Environ. Sci. Technol. 2005, 39, 8099-8107
Thiol and Metal Contents in Periphyton Exposed to Elevated Copper and Zinc Concentrations: A Field and Microcosm Study S EÄ V E R I N E L E F A U C H E U R , RENATA BEHRA,* AND LAURA SIGG Swiss Federal Institute of Aquatic Science and Technology (EAWAG), Ueberlandstrasse 133, CH-8600 Duebendorf, Switzerland
Phytochelatins are metal-binding polypeptides produced by algae under metal exposure. The aim of this study was to investigate the effects of metal concentration variations in natural systems on periphyton at the biochemical level by analyzing its intracellular thiol content, in particular phytochelatins. To that purpose, two field campaigns were conducted in a stream subject to an increase of dissolved metal concentrations (particularly Cu and Zn) during rain events, which results in an increase of their accumulation in periphyton. At background metal concentrations, several thiols were detectable in periphyton, namely, glutathione (GSH), γ-glutamylcysteine (γGluCys), phytochelatins (PC2), and some unidentified thiols, U1 and U2. Glutathione and γGluCys contents were found to vary independently of the rain, as well as U1 and U2, whereas the phytochelatin content increased during the rain events. To investigate whether Cu or Zn may be responsible for this increase, microcosm experiments were carried out with natural water enriched with Cu, Zn, and Cd separately, and Cu and Zn in combination. In this study, GSH, PC2, and U1 were also detected, but not γGluCys. An increase in accumulated Cu content did not induce any changes in thiol content, whereas an increase of the Zn content induced a decrease in GSH content and an increase in phytochelatin content. Zinc rather than Cu may thus induce a phytochelatin content increase in periphyton in the field studies. Addition of Cu and Zn in combination also induced an increase in phytochelatin content. Cadmium was found to be the most effective inducer, with the production of larger phytochelatins (PC3-4). This study is the first one to report changes in thiol content in periphyton in response to an increase of the metal concentration in natural freshwaters.
Introduction Phytochelatins (PCn) are intracellular metal ligands produced by plants, fungi, and algae in response to elevated metal concentrations in their environment (1-3). These small polypeptides with the amino acid sequences (γGluCys)nGly, where n ) 2-11 (PC2-11), are enzymatically produced from glutathione (GSH) and have a strong affinity to metals due * Corresponding author phone: +41-1-823-5119; fax: +41-1-8235311; e-mail:
[email protected]. 10.1021/es050303z CCC: $30.25 Published on Web 09/09/2005
2005 American Chemical Society
to the thiol groups from cysteine. Essential roles of phytochelatins in metal homeostasis as well as detoxification have thus been shown in yeast and in plants (4-6). In algae, phytochelatin induction depends on both the algal species and the metal (7-9), cadmium being generally the strongest inducer. To a less extent, copper and zinc are also found to induce phytochelatins in marine and freshwater algae (812). Phytochelatins have been especially examined in marine phytoplankton. Their content seems to be regulated by metal concentrations in water and was observed to decrease between sites receiving sewage and riverine inputs near harbors and nonpolluted sites (13-15). Only one study has so far examined phytochelatins in freshwater algal communities (16). In this study, phytochelatins were detected in phytoplankton in three lakes, but no clear relationship between metal concentrations in water and phytochelatin concentrations could be found. Periphyton is a complex assemblage of algae, bacteria, and fungi which grow attached to substrata in streams and in the littoral zones of lakes. It plays major ecological roles in freshwater ecosystems as a primary producer (17) and a source of food for grazers and in nutrient cycling (18). Studies on the interaction of metals with periphyton have shown that metal bioaccumulation was tightly linked with metal concentrations in water (19-21). The response of periphyton to variations in environmental metal concentrations was observed to be fast (22) and to depend on metal speciation (21). However, no data are available so far to evaluate whether the phytochelatin content responds to rapid variations in metal accumulation. The aims of our study were (i) to examine whether phytochelatins were detectable in freshwater periphyton communities after short-term exposure to elevated metal concentrations, (ii) to investigate whether Cu, Zn, and Cd may induce phytochelatin production, and (iii) to evaluate if phytochelatins may be useful as indicators of metal exposure. For that purpose, two studies were conducted. The first one was a field experiment, carried out in a small stream where periphyton is subject to increased Cu and Zn concentrations during rain events. In the second study, the effects of Cu, Zn, and Cd on phytochelatin and metal contents were studied using microcosms in which Cu, Zn, and Cd were added separately, and Cu and Zn in combination.
Materials and Methods The field and microcosm studies were conducted at two different sites. The two field studies were performed in the Furtbach (Canton Zu¨rich, Switzerland), a small stream subject to release of dissolved copper and zinc from sediments, especially during rain events (22). Alkalinity ranges from 2 to 6 mM, pH from 7.7 to 8, and dissolved organic carbon (DOC) concentration from 2.6 to 5.4 mg L-1. The microcosm study was carried out at a site located 1.5 km from the outlet of Lake Greifen (Canton Zu ¨ rich, Switzerland), where natural waters from the Glatt River contain a low background of dissolved metal concentrations. Water alkalinity ranges from 2 to 3 mM, pH from 8 to 8.3, and DOC concentration from 3.6 to 4.3 mg L-1(21). Experimental Setup. Artificial substrata used to colonize periphyton in the Furtbach stream were previously described by Meylan et al. (22). Briefly, Teflon racks holding 12 microscope glass slides (76 × 26 mm) were placed directly into the stream for 3-4 weeks. To minimize accumulation of inorganic particles into the biofilm, the racks were placed vertical to the bottom of the stream, parallel to the current, VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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and at about 10 cm under the water surface. Two sampling campaigns were performed, one at the beginning of fall (September 2002) after at least 2 days of sunny weather and the other one in spring (May 2004) after 5 days of sunny weather. Samples were collected before, during, and after a rain event, during daylight, except for one sample collected at 20:00 on Sept 23, 2002. Rainfall was measured at a meteorological station situated 2 km from the sampling site. For the microcosm study, periphyton was colonized in flow-through 50 L aquaria, containing holders with microscope glass slides. Water was continuously pumped from the Glatt River, providing a constant nutrient source to the periphyton. After 5 weeks, a sufficient biofilm was formed and translocated into 5 L tanks containing different added concentrations of Cu, Zn, or Cd, and of Cu and Zn in combination. Two concentrations were used for each metal, 500 and 1500 nM for Cu, and 250 and 1000 nM for Zn. These selected Cu and Zn concentrations were chosen on the basis of a previous study (21) to match the highest free Cu2+ and Zn2+ concentrations in the Furtbach stream (22). Additions of 500 and 1500 nM Cu are estimated to give a pCu around 13 and 12, respectively, and addition of 250 nM Zn is expected to give a pZn around 9. Experiments were also performed with exposure to a combination of these Cu and Zn concentrations. Cd was only studied at 1000 nM. Triplicate tanks were set up for each concentration. Three other tanks that did not receive any addition of metal were used as controls. Metal solutions added in the water tanks were prepared by dilution of Cu, Zn, and Cd stock solutions of 1 g L-1 (from “Baker Analyzed” reagent for atomic absorption spectrometry, Deventer, Holland) and were equilibrated for 1 day before the experiments were performed. Periphyton was exposed for 1 day to the metals before determination of the thiol and metal contents. Communities were regularly examined microscopically and were found to be dominated by algae. Sampling and Analytical Methods. For thiol analysis, periphyton was collected from six glass slides and placed at -80 °C before freeze-drying. Around 150 mg of the lyophilized biofilm was well-mixed with a mortar, resuspended in 1 mL of a solution of hydrochloric acid (HCl) and diethylenetriaminepentaacetic acid (DTPA) (0.12 M/5 mM), and heated for 2 min at 95 °C. The homogenate was then centrifuged for 10 min at 10000g to pellet cell debris. The pellet was resuspended a second time in HCl/DTPA to recover an additional 10% of the total extracted thiols. To analyze thiols with HPLC, the supernatant was derivatized with monobromobimane, which gives a fluorescent tag to SH groups, as already described elsewhere (23). The products were separated on a reversed-phase C18 column (hypersil ODS, 5 µm, 250 × 4 mm) with a precolumn (5 × 4 mm) (MachereyNagel AG, Switzerland) using a linear gradient from 10% to 36% acetonitrile for 65 min with 0.1% trifluoroacetic acid as the aqueous mobile phase. Thiols were detected by fluorescence at 380 nm excitation and 470 nm emission wavelengths. The retention times of phytochelatin oligomers were verified with PC2, PC3, and PC4 standards, provided by the Biochemistry Institute of the University of Zu ¨ rich, Switzerland, and were at 25.4 min for PC2, 33.9 min for PC3, and 39.4 min for PC4. The retention times of PC5 and PC6 were calculated from the linear relationship between the retention time of the standard peptides and log n, where n is the number of γ-GluCys dipeptide pairs. A calibration curve between the GSH peak area and the concentration of standard solutions was used for quantification. Variation due to the extraction and derivatization steps was calculated to be 5% between replicates. The limit of detection (calculated as 3 times the standard deviation of 10 measurements) was calculated as 0.5 µmol of SH/g of chlorophyll a (Chla) and the limit of quantification (calculated as 10 times the standard 8100
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deviation of 10 measurements) as 1.5 µmol of SH/g of Chla. All samples were analyzed within one month after the sampling. The metal content was also determined in periphyton from the Furtbach stream and from the microscosm experiment. In the Furtbach experiments, an EDTA-wash technique described and validated by Meylan et al. (21, 22, 24) was applied to discriminate between adsorbed and intracellular accumulated metal. Periphyton was scratched from the glass slides and resuspended in filtered river water (0.45 µm, Orange Scientific Gyrodisc). Three 10 mL aliquots of periphyton suspension were filtered on acid-washed and preweighed filters (cellulose nitrate, 0.45 µm, Sartorius) and dried for 15 h in an oven at 50 °C. They were then placed in Teflon digestion flasks with 1 mL of H2O2 (30%) and 3 mL of HNO3 (65%). Digestion was performed in a high-performance microwave digestion unit (mls1200 mega) for 15 min. To differentiate between extracellular metal adsorption and intracellular uptake, the periphyton was washed with 4 mM EDTA, pH 8 (final concentration), for 10 min. The intracellular metal concentration was defined as the cellular metal content determined after the EDTA wash. In the microcosm experiments, no discrimination was done between the adsorbed and the intracellular metal contents. The total accumulated metal content was determined in a subsample of the lyophilized periphyton pool used for thiol analysis, digested as described above. The metal concentrations in the digested solutions were determined by ICP-MS (inductively coupled plasma mass spectrometry; Perkin-Elmer Elan 5000 and Thermo Elan 2). The accuracy of the digestion was checked by analyzing a plankton reference material (CRM 414, Community Bureau of Reference, Commission of the European Communities), and the ICP-MS measurements were also checked using SLRS-4 reference water (National Research Council Canada). Satisfactory results were obtained in both cases. Dissolved metal concentrations (Cu, Zn, Cd, Pb, Cr, Co, Mn, Ni) were measured by ICP-MS (Perkin-Elmer Elan 5000 and Thermo Elan 2) in river water, filtered (poly(ether sulfone), 0.45 µm, Orange Scientific Gyrodisc) and acidified to 0.01 M HNO3. The Chla content was determined by the rapid HPLC method from Murray et al. (25) after extraction of about 10 mg dry weight of periphyton in 90% ethanol. Statistics. Significant differences between thiol concentrations at the background level of dissolved metal concentrations and at their maximum concentrations in the Furtbach experiments were tested with ANOVA (post hoc Fisher LSD, R ) 0.05). This test was also applied to test differences in metal accumulation as well as in thiol concentrations between treatments (different concentrations of the same metals and different metals) in the microcosm experiments. All statistical analyses were computed with the software Statistica 6.0.
Results Stream Experiments. The results of dissolved Cu and Zn concentrations as well as their accumulation in periphyton measured in the Furtbach stream during the rain event of September 2002 were previously described elsewhere (22). In May 2004, the experiment was repeated during a 2 day rain period. Compared with those of the first study, background dissolved Cu and Zn concentrations measured before the rain were 7- and 8.6-fold higher, with a concentration of 155 ( 8 nM for Cu and 172 ( 9 nM for Zn (Figure 1). During the rain, their concentrations increased to 245 ( 5 nM for Cu, representing a 1.5-fold increase, and to 385 ( 26 nM for Zn, representing a 2-fold increase. After the rain, both concentrations decreased to values similar to those before the rain. In May 2004, additional metals (Cd, Co, Cr, Mn, Ni,
FIGURE 1. Variation of dissolved Cu and Zn concentrations over a 2 day rain event in May 2004 in the Furtbach stream.
FIGURE 2. Variation of total and intracellular Cu and Zn concentrations in periphyton collected over a 2 day rain event in May 2004 in the Furtbach stream.
TABLE 1. Dissolved Metal Concentrations (nM) Measured in the Furtbach Stream before and during the Rain Event in May 2004 (n ) 3) dissolved metal
min value, before the rain
Cd Cu Co Cr
0.11 ( 0.02 155 ( 8 2.9 ( 0.2 20 ( 2
max value, min value, max value, during dissolved before during the rain metal the rain the rain 0.19 ( 0.01 245 ( 5 3.1 ( 0.0 19 ( 1
Mn Ni Pb Zn
58 ( 7 3.7 ( 0.5 1.0 ( 0.1 172 ( 9
127 ( 4 8.2 ( 0.2 3.9 ( 0.2 385 ( 26
and Pb) were also analyzed (Table 1). During the rain, Cd, Mn, and Ni concentrations increased by a factor of about 2 and the Pb concentration by a factor of about 4, whereas Cr and Co concentrations remained constant. After the rain, the metal concentrations dropped to values similar to those measured before the rain (data not shown). Before the rain, in May 2004, the average total accumulated Cu concentration in periphyton was 0.29 ( 0.02 mmol/g of Chla, which was comparable to values measured in September 2002 (Figure 2). About 81% of the total content was found to be intracellular. During the rain, total and intracellular accumulated Cu concentrations increased by a factor of more than 2 to 0.66 ( 0.04 mmol/g of Chla and to 0.50 ( 0.02 mmol/g of Chla, respectively. The total accumulated Cu concentration during the rain was higher in
September 2002 with a value of 0.81 ( 0.01 mmol/g of Chla, whereas the intracellular Cu concentration was lower with a value of 0.34 ( 0.01 mmol/g of Chla (22). After the rain, both concentrations remained high with values of 0.45 ( 0.02 mmol/g of Chla for the total accumulated and 0.39 ( 0.02 mmol/g of Chla for the intracellular contents. Total and intracellular Zn contents were also found to vary during the rain event. At the beginning of the sampling period, the average total accumulated Zn concentration was 1.31 ( 0.06 mmol/g of Chla and the average proportion of intracellular to total Zn was around 81%. With the rain, the total accumulated Zn content increased to 2.79 ( 0.01 mmol/g of Chla and the intracellular content to 1.64 ( 0.02 mmol/g of Chla. The total and intracellular Zn concentrations were both higher in September 2002 with values of 5.8 ( 0.3 and 2.0 ( 0.2 mmol/g of Chla (22). After the rain, the total and intracellular Zn contents were observed to remain constant for 4 days. Several thiols were detected in periphyton in September 2002 and in May 2004 (Figures 3 and 4). In September 2002, before the rain, GSH, γ-glutamylcysteine (γGluCys), a thiol with an unknown structure (U1) eluting at 22.4 min, and PC2 were detected. Glutathione was the most predominant thiol with a concentration of 79 ( 13 µmol of SH/g of Chla. During the rain, the GSH and γGluCys concentrations remained constant. In contrast, the U1 concentration decreased whereas the PC2 concentration increased significantly (P < 0.05). Two larger phytochelatins, PC3 and PC4, were also detectable but not quantifiable. After the rain, the GSH and γGluCys concentrations remained constant, the U1 concentration decreased until complete disappearance, and PC2 remained at high concentrations. PC3 was also detectable. In May 2004, before the rain, the same thiols as in September 2002 were present in periphyton, namely, GSH, γGluCys, U1, and PC2. An additional unidentified thiol, U2, eluting at 30.5 min, was also detected. However, in May 2004, γGluCys was the major thiol with an average concentration of 32 ( 25 µmol of SH/g of Chla. During the rain, the GSH concentration increased significantly to a maximum value of 124 ( 32 µmol of SH/g of Chla (P < 0.05), becoming the most predominant thiol in the periphyton, whereas the γGluCys concentration decreased and the PC2 concentration remained constant. U1 and U2 concentrations were again observed to decrease, but the decrease was initiated well before the onset of the rain. PC3 and PC4 were again both induced. After the rain, the GSH concentration continuously increased whereas the γGluCys, U1, PC2, U2, and PC3 concentrations remained constant, and PC4 was no longer detectable. Microcosm Experiments. A good reproducibility on the dissolved metal concentrations was obtained between the tanks used to expose periphyton (Tables 2-5). The total accumulated Cu concentration in periphyton increased significantly with the increase of the dissolved Cu concentration in water (Table 2). The total accumulated Zn concentration did not significantly vary by increasing the Zn concentration to 259 ( 11 nM (P > 0.05) but significantly increased (P < 0.05) with exposure to 1043 ( 27 nM Zn (Table 3). Cu and Zn were also added in combination (Table 4). At the lowest Cu and Zn concentrations (458 ( 16 and 303 ( 19 nM), the accumulated Cu and Zn concentrations were similar to those measured in periphyton exposed to the metals separately. At higher Cu and Zn concentrations, accumulation of both metals was higher when added in combination than when added separately, probably due to changes in Cu and Zn speciation in solution. Exposure of periphyton to Cd, carried out as a control for phytochelatin induction, resulted in a 600-fold increase of the Cd content (Table 5). In control microcosms, GSH, U1 (unknown thiol identical to that found in the Furtbach experiment), and PC2 were detected in the periphyton (Figure 5). Glutathione was the VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Thiol content in periphyton colonized in the Furtbach stream during a rain event in September 2002.
TABLE 2. Dissolved Cu and Zn Concentrations Measured in Microcosms and Total Accumulated Cu and Zn Concentrations in Periphyton as a Function of the Concentration of Cu Added (n ) 3)
control Cu1 Cu2
concn of Cu added (nM)
Cu concn measd (nM)
Zn concn measd (nM)
accumulated Cu concn (mmol/g of Chla)
accumulated Zn concn (mmol/g of Chla)
500 1500
25 ( 0 398 ( 8 1130 ( 86
34 ( 11 26 ( 14 32 ( 21
0.29 ( 0.03 0.86 ( 0.03 2.01 ( 0.39
1.27 ( 0.18 1.33 ( 0.18 1.31 ( 0.06
TABLE 3. Dissolved Cu and Zn Concentrations Measured in Microcosms and Total Accumulated Cu and Zn Concentrations in Periphyton as a Function of the Concentration of Zn Added (n ) 3)
Zn1 Zn2
concn of Zn added (nM)
Zn concn measd (nM)
Cu concn measd (nM)
accumulated Cu concn (mmol/g of Chla)
accumulated Zn concn (mmol/g of Chla)
250 1000
259 ( 11 1043 ( 27
23 ( 1 28 ( 1
0.29 ( 0.04 0.23 ( 0.03
1.44 ( 0.29 1.71 ( 0.05
TABLE 4. Dissolved Cu and Zn Concentrations Measured in Microcosms and Total Accumulated Cu and Zn Concentrations in Periphyton as a function of the Concentrations of Cu and Zn Added in Combination (n ) 3)
Cu1/Zn1 Cu1/Zn2 Cu2/Zn1 Cu2/Zn2
concn of Cu added (nM)
concn of Zn added (nM)
Cu concn measd (nM)
Zn concn measd (nM)
accumulated Cu concn (mmol/g of Chla)
accumulated Zn concn (mmol/g of Chla)
500 500 1500 1500
250 1000 250 1000
458 ( 16 467 ( 8 1279 ( 131 1427 ( 35
303 ( 19 1158 ( 31 314 ( 25 1167 ( 20
0.63 ( 0.05 1.27 ( 0.11 2.89 ( 0.21 3.76 ( 0.36
1.14 ( 0.13 3.35 ( 0.37 1.73 ( 0.24 3.33 ( 0.40
predominant thiol with a concentration of 243 ( 18 µmol of SH/g of Chla, whereas the concentrations of U1 and PC2 were much lower with values of 8.2 ( 1.4 and 4.7 ( 0.4 µmol of SH/g of Chla, respectively. With exposure to 500 and 1500 nM added Cu, the GSH, U1, and PC2 concentrations remained constant and another unidentified thiol, U3, eluting at 23.9 min, was detected with a similar concentration of 6.5 ( 0.3 µmol of SH/g of Chla in both Cu treatments (Figure 5a). Exposure to 250 nM added Zn did not induce any changes in the GSH, U1, and PC2 concentrations (Figure 5b). A thiol identical to that detected in Cu treatments, U3, was produced with a concentration around 4 µmol of SH/g of Chla. Higher 8102
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Zn addition provoked a significant decrease of the GSH concentration to 211 ( 9 µmol of SH/g of Chla (P < 0.05). The U1 and U3 concentrations remained constant, and the PC2 concentration significantly increased to 6.7 ( 0.9 µmol of SH/g of Chla (P < 0.05). Addition of both Cu and Zn induced several changes in the thiol content, namely, a decrease in GSH concentration, an increase in U1 and PC2 concentrations, and the appearance of U3 and another unknown thiol, U4, eluting at 36 min (Figure 5c). However, U4 was not present in periphyton exposed to 500 nM Cu and 250 nM Zn added in combination. Exposure to Cd produced a decrease in GSH content to 203 ( 13.1 µmol of SH/g of Chla, whereas the U1
TABLE 5. Dissolved Cd Concentration Measured in Microcosms and Total Accumulated Cd, Cu, and Zn Concentrations in Periphyton as a function of the Concentration of Cd Added (n ) 3)
control Cd
concn of Cd added (nM)
Cd concn measd (nM)
Cu concn measd (nM)
Zn concn measd (nM)
accumulated Cd concn (mmol/g of Chla)
accumulated Cu concn (mmol/g of Chla)
accumulated Zn concn (mmol/g of Chla)
1000
0.2 ( 0.0 1038 ( 11
30 ( 2.3
76 ( 12
(3.92 ( 0.73) × 10-3 2.32 ( 0.63
0.25 ( 0.04
0.98 ( 0.10
FIGURE 4. Thiol content in periphyton colonized in the Furtbach stream during a rain event in May 2004. concentration remained constant and the PC2 concentration increased by a factor of more than 2 (Figure 5d). Periphyton exposed to Cd also contained U3 as in the Cu and Zn
treatments and larger phytochelatins, PC3 and PC4, at concentrations of 5.3 ( 0.4 and 6.4 ( 1.3 µmol of SH/g of Chla. PC5 was also detectable but not quantifiable. VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. (a) Thiol content in periphyton exposed to added Cu in microcosms. (b) Thiol content in periphyton exposed to added Zn in microcosms. An asterisk indicates a significant difference between the control and Zn-exposed periphyton (P < 0.05). (c) Thiol content in periphyton exposed to added Cu and Zn in combination in microcosms. An asterisk indicates a significant difference between the control and Cu- and Zn-exposed periphyton (P < 0.05). (d) Thiol content in periphyton exposed to added Cd in microcosms. An asterisk indicates a significant difference between the control and Cd-exposed periphyton (P < 0.05).
Discussion Rain events of September 2002 (22) and May 2004 induced an increase of dissolved metal concentrations in the Furtbach stream and of accumulated metal concentrations in the periphyton. Variations of bioaccumulated metal concentra8104
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tions followed the same pattern in both studies, but different amounts of adsorbed and intracellular Cu and Zn were found. Differences in the species composition of periphytic samples between studies and as a consequence, in the composition of the matrix in which periphytic organisms were embedded,
are expected to influence both the bioavailability and the cellular distribution of the metals. Thus, accumulation of metals in periphyton collected in the same river can vary among sites, and also temporally within sites (20). Moreover, a previous study showed that the intracellular Cu content in periphyton varied as a function of the labile Cu concentration, whereas the intracellular Zn content was related to the free Zn2+ concentration in the Furtbach stream (24). The thiol content in periphyton also varied during rain. The glutathione concentration was found to be much lower before the rain in May 2004 than in September 2002, but increased to similar values during the rain. These GSH concentrations are comparable to those found in marine communities (15, 26), whereas higher concentrations have been found in laboratory algal cultures of both freshwater and marine species (15, 23, 27). The low GSH content detected in May 2004 may indicate that other stress factors were occurring. Since the GSH content is found to vary depending on nutrient bioavailabity in algae (28), low GSH concentrations might result from nutrient limitations, but no measurements on nutrient concentrations were performed in this study. Differences in light intensity (29) and in temperature (30) may also have influenced the GSH level. Besides being involved in metal homeostasis, GSH participates in the detoxification for oxidative stress and of various xenobiotics, processes that might also deplete its concentration. Glutathione is also the precursor of phytochelatins. Before the rain, the PC2 concentration in May 2004 was about 5 times higher than in September 2002, suggesting that part of the GSH pool was consumed to produce PC2 in May 2004. The GSH concentration increase during the rain may have been due to a nutrient input, which allowed the periphyton to produce more GSH to form PC3-4. Although no data on nutrients were available in May 2004, analyses were performed in September 2002 and showed that nutrient contents increased during the rain event (S. Meylan, personal communication). γ-Glutamylcysteine, the precursor of GSH, was also detected in periphyton, and its concentration did not vary with the rain. In May 2004, its concentration decrease was rather concomitant with the concentration increase of GSH, suggesting its consumption for GSH formation. While no data are available for freshwater communities, comparable γGluCys content values were found in marine phytoplankton (15) whereas higher concentrations were measured in laboratory algal cultures (23, 27). Phytochelatin 2 (PC2) was detectable in periphyton before the rain in both field studies. With the increase of dissolved metal concentrations during the rain, an increase of the phytochelatin content was observed by either the increase of the PC2 content in September 2002 or by the production of larger phytochelatins (PC3-4) in May 2004. The fact that, in May 2004, the PC2 concentration was 5 times higher than in September 2002 before the rain and did not increase during the rain suggests that PC2 production might have reached a plateau. Kinetic studies on metal-induced phytochelatin synthesis showed that PC2 was synthesized and reached a plateau within a few hours in algae exposed to Cd and Pb (8, 31-33). Comparable phytochelatin concentrations were found in freshwater and marine communities (13, 16), as well as in laboratory algal cultures (8, 23). Besides phytochelatins, two unidentified thiols, U1 and U2, were detected in periphyton. The U1 concentration was higher in May 2004 than in September 2002, and U2 was only detected in May 2004. In both field studies, their concentrations were found to decrease over the sampling period, independently of the rain. The occurrence of other small thiols has also been observed in plants and in algae exposed to metals (3, 34, 35). These peptides were phytochelatin homologues, differing from PCn by their carboxy-terminal
amino acid with the presence of β-alanine, serine, or glutamate instead of the glycine or by its lack with the formation of desGly-PCn. Another form of phytochelatin homologues containing one additional cysteine, PCn-Cys, was also reported in the periphytic freshwater alga Stigeoclonium tenue after exposure to Zn (12). The increase of the metal concentrations in water during rain events was found to be accompanied by an increase in phytochelatin content, but no clear relationship could be established among thiol production, the dissolved metal concentrations in water, and the bioaccumulated Cu and Zn concentrations in periphyton. Moreover, in both studies, bioaccumulated metal concentrations were always in large excess of the thiol concentrations, by a factor of 2 for Cu and 10 for Zn, compared to the intracellular content. Several factors may be responsible for that. As discussed for GSH, species composition, light, or nutrients may also influence the production of PCn and of the unidentified thiols. Moreover, the concentrations of other metals, such as Pb or Cd, were also increasing over the sampling period in May 2004 (Table 1). Because Pb and Cd are better phytochelatin inducers than Cu and Zn (8, 33), these metals, even at low concentration, may have also played a role in phytochelatin induction in periphyton. Also, interaction effects between various metals may influence the thiol content in periphyton (15). To study the relationship between phytochelatin content and metal concentrations in water and to distinguish which metal may be responsible for the PC content increase, microcosm experiments were carried out to examine the effects of Cu, Zn, and Cd on phytochelatin induction in periphyton under controlled exposure conditions. Bioaccumulations of Cu and Zn obtained in the control microcosms were comparable to those from the Furtbach studies, although the dissolved background concentrations of Cu and Zn were lower than those in the Furtbach water. These similar bioaccumulations may be due to differences in metal bioavailability or in the composition of the periphyton. Major thiols, namely, GSH, PC2, and U1, were also present in the control periphyton. Cadmium is well-known to be the most effective inducer of phytochelatins in plants and algae. Its effect was thus also examined in the microcosm study as a positive control of the capability of periphyton to produce phytochelatins. As expected, Cd induced a high level of phytochelatins (PC2-4). In the Cu experiment, upon Cu accumulation, there was no change in GSH concentration as already observed in marine algae (27). In our study, no additional phytochelatins were synthesized in response to Cu accumulation compared to the control. The free Cu2+ concentrations studied might thus have been too low to induce phytochelatin synthesis. Induction experiments with freshwater phytoplankton showed that, as in our study, the PCn concentration only slightly increased at pCu between 15 and 11.5 but significantly increased at pCu 9 (16). No significant induction of PCn was observed in several marine species between pCu 13.8 and pCu 12, but induction of PCn was observed at higher pCu (8, 13, 27). Our results indicate that phytochelatins are not indicative of increased Cu exposure in periphyton in the Cu concentration range studied. At the lowest Zn exposure concentrations, no Zn accumulation was observed, suggesting cellular regulation of the Zn content in organisms composing the periphyton, as previously observed in marine and freshwater algae (3638). Higher Zn concentrations slightly increased Zn bioaccumulation, and induced a significant increase of the PC2 content and concomitantly a slight decrease of the GSH content. Phytochelatin induction under Zn exposure was also observed in freshwater and marine algae (10, 11). Metal accumulation in periphyton exposed to Cu and Zn in combination was found to be much higher than that of VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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periphyton exposed to the metals separately, which was most probably due to changes in metal speciation. Indeed, added in combination, the total dissolved metal concentration could have exceeded the concentration of the natural ligands, leading to an increase of the free metal ion concentration and thus of bioavailability. As observed in the periphyton exposed to Cu and Zn separately, the increase of the PC2 content appears to be related to an increase of the accumulated Zn content. The concentrations of the unidentified thiols U1 and U3, which were also detected with exposure to Cu and Zn, seem also to vary as a function of the Zn content rather than of the Cu content. Some conclusions from the microcosm experiments may then be applied to explain some results from the field study. Microcosm experiments showed that Zn, but not Cu, induced phytochelatins in the studied concentration range. Moreover, the presence of Cd at low concentrations may have contributed to the production of PCn. This study is the first one to report the occurrence of glutathione, phytochelatins, and other small unknown thiols in periphyton at metal background concentrations as well as their concentration variations in response to a metal concentration increase. The field and microcosm studies showed that phytochelatins do not simply indicate the level and the presence of specific metals in water. Before phytochelatins are employed as indicators of metal contamination, further studies have to be conducted to better understand the factors regulating the production of phytochelatins.
Acknowledgments We are grateful to Se´bastien Meylan for helpful discussions and assistance in setting up the experiments and to Adrian Amman and David Kistler for ICP-MS measurements. We also thank three anonymous reviewers for their valuable comments.
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Received for review February 15, 2005. Revised manuscript received July 1, 2005. Accepted August 3, 2005. ES050303Z
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