Development of a simple and rapid toxicity bioassay for paralytic shellfish toxins and ciguatoxins in seafoods  

Project Title: Development of a Simple and Rapid Toxicity Bioassay for Paralytic Shellfish Toxins and Ciguatoxins in Seafoods

Applicant: The Hong Kong University of Science & Technology

Total Approved Grant: $1,274,366 (Actual expenditure: $1,272,624.95)

Duration: 1.1.2000 to 31.12.2001

Status: Completed

Scope:
To develop, characterize and standardize the mouse neuroblastoma (MNB) assay for both paralytic shellfish toxins (PST) and ciguatoxins (CTX) to allow for a rapid, sensitive and inexpensive screening for these toxins in fish and shellfish in Hong Kong.

Summary of findings / outcome:

A standard and a rapid mouse neuroblastoma (MNB) assays and a high-performance-liquid chromatographic method were established for use in the monitoring of paralytic shellfish toxins (PSTs) in shellfish and other environmental samples. The C2 toxin and its derivatives were found to be prevalent in the local samples. The cell-based MNB assays, when used properly, may reduce the number of the mouse assays that are currently in use for PSTs. For PSTs, the four different methods gave fairly comparable analytical results. But the four methods cannot substitute for each other due to their differences in specificity and operational complexity. The MNB assays are hundreds of times more sensitive than the mouse assay and only require a small amount of toxin samples for each assay. But they are 10 to 50 times slower than the mouse assay and require high level of laboratory skills. They are not suited for field use. In the present project, techniques were also established for producing sufficient quantities of pure PSTs to be used as analytical standards and for other related studies. Our monitoring results using the methods developed revealed that the C2 toxin and its derivatives are toxins of high environmental health concern and warrant priority attention by the Hong Kong regulatory agencies.

We have successfully completed the scientific aspects of all the five tasks listed in the proposal. The MNB assays established and characterised in this project are useful as a screen tool to reduced the number of the mouse assays needed and to increase the sensitivity of the PST toxicity testing. However, the MNB assays are cell-based assays that can only be performed in a well-controlled laboratory setting and cannot be used in the field. Therefore, it was not feasible to formulate a monitoring strategy for contaminated shellfish based on this assay method as originally conceived in this project. The scope of monitoring work for the identification of environmentally significant PSTs in contaminated shellfish in Hong Kong using the developed analytical methods was done as permitted by the resources available in this project. An extra task not originally proposed was undertaken which added considerable value to the overall contribution of this project. Namely, we established in this project a novel culturing and purification procedure to produce mg quantities of pure C1 and C2 toxins and GTX2 and GTX3. These pure toxins are useful as analytical standards and for other R&D work. The biotechnological procedure developed is useful in large-scale production of marine biotoxins that are of high commercial values.

The objectives achieved-

  1. To develop, characterize and standardize the MNB assay for both PST and CTX with commercially available standards and to confirm its correlation with the mouse bioassay and HPLC analysis.

  2. Establishment of all assays with shellfish extracts and correlation of results of the MNB assay with those obtained for the mouse bioassay and HPLC.

  3. Identification of environmentally significant PSTs in contaminated shellfish in Hong Kong using the developed analytical methods.

In addition to the above objectives listed in our original proposal, we achieved the establishment of culture techniques of marine dinoflagellates for large-scale production of C2 toxin and its derivatives and purification techniques for obtaining mg quantities of the toxins for research and development uses. These toxins in pure forms are otherwise prohibitively expensive from commercial sources. The techniques developed in this project are useful in alleviating the problems of costly toxin supplies, and therefore will stimulate and facilitate the further research related to these biotoxins.