These plates were placed in a 12°C culture chamber overnight. Receptacles 
were then placed in sterile charcoal filtered seawater the next morning. Eggs 
and sperm were immediately released, and fertilization was observed within 15 
to 20 minutes. Zygotes were immediately pipetted into culture dishes (60 x 1 5 
mm plastic petri dishes, with 2 mm square grids — Falcon Plastics) while 
keeping the eggs suspended in seawater by stirring. 24 hours after dispensing 
into culture dishes, the toxicant was introduced by allowing the zygotes to 
settle on the bottom of the dish, the seawater removed, and replaced with 
seawater containing the toxicant at the test levels. In a few cases, the tips were 
pre-treated with the toxicant, and in these cases, the above sequence was 
suitably modified. 
The growth medium for the Fucus assays was, in all cases, sterile charcoal 
filtered seawater. The parameter measured for these experiments was the 
increase in length after 12 days of growth. 
Methods of procuring Laminaria spores were similar to those of Fucus 
gametes. Sporogenous plants were collected and then washed in deionized 
water to remove surface contaminants. Small pieces (2-3 cm square) of 
sporogenous tissue were placed into moist chambers overnight. These pieces 
were placed into sterile seawater the following morning, and spores were 
released in abundance. Spores were dispensed into culture dishes at 
concentrations sparse enough to allow counting and to prevent overcrowding, 
but dense enough (ca. 100-260 eggs) for good statistical data. In all cases, the 
culture medium was Provasoli’s Enriched Seawater (6). The parameter 
measured was the increase in diameter of the gametophyte after 21 days of 
growth. 
All assays were conducted at 400 ft-c of continuous cool white fluorescent 
light. Except for the first series of experiments to determine the optimal 
temperature salinity combinations for the assays, the temperature and salinity 
were 18°C and 30 o/oo for Fucus and either 12 or 18°C and 30 o/oo for 
Laminaria. For the various tests, the petroleum product (either No. 2 fuel oil, 
JP-4, JP-5, and Willamar crude) at concentrations ranging from 0-2000 ppm 
was equilibrated with seawater, proper dilutions made, and added to the 
cultures. The oil-seawater mixtures were analyzed by infrared 
spectrophotometry (Perkin-Elmer Model 621) to determine the amount of 
dissolved product causing toxicity. 
Observations and measurements of Fucus zygotes and Laminaria 
gametophytes were made with a Unitron inverted microscope (Model BMIC). 
Approximately 10-20 individuals were measured per dish. Four replicates were 
performed for each treatment. 
104 
