Following
spawning, the egg and sperm bundles of two isolated
colonies were collected separately by gentle suction into
200 ml containers. The buoyant nature of the eggs
allowed separation into individual containers for the
fertilization experiment. The fertilization inhibition
experiments were carried out according to the method
of Negri and Heyward (2000). 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.