Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands

Abstract

The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml⁻¹ according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC₂₀). The LOQ was estimated as 0.0096 ng CTX1B eq. g TE⁻¹ with 23–31% variability between experiments. These values were deemed sufficient even though quantification given at the IC₅₀ (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17–19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g⁻¹ of TE ranging from 0.058 (±0.012) to 6.23 (±0.713) ng CTX1B eq. g TE⁻¹. The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

Keywords:

• bioassay
• risk assessment
• screening – cell culture
• toxicology – cytotoxicity
• ciguatoxins
• fish

Acknowledgements

The authors thank the Spanish Ministry of Education and Sciences for financial support through INIA Project RTA2009-00127-00-00 and through an INIA PhD scholarship to A. Caillaud; and the Health Directory of Canary Islands (Tenerife) for providing samples. The authors also acknowledge the IRTA staff for technical assistance in fish sample preparation.