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Please Click Here for a print version of the handling notes for Oysters.

Practical Notes on Oyster Embryo Bioassay

These notes can be used in addition to Environment Agency procedures1, to provide a few extra tips on undertaking a successful test.
Plan to strip oysters on day of arrival.  
Ideally oysters should be used not later than 24hours out of water, but tests can be performed successfully if oysters have either arrived a day or two late or if the test needs to be delayed provided the following procedures are undertaken: 

• If delivered late place oysters in cool sea water (10-15°C ) with some aeration for an hour to recover.  If they are placed in water at a temperature of 20°C or above there is a danger of oysters spawning.  If this is unavoidable then we suggest placing each oyster in separate 1 litre containers and keeping an eye on them  –
• If a test needs to be delayed it is possible to hold oysters for 2 to 3 days in cool (10-12°C) aerated or running seawater.  Otherwise the oysters can be held out of water for up to 24 hours in damp conditions at 6-10°C and then allowed to recover the next day as described above.

Selection of oysters.  Following Environment Agency procedures1 for obtaining and fertilising gametes, select the best oysters and gametes before pooling them.  Good oysters have a large area of creamy coloured gonad and are particularly good when the surface is veined. Gametes should ooze out easily when incised.  Under the microscope good eggs have uniform pear shapes which round up on contact with seawater. Good sperm becomes active on contact with sea water
Tissue debris in gametes: A 90-100 micron screen removes large tissue debris in the gametes, but if there is a lot of very small debris this should be removed by screening eggs on a 17 to 20 micron screen (small eggs and debris will fall through whilst eggs will collect on the screen)
Sperm Quality and quantity: Sperm can vary greatly in concentration and activity and it is sometimes difficult to know how much to add to fertilize eggs.  We suggest that eggs are examined 5 to 10 minutes after inoculation to check that there are about 3 to 10 sperm cells per egg, adding a bit more if there is any doubt. Sometimes sperm is inactive either because the oysters have got very cold in transit or they have been out of water too long. If this is the case a higher concentration of sperm than normal may be needed. 
30 minutes after fertilization Check eggs again.  If there are less than 80% showing first signs of cleavage or with polar bodies start the test again with fresh gametes There is no need to wait for 2 hours before doing so. Start the test again this time taking eggs from different oysters and/or different parts of the gonad and take extra care with the sperm.
A test failure with EA and WRC is for 40% and above abnormality.
The ICES protocol says 20% abnormal is acceptable and 40% not uncommon in controls
See our separate notes “Oyster embryo test: Troubleshooting”

REFERENCES

THAIN J.E. (1991) Biological effects of contaminants: the oyster (C. gigas) embryo bioassay In: Techniques in Mar.Env. Sci., ICES
Environment Agency The direct toxicity assessment of aqueous environmental samples using the oyster (Crassostrea gigas) embryo-larval development test (2007)

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