Bioassay: Use of Cryopreserved Larvae
Report No FR/D0019
J Widdows and P N Salkeld
- 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.
- 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.
- 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.
- 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.
- 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 -196°C (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.
- 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.
- 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 µg Cu l-1),
whereas the embryo and early D larval stages showed lethal effects
(LC50) between 30 and 100 µg Cu l-1, and yet larval settlement was
markedly enhanced at 100 µg Cu -1.
- 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 µg
fluoranthene l-1 (maximum solubility), whereas larval growth rate
was significantly reduced above 45 µg fluoranthene l-1. However,
there was no evidence of lethal effects on larvae at the highest
concentration of 180 µg l-1 over a period of 96 hours.
- The response to the organotin, tributyltin, showed that larval
growth could detect significant effects at 0.1 µg TBT l-1, and
embryo survival was significantly lowered at 0.3 µg TBT l-1.
However, larval survival was not affected even after 96h exposure to
3 µg TBT l-1.
- 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
- 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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