Rapid Assessments of Metal Bioavailability in Marine Sediments

Jun 7, 2013 - (1, 2) However, due to the extremely complex and variable composition of sediments, measuring ..... Zn, 140, 68.7, 3530, 25.2, 51.4, 568...
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Rapid Assessments of Metal Bioavailability in Marine Sediments Using Coelomic Fluid of Sipunculan Worms Qiao-Guo Tan, Caihuan Ke, and Wen-Xiong Wang* State Key Laboratory of Marine Environmental Science, College of Environment and Ecology, College of Oceanography and Earth Science, Xiamen University, Xiamen 361005, China S Supporting Information *

ABSTRACT: A suitable test organism for assessing the bioavailability of sediment-bound metals should accumulate metals mainly from the sediment instead of other sources such as water. The deposit-feeding sipunculan worms, which indiscriminately ingest sediment particles and have a very low uptake rate of dissolved metals, appear to be such good candidates. The worms have additional advantage due to simple anatomy and are like little sacs full of liquid, that is, coelomic fluid, which can be easily collected for metal analysis after simple sample treatment. We measured the metal concentrations in a sipunculan worm, Phascolosoma arcuatum and in sediments collected from intertidal zones of Xiamen City, China. Significant correlations were found for the concentrations of chromium, nickel, copper, zinc, cadmium, and lead in sediments and their concentrations in both somatic tissue and coelomic fluid of the worms. Analyzing the metals in coelomic fluid led to similar results as the somatic-tissue metals for assessing the bioavailability of sediment-bound metals and the spatial pattern of sediment-bound metal contamination. Therefore, measuring coelomic-fluid metal concentrations can be used to provide a rapid assessment of metal bioavailability in marine sediments.

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INTRODUCTION

their gut contents by dissection is also difficult due to the anatomy of these animals. The burrowing sipunculan worms appear to be good candidates to overcome the shortcomings of these popular biomonitors. First, the worms are deposit-feeders and indiscriminately ingest large amount of sediments for food.10 Second, the uptake rate of metals from water was remarkably low in sipunculan worms and was among the lowest in depositfeeders, which was partly attributed to their lower ventilation rate.11 Therefore, sediment is the dominant and presumably only source of metals to sipunculan worms, and the bioavailability of sediment-bound metals can be reflected by metal accumulation in the worms with much less confounding effects. Third, sipunculan worms have a very simple anatomy, and their gut contents can be easily removed for metal analysis. Moreover, the worms are rich in coelomic fluid, which can be readily collected for a rapid metal analysis. The worms do not possess the blood vascular system, and the coelomic fluid fills their spacious body cavity and serves the function of blood, including gas exchange and nutrient and waste transport.12,13 Thus, measuring the metal concentrations in coelomic fluid may be a feasible method for assessing the bioavailability of

In aquatic environments, sediments are the major pool of metal contaminants. Understanding metal bioavailability in sediments is thus crucial for assessing the associated ecological risks.1,2 However, due to the extremely complex and variable composition of sediments, measuring metal concentration in sediments usually provides very limited knowledge of metal bioavailability.1−3 Therefore, finding suitable organisms that can respond mainly to metals in sediment is important not only for monitoring the potential adverse effects, but also for studying the effects on bioavailability of various physicochemical factors (e.g., particle size, organic matter, acid-volatile sulfide-AVS). Sediment dwelling polychaetes and bivalves are the most frequently used biomonitors in the studies of marine sediments.2,4−6 However, most bivalves are suspension feeders, thus phytoplankton, suspended particles, and water may contribute more importantly than sediments to their metal accumulation.7,8 In this regard, deposit-feeding polychaetes may be more suitable as biomonitor, but they are delicate and fragile, therefore can cause logistic difficulty during sampling and handling and increase the chance of sample contamination. Moreover, the gut contents of bivalves and polychaetes may bias the measurement of metals accumulated in the biological tissues.9 Depurating the animals for a certain period of time may alleviate this problem, but its effectiveness is doubtful and may lead to considerable loss of accumulated metals. Removing © XXXX American Chemical Society

Received: March 13, 2013 Revised: May 23, 2013 Accepted: June 7, 2013

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sediment-bound metals, but this has never been tested in previous studies. In intertidal zones of East and South China, Phascolosoma arcuatum (also frequently mentioned as P. esculenta) is a widely distributed sipunculan species. Indeed, it is famous seafood in Southeast China, and large scale mariculture of this economic species is already in place.14 Like many other burrowing worms, this sipunculan worm has limited range of movement and their metal concentrations should be representative of the site where they were sampled. Previous surveys showed that metal contamination in coastal sediments of Xiamen, located in Southeast China, was generally low, but one station (i.e., Maluan Bay) had elevated concentrations of Cr (134.3 mg kg−1), Ni (62.0 mg kg−1), Cu (71.7 mg kg−1).15,16 However, exceedingly high metal concentrations were recently recorded in oysters collected from the nearby Jiulong River Estuary; the oysters were blue or green in color due to their high Cu concentrations up to 14380 μg g−1 dry weight.17 Such contamination in the oysters was largely due to the releases of industrial effluents directly into the estuary and the nature of hyperaccumulation of Cu and Zn by the oysters. The coastal areas of Xiamen were known to be the natural habitats of sipunculan worms, and a gradient of metal concentration in the sediments was expected considering the distribution of point sources of pollution. In the present study, we therefore collected sediments and the sipunculan worm, P. arcuatum, from the intertidal areas of Xiamen. Metal concentrations in the sediments and the somatic tissue and coelomic fluid of the worms were measured. Specifically, we tested the hypothesis that metals in the somatic tissue and especially coelomic fluid can reflect the bioavailability of sediment-bound metals. The objective was to prove that the worm with the many attributes of a good biomonitor was indeed a suitable tool to rapidly assess marine sediments. Sequential extraction was also conducted for the sediments, and the metal concentrations in the different fractions were correlated to those measured in the worms to test the usefulness of the extraction method in assessing the bioavailability of metals.

Upon arrival, the worms were immediately dissected after being thoroughly rinsed with sand filtered seawater and then deionized water (18.2 MΩ·cm). Efforts were made to remove the sediment adhered on the surface of worm body by gentle rubbing with gloved hands. The sediments were stored at −20 °C for less than 4 weeks before further handling. Sample Preparation and Metal Analysis. For each station, 10 sipunculan worms of similar size were selected for metal analysis. Each worm was dissected into two fractions, that is, coelomic fluid and somatic tissue. About 200 μL of coelomic fluid was collected into a 2.5 mL eppendorf tube, into which 200 μL of concentrated nitric acid and 100 μL of 30% hydrogen peroxide were added. After 4 h digestion at 85 °C, the mixture was diluted to 2 mL with deionized water and a clear liquid was obtained, which was submitted for metal analysis without further processing. The somatic tissue of worms was microwave digested. Around 200 worms were sampled from Station 10 for analyzing metal concentrations in the digestive fluid, which were then compared with those measured in the sediments and coelomic fluid. The digestive fluid collected from worms living in relatively clean areas was used in previous studies as a medium for extracting metals from contaminated sediments in vitro to indicate the metal bioavailability.11,18 Metal concentrations in the digestive fluid were analyzed following the same method as for coelomic fluid. The sediments were wet sieved through a 63 μm nylon mesh. Fine fraction ( Zn, Ni, Cu, As > Co > Cr, Pb (not statistically tested). This order was in good agreement with previous results derived from in vitro extraction experiments. Specifically, Wang et al.18 reported that 12.9 (±6.3)% of Cu, 7.9 (±5.2)% of Zn and −0.1 (±1.9)% of Pb in sediments of Pearl River Estuary were extracted by the digestive fluid of another sipunculan worm, Sipunculus nudus. In another study, 53.4 (±6.9)% of Cd, 33.9 (±8.8)% of Zn and only 2.7 (±1.0)% of Cr could be extracted by the digestive fluid of S. nudus from radiolabeled sediments,11 which explained the differences in the assimilation efficiency of these metals by the worm. Measuring metal concentrations in the digestive fluid of sipunculan worms collected from field should reflect the bioavailability of sediment-bound metals more accurately than the in vitro extraction, because the conditions (e.g., redox and pH) of the digestive fluids might have been altered due to centrifugation and handling. However, the small volume of

Figure 2. The correlation between metal concentrations in the somatic tissue and coelomic fluid of P. arcuatum. r: Pearson correlation coefficient (**p < 0.01).

terms of metal transfer up the food chain. For a typical worm (i.e., 0.1 g of tissue and 400 μL of coelomic fluid), >80% of As and Cd and >90% of other studied metals were distributed in the tissue. The between-station variation of metal (except Zn and Pb) concentrations in biological samples was higher than in sediments (SI Figure S2, Table S3), suggesting that the differences in sediment-bound metal concentration led to

Table 1. Comparison of Metal Concentrations in Sediment (Total or Non-Residual), Digestive Fluid (DF) and Coelomic Fluid (CF) of the Worm P. arcuatum Collected from Station 10a sediment (mg kg−1)

a

DF: sediment (g L−1)

metal

total

nonresidual

DF (μg L−1)

total

nonresidual

CF (μg L−1)

CF: DF

Cr Mn Fe Co Ni Cu Zn As Cd Pb

39.6 606 46 100 10.1 20.2 57.3 140 13 0.24 49.7

10.7 nd nd 4.95 4.67 37.3 68.7 2.54 nd 37.9

32.4 1810 6650 48.8 350 973 3530 208 428 10.8

0.819 2.99 0.144 4.85 17.3 17.0 25.2 16.0 1809 0.217

3.04 nd nd 9.85 75.0 26.1 51.4 81.9 >1809 0.285

8.89 1000 15 200 32.1 257 375 568 274 12.8 9.58

0.275 0.552 2.29 0.658 0.735 0.385 0.161 1.32 0.03 0.887

Median metal concentrations were presented for the coelomic fluid. nd = not determined. D

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digestive fluid collected from individual worms limited the applicability of this method. In contrast, sufficient amount of coelomic fluid can be sampled from a moderately sized worm for metal analysis. Beside the logistic advantages, metals in the coelomic fluid are assimilated while the metals dissociated from sediment particles into digestive fluid may not necessarily be absorbed, therefore measuring the former better serves the purpose of indicating bioavailability. The PCA graphically summarized the complex data (i.e., concentrations of multiple metals in worms) with two principal components. One principal component had high loadings of Cr, Ni, Cu, and Zn, the other had high loadings of Mn, Co, As, Cd, and Pb (SI Figure S4, left panel). Therefore, it is possible to divide the metal contaminants into two groups, and the contaminants in the same group tend to occur together. The characteristics of metal pollution of each station became clear by plotting their scores registered on the two components (SI Figure S4, right panel, see further descriptions in SI). The results were similar for somatic tissue and coelomic fluid. Strong correlation between As and Fe was observed in somatic tissue (r = 0.959, SI Figure S5), but such correlation did not exist in the coelomic fluid. The high Pb concentration in the sipunculan worms was remarkable. For example, at station 9 to 11 (Jiulong River Estuary), the median Pb concentration in worm tissue was 69.7, 36.2, and 69.1 μg g−1, respectively. However, Pb concentration in the sediments (46.3−49.7 mg kg−1) did not exceed the criteria of 60 mg kg−1 according to the Marine Sediment Quality of China (GB 18668-2002). In the oysters (Crassostrea angulata) collected from the same estuary in 2010, Pb concentration was much lower, that is, 1.5−7.5 μg g−1.17 Pb concentration in polychaetes collected from sediments covering a wide range of Pb concentrations was found to be 0.3−13 μg g−1 (SI Figure S3). These comparisons indicate that the sipunculan worm is a very efficient Pb accumulator. The median Pb concentration in the worms from all stations well exceeded the maximum limit of 0.3 μg g−1 wet weight for fish recommended by the Alimentarius Commission (assuming a wet weight to dry weight ratio of 7 for the worm).27 As the sipunculan worm is a delicacy in Southeast China, its high Pb concentration may cause considerable health risks. Correlation between Metal Concentrations in Sediment and Worms. For Cr, Ni, Cu, Zn, Cd, and Pb, metal concentrations in both worm tissue and coelomic fluid correlated significantly to the total and nonresidual concentration of metals in sediment (Figure 3, SI Table S2). Although Cr and Ni were mostly in the residual fraction, significant correlations were still observed due to their relatively constant residual concentration among stations. There was no significant correlation for the total concentration of Co and As, but significant correlations were observed for the F1 fraction. Using metal concentrations in tissue or coelomic fluid for the correlation analysis led to similar results in most cases, which again showed the feasibility of analyzing coelomic fluid metals for assessing the sediment−metal bioavailability. In many previous studies, the correlation between metal concentration in organisms and sediments was usually found to be weak.1,3,28 The lack of correlation is not unexpected if the geochemical properties of sediments varied substantially among sites and sediments may not be the major source of metals for the investigated organisms. In the present study, however, good correlation existed between the metal concentration in sediment and the sipunculan worm for at least Cr, Cu, and

Figure 3. The correlation between metal concentrations in the somatic tissue (or coelomic fluid) of P. arcuatum and in nonresidual fraction of sediments. For Cd, total concentration was used since the nonresidual concentration was not determined. r: Pearson correlation coefficient (* p < 0.05, ** p < 0.01).

Zn. Such good correlation could also be observed for other metals with a more contrasting and normally distributed gradient of sediment concentration. The good correlation was a result of the similar geochemical characteristics of sediments and the special ecology and physiology of sipunculan worm, which both reduced the confounding effects. Specifically, the sediments were collected from a restricted area and had similar geochemical properties including the concentrations of Fe, Mn, Al, and organic matter and metal concentrations in the residual fraction (SI Table S1, Figure 1). In addition, the sipunculan worm is very suitable for studying the bioavailability of sediment-bound metals, because they accumulate metals predominantly from sediment,11 and do not strongly regulate their body concentration of metals (possibly except Zn in coelomic fluid, SI Figure S2). Although it is usually in sediments with very different geochemistry that metal bioavailability is not well predicted by total concentration and thus the use of a suitable biomonitor is necessary, using sediments of similar geochemistry is preferable for testing the suitability of the sipunculan worm as a biomonitor. For these geochemically similar sediments, bioavailability is expected to be roughly proportional to the total (or nonresidual) metal concentrations. Consequently, the suitability of the worm can be verified by looking at the correlation between metal concentrations in the worms and in the sediments. After this verification, the sipunculan worm can E

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sediment bioconcentration factor in frequently used biomonitors. This material is available free of charge via the Internet at http://pubs.acs.org..

then be used to study metal bioavailability in sediments of contrasting geochemistry. There are several other advantages of the sipunculan worm making it suitable for assessing sediment−metal bioavailability. First, P. arcuatum can live in sediment without overlying water, which can minimize the confounding effects of aqueous uptake in bioassays. Second, the worm has muscular body wall, making it resistant to damage during handling. Third, the surface of the worm is relatively smooth and easy to clean, which can help minimize contamination in sample preparation. Beside in China, this species is also widely distributed in northeastern India, northern Australia, and Southeast Asia.13 In other regions, ecologically and physiologically similar sipunculan worms may be available. The sipunculan worm P. arcuatum can be used as a screening tool for marine sediment contamination. Sipunculan worms survive well under laboratory conditions.13,29 For example, it was found that after being kept in aerated seawater for 10 months, only 2 in 50 individuals of P. arcuatum died.29 In addition, 75 individuals of P. arcuatum were exposed to the sediment collected from the contaminated site (station 9) for two weeks, 3 and 2 individuals died only on the first and second day, respectively. This sipunculan worm was relatively tolerant to metals; high 96 h 50% lethal concentrations of Zn (10.9 mg L−1), Cd (4.5 mg L−1), and Pb (10.6 mg L−1) were reported.30 Therefore, worms collected from field or supplied by mariculture farms can be exposed in laboratory to sediments sampled from the sites of concern, and bioavailability can be assessed by measuring the elevation of metal concentration in coelomic fluid or somatic tissue. Although the chemical extraction methods including the BCR extraction method are continuously criticized for the lack of specificity of extractants and redistribution during extraction,31,32 these methods can roughly separate the more labile metals from sediments and may reduce uncertainty in bioavailability prediction.3,33 In the present study, the BCR extraction method provided marginally better prediction of the bioavailability of Cr, Ni, Co, and As, and predicted the bioavailability of Cu, Zn, and Pb similarly (SI Table S2). For sediments with similar geochemical properties, the benefits of the extraction approach might have been counteracted by the uncertainty derived from the extraction procedures. To conclude, elevated Cr, Ni, Cu, and Zn concentrations were observed at particular sites from the studied coastal areas. Such spatial pattern of metal contamination was reflected by that found in the sipunculan worm, P. arcuatum. The sipunculan worm is a suitable organism for monitoring and assessing the bioavailability of marine sediment-bound metals, which is attributed to its special ecology and physiology. Measuring metal concentrations in the coelomic fluid can generate similar results in assessing bioavailability as measuring somatic-tissue metals; however, the former is logistically more convenient and may provide a more immediate measure of metal bioavailability in bioassays. Therefore, measuring metals in the coelomic fluid of the sipunculan worm can provide a tool to rapidly assess metal bioavailability in marine sediments.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Xiaoyu Guo, Yubo Yang, Riwei Tu, and Ting Ding for their assistance in field sampling, and Dr. Ke Pan for metal analysis. This study was supported by the grant (2011M500072) from China Postdoctoral Science Foundation to Q.-G.T. and the National Natural Science Foundation of China (Grant No. 21237004) to W.-X.W.

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ASSOCIATED CONTENT

* Supporting Information S

Map of sampling sites and site description; methods of sample preparation, metal analysis and PCA; further results and discussion on the concentration of metals in worms, PCA, correlation between metals in worms and sediments, correlation between As and Fe in somatic tissue; review of F

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