Annual report, NSF grant no. GX37345, February 15, 1973 to February 14, 1974 : marine petroleum pollution : biological effects and chemical characterization




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The goal of the chemical section of this project is to characterize a few of the more toxic agents found in several representative test oils. Our experimental design is to use several physical and chemical methods to fractionate test oils while following toxicity by the bioassays described in the biological sections of this report. Accomplishment of this goal may demonstrate that a few key compounds are responsible for much petroleum toxicity. The concentration (and inferred toxicity) of a few specific compounds could probably be measured more accurately in the environment than the concentration (and inferred toxicity) of 'oil.'
Exploratory studies were made of the possible effects of petroleum oils on marine animals. Materials tested were: No. 2 fuel oil, Kuwait crude, Southern Louisiana crude, Bunker C, ship diesel fuel, five fractions of No. 2 fuel oil, biphenyl, naphthalene, methyl naphthalene, dimethyl naphthalene, durene and fluorene. Oil stock was made up in sea water or Ringer solutions, 1:8; single compounds, 15 mg %. Animals and tissues used for assay were eggs and sperm of sand dollars Melitta quinquesperforata; eggs and larvae of littoral barnacles Chthamalus fragilis and Balanus amphitrite niveus; larvae of crabs, viz. spider crab Libinia dubia, striped hermit crab Menippe mercenaria; porcelain crabs Petrolisthes armatus; sea catfish Arius felis; isolated spiral valve and cornea of stingarees Dasyatis sabina; and isolated gills of pinfish Lagodon rhomboides. Development of eggs of the barnacle Chthamalus fragilis in vitro was affected adversely by fuel oil at levels of 20% or more. The egg case seemingly afforded the embryos some protection against oil, late embryos hatched at concentrations of 50% oil, but larvae were quickly killed. Phototactic responses of barnacle larvae (Balanus amphitrite) were weaked and abolished in fuel oil and the single compounds listed above. Concentrations affecting half the larvae were; oil, 10% of stock solution; naphthalene, 20%; methyl naphthalene, 50%; dimethyl naphthalene, 10%. Hermit crab zoeae survived and developed in 2% fuel oil (11 days). About half the larvae were killed in 8% oil. Spider crab larvae developed to young crabs (10 days) in 2% oil; development was somewhat retarded in 4%, and greatly retarded in 10% oil. Survival of stone crab zoeae (5 days) was reduced in oil (4 to 50%), the mortality increasing with the concentration and duration of exposure. Catfish sought to escape from fuel oil (levels of 190 ppm or greater). Fish were killed by oil; half mortality level at 48 h was 140 ppm. Fins and gills were damaged, feeding responses deteriorated, food was regurgitated. The heart rate was slowed temporarily following addition of oil, 100 ppm. Oxygen consumption of porcelain crabs, gills of pinfish and spiral valve of stingarees was not depressed by oils (2-3 h). But it was reduced in the cornea of the stingaree. The results are related to findings reported in the literature dealing with oil pollution.
Sea water when equilibrated with a sample of #2 fuel oil becomes toxic in varying degrees to growth of representative types of microalgae, two blue-greens, a diatom, two greens, and a dinoflagellate. For a sensitive organism such as Chlorella autotrophica, strain 580, 2ml of sea water equilibrated with oil (15μg of organics ml⁻¹) in 20 ml of growth medium is lethal, or roughly in the range of 15-150 ppb if the toxic material(s) constitute 1-10% of the sample. This fuel oil-equilibrated sea water also immediately stops photosynthesis in organism 580. For the other microalgae tested similar effects on growth and photosynthesis were found but required higher concentrations of the oil-equilibrated sea water. Water solubles from Kuwait or Southern Louisiana crudes were not toxic; however, growth experiments in open or closed growth systems showed that organism 580 would not grow above 5μl of Southern Louisiana/25 ml of medium, or 10μl of Kuwait/25 ml of medium (oil in direct contact with algae). With both the sea water equilibrated with fuel oil and the crudes, the toxic activity is mainly localized in higher boiling fractions derived from distillation cuts from these materials. To date, it is clear, and not surprising, that water solubles and whole oils were toxic to the microalgae. We plan to extend this survey work to more petroleum samples. In the case of the #2 fuel oil sample we have begun attempts at chemical separation which together with bioassays may give us information on the amount and types of toxic material(s) present.