Cd and Zn Uptake Kinetics in Daphnia magna in

The uptake kinetics of Cd and Zn in a freshwater cladoceran. Daphnia magna after exposure to different concentrations of Cd for various durations was ...
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Environ. Sci. Technol. 2004, 38, 6051-6058

Cd and Zn Uptake Kinetics in Daphnia magna in Relation to Cd Exposure History RUI GUAN AND WEN-XIONG WANG* Atmospheric, Marine, Coastal Environment Program, and Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

The uptake kinetics of Cd and Zn in a freshwater cladoceran Daphnia magna after exposure to different concentrations of Cd for various durations was quantified. The accumulated Cd concentrations increased with ambient Cd concentration and exposure duration. As a detoxification mechanism, metallothioneins (MTs) were induced when the Cd preexposure condition was beyond the noneffect threshold. The MT induction was dependent on both Cd concentration and duration of preexposure. Increasing the Cd exposure concentration to 20 µg L-1 for 3 d caused a 44% reduction in Cd assimilation efficiency (AE, the fraction assimilated by the animals after digestion) by the daphnids from the dietary phase, but a 2.4-fold increase in Zn AE. Generally, the dissolved metal uptake rate was not significantly affected by the different Cd preexposure regimes, except at a much higher Cd concentration (20 µg L-1) when the Zn influx was enhanced. Significant effects from Cd exposure on the ingestion rate of the daphnids were also observed. When the MT synthesis was not coupled with the accumulated Cd tissue burden (e.g., a delay in MT synthesis), apparent Cd toxicity on the feeding behavior and the Cd AE was observed, thus highlighting the importance of MTs in modifying the metal uptake kinetics of D. magna. Overall, daphnids responded to acute Cd exposure by reducing their Cd AE and ingestion, whereas they developed a tolerance to Cd following chronic exposure. The bioavailability of Zn was enhanced as a result of Cd preexposure. This study highlights the important influences of Cd preexposure history on the biokinetics and potential toxicity of Cd and Zn to D. magna.

Introduction The freshwater zooplankton, Daphnia magna, has been widely used in ecological and environmental research. Its sensitivity to low metal concentrations (1, 2) makes it an ideal species to assess freshwater quality. Toxicity testing using Daphnia has provided an important foundation for the establishment of freshwater quality criteria in many countries. Over the past decades, there have been many studies on trace metal bioaccumulation in D. magna as influenced by chemical (e.g., metal concentration, Ca2+, pH, SO42-) and biological factors (3-8). Among the many factors leading to the understanding of trace metal uptake, the influences of preexposure to metals have seldom * Corresponding author phone: +852 2358 7346; fax: +852 2358 1559; e-mail: [email protected]. 10.1021/es049562z CCC: $27.50 Published on Web 10/15/2004

 2004 American Chemical Society

been considered for D. magna. A few recent studies have examined metal toxicity in multiple generations of cladocerans (9-11). Metal exposure may induce the acclimation (obtained over a relatively short time exposure with changes in the physiological process in the organisms) or adaptation (obtained after generational exposure with genetic changes increasing tolerance to the metals) of daphnids in the natural environment (12-14). Bodar et al. (15) observed the development of resistance (i.e., the ability to withstand a toxicant exposure that ultimately results in death) in a single generation of D. magna during exposure to sublethal concentrations of Cd in the laboratory. Bossuyt and Janssen (11) found a 2-fold increase in acute Cu tolerance (i.e., the ability to withstand a toxicant exposure to a given concentration for an indefinite period of time) in the second and third generations of daphnids in a successive generational exposure experiment. The responses of Daphnia to metal pollution are the result of either physiological acclimation or adaptation (16). There have been a few studies on the adaptation of bivalves to metal pollution with subjects collected from contaminated or uncontaminated sites (17, 18). Blackmore and Wang (19) reported that Cd assimilation increased significantly after preexposure of the green mussel Perna viridis to Cd, as a result of the increase in the Cd body burden and the association of Cd with metallothionein-like proteins. Shi et al. (20) showed that preexposing the green mussels to Ag caused potential physiological changes and subsequently affected the Ag uptake kinetics in the animals. These recent evidences strongly imply that aquatic animals are able to modify the biokinetics of their metal accumulation in response to metal exposure. Little is known about whether the past history of metal contamination would influence the physiology of cladocerans and their uptake kinetics. Variations in metal exposure histories can cause differences in population sensitivities. The lack of such knowledge may hinder the science-based assessment of the potential impact of metals on aquatic environments (21). While it is well-established that cladocerans may develop tolerance to metal exposure, whether a change in animals’ metal biokinetics may account for such tolerance is a matter of speculation. Clearly, understanding the metal uptake kinetics in response to metal exposure has implications on interpreting toxicity and tolerance when chronic or multigenerational exposure has occurred. In this study, we quantified the effects of varying Cd exposure regimes on Cd and Zn uptake and elimination in the cladoceran D. magna. Cd and Zn are chemically similar and often share similar uptake pathways (22, 23), but Cd is not essential while Zn is essential to daphnids. The parameters quantified in this study included food ingestion, the metal assimilation efficiency from the dietary phase, the metal influx rate from the dissolved phase, the efflux rate constant, and metallothionein (MT) induction, as well as the resultant Cd body burden after Cd exposure. These physiological parameters are critical to understand the uptake kinetics of metals in the metal-exposed animals. We further examined the influences of different exposure regimes resulting in comparable Cd body burdens through a combination of exposure concentrations and durations on the metal uptake by the daphnids.

Materials and Methods Experimental Organisms and Metals. A clone of D. magna that had been cultured in our laboratory for 5 years was used in this study. The animals were cultured in glass-fiber-filtered VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(GF/C Whatman) pond water at 23 °C with a light to dark cycle of 14:10 h. Mixture of the green algae Chlamydomonas reinhardtii and Chlorella vulgaris was fed to the daphnids at a concentration of 1-2 × 106 cells mL-1 on a daily basis. These two algal strains were grown in artificial WC medium (CaCl2: 0.25 mM, MgSO4: 0.15 mM, NaHCO3: 0.15 mM, K2HPO4: 0.05 mM, NaNO3: 1 mM, H3BO3: 0.1 mM, and trace metals and vitamin) (24) and collected at exponential growth stage and centrifuged to remove the growth medium before being fed to the animals. The water for culture of organisms and used for all experiments (with a pH of 8.2, DOC concentration of 104 µM, Ca2+ of 600 µM) was collected from a university pond. Background Cd and Zn concentrations in the pond water, quantified using the trace metal clean technique, were 0.016 µg L-1 (0.14 nM) and 1.6 µg L-1 (25 nM), respectively. All experiments were conducted in 0.22 µm filtered water at 23 °C. Two metals, Cd and Zn, were considered in this study. The biokinetics of both metals were traced using the gamma radiotracers 109Cd (in 0.1 N HCl, obtained from New England Nuclear, Boston) and 65Zn (in 0.1 N HCl, from Riso National Laboratory, Denmark). The radioactivity was determined by a Wallac 1480 NaI(T1) gamma detector (Turku, Finland), at 88 keV for 109Cd and 1115 keV for 65Zn. The counts were related to appropriate standards and calibrated for spillover. Counting times were 3 min and were sufficient to yield propagated counting errors of less than 5%. Preexposure of D. magna to Cd. Daphnia were exposed to different concentrations of dissolved Cd for different durations in four independent exposure experiments. Cd (as CdCl2) and NaOH (to neutralize the pH because CdCl2 was dissolved in 0.1 N HCl) were spiked into GF/C filtered pond water before the green algae C. reinhardtii (at 105 cells mL-1) and the cladoceran D. magna were added. The green algae had been previously cultured in modified WC medium without Zn, Cu, and EDTA and were collected by centrifugation. In each treatment, the daphnids at the same age were exposed to the Cd-spiked solution at a density of 0.1 individual mL-1. In experiments with different durations of exposure, the starting dates of the exposure were adjusted for different treatments to attain the same ending date, such that all the experimental daphnids were identical in age (21-23 d) after the preexposure, and the biokinetics measurements were conducted simultaneously. The influence of different initial ages of preexposure on metal uptake was not quantified in this study. In this study, no effort was made to separate the dietary and dissolved pathways of Cd uptake by D. magna during the preexposure period. The dietary source was due to the adsorption of aqueous Cd to algae or internalization of Cd by the algal cells. On the basis of our preliminary study, however, less than 4% of ambient Cd was accumulated by the algal cells within 24 h of exposure. Thus, the dissolved uptake was the dominant pathway for Cd uptake by D. magna during the exposure period. The exposure medium (containing the same Cd concentration) and food were renewed every day during the preexposure. Four different exposure experiments were conducted to examine the biokinetics of Daphnia following (1) exposure to different Cd nominal concentrations for a fixed time (3 d) (Expt. 1 or Conc-3d); (2) exposure to a high but sublethal Cd concentration (5 µg L-1) for different durations (1-13 d) (Expt. 2 or Time-5 µg L-1); (3) exposure to a low Cd concentration (0.5 µg L-1) for different durations (1-13 d) (Expt. 3 or Time0.5 µg L-1); and (4) exposure to different regimes (combinations of different concentrations and durations) to result in the same Cd body concentrations (Expt. 4 or Same-Burden). In the last experiment, two levels of concentration × duration were designed as (i) 1 µg L-1 Cd for 9 d versus 3 µg L-1 Cd for 3 d and (ii) 5 µg L-1 Cd for 9 d versus 15 µg L-1 Cd for 3 6052

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d. Thus, two different Cd body burdens were attained in these two combinations. In each exposure experiment, there was a control treatment without an additional Cd spike. Metal Body Burden and Metallothioneins. By the end of the preexposure, a group of daphnids from each treatment was depurated in clean pond water for 1-2 h to evacuate the gut Cd. They were subsequently dried at 80 °C to a constant weight and digested in concentrated (70%) nitric acid (HNO3, Aristar grade, BDH Ltd.) at 110 °C. These digests were transferred to a volumetric flask with Nanopure purified water and diluted to make the metal concentration in an appropriate range for analysis by inductively coupled plasmamass spectroscopy (ICP-MS) (Perkin-Elmer, Elan 6000). The metal concentrations were expressed as µg g-1 dry wt. For MT measurements, a group of preexposed animals (five to eight individuals) was first weighted after removing any water from their body surfaces. The MT was then measured using a modified silver saturation assay as described by Scheuhammer and Cherian (25). The daphnid tissues were homogenized by ultrasonication in 0.5 mL of sucrose buffer (0.25 M) and centrifugated at 16 000g in a refrigerated centrifuge for 20 min. The supernatant fluid was mixed with 0.3 mL of glycine buffer (0.5 M) and 0.5 mL of 20 µg Ag+ mL-1 glycine solution labeled with 0.05 µCi 110mAg. Following 10-20 min of incubation at room temperature, 0.1 mL of red blood cell hemolysate was pipetted into the mixture. New homogenized solution was heated in a boiling water bath for 5 min and subsequently centrifuged at a speed of 1200g for 5 min. The process from hemolysate addition to centrifugation was repeated another 2 times before collection of the final supernatant by a 5 min centrifugation at 16000g. Three replicates from each treatment and one blank (without the daphnids) were included in the analysis. The gamma emission of 110mAg was detected at 658 keV. The amount of MT in the sample was calculated by the following equation (25):

µg of MT per g of tissue ) (RAg - Rb)/R0 × C0 × V0 × 3.55/WW where RAg, Rb, and R0 are the radioactivity of 110mAg in the final supernatant, supernatant of the blank, and the initial mixture, respectively; C0 is the stable Ag concentration in the initial mixture; V0 is the total volume of the stable Ag in the initial mixture; and WW is the wet weight of the sample. Dissolved Influx Rate and Dietary Assimilation Efficiency. The influx rate from the dissolved phase describes the rate at which the animals take up the metals from the dissolved phase. Fifteen individuals from each replicate (3 replicates per treatment) were added into 150 mL of 0.22 µm filtered pond water spiked with the stable metals (1 µg L-1 for Cd and 5 µg L-1 for Zn) and radiotracers (109Cd: 22.2 kBq L-1 and 65Zn: 55.5 kBq L-1) for 8 h without food addition. Every 2 h, the animals from the three replicates of each treatment were picked out by pipets and placed in the filtered water to remove the loosely adsorbed metals. The animals were then measured for their radioactivity and subsequently transferred back into the original water for further dissolved uptake measurements. At the end of 8 h dissolved uptake, the animals were dried at 80 °C to a constant dry weight. The influx rate was calculated as the slope of the linear regression between the accumulated metal concentration and the time of exposure (2-8 h). The metal assimilation efficiency (AE) from the dietary phase was quantified using the well-established radiotracer pulse-chase feeding technique (26, 27). The green algae C. reinhardtii were radiolabeled with 148 kBq L-1 109Cd and 370 kBq L-1 65Zn for 6-7 d. The uniformly radiolabeled cells were subsequently collected before being fed to the daphnids. The preexposed animals were first placed in 0.22 µm filtered

TABLE 1. Cd Body Burden, Metallothionein (MT) Concentration, Ingestion Rate, Cd and Zn Assimilation Efficiency (AE), and Influx Rate from the Dissolved Phase in D. magna Following Exposure to Cd for Different Durations in Different Experimentsa P-E duration expt (d) 1

3 3 3 3

2

P-E concn (µg L-1)

MT (µg g-1 wet wt)

0 (control) 4.1 ( 0.5 15.4 ( 0.7b 5 37.1 ( 2.1c 20 85.8 ( 8.3d

15.7 ( 17.0 ( 3.0a 29.5 ( 4.8b 48.1 ( 6.1c

0.10 ( 0.11 ( 0.02a 0.09 ( 0.01a 0.15 ( 0.02b

0.33 ( 0.35 ( 0.01a 0.35 ( 0.06a 0.82 ( 0.16b

16.7 ( 1.8a 18.9 ( 5.0a,b 21.2 ( 1.9b 63.7 ( 4.3c 62.9 ( 4.0c

0.08 ( 0.01a 0.09 ( 0.01a 0.08 ( 0.00a 0.10 ( 0.02a

0.28 ( 0.02a,b 1.9 ( 0.1a 0.26 ( 0.02a 4.8 ( 2.5b 0.23 ( 0.00c 19.1 ( 2.6c 0.32 ( 0.03b 15.3 ( 6.4c

0.4a

1.8 ( 0.2a 49.5 ( 3.0b 46.4 ( 0.8b 93.6 ( 8.2c 97.5 ( 2.1c

2.1a

1 3 7 13

0 (control) 5 5 5 5

1 3 7 13

0 (control) 1.3 ( 0.1a 0.5 7.3 ( 0.0b 0.5 12.3 ( 0.6c 0.5 21.0 ( 1.2d 0.5 25.0 ( 1.8e

18.0 ( 0.8a 18.2 ( 2.4a 25.4 ( 1.0b 27.4 ( 1.5b 28.8 ( 3.7b

2.7 ( 0.2a 21.8 ( 0.2b 24.2 ( 0.0c 58.0 ( 1.6d 60.6 ( 3.5d

21.6 ( 4.0a 34.5 ( 1.9b 28.7 ( 2.5c 66.0 ( 3.8d 45.4 ( 3.0e

3

4 9 3 9 3

influx rate (µg g-1 dry wt h-1) Cd Zn

Cd body burden (µg -1 g dry wt)

0 (control) 1 3 5 15

0.02a

0.03a

ingestion rate (mg g-1 dry wt h-1)

Cd

Zn

28.7 ( 24.9 ( 12.3a,b 15.3 ( 5.5b 1.0 ( 0.9c

58.6 ( 61.2 ( 3.5a 57.5 ( 9.5a 32.9 ( 1.6b

2.5 ( 0.7a 3.5 ( 1.6a 6.0 ( 0.6b 8.4 ( 2.3b

32.3 ( 0.6a 46.6 ( 5.4b 66.3 ( 1.3c 64.1 ( 11.0c

15.1 ( 0.8a 10.7 ( 4.2a,b 8.1 ( 3.3b 17.7 ( 1.2c

62.4 ( 1.5a 72.2 ( 1.8b 65.7 ( 3.7a 66.7 ( 7.0a,b

5.4 ( 0.3a 9.2 ( 1.6c 6.5 ( 1.0a,b 7.9 ( 1.4b,c

45.7 ( 2.3a 39.0 ( 3.2b 42.6 ( 2.6a,b 57.9 ( 0.9c 55.3 ( 6.9c

14.7 ( 1.1a 11.5 ( 0.6b 13.7 ( 1.8a,b 21.5 ( 0.1c 30.2 ( 8.0d

6.2a

14.4 ( 3.0a,b 13.4 ( 1.2a,b 15.3 ( 1.2a 10.5 ( 2.0b 0.12 ( 0.01a,b 0.11 ( 0.00a 0.08 ( 0.01c 0.14 ( 0.03a,b 0.13 ( 0.01b

0.37 ( 0.02a 24.9 ( 6.0a 0.47 ( 0.06b,c 18.2 ( 0.9a 0.35 ( 0.09a,b 18.2 ( 4.3a 0.55 ( 0.08c 9.6 ( 0.9b 0.49 ( 0.03c 1.9 ( 0.1c

AE (%) 3.9a

a The Cd concentration in the water is the nominal concentration. P-E, preexposure; blank values, data not measured. Data are mean ( standard deviation (n ) 3). Different letters in the superscripts represent statistically significant difference between treatments (t-test).

water at a density of 0.1 individual mL-1 to evacuate their guts for 1-3 h without the presence of food particles. The radiolabeled algal cells were subsequently added into the beakers containing 50 mL of 0.22 µm filtered pond water at a concentration of 105 cells mL-1. A total of 3 replicates were used for each treatment. Five Daphnia were then added to each beaker and were allowed to feed on the radiolabeled algae for 20 min in the dark, which was comparable to or shorter than the gut passage time. Thus, the short-term feeding minimized the defecation of radioactive feces (after digestion) within the radioactive feeding period. After radioactive feeding, the radioactivity in the animals was immediately counted, and the animals were returned to 50 mL of new filtered pond water containing 105 cells mL-1 of the same food but without the metal spikes, to depurate their ingested radiolabeled food. Any feces egested during the 20 min feeding period was also immediately collected by filtering the radioactive water through a 40 µm nylon mesh and assayed for the radioactivity. The total amount of radioactivity ingested by the animals during the radioactive feeding period was calculated as the sum of the radioactivity retained in Daphnia and in the feces immediately after the 20 min radioactive feeding period. The ingestion rate of D. magna during the 20 min pulse feeding was calculated based on the radioactivity ingested by the daphnids and the radioactivity in the green algae. The animals were depurated of their ingested radioactive food for 24 h. The amounts of radioactivity retained in the daphnids during this period were measured at time intervals. The pond water and food were replaced each time during the counting of the radioactivity of Daphnia. The metal assimilation efficiency (AE) was calculated as the percentage of the ingested metals retained in the animals after 12 h of depuration (27). Rates and Routes of Elimination. In a separate experiment, the rates and routes of elimination of Cd and Zn by the preexposed D. magna were quantified by exposing the animals to different Cd concentrations (0.5, 5, and 20 µg L-1), with spikes of 109Cd (55.5 kBq L-1) and 65Zn (122 kBq L-1). The animals were exposed for a total of 3 d with a renewal of water and food on a daily basis. On the fourth day, D. magna were removed and counted for their radioactivity.

Three replicates of 15 individuals from each concentration treatment were then depurated in 150 mL of filtered pond water with the addition of the green algae at 105 cells mL-1. The radioactivity retained in the animals was measured every 12 h within the first 2 d and once every day for the following 5 d. The water and food was renewed every 12 h. Each time when the water was renewed, all molts, released neonates, and feces were collected, and their radioactivity was measured (27). A 10 mL, a water sample was also taken for radioactivity counting, which was considered to represent the excretion pool in the water (e.g., excreted in the soluble form).

Results Cd Accumulation and MT Concentration. Preexposed D. magna in all experiments exhibited significant elevation of Cd body burden (p < 0.01 for Expt. 1 and p < 0.001 for Expts. 2-4, t-test) as compared to control groups (Table 1). The background Cd tissue concentrations in Daphnia were 1.34.1 µg g-1 dry wt. In Expt. 1 (Conc-3 d) in which the animals were exposed to different Cd concentrations for 3 d, there was a linear increase of Cd body burden (Cb) with increasing ambient Cd concentrations (Cw), as described by the equation Cb ) 11.3 + 3.8Cw (r2 ) 0.973). No saturation of Cd accumulation was observed at the tested Cd concentrations up to 20 µg L-1 in this experiment. In Expts. 2 and 3 in which the animals were exposed to a constant high (5 µg L-1) and low Cd (0.5 µg L-1) concentrations, a steady-state condition (e.g., no further or only a small increase in the Cd tissue concentration) was gradually reached after 7 d of exposure. In Expt. 4 (Same-Burden), similar Cd body burdens (21.8-24.2 µg g-1 dry wt for the low-level pair and 58.0-60.6 µg g-1 dry wt for the high-level one) were achieved through different Cd-exposure regimes (exposure concentration × exposure time), suggesting that the accumulation of Cd was proportional to Cd dissolved concentration and exposure time in this experiment. Daphnids at the higher exposure regimes (5 µg L-1 for 9 d or 15 µg L-1 for 3 d) accumulated 2.4-2.8fold body Cd concentrations of those exposed at the lower exposure regimes (1 µg L-1 for 9 d or 3 µg L-1 for 3 d). Generally, the induction of MT depended on the Cd exposure concentration, Cd tissue burden, and duration of VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Newly accumulated Cd and Zn concentrations by D. magna over 8 h dissolved exposure to 1 µg L-1 for Cd or 5 µg L-1 for Zn after the animals were preexposed to different Cd concentrations for different durations. Expt. 1 (Conc-3d): (b): control; (O): 0.5 µg L-1; (1): 5 µg L-1; (3): 20 µg L-1. Expt. 2 (Time-5 µg L-1): (b): 1 d; (O): 3 d; (1): 7 d; (3): 13 d. Expt. 4 (Same-Burden): (b): control; (O): 1 µg L-1 , 9 d; (1): 3 µg L-1, 3 d; (3): 5 µg L-1, 9 d; (9): 15 µg L-1, 3 d. Detailed Cd preexposure regimes are shown in Table 1. Mean ( standard deviation (n ) 3). exposure (Table 1). With only 1 d exposure (Expt. 2 and Expt. 3), MT was not induced significantly (p > 0.05, t-test). Beyond these thresholds, the MT concentration in the animals increased with increasing Cd concentration or exposure time. Furthermore, despite the significant increase in Cd tissue accumulation after exposure to 5 µg L-1 for 1 d (Expt. 2), the MT concentration was comparable to that of the control, suggesting that MT synthesis was not synchronous with the accumulated Cd. In Expt. 4 (Same-Burden), the MT concentrations in the Cd-exposed animals were significantly higher (p < 0.05, t-test) than those in the control. MT synthesis was also time-dependent in this experiment. A longer exposure tended to induce more MT than the corresponding high concentration exposure. Dissolved Uptake. In all the treatments, the daphnids took up Cd and Zn from the dissolved phase in a linear pattern during the 8 h exposure period (Figure 1). Table 1 shows the dissolved uptake rates of different Cd-exposed daphnids. In all experiments, the influx rates of Cd from the dissolved phase did not vary considerably (0.08-0.15 µg g-1 dry wt h-1). The highest uptake rate (0.15 µg g-1 dry wt h-1) was observed in Expt. 1 (Conc-3 d) when the daphnids were exposed to the highest Cd concentration (20 µg L-1) for 3 d. At the tested Zn concentration, the Zn uptake rates ranged between 0.23 and 0.82 µg g-1 dry wt h-1 in all experiments. In contrast to Cd uptake, increasing the Cd exposure concentration (up to 20 µg L-1) or the exposure duration (up to 9-13 d) enhanced the dissolved Zn uptake rate. For example, the Zn uptake increased by 2.5-fold following exposure of the animals to 20 µg Cd L-1 for 3 d (Expt. 1), with a resulting increase in Cd tissue burden by 20.9-fold. In Expt. 4 (Same-Burden), the Zn influx rates at the higher exposure regimes were also significantly higher (p < 0.01, t-test) than those in the control treatment. Ingestion Rate. Generally, increasing the Cd concentration during the preexposure period decreased the dietary 6054

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FIGURE 2. Retention of pulse-ingested Cd and Zn in D. magna after the animals were preexposed to different Cd concentrations for different durations. Expt. 1 (Conc-3 d): (b): control; (O): 0.5 µg L-1; (1): 5 µg L-1; (3): 20 µg L-1. Expt. 2 (Time-5 µg L-1) and Expt. 3 (Time-0.5 µg L-1): (b): 1 d; (O): 3 d; (1): 7 d; (3): 13 d. Expt. 4 (Same-Burden): (b): control, (O): 1 µg L-1, 9 d; (1): 3 µg L-1, 3 d; (3): 5 µg L-1, 9 d; (9): 15 µg L-1, 3 d. Detailed Cd preexposure regimes are shown in Table 1. Mean ( standard deviation (n ) 3). ingestion of D. magna, but a further increase in the preexposure period recovered the feeding decrease to some degree. A 3 d exposure to 5 and 20 µg L-1 of Cd resulted in a 46-47 and 92-97% feeding depression, respectively (Expt. 1). In Expt. 2 (Time-5 µg L-1), there was a major decrease when the animals were acutely exposed to 5 µg L-1 for 1 d. With a further extension of exposure to 3, 7, or 13 d, the ingestion increased by 3-, 11-, and 9-fold as compared to that for 1 d exposure. At a lower Cd exposure concentration (0.5 µg L-1), the ingestion rate appeared to be somewhat constant among the different exposure duration treatments (Expt. 3). The inverse influence of preexposure concentration and duration on ingestion rate of D. magna was more significant with the increasing resultant Cd tissue burden in Expt. 4 (Same-Burden). In addition, there was notable difference in the quantified ingestion rate for the same Cd exposure regime but in different experiments (e.g., 0.5 µg L-1 for 3 d in Expts. 1 and 3, and 5 µg L-1 for 3 d in Expts. 1 and 2), possibly due to the different responses to Cd exposure from different batches of daphnids. Assimilation Efficiency (AE). The depuration of Cd and Zn following the pulse ingestion of radiolabeled food typically followed a two-phase loss and was comparable among the different independent experiments (Figure 2). Cd was continuously lost from the daphnids following the initial

FIGURE 3. Relationships between Cd assimilation efficiency (AE) and ingestion rate (IR) of D. magna and the Cd tissue burden and Cd:MT ratio. (b): Expt. 1 (Conc-3 d); (O): Expt. 2 (Time-5 µg L-1); (1): Expt. 3 (Time-0.5 µg L-1). Detailed Cd preexposure regimes are shown in Table 1. Mean ( standard deviation (n ) 3). The solid lines are the correlations between the IR/AE and the Cd tissue burden or Cd:MT ratio in each experiment. The dotted line is drawn by eye. assimilation in Expt. 2 (Time-5 µg L-1). Typically, about 10% of Zn remained in the daphnids after 12 h of depuration, and Cd retention (30-70%) was much higher than that of Zn. The AEs of Cd (57-62%) were comparable at Cd concentrations of less than 5 µg L-1 but then decreased to 33% when the concentration was elevated to 20 µg L-1 (Table 1). In contrast, the daphnids preexposed to 5 µg L-1 of Cd for 1 and 3 d assimilated much less Cd (32 and 47%, respectively) than those exposed for 7 and 13 d (64-66%). A lower Cd concentration (0.5 µg L-1) had no significant influence on the Cd AE. In Expt. 4 (Same-Burden), the two lower-concentration exposures resulted in a comparable or slightly lower Cd AE than the controls. In contrast, the two higher-concentration exposures caused an increase in the Cd AEs. There was no significant difference in the Cd AEs between the two different preexposure regimes with comparable Cd tissue burdens. The AEs of Zn in all experiments were generally very low (7 d) following an initial linear increase over time. Such a relationship between tissue Cd concentration and exposure time is typical for many organisms (28), such as the freshwater oligochaete Tubifex tubifex, which was characterized by onecompartment, first-order kinetic uptake (29). The Cd preexposure concentration had a stronger impact on the elevation of Cd body burden than did the exposure duration, although the higher exposure concentration may have a stronger toxic effect on the animals (growth inhibition, Guan and Wang, unpublished data). Therefore, Cd body burden alone may not provide sufficient information on the health condition of the daphnids and the potential metal accumulation by the animals. The accumulated metals are distributed in different subcellular fractions of the animals. Our study demonstrated a significant induction of MT in response to Cd exposure, similar to earlier findings on Daphnia (13). Previous studies suggest that MT is not only induced by metal exposure but also by handling stress (30, 31), changes in temperature (32), or reproductive status (33). MT can bind and exchange essential trace metals such as Zn and Cu as well as sequester toxic metals such as Cd and Hg (34). MT was the only subcellular fraction quantified in this study, but our preliminary study indicated that it was the dominant fraction in sequestering the internal Cd in D. magna (Guan and Wang, unpublished data). The relationship between MT induction and Cd tissue burden appears to be somewhat complicated. There was a certain trigger level for MT induction in response to Cd exposure. A low Cd exposure or short-term (60 µg g-1 dry wt) in which the Zn uptake increased significantly (for Expt. 1 and 4). However, the Zn uptake rate was only slightly impacted by the Cd body burden at the level as high as 90 µg g-1 dry wt in Expt. 2 (time-5 µg L-1). These data clearly suggested that the response of Zn uptake to Cd exposure was dependent on the Cd exposure regime (e.g., Cd exposure concentration and duration). The enhancement of the dissolved uptake potential of Zn may have been due to MT synthesis, which may make more ligands available for metal binding. An earlier study has indicated that Zn accumulation in Daphnia is dominated by its uptake from the dissolved phase as a result of its very low assimilation from the ingested dietary source (7). Given the significant enhancement of Zn dissolved uptake after the acute Cd exposure (e.g., 2.5-fold higher after exposure to 20 µg L-1 for

3 d), such results will likely affect the interpretation of the toxicity testing results, especially when a mixture of metals is tested. Increasing the preexposure concentration significantly exacerbated the feeding inhibition of daphnids after the exposure. This feeding inhibition should be due to the toxic effect of accumulated Cd in the animals. In contrast to the influence of Cd tissue burden on daphnid feeding behavior, the feeding activity of the animals was less responsive to the Cd load in ingested particles (35, 36). Taylor et al. (35) found that the feeding inhibition of D. magna to Cd was almost entirely due to Cd bound to the food (algae) rather than to the dissolved Cd. Guan and Wang (36) recently found that feeding inhibition in Daphnia was observed at an ambient Cd concentration of 917 nM (100 µg L-1). In this study, significant reduction in feeding rate was observed at a Cd tissue concentration about 20 µg g-1 dry wt (an 8.1-fold increase in tissue Cd burden, Expt. 4) following exposure to dissolved Cd 1 µg L-1 for 9 d. Feeding inhibition has been previously suggested to be a good biomarker to assess Cd toxicity in cladocerans (37) and rotifers (38). Significant feeding recovery was observed in Expt. 2 with increasing Cd preexposure duration at 5 µg L-1 with no apparent damage on the digestion system. The short-term exposure (1-3 d) to this Cd level may cause the animals to reject food instantly due to the detection of stress through chemoreceptors (39, 40). With a further extension of Cd exposure, the ingestion rate indeed increased as a result of recovery, indicating the development of tolerance with increasing preexposure time. Significant MT induction may partially account for such tolerance development. For example, the MT levels were comparable to the control level within the first 3 d of exposure (as a result of delay in MT synthesis) but then increased by a factor of 3.8 with increasing the exposure duration to 7-13 d. However, MT induction was not sufficient to protect the animals from Cd toxicity in other experiments; thus, the recovery of ingestion was probably not solely dependent on MTs but may also be related to other unknown mechanisms (e.g., defense against toxic concentration and may result in the development of tolerance). Our study also suggests that the ratio of Cd body burden to MT may better predict the Cd toxicity to Daphnia than the Cd tissue burden alone. In Expt. 2 (Time-5 µg L-1), the depression of ingestion activity by Cd preexposure was significantly correlated with the Cd/MT ratio instead of the Cd tissue burden due to the development of tolerance including MT induction. An increasing ratio of Cd/MT may indicate that a spillover of Cd from MT and an increasing potential of Cd toxicity. This ratio is particularly useful when considering the dynamic change of MT in the animals as a function of Cd tissue burden. Few studies have been conducted on the effect of metal preexposure on metal assimilation efficiency (19, 20), especially for freshwater invertebrates. The Cd AE of D. magna decreased significantly after exposure to high Cd concentration (up to 20 µg L-1) for 3 d, indicating the acute toxic effect of Cd stress on the digestion system. Similar toxic effects were also observed in Expt. 2 (Time-5 µg L-1) when the animals were acutely exposed to 5 µg L-1 Cd for 1-3 d. The Cd/MT ratio appears to be a better predictor of Cd toxicity on the digestion systems than the Cd tissue burden alone. Among the three experiments, the apparent Cd toxicity on Cd assimilation was observed at a Cd/MT ratio >1.2, again highlighting the protective role of MT in the animals. Our present results on Daphnia were thus in strong contrast to marine mussels (18, 19) in which the Cd assimilation from the dietary phase increased substantially following Cd preexposure, likely caused by an induction of MT.

In Expt. 4 (Same-Burden), there was an increase in Cd AE when the animals were exposed to Cd at 5 µg L-1 for 9 d or 15 µg L-1 for 3 d (46% in the control vs 55-59% in the exposed group). However, it is difficult to conclude whether the increase in Cd AE was directly a result of Cd exposure or due to the suppression of the ingestion. An earlier study has indicated that the Cd AE was inversely related to the food concentration (e.g., from 77 to 56% with increasing C. reinhardtii concentration from 0.136 to 2.73 mg C L-1 (27)). Thus, there may be a compensation effect on the Cd AE (a potential increase) in D. magna through ingestion depression. Conversely, the toxic influence on Cd AE observed in this study would become more pronounced if the inverse relationship between Cd AE and ingestion rate was also considered. The AEs of Zn by daphnids were much lower than those for Cd, and the influences of preexposure were also less obvious. However, the Zn AEs generally increased in response to Cd exposure and increasing Cd tissue burden. For example, the Zn AEs increased by a factor of 3.4 (from 2.5 to 8.4%) when the animals were exposed to 20 µg L-1 for 3 d, which was also accompanied by a major increase in the Zn influx rate from the dissolved phase (Expt. 1). In Expt. 4 (Same-Burden), the Zn AE was as high as 30% at the high exposure regime (15 µg L-1 for 3 d), which was 2-fold of the control (14.7%), and the highest measured among different experiments. Thus, the Zn bioaccumulation from both dissolved and food sources was elevated in response to Cd exposure. In contrast to the variable influx parameters, the efflux rate constants were comparable with different Cd body burdens in the daphnids. The measured efflux rate constants were also comparable to our previous measurements (27, 36). Comparable efflux rates observed among different Cd exposures may have been due to the dominance of MT in binding with Cd and Zn following their accumulation. Similar to our previous studies (27, 36), the dissolved excretion was the predominant route for Cd loss from the daphnids, whereas neonate production (e.g., maternal transfer) was also an important pathway for Zn loss. The higher percentage of Zn lost from the animals at a higher exposure was primarily as a result of increasing amount of neonates reproduced. It appears that the daphnids responded to increasing Cd exposure by allocating more energy into reproduction (e.g., more neonates) rather than to the growth, and consequently, the loss of body Zn by neonate production increased. The amount of Zn allocated to each individual neonate, however, remained comparable among different Cd preexposure treatments. Because Cd was barely transferred through reproductive activity, the relative importance of each pathway remained unchanged. MTs play an important role in the effect of Cd preexposure. Metal binding with MTs reflects detoxification and may result in metal tolerance and resistance (16, 41-43). However, the duration of exposure may be critical in MT synthesis and may cause modification of metal uptake kinetics and toxicity. An acute exposure at a high Cd concentration may cause significant toxic effects on the dietary Cd uptake through food rejection and AE depression and would dramatically reduce the Cd bioavailability. Given the significant responses of daphnids to Cd exposure, the different discharges in the real environment may potentially influence the acute toxicity and long-term bioavailability of metals to relevant aquatic biota. Acute effects are certainly worthy of serious attention, but the chronic accumulation of metals also requires sufficient consideration for its potential risk during trophic transfer. Recent studies indicated that MT-bound metals were more bioavailable to predators than were metals bound to insoluble fractions (i.e., cell walls, exoskeleton, and metal concretions) (44). Therefore, metal preexposure studies have VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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important implications on environmental risk assessments. Attention should also be paid to the fact that preexposure effects are specific to metals and organisms, both of which make the potential influences more complicated.

Acknowledgments We are very grateful to the anonymous reviewers for their very detailed and constructive comments and suggestions. This work is supported by a Competitive Earmarked Research Grant from the Hong Kong Research Grants Council (HKUST6097/02M) to W.-X.W.

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Received for review March 19, 2004. Revised manuscript received July 12, 2004. Accepted August 31, 2004. ES049562Z