Determination of heavy metal toxicity by using a micro-droplet hydrodynamic voltammetry for microalgal bioassay based on alkaline phosphatase Md. Saiful Islam a , Kazuto Sazawa a , Noriko Hata a , Kazuharu Sugawara b , Hideki Kuramitz a, * a Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Gofuku 3190, Toyama, 930-8555, Japan b Maebashi Institute of Technology, Maebashi, 371-0816, Gunma, Japan highlights graphical abstract  Alkaline phosphatase enzyme inhi- bition bioassay using Chlamydomonas reinhardtii was developed.  The induction of alkaline phospha- tase activity was successfully carried out.  A 50 mL micro-droplet was used as reaction medium.  Electrochemical measurements were carried out using Rotating disk elec- trode (RDE).  The enzyme inhibition test using RDE shows the most sensitive response to heavy metals than some common methods. article info Article history: Received 18 May 2017 Received in revised form 29 August 2017 Accepted 2 September 2017 Available online 4 September 2017 Handling Editor: Frederic Leusch Keywords: Microalgal bioassay Chlamydomonas reinhardtii Alkaline phosphatase Hydrodynamic voltammetry Droplet Rotating disk electrode abstract We developed an electrochemical microalgal bioassay for the determination of heavy metal toxicity in water on the basis of the alkaline phosphatase (ALP) enzyme inhibition of Chlamydomonas reinhardtii. Five heavy metals were chosen as toxicants: Hg, Cd, Pb, Zn, and Cu. The induced ALP activity of C. reinhardtii was inhibited using the phosphate starvation method, and the results were evaluated by measuring the electrochemical oxidation of p-aminophenol (PAP) following the enzymatic conversion of p-aminophenyl phosphate (PAPP) as a substrate. The rapid determination of enzymatic activity was achieved using hydrodynamic voltammetry in a 50 mL micro-droplet with a rotating disk electrode (RDE). Enzymatic activity over a PAPP substrate is affected by heavy metal ions, and this phenomenon decreases the chronoamperometric current signal. The concentrations of Hg, Cd, Pb, Zn, and Cu in which the ALP activity was half that of the control (EC 50 ) were found to be 0.017, 0.021, 0.27, 1.30, and 1.36 mM, respectively. The RDE system was demonstrated to be capable of detecting enzymatic activity by using a small amount of regent, a reaction time of only 60 s, and a detection limit of 5.4  10 7 U. © 2017 Elsevier Ltd. All rights reserved. * Corresponding author. E-mail address: kuramitz@sci.u-toyama.ac.jp (H. Kuramitz). Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere http://dx.doi.org/10.1016/j.chemosphere.2017.09.008 0045-6535/© 2017 Elsevier Ltd. All rights reserved. Chemosphere 188 (2017) 337e344