Improved approaches to ecotoxicity testing of petroleum products

Crude oils produced in the North West shelf of Western Australia are highly volatile, a characteristic not shared by most of the Northern Hemisphere crude oils on which internationally accepted toxicity test protocols were developed. Because of this volatility and some other factors, the LC50 and EC50 values obtained from acute toxicity tests will be significantly affected by the changes of toxicant concentration in test solutions during the period of exposure. To address these issues all steps of a standard protocol for crude oil toxicity testing have been revised. A systematic study has been performed on factors which affect petroleum hydrocarbon solubilisation in aqueous systems during test solution preparations. The influence of mixing time, agitation energy and volume/interface ratio on a hydrocarbon concentration in a water-soluble fraction (WSF) was studied for heavy, medium and light crude oils. A study of the sensitivity of marine unicellular algae to WSF of crude oils was conducted with Isochrysis sp., Nannochloropsis-like sp. and Nitzchia closterium. Total concentrations of hydrocarbons dissolved in test solutions were estimated by UV-spectrometry and GC/FID chemical analyses. When the toxicant concentration decreased during the exposure period, the EC50 values derived from initial or final concentrations either underestimate or overestimate toxicity, respectively. Therefore, weighted average concentrations (WAC) calculated for the whole test period were recommended for expressing hydrocarbon concentrations in test solutions of crude oils. Toxicity indices calculated from WAC of total hydrocarbons for different crude oils can be compared regardless of the rates of hydrocarbon loss.     

Toxicity of Crude Oil to the Survival of the Fresh Water Fish Puntius sophore (HAM.)

The exposure of Puntius sophore to different concentrations of crude oil (200… 4000 ppm) revealed that 4000 ppm was acutely lethal to the fish and mortality did not occur at <500 ppm at 21±1 °C for up to 15 days. The increase in the opercular frequency during exposure to crude oil has been recorded almost parallel to toxicant concentrations. The objectionable odour of the crude oil and the formation of coagulated mucous film over the body and gills were the main factors causing mortality among the experimental fish. The 24, 48, 78 and 96-h TL 50 values have been 3275, 1750, 1650 and 1450 ppm. The corresponding regression coefficients b were –1.8056, –1.5849, –1.6037 and –1.6497.     

Inhibition of phytoplankton photosynthesis by the WSF of used lubricating oil

The photosynthetic rates of five phytoplankton species were inhibited following treatment with the water-soluble fractions (WSF) of used lubricating oil. Exhaust oil from an outboard motor inhibited photosynthesis in three out of the five species tested which indicates a species specific response in the case of this form of oil pollution. Water-soluble fractions from Qatar crude had no inhibitory effect on any of the species tested but had a stimulatory effect on the photosynthetic rate of one species. Both oxygen evolution and carbon assimilation were inhibited by the water-soluble fraction of used lubricating oil. In four out of five species, used lubricating oil WSF caused a net oxygen consumption during the light phase rather than a net oxygen release. These data indicate that used lubricating oil water-soluble fractions are especially toxic to phytoplankton.     

Impact of adsorbed petroleum hydrocarbons on marine organisms

Adsorptions of south Louisiana crude oil from seawater by clays, non-clay minerals and sediments were conducted in the laboratory. Effect of sediment-adsorbed and water dispersed crude oil on adult oysters were investigated in aquaria. Hydrocarbons in oyster tissues and surrounding water were identified by gas chromatography. Field specimens from an area of a new oil spill and an area five months after an oil spill were also analysed. Evidence of secondary chemical effect of aged oil in sediments on oyster mortality is presented. Oil coated asbestos surfaces severely reduced recruitment of sedentary larval organisms.     

Study on the fate of petroleum-derived polycyclic aromatic hydrocarbons (PAHs) and the effect of chemical dispersant using an enclosed ecosystem,mesocosm

Polycyclic Aromatic Hydrocarbons (PAHs) are one of the components found in oil and are of interest because some are toxic. We studied the environmental fate of PAHs and the effects of chemical dispersants using experimental 500 l mesocosm tanks that mimic natural ecosystems. The tanks were filled with seawater spiked with the water-soluble fraction of heavy residual oil. Water samples and settling particles in the tanks were collected periodically and 38 PAH compounds were analyzed by gas chromatography-mass spectrometry (GC-MS). Low molecular weight (LMW) PAHs with less than three benzene rings disappeared rapidly, mostly within 2 days. On the other hand, high molecular weight (HMW) PAHs with more than four benzene rings remained in the water column for a longer time, up to 9 days. Also, significant portions (10-94%) of HMW PAHs settled to the bottom and were caught in the sediment trap. The addition of chemical dispersant accelerated dissolution and biodegradation of PAHs, especially HMW PAHs. The dispersant amplified the amounts of PAHs found in the water column. The amplification was the greater for the more hydrophobic PAHs, with an enrichment factor of up to six times. The increased PAHs resulting from dispersant use overwhelmed the normal degradation and, as a result, higher concentrations of PAHs were observed in water column throughout the experimental period. We conclude that the addition of the dispersant could increase the concentration of water column PAHs and thus increase the exposure and potential toxicity for organisms in the natural environment. By making more hydrocarbon material available to the water column, the application of dispersant reduced the settling of PAHs. For the tank with dispersant, only 6% of chrysene initially introduced was detected in the sediment trap whereas 70% was found in the trap in the tank without dispersant.     

In situ impact of petrochemicals on the photosynthesis of the seagrass Zostera capricorni

We used photosynthetic activity (measured as chlorophyll a fluorescence) and photosynthetic pigment concentrations to assess the effect of pulsed exposures of aged crude oil (Champion Crude), dispersant (VDC) and an oil+dispersant mixture on the seagrass Zostera capricorni Aschers in laboratory and field experiments, using custom-made chambers. Samples were exposed for 10 h to 0.25% and 0.1% concentrations of aged crude oil and dispersant as well as mixtures of 0.25% oil+0.05% dispersant and 0.1% oil+0.02% dispersant. During this time and for the subsequent four day recovery period, the maximum and effective quantum yields of photosystem II (Fv/Fm and DeltaF/Fm' respectively) were measured. In the laboratory experiments, both values declined in response to oil exposure and remained low during the recovery period. Dispersant exposure caused a decline in both values during the recovery period, while the mixture of aged crude oil+dispersant had little impact on both quantum yields. In situ samples were less sensitive than laboratory samples, showing no photosynthetic impact due to dispersant and oil+dispersant mixture. Despite an initial decline in DeltaF/Fm', in situ oil-exposed samples recovered by the end of the experiment. Chlorophyll pigment analysis showed only limited ongoing impact in both laboratory and field situations. This study suggests that laboratory experiments may overestimate the ongoing impact of petrochemicals on seagrass whilst the dispersant VDC can reduce the impact of oil on seagrass photosynthesis.     

Effects of surface and sunken crude oil on the behaviour of a sea urchin

Studies have been made of the effects of exposure to various forms of crude oil on the righting behaviour of Paracentrotus lividus and its reactions towards the presence of oil. Prolongation of the righting response was recorded in animals exposed to contact with surface or sunken fresh crude oil or to their water soluble fractions. No such effect was recorded on exposure to weathered oils and results indicate that the more volatile components of crude oil were responsible for this effect. Paracentrotus showed no avoidance reaction to the presence of sunken oil in its vicinity. The likely ecological significance of these results is discussed.     

Oil source analysis of Upper Tertiary Shawan Formation in Chepaizi area,in the northwest margin of Junggar basin

Well che89, located in the Chepaizi area in the northwest margin of Junggar basin, acquires high production industrial oil flow, which is an important breakthrough in the exploration of the south foreland slope area of Junggar basin. The Chepaizi area is near two hydrocarbon generation depressions of Sikeshu and Shawan, which have sets of hydrocarbon source rock of Carboniferous to Jurassic as well as Upper Tertiary. Geological and geochemical parameters are proper for the accumulation of mixed source crude oil. Carbon isotope, group composition and biomarkers of crude oil in Upper Tertiary of well Che89 show that the features of crude oil in Upper Tertiary Shawan Formation are between that of Permian and Jurassic, some of them are similar to these two, and some are of difference, they should be the mixed source of Permian and Jurassic. Geochemical analysis and geological study show that sand extract of Lower Tertiary Wulunguhe Formation has the same source as the crude oil and sand extract of Upper Tertiary Shawan Formation, but they are not charged in the same period. Oil/gas of Wulunguhe Formation is charged before Upper Tertiary sedimentation, and suffered serious biodegradation and oxidation and rinsing, which provide a proof in another aspect that the crude oil of Upper Tertiary Shawan Formation of well Che89 is not from hydrocarbon source rock of Lower Tertiary.     

Histochemical Observation on Crude Oil Poisoning in the Respiratory Epithelium of Puntius sophore

The sensitivity of respiratory mucus cells to the aqueous extracts of 200… 2000 ppm of crude oil has been determined in Puntius sophore using histochemical techniques. The results indicate the mucus cells to be highly susceptible to the crude oil poisoning in both lethal and sublethal concentrations. Sublethal extracts induce excessive mucus production and form a protective covering over branchial epithelia. Lethal extracts, however, reduce the number and size of the mucus cells causing scanty mucus secretion in short-term exposure (≦4 h), but degenerate the mucus cells when the exposure period is prolonged (≧12 h).     

The comparative effects of oil dispersants and oil/dispersant conjugates on germination of the marine macroalga Phyllospora comosa (Fucales: Phaeophyta)

Germination inhibition of the marine macrophyte Phyllospora comosa was utilized as a sub-lethal end-point to assess and compare the effects of four oil dispersants and dispersed diesel fuel and crude oil combinations. Inhibition of germination by the water-soluble fraction of diesel fuel increased following the addition of each of the dispersants; the nominal 48-h EC₅₀ concentration of diesel fuel declined from 6800 to approximately 400 μl l⁻¹ nominal for each dispersed combination. This contrasted with crude oil, where the addition of two dispersants resulted in an enhanced germination rate and an increase in nominal EC₅₀ concentrations from 130 μl l⁻¹ for the undispersed crude to 4000 and 2500 μl l⁻¹. The results indicate that, while germination inhibition of P. comosa may be enhanced by the chemical dispersal of oil, the response varies with type of both oil and oil dispersant.     

Dispersant Use and a Bioremediation Strategy as Alternate Means of Reducing Impacts of Large Oil Spills on Mangroves: The Gladstone Field Trials

Over a three-year period (1995–1998), we studied short-term effects of dispersant use and a bioremediation strategy in two consecutive field trials in sub-tropical Australian mangroves. In each case, weathered oil was applied, and a large spill simulated, in mature Rhizophora stylosa trees around 4–9 m tall. In the first trial, we used Gippsland light crude oil with or without dispersant, Corexit 9527. In the second, a bioremediation strategy followed application of Gippsland oil or Bunker C fuel oil. Bioremediation involved forced aeration with supplemental application of nutrients. Dispersant use had an overall positive benefit shown as reduced tree mortality. By contrast, there was no apparent reduction in mortality of trees with bioremediation. However, one year after oiling, leaf densities of surviving trees were greater in bioremediation plots than in controls, and less in oil-only plots. These and other results have been incorporated into spill response management strategies in Australia.     

Oil source analysis of Upper Tertiary Shawan Formation in Chepaizi area,in the northwest margin of Junggar basin

Well che89, located in the Chepaizi area in the northwest margin of Junggar basin, acquires high production industrial oil flow, which is an important breakthrough in the exploration of the south foreland slope area of Junggar basin. The Chepaizi area is near two hydrocarbon generation depressions of Sikeshu and Shawan, which have sets of hydrocarbon source rock of Carboniferous to Jurassic as well as Upper Tertiary. Geological and geochemical parameters are proper for the accumulation of mixed source crude oil. Carbon isotope, group composition and biomarkers of crude oil in Upper Tertiary of well Che89 show that the features of crude oil in Upper Tertiary Shawan Formation are between that of Permian and Jurassic, some of them are similar to these two, and some are of difference, they should be the mixed source of Permian and Jurassic. Geochemical analysis and geological study show that sand extract of Lower Tertiary Wulunguhe Formation has the same source as the crude oil and sand extract of Upper Tertiary Shawan Formation, but they are not charged in the same period. Oil/gas of Wulunguhe Formation is charged before Upper Tertiary sedimentation, and suffered serious biodegradation and oxidation and rinsing, which provide a proof in another aspect that the crude oil of Upper Tertiary Shawan Formation of well Che89 is not from hydrocarbon source rock of Lower Tertiary.

     

Drilling mud-evoked hydranth shedding in the hydroid Tubularia crocea

At 100 000 ppm, the suspended particulate and liquid phases of a reference drilling mud and a used production mud significantly increase the hydranth shedding proportion in the hydroid coelenterate Tubularia crocea after 48 h. At 10 000 ppm only the liquid phase of the synthetic drilling mud significantly increased hydranth shedding. At 1000 ppm the 3% iron and calcium forms of the five lignosulphonates tested were found to be toxic. Other organic constituents of drilling muds found to be toxic included, tannic acid (100 ppm), paraformaldehyde (1 ppb), and water-soluble fractions of crude oil and No. 2 fuel oil (approximately 20 ppm). Mercury, cadmium, copper, cobalt, zinc and nickel were found toxic at 100 ppm. Exposure to 0.1−0.001 ppm mercury, 1% WSF of crude oil, and 10 ppm cadmium resulted in a significant decrease in the hydranth shedding proportion. This reduction in hydranth shedding proportion may reflect hormesis, a stimulation of growth by low-level exposure to a pollutant, but cannot be conclusively demonstrated until growth measurements are made.     

Some Haematological Effects of Crude Oil on Freshwater Catfish,Heteropneustes fossilis (BLOCH)

The exposure of catfish, Heteropneustes fossilis to different concentrations (200, 500, 700, 1000, 1500, 2000 ppm) of crude oil extract for a varying period (for up to 48 h in lethal extract and 360 h in sublethal one) resulted into a number of haematological changes. All the parameters taken here were found to have been increased except the haemoglobin level which fell down to 14.3 % in an acute lethal concentration (2000 ppm) after 48 h of exposure. The haematocrit value increased significantly (12 %) in 2000 ppm. The blood sugar level showed hyperglycemia in all the concentrations. The increase in ascorbic acid (28 %) was pronounced in higher concentrations. Changes occurring in different parameters seemed to be reversible as all the parameters returned to their normal levels after returning the fish to normal media, except the nuclear swelling which did not resume the normal functioning even after a prolonged treatment (1 month or more) in the recovery jar. The haematological effects shown by the crude oil resembled partly those kept in severe hypoxic conditions and partly to the fishes poisoned by heavy metals (Cu and Zn). On the basis of results obtained in this investigation, crude oil may be categorised as a complex toxic agent.     

Oil spill clean-up: The effect of three dispersants on three subtropical/tropical seagrasses

Three seagrasses found throughout the Greater Caribbean tropical/subtropical region as major critical habitat organisms were tested in the laboratory for toxicity limits to three dispersants commonly stockpiled in the region. At concentrations in the recommended dosage level, that is, below 1 ml dispersant with 10 ml oil in 100 000 ml seawater, even for 100 h no large mortality occurred (15–18 barrels per acre as calculated by Exxon, 1985). At an order of magnitude higher, especially for longer time periods, the more sensitive seagrasses Syringodium filiforme and then Halodule wrightii succumbed. The dispersants had widely differing effects, with Corexit 9527 and Arcochem D609 having far less toxic effect than Conco K(K) at the same exposure time and concentration. There was comparatively little difference between effects of oils (Louisiana crude versus Murban). Types and brands of dispersants should be referred to specifically in oil spill contingency plans since such widely varying ecological toxicity occurs among various dispersants. Use of the word ‘dispersant’ as a policy tool should be used with caution, realizing that dispersants vary widely in toxicity effects. Further testing of seagrasses in other ocean basins and those dispersants to be used there is highly recommended.     

Effects of sheens associated with offshore oil and gas development on the feather microstructure of pelagic seabirds

Operational discharges of hydrocarbons from maritime activities can have major cumulative impacts on marine ecosystems. Small quantities of oil (i.e., 10 ml) results in often lethally reduced thermoregulation in seabirds. Thin sheens of oil and drilling fluids form around offshore petroleum production structures from currently permissible operational discharges of hydrocarbons. Methodology was developed to measure feather microstructure impacts (amalgamation index or AI) associated with sheen exposure. We collected feather samples from two common North Atlantic species of seabirds; Common Murres (Uria aalge) and Dovekies (Alle alle). Impacts were compared after feather exposure to crude oil and synthetic lubricant sheens of varying thicknesses. Feather weight and microstructure changed significantly for both species after exposure to thin sheens of crude oil and synthetic drilling fluids. Thus, seabirds may be impacted by thin sheens forming around offshore petroleum production facilities from discharged produced water containing currently admissible concentrations of hydrocarbons.     

Impaired osmoregulation in the shrimp Palaemon adspersus exposed to crude oil extract

Palaemon adspersus is a hyper- and hypo-osmoregulating shallow-water shrimp. For a maximum period of 34 days P. adspersus were exposed to 20, 70, 100 and 200 ppb WSF of North Sea crude oil. The osmotic concentration of the haemolymph was measured by freezing point depression once a week. The ability to maintain hyper-osmolality was significantly decreased (p<0.05) after one, two and three weeks exposure to 200, 70 and 100 ppb, respectively. No effect was observed in 20 ppb. After an accidental exposure of shrimps to 24 h acute oil pollution, they were placed in pure sea water. The ability to maintain hyper-osmolality was reduced one week after the accidental oil exposure, but was regained within three weeks.     

Evidence for reduced post-spill recovery by the halophyte Sporobolus iocladus (Nees ex Trin.) Nees in oil-contaminated sediments

The germination behavior of Sporobolus iocladus seeds including germination percentage, accumulated germination percentage, the average incubation period to germination and germination velocity was studied under laboratory conditions. Treatments included six salinity regimes (0, 70, 140, 210, 280 and 350 mM NaCI) and three sources of oil hydrocarbons; Light Arabian Crude, polynuclear aromatic hydrocarbons (PAHs) including diaromatic or triaromatic hydrocarbons (in crude oil equivalent concentrations, COEC). The average incubation period needed for seeds to germinate was significantly longer for seeds germinated in 350 mM NaCl (6 +/- 1.16 days) compared with the control (4 +/- 00 days). The accumulated germination percentage gradually decreased with increasing salinity (control: 90 +/- 10, while 350 mM NaCl: 63 +/- 8.8). Oil hydrocarbons significantly affected all germination parameters of S. iocladus seeds regardless of salinity levels. COEC of di- and triaromatic hydrocarbons suppressed seed germination more than crude oil. Seeds exposed to diaromatic hydrocarbons failed to germinate. Hydrocarbon's salinity interaction significantly reduced the number of germinated S. iocladus seeds. It is concluded that hydrocarbon pollutants adversely affect S. iocladus through reducing germination. It is also suggested that the toxic effect of hydrocarbons on seeds is not solely mediated through their interaction with salinity. The ecological implications of these findings are discussed in relation to other studies on the post-spill recovery of halophytes.     

Effects of water-soluble fractions of crude oil and dispersants on nitrate generation by sandy beach microfauna

Effects on sandy beach microfauna of soluble pollutants, such as might be associated with an oil spill, were investigated in terms of nitrate generation. Nitrate generation by the microfauna in small sand columns in the laboratory was severely inhibited by water-soluble fractions of crude oil, dispersant and oil/dispersant mixtures in order of increasing effects. Short-term effects of such pollutants on nutrient regeneration by exposed sandy beaches are discussed.     

Development of a laboratory exposure system using marine fish to carry out realistic effect studies with produced water discharged from offshore oil production

A biotest system for environmentally realistic exposure of fish to produced water (PW) was developed and tested. Authentic PW was collected at an oil production platform in the North Sea and preserved by freezing in multiple aliquots a 25 L. After transport to the test laboratory onshore, daily PW aliquots were thawed, homogenised and administered to the test fish, Atlantic cod (Gadus morhua), in two diluted exposure concentrations, 0.1% and 0.5%, during a 15 d period, using a continuous flow-through exposure setup. Positive control groups were exposed to two crude oil treatments for comparison. Chemical analyses showed that alkylphenol (AP) and PAH concentrations in PW exposure waters were very low. Observations of significantly increased AP and PAH metabolite levels in PW exposed fish demonstrated the suitability of the biotest system for its use in biological exposure/effect studies of PW, and it also demonstrated the sensitivity of bile metabolites as PW exposure markers in fish. The relevance of the biotest system for PW effect studies and for validating modelled environmental risk estimates of PW dischargers from offshore oil production is discussed.