Toxicity of a fuel oil to the eggs of parhyale hawaiensis and amphithoe valida (amphipoda)

Experiments were carried out on the toxicity of a fuel oil to developing embryos and juveniles of two marine amphipods. Parhyale hawaiensis eggs were able to develop from early germinal disc stage (early eggs) to juvenile form in concentrations of 10 (2 ppm) up to 40% (8 ppm) WSF, but deleterious effects were evident in hatching success of eggs and survival of juveniles at concentrations ≥ 10% WSF. The juveniles that hatched from those eggs in higher WSFs had greater mortality than the juveniles that hatched from the eggs exposed to lower WSFs. Survival of juveniles from late eggs (cephalic region differentiated) was also greater than those from early eggs. Amphithoe valida eggs were more sensitive to the WSF of fuel oil than the eggs of P. hawaiensis; < 10% of the test eggs hatched in the 4 ppm WSF compared with 66% of P. hawaiensis eggs at the same concentration. The toxicity of fuel oil to the two amphipod eggs is therefore dependent on the concentration and duration of exposure on the one hand, and on the developmental stages and probably the nature of the egg case on the other.     

Effect of oil and dispersants on the growth of Mussels

Mussels (Mytilus edulis L.) were exposed to North Sea crude oil, microencapsulated oil and dispersants, singly and in combination, and growth rates measured at 24–48 h intervals.Exposure to microencapsulated pure oil (2·0–2·1 mg litre⁻¹) and to microencapsulated mixtures of oil (2·2−2·5 mg litre^-1+5 % of the different dispersants (FINASOL OSR 5, COREXIT 9527, DISPOLENE 36 S) gave approximately the same reduction in growth rate (80–90%) within 170h.Oil chemically dispersed with DISPOLENE 36 S and a pure oil mechanically dispersed in water were significantly less toxic. In high concentrations (2 mg litre⁻¹) all disperants are toxic, DISPOLENE 36 S ssignificantly more than the others.Mussels exposed for 170 h to microencapsulated oil and to microencapsulated oil dispersant mixtures recovered to control growth within 300 h in clean seawater, while in those given pure oil-in-water suspension, the recovery was slower.It is concluded that the toxicity of oil is mainly related to size and concentration of oil particles, while the effect of 5% dispersants added is negligible.     

Effects of dietary and water-borne oil exposure on larval pacific herring (Clupea harengus pallasi)

Pacific herring (Clupea harengus pallasi) larvae were exposed either directly to the water-soluble fraction (WSF) of crude oil or indirectly via oil-contaminated prey (OCP) (Artemia salina) for <- 28 days, to determine the relative effects of diet and water as routes of contamination. Larvae were affected rapidly by 0·9 ppm WSF, which caused high larval mortality, reduced swimming ability and rapid reductions in feeding rates. Larval length was significantly reduced by 0·7 ppm WSF in 7 days and by 0·3 ppm WSF within 14 days. The WSF exposure also caused similar reductions in larval weights. Highly contaminated prey (6 ppm prey exposure) caused significant mortality, but surviving larvae appeared robust. The OCP did not affect swimming, feeding, or growth. Exposure of larvae was not significantly extended by OCP, which rapidly depurated WSF in clean water (98% in 1 day). Therefore, OCP is probably not an important source of low molecular weight petroleum contamination to larval fish in the marine environment.     

Sensitivity of three microalgae to crude oils and fuel oils

Four crude oils and five fuel oils have been tested for toxicity with three microalgae—a blue-green, a green and a diatom. The oils were absorbed on filter paper pads and the pads submerged in the growth medium. The crude oils were less inhibitory than equal amounts of fuel oils. Despite initial growth lags, the crude oils allowed growth at 30 μl/20 ml of culture medium (10⁵ cells/ml) while fuel oils were lethal at 10 μl/20 ml. The toxicity patterns of two of the whole fuel oils were different from that seen with their water soluble fractions (WSF); for example, the Baton Rouge fuel oil sample was very toxic to growth of the three test organisms whereas its WSF was relatively innocuous. Photosynthesis of a sensitive organism Chlorella autotrophica, strain 580 (10⁷ cells/ml), was only temporarily depressed by the crude oils (30 μl/20 ml). Four of the fuel oils inhibited photosynthesis, O₂ output decreasing to zero without recovery. When the fuel oils were heated in a stream of helium they were detoxified. Chemical analyses of two of the toxic fuel oils before and after heating were compared with analyses of the Montana sample, a largely non-toxic fuel oil, in an effort to determine what types of compounds might be involved. Classes of aromatic compounds which were not accountable for the toxicity observed here include naphthalene, methylnaphthalenes, dibenzothiophenes, phenanthrenes and compounds with volatilities greater than methylnaphthalenes. Paraffinic and asphaltic fractions of fuel oil were also non-toxic. The accumulated chemical data suggest that the toxicity of whole fuel oils is due to the less water soluble types of compounds in the higher boiling aromatic fraction.     

The effects of a water-soluble petroleum fraction on the neuroid electrical activity of the hydroid coelenterate Tubularia crocea

Several properties of the excitable epithelium of the hydroid coelenterate, Tubularia crocea, are affected by a water-soluble fraction (WSF) of Platform Holly, Monterey Formation, crude oil (100% WSF= 20 ppm total hydrocarbons). In a flowing seawater system, 0·5 ml pulses of 1 WSF cause a significant increase in the epithelial pulse activity, while a 10·min exposure to 0·1% WSF produces a significant increase in the epithelial pulse frequency. Electrophysiological responses to the volatile and non-volatile subfractions are equal, but significantly lower than those to the whole WSF. Behaviorally, the 100% WSF elicits a more vigorous proximal tentacle closure than either its volatile or non-volatile subfraction. However, the closure response to the volatile subfraction exceeds that for the non-volatile component.Of the two epithelial neuroid pulse systems, the hydranth pulse (HP) system is affected to a greater extent than the neck pulse (NP) system. During 10 minutes' treatment with 100% WSF the amplitude of the neck pulses and hydranth pulses declined by 50% and 80%, respectively. The maximum ³H-toluene uptake from 100% WSF also occurs within this period, and sodium and calcium concentrations fluctuate rapidly. These results suggest that a rapid bioaccumulation of hydrocarbons by T. crocea alters membrane permeability, upsetting critical ion gradients which, in turn, alter the firing frequency of the epithelial conducting systems. This study demonstrates that concentrations of WSF approaching environmentally realistic levels (in areas of chronic pollution) alter the epithelial conducting systems that co-ordinate important elements of behavior in Tubularia. This system provides a sensitive neuroid-behavioral assay for aquatic contaminants.     

The combined effect of salinity,temperature and oil on the growth pattern of embryos of the killifish, Fundulus heteroclitus Walbaum

Embryos of the estuarine teleost, Fundulus heteroclitus, were exposed during development to different concentrations of the water-soluble fractions (WSF) of a No. 2 fuel oil under various temperature-salinity combinations. Temperatures and salinities were, respectively: 20, 25 or 30°C and 10, 20 or 30%S. The WSF were extracted from No. 2 fuel oil daily and diluted to concentrations of 15, 20 or 25% of the original mixture. Controls were exposed to seawater without WSF at each temperature-salinity combination.The length of eleutheroembryos was markedly decreased by exposure to WSF. Temperature exerted an effect on growth at low WSF concentrations (0 and 15% WSF) but not at higher ones. The two highest WSF concentrations had a predominant effect on growth that was not further modified by physical factor influences. Conversely, as the WSF exposure increased, the yolk diameter increased, indicating a decreased utilisation of yolk material during development. The mean number of vertebrae in the eleutheroembryos decreased at high WSF concentrations and at high temperatures. Morphological abnormalities consisted predominantly of spinal deformations (scoliosis and lordosis). The data indicate that the survivors of chemical toxicity during embryo development exhibit sublethal responses which would reduce their survival potential in later life.     

Effects of oil exposure on oxygen consumption of cod eggs and larvae

The oxygen consumption reflects the aerobic energy production of an animal. For fish eggs and larvae the normal oxygen consumption represents the optimal conditions for growth and development. Under the stress of environmental pollution gross deviation in oxygen uptake rate of the developing fish embryo may reflect metabolic disturbances and impede the development. Since the eggs and larvae of cod (Gadus morhua L.) are likely to be exposed to oil pollution from offshore installations in the North Sea we decided to investigate the effects of the water-soluble fraction (WSF) of North Sea crude oil on the oxygen consumption of cod eggs and larvae. The results showed that oxygen consumption of the larvae at the time of final yolk absorption (5–7 days post hatching at 5°C) is strongly suppressed by oil exposure at concentrations down to 50 ppb. No effect on the oxygen uptake, however, was found during the egg stage.     

Total oxyradical scavenging capacity and cell membrane stability of haemocytes of the Arctic scallop, Chlamys islandicus, following benzo(a)pyrene exposure

Industrial activities, notably oil and gas industries, are expanding in the Arctic. Most of the biomarkers were developed using temperate organisms living at temperatures above 10 °C. Little is known about the biomarker responses of organisms living between −1.88 and 5 °C. Therefore, assessment of the toxicity of chemicals to cold-water adapted species is required. In this study, the Arctic scallop, Chlamys islandicus, was selected as a key species for bio-monitoring because of wide distribution in Arctic waters and its commercial value. Test animals, stored in seawater at 2 °C, were injected with benzo(a)pyrene (diluted in cod liver oil 5 mg ml⁻¹) in the adductor muscle every 24 h for four days giving a final dose of 0, 74 and 90.6 mg kg⁻¹ wet weight for control, low and high dose, respectively. The biomarkers used were total oxyradical scavenging capacity (TOSC) in the digestive gland and cell membrane stability of haemocytes. TOSC values were significantly reduced (ca. 30%) in exposed groups (P<0.05), indicating a depletion in oxyradical molecular scavengers. The antioxidant defences appeared to be overwhelmed by the reactive oxygen species as the plasma membranes of haemocytes were destabilised (P<0.05) probably due to lipid peroxidation. These data indicate that reactive oxygen species (ROS) were produced by Arctic scallops via the metabolisation of benzo(a)pyrene at 2 °C.     

Degradation of crude oils byPseudomonas sp. in enriched seawater medium

Microbial degradation of five crude oils such as Arabian light, Berri, Murban, Khafji and Minas crude oil by a pure bacterial strain,Pseudomonas sp. isolated from the sea water sampled at Kawasaki Harbor in Tokyo Bay, was studied experimentally in the enriched seawater medium.The degradation of crude oils was determined in total residual oil and in four fractions of saturated, aromatics, asphaltene and column residue by use of the column chromatography with activated alumina.The saturated fraction was shown to be most biodegradable. The aromatics followed for all five crude oils examined and the asphaltene was biodegradable to some extent. The column residue was not apparently degraded byPseudomonas sp. within 30 days. Each of Arabian light, Berri or Murban crude oil was degraded from 59 to 63.5 %, Khafji crude oil 49 % and Minas crude oil, solid at room temperature, only 33 %. Degradation rate of the five crude oils was determined to be in a range from 2.88 to 17.3 mg-oil 1^–1 hr^–1 or from 6.0×10^–12 to 1.56×10^–10 mg-oil cell^–1 hr^–1. Relative degradation ofn-paraffins of different carbon numbers in the saturated fraction was found to be similar regardless of carbon number for the five crude oils.     

Non-volatile Hydrocarbon Chemistry Studies Around a Production Platform on Australia's Northwest Shelf

In September 1994 and 1995, scientists from the Australian Institute of Marine Science (AIMS) and the Australian Geological Survey Organization (AGSO) conducted surveys aboard the RV Lady Basten to determine the dispersion, fates and effects of produced formation water (PFW) discharged from the ‘ Harriet A ’ oil production platform near the Montebello Islands. This report is one of four related papers and describes the non-volatile hydrocarbon chemistry studies. The dispersion of the PFW into dissolved and particulate fractions of seawater were measured using moored high volume water samplers, surface screen samplers and moored and drifting sediment traps. Bio-accumulation was studied using transplanted oysters, and dispersion measured into sediment with benthic grabs.Results showed enrichment in non-volatile hydrocarbons in surface microlayer samples to a distance of 1·8 km in the direction of tidal flow. Concentrations in surface microlayers near the platform varied by an order of magnitude and corresponded to when a surface slick was visible or not visible. Concentrations of oil in seawater ranged from 2·0 to 8·5 μg l⁻¹at near stations to 1·3 μg l⁻¹at 1·8 km. Water column samples showed the processes of desorption from particles for soluble components occur within the range of 1·8 km. Most particulate hydrocarbons drop out of suspension within c. 1 to 2 km from the platform. Fluxes of particulate hydrocarbons through the water column at c. 1 km, as estimated by moored sediment traps in 1995, were 138 to 148 ng cm⁻²day⁻¹. A decrease in sediment concentrations within c. 1 km of the platform was measured as 2·45±1·29 μg g⁻¹dry wt (n=15) in 1994 to 0·86±0·54 μg g⁻¹dry wt (n=21) in 1995, after the platform installed a centrifugal separator in the discharge treatment process. Thus the residence time of this relatively low molecular weight oil was estimated in the coarse aerobic sands surrounding the platform to be less than one year. Oysters suspended near the platform bio-accumulated hydrocarbons and other lipophilic organics in their tissues. Uptake rates and bio-concentration factors of hydrocarbons indicated potential toxicity at the near-field stations within c. 1 km radius.A mass balance was constructed to show the partitioning of the input of hydrocarbons from the PFW into the surrounding marine ecosystem. The rates of dissipation processes were estimated as follows: dilution from tidal currents>degradation in the water column>sedimentation>evaporation. The calculations based on maximum concentrations measured in the environmental samples accounted for 85% of the daily input suspended within a 1 km radius.It is estimated that the potential zone of toxic influence in the water column extends to a distance of approximately 1 km. Concentrations of oil in sediments were too low to indicate potential toxicity. By the collaborative application of oceanographic and geochemical techniques to marine environmental problems, we endeavour to provide effective feedback to the oil industry to gauge the effectiveness of their operational strategies in minimizing impact in these pristine regions.     

The influence of dissolved petroleum hydrocarbon residues on natural phytoplankton biomass

The effect of dissolved petroleum hydrocarbons in the environment on phytoplankton biomass measured as chlorophyll a was studied near the oil tanker route in the southern Bay of Bengal. In the transect from 5° N, 77° E to 5° N, 87° E the concentrations of dissolved petroleum hydrocarbons were negatively correlated with phytoplankton biomass, whereas in the 0° N, 87° E to 1° N, 79° E transect they were positively correlated with phytoplankton biomass. The mean petroleum hydrocarbon concentrations in the two transects were 12·12 ± 4·67 μg litre⁻¹ and 11·23 ± 4·5 μg litre⁻¹, respectively.It is surmised that the effect of dissolved petroleum hydrocarbons on phytoplankton biomass varies depending on the nature rather than the quantity of petroleum hydrocarbons present. Culture studies with unialgal Nitzschia sp. in seawater collected from selected stations in the study area as well as in artificial seawater spiked with the water-soluble petroleum hydrocarbon fraction of light Arabian Crude support this.     

Toxicity of Santa Barbara seep oil to starfish embryos: Part 1—hydrocarbon composition of test solutions and field samples

The composition and concentration of the water-soluble fraction (WSF) of four oils (Prudhoe Bay Crude, Holly Monterey Crude, Holly Rincon Crude and Isla Vista Seep Oil) are described. Changes in concentration and composition with exposure time in our bioassay chambers are described. Rapid concentration decreases and an unusual compositional relationship point to the importance of performing chemical analyses in conjunction with bioassay experiments.Comparison of the chemical composition of dissolved hydrocarbons in seawater at the Isla Vista Seep Oil site with the WSF of the seep oil shows many similarities, although the concentration is lower than the WSF by approximately 1000-fold.     

Effects of long-term exposure to silver or copper on growth, bioaccumulation and histopathology in the blue mussel Mytilus edulis

The purpose of this study was to determine the long-term accumulation of either silver or copper from low concentrations in seawater by blue mussels, Mytilus edulis. Mussels raised from eggs in the laboratory to the age of 2·5 months (approximately 4·5 mm in length) were continuously exposed to 0, 1, 5 and 10μg/liter of either silver (nitrate) or copper (chloride) and sampled at 12, 18 and 21 months for growth studies, measurements of metal accumulation and histopathological examination.Whole-body soft tissues were analyzed for the presence of both silver and copper, as background levels of copper in the incoming seawater averaged 2–4 μg/liter. Mussels exposed to silver had accumulated significant amounts of silver only at the highest test concentration (10 μg/liter Ag) after 12 months, but at 18 and 21 months significant levels were accumulated at all three test concentrations. Mussels exposed to copper accumulated significant amounts of copper at 5 and 10 μg/liter Cu after all three sampling periods, but not at 1μg/liter. Silver-exposed animals also accumulated significantly greater amounts of copper than control animals.In a comparative study, field-collected juvenile mussels (approximately 16·1 mm in shell length) and adult mussels (approximately 53·4 mm in shell length) were exposed for 12 months to 0, 5, 25 and 50 μg/liter silver only and subsequently sampled for metal-accumulation analyses and growth measurements. Juvenile mussels accumulated significant amounts of silver at all test concentrations, with the exception of mussels exposed to 5 μg/liter Ag for 6 months. Copper accumulation in the silver-exposed juveniles was significant only at 50 μg/liter Ag after 6 months, but at all test concentrations after 12 months. Adult mussels exposed to silver accumulated significant levels of both silver and copper, but at somewhat lower levels than juveniles.In the growth study, silver had no effect on laboratory reared mussels at the highest concentration of 10 μg/liter tested, whereas copper at 10 μg/liter did appear to affect growth as early as 4 months after the start of experimental exposure. Field-collected juvenile mussels did show inhibition in growth after 6 months' exposure to 25 and 50 μg/liter Ag, with some growth occurring after 12 months. Adults also showed inhibition in growth after 6 months but not at 12 months.Histopathological examination of mussels exposed to either 5 or 10 μg/liter of copper for 18 months showed changes in the digestive diverticula, gastrointestinal tract, reproductive tract and muscle tissues. These changes were more noticeable in mussels exposed to 5 μg/liter Cu than in those exposed to 10 μg/liter. Mussels exposed to silver for 21 months showed yellowish to black particulate deposition in the basement membrane and connective tissue of the various organs and tissues. Silver deposition increased with increasing test concentration.     

Impact of environmental factors on in situ determination of iron in seawater by flow injection analysis

A sensitive method for iron determination in seawater has been adapted on a submersible chemical analyser for in situ measurements. The technique is based on flow injection analysis (FIA) coupled with spectrophotometric detection. When direct injection of seawater was used, the detection limit was 1.6 nM, and the precision 7%, for a triplicate injection of a 4 nM standard. At low iron concentrations, on line preconcentration using a column filled with 8-hydroxyquinoline (8HQ) resin was used. The detection limit was 0.15 nM (time of preconcentration = 240 s), and the precision 6%, for a triplicate determination of a 1 nM standard, allowing the determination of Fe in most of the oceanic regimes, except the most depleted surface waters. The effect of temperature, pressure, salinity, copper, manganese, and iron speciation on the response of the analyser was investigated. The slope of the calibration curves followed a linear relation as a function of pressure (C_p = 2.8 × 10^{− 5}P + 3.4 × 10^{− 2} s nmol^{− 1}, R² = 0.997, for Θ = 13 °C) and an exponential relation as a function of temperature (C_Θ = 0.009e^0.103Θ, R² = 0.832, for P = 3 bar). No statistical difference at 95% confidence level was observed for samples of different salinities (S = 0, 20, 35). Only very high concentration of copper (1000 × [Fe]) produced a detectable interference. The chemical analyser was deployed in the coastal environment of the Bay of Brest to investigate the effect of iron speciation on the response of the analyser. Direct injection was used and seawater samples were acidified on line for 80 s. Dissolved iron (DFe, filtered seawater (0.4 μm), acidified and stored at pH 1.8) corresponded to 29 ± 4% of Fe_a (unfiltered seawater, acidified in line at pH 1.8 for 80 s). Most of Fe_a (71 ± 4%) was probably a fraction of total dissolvable iron (TDFe, unfiltered seawater, acidified and stored at pH 1.8).     

Toxicological effects of potash brine on bay of Fundy marine organisms

The acute lethality and sublethal heavy metal contamination potential to marine organisms of a waste brine solution from a potash mining operation were determined. The brine was acutely lethal to marine organisms of the Bay of Fundy, Canada, at concentrations between 47 and 55‰ salinity (96-h LC₅₀ values: threespine stickleback, Gasterosteus aculeatus, 47·7‰; polychaete worm, Nephtys incisa, 52·5‰; blue mussel, Mytilus edulis, lethal to whole animal and reduced ciliary activity of gills above 55‰).The brine diluted to sublethal concentrations (≤40‰) contained manganese (≤ 49·5 ×), copper and zinc (≤ 5·2 ×), lead (≤ 2·6 ×) and cadmium (≤ 0·7 ×) in decreasing order of enrichment relative to control seawater. Copper was accumulated in mussels exposed to these solutions over 44 days to the greatest degree (4 ·4 ×), followed by manganese and lead. Tissue lead concentration reached a steady value within 44 days and cadmium was not accumulated.     

Solubility of calcite in the ocean

The apparent solubility product K′_sp of calcite in seawater was measured as a function of temperature, salinity, and pressure using potentiometric saturometry techniques. The temperature effect was hardly discernible experimentally. The value of K′_sp at 25°C was 4.59·10⁻⁷ mole²/(kg seawater)² at 35‰S, 5.34·10⁻⁷ at 43‰S, and 3.24·10⁻⁷ at 27‰S. The apparent partial molal volume was found to be −34.4 cm³ at 25°C and −42.3 cm³ at 2°C from a linear fit of log(K′_sp ^P/K′_sp ¹). These results were used in conjunction with field data to calculate the degree of saturation in the oceans and showed undersaturation at shallower depths than previously reported.     

The behavior of fly-ash derived arsenic in seawater

With the rising cost of oil the electric power generating companies are turning to coal as a fuel source. Large amounts of fly ash are produced as a by product of coal combustion. This fly ash must then be disposed of, with the oceans being considered an alternative to land fill disposal. This research investigated the sorptive behavior of the surface-associated arsenic and utilized the results to project arsenic's impact on the water column during the ocean disposal of fly ash.Several acid digests were investigated to determine an effective method of arsenic recovery from fly ash. Of these, the HCl digest was the most effective technique, yielding 100% arsenic recoveries from fly-ash particles. The arsenic content of the fly ashes studied varied from 69 ± 11 μg g⁻¹ to 323 ± 24 μg g⁻¹, reflecting differences in the arsenic content of the source coal. In both seawater and freshwater there is an increase in arsenic desorption with increasing pH. The greatest release of arsenic occurred at pH 12 with generally over 80% of the surface arsenic released.Fly ash in contact with seawater and freshwater can exhibit either acidic or alkaline tendencies depending upon the soluble elemental composition on the surface of the flyash particle. The acidic ashes were shown to leach a greater percentage of arsenic (16.9%) than the more alkaline ashes (8.2%). During these leaching studies in seawater, arsenic was found to leach in both the pentavalent and trivalent oxidation state. The pentavalent state was predominant, comprising 77% of the arsenic initially desorbed.The dissolution in seawater of arsenic was utilized to assess the possible impact of the ocean disposal of fly ash. Based upon these data it appears that the natural levels of arsenic in the water column would not be significantly increased. Further research is needed on the fate of fly-ash particles in marine sediments.     

Toxicity of Santa Barbara seep oil to starfish embryos: Part 3—influence of parental exposure and the effects of other crude oils

We exposed the bat starfish, Patiria miniata, to the water-soluble fraction (WSF) of oil from a natural seep near Santa Barbara, California, during the first 48 hours of embryological growth. There were no consistent differences among populations whose parents were from areas with differing levels of hydrocarbon exposure. In a second experiment, the order of decreasing toxicity of crude oils to the embryos was: Monterey Zone > Rincon Zone > Prudhoe Bay > Isla Vista Seep. The results are discussed in relation to the effects of natural seeps and oil characteristics.     

Metabolic conversion of cyclohexane by Pacific salmon microsomal preparations

Hydrocarbons are being introduced into the marine environment from a variety of sources including combustion processes, crude oil spills, fuel oil spills, and controlled disposal such as processed ballast water. Even in the relatively clean waters of Alaska hydrocarbons are being released at an alarming rate. The purpose of this study was to determine the optimum conditions for the metabolism of a representative hydrocarbon by Coho Salmon Oncorhynchus kisutch liver microsomes. The product of cyclohexane metabolism in the salmon microsomal system was identified by gas chromatography-mass spectrometry as cyclohexanol. Conditions of the microsome incubation were varied systematically to determine the optimum temperature, pH, and ionic strength for cyclohexanol production. Cyclohexanol was quantified by capillary column gas chromatography. Maximum cyclohexanol formation was achieved at 20°C, a pH of 8·0–8·5 and an ionic strength of 0·026. A linear rate of cyclohexanol formation is seen from 0–60 min of incubation and there is an apparent decrease in the rate from 60–90 min. Poor stability of the microsomal preparation from the species studied was also identified and several stability studies have been undertaken using cyclohexane metabolism as a monitor.