The Role of Temperature and Heterotrophism in Determining Crude Oil Toxicity
Authors: Graham J.E.S. , Hutchinson T.C. 
Published: March 2019

Abstract
Crude oil spills are increasingly likely to occur from drilling, pumping and transportation activities as oil development proceeds at a rapid pace. These spills may occur over the wide range of climatic conditions which obtain in Canada. Little is known of oil toxicity at different temperatures; consequently, laboratory studies were made of the variability of the toxicity of aqueous extracts of a Norman Wells crude oil to freshwater algae over the temperature range 5°C to 35°C. Two unicellular green algae were studied: Chlamydomonas eugametos and Chlorella vulgaris. Their response (measured by cell numbers) varied with temperature and species. Whereas Chlamydomonas eugametos showed a general pattern of growth inhibition by oil at all temperatures with maximum inhibition at 25°C, Chlorella vulgaris showed general growth stimulation by oil with maximum stimulation at 25°C, this temperature was chosen for all further experimentation.
All experiments were done using unialgal cultures and sterile technique. Cells were grown in 50 ml of nutrient medium (BBM) in 125 ml Erlenmeyer flasks. Such flasks allow gas exchange and permit loss of volatile hydrocarbons. Aqueous extracts were made by slowly stirring 5% crude oil with the nutrient medium for six hours using a magnetic mixer. The extract was then allowed to sit for two to four hours before the lower fraction was drawn off for use. Experiments were carried out in controlled environment chambers (±2°C) with a twelve hour light-dark cycle. All further experiments used a similar methodology. (Note: Chlamydomonas eugametos experiments were carried out on a rotary shaker at 125 rpm.)
An attempt was made to determine the reason for the remarkable stimulation in growth of Chlorella vulgaris #29 at 25°C. This organism has been described in the literature as heterotrophic. Thus three reasons for stimulation seemed possible:
1. heterotrophic uptake of hydrocarbons directly from solution;
2. heterotrophic uptake of organic compounds formed or released by microbial breakdown of hydrocarbons (the aqueous extract of crude was not sterile); or
3. the use of CO2 released to solution by microbial respiration.
The original experiment was repeated in the dark at 20°C to determine if stimulation still occurred. It did not, since cells exposed to the aqueous extract decreased in numbers. However, after two weeks the cells were illuminated and even though experimental flasks started off with depleted populations, they outgrew the control cells within two weeks. This suggested that if stimulation was related to heterotrophism, it must, at least in this case, have been the unusual case of photoheterotrophism.
The reasons for this stimulation of growth are currently under investigation. Several methods are being employed to investigate the suspected heterotrophism. Experiments will be done to determine whether light energy is essential to the stimulation. Two varieties of Chlorella vulgaris, i.e. #29 and #260 are heterotrophic and autotrophic respectively, are to be used in experiments. Sterile aqueous extracts made by pressure ultrafiltration will be used. These experiments should determine whether algal growth stimulation is related to heterotrophism or whether microbial degradation of hydrocarbons is the real source of stimulation.
Although the toxicity of crude oil may be rapidly ameliorated by physical and/or biological phenomena, one must still be aware of the possibility of a large input of organic carbon causing extensive eutrophication. Thus both toxicity and eutrophication will cause a selection, in terms of survival, in a natural environment. It is evident that although an oil spill may not totally destroy an ecosystem, it will certainly alter its natural composition considerably.