DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF PORT HARCOURT, PORT HARCOURT.

A comparison of the sensitivity and rapidity of the inhibition of nitrification and respiration processes with mortality, as indices of bacterial toxicity assessment was performed. The toxicants employed were four drilling fluids: oil-based Paradril, a synthetic based IMCO-O, water–based IMCO-W and Gel/KCL/Polymer. The static test for acute toxicity assessment was employed for a 48h period. The oxidation of Ammonia to Nitrites by Nitrosomonas, and the oxidation Nitrites to Nitrates by Nitrobacter were the indices for the nitrification process. Respiration was monitored by changes in the carbon (IV) oxide evolution by Escherichia coli. Mortality was monitored using changes in the aerobic colony count of viable cells. The 8h median lethal concentrations (LC₅₀) of drilling fluids to all three-test organisms were significantly higher than the corresponding 8h median effective concentrations (EC₅₀). The 8h EC₅₀ of Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer to Nitrosomonas were 0, 84.4, 690.1 and 0 mg/L and for Nitrobacter were 0, 0, 134.2 and 0 respectively. The 8h EC₅₀ of these toxicants to E. coli were 471.4, 0, 192.9 and 75.3 mg/L respectively. The 8h LC₅₀ of these toxicants to Nitrosomonas were 1190.7, 964.7, 4673.2 and 844.9mg/L respectively. The 8h LC₅₀ of these toxicants to Nitrobacter were 1410.2 0, 639.0 and 2065.9mg/L and for E. coli 414.8, 1374.1, 971.8 and 1900.8mg/L respectively. Results of 8h EC₅₀ for all test organisms revealed differences in the sensitivities of the organisms to the toxicants while 8h LC₅₀ results of toxicants for test organisms were not as definite. However 36h LC₅₀ results exhibited definite differences in the sensitivities of the organisms to the toxicants. Results showed that inhibition of nitrite oxidation by Nitrobacter was more sensitive than inhibition of nitrite formation by Nitrosomonas and carbon VI oxide evolution by E coli. Inhibition of nitrite formation and CO₂ evolution displayed similar levels of sensitivities at 95% confidence levels. These results indicate that monitoring inhibition of metabolic processes rather than mortality was a more rapid and sensitive tool for ecotoxicological evaluation of chemicals employed in the petroleum industry in the Niger Delta.

There has been a growing awareness of the ecotoxicological implication of the multitude of new chemicals being produced and eventually discharged into the environment (Dutka et al, 1988). Detailed toxicity assessment of all existing chemicals using conventional animal is space and time consuming as well as high in operational cost (Liu et al, 1989). With the high demand of rapid simple screening test for evaluating the acute toxicity of chemicals in the environment, the use of microorganisms in short–term assays has become very important (Okpokwasili and Odokuma, 1994, 1996a, 1996b, Odokuma and Ogbu 2002, Odokuma and Ikpe 2003, Odokuma and Kindzeka, 2003). Such need has led to the development of a wide array of short–term bioassay using bacteria, yeast, fungi, protozoa, and algae for screening ad monitoring toxicant effects (Bitton, 1983, Dutka ad Bitton, 1986).

Over the years, bacteria have become particularly important in ecotoxicological assessment. This is because they are easy to handle and respond quickly to changes in their environment. Bacterial tests are quite inexpensive and need only small amounts of test substances (Dutka and Kwan, 1982). Bacteria represent the initial steps in most food chains, they are ubiquitous in nature and many of them are able to absorb (Brown and Lester, 1982) to accumulate (Strandberg and Arnold 1988) or to transform metals. Also, it is easy to standardize bacteria for toxicity tests in comparison to many eukaryotic organisms (Bauda and Block, 1985). In addition, their short life cycle means fast experimental results (Wang, 1984). Bacteria are also important in maintaining the ecological balance of nature (Biogeochemical cycles).

Bioassay tests employing bacteria are based on the inhibition of some vital metabolic function. These include inhibition of nitrite utilization by Nitrobacter (Okpokwasili and Odokuma, 1994 and 1996a, b), microtox tests (Gusy et al., 1988), induction of mutations (Vandermeulen, 1986) and inhibition of inducible extracellular enzymes (Dutton et al., 1990, Odokuma and Okpokwasili 2003 a, b). The toxicity of a chemical to microorganisms could also be measured in terms of growth inhibition (Narkis and Zur, 1979), oxygen consumption (Stabbert and Grabons, 1986), ATP production (Parker ad Priyle, 1984), enzyme activity (Bitton et al 1986), mutagenic potency (Xu et al, 1987) or colony formation on agar plate (Anderson and Abdelgbani, 1980). Odokuma and Kindzeka (2003) however reported that each toxicity screening test appeared to have its own sensitivity pattern. It would be unwise to assess the ecotoxicological importance of a chemical substance by a single specie test. Thus for a more accurate and comprehensive picture to be obtained different bacterial genera and different higher organisms occupying ecological niches in the same ecosystem should be employed.

In this study, three different bacteria namely Nitrosomonas, Nitrobacter and E. coli, occupying different ecological niches in the Niger Delta ecosystem were subjected to four drilling fluids: oil-based Paradril, a synthetic based IMCO-O, water–based IMCO-W and Gel/KCL/Polymer. Nitrosomonas and Nitrobacter are aerobic bacteria and play important rules in the nitrification process of the Nitrogen cycle. Nitrosomonas oxidizes ammonia into nitrites while Nitrobacter oxidizes nitrites into nitrates (Stanier et al., 1982). Escherichia coli a facultative anaerobic bacterium is capable of reducing nitrates into nitrites in the Nitrogen cycle (Stanier et al, 1982). However in an aerobic environment E. coli will respire aerobically producing carbon dioxide as by- product. The effects of four drilling fluids Paradril, IMCO–O, IMCO–W and Gel/KCl/Polymer on the inhibition of the nitrification processes of Nitrosomonas and Nitrobacter, inhibition of aerobic respiration by E. coli and their ultimate survival (mortality) were evaluated. The first objective was to determine which of the processes, nitrification or respiration (Effective Concentration) or survival (lethal concentration) was more sensitive to these toxicants. A second objective was to determine which test was more rapid in obtaining results. The final objective was to put forward this method as one to be considered in the battery of tests being evaluated for use in ecotoxicological studies in the Nigerian petroleum industry.

These were Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer. Paradril and Gel/KCL/Polymer were obtained from Magcobar Manufacturing Company Port Harcourt, Nigeria while IMCO–O and IMCO–W were obtained from the Nigeria Agip Oil Company Port Harcourt, Nigeria.

Three test organisms, Nitrosomonas, Nitrobacter and Escherichia coli were employed in this study. The organisms were all isolated from soil within the University of Port Harcourt, Rivers State, Nigeria. Surface soil samples (0-15cm depth) were collected from soil using a sterile auger drill. The soils were transferred into sterile polyethylene sachets and immediately taken to the laboratory for analysis. The methods used for the isolation of bacteria (Nitrosomonas and Nitrobacter) from soil were adopted from Colwell and Zambruski (1972) Nitrosomonas was isolated using Winogradsky medium for nitrification phase 1. The medium had the following composition (NH₄)₂SO₄, 2.0g; K₂HPO₄, 1.0g; MgSO₄.7H₂O, 0.5g; NaCl, 2.0g; FeSO₄.7H₂O, 0.4g; CaCO₃, 0.01 agar 15.0g; distilled water 1000ml. Nitrobacter was isolated using Winogradsky medium phase 2. It had the following composition, KNO₂ 0.1g; Na₂ – CO₃, 1.0g; NaCl, 0.5g; FeSO₄.7H₂O, 0.4g; agar 15.0g; distilled water 1000ml. Escherichia coli was isolated using methylene blue agar. The media were autoclaved and aseptically transferred to sterile Petri dishes after cooling to about 45⁰C. The Petri dishes were then inoculated with soil filtrate and incubated aerobically 4 days at for room temperature (28 ± 2⁰C) for Nitrosomonas and Nitrobacter and 18 to 24h at 42⁰C for E. coli. Further identification and characterization of pure cultures of these bacteria were undertaken using criteria of Krieg and Holt (1994). The broth media used for Isolation of the test organisms also served as diluent for producing the various toxicant concentrations.

Discrete colonies from each of the different culture media were subcultured into fresh media. These were transferred into slants and stored at 4⁰C. The slant cultures served as stock cultures. The standard inocula were prepared from the stock cultures. Each of the isolates were picked from the respective stock cultures and inoculated aseptically into 100ml of appropriate broth in a 250ml Erlenmeyer flask. They were then, incubated at 37⁰C for 24h. One millilitre was transferred from the respective flasks and ten-fold serial dilutions were made up to 10³. An amount (0.1ml) of the 10³ dilutions was plated into appropriate sterile agar plates. Incubation under appropriate conditions of the isolates followed immediately. Plates containing 45 – 70 colonies were selected for the toxicity test.

Toxicity tests were carried out to determine the toxicant effect on nitrite accumulation by Nitrosomonas, nitrite consumption by Nitrobacter and evolution of carbon IV oxide by E. coli. Mortality was also another parameter employed in assessing the toxicity of the drilling fluids to the three organisms.

Five logarithmic concentrations of each of the drilling fluids: 0.01, 0.1, 1.0, 10.0 and 100.0mg/L were prepared using appropriate broth: Winogradsky phase I, Winogradsky phase II and lactose broth were used as diluents in tests with Nitrosomonas, Nitrobacter and E. coli respectively. One hundred milligrams of each of the drilling muds were weighed. The volume of this weight was noted. This volume was then transferred into a 1litre conical flask. For water soluble drilling muds, the volume was then made up to 1000ml with diluent (broth). Subsequent concentrations, 10, 1.0, 0.1 and 0.01mg/L were obtained by further ten-fold serial dilution. The procedure for water insoluble muds differed slightly. The volume equivalent of 100mg of mud was transferred to a 1L flask and the volume made up to 1000mL with diluent. The same was repeated to achieve 10, 1.0, 0.1 and 0.01mg/L by dividing the equivalent volume of 100mg/L of mud by 10, 100, 1000 and 10,000 respectively and making up the volume to 1000ml with diluent. No solvent was employed to dissolve the water insoluble drilling muds. This was done deliberately to avoid synergistic or antagonistic effects of solvents.

The nitrite accumulation test was adapted from APHA (1998). The standard inoculum (0.1ml) of Nitrosomonas was added to each of the toxicant concentrations contained in 250ml Erlenmeyer flask. Nitrite concentration accumulated by Nitrosomonas was determined by coupling of diazotised sulfanilic acid with a –napthyl–ethylene–diamine dihydrochloride (NED) (APHA, 1998). Controls containing Winogradsky phase I broth and organism without toxicant was treated in the same manner. Increase in the concentration of nitrite with time indicated a positive result (the ability of the organism to convert ammonia to nitrite).

The nitrite consumption test was adapted from (APHA, 1998). The standard inoculum (0.1ml) of Nitrobacter was added to triplicate sets of the toxicant concentration as in nitrite accumulation tests. The mixture was incubated at room temperature (28 ± 2⁰C) for 72h. At exposure time of 0h, 2h, 3h, 4h, 8h, 12h, 24h, 36h ad 48h nitrite content was determined as in nitrite accumulation test.

The method of Allen et al. (1985) was adapted for the determination of evolution of carbon IV oxide by E. coli. The standard inoculum (0.1ml) of E. coli was inoculated into each 250ml Erlenmeyer flasks containing sterile lactose broth with various toxicant concentrations. The flasks were corked with sterile wooden corks with holes through which glass delivery tubes were passed into another 250ml Erlenmeyer flask containing 200ml of 0.01M calcium hydroxide solution. Incubation followed immediately at 37⁰C for 48h. At exposure times of 0, 1, 2, 3, 4, 8, 12, 24 and 48h, 10ml of Ca (OH)₂ were collected from each of the receiving flasks and 0.02m tetraoxosulphate IV acid (H₂SO₄) was used to titrate against the base. Methyl-red was used as indicator. The amount of carbon IV oxide (in moles) evolved were equated to the number of moles of base used in titration. It was converted to volumes by multiplying by 22.4dm³. The stoichiometric equation of the reaction is given below;

From step II 1 mole of CO₂ = 1mole of CaHCO₃)₂

Mole of Ca(HCO₃)₂ = C_AV_A/V_B = M_C

Where

C_A = Concentration of acid = 0.02

V_A = Volume of acid used in titration

V_B = Volume of base used = 10ml

M_C = moles of CO₂

Actual volume of CO₂ = M_C x 22.4dm³

The effective concentration (EC₅₀) values were used as a measure of the inhibition of nitrite accumulation, nitrite consumption and carbon IV oxide evolution. The values were extrapolated from the graph of percent (%) inhibition versus concentration. Percent inhibition was obtained using Probit’s Formula (Finney, 1978).

= (Value of each treatment/value of control) x 100/1

A reduction in the total viable cells (death) was also one of the criteria used in assessing toxicity of the drilling fluids to the three test bacteria. Mortality was used to determine the lethal concentration (LC₅₀) of each of the drilling fluids after a duration of 48h. This was obtained by plotting a Probit graph of percentage mortality versus concentration of toxicants (Finney, 1978). Regression analysis was used to obtain the line of best fit. The one–way analysis of variance and the least significant difference test (LSD) were employed for analysis of data (Finney, 1978).

In Table 1 the chemical composition and physical characteristics of the drilling fluids are presented. IMCO–O and IMCO–W displayed similar chemical composition. However IMCO–O contained calcium hydroxide and a synthetic fluid derived from mineral oil as its continuous phase. IMCO–W contained sodium hydroxide and water as its continuous phase. Paradril contained amines and diesel as its continuous phase while Gel/KCL/Polymer contained carboxyl methyl cellulose and water as its continuous phase.

In Table 2, 3 and 4 the EC₅₀ of the drilling fluids on Nitrosomonas Nitrobacter and Escherichia coli are presented. The EC₅₀ of all drilling fluids decreased with increase in exposure period for all test organisms. The 8h EC₅₀ of Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer to Nitrosomonas was 0, 84.4, 690.1 and 0mg/L respectively while 8h EC₅₀ of Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer to Nitrobacter was 0, 0, 134.2 el 0mg/L respectively The 8h EC₅₀ of Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer to E. coli was 471.4, 0, 192.9 ad 75.3 mg/L respectively. These results showed the following trend in sensitivity to the drilling fluids; Nitrobacter>E. coli ³ Nitrosomonas indicating inhibition of nitrite consumption to be the most sensitive of the three options.

In Table 5, 6 and 7 the LC₅₀ of the drilling muds to Nitrosomonas, Nitrobacter and E coli are presented respectively. The LC₅₀ of all toxicants decreased with increase in exposure period for all three bacteria. The 48h LC₅₀ of Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer to Nitrosomonas was 0, 91.17 0, ad 0 mg/L respectively. The 48h LC₅₀ of Paradril, IMCO–O, IMCO–W and Gel/KCL/Polymer to Nitrobacter was 23.62, 0, 0 and 0mg/L respectively while the 48h LC₅₀ of these fluids to E. coli was 0, 458.2, 228.8 and 428.9 mg/L respectively. These results revealed the following trend of sensitivity of bacteria the drilling fluids, Nitrobacter ³ Nitrosomonas > E. coli

When comparison of the 48h LC₅₀ with the 8h EC₅₀ of the drilling fluids to the three test organisms (Table 8) was made results showed that the EC₅₀ values were significantly smaller than the LC₅₀ values. These results were significant at 95%, confidence levels (calculated F value was 4.76 while tabulated F was 1.08 at 0.05 probability level).

Differences in the chemical composition of drilling muds elicited varying responses from the test organisms. Similar observations have been made by Odokuma and Ikpe (2003). They observed that oil–based (mineral or its fractions as a continuous phase) drilling muds were more toxic to Nitrobacter Desmocaris trispinosa and Metylus edulish and than water – based drilling muds. Paradril is an oil–based drilling mud with diesel as its continuous phase. IMCO-W and Gel/KCL/Polymer are water–based drilling muds, while IMCO–O has a synthetic fluid as its continuous phase. It was expected that Paradril would be the most toxic to the test organisms of the four drilling fluids. Both LC₅₀ and EC₅₀ values did not reflect this. Different organisms exhibited different responses to Paradril. The composition of IMCO – O and IMCO – W were similar. However they differed in the following; while IMCO-O contained lime (Ca(OH)₂) and a synthetic fluid as its continuous phase, IMCO–W contained caustic soda (NaOH) and water as its continuous phase. These differences were enough to elicit different responses to these toxicants by the test organisms. It was expected that IMCO–O would be more toxic because of its synthetic continuous phase originating from mineral oil. This was generally the case. All the muds contained barite, bentonite (viscosifier) lignosulfonates, lubricants detergents and emulsifiers. The asphalt or its derivatives (polycyclic hydrocarbons) present in IMCO–O are known mutagens (Okpokwasili and Nnubia, 1995). Emulsifying agents in drilling fluids have been reported to dissolve the lipid components of cell membranes (Odokuma and Okpokwasili 1996a). Chrome lignosulfonate and lignite contain chromium which is toxic in its hexavalent state (Cr⁶⁺). Quaternary ammonium compounds and carbamates present in some viscosifiers have biocidal actions. High sodium chloride (brine) could shrink the test organisms due to differences in the osmotic pressure since they were not halophytes these various constituents contributed to the varying and sometimes unpredictable response exhibited by test organisms. Within 8h of exposure the EC₅₀ of test organism showed the following trend Nitrobacter > E. coli ³ Nitrosomonas. These results indicated the following:

The sensitivity of Nitrobacter to toxicants has been attributed to the sensitivity of the constitutive enzyme nitrite enzyme mediating the oxidation of nitrate to nitrates in Nitrobacter (Odokuma and Okpokwasili 2003 a, b).

Inhibition of the nitritase function by toxicants, apart from cell wall disruption, may be due to the high permeability of Nitrobacter's outer membrane (Stanier et al, 1982). The membrane being the site of the nitritase enzyme complex, mediated respiration (Stanier et al, 1982) and thus, their inhibition affected the respiration process.

Previous reports by Dutton et al, 1990; Odokuma and Okpokwasili, 2003 a,b) have also presented E. coli as having little or no response to toxic chemicals. In these studies, the inability of the test chemicals to inhibit the synthesis of the intracellular enzyme b -galactosidase, which mediated the breakdown of lactose into glucose and galactose, was shown. They attributed this to the lack of permeases to transport the toxicant across the cell membrane, thus making this enzyme operon in E. coli a poor toxicity index. The present study revealed a high sensitivity of E. coli to the drilling fluids. The CO₂ evolved by E. coli was as a result of fermentation of lactose present in the lactose broth. Inhibition of carbon dioxide evolution was due to prevention of this fermentation process. The cell membrane of all three test organisms do not seem to be fundamentally different as all are Gram negative (Stanier et al, 1982) However E. coli has a smaller specific membrane area, than most other Gram negative bacteria (Prosser, 1989). This may contribute to its sensitivity. Unlike the location of nitritase in Nitrobacter, ammonia monooxygenase enzyme responsible for the oxidation of ammonia to nitrite in Nitrosomonas is not resident on the cell membrane. Thus the interaction with the drilling fluids and the monooxygenase enzyme is reduced compared with that of the nitritase enzyme in Nitrobacter. This may have contributed to the higher EC₅₀ values of Nitrosomonas,

Although this present study, was not designed to study, the detailed mechanism of drilling fluid toxicity, a general mechanism may be, increasing membrane permeability that causes dissipation of ion gradient and membrane potential leakage of essential cell constituents. Such a mechanism has previously been suggested to explain the cellular effects in Bacillus subtilis (Yamada, 1979), Nitrosomonas and Nitrosospira strains (Brandt et al, 2001) challenged with linear alkyl benzene sulfonate.

The LC₅₀ at 8h exposure period to toxicants were generally much higher than the corresponding LC₅₀ values. These results indicated that it took a higher concentration of toxicant to cause actual death of the organism than inhibition of either nitrification or respiration. These results showed that EC₅₀ results were faster to achieve and more sensitive than LC₅₀ (mortality) results. Within 8h of exposure to drilling fluids, acute toxicity result using EC₅₀ measurements could be determined.

The use of inhibition of nitrite consumption by Nitrobacter or carbon IV oxide evolution by E. coli are indices worth considering in the development of a toxicity protocol for the evaluation of the toxicity of drilling fluids in the Niger Delta. This is because of the rapidity in which results are achieved and the toxicity of these fluids to the both metabolic processes in these organisms. Both organism Nitrobacter and E. coli occupy very important trophic levels in ecosystems in the Niger Delta. Very little space and inexpensive equipments are required for the tests. The organisms are very easy to obtain from the soil or aquatic environment. These are some of the major the requirements needed for excellent tools for ecotoxicological assessment.