Reproductive Responses and Detoxification of Estuarine Oyster

Feb 7, 2015 - ... Hong Kong University of Science and Technology, Clearwater Bay, ... Dynamics of maternally transferred trace elements in oyster larv...
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Reproductive Responses and Detoxification of Estuarine Oyster Crassostrea hongkongensis under Metal Stress: A Seasonal Study Nanyan Weng† and Wen-Xiong Wang*,†,‡ †

Key Laboratory of Coastal and Wetland Ecosystems, Ministry of Education, College of the Environment and Ecology, Xiamen University, Xiamen, Fujian 361102, People’s Republic of China ‡ Division of Life Sciences, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong S Supporting Information *

ABSTRACT: Understanding the impacts of metal stress on the reproduction of dominant species, such as oysters, in seriously contaminated estuarine environments has great ecological implications. In the present study, the reproductive conditions were examined monthly for 1 year in oysters Crassostrea hongkongensis from a heavily metalcontaminated site (Baijiao, mainly by Cu and Zn) in the Jiulong River estuary and a relatively clean nearby estuary (Jiuzhen). Oysters sampled in the contaminated site showed a delayed gametogenesis, a relatively shorter spawning period, and a lower gonad condition index in comparison to the oysters sampled in the reference site. In particular, we found that the proportion of females increased significantly in the contaminated oysters, which provided the first evidence that the feminization in wild oyster populations could be related to trace metal pollution. Additionally, the potential detoxification mechanism of trace metals in oysters was also investigated. Compartmentalization of trace metals in membrane-limited vesicles in hemocytes could be an important detoxification mechanism for the contaminated oysters. Our findings indicated that the long-term metal exposure may greatly influence the reproduction of the oysters and finally affect the recruitment and population of this species.

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reduced growth in oysters (C. hongkongensis).11,12 Among the many adverse effects, impairment of reproductive activity is one of the most devastating consequences of environmental pollution and can lead to a decline in species and, consequently, affect the continuation of populations. Therefore, understanding the impact of metal contamination on the reproduction of dominant species, such as oysters, has great implications for these estuarine ecosystems. Indeed, the population numbers of shellfish in the Jiulong River estuary have greatly decreased in recent years, which may be attributed to the metal pollution. Also, several previous studies have demonstrated that trace metals, such as cadmium, can greatly affect the reproduction and recruitment of clams.13−17 Moreover, the fact that blue-colored oysters accumulated such high concentrations of Cu and Zn while still surviving indicated that these oysters must possess efficient strategies of metal detoxification. Structure compartmentation of metals in membrane-bound vesicles (granules) has been documented as an important detoxification mechanism of metals in bivalves.18−22 Whether there is a similar detoxification strategy for the blue-colored oysters is still unclear. Only one relevant

INTRODUCTION Benthic bivalves play a key role in community structure and maintenance of estuarine ecosystems, because they are the important primary consumers and can control the abundance of primary producers, such as phytoplankton.1,2 Reduction of these dominant species in the estuarine habitat can result in great changes of the ecosystems. It is thus important to identify the environmental stress and potential impacts on these species. With rapid industrial development, trace metal pollution has become one of the most important threats to marine organisms in many coastal and estuarine environments.3−6 A recent study has shown that the Jiulong River estuary located in Fujian province was severely polluted by trace metals.7 Blue-colored oysters (Crassostrea hongkongensis) that accumulated extraordinary high concentrations of Cu (14 000 μg g−1 of dry weight) and Zn (24 000 μg g−1 of dry weight) were found in a contaminated site of this estuary.7 The oyster C. hongkongensis is one of the major sedentary species in contaminated estuaries in south China. Although many studies have been conducted to investigate the metal pollution levels in coastal environments and bivalves in south China,8−10 little is known about the effects of long-term metal pollution on the health conditions of oyster populations, especially in these severely contaminated environments. Two relevant studies suggested that metal pollution in these environments can result in cellular dysfunctions as well as © XXXX American Chemical Society

Received: November 11, 2014 Revised: February 6, 2015 Accepted: February 7, 2015

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observed with an Olympus BX5 light microscope, and images were taken with the Olympus BX51 digital camera. All oysters were observed microscopically to assess the gonad developmental stage according to the qualitative criterion proposed earlier.24,25 Five progressive stages of gonad development were distinguished as follows. Stage 0: undifferentiated gonad (see Figure S1A of the Supporting Information). Gonadal acini are rare, small, and poorly branched. Clutches of gonial cells can be found near gonaduct and surrounded by vesicular connective tissue. In this stage, it is not possible to distinguish the male and female gonad. Stage 1: developing gonad (see panels B and F of Figure S1 of the Supporting Information). The vesicular connective tissue is reduced; the follicles are expanded; and early vitellogenic oocytes or spermatocytes can be observed on the follicular walls. Stage 2: ripe gonad (see panels C and I of Figure S1 of the Supporting Information). Mature oocytes are filled with the whole follicles of females, characterized by a polygonal shape and discrete nucleus. In the males, the spermatozoa are arranged in a radial manner and occupy most of the spermatogenic follicle. Stage 3: spawning gonad (see panels D and H of Figure S1 of the Supporting Information). Partial liberation of mature gametes are characterized by loosely packed gametes in the follicles; free ripe oocytes are prominent in the follicles; and the radial arrangement of the spermatozoa is lost. Stage 4: spent or resting gonad (see panels E and G of Figure S1 of the Supporting Information). Almost all of the gametes have been released or resorbed (spent); the follicles are degenerated; and often few residual gametes remain. The area occupied by the gonad was also determined using an image system analyzer (Image Pro Plus, version 4) at 4× from a mean of three different slices from each specimen. The image analysis was based on the intensity of the tissue-specific color, and the gonad and visceral area were automatically calculated in pixels and expressed in millimeters squared. The area reported as the GCA (%) was calculated as GCA = (gonad occupation area/total area) × 100. Moreover, GSI as a specific parameter for a quantitative estimation of the reproductive state of bivalves was determined.17 Specifically, the remaining oyster tissues of gonad development examination were dissected into gonadal tissue and somatic tissue and dried at 80 °C to obtain the dry weights. The oyster GSI was calculated following the method by Liu et al. as GSI (%) = (weight of gonad/total tissue weight) × 100.26 The sex of oysters was identified by microscopic examination according to different histological characteristics of gonad tissue between females and males, as described above (n = 33 for each site each month). The oysters with gonad in the undifferentiated stage, which is difficult to be distinguished, were classified as undeterminated individuals. The sex ratio was calculated as the percentage distribution of males, females, and undeterminated individuals each month for oysters in two sites. Histological and Ultrastructure Examination of Mantle Tissue. Because the mantle tissue of contaminated oysters showed an abnormal blue color and a high metal bioaccumulation, histological and ultrastructure examinations were conducted for this tissue to explore the potential metal detoxification mechanisms in oysters. The procedure of histological examination of mantle tissue was the same as the gonad examination. For ultrastructural observation, tissues were

study demonstrated that a large proportion of Cu and Zn was distributed in the cellular debris fraction of blue-colored oysters.7 In the present study, oysters (C. hongkongensis) were therefore collected monthly in the Jiulong River estuary and a relatively clean nearby Jiuzhen estuary for a 1 year period. The reproductive cycle of C. hongkongensis populations from both sites was identified through histological observations. Several physiological reproductive end points, such as gonad development stage, sex ratio, gonadosomatic index (GSI), and gonadal coverage area (GCA), as well as metal concentrations in gonad tissues were compared between the two oyster populations. The potential effects of long-term exposure of trace metals on the reproduction of oysters in the field were then examined. Meanwhile, the tissue distribution of metals was assessed during the sampling period, and the histological and ultrastructure observations of mantle tissues were conducted to explore the possible detoxification mechanisms of trace metals in contaminated oysters.

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MATERIALS AND METHODS Study Sites and Oyster Sampling. This study was carried out in a major oyster species C. hongkongensis from two estuaries (Jiulong River estuary and Jiuzhen estuary) of Fujian province, southern China. The reference site (Jiuzhen) is located in an oyster farm in the relatively clean Jiuzhen estuary in Fujian province and remote from any industrial activity. The contaminated site (Baijiao) is located in the Jiulong River estuary, which was about 50 km away from the reference site, and is highly contaminated with trace metals, especially Cu and Zn.7 This location was close to sources of industrial effluent discharges. Monthly monitoring indicated that these two estuaries were similar in their physicochemical (temperature, salinity, dissolved oxygen, and pH) and biological (phytoplankton abundance) conditions (see Table S1 of the Supporting Information).23 Oysters were collected from the oyster reefs during low tides. The sampling were conducted monthly in both sites at the same location for a 1 year period (September 2011−August 2012: spring, March−May; summer, June−September; fall, October−November; winter, December−February). Upon arrival in the laboratory, the shell length of oysters was measured and only individuals (about 1.5−2 years old) with similar size (10−12 cm) were included in the analysis (see Table S2 of the Supporting Information). A total of 40 oysters from each site were selected each month, and seven of them were analyzed for metal concentrations in different soft tissues of oysters. Their mantle, gill, digestive gland, and gonad were dissected separately. The remaining oyster samples (n = 33) were used to examine the gonad developmental stage, sex ratio, GSI, and GCA. Histological Examination of Gonad Development. A fraction of gonad tissue (0.5 × 0.5 × 0.5 cm) including a portion of digestive gland was dissected dorsoventrally in the middle body of the oyster C. hongkongensis (n = 33 for each site each month) and then fixed for 48 h in aqueous Bouin’s fixative for histological determination. The remaining tissue was stored at −80 °C for the following experiments. After dehydration through increasing alcohol concentrations (70, 80, 90, and 95%), the tissues were embedded in paraffin. Serial sections were cut at 6 μm using the HM315 microtome and then treated with hematoxylin−eosin staining solution. The tissues were finally mounted under a coverslip in neutral resins. Slides were B

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Figure 1. Percentage of females and males in each stage of the reproductive cycle of C. hongkongensis sampled from Baijiao (the contaminated site) and Jiuzhen (the reference site) from September 2011 to August 2012. “W” is for winter (n = 33 for each month).

prepared by several procedures as described previously.27 In brief, the small pieces of mantle tissue of oysters (n = 3 for each site) from two sampling sites were fixed with 0.2 M normal phosphate-buffered saline (PBS, pH 7.4) containing 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde. After 2 h of fixation, the tissue was rinsed with 0.1 M PBS buffer solution for 15 min and repeated 3 times. Then, the samples were postfixed in 1% osmic acid, dehydrated through an ethanol series, and embedded in Spurr’s resin. Gold sections (approximate thickness of 80 nm) were mounted on copper grids, stained in uranyl acetate and lead citrate, and then examined using a JEM1230 transmission electron microscope. Metal Analysis. Samples of oyster tissues (n = 7 for each site each month) were dried at 80 °C to obtain the dry weights and then digested with nitric acid (70%, Merck Suprapur) and H2O2. For the analytical quality control, a standard reference material [SRM 1566b oyster tissues, National Institute of Standards and Technology (NIST), Gaithersburg, MD] was digested simultaneously. The concentrations of Cu, Zn, Cr, Ni, Cd, Ag, and Pb in all digested samples were determined by inductively coupled plasma−mass spectrometry (ICP−MS, Agilent 7700x). ICP−MS was calibrated with the external standards, and appropriate internal standards (45Sc, 72Ge, and 118 In) were selected to correct the instrumental drift and sensitivity change. A quality control sample was repeatedly measured after each 10 samples, and the relative standard deviations were less than 10%. For SRM 1566b oyster tissues, the recovery of all metals was within 10% deviation from the certified values, except Cr, of which the certified concentration was not available. Reported results were not adjusted for recovery. Statistical Analysis. A one-way analysis of variation (ANOVA) was performed to evaluate the statistically significant difference of metal concentrations (Cu, Zn, Cr, Ni, Cd, Ag, and

Pb) in different tissues, GCA, and GSI between sites and months. Prior to the ANOVA, the normality and homogeneous variances were tested with untransformed data. Data were then submitted to a S−N−K test to confirm the critical difference between groups. The differences of the sex ratio between sites and months were tested by a χ2 test, and the relative effects of site and month on the GSI, GCA, and sex ratio were evaluated by the generalized linear model. All statistical analyses were performed using SPSS 16.0, and p < 0.05 was accepted as significant. The plots were drawn in Sigma Plot 10.0.

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RESULTS Reproductive Cycle. Figure 1 shows the frequency distribution of different gonad development stages in oysters from both sites during the 1 year sampling period. In the reference (Jiuzhen) site, both male and female gonads began to develop in January, with increasing development from February to April. Ripe oysters were observed as early as April when 38.9% of the oysters were ripe and 46.2% of the female oysters had partially spawned. Spawning individuals of males were first found in May, and more than 70% of oysters (both female and male) sampled in June were in spawning. The major spawning season lasted from April to September. Oysters in spent stage (stage 4) were observed from July to December, with a maximum in October (57.2% for females and 80% for males). Sexually undeterminated individuals were evident during November and March. For oysters from the contaminated site (Baijiao), developing gonads were first documented in February for females and March for males. In April, 31.6% of females and 44.4% of males were ripe. Spawning oysters first appeared in May for females and June for males. The period from May to September was the major spawning season. During the period of October and December, almost all of the individuals were in spent stage. C

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similar monthly change pattern of GSI and GCA was also observed in oysters from the contaminated site (Baijiao). Percentages of males, females, and undeterminated individuals at each month from both sites are illustrated in Figure 3. A relatively high proportion of females was found in all of the sampling months in both sites, especially during the period from April to September (ripe and spawning seasons). During winter and early spring seasons, the gender of most of the individuals could not be determined, especially for the contaminated site. In comparison between sites within months, the oyster population from the contaminated site (Baijiao) presented a significantly increasing proportion of females (χ2 test; p < 0.05) in most months (except January, February, and March) of the reproductive cycle. In May and August, about 90.1 and 88.9% of individuals sampled from the Baijiao site were females, whereas 63.6 and 55% of individuals were females from the Jiuzhen site. Using all of the individuals for calculation (n = 396 for each site), the sex ratio (females/males = 2.54) of Baijiao oysters was also significantly higher than that of Jiuzhen oysters (females/males = 1.43) (χ2 test; p < 0.05). As described in Table S3 of the Supporting Information, both site and month significantly influenced the GSI, GCA, and sex ratio of oyster populations; however, the relative effects of the site were much more important than that of the month and their interactions according to the higher F values. Metal Concentration and Distribution in Different Tissues. Annual means and ranges of the metal concentration in different tissues of oysters from two sites are shown in Table 1. In comparison between sites within months, concentrations of all metals (Cu, Zn, Cr, Ni, Cd, Ag, and Pb) in different tissues (gill, mantle, digestive, and gonad) of oysters from the contaminated site (Baijiao) were significantly higher than those from the reference site (Jiuzhen), especially for Cu and Zn (ANOVA; p < 0.05). For instance, the annual mean concentrations of Cu of Baijiao oysters were 23.8-fold higher in the gills, 26.4-fold higher in the mantle, and 21.9-fold higher in the digestive gland, and 21.9-fold higher in the gonad than those of Jiuzhen oysters. There was also a large variation of metal concentrations in oysters given the rather small replicate of samples measured (n = 7). Temporal variations of the metal concentration in different tissues of oysters from both sites are given in Figure S2 of the Supporting Information. All of the metals (Cu, Zn, Cr, Ni, Cd, Ag, and Pb) displayed obvious monthly fluctuations in different oyster tissues of two sites (ANOVA; p < 0.05), except Cd, Ni, and Ag in gills. Metal concentrations in the digestive and gonad tissues showed greater monthly variations than those in the gill and mantle. Both tissues showed a similar monthly change pattern for most of the metals, i.e., a great decrease in the spring season (from March to May), followed by an increase in the summer season (from June to September), and finally, a decrease but relatively high concentration in winter. During winter and early spring seasons, the highest and lowest metal concentrations were always found in gills and gonads from both sites for all metals, except Pb, for which the tissues of the digestive and gonad exhibited much higher concentrations. For Cu and Zn, the highest concentrations were always found in gills and mantle and gonad tissues kept the lowest metal concentrations from both sites during the whole sampling period. The relative distribution of each metal in each tissue is shown in Figure 4. The results were based on metal contents in different tissues as well as relative tissue mass. The distribution

Sexually undeterminated individuals were obvious (more than 60% of both females and males) from January to March. In general, both males and females of oysters from both sites exhibited a synchronism in gametogenic evolution and spawning throughout the period studied. However, differences between sites were also observed. First, the contaminated oysters experienced a delay in gametogenesis about 1 month for females and 2 months for males. Second, the main spawning period of contaminated oysters covered only 4 months from June to September, whereas an extended spawning period was observed in the reference site, especially for males lasting eight months from May to December. Moreover, an increasing proportion of individuals was in the spent stage (stage 4), especially during the period from October to December. Gonad State and Sex Ratio. The GSI and GCA values of oysters from both sites were comparable between females and males during the sampling period; therefore, the results presented in Figure 2 include both males and females. In

Figure 2. Temporal change of GSI and GCA of C. hongkongensis sampled from Baijiao (the contaminated site) and Jiuzhen (the reference site) from September 2011 to August 2012. Data are the mean ± standard deviation (SD) (n = 33 for GSI and GCA for each month). Different letters indicate a significant difference between months, with the uppercase letters used for the Baijiao site and the lowercase letters used for the Jiuzhen site. “∗” indicates a significant difference between sites according to the corresponding month (p < 0.05 in all cases).

comparison between sites within months, the GSI and GCA values of oysters from the reference site (Jiuzhen) were significantly higher than those from the contaminated site (Baijiao) for most of the sampling months (ANOVA; p < 0.05). Significant monthly changes of GSI and GCA were observed in the oysters from both sites (ANOVA; p < 0.05). In the reference site, both GSI and GCA displayed an increase from December, reached the maximum value in April, followed by a decrease in April−June, then an increase to another peak in August, and then decreased to the lowest value in November. A D

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Figure 3. Sex proportion (%) of females, males, and indeterminate individuals of C. hongkongensis sampled from Baijiao and Jiuzhen from September 2011 to August 2012 (n = 33 for each month).

(including the temperature, salinity, dissolved oxygen, chlorophyll a content, and pH) had no significant difference between the two sampling sites around the year (see Table S1 of the Supporting Information).23 Therefore, we hypothesized that environmental contamination may be the major factor contributing to the changed reproductive cycle of oysters from the Jiulong River estuary. Similarly, a recent study on oyster Crassostrea angulata in the coast of Taiwan also demonstrated that the oyster population from contaminated areas showed a delay of gonad development and a high prevalence of individuals with undifferentiated and spent gonads.25 To explore the potential influence of metal contamination on oyster reproduction, metal concentrations in different oyster tissues were determined each month in both sites. We found that all of the tissues of oysters from the contaminated site had higher metal accumulation (Cu, Zn, Cr, Ni, Cd, Ag, and Pb) than those from the reference site around the whole reproductive cycle. In particular, the gonad of oysters from the contaminated site had high metal accumulation, even though it was not the target organ of metal accumulation in bivalves.32,33 For example, the annual mean concentrations of Cu, Cr, Ag, and Cd in gonad of Baijiao oysters were up to 21.9-, 17.9-, 7.2-, and 3.8-fold higher than those of Jiuzhen oysters, respectively. The high metal accumulation in gonad tissue of oysters from the contaminated site may indicate the potential for adverse effects on the reproduction of oysters. AdjeiBoateng et al. showed that gonadal development of the clam Galatea paradoxa was negatively correlated with Zn and Fe accumulation in gonad tissue.34 Brown et al. found that the reproductive capacities of Potamocorbula amurensis in San Francisco Bay were significantly and negatively correlated with the bioaccumulation of Ag.35 Similarly, long-term environmental monitoring conducted in the contaminated areas of south San Francisco Bay demonstrated that the population of Macoma balthica continued to decline and only a few individuals were able to produce mature gametes from 1970 to 1980 as a result of metal contamination (especially Cu). The reproductive capacity then gradually recovered from 1980 to 1991 with the decreased input of trace metals.36 Therefore, long-term metal exposure of the oyster population in the contaminated site may contribute to their changes in the reproductive cycle. To further assess the reproductive capacity of oysters, we also evaluated the GSI, GCA, and sex ratio of the two oyster

patterns were similar for both sites, with mantle always containing the largest percentage, especially for Cu and Zn. The relative proportion of metals in different tissues followed the order of mantle > gill > digestive > gonad for all of the metals, except Ni, in Jiuzhen oysters, for which much more Ni was found in the gills. Because a larger proportion of metals was distributed in mantle tissue, which also showed an abnormal blue color from Baijiao, histological and ultrastructural observations were conducted for mantle tissue of oysters from both sites to explore the possible detoxification mechanisms of trace metals. Figure 5 shows a large number of hemocytes in the mantle of Baijiao oysters (panels A and B of Figure 5), whereas only a few hemocytes were found in the mantle of Jiuzhen oysters (panels C and D of Figure 5). Furthermore, numerous amorphous, round, and limited membranes of vesicles with 0.5−1.0 μm in diameter were observed in the hemocytes of mantle tissues of contaminated oysters (panels E and F of Figure 5). The edges of vesicles were also characterized by electron-dense inclusions.

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DISCUSSION The oyster C. hongkongensis is one of the main sedentary species in estuaries of south China. The possible influences of metal pollution on the reproduction of this species have seldom been considered, especially in severely contaminated environments. In the present study, the reproductive cycle of C. hongkongensis from both contaminated and reference sites was investigated through histological examination. We found that the oyster C. hongkongensis had an annual reproductive cycle, with the beginning of gametogenesis in January for the reference site (Jiuzhen). The main ripe and spawning periods were from April to September (spring to summer), followed by the spent and undifferentiated stages for the rest of the year. Significant variations of the reproductive cycle and gonad conditions were observed between the two oyster populations. In the contaminated site, oyster populations exhibited a delayed gametogenesis, a relatively shorter spawning period, and an increase proportion of individuals in the spent stage when compared to the reference site. In natural conditions, the reproduction of bivalves has been regulated by many environmental factors, such as the temperature, salinity, and food availability.28−31 Possible interference of contaminants has been demonstrated in many previous studies.14−16 According to our previous study, the basic environmental conditions E

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(128−337) (1170−3500) (0.17−0.67) (0.15−2.23) (1.26−8.95) (0.47−2.88) (0.78−2.44)

populations. We found that the GSI and GCA in both sexes from the contaminated site were significantly lower than those from the reference site for most months during the reproductive cycle, indicating that long-term metal exposure may cause decreased production of gametes in these oysters. Several previous studies demonstrated that exposure to metals may impact the energy balance of bivalves attributing to increased maintenance cost, which finally led to a reduction in energy available for reproduction.13,37,38 More importantly, we found that oysters collected from the contaminated site showed an increased proportion of females in most months of the year. For instance, the proportion of females in May was 90.5% compared to 63.7% in the reference site. The imbalanced sex ratio of the oyster population may be caused by several reasons, such as size, poor environmental conditions, and pollution.25,26 In our study, the size of oysters and environmental conditions were similar between the two sites. A growing body of studies highlighted the potential role of contaminants with endocrine-disrupting properties on the sex determination of marine organisms.39−41 Among the various toxic effects, metals are suspected of estrogenic effects in wildlife, such as the estuarine crab Chasmagnathus granulata.42 High estrogenicity of Cd, followed in effect level by Co, Pb, Cr, and Cu in vitro (human breast MCF-7 cells), was verified by Choe et al.43 using an estrogen screen assay, and these metals are considered as a new class of non-steroidal environmental estrogens. The increased expression of the estrogen-regulated genes for the progesterone receptor induced by Co, Cu, Ni, Pb, Hg, and Cr was also observed in previous studies.17,44 Thus, the feminization of oysters from the contaminated site may be attributed to the endocrinedisrupting effect of metals. Cadmium is one of the trace metals that has been verified as an endocrine-disrupting chemical in marine bivalves.13−15 Recently, Liu et al. demonstrated that 30day sublethal exposure of Cu, Zn, Pb, and Cd in the laboratory can significantly change the sex ratio of the blood clam Tegillarca granosa.26 However, they found an increasing proportion of males in the clams following metal exposure, in contrast to our present finding. The results indicated that the endocrine-disrupting effects of metals may be species-dependent. In addition to trace metals, organic pollutants, such as pesticide (DDTs), bisphenol A, and organotins, have also been shown to have endocrine-disrupting effects on marine mollusks.39−41 For instance, the xenoestrogens, bisphenol A, resulted in superfeminization of prosobranch snails.40 The oysters sampled from the contaminated site (Baijiao) in the Jiulong River estuary are considered to be mainly contaminated by multi-metals. We did not specifically quantify levels of organic contaminants in the oysters. Zhang et al. showed that the levels of 17 organic phosphorus pesticides (OPs) and 18 organic chlorine pesticides (OCs), including DDTs, in seawater from the Jiulong River estuary were low compared to other estuaries.45 Evidence also suggested that the other trace organic pollutants, such as polychlorobiphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), were relatively low in the sediment of the Jiulong River estuary, and the concentrations of these organic pollutants in bivalves, such as mussels and oysters, were below the limits of food criteria.46 More recently, Zhang et al.47 measured the concentrations of estrogenic compounds in the same estuary and found that the levels of these compounds (bisphenol A, diethylstilbestrol, and nonylphenol) were very low in the water in stations close to our sampling site

Cu Zn Cr Ni Cd Ag Pb

17100 20900 10.9 14.3 11.5 16.5 1.34

(9710−26900) (13800−27200) (5.89−18.7) (9.51−21.1) (8.10−14.7) (10.9−27.6) (0.89−1.77)

13300 18800 5.37 4.23 9.72 13.5 1.38

(5700−24700) (9210−27200) (2.51−10.2) (1.25−7.80) (6.59−13.5) (6.61−20.7) (1.08−2.03)

8370 12100 7.51 3.36 14.3 9.92 2.33

(3430−14500) (5580−18700) (1.71−13.2) (1.42−8.03) (2.98−22.5) (3.14−26.4) (0.93−3.36)

5300 8040 7.00 3.02 15.1 10.8 2.33

(1270−8620) (2230−14500) (3.32−14.5) (0.73−5.28) (6.66−22.3) (3.20−20.0) (1.49−3.45)

720 7650 0.51 6.67 4.48 2.64 0.70

(497−1080) (5150−11700) (0.38−0.76) (3.61−12.2) (3.57−5.88) (0.44−5.50) (0.53−0.95)

503 5870 0.26 1.04 2.95 1.68 1.14

(293−785) (3800−7850) (0.19−0.38) (0.39−2.46) (1.73−4.21) (0.51−2.99) (0.68−1.57)

382 4560 0.37 0.90 4.03 1.42 1.43

(196−699) (2430−7480) (0.18−0.54) (0.16−2.85) (1.04−6.71) (0.45−2.72) (0.58−2.10)

242 2560 0.39 0.80 3.98 1.49 1.44

gonad digestive Jiuzhen mantle gill gonad digestive Baijiao mantle gill

Table 1. Annual Mean and Range of Metal Concentrations (μg/g of Dry Weight) in Different Tissues of Oysters C. hongkongensis Sampled from Baijiao and Jiuzhen during a 1 Year Period

Environmental Science & Technology

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Figure 4. Percentage of metal distribution in different tissues of C. hongkongensis sampled from Baijiao (the contaminated site) and Jiuzhen (the reference site) from September 2011 to August 2012. Data are the mean values (n = 7 for each month).

Figure 5. (A−D) Light micrograph and (E and F) electron micrograph of mantle tissue of C. hongkongensis sampled from Baijiao (the contaminated site) and Jiuzhen (the reference site). Panels A (40×) and B (100×) are for the mantle tissue from Jiuzhen, and panels C (40×), D (100×), E (6000×), and F (12000×) are for the mantle tissue from Baijiao. N, nuclei; V, vesicles. Arrows show the membrane-limited vesicles (n = 3 for each site).

(Baijiao). The concentrations of OP, estrone (E1), 17βestradiol (E2), and 17α-ethynylestradiol (EE2) in the surface sediments were below the detection limits. Overall, the metal contamination was implicated as the major cause of the imbalanced sex ratio in contaminated oysters, and our results provided the first evidence of feminization of wild oyster populations, which could be related to a long-term metal exposure in the field. To further confirm our conclusion, studies of a longer period (e.g., several years) focusing on more populations and oyster species are needed. The long-term metal exposure appeared to greatly influence the reproduction of oyster populations from the contaminated site, by changing the reproductive cycle, reducing the gonad conditions, and altering the sex ratios. Nevertheless, the contaminated oysters still maintained a complete reproductive cycle, and the spawning of gametes was synchronized in males and females, which reflected the high ability of oysters to

conquer metal stress. Therefore, the potential detoxification mechanisms of trace metals were further explored. Among the different tissues of oyster, the mantle tissue contained the largest proportion of trace metals. Interestingly, despite the very high metal concentrations accumulated by oysters from the contaminated site, the proportion of tissue metal levels remained relatively constant, indicating that these oysters were able to physiologically manage these high metal levels. Wang et al.7 also demonstrated that mantle and gill were the major storage organ in oyster C. hongkongensis sampled from the Jiulong River estuary. Histological observation of mantle tissue found a large number of hemocytes in the mantle of oysters from the contaminated site; however, the reference oyster mantle contained very few hemocytes. In a much earlier study, Ruddell and Rains48 found that the concentrations of trace metals in mantle were positively correlated with the number of amoebocytes (hemocytes) in Crassostrea gigas and G

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Environmental Science & Technology

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Crassostrea virginica. These results indicated the potential roles of hemocytes in the storage of metals in contaminated oysters. Furthermore, the ultrastructure of hemocytes in contaminated oyster mantle showed many membrane-limited vesicles with a diameter of about 0.5−1.0 μm in the hemocytes of contaminated oyster mantle, and the edge of vesicles was highly electron-dense. Similar vesicles (granules, 0.2−0.8 μm in diameter) were also found in the amoebocytes (hemocytes) of contaminated Pacific oyster C. gigas.21 In their study, about 90% of Cu and Zn in mantle tissue were accumulated in the amoebocytes and, in almost all of the amoebocytes, Cu and Zn were present in these vesicles through the X-ray analysis. George et al.49 also demonstrated that the detoxification of metals in the green oyster Ostrea edulis heavily contaminated with Cu and Zn was related to isolation of metals into the membrane-limited vesicles of granular amoebocytes (hemocytes). Thus, isolation of trace metals in the membrane-limited vesicles in hemocytes may be an important detoxification strategy for the contaminated oysters. Our study demonstrated that the long-term metal exposure may greatly influence the reproduction of oyster C. hongkongensis and finally affect the recruitment of this species in an estuarine ecosystem. The contaminated oysters exhibited a delayed gametogenesis, a relatively shorter spawning period, and a lowered gonad condition index. In addition, the feminization of oyster populations was first observed in the natural environment as a result of possible metal exposure. Compartmentation of trace metals in membrane-limited vesicles in hemocytes could be an important detoxification mechanism for the contaminated oysters. Our findings may have great implications for the ecological conservation of bivalve species in metal-contaminated estuaries.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Seasonal trends and annual mean values of general hydrological parameters in Baijiao (BJ) and Jiuzhen (JZ) sites during a 1 year period (Table S1), shell size of C. hongkongensis sampled from Baijiao (the contaminated site) and Jiuzhen (the reference site) from September 2011 to August 2012 (Table S2), results of the relative effects of site and month on the gonad somatic index, gonad coverage area as well as sex ratio of oysters C. hongkongensis evaluated by the generalized linear model (Table S3), gonad development stages for female and male C. hongkongensis (Figure S1), and temporal change of metal concentrations in different tissues of C. hongkongensis sampled from Baijiao and Jiuzhen from September 2011 to August 2012 (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the anonymous reviewers for their very detailed comments. This study was supported by a Key Project from the National Natural Science Foundation (21237004) and a General Research Fund grant from the Hong Kong Research Grants Council (662813). H

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