ECOTOXICITY OF DEGREASER AND DRILLING FLUID … · ecotoxicity of degreaser and drilling fluid used in upstream sector of nigeria petroleum industry on fresh water higher organism

ECOTOXICITY OF DEGREASER AND DRILLING FLUID USED

IN UPSTREAM SECTOR OF NIGERIA PETROLEUM INDUSTRY

ON FRESH WATER HIGHER ORGANISM (TILAPIA

GUINEENSIS) AND MICROORGANISM (NITROBACTER SP.)

NRIOR R. R. AND

ODOKUMA L. O.

ABSTRACTThe ecotoxicological evaluation of degreaser: Rigwash KLYNE 025A and Aquabreak PX and drilling fluids:

Addax-WBF (water base) and Addax-OBF (oil base) to higher organism - fresh water fish Tilapia guineensis and

microorganism Nitrobacter sp. were examined. The static with renewal option for acute toxicity assessment at

concentrations 0, 10, 100, 1000, 10000 and 100000ppm concentrations were employed. Mortality of the fish

(LC ) was used as indices to monitor toxicity. The results showed that the oil-based drilling fluid were more toxic 50

to the test organisms than the water-based drilling fluid. Degreaser LC revealed that Aquabreak were more toxic 50

than Rigwash. The 96 h LC range were as follows: Aquabreak (1.29ppm) > Rigwash (12.26ppm) > Oil base 50

(12463.01ppm) > Water base (25390.00ppm) (the lower the LC the more toxic the toxicant). The 24h Median 50

Lethal Concentration (LC ) of the sensitivity of the bacterium Nitrobacter to the toxicity of Drilling fluid - oil 50

based and water based and Degreaser - Rigwash and Aquabreak in freshwater were Aquabreak (24193.88ppm) >

Rigwash (30158.66ppm) > Water base (40160.96ppm) > Oil base (40414.20ppm). Comparatively, fresh water

fish Tilapia guineensis was more susceptible than the bacterium Nitrobacter sp.to both degreaser (Rigwash

KLYNE 025A and Aquabreak PX) and drilling fluids (Addax-WBF water base and Addax-OBF oil base).

Degreaser Aquabreak PX to Tilapia guineensis showed the highest level of toxicity (LC 1.29ppm). The test 50

organisms displayed dissimilar levels of sensitivities to the four test toxicants at 95% probability levels. Findings

from the study revealed that microorganism (Nitrobacter sp.) are more resistant to drilling fluid and degreaser

than semi higher organism (Tilapia guineensis). Based on these findings; it is advocated that ecotoxicological

evaluation of chemical used in aquatic environment should not only be assessed using microorganism but in

conjunction with higher organism.

Key words: Degreaser, Drilling fluid, Tilapia guineensis, Nitrobacter sp.

INTRODUCTION

Trichloroethylene (TCE) is one of the compounds that is mainly used as degreaser. Chlorinated

aliphatic hydrocarbons (CAH ) may enter into water bodies and contaminate water sources and S

affect human health in a direct or indirect manner (Mesdaghinia et al., 2005). Many efforts have

reported the removal of organic material from aqueous solutions (Mesdaghinia et al., 2005;

Naghizadeh et al., 2008). Because of improper handling and disposal practices, TCE has been

frequently detected in groundwater. TCE is considered as a probable carcinogenic chemical

(Group B ) to human (Wartenberg et al., 2000) it has also many other adverse effects on human 2

and animals (Wartenberg et al., 2000; CEPA, 1993). Due to its serious health effects, U.S

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Environmental protection Agency (U.S EPA) has set the maximal contaminant level (MCL)

and maximum contaminant level goal (MCLG) for TCE as 0.005mg/L and zero respectively.

Most conventional treatment processes such as coagulation, sedimentation, precipitate

softening; filtration and chlorination are not efficient in removal of TCE.

Degreasers are chemicals that are used to clean metal by washing dirt, grease and oil from auto

engine parts. A degreaser can either be oil-based or water-based. Oil-based degreasers are

usually toxic and flammable. Even small amounts entering surface or groundwater can result in

serious pollution (Schlemmer et al., 2002; Nrior and Odokuma, 2015). Many oil-based

degreasers readily evaporate and contribute to smog or ground level ozone. Water based

cleaners are generally safer for the user and the environment. They are less toxic than oil based

degreasers and small amounts can be broken down in sewage treatment facilities. Drilling fluids

went through major technological evolution, since the first operations performed in the US,

using a simple mixture of water and clays, to complex mixtures of various specific organic and

inorganic products used nowadays. These products improve fluid rheological properties and

filtration capability, allowing penetrating heterogeneous geological formations under the best

conditions (Schlemmer et al., 2002) but the negative environmental impact is on daily toll

increase, Estuaries are highly sensitive zones subjected to the heavy industrialization and

overpopulation. The individual components of the chemical additives in the mud may pose

toxicity problems. The complexity of the problems met in petroleum drilling has led to

emerging techniques for the formulation of appropriate fluids. There are two main types of

drilling fluids: Water based fluid and Oil based fluid.

Drilling mud (drilling wastes) are sometimes unintentionally or intentionally released into

water bodies and can damage the gills of prawn, shrimp and other bottom dwellers at post larval

stages. For most aquatic animals, the gills are major sites through which waterborne pollutants

can enter the body and are often affected by such substances (Soegianto et al., 2008).

Several substances or waste materials introduced into the environment as a result of petroleum

exploration activities may be toxic and persist in their immediate environments. These toxic

effects may be acute or chronic (Rhodes and Hendricks, 1990; Okpokwasili and Odokuma,

1096). Acute (short-term) and chronic (long-term) health impacts can occur through

bioaccumulation of oil, metals and other products in aquatic species that are consumed by

humans. In many countries, muds and cuttings are discharged on site into the ocean. However,

the regulators in Nigeria [Federal Ministry of Environment (FMEnv) and Department of

Petroleum Resources (DPR)] require that a toxicity test be conducted on any drilling mud to

ascertain their safety before release into the environment or deep wells (EGASPIN, 2002). The

lethal concentration (LC ) is a standard toxicity test to determine the concentration of the 50

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Nigerian Journal Of Oil And Gas Technology - Vol 3 No. 1

substance which will prove lethal to 50% of a test population of the organisms in a specified

duration (Landis and Yu, 2004).

In the Niger Delta, use of drilling fluids during operations is a common practice and their uses

pose a lot of stress to the environment (Nrior and Odokuma, 2015). Thus the objective of this

study was to assess the toxicity of degreaser and the two main classes of drilling fluids (water

based and oil based) commonly employed in drilling operations in the Niger Delta, Nigeria on

fresh water higher organism (Tilapia guineensis) and microorganism (Nitrobacter sp.) These

organisms were chosen because they produce consistent and reproducible response to toxicants

and could be transported and maintained in the laboratory with relative ease, and also due to the

economic importance of the selected fish to the local communities.

MATERIALS AND METHODS

Source of test organisms

Drilling Fluids: Drilling fluids employed in this study were of two types the water- based and

Oil-based drilling fluids; obtained from Addax Petroleum Development Company, Nigeria.

Degreasers: Degreasers were KLYNE 025A Rigwash and Aquabreak PX purchased from

Offshore Chemicals, Port Harcourt, Nigeria.

Source of test organisms

Higher organism: Tilapia guineenis fresh water fish were obtained from Asarama stream,

Andoni, Rivers State, Nigeria.

Microorganism: Nitrobacter sp. was isolated from same habitat.

Collection of higher organism (Tilapia guineensis)

Juvenile fish of fairly equal size were randomly caught with a hand net of mesh 0.5mm and

transferred into the test vessel. The fish were not touched with hand during the selection so

as to avoid stress due to handling only active and healthy fish were selected.

Average weight per organism: Tilapia guineenis = 191 ± 5mg

Average length of test species: Tilapia guineenis = 2.5 ± 0.5cm

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Ecotoxicity Of Degreaser And Drilling Fluid Used In Upstream Sector Of Nigeria Petroleum Industry On Fresh Water Higher Organism (tilapia Guineensis) And Microorganism (nitrobacter Sp.)

Isolation of microorganism (Nitrobacter sp.)

Aliquot (0.1ml) of the water samples was transferred onto sterile Winogradsky's agar plates in

duplicates. Uniformly spread with sterile glass spreader (spread plate method) and incubated in 0inverted position at 30 C for 72-96hours. Creamy, mucoid, flat colonies were suggestive of

Nitrosomonas species. Gram staining of the colonies revealed Gram negative short-rods

indicative of Nitrosomonas. The colonies were aseptically subcultured unto fresh

Winogradsky's agar plates. Grayish, mucoid, flat colonies were suggestive of Nitrobacter.

Gram staining of the colonies revealed pear-shaped organisms indicative of Nitrobacter.

Suspected Nitrosomonas and Nitrobacter species were used to inoculate sterile Winogradsky 0

broth containing ammonium sulphate and sodium nitrite respectively and incubated at 30 C for

2-6days. After 48hours of incubation, 1ml each of sulfanilic acid and dimethyl-naphthalamine,

and a little zinc dust were added to the respective medium. Nitrite production from ammonium

sulphate indicated by red colouration was confirmatory of Nitrosomonas species. Nitrate

production from sodium nitrite indicated by red colouration was confirmatory of Nitrobacter

species.

Ecotoxicity procedure of degreaser and drilling fluid on Nitrobacter sp

Preparation test medium

The effluent was prepared following the procedure outlined in APHA, (1998), 10ppm, 100ppm,

1000ppm, 10000ppm and 100000ppm concentrations of the toxicants were prepared using

0.5dilution factor respectively.

Preparation of test organism (Nitrobacter sp.)

A loopful of the test organism was transferred into 10ml sterile appropriate broth. This was 0 0

incubated for 2-4days at room temperature (28±2 C) and stored in refrigerator at 4 C. Aliquot

(1ml) of the 24h culture was transferred into fresh sterile broth (10ml), incubated for 24h (to

ensure that actively growing organisms were used for toxicity test) and preliminary standard

Inoculum determined (APHA, 1998)

Preparation of standard bacterial inoculum

Tenfold serial dilution of the organism was made and aliquot (0.1ml) was inoculated onto

Winogradsky agar in triplicates using spread plate technique. The plates were incubated for 4

days for Nitrobacter sp. After the incubation periods, the plates were examined for discrete

colonies.

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The dilution that gave between 200 and 300 colonies was noted and used as reference dilution

to obtain the standard Inoculum for the toxicity bioassay.

Test procedure for the bacterium: Nitrobacter sp.

Five milliliter (5ml) of the test organisms was added to 45ml of each toxicant concentration

(10ppm, 100ppm, 1000ppm, 10000ppm and 100000ppm respectively), and plated out

immediately after inoculation on appropriate media. This is known as zero hour count plating. 0These were incubated at room temperature (28±2 C). Aliquots (0.1ml) of each concentration

of the toxicants (drilling fluid and degreaser) were then plated out after 4 hours, 8 hours, 12

hours and 24 hours on Winogradsky agar. This was followed by incubation for 96hrs for

Nitrobacter. The plates were then counted and average colonies taken, then Colony Forming

Unit per milliliter (cfu/ml) calculated.

Ecotoxicity procedure of degreaser and drilling fluid on Tilapia guineensis

Acclimatization: The test organisms were acclimatized separately in glass tanks shortly after

sampling at room temperature for ten days. The water in the acclimatization units was replaced

with water from the organism's habitat water once daily. A maximum of fifty organisms were

kept in each tank. This number was kept like this to prevent crowding. The dimensions of

holding tanks were 2 x 6 x 6m.

Toxicity Testing: The test vessels had the following dimensions, 1m x 1m x 1m. The vessels

were wrapped with dark polyethylene. The vessels contained estuarine water from Bonny

River and brackish water from Trans-Amadi River for shrimps. Six logarithmic

concentrations of the test chemicals; 0, 10, 100, 1000, 10000 and 100000 were prepared using

water from the habitat of the test organism, as diluents. A preliminary range finding test was

first performed before these concentrations were arrived at. The 96h acute toxicity bioassay

was carried out on Mysidoposis bahia and Palaemonetes africanus using the procedure of

APHA 1998. Seven different toxicant concentrations 0, 10, 100, 1000, 10000 and 100000ppm

were prepared for the experiment with controls of filtered clean water from the habitat of the

test organisms (dilution water). Ten shrimps of fairly equal sizes were randomly caught with

hand net and carefully transferred into each test vessel. The organisms were not touched with

hand during the selection so as to avoid stress due to handling. Only healthy and active

organism was selected.

Mortality was recorded after 4, 8, 12, 24, 48, 72 and 96 hours. Dead shrimps were removed at

each observation. Mortality was plotted against the concentration on a log graph. Regression

analysis was used to obtain the line of best fit. To obtain the median lethal concentration (LC ), 50

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a horizontal line is drawn from the 50% mortality point to intersect the graph; the point to

intersection is extrapolated on the abscissa by dropping a vertical line on it and given the LC 50

for the shrimps. The one way analysis of variance and the least significance difference test

(LSD) were employed for analysis of data (Reish and Oshida, 1987).

Percentage log survival of Nitrobacter sp.

The percentage log survival of the bacterial isolates in the toxicant used in the study was

calculated using the formular adopted from Williamson and Johnson (1981). The percentage

log survival of bacterial isolates in the toxicant was calculated by obtaining the log of the count

in each toxicant concentration, dividing by the count in the zero toxicant concentration and

multiplying by 100.

Thus; % log survival = Log C x 100

Log c

Where Log C = log of the count in each toxicant concentration,

Log c = log count in the zero toxicant concentration

Percentage mortality of fresh water juvenile test specie Tilapia guineenis.

Mysidoposis bahia (marine water shrimp), Palaemonetes africanus (brackish water

crustacean) and fresh water fish (Tilapia guineensis) were used in the study as a specimen of

higher organism to assess the probable toxic effect drilling fluid, oil spill dispersant, degreaser

and industrial detergents could have on fishes and other higher organisms in the aquatic

environment. The formular for the percentage mortality was adopted from APHA (1992). The

percentage mortality was done by dividing the number of organisms that died at each exposure

hour by the total test organism and multiplying by 100.

Thus; % Mortality = Number of organisms dead X 100

Total number of organism

Statistical analysis and Median Lethal Concentration (LC )50

Data representing % mortality and concentration from semi-static bioassay were analysed

using the probit analysis software to determine the LC values.50

The results obtained from toxicity screening were subjected to statistical analysis using

Analysis of Variance (ANOVA) and student t-test at 0.05 confidence limit (Reish and Oshida,

1987) to determine the significant difference between the susceptibility of the Nitrobacter sp.

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(test bacteria) and fresh water fish Tilapia guineenis to the test toxicants (drilling fluid and

degreaser).

RESULT AND DISCUSSION

The 96h median Lethal concentration (LC ) of higher organism Tilapia guineensis and 24h 50

LC of microorganism Nitrobacter sp. to the drilling fluids (water-based and oil-based) and 50

degreaser (Rigwash and Aquabreak) in fresh water system were presented in Fig. 1. The results

showed that the oil-based muds were more toxic to the test organisms than the water-based

muds. Degreaser LC revealed that Aquabreak were more toxic than Rigwash. Aquabreak is 50

most toxic of the test toxicants. The 96 h LC range were as follows: Aquabreak (1.29ppm) > 50

Rigwash (12.26ppm) > Oil base (12463.01ppm) > Water base (25390.00ppm) (noting the

lower the LC the more toxic the toxicant).50

The 24h Median Lethal Concentration (LC ) of the sensitivity of the bacterium Nitrobacter to 50

the toxicity of Drilling fluid - oil based and water based and Degreaser - Rigwash and

Aquabreak in freshwater were Aquabreak (24193.88ppm) > T-pol (28617.98ppm) >

OSD/Seacare (29251.72ppm) > Rigwash (30158.66ppm) > Gamazyme BTC (31145.60ppm)

> Water based (40160.96ppm) > Oil based (40414.20ppm) > OSD/LT (47184.72)(noting the

lower theLC , the more toxic the toxicant). 50

Fig. 1: LC of degreaser and drilling fluid on fresh water macro and micro organisms50

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The low LC values of degreaser – Aquabreak toxic than Rigwash and oil -based fluid than 50

water based fluid may be related to their chemical composition (Odokuma and Okpokwasili,

1992). The presence of mineral oil in their liquid phase may be responsible for their increased

toxicity. Nrior and Odokuma, (2015) have reported that water-based fluids remain non- toxic

even at very high concentrations (10,000ppm). This result revealed that toxicity of oil-based

fluids, may also contribute to persistence of these fluid/muds in environments in addition to the

reduced biodegradability. However, it is interesting that not all the species, (even

physiologically similar group of micro-organisms), are similarly influenced by the presence of

a given toxic chemical in their environment. The nature of interaction between effluents

(toxicity set up) and microorganisms is complex due to various reactions taking place during a

prolonged or previous exposure (Obire and Nrior, 2014). Oil based drilling fluid in marine

water alone is toxic to the tested species at concentrations that are significantly greater than the

levels of pollutant commonly detected in the environment (Fig. 2-5).

Fig. 2: Lethal toxicity of drilling fluid - Water base on Nitrobacter sp. in fresh water

Fig. 3: Lethal toxicity of drilling fluid - Oil base on Nitrobacter sp. in fresh water

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Fig. 4: Lethal toxicity of degreaser – Rigwash on Nitrobacter sp. in fresh water

Fig. 5: Lethal toxicity of degreaser - Aquabreak on Nitrobacter sp. in fresh water

Degreaser and drilling fluid toxicity to Nitrobacter sp. results showed that certain toxicant concentrations were stimulating (increase the activity) while others were inhibiting to nitrite utilization by nitrobacter. Similar observations have been reported (Wang, 1984). A good increase in the loss of nitrites with increasing exposure time was observed with the degreasers. The increase in nitrite utilization with increase in toxicant exposure time was not in uniform with the degreaser. Aquabreak degreaser was more toxic than Rigwash degreaser; oil based drilling fluid is more toxic in comparism to water based drilling fluid. At 24 hours 20%

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reduction in the viable count of Nitrobacter exposed to Rigwash degreaser was observed and 0% reduction in the viable count was also observed on the exposure to Aquabreak degreaser. This might have resulted from degreaser effect on any of the target sites. It is known that the site of action of a toxicant is a function of the nature of the toxicant (Obire and Nrior, 2014).

The percent log survival of Nitrobacter during 0h, 4h, 8h, 12h and 24h exposure periods to degreasers and drilling fluid (water based and oil based) showed that the degreaser Righwash exhibited little effect while Aquabreak showed significant effect on the percent log survival of the organism. Careful examination showed that Aquabreak reduced the percent log survival more than Righwash; oil based drilling fluid more than water based fluid (Fig. 2-5).

The results of this study suggest that degreaser (Aquabreak) caused cell mortality. This has led to the reduction in the viable counts. This may be due to inhibition of the nitrification process within the 24hour exposure period. The degreaser Righwash did not elicit any appreciable reduction in viable counts though Aquabreak did. This clearly suggests that the mode of action of chemical degreaser is not limited to inhibition of the nitrification process by Nitrobacter but also involved cell death.

The percentage mortality of the test organisms Tilapia guineensis - fresh water fish taken during toxicity of Drilling fluid - oil based and water based in marine and brackish water were shown in Fig. 6-9.

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In this research, concentration range between 0 to 100000; which showed that water based

drilling fluid had low toxicity on fresh water fish (Tilapia guineensis) and Nitrobacter sp. did

not have significant effects on the organism's mortality in comparism to oil based drilling fluid .

These shrimps have perfect resistance against pollution of drilling fluids and the results

indicated that Tilapia guineensis could be used for toxicity tracing drilling fluid in fresh water.

Therefore, drilling fluids and concentration of pollution in certain times of the year may cause

mortality for a species of animals and does not have any effect on the others . In general there

was significant difference between the sensitivity of the bacterium Nitrobacter and the higher

organism Tilapia guineensis used for the test as indicated by ANOVA used for analysis. The

difference in response of these bacterium and higher organism to the three different toxicants

could be due to difference in genetic make up, prolonged or previous exposure to the effluent

(Nrior and Odokuma, 2015), mutation (Zelibor, 1987).

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ECOTOXICITY OF DEGREASER AND DRILLING FLUID … · ecotoxicity of degreaser and drilling fluid used in upstream sector of nigeria petroleum industry on fresh water higher organism