Bioassay: Use of Cryopreserved Larvae
Report No FR/D0019

J Widdows and P N Salkeld

Mar 1995


  1. One of the Department of the Environment's main concerns is the assessment and improvement in the environmental quality of marine and coastal waters. Recently, the Marine Pollution Monitoring Group included the oyster embryo test in the UK National Monitoring Plan as one of several techniques for monitoring the biological effects of pollution in the marine environment.

  2. The present oyster embryo test, however, suffers from a number of limitations, namely, the availability of mature oysters containing 'high quality' gametes throughout the year, the use of 'controls' with low survival rates, as well as the relatively low sensitivity of the test.

  3. Recently developed and patented techniques (Cell Systems Ltd) for the cryopreservation of bivalve larvae offer the potential of providing a uniform stock of high quality larvae which are available throughout the year, thus overcoming many of the limitations in the oyster embryo test.

  4. Therefore, the primary objectives of this study were: (i) To develop procedures for the use of cryopreserved larvae and the measurement of larval growth for the purposes of water quality and toxicity testing, and (ii) To compare the sensitivities of the 'early embryo survival' and 'growth of natural and cryopreserved larvae' in response to selected contaminants.

  5. As a result of this study the authors have demonstrated that the technique of cryopreservation of bivalve larvae has not been adequately developed and at present cannot be adopted as a routine method. They obtained a degree of recovery from -196C (i.e. larvae would swim for several days), but despite considerable additional research effort (this was not anticipated or built into this DoE contract) they were unable to obtain long-term survival or growth of cryopreserved larvae.

  6. Due to the lack of viable cryopreserved larvae over the period of the contract, the authors therefore focused on the other aspect of the project, namely to quantify changes in the sensitivity to selected toxicants during ontogenetic development.

  7. The responses of different developmental stages to Cu indicated a non-linear relationship between Cu concentration and ontogenetic development. The settlement stage showed a slight inhibition of settlement at low Cu concentrations (i.e. 10 and 30 g Cu l-1), whereas the embryo and early D larval stages showed lethal effects (LC50) between 30 and 100 g Cu l-1, and yet larval settlement was markedly enhanced at 100 g Cu -1.

  8. Fluoranthene, a toxic hydrocarbon with a relatively low volatility, was used to test the effects of an organic toxicant. Oyster embryo development was only inhibited by exposure to 180 g fluoranthene l-1 (maximum solubility), whereas larval growth rate was significantly reduced above 45 g fluoranthene l-1. However, there was no evidence of lethal effects on larvae at the highest concentration of 180 g l-1 over a period of 96 hours.

  9. The response to the organotin, tributyltin, showed that larval growth could detect significant effects at 0.1 g TBT l-1, and embryo survival was significantly lowered at 0.3 g TBT l-1. However, larval survival was not affected even after 96h exposure to 3 g TBT l-1.

  10. Oyster larval growth was able to detect and quantify differences in water quality at two field sites (Sutton Harbour and Newton Ferrers), but no significant differences were detected between surface microlayer and sub-surface samples, and no significant effects were detected using embryo survival as the biological endpoint.

  11. In conclusion, the results of this study suggest that the sub-lethal response of larval growth offers greater sensitivity and reproducibility compared with the embryo survival test. Consequently, the authors recommend that further effort should be de voted to the use of larval growth as a measure of water quality and the cryopreservation methodology should be further developed in order to provide a readily available stock of high quality larvae for water quality testing.

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