Evaluating The Effectiveness Of Copper-based Algaecide On Microcystis Aeruginosa Using SC-ICP-MS

Microcystis aeruginosa（M.aeruginosa）distributes in worldwide and usually proliferates crazily in eutrophic freshwater to form harmful algal blooms（HABs）.Copper sulfate is usually used to control HABs in freshwater system due to its effectiveness and low cost.There is marked cell heterogeneity in one cell population,quantifying cellular contents at single cell level will display cell heterogeneity and provide the most authentic results.Single cell-inductively coupled plasma mass spectrometry（SC-ICP-MS）is a novel analysis method for directly quantifying intracellular element in an individual cell level.This study aimed to simplify the sample preparation procedure and develop a SC-ICP-MS analysis method for quantifying intracellular metal of unicellular algae,M.aeruginosa.Then,the developed method was applied to explore the variation of cell status and copper uptake of M.aeruginosa after exposed to two copper-based algaecides(CuSO₄ and EarthTec^?algaecide)at different copper concentrations（0,30,60,100 and 200 μg/L）,for expanding the application of SC-ICP-MS and providing useful information for determining the dosage of copper-based algaecide in HABs control.Firstly,based on the principle of SC-ICP-MS,the method parameters were preliminarily determined,the abundant endogenous magnesium of M.aeruginosa was selected as an indicator for cell status.Then,the method for determining transport efficiency was developed by using fresh M.aeruginosa cell suspension with known concentration as standard.Finally,several method parameters,including dwell time（100μs）,scan time（100 s）,sampling flow rate（21～22 μL/min）,and the concentration of standard cell suspension（～500,000 cells/mL）,were optimized to obtain high and stable transport efficiency.After method optimization,the quantification limit of intracellular copper and intracellular magnesium contents were 65 ag/cell（RSD is 10.05%）and 98 ag/cell（RSD is 11.78%）respectively,and the quantification limit of cell concentration was 3,000 cells/mL（RSD is 4.23%,recovery is 95.11%）.Compared with other SC-ICP-MS analysis methods,the developed analysis method had simpler sample preparation procedure,and its transport efficiency was up to 63.65%.Moreover,in copper-based algaecides treatment experiment,the variation of intact cell concentration monitored by flow cytometer was consistent with the variation monitored by SC-ICP-MS,which verified the reliability of developed SC-ICP-MS analysis method.The analysis results of SC-ICP-MS showed,the CuSO₄ and EarthTec^?algaecides at copper concentration of 30 μg/L were nontoxic to M.aeruginosa respectively;M.aeruginosa released endogenous magnesium and decreased intact cell concentrations after treated with CuSO₄ and EarthTec^?algaecides at copper concentration of 60,100 and 200 μg/L for certain time,and the higher copper concentration of copper-based algaecide was used to treat cells,the faster the speeds of endogenous magnesium release and intact cell concentration decrease,indicating that the control efficiency of copper-based algaecide was related with both dosage and exposure time.The mean intracellular copper concentration of M.aeruginosa and the concentration of cells with detectable copper level increased over time after exposed to CuSO₄ and EarthTec^?algaecides both with a copper concentration of 30μg/L;in 60,100 and 200μg copper/L of CuSO₄ and EarthTec^?algaecides treatment groups,the mean intracellular copper concentration of M.aeruginosa increased at first and then decreased over time except 60 and 100 μg copper/L of CuSO₄ groups continuous increase,the concentration of cells with detectable copper level increased at first and then decreased over time in all treatment groups,and the decrease of concentration of cells with detectable copper level occurred almost simultaneously with the decrease of concentration of intact cells,indicating that M.aeruginosa lysed after absorbed a certain amount of copper ions.Moreover,the copper concentration of copper-based algaecide was corresponded with the copper uptake rate more,instead of copper uptake;and the intracellular copper mass distribution of M.aeruginosa displayed marked cell heterogeneity in copper absorption.