The Detection of Ciguatera Toxins in NSW Spanish Mackerel

Ciguatera Poisoning (CP) is an illness through the consumption of fish containing naturally occurring toxins, and is considered a high risk for Australian seafood safety. Ciguatoxins (CTXs) are produced by benthic microalgae (Gambierdiscus spp). In Australia, CP cases are related to fish caught in Queensland (QLD), Northern Territory (NT) and New South Wales (NSW) waters. Spanish Mackerel (Scomberomorus commerson) is the main species that has resulted in CP cases from fish caught in NSW and sub-tropical QLD, and is an important commercial species. An inability to address the risks of CTXs has led to illnesses, with the potential to damage public perceptions of seafood with economic losses to industry. Currently no validated monitoring or measurement methods are available. Prevention methods used internationally are to avoid larger fish of certain species, avoid certain fish species altogether, or avoid fish from certain regions.

A previous project, FRDC 2014-035, set up a facility to determine CTX, and found ~1% of flesh and 7% of liver samples contained detectable CTXs in Spanish Mackerel from NSW waters. A higher proportion of fish from QLD waters contained detectable CTX, with no significant pattern in relation to fish size. As that study tested one analogue of CTXs, Pacific Ciguatoxin 1B (P-CTX1B, or commonly CTX1B), over one year of sampling (n=71 fish), it was recommended that sampling be expanded geographically and temporally to examine biological and environmental variables that could correlate to CP risk. A further recommendation from 2014-035 was that results from liquid chromatography mass spectrometry (LC-MS/MS) be compared against rapid toxicity assays. Discussions with the seafood industry as part of the National Ciguatera Workshop held in 2019 found that rapid, low cost detection of CTXs was a priority. A priority from the NSW RAC in April 2019 for a “A tool to determine the presence of ciguatera in NSW caught Spanish Mackerel”.

The present study aimed to: 1) determine industry CTX analysis needs and conduct a viability assessment of CTX measurement tools against these needs; 2) Obtain samples ~300 individual Spanish Mackerel of all sizes caught in industry relevant regions of NSW and QLD waters and measure CTX1B and other available CTXs; and 3) Analyse CTXs in Spanish Mackerel in comparison to biological and environmental variables and 4) Develop recommended options for food safety risk management for Spanish Mackerel to allow for a viable industry while protecting public health.

The analysis of liver and muscle tissue from 249 Spanish Mackerel caught in NSW and QLD waters over 2 fishing seasons (2020/21 and 2021/22) found a lower CTX detection rate using LC-MS in the 2020/21 and 2021/22 seasons in comparison with that of the 2014/15 season. No fish collected during 2020/21 and 2021/22 showed quantifiable levels of CTXs using LC-MS. Using ELISA testing for comparison, from the 2021/22 fishing season, 35 fish of 148 showed a CTX detection but below the level of quantification, and 10 fish had CTX levels that were quantifiable, with the highest at 0.012 µg/kg. Three flesh and liver samples were ≥ 0.01 µg/kg, which is the US FDA CTX guidance level. Fish caught in QLD were considerably more likely to contain CTXs than fish caught in NSW over the 3 fishing seasons. Using the sensitive ELISA method on all fish collected in the 2021/22 fishing season, no fish caught in NSW waters (0 of 32) were found to contain CTXs, whereas 35 of 116 fish (30%) from QLD were found to contain some CTXs, usually below the level of quantification. These CTX+ fish were collected from the vicinity of Fraser Island, Hervey Bay, Rockhampton, Wigton Islands and Coolum.

The lower CTX prevalence in Spanish Mackerel from 2020/21 and 2021/22 fishing seasons in comparison to CTX prevalence in 2014/15, as measured using LC-MS analysis, parallels reports of CP cases, which peaked in 2014-2016 in NSW and QLD, and have been lower in more recent years. Since 2019, 19 CP outbreaks in QLD caused by Spanish Mackerel were reported, and the CTX levels in 10 remaining meal samples of fish were measured using LC-MS by QLD Health. Of fish with CTXs tested, weight and length data were added to our dataset from project 2014-035 to determine the relationship between CTXs and Spanish Mackerel weight or length. With the addition of data, there remains no significant correlation between the prevalence of detectable CTX concentration using LC MS or ELISA and Spanish Mackerel length or weight.

In an analysis of the environmental, spatial or other correlates of CTXs in Spanish Mackerel and CP cases, an observation was made in relation to annual cyclone frequency and intensity and annual CP cases in QLD and xii NSW with a lag time of ~1 year, a relationship that has been previously noted in a study in the Pacific. This requires further analyses to determine its consistency.

From the comparison of CTX detection methods, results from the enzyme-linked immunosorbent assay (ELISA) were well correlated with those from either LC-MS or neuroblastoma cell-based assay (N2a) (r2 of 0.68 and 0.98, respectively). However, the ELISA and the N2a assay were more sensitive and able to detect the presence of lower level CTXs that were below the level of detection of the LC MS method. The ELISA test was found to be potentially viable for use at a central site with laboratory facilities. However, CTX extraction from fish tissue currently requires time frames of 6-12 h and complex laboratory equipment to complete. Therefore it is not currently suitable to be performed on site or rapidly. Further development of fish tissue extraction methods would be necessary to determine if ELISA or other more rapid methods are suitable for inclusion in the suite of provisions for CP risk management. Based on our new data, we recommend that current CP regulations remain in place. Recommendations are given for future approaches, including those relevant to public health, analytical advances, and additional environmental and fisheries studies.