revised) 2 Simultaneous determination of malachite green, crystal violet and 3 their leuco-metabolites in aquaculture water samples using 4 monolithic fiber-based solid-phase microextraction coupled with high 5 performance liquid chromatography 6 7 Abstract: In this work, a convenient, sensitive and environmentally friendly method 27 for simultaneous monitoring of malachite green (MG), crystal violet (CV), 28 leucomalachite green (LMG) and leucocrystal violet (LCV) in aquaculture water 29 samples was developed by coupling monolithic fiber-based solid-phase 30 microextraction (MF/SPME) to high performance liquid chromatography with visible 31 and fluorescence detection. The MF/SPME based on dual functional monomers was 32 used to concentrate target analytes. Because there were multiple interactions between 33 sorbent and analytes, the MF/SPME exhibited a high extractive capability toward 34 analytes. In the optimized process, several key parameters including desorption 35 solvent, pH value and ionic strength in sample matrix, extraction and desorption time 36 were investigated. Under the optimized extraction conditions, the limits of detection 37 (S/N=3) and limits of quantification (S/N=10) of the developed method were 38 0.013-0.30 µg/L and 0.043-0.99 µg/L, respectively. Satisfactory linearity was 39 achieved for analytes with the correlation coefficients (R 2 ) above 0.99. Acceptable 40 method reproducibility was achieved by evaluating the repeatability, intermediate 41 precision, fiber-to-fiber and batch-to-batch reproducibility with relative standard 42 deviations (RSD) of all less than 11%. Finally, the proposed method was successfully 43 applied to the determination of MG, CV, LMG and LCV in several aquaculture water 44 samples. Recoveries obtained for the determination of the target analytes in spiking 45 samples ranged from 81.0% to 111%, with RSD below 11% in all cases. 46 Keywords: Monolithic fiber-based solid-phase microextraction; malachite green; 47 crystal violet; triphenylmethane dyes; aquaculture water 48 49 50 Malachite green (MG) and crystal violet (CV) belong to triphenylmethane dyes. 52 Since the 1930s, MG and CV have been widely used as effective antifungal and 53 antiparasitic agents in aquaculture [1-3]. MG and CV are readily converted into 54 leucomalachite green (LMG) and leucocrystal violet (LCV), respectively, Through 55 biological effect. Because of carcinogenic and mutagenic properties, in many 56 countries, MG and CV have been banned for use as fungicides and antiseptics in 57 aquaculture and fisheries [4, 5]. However, as low-cost and available agents, MG and 58 CV probably are illegally used in aquaculture and fisheries [6]. Therefore, it is 59 necessary to develop a simple, sensitive and reliable method for determination of MG, 60 CV and their leuco metabolites in aquatic systems. 61 So far, several methods including high-performance liquid chromatography (HPLC) 62 [7, 8], liquid chromatography-mass spectroscopy (LC-MS) [9], gas 63 chromatography-mass spectroscopy (GC-MS) [10] and ...