Fig. 2. Drawing ofPyrocvi/is/H/iH/ii cells; the long axis ib> approximately 100 

 microns. 



preparing dilution water. A 500-ml sample of the sediment was 

 poured into a 2,000-ml flask and seawater was added to bring 

 the final volume to 1 1. This suspension was stirred for 5 min 

 with a magnetic stirbar and the suspension allowed to settle for 

 1 h at 25°C. The liquid phase, which is the suspended 

 particulate phase, was then poured into another 2,000-ml flask 

 and stirred for 5 min; then the pH and dissolved oxygen (DO) 

 were recorded. The pH was adjusted to 7.8, and if the DO was 

 below 4.9 parts per million (ppm), the phase was aerated for 

 5 min. At that point, pH and DO were recorded for 20 min at 

 5-min intervals. This reaeration continued until DO was 

 stabilized above 4.9 ppm. This test medium was used for 

 toxicity testing (Federal Register, 1985). 



Artemia salina Toxicity Test 



The A. salina toxicity test was modified from several 

 sources ( Peltier & Weber, 1985; Vanhaecke&Persoone, 1984; 

 Persoone & Castrisi-Catharios, 1989; Persoone et al., 1989) 

 and ARTOXKIT M (Persoone, State University of Ghent, 

 Belgium). The following is a brief description of the procedure 

 used. 



Artemia salina cysts were obtained from Aquarium 

 Products (Glen Bumie. MD), with its cyst origin identified as 

 Columbia. Filtered ( 1 |a-Whatman) 3.8% seawater was used 

 for hatching the Artemia cysts and preparing the dilution water 

 (Persoone (feCastritsi-Catharios, 1989). Hatching was initiated 

 48 h before the start of the toxicity test. A 2,000-ml separator/ 

 funnel was used as an incubation chamber where 15 to 20 ml 

 of Artemia cysts were added and mixed vigorously using an air 

 stream for 24 h at 27°C under continuous illumination. Upon 

 hatching, the nauplii were allowed to settle to the bottom of the 

 separatory vessel and drained into a 250-ml beaker containing 

 fresh dilution water. At the end of the 24-h incubation period, 

 the larvae are at instar 1 of their life cycle (Fig. 1 ). 



The inslar I nauplii were incubated for another 24 h, in 

 continuous light, at 27°C. Their positively phototactic response 

 permits them to be concentrated near the vicinity of a light 

 beam and allows for easier pipetting ( Peltier & Weber, 1 985 ). 

 At this point, and for the duration of the exposure to the test 



medium, the larvae were at instar II-III stage (Fig. 1). The 

 larvae were not fed during the entire procedure and no mortality 

 was observed due to starvation. 



Two multiwell plates each consisting of 24 individual 

 wells (3 ml) were used to test the elutriate produced from the 

 processing of each sediment sample. Two replicates for each 

 sediment sample were produced. In all, forty-four wells 

 (20 experimental and 4 seawater controls) were used and 

 positions within the multiwell plates were randomly assigned. 

 One ml of the test elutriate was added to a well and 10 Artemia 

 nauplii were added by micropipetting. The wells were then 

 placed for 24 h in an incubator without light at 27°C. After the 

 incubation period, each well was examined under a dissecting 

 microscope using a xlO magnification and the live Artemia 

 were counted. Mortality was determined if a larva was not 

 observed moving for 10 seconds (Vanhaecke & Persoone, 

 1984). If any of the sediment elutriate samples showed any 

 signs of mortality, a LC50 was calculated (Peltier & Weber, 

 1985). 



Pyrocystis lunula Toxicity' Test 



Pyrocystis lunula, a marine dinoflagellate, was maintained 

 in f/2 medium, its composition given in Table 1 (Guillard & 

 Ryther, 1962). Cultures of P. lunula were maintained at 20°C 

 and illuminated with cool white fluorescent lamps shaded to a 

 light intensity of 17 u-einsteins/cm-. The illumination cycle 

 was 1 2 h light and 1 2 h dark. 



TABLE 1 



Composition of f/2 Medium. 



Constituent 



Concentration 



NaNO, 



NaH,P04«H,0 

 Fe sequestrene' 

 Na.SiOj'QHp 



Vitamins; 



Thiamine«HCL 

 Biotin 



Trace Metals: 



CuS0j'5H,0 

 ZnS0/7H_,0 • 

 CoCL,'6H,0 

 MnCL,'4H,0 

 Na_,Mo04«2H,0 



Seawater* 



150 nig 



10 mg 



10 mg (1.3 mg Fe) 



30-60 mg (3-6 mg Si) 



0.2mg 

 0.001 mg 

 0.001 mg 



0.0196 mg (0.005 mgCu) 

 0.044 mg (0.01 mgZn) 

 0.020 mg(0.00.'i mgCo) 

 0.360 mg (0.1 mg Mn) 

 0.0126 mg (0.005 mg Mo) 



To 1 liter 



The medium is modified by the omission of silicate and the 

 addition of TRIS buffer to increase the final pH to 7.6. 



■ Sodium iron salt of ethylene dinilriloletraacetic acid (EDTA). 

 " Artificial seawater is prepared from the formula of Lyman and 

 Fleininy (1940). 



374 



