Note A new bioassay for the inspection and identification of TBT-containing antifouling paint Hervé Gueuné, Gérald Thouand, Marie-José Durand * University of Nantes, UMR CNRS 6144, GEPEA, CBAC Group, IUT de la Roche sur Yon, 18 bd. Gaston Defferre, 85035 La Roche sur Yon, France article info Keywords: Organotin Paint Bioluminescence Bacteria Detection Inspection abstract Since the 1960s tributyl (TBT)-based antifouling paints are widely applied to protect ship’s hulls from biofouling. Due to its high toxicity to aquatic ecosystem most of the countries (28 nations in 2008) signed the AFS convention to control the use of harmful antifouling systems on ships. Nevertheless there is cur- rently no simple method to control the presence of organotin in paint. In this study, we propose a bioas- say based on the use of a recombinant bioluminescent bacteria to detect directly in paint the presence of TBT. We also propose a simple device as an inspection system to control the absence of organotin in the ship’s hull paint. The presence of organotin could be revealed in less than three hours. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Organotin compounds, especially tributyltin (TBT), have been widely used as paint ingredients to prevent biofouling on ship hulls. Since the early 1980s the toxic effect of organotin compound on marine organisms has been extensively reported in the litera- ture (Fent, 1996; Hoch, 2001; Alzieu, 2000 for review). Due to its high toxicity, the International Marine Organisation (IMO) adopted the antifouling systems convention in 2001 (AFS). This convention prohibited the application of TBT based antifouling paints by 1st January 2003 and the presence of such paints on the surface of ship hulls by 1st January 2008 (IMO, 2005). Even if the major antifouling paint companies have decided to comply with the regulations and propose only TBT free paint, there is no simple way to govern how the legislation is applied and, as noted by Kotrikla, there is a possibility that a part of the world’s fleet will continue to use organotin compounds (Kotrikla, 2009). Several analytical methods are available to detect organotin species. Gas chromatography (GC) in combination with mass spec- trometry (MS) or flame photometric detection (FPD, tin selection) are the most popular techniques (Abalos et al., 1997 for review). Even if these conventional methods are sensitive, they are time consuming, expensive and they need complex sample preparation. Moreover with these methods, on-site analysis is not possible be- cause the instruments are not portable, therefore they cannot be employed in screening the presence of TBT-antifouling paint. Senda et al. (2003) suggested using X-ray fluorescence analysis (XRF) to distinguish between TBT-antifouling paint and TBT-free paints. They proposed using an abrasive paper to sample paint frag- ments before analysis by X-ray fluorescence. Another way to detect the presence of organotin is to use a recombinant bioluminescent bacteria. Microbial toxicity tests based on recombinant bacteria are widely used to sense the presence of pollutants. In the major cases the lux genes (lux A and lux B or the full operon lux CDABE) minus its promoter, are inserted downstream of the promoter of a gene involved in a resistance to a metal, the biodegradation of an organic compound or a stress (Billard and DuBow, 1998; Köhler et al., 2000). An other strategy to obtain specific bioluminescent bacteria is to identify Escherichia coli genes that are transcriptionally regulated by cellular response to a specific compound. For example, a luxAB genes transcription fusion library have been constructed in E. coli and screened for bioluminescence in the presence and ab- sence of TBT. One clone called TBT3, whose luminescence was in- creased in a dose-dependent manner upon exposure to TBT and DBT was selected (Briscoe et al., 1996; Durand et al., 2003). The genetic construction and the localisation of the luxAB genes in the chromosome of the TBT3 bacteria have been recently de- scribed by Gueuné et al. (2008). The promoter activated by TBT and DBT is located upstream of two co-transcribed orphan genes, ygaV and ygaP. The TBT3 strain has been applied to detect TBT and DBT in sea water with a detection limit of 0.08 lM and 0.1 nM, respectively. TBT3 is not sensitive to the other organotin compound or to organometallic compounds (Durand et al., 2003). Due to its high specificity and sensitivity to TBT and DBT, in this study we propose to use TBT3 strain to detect TBT in antifouling paint. A simple device for the inspection of a ship hull in order to ensure the effectiveness of the international convention has been developed. We also applied this bioassay to waste water from shipyards. 0025-326X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2009.09.012 * Corresponding author. Tel.: +33 2 51478442; fax: +33 2 51478456. E-mail address: Marie-Jose.Durand-Thouand@univ-nantes.fr (M.-J. Durand). Marine Pollution Bulletin 58 (2009) 1734–1738 Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul