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A new polymeric biocide polyhexamethylene guanidine (PHMG) molybdate has been synthesized. The obtained cationic polymer has limited water solubility of 0.015 g/100 mL and is insoluble in paint solvents. The results of acute toxicity studies indicate moderate toxicity of PHMG molybdate, which has a median lethal dose at 48 h of 0.7 mg/L for Daphnia magna and at 96 h of 17 mg/L for Danio rerio (zebrafish) freshwater model organisms. Commercial ship paint was then modified by the addition of a low concentration of polymeric biocide 5% (w/w). The painted steel panels were kept in Dnipro River water for the evaluation of the dynamics of fouling biomass. After 129-d exposure, Bryozoa dominated in biofouling of tested substrates, forming 86% (649 g/m2) of the total biomass on control panel surfaces. However, considerably lower Bryozoa fouling biomass (15 g/m2) was detected for coatings containing PHMG molybdate. Dreissenidae mollusks were found to form 88% (2182 g/m2) of the fouling biomass on the control substrates after 228 d of exposure, whereas coatings containing PHMG molybdate showed a much lower biomass value of 23.6 g/m2. The leaching rate of PHMG molybdate in water was found to be similar to rates for conventional booster biocides ranging from 5.7 μg/cm2/d at the initial stage to 2.2 μg/cm2/d at steady state. Environ Toxicol Chem 2017;36:2543-2551. © 2017 SETAC [ABSTRACT FROM AUTHOR]