The gametes were
exposed to varying concentrations of each of the WAF
types in sterile 6-well polystyrene tissue culture plates
(NuncTM, Denmark). Each well contained approximately
100 eggs, 106 sperm and various doses of WAF
made up to 10 ml with 0.2 mm filtered seawater. Fertilization
was stopped after 4 h by the addition of 400 ml
fixative (10 g ml1 sodium b-glycerophosphate, 4%
formaldehyde buffered at pH 7) which maintained cell/
embryo integrity. Samples of sperm were also fixed for
enumeration using a haemocytometer (Improved Neubauer).
The proportions of fertilized eggs were counted
using a dissecting microscope.