, Volume 21, Issue 4, pp 1102–1111 | Cite as

Effects of metal pyrithione antifoulants on freshwater macrophyte Lemna gibba G3 determined by image analysis

  • Hideo Okamura
  • Luvsantsend Togosmaa
  • Takuya Sawamoto
  • Keiichi Fukushi
  • Tomoaki Nishida
  • Toshio Beppu


Copper pyrithione (CuPT2) and zinc pyrithione (ZnPT2) are two popular antifouling agents that prevent biofouling. Research into the environmental effects of metal pyrithiones has mainly focused on aquatic animal species such as fish and crustaceans, and little attention has been paid to primary producers. There have been few reports on residues in environmental matrices because of the high photolabile characteristics of the agents. Residue analyses and ecological effects of the metabolites and metal pyrithiones are not yet fully understood. This study was undertaken to assess the effects of CuPT2, ZnPT2, and six metabolites (PT2: 2,2′-dithio-bispyridine N-oxide, PS2: 2,2′-dithio-bispyridine, PSA: pyridine-2-sulfonic acid, HPT: 2-mercaptopyridine N-oxide, HPS: 2-mercaptopyridine, and PO: pyridine N-oxide) on a freshwater macrophyte. A 7-day static bioassay using axenic duckweed Lemna gibba G3 was performed under laboratory conditions. Toxic effects of test compounds were assessed by biomass reduction and morphological changes were determined in image analysis. Concentrations of ZnPT2 and CuPT2 and those of PT2 and HPT in the medium were determined by derivatizing 2,2′-dithio-bispyridine mono-N-oxide with pyridine disulfide/ethylene diamine tetra-acetic acid reagent that was equimolar with pyrithione. The toxic intensity of the compounds was calculated from the measured concentrations after 7-day exposure. ZnPT2, CuPT2, PT2, and HPT inhibited the growth of L. gibba with EC50 ranging from 77 to 140 μg/l as calculated from the total frond number as the conventional index, whereas the other four metabolites had less effect even at 10 mg/l. The presence of the former four toxic derivatives resulted in abnormally shaped and unhealthily colored fronds, whose size was about 20% of the control fronds. EC50, calculated from the healthy frond area determined in image analysis, ranged from 10 to 53 μg/l. Thus, image analysis as part of a duckweed bioassay can detect the toxic effects of pyrithione derivatives with 3–10 times higher sensitivity than the traditional index.


Abnormal Copper Duckweed Frond area Frond color Mercaptopyridine 


AFS treaty

International Convention on the Control of Harmful Anti-Fouling Systems on Ships


Copper pyrithione, 2-mercaptopyridine N-oxide copper salt




Dimethyl sulfoxide


50%-Effect concentration


Ethylene diamine tetra-acetic acid


Healthy frond area


Healthy frond number


High-performance liquid chromatography




2-Mercaptopyridine N-oxide


Instrumental quantification limit


Individual frond area, same as frond size




No-observed-effect concentration


Pyridine disulfide, same as PS2


Pyridine N-oxide


2,2′-Dithio-bispyridine mono-N-oxide




Pyridine-2-sulfonic acid


2,2′-Dithio-bispyridine N-oxide


Specific growth rate


Total frond area


Total frond number


Zinc pyrithione, 2-mercaptopyridine N-oxide zinc salt



This study was supported by a Grant-in-Aid for Scientific Research (B20310019) from Japan Society for the Promotion of Science (JSPS).


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Hideo Okamura
    • 1
  • Luvsantsend Togosmaa
    • 2
  • Takuya Sawamoto
    • 2
  • Keiichi Fukushi
    • 2
  • Tomoaki Nishida
    • 3
  • Toshio Beppu
    • 4
  1. 1.Research Center for Inland Seas, Kobe UniversityKobeJapan
  2. 2.Graduate School of Maritime Sciences, Kobe UniversityKobeJapan
  3. 3.Faculty of Agriculture, Shizuoka UniversityShizuokaJapan
  4. 4.Faculty of Life and Environmental Science, Teikyo University of ScienceYamanashiJapan

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