Environ. Sci. Technol. 2006, 40, 4025-4030
Acute Toxicity of Mercury to Daphnia magna under Different Conditions MARTIN T. K. TSUI AND WEN-XIONG WANG* Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong
We investigated the variations of acute toxicity of mercury (Hg) in Daphnia magna under different temperatures, population origins, body sizes, and Hg pre-exposures. We measured Hg concentrations in the water and in the surviving daphnids, and used the subcellular fractionation approach to determine Hg in the metal-sensitive fraction (MSF) to predict Hg toxicity. The 24-h median lethal concentrations and 24-h lethal body burden were 12-55 µg L-1 and 10-26 mg kg-1 wet wt, respectively. High Hg tolerance accompanied by reduced Hg uptake occurred in the daphnids under extreme conditions (low temperature and high pre-exposure to Hg). Correlating Hg levels in different compartments and daphnid survival resulted in the following order of sequence: aqueous Hg > whole body Hg > Hg in the MSF. However, the threshold lethal concentration of Hg (concentration causing 1% mortality) based on the concentration of Hg in the MSF was the best indicator of Hg toxicity. Therefore, the subcellular fractionation approach is less useful in explaining acute toxicity than is sub-lethal Hg toxicity. The number of Hg binding sites in the animals varied under different conditions but the affinity of the transporter to Hg generally decreased as the animals’ tolerance increased. Mercury tolerance under different conditions could be enhanced by reducing the Hg uptake, enhancing the intrinsic tolerance, and/or increasing the detoxification activity.
Introduction Conventionally, toxicity testing in aquatic environments is conducted using water-only exposure and toxicity is expressed in terms of the no observable effect concentration (NOEC) and/or the median lethal concentration (LC50). It is well-known that abiotic (e.g., temperature) and biotic factors (e.g., different populations) influence metal toxicity in aquatic organisms (1, 2). Most studies have not concurrently measured metal accumulation in the tissues and the cytosolic fraction that may potentially affect metal toxicity (3, 4), partly due to the difficulty in determining contaminant concentrations in the limited amount of tissue available from the surviving individuals. Recently, Heugens et al. (5) performed acute toxicity and accumulation tests of cadmium (Cd) in the freshwater cladoceran, Daphnia magna, and showed that temperature-dependent Cd toxicity was due to changes in both Cd accumulation and the animals’ sensitivity to Cd toxicity. Nevertheless, they did not measure the Cd content in the acutely exposed daphnids but estimated the * Corresponding author phone: (852) 23587346; fax: (852) 23581559; e-mail:
[email protected]. 10.1021/es052377g CCC: $33.50 Published on Web 05/06/2006
2006 American Chemical Society
Cd body burden indirectly from separate accumulation experiments. Redeker and Blust (6) reported that the LC50s of Cd in the freshwater oligochaete (Tubifex tubifex) varied with exposure times but the lethal body burden (LBB) was relatively constant over time (∼36 mg kg-1 wet wt). The same amount of Cd was therefore required to exert the observed toxicity regardless of exposure duration. Previous studies indicated relatively constant LBBs of nonpolar organics in aquatic organisms under different conditions (7). Since nonpolar organics mainly accumulate in the lipid fraction, accumulation of these compounds on a lipid-weight basis (rather than the ambient concentration) can be an excellent indicator of toxicity under different conditions (7). In contrast, metals are partitioned into different cytosolic pools, such as the metal-sensitive fraction (MSF) and the biologically detoxified metal (BDM) (3). Wallace et al. (3) operationally defined that the MSF contains organelles and enzymes while the BDM contains metal-rich granules and heat-stable proteins (or metallothionein). Consequently, toxicity of metals based on tissue concentration is more complicated and difficult to predict than that of nonpolar organics. The present study represents the first attempt to test the concept of subcellular fractionation approach to study the metal toxicity in aquatic invertebrates. Daphnia magna are widely used in toxicity testing; they are sensitive to metal toxicity and provide a conservative estimate of metal impacts in aquatic ecosystems. In this study, we hypothesized that the different LC50s of Hg in D. magna measured under different conditions are simply caused by varied Hg accumulation in D. magna (i.e., lower Hg uptake causes a higher LC50 of Hg) and therefore the lethal body burden of Hg should be the same in all cases. To investigate the role of different subcellular pools in mitigating Hg toxicity, we fractionated the tissues of surviving daphnids after acute Hg exposure, measured the Hg distribution in these pools, and attempted to link the subcellular Hg concentrations to the observed toxicity. We chose the four variables in this study (temperature, origin of population, animal body size, and pre-exposure history) because they are realistic variables for natural zooplankton populations and did cause different LC50s in D. magna in our preliminary experiments.
Experimental Section Daphnids and Mercury. Three populations of Daphnia magna were used in this study. The primary population was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (abbreviated CAS) (Wuhan, People’s Republic of China). The other two populations originated from LFS Cultures (University, MS) (abbreviated LFS) and the Carolina Biological Supply Company (Burlington, NC) (abbreviated CBSC). Daphnids from different sources were separately maintained under the same conditions in the laboratory. Routinely, the daphnids were reared in moderately hard pond water (pH 8.3, hardness 76 mg CaCO3 L-1, DOC 1.1 mg L-1, and background Hg 0.15 ng L-1) and fed Chlamydomonas reinhardtii at 105 cells mL-1 daily. The pond water was collected from an uncontaminated freshwater pond within the campus of HKUST, filtered through Whatman GF/C glassfiber filter paper (Maidstone, UK), and aerated vigorously overnight prior to the medium renewal. Half of the culture medium for the waterfleas was renewed every other day. Algal and animal cultures were maintained in an environmental incubator at 23.5 °C under a light/dark regime of 14:10. The background Hg body burden in the daphnids was ∼45 µg kg-1 wet wt as determined using the concentrated HNO3 digestion method. VOL. 40, NO. 12, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Both radioactive and stable HgCl2 were used together and the Hg concentrations in the water and animals were determined by radioassaying these compartments nondestructively. The 203HgCl2 was purchased from Risø National Laboratory (Roskilde, Denmark) while stable Hg was obtained from Sigma (St. Louis, MO). A stock of Hg solution (20 µg mL-1) was prepared by dissolving stable HgCl2 in 1 M ultrapure HCl (BDH, Poole, UK) and spiking it with a radiotracer (203Hg) at 3.7 kBq mL-1. The solution was stored in the dark before use. Acute Toxicity Tests. Each toxicity test consisted of 5-8 Hg concentration treatments and a control without added Hg. There were three replicates for each concentration and 10-15 daphnids in each beaker (100-150 mL of solution). The pond water was vigorously aerated and filtered through a Millipore 0.22-µm membrane, which was then spiked with different Hg concentrations (ranging from 5 to 105 µg L-1). The spiked water was kept in polypropylene beakers and equilibrated overnight before the tests. The daphnids were fed C. reinhardtii for 1-2 h prior to the bioassays to minimize the degree of starvation during the exposure period (performed without food addition). Daphnids from the CAS population were used throughout all experiments; the LFS and CBSC populations were used in the experiments to compare Hg toxicity among different populations (see below). After 24 h of exposure, the number of surviving animals in each beaker was counted and the animals without response upon gentle agitation were considered dead. Aliquots of water samples in duplicate were also collected at the beginning and end of each toxicity test from each beaker to measure actual Hg concentrations. The averaged Hg concentration was used for all subsequent calculations. The toxicity tests lasted for 24 h, instead of 48 h, because the difference between the 24-h and 48-h LC50 of Hg was insignificant under our experimental conditions, indicating that the toxic action was exerted within the first 24 h of exposure (8). To measure the Hg accumulation, surviving individuals were collected by a disposable pipet, rinsed gently with ultrapure water, and depurated in filtered pond water in a glass beaker for 10 min in order to depurate Hg in the carapace fluid (9), which was not considered as true Hg uptake. The depurated animals were radioassayed for Hg content, dabbed dry with Kimwipe paper, and transferred into preweighed 2-mL microcentrifuge tubes for wet weight measurements using an electronic microbalance (to the nearest 0.1 mg) (Sartorius, Goettingen, Germany). The weighed tissue samples were stored in a freezer at -80 °C until further processing. Influences on Hg Toxicity. The following four variables were examined in this study. (i) Temperature: 10, 24, and 32 °C. All the animals were kept at the same temperature after birth (i.e., 23.5 °C) until they were 3 days old. They were then transferred to the respective testing temperature for acclimation for another day before the bioassay. This ensured that the animals had similar body sizes under different temperatures at the beginning of the test (5). (ii) Population: CAS, LFS, and CBSC. Four-day-old juveniles were tested for their sensitivity to Hg toxicity at 24 °C. (iii) Body size: 4, 12 and 28 days old (carapace lengt: 2.4, 2.9, and 3.2 mm, respectively). They were tested under the same conditions as mentioned before. (iv) Pre-exposure: PE I, PE II, and PE III with different combinations of exposure length and Hg concentration (stable Hg only). In PE I, 32 °C. Among different populations, the LFS population showed significantly higher
24-h LC50 than did the other two populations and the survival curve for the LFS population was slightly shifted to the right at ∼25 µg L-1. Larger daphnids were less sensitive to Hg toxicity. Daphnids with carapace length of 3.2 mm showed significantly higher 24-h LC50 than the other two sizes (i.e., 2.4 and 2.9 mm). Pre-exposure to Hg elevated the 24-h LC50s, but only daphnids under PE III had approximately 2-fold of 24-h LC50 as the daphndis without PE, while the survival VOL. 40, NO. 12, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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curve of the PE III-treated animals shifted to the right relative to the animals without PE or animals treated with PE I and PE II. Mercury Accumulation. Generally, Hg uptake by the surviving daphnids increased as a function of the aqueous Hg concentration (Figure 1b). Mercury uptake increased without saturation for the daphnids at 24 and 32 °C but Hg uptake at 10 °C became relatively constant (∼10-14 mg kg-1 wet wt) when the aqueous Hg concentration was >27 µg L-1. The CBSC population accumulated lower levels of Hg when compared to the other two populations at 19-29 µg L-1. Larger daphnids (3.2 mm) had much lower Hg body burden over a range of aqueous Hg concentrations than did the other two sizes (2.4 and 2.9 mm). Animals without PE appeared to accumulate slightly lower Hg body burdens than did all the pre-exposed animals. Mercury uptake by the PE III-treated animals became relatively constant (19-26 mg kg-1 wet wt) when the aqueous Hg concentration was >39 µg L-1. The maximal Hg tissue concentrations in the surviving daphnids were estimated by the Michaelis-Menten kinetics (details given in the Supporting Information III). Table S1 (Supporting Information) shows the estimated maximal Hg tissue concentrations (i.e., [HgT]max) and the half-maximal saturation constant (Kd). Increasing the temperature enhanced the [HgT]max while the CBSC population had lower [HgT]max than the other two populations. However, the influence of body size on [HgT]max could not be assessed since the [HgT]max for animals with 2.9 mm carapace length could not be estimated with accuracy. In addition, the PE III-treated daphnids had much lower [HgT]max than the animals without PE and PE I- and PE II-treated daphnids. According to Buchwalter and Luoma (19), the [HgT]max and Kd can be interpreted as the number of metal transporters on the animal membrane and the affinity of metals to the transporter, respectively. Daphnids with increasing 24-h LC50s were usually associated with decreasing numbers of transporters for Hg as well as decreasing affinity of the transporters to Hg. Lethal Body Burden of Hg. The survival curves of the daphnids as a function of Hg body burden and the 24-h LBB50s of Hg under different conditions are shown in Figure 1c and Table 1, respectively. Survival at 10 °C decreased drastically even though the Hg body burdens were relatively constant at ∼9-11 mg kg-1 wet wt. At 32 °C, the animals appeared to be intrinsically more sensitive to Hg toxicity than at 10 and 24 °C, with significantly lower 24-h LBB50. Among different populations, the LFS population tolerated higher Hg body burden and had the highest 24-h LBB50, while the CBSC population was more intrinsically sensitive to Hg toxicity than the other two populations. Larger animals (3.2 mm) were more intrinsically sensitive to Hg toxicity (and also had lower 24-h LBB50) than the smaller animals (2.4 mm and 2.9 mm). The pre-exposed daphnids under the PE I and PE II regimes had survivorship similar to that of the animals without PE at Hg body burden
