Arsenic speciation in freshwater fish: Focus on extraction and mass balance

Arsenic (As) speciation in muscle tissues of freshwater fish was investigated with special emphasis on extraction yields and mass balances. For the quantification of water-soluble As compounds, samples were extracted using a 1:1 (v/v) methanol/water mixture. Various extraction parameters, such as extraction volume, number of additional extractions, intermediate and final volume in sample preconcentration were optimized so as to improve the extraction efficiency. Arsenic compounds were determined by HPLC with online ICP-MS, using both cation and anion exchange separation. The species studied were eel (Anguilla anguilla L.), flathead grey mullet (Mugil cephalus L.), chub (Leuciscus cephalus L.), and carp (Cyprinus carpio L.). Mean total As concentrations ranged from 354 μg kg−1 dry weight (carp) to 1804 μg kg−1 dry weight (mullet). Under optimized conditions, the percentage of total As extracted ranged from 64% for carp, to 82%, 84%, and 89% for grey mullet, eel and chub, respectively. Extraction of lipid-rich eel with n-hexane recovered some additional 3% of total As. The sizeable effect of sample matrix on HPLC retention time of some organoarsenicals in gradient elution cation exchange chromatography was exploited to achieve separation of coeluting compounds by analysing the same sample at different dilutions. The recovery of As from chromatographic columns was 101%, 102%, 103% and 104% for carp, chub, mullet and eel, respectively, indicating that no As was retained during chromatography. Arsenobetaine (AB) was the dominating As compound, but several other arsenicals, including arsenous acid (Asiii), arsenic acid (Asv), methylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), arsenocholine ion (AC), tetramethylarsonium ion (TETRA), oxo-arsenosugar-glycerol (AS1), oxo-arsenosugar-phosphate (AS2), oxo-arsenosugar-sulfate (AS4), thio-arsenosugar-phosphate (ThioAS2), and three unknown As compounds, were found. Arsenic speciation in carp was different compared to the other fish species, and a lower proportion of AB along with a high contribution of AS2 and ThioAS2 was found. DMA and TMAO were noteworthy minor compounds in eel and carp, respectively. Arsenic speciation and the chemical composition of fish muscle both appeared to affect the extraction yield.