Environ. Sci. Technol. 1999, 33, 2905-2909
Significance of Trophic Transfer in Predicting the High Concentration of Zinc in Barnacles WEN-XIONG WANG,* JIAN-WEN QIU, AND PEI-YUAN QIAN Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong
Barnacles are known to accumulate Zn to a phenomenal concentration, but physiological processes governing Zn accumulation are poorly defined. We determined the assimilation efficiency and efflux rate constant of Zn in barnacles (Balanus amphitrite) using radiotracer technique. Assimilation efficiency of Zn from ingested food ranged between 76 and 87% for the diatom diets and between 86 and 98% for the zooplankton preys. These AEs were the highest measured among aquatic invertebrates. Varying distribution in the soft tissues of zooplankton did not account for the variability of Zn AE observed among different zooplankton preys. Most Zn was distributed in the guts of the animals, presumably associated with the numerous granules beneath the gut epithelium. The efflux rate constant was 0.003 d-1, and the calculated biological retention halftime was about 230 days. Using a simple bioenergeticbased kinetic model, we demonstrated that trophic transfer can account for such a high Zn concentration in barnacles. The predicted Zn concentrations in barnacles (261011 560 µg g-1) were directly comparable to the concentrations measured in Hong Kong coastal waters (3100-11 000 µg g-1). The high Zn concentration is related to its very efficient assimilation in barnacles coupled with a very low efflux rate. Biological variability must be fully appreciated before barnacles can be designated as an appropriate biomonitor of Zn contamination in coastal waters. Our study suggests that metal concentration in aquatic animals can be predicted only when both physiological and geochemical processes are considered.
Introduction Marine organisms can develop remarkable strategies for accumulating trace elements from the ambient environments. In decapod crustaceans, zinc accumulation is frequently regulated, and its typical concentration is maintained in the animals within a relatively narrow range (about 100 µg g-1) (1, 2). However, the phenomenal Zn concentrations in barnacles are probably the highest recorded among aquatic animals. Concentrations as high as 16 000 µg g-1 in Balanus amphitrite, or equivalent to 1.6% of their dry tissue weights, have been reported (3, 4). Earlier studies demonstrate that Zn in barnacles is mostly associated with the numerous granules consisting of zinc phosphate in the cell layer beneath the midgut epithelium (e.g., stratum perintestinale) (5-7). * Corresponding author phone: 852-2358-7346; fax: 852-23581559; e-mail:
[email protected]. 10.1021/es990149e CCC: $18.00 Published on Web 07/23/1999
1999 American Chemical Society
Formation of insoluble Zn-containing granules is an important detoxification mechanism and may explain the low efflux rate in barnacles (1). Despite such a high Zn concentration in barnacles, physiological processes governing its uptake are not well defined. No attempt has been made to quantitatively interpret the high Zn concentration in barnacles. In several Indo-Pacific regions, barnacles have been used as biomonitors of metal contamination in coastal waters (1, 3, 4, 8, 9). In biomonitoring programs, it is assumed that metal concentration in the animals readily reflects the bioavailable metal level in the ambient environments, but this assumption has not been rigorously tested under realistic conditions. Most biomonitors are exposed to metals both in the dissolved and particulate phases, yet few studies consider metal uptake from the food source (e.g., trophic transfer). Consequently, any relationship between metal concentration in tissue residues and the bioavailable metal level in the environments may be confounded by the biological processes. Many studies have focused on the chemical/ geochemical controls on metal accumulation and availability in aquatic invertebrates (10, 11), but it is equally important to understand the biological variables affecting metal accumulation. Several approaches, most notably the geochemical and kinetic modeling approaches, are now employed to predict metal concentrations in aquatic invertebrates (12-15). Geochemical approach considers how changes in aquatic chemistry can affect metal accumulation in aquatic organisms (e.g., the free metal ion speciation model) (10). This approach is used to interpret Cd concentrations in aquatic insects collected from different lake systems in Canada (12, 13). Another approach, as evidenced by the development of a bioenergetic-based kinetic model (16, 17), considers both physiological and geochemical processes affecting metal uptake. The model has been recently applied to predict metal concentrations in marine bivalves (clams and mussels) and copepods (14, 15, 18, 19). It has been consistently shown that the metal concentrations in the animals predicted by the kinetic model are comparable to the actual concentrations. Furthermore, the kinetic model can be used to assess the significance of trophic transfer in aquatic invertebrates (20). In this study, we quantified the trophic transfer of Zn in barnacle (Balanus amphitrite) from planktonic preys. This species has been used as biomonitors in Hong Kong and Southern China (1, 3, 4, 8, 9). We measured the assimilation efficiency (AE) of Zn from ingested food. AE is a critical physiological parameter quantifying metal trophic transfer and metal bioavailability from food particles (21). We compared the bioavailability of Zn from phytoplankton and zooplankton food and examined the mechanisms controlling Zn bioavailability from different food sources. A bioenergeticbased kinetic model was then employed to predict Zn concentration and to assess the significance of trophic transfer in barnacles.
Materials and Methods Barnacles B. amphitrite were collected from Sai Kung, Hong Kong, and induced to spawn under laboratory conditions. Larvae were cultured as described in Qiu and Qian (22), and cyprid larvae were allowed to settle in plastic Petri dishes. Juveniles were cultured to the adult stages by feeding them brine shrimp Artemia salina larvae daily. Adult barnacles (6-9 mm size, 2-6 mg dry tissue weight) from the same broodstock were used in this study. Prior to radioactive uptake VOL. 33, NO. 17, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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experiments, barnacles were individually separated and thoroughly cleaned off any epibionts. All experiments were conducted at 23 °C and a salinity of 30 ppt. Measurements of Assimilation Efficiency. Five planktonic foods were used in this study, including two diatoms (Skeletonema costatum and Chaetoceros muelleri) and three zooplankton (brine shrimp A. salina larvae, copepods Canthocalanus pauper, and copepod Temora turbinata). Diatoms were obtained from Provasoli-Gulliard phytoplankton collection center, and copepods were collected by net tows from Clear Water Bay, Hong Kong. Radioisotope 65Zn (in 0.1 N HCl) was used as radiotracer in this study. Diatoms were radiolabeled as described in ref 23. Cells in the exponential growing phase were filtered onto polycarbonate membranes and resuspended into 150 mL of 0.2 µm filtered seawater enriched with f/2 levels of N, P, Si, vitamins, and f/20 levels of trace metals minus EDTA, Cu, and Zn (24). Radioisotope additions were 123 kBq L-1 for 65Zn (corresponding to 15 nM). The initial cell density in the medium was about 20 000 cells mL-1. The cells had undergone 4-6 divisions after 4-5 days and were considered to be uniformly radiolabeled. They were then filtered and resuspended twice in 0.2 µm filtered water before being fed to the barnacles. Newly hatched A. salina larvae were radiolabeled with 65Zn either in the dissolved phase or by feeding on diatom S. costatum. In the dissolved uptake experiments, radioisotope additions were 185-370 kBq L-1 (corresponding to 2244 nM). Larvae were labeled with dissolved 65Zn for 1, 2, or 3 days, respectively. In the food uptake treatment, larvae were fed radiolabeled S. costatum 4 times a day for 2 days. A. salina larvae were then collected by a mesh, rinsed, and placed in nonradioactive water before being fed to the barnacles. Copepods (about 1000 individuals in 400 mL) were radiolabeled with dissolved 65Zn for 2 days, after which they were collected and rinsed before being fed to the barnacles. The distributions of metals in the cytoplasm of diatom cells were determined as described in refs 25 and 23. Metal fractionation in the soft tissues of A. salina larvae and copepods was determined as described in ref 26. Briefly, zooplankton was extracted with 4 mL of 0.2 N NaOH at 65 °C for 4 h and then filtered through 10 µm polycarbonate membrane to remove the exoskeleton. Chloroform (2 mL) was added to separate the polar and nonpolar biochemical fractions of extracts (i.e., soft tissues). Pulse-chase feeding technique was employed to determine Zn assimilation efficiency (21). Barnacles were placed individually in 100 mL of filtered seawater. Radiolabeled foods were added at a concentration of 20 000 cells mL-1 for S. costatum or C. muelleri and two individuals mL-1 for A. salina larvae or copepods. We placed a light source on top of the beakers to stimulate the feeding by the animals. Individual barnacles were fed for 0.5-1 h, during which there was no egestion of radioactive feces. Barnacles were then immediately rinsed with nonradioactive water, and their radioactivity counted for 2 min (described below). They were placed individually into 120 mL of filtered seawater containing A. salina larvae at a density of 2 larvae mL-1. During the initial 8 h depuration, any egested feces was pipetted out on an hourly basis to minimize radioisotope desorption from the feces into the dissolved phase. The radioactivity in barnacles was monitored every 6-10 h for 68 h. At each time interval, the water and food were also renewed. Molts of exoskeleton were collected and radio-assayed but contained negligible radioactivity. Measurements of Efflux Rate Constant. Brine shrimp A. salina larvae and diatom C. muelleri were radiolabeled as described above. Each day, radiolabeled foods were collected and resuspended into nonradioactive water, and a mixture of brine shrimp larvae and diatom was used to feed the barnacles for 1 h. Barnacles were removed following the 2906
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FIGURE 1. The retention of Zn in the barnacle Balanus amphitrite over time following radiolabeled pulse feeding on different food particles. Only means are shown: (b), Artemia salina larvae radiolabeled for 1 day; (O), A. salina larvae radiolabeled for 2 days; (1), A. salina larvae radiolabeled for 3 days; (3), copepod Canthocalanus pauper; (9), copepod Temora turbinata; (0), A. salina larvae fed with radiolabeled Skeletonema costatum for 2 days; ([), diatom S. costatum; and (]), diatom Chaetoceros muelleri. radioactive feeding and placed into nonradioactive water containing A. salina and C. muelleri. There were 10 replicate individuals for efflux rate measurement. Barnacles were repeatedly fed under these conditions for 9 days, after which they were depurated in 240 mL of nonradioactive water for 38 days. Two individuals were dissected to determine the distribution of Zn in different bodies on the beginning of depuration. The radioactivity retaining in the barnacles was monitored at time intervals. Water and food were renewed daily. By the end of the 38-day depuration, barnacles were dissected, and radioactivity in different parts of their bodies was measured. Modeling Zn Concentration and Trophic Transfer in Barnacles. With only trophic transfer involved, Zn accumulation in barnacles can be described by the following first-order bioenergetic-based kinetic model (14, 16, 17, 26)
dC/dt ) (AE‚IR‚Cf) - (ke + g)‚C
(1)
where C is the Zn concentration in the animals (µg g-1 ), t is the time of exposure (d), AE is the Zn assimilation efficiency from ingested particles, IR is the ingestion rate of the animal (g g-1 d-1), Cf is the Zn concentration in ingested particles (µg g-1), ke is the Zn efflux rate constant (d-1), and g is the growth rate constant (d-1). Under steady-state condition, C can be calculated as
Css ) (AE‚IR‚Cf)/(ke + g)
(2)
where Css is the Zn concentrations in the animals (µg g-1) obtained from food. Five parameters (AE, IR, Cf, ke, and g) are needed in the kinetic model to calculate the likely Zn concentration in barnacles due to trophic transfer. Radioactivity Measurements. Radioactivity of 65Zn in the samples was measured by a Wallac 1480 NaI(Tl) γ detector. The γ emission of 65Zn was detected at 1115 keV. Radioactivity was calibrated for radioactive decay and counting efficiency using standard. Counting time was adjusted to yield a propagated counting error 50% of Zn in mussels and copepods is predicted to derive from ingestion of food materials (14, 26). The significance of trophic transfer is primarily due to the high AE and high Cf, despite the fact that ku is also high because Zn transport can be greatly facilitated by binding with protein ligand. In a marine deposit feeder (Nereis succinea), nearly all Zn in body residues are predicted to be from ingested sediments as a result of the high IR (43). Consequently, various physiological processes (AE and IR) and geochemical processes (Cf) contribute to the significance of Zn trophic transfer. Among the trace metals examined so far, Zn and Se accumulation in diverse aquatic invertebrates (bivalves, copepods, polychaetes, and barnacles) have consistently been shown to be derived dominantly from trophic transfer (20).
Our study suggests that Zn concentrations in barnacles can be predicted with reasonable accuracy by a simple bioenergetic-based kinetic model if important metal physiological parameters (AE and efflux rate constant) and animal’s physiological parameters (feeding rate and growth rate) are properly identified and determined. Geochemical approaches such as the free metal ion speciation model have been applied to predict Cd concentration in aquatic insects by considering Cd free ion speciation, complexation with dissolved organic matter, and competition with H+ for biological uptake sites (12, 13). Contrary to the free ion speciation model, the kinetic model can be used to assess the significance of trophic transfer and to understand the important biological processes governing metal accumulation in animals. It should be noted that the parameters identified in the kinetic model are not constant but are dependent on various ecological conditions. In this study we employed the best estimates of each physiological and geochemical parameter in the calculation. We considered a range of Zn Cf (varying by a factor of 1.7), Zn AE (ranging from 75 to 95%), Zn ke (varying by a factor of 7), and g (varying by a factor of 5) in the model. A maximum IR (40% of the dry tissue weight a day) was used in the calculation, but it is recognized that IR is dependent on the food quantity/quality or other environmental conditions (e.g., season) (34). Unfortunately, there are few studies on the feeding rate of barnacles in the field to allow extensive modeling of this physiological parameter. Our results also show that the growth rate constant and efflux rate constant are critical in affecting Zn concentration in barnacles. Zn concentrations in plankton are within a relatively narrow range in different coastal regions (37, 38). Regulation of Zn uptake is well documented in a variety of aquatic organisms (13, 44). Consequently, variation of Zn concentrations in barnacles may simply reflect the influence of barnacles growth rate, feeding rate, efflux rate, and AE, which are in turn dependent on food availability, season, and other environmental conditions. Zn concentrations in barnacles show considerable variations (up to 10 times) among samples collected from the same location (where Cf can be assumed to be the same for different individuals) (4, 5, 8), further suggesting that biological factors (e.g., individual variability) must be fully appreciated before barnacles can be designated as good indicators of Zn contamination in coastal waters. Similarly, caution must be exercised in choosing biomonitors in which metals are mainly accumulated from ingested food. Metal uptake may be more dependent on the physiological conditions of the animals than on the metal concentration in ingested food particles, particularly for metals such as Zn, which may be regulated in prey organisms.
Acknowledgments We are grateful to Dr. L. Guo and three anonymous reviewers for their helpful comments. Discussion with Prof. Phillip Rainbow was also very helpful. This study was supported by a DAG/RGC grant and the start-up fund to W.-X.Wang.
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Received for review February 10, 1999. Revised manuscript received May 18, 1999. Accepted May 24, 1999. ES990149E
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