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 15 

 mm plastic petri dishes, with 2 mm square grids — Falcon Plastics) while 

 keeping the eggs suspended in seawater by stirring. Twenty four hours after dispens- 

 ing 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 1 2 days of growth. 



Methods of procuring Laminaria spores were similar to those of Fuais 

 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 



