of amino acids on larval metamorphosis of the polychaete Hydroides elegans. Mar. Ecol.: Prog. Ser. 2004, 267, 209–218. (47) Yen, J. H.; Liao, W. C.; Chen, Y. S.; Wang, Y. S. Interaction of polybrominated diphenyl ethers (PBDEs) with anaerobic mixed bacterial cultures isolated from river sediment. J. Hazard. Mater. 2009, 165, 518–524. (48) Gray, J. S.; Wu, R. S. S.; Or, Y. Y. Effects of hypoxia and organic enrichment on the marine coastal environment. Mar. Ecol.: Prog. Ser. 2002, 238, 249–279.
ES1012615
VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7137
