Temperature-Dependent Sensitivity of a Marine Diatom to Cadmium

Oct 22, 2008 - of a Marine Diatom to Cadmium. Stress Explained by Subcelluar. Distribution and Thiol Synthesis. MENG-JIAO WANG AND. WEN-XIONG ...
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Environ. Sci. Technol. 2008, 42, 8603–8608

Temperature-Dependent Sensitivity of a Marine Diatom to Cadmium Stress Explained by Subcelluar Distribution and Thiol Synthesis MENG-JIAO 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 May 28, 2008. Revised manuscript received September 1, 2008. Accepted September 10, 2008.

This study examined the potential bioaccumulation and biochemical mechanisms (phytochelatin and other thiols induction) in the temperature-dependent sensitivity of a marine diatom Thalassiosira nordenskioeldii to cadmium (Cd) stress. A higher environmental temperature increased the sensitivity of diatoms to Cd toxicity. Either increased cellular Cd accumulation or a poorer detoxification ability was responsible for the higher concentration of metal-sensitive fraction (MSF)Cd in the diatoms and subsequently the higher Cd sensitivity with increasing temperature. In addition, N-deficiency or glutathione depletion may partly explain the highest sensitivity at the highest tested temperature (30.5 °C). Although temperature affected the biochemical composition (e.g., the N/C ratio and phytochelatin induction), physiological processes (e.g., the growth rate, photosynthesis, Cd uptake, accumulation, and subcellular distribution) and the resulting differential tolerances, Cd concentration in MSF or organelles served as the best indicators of Cd toxicity in diatoms at different temperatures. Phytochelatins (PCs) were induced by increasing [Cd2+] and the significant relationship between the intracellular Cd and PC-SH concentration suggested that PC-SH is a biomarker for cellular metal stress. However, the intracellular Cd/ PC-SH ratio did not always explain the temperaturedependent metal tolerance. The functions of PCs other than metal chelation and detoxification need to be further examined.

Materials and Methods

Introduction Cadmium (Cd) may cause serious environmental problems when released into aquatic systems. Apart from creating oxidative damage by reducing cellular antioxidant capacity, Cd exerts toxicity mainly by nonspecifically binding to sulfhydryl groups of physiologically important proteins and displacing the essential metals in metalloenzymes (1). To help manage the environmental impact of Cd, a reliable Cd toxicity indicator is necessary. Environmental conditions control the biochemical and physiological processes of phytoplankton and can lead to differential Cd sensitivities. To minimize environmental influences on Cd toxicity, recent studies used the subcellular partitioning approach (a further development of the tissue residue approach) to examine Cd toxicity in aquatic organisms. In a recent study (2), we found * Corresponding author phone: (852) 2358-7346; fax: (852) 23591559; e-mail: [email protected]. 10.1021/es801470w CCC: $40.75

Published on Web 10/22/2008

that Cd concentration in the metal sensitive fraction (MSF, organelles plus heat-denatured proteins) and organelles in the marine diatom Thalassiosira nordenskioeldii may serve as reliable Cd toxicity indicators when quantified under different irradiances. Other than irradiance, temperature is another important environmental factor exerting strong influence on toxic responses to metal stress in algae. Temperature may cause significant changes of physiological and biochemical processes of phytoplankton, and may be a potential factor when interpreting the toxicity testing results based on algae. To our knowledge, there has been no research on the underlying mechanism leading to Cd toxicity at different temperatures using the subcellular partitioning approach. In response to metal stress in phytoplankton, phytochelatins (PCs) are synthesized enzymatically by transferring the γ-Glu-Cys (γ-EC) group from one glutathione (GSH) molecule to another GSH or previously synthesized PCn-1 molecule (3), and Cd is generally thought to be the strongest PC activator (4). The general structure of a PC is (γ-EC)n-Gly (PCn), with n ) 2-11 (5), while PC2-4 are the PC predominant structures in phytoplankton (6). PCs are rich in cysteine and chelate metals through thiol groups in their amino acids, playing an important role in metal detoxification and tolerance in plants (7). Other studies suggest that greater PC production is not always instrumental in increasing metal tolerance in higher plants or phytoplankton (8, 9). The binding stoichiometry of thiol groups to Cd ranges from 2 to 4 (10); this ratio may be useful in resolving the controversy but it has been the subject of few studies (6, 11). GSH is also considered as a metal chelator apart from being a substrate of PC (3, 12). Thus, the examination of GSH, PC2-4, and their precursor cysteine and γ-EC may provide useful information for understanding the importance of metal detoxification and tolerance in phytoplankton. Such measurements may also test whether PCs can become a biomarker for metal stress. In this study, we examined (1) the effects of temperature on the sensitivity of T. nordenskioeldii to Cd (based on inhibition of the growth rate and photosynthesis-related PAM parameters), Cd accumulation, and subcellular distribution; (2) if the MSF-Cd and organelle-Cd concentrations could predict Cd toxicity at different temperatures; (3) if PCs play an important role in Cd detoxification and temperaturedependent Cd sensitivity; and finally, (4) if PCs in diatoms are a reliable biomarker of Cd stress at different temperatures.

 2008 American Chemical Society

Phytoplankton Cultures and Cd-Exposure Medium. The phytoplankton culture conditions and the Cd-exposure medium were similar to those in our previous study (2). The isolated Hong Kong local marine diatoms T. nordenskioeldii were cultured in the f/2 medium (pH ) 8.2 ( 0.1) at 23.5 °C and 170 µmol photons m-2 s-1 with a 14:10 h light/dark (L/ D) cycle. For the preparation of the experimental medium, 0.22 µm filtered and chelexed nutrient stocks (N, P, vitamin, and Si) were added into the similarly treated artificial seawater (AQUIL, (13)) at f/2 levels, while the filtered trace metals were added at f/10 levels. Nitrilotriacetate (NTA, at a fixed concentration of 0.1 mmol L-1) and Suprapure NaOH (1 mol L-1) were used to control the free Cd ion concentrations ([Cd2+]) and medium pH (8.2 ( 0.1), respectively. Toxicity experiments were performed at three different temperatures (18, 24, and 30.5 °C) at seven [Cd2+] levels (10-11, 9 × 10-8, 3.2 × 10-7, 10-6, 1.7 × 10-6, 3 × 10-6, and 8.1 × 10-6 mol L-1 VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Decrease of average growth rate (µ) (A), cellular N/C ratio (B), maximum PSII quantum yield (ΦM) (C), and operational PS II quantum yield (Φ′M) (D) in the diatom Thalassiosira nordenskioeldii with increasing [Cd2+]. Data are means ( SD (n ) 3). in treatments A-G, respectively). Three replicated beakers had only stable Cd added and two replicated beakers had both stable Cd and radioactive 109Cd (18.5-148 kBq L-1, in 0.1 mol L-1 HCl) added. The corresponding total dissolved Cd concentrations were calculated with the MINEQL+ software for different temperature treatments. All polycarbonate flasks and beakers were soaked in HCl (1 mol L-1) for at least 24 h before the experiments, and then rinsed with Milli-Q water (18.2 mΩ) 7 times. Measurement of Toxicity Parameters. All experimental procedures were similar to our previous study (2). T. nordenskioeldii were transferred from the f/2 culture medium into the f/10 trace metal medium for acclimation for 2 weeks at different temperatures (18, 24, or 30.5 °C), under the light conditions of 50 µmol photons m-2 s-1 with a L/D cycle of 14:10. Then, the cells at the midexponential growth phase were collected by centrifugation and transferred into the Cdexposure medium under the same conditions as during the acclimation, with an initial cell density of 1-8 × 104 cells mL-1. The exposure lasted for 72 h. The cell density was quantified every 24 h with a Coulter Particle Counter and the average growth rate (µ) was calculated as the slope of the linear regression of the natural logarithmically transformed cell density versus time. At the end of the experiment, the maximal PSII quantum yield (ΦM) and operational PSII quantum yield (Φ′M) were calculated from the fluorescence induction curve performed by the PAM 101/103 fluorometer (14). Simultaneously, a 40 mL sample was filtered gently and subjected to particulate organic carbon (POC) and nitrogen (PON) measurements with a CHNS/O analyzer 2400. Intracellular Cd Concentration, Subcellular Cd Fractionation, and Low Molecular Weight Thiols Measurement. At 72 h, a 200 mL aliquot from each radioactive replicate was filtered, rinsed 3 times with filtered artificial seawater, and then resuspended in 10 mL of 8-hydroxyquinoline-5-sulfonate to remove any Cd loosely adsorbed on the cell surfaces. Afterward, the cells were centrifuged again and resuspended in 3 mL of Tris-buffer for intracellular Cd measurement and subcellular Cd fractionation, while 1 mL of remaining 8-hydroxyquinoline-5-sulfonate was counted for radioactivity as surface-adsorbed Cd. The Cd subcellular fractionation was quantified using the described methods (2, 15), with some modifications. The centrifugation to separate S1 (combination of organelles, heat-denatured proteins, and heat-stable proteins) and P1 (metal-rich granules and cellular debris) was operated at 1400g instead of 800g. We also added the antioxidant 2-mercaptoethanol (5 mmol L-1) and freshly 8604

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prepared antiprotease phenylmethylsulfonyl fluoride (PMSF) (0.1 mmol L-1) into the Tris-buffer to protect the proteins against oxidation or degradation. At the same time, a 200 mL sample was harvested for measurement of cysteine, γ-EC, GSH, and PC2-4, following the method described by Wei et al. (16), but with some modifications of the mobile phase and flow gradient. Briefly, cells were filtered onto a precombusted GF/F membrane, immediately frozen in liquid nitrogen and placed directly into 2 mL of 70 °C methanesulfonic acid (MSA, 10 mmol L-1). The mixture was then homogenized with a tissue grinder and, afterward, centrifuged at 13,800g for 10 min. A 0.8 mL portion of the supernatant was removed, with the pH adjusted to 9.0 using borate buffer (100 mmol L-1, containing 10 mmol L-1 diethylenetriaminepentaacetic acid). After reacting with excess dithiothreitol (DTT, 15 mmol L-1) for 10 min, the mixture was labeled with monobromobimane (mBrB, 50 mmol L-1). After another 10 min, excess DTT (15 mmol L-1) was added to react with the remaining mBrB. Finally, the mixture was stabilized with MSA (1 mol L-1) and transferred into an autosampler vial for HPLC analysis. All operations were done at 4 °C. The column used was a Supelco Discovery RP Amide-C16 HPLC column (4.6 × 250 mm). IC50 Calculation and Data Analysis. The median inhibition concentrations (IC50) based on µ inhibition were calculated for different Cd concentrations ([Cd2+], intra-Cd, or subcellular Cd) with the ICPIN software version 2.0 (USEPA, Duluth, MN, using linear interpolation method). Significant differences were accepted if p < 0.05 by one-way ANOVA with post hoc multiple comparisons (SPSS 11.0 software).

Results Cell Growth and PAM Parameters (ΦM and Φ′M) at Different Temperatures. Changes in µ, ΦM, and Φ′M as a function of [Cd2+] at different temperatures are shown in Figure 1. In the control treatments, the cells grew faster at higher temperature. With the increase of [Cd2+], µ decreased significantly above 10-11 mol L-1 at the two higher temperatures, while above 9 × 10-8 mol L-1 for the lowest temperature group. The growth at 30.5 °C decreased so precipitously that it was almost completely inhibited at 10-6 mol L-1. At the other two temperatures, such serious growth inhibition was only observed at 8.1 × 10-6 mol L-1, suggesting a higher sensitivity of diatoms to [Cd2+] with increasing temperature. Correspondingly, the IC50 value for [Cd2+] was lower at a higher temperature (Table 1). Similarly, the general trend of ΦM and Φ′M inhibition by [Cd2+] suggested a higher sensitivity

TABLE 1. Median Inhibition Concentration (IC50) Values of Growth Inhibition at Different Temperatures, As Well As Regression Analysis (r2 Values) of Different Toxic Responses for [Cd2+] (µmol L-1) and Different Cellular Cd Concentrations (µmol mol C-1)a

[Cd2+] total cellular intracellular MSF organelles HDP BDM MRG HSP cellular debris surface adsorbed

18 °C

24 °C

30.5 °C

max/min

r2 (µ)

r2 (ΦM)

r2 (Φ′M)

2.59 ( 0.08 658 ( 18.1 523 ( 14.5 96.4 ( 2.72 91.3 ( 2.50 6.05 ( 0.00 400 ( 11.6 99.7 ( 4.26 300 ( 7.06 26.8 ( 0.20 135 ( 3.35

1.87 ( 0.06 841 ( 21.1 718 ( 18.6 124 ( 4.05 105 ( 3.38 19.8 ( 0.76 568 ( 13.3 94.7 ( 2.52 473 ( 11.6 25.7 ( 0.55 123 ( 2.45

0.30 ( 0.01 241 ( 2.83 211 ( 2.34 63.5 ( 1.20 57.6 ( 1.14 6.94 ( 0.10 145 ( 1.15 51.4 ( 0.30 93.2 ( 0.84 8.70 ( 0.00 29.3 ( 0.55

8.49 3.49 3.40 1.96 1.82 3.27 3.93 1.94 5.08 3.08 4.60

0.694*** 0.709*** 0.707*** 0.854*** 0.869*** 0.514*** 0.663*** 0.800*** 0.579*** 0.559*** 0.630***

0.413** 0.359** 0.340* 0.460** 0.495** 0.272* 0.310* 0.459** 0.273* 0.332* 0.410**

0.507*** 0.410** 0.384** 0.486** 0.523*** 0.282* 0.353** 0.495** 0.316* 0.427** 0.491**

a MSF: metal sensitive fraction; HDP: heat-denatured protein; BDM: biologically detoxified metals; MRG: metal-rich granules; HSP: heat stable protein. r2(µ): r2 value based on µ inhibition. r2 (ΦM): r2 value based on ΦM inhibition. r2 (Φ′M): r2 value based on Φ′M inhibition. * ) p < 0.05, ** ) p < 0.01, *** ) p < 0.001.

of the photosynthetic PSII system with increasing temperature, despite a few unexpected fluctuations in the two lower temperature groups. The N/C ratio in the control treatments followed a 24 °C > 30.5 °C > 18 °C pattern. However, this ratio at 30.5 °C dropped so sharply that it was much lower than the other two groups at [Cd2+] g 9 × 10-8 mol L-1. Cellular Cd Accumulation and Subcellular Distribution. After normalization by POC, there was an obvious linear log-log relationship between [Cd2+] and either intracellularCd (intra-Cd, which may also contain some tightly membrane-bound Cd) or total cellular-Cd (total-Cd, the combination of intra-Cd and loosely surface-adsorbed Cd) (Supporting Information Figure S1). For both intra-Cd and total-Cd concentrations, there was no significant difference among the control treatments of different temperatures. With the increase of [Cd2+], Cd bioaccumulation was the highest at 24 °C and the lowest at 18 °C. The bioaccumulation at 30.5 °C was comparable to that at 24 °C when [Cd2+] < 3.2 x 10-7 mol L-1, but became comparable to that at 18 °C at a higher [Cd2+]. The percentage of surface-adsorbed Cd generally increased with [Cd2+] in all temperature groups (Supporting Information Figure S2). The heat-stable protein (HSP)-Cd was the major subcellular compartment, followed by metalrich granule (MRG)-Cd and organelle (org)-Cd, with heatdenatured protein (HDP)-Cd the least important. In the 18 and 24 °C groups, the percentage of org-Cd decreased with [Cd2+] till 10-6 mol L-1 and then leveled off. The percentage of HSP-Cd decreased with [Cd2+] continually for the 30.5 °C group (from 54% to only 40%), while it increased from 49% to 65% (at 10-6 mol L-1) followed by leveling off for the 24 °C group. At 18 °C, it increased sharply from 48% to 68% (at 9 × 10-8 mol L-1), followed by a smooth decrease. The relationships between each Cd concentration and µ, ΦM, or Φ′M inhibition were all significant (p < 0.05), especially with growth inhibition (Figure 2). Among all the correlations, the r2 values based on µ inhibition with org-Cd and MSF-Cd were the highest. Correspondingly, the IC50 values of µ inhibition (Table 1) varied the least with the different temperatures when based on org-Cd, MSF-Cd, and MRG-Cd (1.82-1.96×), but they varied the most when based on [Cd2+]. Since ΦM and Φ′M were not inhibited by more than 50%, their IC50 values were not calculated. Cysteine and Low Molecular Weight (LMW) Thiol Induction. Typical HPLC profiles of different thiol compounds are shown in Supporting Information Figure S3. Cysteine and LMW thiols concentrations were normalized to POC values. Of the 6 thiol compounds, γ-EC generally had the highest concentration, while cysteine always had the least (Figure 3). In the control treatments, the concentration of

GSH and the three PCs followed a similar trend (GSH > PC4 > PC2 > PC3). With an increase of [Cd2+], T. nordenskioeldii at different temperatures had difference of PC induction patterns (Figure 3). There was no significant difference in the induced concentration of GSH and PC-SH (2×PC2 + 3×PC3 + 4×PC4) among the control treatments. With increase of [Cd2+], GSH of all groups decreased, with a few exceptions (at 24 °C). PC-SH of all groups increased with [Cd2+], except for a slight decrease when [Cd2+] > 3 × 10-6 mol L-1 at 18 °C. There was a sharp increase of PC-SH at 24 °C when [Cd2+] > 1.7 × 10-6 mol L-1. A significant relationship was found between intraCd and PC-SH, but less so for [Cd2+] (Figure 4A and B). We also computed the ratios of intra-Cd to cysteine, γ-EC, GSH, and PC-SH. Intra-Cd/PC-SH generally increased with [Cd2+] (Figure 4C). The last two data points at 30.5 and 24 °C interrupted the expected strong relationship between intra-Cd/PC-SH and µ inhibition (Figure 4D). However, a strong linear relationship between intra-Cd/PC-SH and µ inhibition at 18 °C was observed (p < 0.001) (Figure 4D). There was no significant relationship between intra-Cd/cys, intra-Cd/γ-EC, or intra-Cd/GSH and µ inhibition (data not shown).

Discussion Temperature Influence on Diatom Sensitivity to [Cd2+] Exposure. Cadmium exerted obvious inhibition on growth and photosynthesis PSII system in T. nordenskioeldii, which were also sensitive to temperature. Either cellular Cd accumulation or subcellular Cd distribution may account for this temperature sensitivity. When we compared cells in the control treatment at 24 °C with those at 18 °C, there was no significant difference in the Cd cellular burden. However, when [Cd2+] continued to increase, T. nordenskioeldii at 24 °C showed a much higher intracellular Cd concentration, indicating its higher accumulation ability. Thus, even though there was no significant difference in the percentage of MSFCd between these two groups, cells at 24 °C had a higher MSF-Cd concentration and higher sensitivity. We operationally separated intra-Cd into five subcellular compartments (MRG, cellular debris, organelles, HSP, and HDP). Consistent with our previous study (2), BDM (metalrich granules plus heat stable proteins) was the dominant pool for intra-Cd. Interestingly, the pattern of subcellular Cd distribution versus [Cd2+] was totally different between 30.5 °C and the two lower temperatures, which may directly lead to its highest Cd sensitivity. At 30.5 °C, there was an obvious “spillover” of Cd from the BDM pool into the MSF pool (mainly organelles) at [Cd2+] > 9 × 10-8 mol L-1, which resulted in the much higher MSF-Cd and org-Cd percentage, VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Growth inhibition of diatoms Thalassiosira nordenskioeldii with [Cd2+] (mol L-1) and different cellular Cd concentrations (µmol mol C-1). MSF: metal sensitive fraction; Organ: organelle; BDM: biologically detoxified metals; MRG: metal-rich granules. Data are means ( SD (n ) 2). implying a weaker detoxification ability at 30.5 °C. Thus, although diatoms at 30.5 °C did not accumulate more cellular Cd than at 24 °C, more of its intra-Cd found its way into the biologically sensitive pool and led to the highest sensitivity. In the other two groups, however, there was no such “spillover effect”, showing a much better detoxification ability. With the lowest Cd accumulation ability as well as a higher BDM detoxification ability, cells at 18 °C were the most tolerant to Cd stress. Besides, N-deficiency or GSH depletion may also be partly responsible for the highest Cd sensitivity at 30.5 °C. Considering the importance of N in detoxification and metabolism, cells at 30.5 °C with much lower N/C ratios (while the cellular quota of both C and N increased at this temperature) may not be able to provide sufficient N for some lifemaintaining amino acids or proteins, e.g., GSH, as indicated by the much lower GSH contents at higher [Cd2+]. GSH is important in metal chelation, defense against reactive oxygen derivatives, redox state regulation, or transport of amino acids (17). Even if the system were successful in detoxifying Cd at 30.5 °C, the cells may still be in an N-thirsty condition or unbalanced redox state, which led the cells to be more vulnerable to Cd stress. Prediction of Cd Toxicity with Organelles-Cd and MSFCd. In this study, we found that there was a significant relationship between µ inhibition and different Cd concentrations (p < 0.001), among which IC50 values still exhibited the least variation based on org-Cd (1.8×) and MSF-Cd (1.9×). Correspondingly, MSF-Cd and org-Cd also served among the best ones from the regression relationship between ΦM or Φ′M inhibition and different Cd concentrations, although PAM parameters were not as sensitive as growth inhibition in terms of IC50. Interestingly, the org-Cd-IC50 and MSF8606

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FIGURE 3. Concentration of cysteine (cys), γ-EC, GSH, and PC2-4 in the diatom Thalassiosira nordenskioeldii after 72-h Cd exposure in different temperature conditions: (A) 10-11 mol [Cd2+] L-1; (B) 9 × 10-8 mol L-1; (C) 3.2 × 10-7 mol L-1; (D) 10-6 mol L-1; (E) 1.7 × 10-6 mol L-1; (F) 3 × 10-6 mol L-1; (G) 8.1 × 10-6 mol L-1. Data are means ( SD (n ) 3).

FIGURE 4. Change of PC-SH (2×PC2 + 3×PC3 +4×PC4) concentration in the diatom Thalassiosira nordenskioeldii (µmol mol C-1) with increasing [Cd2+] (A) and intracellular-Cd (B); relationship between [Cd2+] and intra-Cd/PC-SH ratio (C), as well as between this ratio and growth inhibition (D). Data are means ( SD (n ) 3). Regression line in D is only for 18 °C group. Cd-IC50 values are directly comparable to our previous results (88.6 ( 2.58 µmol mol C-1 for org-Cd-IC50 and 119 ( 0.22 µmol mol C-1 for MSF-Cd-IC50) (2) quantified under the same conditions (i.e., 24 °C, 50 µmol photons m-2 s-1), which further supports the reliability of this separation method. The bioavailability and toxicity of metals are highly dependent on the ambient physicochemical (e.g., temperature, salinity, and dissolved organic carbon) and biological conditions (18). Direct measurements of metal accumulation may provide more complete information on the potential toxicity of the metals. The subcellular approach can be considered as a further step of the biotic ligand model (BLM, which only addresses the total accumulation at the biological site of action, e.g., fish gills or whole cells), although it has some inevitable ambiguities such as the potential overlaps among some fractions. However, the subcellular approach is more biologically meaningful to some extent and can provide a further means to understand metal toxicity. This study directly corroborates our previous study (2) that metal toxicity in diatoms is mainly in the metal sensitive fractions and provides additional proof that metal concentration in MSF could be used to predict metal toxicity in marine phytoplankton exposed to Cd. Surprisingly, MRG-Cd-IC50 was also found to have as little variation as org-Cd-IC50 and MSF-Cd-IC50. A close examination of the growth inhibition versus MRG-Cd (Figure 2) showed that the last point of the 30.5 °C group deviated obviously from the regression line, leading to the lower r2 value than that of MSF-Cd or org-Cd plot. Thus, MRG-Cd does not serve as a good indicator in all conditions. Function of PCs in Cd Detoxification and Stress Indicator. Induction of PC2-4 by Cd stress was observed at all temperatures. PCs are thought to alleviate toxic effects by reducing intracellular free metal ion concentrations through chelation (6). Based on this detoxification role, the cells would detoxify more efficiently with higher PC induction and lower intra-Cd/PC-SH ratios. Thus, it is expected that the intraCd/PC-SH ratio (or Cd to PC-SH difference) could be used to indicate toxicity as well as to explain the temperaturedependent sensitivity. However, our results are only partly in agreement with these assumptions, i.e., the intra-Cd/PC ratio may serve as a good toxicity indicator at 18 °C, but the ability to produce PCs in greater amounts is not instrumental in controlling temperature-differential metal tolerance.

When we focused only on 18 °C and a few treatments at the other two temperatures ([Cd2+] e 1.7 × 10-6 mol L-1 at 24 °C, and e9 × 10-8 mol L-1 at 30 °C), the PC-SH contents increased as a response to Cd stress, but they did not keep pace with the increase of intracellular Cd accumulation, which led to the increasing intra-Cd/PC-SH ratio and the corresponding higher toxic effects. A good linear regression was even observed between the intra-Cd/PC-SH ratio and growth inhibition at 18 °C. However, the sharp decrease in the intra-Cd/PC-SH ratio at [Cd2+] > 1.7 × 10-6 mol L-1 at 24 °C, which was due to the dramatic increase of PC3 and PC4, could not account for the deterioration of growth and the PAM parameter inhibition. Besides, the PC-SH concentration at 30.5 °C was similar (at [Cd2+] e 9 × 10-8 mol L-1) or even significantly higher than the other two groups (mainly because of the higher PC4 content). Thus, cells at 30.5 °C had similar or even lower intra-Cd/PC-SH ratios compared with the other two groups, but showed a much higher sensitivity. Our results suggested that diatoms did not increase or at least maintained their tolerance with increasing PCs or decreasing intra-Cd/PCSH/ ratio; similar results were also mentioned previously (8). All these observations pointed out that the intra-Cd/ PC-SH ratio can not be used to indicate Cd toxicity under all conditions or to explain the differential temperaturedependent Cd sensitivity. Several mechanisms may explain our observations. First, a higher PC concentration does not always indicate an efficient sequestration ability. The PC turnover (induction and degradation) may be more important than the absolute PC concentration in determining metal detoxification. Second, binding to PCs may not be the only decisive pathway for detoxification under all conditions. Other important pathways have also been suggested previously, such as starch granule deposit (9), proline (19), or some unknown thiols such as (γEC)n-Ser, (γEC)n-Glu, and (γEC)n-β-Ala (20) (which may be corresponding to the unidentified peaks in our HPLC analysis). With the increase of temperature and [Cd2+], such pathways may not be able to cooperate with PCs to detoxify metals. Therefore, even when the intra-Cd/PC-SH decreased or leveled off, the toxic effects still deteriorated. Third, the PCs also play other important roles apart from metal detoxification. Considering the low levels of PCs present in many species of phytoplankton even at very low Cd conVOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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centrations, they appear to play a role in metal homeostasis (21). PCs may also be involved in the sulfur incorporation pathway and Fe/S metabolism (21). In the different temperature conditioned T. nordenskioeldii, the dominance of different PCs was diverse, which indirectly implied the different functions of various PCs (other than increasing PCCd complex stability with the length of PCs). Although PCs do not contribute to the temperature sensitivity of diatoms to Cd toxicity in all conditions, the significant relationships between intra-Cd with PC-SH support the observed role of PCs as indicators of environmental metal pollution (22, 23) or, better, cellular metal stress. It has been proposed that some detoxification pathways, such as the synthesis of metallothioneins can be used as biomarkers of metal exposure in well-designed environmental monitoring programs (24). Phytochelatins, the enhanced cellular concentration of which has been found in natural phytoplankton collected in polluted waters (25, 26), may also be considered as suitable indicators for metal exposure. Although several algae species have been reported to be able to synthesize PCs in response to toxic metals (23), only a few attempts have been made to use phytochelatins as biomarkers (16, 27). To conclude, higher temperatures increased the diatom’s sensitivity to Cd stress through various mechanisms. Either higher cellular Cd accumulation at 24 °C or poorer detoxification ability at 30.5 °C was responsible for the higher MSF-Cd concentration and the resulting higher sensitivity with increasing temperature. Besides, N-deficiency or GSH-depletion may also partly explain the highest sensitivity at 30.5 °C. Cd concentration in MSF or organelles serves as the best indicator of Cd toxicity in diatoms at different temperatures. PCs were induced by increasing [Cd2+] and the significant relationship between intra-Cd and PC-SH may support the assumption that PCs could serve as indicators of cellular metal stress. Although the intra-Cd/ PC-SH ratio does not explain the temperature-dependent metal tolerance or toxicity, it may serve as a good predictor of toxicity at 18 °C.

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. Wang.

Supporting Information Available Intracellular and total cellular Cd accumulation in the diatom Thalassiosira nordenskioeldii with the increase of [Cd2+], Cd relative distribution between the surface adsorbed and intracellular pool of the diatoms Thalassiosira nordenskioeldii and in five subcellular compartments, and high-performance liquid chromatography of mBrB-labled reagent blank and cell extract. This information is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Wang, W.-X.; Rainbow, P. S. Subcellular partitioning and the prediction of cadmium toxicity to aquatic organisms. Environ. Chem. 2006, 3, 395–399. (2) Wang, M. J.; Wang, W.-X. Cadmium toxicity in a marine diatom as predicted by the cellular metal sensitive fraction. Environ. Sci. Technol. 2008, 42, 940–946. (3) Grill, E.; Loffler, S.; Winnacker, E. L.; Zenk, M. H. Phytochelatins, the heavy metal binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci. USA 1989, 86, 6838–6842. (4) Ahner, B. A.; Morel, F. M. M. Phytochelatin production in marine algae. 2. Induction by various metals. Limnol. Oceanogr. 1995, 40, 658–665. (5) Grill, E.; Winnacker, E. L.; Zenk, M. H. Phytochelatins: the principal heavy metal complexing peptides of higher-plants. Science 1985, 230, 674–676. 8608

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