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[AOS13-P02] Glutathione as Potential Biomarker for Copper Stress in Marine Phytoplankton: Preliminary Results from Incubation Experiments and Field Studies
Keywords:Glutathione, Biomarker, Ariake Sea
Introduction
Phytoplankton serves as primary producer in aquatic ecosystems and plays an important role in maintaining ecosystem health. Copper is a heavy metal that is toxic to phytoplankton at high concentrations and is therefore considered a toxic substance. In natural seawater, the bioavailability of copper to phytoplankton primarily depends on the free cupric ion (Cu2+). However, its concentration in natural seawater is significantly reduced by complexation with organic ligands. Consequently, the total copper concentration in seawater cannot be used to reliably assess the risk of copper toxicity posed to organisms because a substantial portion of the copper exists as non-bioavailable organic complexes. Current methods for assessing copper toxicity are cumbersome. Therefore, we aim to develop a simple and accurate technique for toxicity assessment by measuring glutathione (GSH), which is produced as part of the phytoplankton's defense mechanism against heavy metals such as copper. Natural seawater was collected in the Ariake Sea, and GSH content was calculated in the same manner.
Research Method
Cyanobacteria (Synechococcus sp.) and diatoms (Thalassiosira nordenskioeldii), which are both marine phytoplankton, were cultured in f/2 medium. Free copper concentrations in the culture media were set to 0, 1, and 10 pM, and the cultures were incubated for 14 days. At the end of the culture period, the samples were gently filtered under negative pressure through a 0.7 µm pore size GF/F filter paper. Particulate glutathione (GSH), a type of thiol commonly produced by phytoplankton, was extracted under weak acid conditions and derivatized using monobromobimane. Additionally, chlorophyll a (Chl a) was extracted from the particulate samples using acetone. Both GSH and Chl a were quantified using HPLC separation and fluorescence detection. The GSH content of the phytoplankton community is calculated by dividing the GSH concentration by the Chl a concentration in each sample.
Results and Discussion
Tthe GSH content of Thalassiosira nordenskioeldii did not change significantly following copper addition, and was in the range of 0.35 to 0.45 mmol /g Chl a. Conversely, the GSH content of Synechococcus sp. increased significantly, from approximately 0.13 mmol/g Chl a to 43 mmol/g Chl a.
The variations in GSH contents of Thalassiosira nordenskioeldii and Synechococcus sp. can be attributed to differences in copper tolerance among different phytoplankton species. Previous studies have indicated that diatoms are generally more tolerant to copper compared to cyanobacteria. Specifically, opper concentrations of approximately 10-10 and 10-12 M can impact the growth of diatoms and cyanobacteria, respectively. Our data show that in the absence of copper, both diatoms and cyanobacteria have GSH contents of less than 1 mmol/g Chl a. This is because GSH is used for metabolic processes in phytoplankton cells even under normal conditions as a transporter and to maintain the internal homeostasis. Thus, 1 mmol/g Chl-a should be considered as background level of GSH content in the samples. Therefore, it is necessary to account for this background level when assessing GSH content in the samples.
The GSH content in the particulate samples of Ariake Sea was relatively low at 1.5 mmol/g Chl a at most stations. This value agrees with the GSH contents of diatoms and cyanobacteria incubated under no copper exposure, indicating that the phytoplankton communities were not likely affected by copper toxicity. At two of the stations, the GSH contents exceeded 30 mmol/g Chl a and the Chl a concentrations were very low compared to the other stations, which could be attributable to environmental stress.
Phytoplankton serves as primary producer in aquatic ecosystems and plays an important role in maintaining ecosystem health. Copper is a heavy metal that is toxic to phytoplankton at high concentrations and is therefore considered a toxic substance. In natural seawater, the bioavailability of copper to phytoplankton primarily depends on the free cupric ion (Cu2+). However, its concentration in natural seawater is significantly reduced by complexation with organic ligands. Consequently, the total copper concentration in seawater cannot be used to reliably assess the risk of copper toxicity posed to organisms because a substantial portion of the copper exists as non-bioavailable organic complexes. Current methods for assessing copper toxicity are cumbersome. Therefore, we aim to develop a simple and accurate technique for toxicity assessment by measuring glutathione (GSH), which is produced as part of the phytoplankton's defense mechanism against heavy metals such as copper. Natural seawater was collected in the Ariake Sea, and GSH content was calculated in the same manner.
Research Method
Cyanobacteria (Synechococcus sp.) and diatoms (Thalassiosira nordenskioeldii), which are both marine phytoplankton, were cultured in f/2 medium. Free copper concentrations in the culture media were set to 0, 1, and 10 pM, and the cultures were incubated for 14 days. At the end of the culture period, the samples were gently filtered under negative pressure through a 0.7 µm pore size GF/F filter paper. Particulate glutathione (GSH), a type of thiol commonly produced by phytoplankton, was extracted under weak acid conditions and derivatized using monobromobimane. Additionally, chlorophyll a (Chl a) was extracted from the particulate samples using acetone. Both GSH and Chl a were quantified using HPLC separation and fluorescence detection. The GSH content of the phytoplankton community is calculated by dividing the GSH concentration by the Chl a concentration in each sample.
Results and Discussion
Tthe GSH content of Thalassiosira nordenskioeldii did not change significantly following copper addition, and was in the range of 0.35 to 0.45 mmol /g Chl a. Conversely, the GSH content of Synechococcus sp. increased significantly, from approximately 0.13 mmol/g Chl a to 43 mmol/g Chl a.
The variations in GSH contents of Thalassiosira nordenskioeldii and Synechococcus sp. can be attributed to differences in copper tolerance among different phytoplankton species. Previous studies have indicated that diatoms are generally more tolerant to copper compared to cyanobacteria. Specifically, opper concentrations of approximately 10-10 and 10-12 M can impact the growth of diatoms and cyanobacteria, respectively. Our data show that in the absence of copper, both diatoms and cyanobacteria have GSH contents of less than 1 mmol/g Chl a. This is because GSH is used for metabolic processes in phytoplankton cells even under normal conditions as a transporter and to maintain the internal homeostasis. Thus, 1 mmol/g Chl-a should be considered as background level of GSH content in the samples. Therefore, it is necessary to account for this background level when assessing GSH content in the samples.
The GSH content in the particulate samples of Ariake Sea was relatively low at 1.5 mmol/g Chl a at most stations. This value agrees with the GSH contents of diatoms and cyanobacteria incubated under no copper exposure, indicating that the phytoplankton communities were not likely affected by copper toxicity. At two of the stations, the GSH contents exceeded 30 mmol/g Chl a and the Chl a concentrations were very low compared to the other stations, which could be attributable to environmental stress.