Environ. Sci. Technol. 2008, 42, 940–946
Cadmium Toxicity in a Marine Diatom as Predicted by the Cellular Metal Sensitive Fraction MENGJIAO WANG AND WEN-XIONG WANG* AMCE and Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong
Received August 2, 2007. Revised manuscript received October 21, 2007. Accepted November 29, 2007.
Prediction of metal bioaccumulation and toxicity in aquatic organisms has been based on the free ion activity (e.g., FIAM) or more recently on the binding with the biological/toxicological sites of action (e.g., biotic ligand model). However, metals are bound to various intracellular ligands that may control metal toxicity.Inthisstudy,weexaminedthebioaccumulation,subcellular distribution, and toxicity of Cd in a marine diatom, Thalassiosira nordenskioeldii, under different irradiance levels. The diatoms accumulated more cellular Cd under higher irradiance at low [Cd2+] concentrations, but the accumulation slowed down when the [Cd2+] concentration further increased, implying that cellular binding saturation had been reached. Among the five operationally defined subcellular fractions (metal-rich granule, cellular debris, organelles, heat-denatured protein [HDP], and heat-stable protein [HSP]), Cd was most bound to HSP, whereas it was least bound to HDP. Cd was redistributed with increasing [Cd2+] concentration from the biologically detoxified pool to the presumed metal-sensitive fractions (MSF, a combination of organelles and HDP), which led to higher cellular Cd accumulation, toxicity, and sensitivity. Although diatom growth inhibition was significantly related to [Cd2+] concentration and Cd cellular bioaccumulation, the calculated inhibition concentrations based on MSF or organelles exhibited the least difference, strongly suggesting that MSF can provide the better predictor of Cd toxicity under different irradiance levels compared with [Cd2+] concentration or cellular accumulation. Our results demonstrated that models predicting metal toxicity need to address the subcellular fates of metals and how they respond to external and internal conditions.
Introduction Metal contamination in aquatic environments is a widespread environmental problem. It is thus critical to understand the physical-chemical and biological behaviors of metals. Cadmium (Cd) is a priority pollutant, and its toxicity is mainly related to binding to sulfhydryl groups of proteins or displacement of essential metals in metalloenzymes. Over the past decades, considerable attention has been paid to Cd bioaccumulation and toxicity in aquatic organisms, and several models have been proposed (1). The free ion activity model (FIAM) attempts to explain metal accumulation and toxicity based on the free metal ion activity in a bulk solution * Corresponding author phone: (852) 2358-7346; fax: (852) 23591559; e-mail:
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(2). The more recently proposed biotic ligand model (BLM) predicts metal toxicity based on the binding of metals to the biological site of action (e.g., fish gills, 3). The applicability of FIAM has been tested in a wide variety of aquatic organisms, and conclusions are far from consistent (1). Currently, there is no specific BLM for Cd in aquatic organisms. Recently, a new approach, namely the subcellular partitioning model (SPM), which takes into account the cellular fates of metals, has been proposed to predict metal toxicity in aquatic organisms (1). This approach considers how metal accumulation and subsequent redistribution are directly related to metal toxicity (4), and it takes advantage of a recently introduced conceptual model that groups individual subcellular fractions (e.g., metal-rich granules [MRG], cellular debris, organelles, heat-denatured proteins [HDP], and heatstable protein [HSP]) into ecotoxicological relevant compartments (5, 6). For instance, organelles and HDP are grouped as metal-sensitive fraction (MSF), and HSP and MRG are grouped as biologically detoxified metal (BDM). In marine diatoms, the heat-stable protein may include phytochelatins and glutathione. A few recent studies have been conducted on several aquatic organisms (e.g., phytoplankton, bivalves, and fish) to test whether there was any correlation between metal toxicity and subcellular distribution of metals (7–10). Miao and Wang (7) showed that most Cd was distributed in the insoluble fraction (a combination of metal-rich granules, cellular debris, and organelles) in the diatom Thalassiosira weissflogii. Toxicity differences among the different nutrientconditioned cells were the smallest when the Cd concentration in the soluble fraction (a combination of HDP and HSP) was used, suggesting that intracellular soluble Cd may be the best predictor of Cd toxicity under different nutrient conditions. Studies in bivalves and fish, however, indicated the difficulty in assigning subcellular metal partitioning to any predictive role in an animal’s health (9, 10). Metal uptake by marine phytoplankton has been examined under different environmental conditions, such as temperature, irradiance, and nutrients (11, 12). The influences of these environmental conditions on metal toxicity in marine phytoplankton have remained relatively less well studied. Light irradiance may affect the biochemical composition (e.g., the C:N ratio), cell size, metabolism, and other physiological processes, such as photosynthesis and growth rate (13–15). These induced biochemical and physiological changes may potentially affect metal uptake and subcellular distribution and result in different levels of response and sensitivity to metals. To our knowledge, there has been no study of the effects of irradiance on subcellular Cd sequestration and induced toxicity effects. In this study, we examined the effects of irradiance on Cd accumulation, subcellular distribution, and toxicity in a marine diatom, Thalassiosira nordenskioeldii. We explored whether patterns in cellular or subcellular Cd distributions could be a good predictor of Cd toxicity in marine diatoms. In contrast to the previous study by Miao and Wang (7), which separated the cellular Cd into soluble and insoluble fractions, we further separated the Cd in the diatoms into the five biologically relevant fractions (namely MRG, cellular debris, organelle, HDP, and HSP) and also considered the partitioning of Cd to two subcellular compartments comprised of these fractions (MSF and BDM) (6).
Materials and Methods Diatom Cultures. The diatoms T. nordenskioeldii, originally isolated from the surface waters of Port Shelter, Eastern Hong Kong, were cultured in f/2 medium (16) within a growth 10.1021/es0719273 CCC: $40.75
2008 American Chemical Society
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chamber with a light intensity of 170 µmol photons m-2 s-1 under a 14:10 h light:dark cycle (23 ( 1 °C). The pH of the medium was 8.2 ( 0.1. The seawater (originally collected 10 km off the Eastern Hong Kong shore) was filtered through a 0.22 µm GP Express PLUS Membrane (Sericup, Millipore Corporation) before use. Stocks for the f/2 medium (N, P, Si, vitamins) were removed of any background metals by passing through a Chelex 100 ion-exchange resin (BIO-RAD Laboratories) column before use. All the polycarbonate beakers and bottles were soaked in 1 mol L-1 HCl for at least 24 h before the experiments and then rinsed with Milli-Q water (18.2 MΩ). All metal additions were conducted within a class100 clean bench. Toxicity Measurements. Artificial seawater medium (AQUIL, 17) was used throughout the diatom toxicity testing. The prepared artificial seawater was 0.22 µm filtered and passed through the Chelex 100 ion-exchange resin column to remove any background metals. The nutrients (N, P, Si, and vitamins) were then added at f/2 levels, and the trace metals were added at f/10 levels. Nitrilotriacetate (NTA, 0.1 mmol L-1) was used to control the free Cd ion concentrations ([Cd2+]) in the medium. The total dissolved Cd concentrations for the six treatments were 2.0 × 10-9 (control), 1.5 × 10-6, 8.0 × 10-6, 5.8 × 10-5, 1.5 × 10-4, and 3.6 × 10-4 mol L-1, respectively. The calculated [Cd2+] (by the MINEQL+ software) for each corresponding treatment was 9.41 × 10-12, 9.79 × 10-9, 10-7, 10-6, 2.94 × 10-6, and 8.06 × 10-6 mol L-1, respectively. Suprapure NaOH (1 mol L-1) was added to keep the pH of all media at 8.2 ( 0.1. Bioaccumulation and toxicity of Cd in the diatoms were studied at three different irradiance levels (100, 50, or 25 µmol photons m-2 s-1). For each irradiance level, there were six Cd concentration treatments, and each treatment had five replicates. Three of the replicates were spiked with stable Cd, to be used for growth rate and photosynthesis measurements. Another two replicates were spiked with both stable Cd and radioactive 109Cd (18.5–148 kBq L-1, in 0.1 mol L-1 HCl), to be used for Cd accumulation and subcellular fractionation measurements. T. nordenskioeldii cells were first transferred from the f/2 medium to the f/10 levels of trace metals and acclimated at 23 ( 1 °C, with different light irradiances (100, 50, or 25 µmol photons m-2 s-1) and light:dark cycles (14:10 h) for 3–5 days. During the acclimation period, cell density was measured daily with the Coulter particle counter Z1. The midexponentially growing cells were then collected by centrifugation (4000 rpm, 24 °C, 15 min) and immediately resuspended into the Cd testing medium, at a cell density of 1–8 × 104 cells/mL. The total incubation time was 72 h. The cell density was measured every 24 h with the Coulter particle counter Z1. The average growth rate (µ) was determined from the slopes of the linear regression between the logarithmically transformed cell density and the time. At the same time, the pulse-amplitude-modulated (PAM) fluorescence measurements were performed with a PAM 101/103 fluorometer equipped with an ED-101 PM emitter-detector-cuvette unit (Heinz Walz, Effeltrich, Germany), following the method described (18, 19). The maximal photosynthetic system II (PSII) quantum yield (ΦM) and operational PSII quantum yield (Φ′M) were calculated from the fluorescence induction curve (18). At the end of the experiment (72 h), 40 mL of the cell culture was collected from each replicated bottle and gently (10-6 M, in contrast to Cd in organelles. The ‘spillover’ of Cd from HSP to organelles 944
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may occur between 10-6 M and 3 × 10-6 M [Cd2+]. From 3 to 8 × 10-6 M [Cd2+], the decrease of Cd in HSP was accompanied by an increase of Cd in debris without significant change in organelles. Cd in the organelle fraction also remained unchanged from 9.41 × 10-12 to 9.79 × 10-9 M [Cd2+], while Cd in HSP decreased and the fraction of Cd in MRG increased. The ‘spillover’ of Cd from these detoxified fractions to other subcellular fractions, i.e., redistribution of Cd to the sensitive sites, may cause deleterious effects on the photosynthetic PSII system and the growth of the diatoms. Earlier studies demonstrated the increase in the C:N ratio in phytoplankton with increasing irradiance, which may be explained by the light-nutrient hypothesis (14, 15). Light and nutrients may serve as complementary resources for phytoplankton. Under limitation by one resource, phytoplankton may use the other more available resource to partially compensate for the lack of the limiting factor (13). Thus, phytoplankton may take up excess nutrients (N) at low irradiance to partially compensate for the low light, resulting in a lower C:N ratio. A higher uptake of N may allow phytoplankton to build the cellular machinery needed for the increased light absorption (13), and presumably more N would be available for HSP synthesis. Consequently, the higher C:N at high irradiance may lead to a lower level of HSP and the resulting ‘spillover’ effect. This hypothesis is consistent with our observation that the percentage of Cd in HSP was much lower with higher irradiance than with lower irradiance. Cd may have overflowed into the metal-sensitive pool and induced higher toxic response (PSII activity and growth inhibition). Conversely, the lower C:N ratio and possible higher HSP levels may have alleviated the Cd toxicity in the diatoms under low and medium irradiance. The IC50 values based on different Cd concentrations were always the lowest with the high irradiance, suggesting that T. nordenskioeldii was the most sensitive to Cd toxicity under high irradiance. The toxic end points also decreased sharply at the two highest tested [Cd2+] levels. At the same lower [Cd2+], the diatoms accumulated more Cd under high irradiance; thus, the higher Cd burden may have accounted for the high Cd toxicity in this treatment. Another likely explanation for the high sensitivity of diatoms is the inhibition of the synthesis of detoxifying ligands in the diatoms. It was likely that less cellular N was available for synthesis of inducible glutathione and phytochelatins (HSP), which may act to detoxify the incoming Cd. Prediction of Cd Toxicity in Diatoms. Metabolic and physiological changes may affect the toxic responses of diatoms under different light conditions. A good toxicity predictor should be rather constant under different environmental/physiological conditions. Although good correlation between [Cd2+] and growth inhibition was observed, the calculated IC50s differed by 3.9-fold among the different irradiance treatments, suggesting that differences in Cd
toxicity cannot be entirely explained by [Cd2+], primarily because the diatoms displayed different bioaccumulation potentials. A few previous studies also suggested that FIAM could not be used universally to predict Cd accumulation and toxicity in different groups of aquatic organisms (1, 28). The biotic ligand model, in principle, uses the bioaccumulation to predict metal toxicity by considering the toxicological site of action (e.g., in the whole organism, such as unicellular algae and zooplankton, and in fish gills). In our study, there was similarly a strong correlation between the cellular accumulation (surface-adsorbed Cd, total cellular Cd, and intracellular Cd) and the growth rate inhibition, and the differences of IC50s based on these parameters were smaller than those of [Cd2+]. Thus, the BLM may be reasonably used to predict Cd toxicity in marine diatoms. Miao and Wang (7) questioned whether intracellular Cd is a good predictor of Cd toxicity in marine phytoplankton under different nutrient conditions because of the time lag effects of Cd on phytoplankton. In their study, the relative changes of µ with intra-Cd were significantly different (p < 0.05) between two time points of measurements, even at comparable intracellular Cd levels. It appears that Cd may need some time (hours) after entering the cells to reach the toxic binding sites and trigger deleterious effects. As suggested by Wallace et al. (6), it would be expected that toxicological end points exhibited by diatoms in our study would be more closely related to partitioning of Cd to organelles and enzymes (the metal-sensitive fractions) than to surface-absorbed or total cellular Cd. The results from the IC50s indeed support this prediction, i.e., the IC50s based on MSF and organelles exhibited the least variation under different levels of irradiance. Moreover, the correlation between the growth rate inhibition and MSF or organelles was the most significant. These data strongly demonstrated that Cd toxicity in diatoms was best predicted by its distribution in MSF. Cd in the organelles worked as well as that in MSF as a predictor, mainly because the organelles are the principal component (>87% in most treatments) in MSF. Since the HDP only made up a small fraction of MSF and it was difficult to measure given its small quantity, it was difficult to determine if Cd in HDP is a good predictor of Cd toxicity. Despite the proposed usefulness of MSF by Wallace et al. (6), there has been essentially no experimental data to supports the MSF hypothesis (i.e., metals in MSF can predict metal toxicity in a variety of aquatic organisms). Tsui and Wang (29) for the first time used the subcellular approach to predict the variations of acute Hg toxicity in Daphnia magna under different temperatures, population origins, body sizes, and Hg pre-exposures. In their study, correlating Hg levels in different compartments with daphnid survival rates (as indicator of Hg toxicity) resulted in the following order of sequence: aqueous Hg > whole body Hg > Hg in the metal sensitive fraction. 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 may be more useful in explaining the sublethal (chronic) Hg toxicity than the acute toxicity. Our present study provided the first experimental evidence that MSF can predict Cd toxicity in marine phytoplankton. Interestingly, growth inhibition exhibited the weakest relationship with binding of Cd to the BDM compartment. Ecotoxicity testing using marine algae (e.g., Skeletonema costatum) has generally employed different light levels ranging from 30 to 300 µmol photons m-2 s-1 for different species (30). The recommended light intensity is usually between 80 to 120 µmol photons m-2 s-1, it is and lower for marine algae than for freshwater species. When the toxicity results are applied to field situations, it is expected that they should be sufficiently conservative to protect most species
(e.g., 95%). In the field, marine phytoplankton are exposed to much lower light irradiance than are those used in algal toxicity testing performed in the laboratory. Given our findings that diatoms are more sensitive to Cd toxicity at high irradiance, it is possible that the laboratory toxicity testing employing high irradiance may overprotect the phytoplankton species in the field. In conclusion, the irradiance affected the biochemical composition (e.g., the C:N ratio), photosynthesis, Cd accumulation and subcellular distribution, and the resulting Cd toxicity in T. nordenskioeldii. The diatoms accumulated more cellular Cd under higher irradiance at low [Cd2+], but then this accumulation slowed down when [Cd2+] further increased, implying that cellular binding saturation had occurred. HSP was the most important subcellular fraction for Cd accumulation in diatoms. With high irradiance, Cd may have spilled over with increasing [Cd2+] from the biologically detoxified pool to the metal-sensitive fractions, which in turn led to higher toxicity and sensitivity. Furthermore, Cd in MSF or organelles served as a best predictor of Cd toxicity under different irradiance levels, and the calculated IC50s based on these subcellular pools exhibited the least difference. Thus, metal subcellular partitioning, which incorporates the subcellular fates of metals such as binding to MSF (and BDM), may provide a better means to predict metal toxicity as compared to metals in the ambient environments (e.g., FIAM) or the metal body residue (e.g., BLM). A further challenge will be to test this predictive ability in other aquatic organisms.
Acknowledgments We are grateful to the anonymous reviewers for their very helpful comments on this work. This study was supported by a Competitive Earmarked Research Grant from the Hong Kong Research Grants Council (HKUST6420/06M) to W.X.W.
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