Exotoxicity of local and industrial refined kerosene on key environmental pollution monitor, Nitrobacter sp. In tri-aquatic systems in Nigeria

Abstract

Nitrification process involves Nitrobacter species and their growth and activities in the microenvironment when impacted negatively would consequently adversely affect soil fertility. In view of the significance of this process, the toxicity of local refined kerosene (LRK) and industrial refined kerosene (IRK) on a key environmental pollution monitor, Nitrobacter was investigated. LRK and IRK were apportioned into six sets for each of the experiments using tri-aquatic systems or microcosms of freshwater (FW), marine water (MW) and brackish water (BW) at percentage concentrations of; 0, 3.25, 6.5, 12.5, 25 and 50 into which the test organism (Nitrobacter sp.) was inoculated at intervals of; 0, 4, 8, 12 and 24hours. Toxicity results indicated that the sensitivity of the test organism was a function of both the contact time and concentrations, and also reflected lethal effects of the pollutants/toxicants (kerosene). The outcome of percentage median lethal concentration (%LC50) on Nitrobacter sp. in the triaquatic microcosms with pollutants were as follows; in IRK + FW 34.41 < in IRK + MW 37.89 < in LRK + FW 39.43 < in IRK + BW 40.99 < in LRK + MW 41.56 < in LRK + BW 45.35. This study revealed that IRK + FW microcosm was the most toxic (LC50) whereas LRK + BW microcosms was the least toxic. The inability of the organism to thrive well at kerosene concentration above 1% (v/v) is a warning signal of serious environmental pollution problem which could affect aquatic life forms and eventually humans. However, due to high fatality rate inherent from the use of LRK (though not reported here) and its toxicity to microbial life, it is hereby advocated that the public should rather resort to use IRK products.

Full text

International Research Journal of Public and Environmental Health Vol.4 (9),pp. 199-204, October 2017
Available online at https://www.journalissues.org/IRJPEH/
https://doi.org/10.15739/irjpeh.17.024
Copyright © 2017 Author(s) retain the copyright of this article ISSN 2360-8803
Original Research Article
Ecotoxicity of local and industrial refined kerosene on
key environmental pollution monitor, Nitrobacter sp. in
tri-aquatic systems in Nigeria
Received 3 August, 2017 Revised 20 September, 2017 Accepted 28 September, 2017 Published 20 October, 2017
Renner R. Nrior1*,
Nathaniel N. Ngerebara2,
Regina T. Baraol3
and
Lawrence O. Amadi4
1, 3Department of Microbiology,
Faculty of Science, Rivers State
University, Port Harcourt,
Nigeria.
2, 4Department of Science
Laboratory Technology, School of
Applied Sciences, Ken-Saro Wiwa
Polytechnic, P.M.B.20, Bori,
Rivers State, Nigeria.
*Corresponding Authors E-mail:
renner4nrior@gmail.com,
lawrenceamadi@ymail.com
Nitrification process involves Nitrobacter species and their growth and
activities in the microenvironment when impacted negatively would
consequently adversely affect soil fertility. In view of the significance of this
process, the toxicity of local refined kerosene (LRK) and industrial refined
kerosene (IRK) on a key environmental pollution monitor, Nitrobacter was
investigated. LRK and IRK were apportioned into six sets for each of the
experiments using tri-aquatic systems or microcosms of freshwater (FW),
marine water (MW) and brackish water (BW) at percentage concentrations
of; 0, 3.25, 6.5, 12.5, 25 and 50 into which the test organism (Nitrobacter sp.)
was inoculated at intervals of; 0, 4, 8, 12 and 24hours. Toxicity results
indicated that the sensitivity of the test organism was a function of both the
contact time and concentrations, and also reflected lethal effects of the
pollutants/toxicants (kerosene). The outcome of percentage median lethal
concentration (%LC50) on Nitrobacter sp. in the triaquatic microcosms with
pollutants were as follows; in IRK + FW 34.41 < in IRK + MW 37.89 < in LRK +
FW 39.43 < in IRK + BW 40.99 < in LRK + MW 41.56 < in LRK + BW 45.35.
This study revealed that IRK + FW microcosm was the most toxic (LC50)
whereas LRK + BW microcosms was the least toxic. The inability of the
organism to thrive well at kerosene concentration above 1% (v/v) is a
warning signal of serious environmental pollution problem which could
affect aquatic life forms and eventually humans. However, due to high
fatality rate inherent from the use of LRK (though not reported here) and its
toxicity to microbial life, it is hereby advocated that the public should rather
resort to use IRK products.
Key words: Toxicity, Percentage Median Lethal Concentration (%LC50), local and
lndustrial refined kerosene, Nitrobacter
INTRODUCTION
Kerosene is one of the most predominantly used energy
sources for households and bush burning by subsistence
farmers in Nigeria. It is one of the refined petroleum
products derived from crude oil by fractional distillation
(Kobeticova et al., 2012). The number of carbon in kerosene
(paraffin) ranges from 10-14 with boiling point of 1650-
2000 (Nihad and Mutlak, 2011). Unlike the conventional
IRK, LRK production is governed by illegality, lack of
expertise and improper processing (i.e., the simple
distillation process) resulting in coloured poor quality
product which poses serious threat to consumers and
equipment. The simple distillation process involves the use
of crude oil, wood fire (heat energy source), galvanized
pipes (of about one inch are connected to the metal drum as

Int. Res. J. Public Environ. Health 200
conductors) immersed in a water bath as condenser. The
first distillation product is collected as petrol, then
kerosene and lastly diesel, the rest is disposed off as waste.
Thus, these mixtures of petrol or diesel in kerosene or
diesel in petrol are the resulting by-products. Because it is
not fractionalized these coloured by-products are relatively
hazardous.
IRK is a clear liquid fuel with a mixture of hydrocarbon
containing 6 - 16 carbon atoms in length. It is a middle
distillate of petroleum refining process, defined as the
fraction of crude oil that boils between 145 and 3000 . It is
a complex mixture of branched and straight chained
compounds; paraffin (55.2%), naphthalene (40.9%), and
aromatics (3.99%) (USEPA, 2011). Refined petroleum
products from crude oil fractionalization include kerosene.
The main sources of IRK discharge to the environment are
leakage from surface or submerged of storage tanks and
spillages due to accidental discharge during transportation
(Raina et al., 2009). Spillages arising from kerosene
especially LRK into our environment is becoming a visible
problem and may be toxic to nitrifying bacteria and other
autochthonous soil microorganisms, thus, influencing their
growth and survival in the ecosystem. Microorganisms play
a fundamental role in the biogeochemical cycles in nature
by re-mineralizing organic matter to carbon dioxide, water
and various inorganic salts. Nitrobacter is a genus of mostly
rod-shaped, gram-negative, aerobic-nitrifying and
chemoautotrophic bacteria and cells normally reproduce by
budding (Willey et al., 2011; Holt et al., 1994). The
conversion of ammonia to nitrate is achieved by two groups
of nitrifying bacteria; the ammonia-nitrifying bacteria and
nitrate-oxidizing bacteria and depends on the activities of
at least two different genera. The first stage in ammonia
oxidation involves Nitrosomonas, Nitrosococcus,
Nitrosospira, Nitrosocystis and Nitrosogloea whilst the
second stage involves the conversion of Nitrite to Nitrate by
the genera Nitrobacter, Nitrocystis, Nitrococcus, Nitrospina,
Nitrospira (Scragy, 2005).
Nitrobacter and nitrifying bacteria play a very important
role in soil mineralization and fertility. The nitrogen
required in large quantity by plants is supplied in the form
of nitrate ion by the activities of nitrifying bacteria through
the process of nitrification (Bona et al., 2011). Driven by the
roles of Nitrobacter and nitrifying bacteria in soil and waste
water treatment plants, assessment of Nitrobacter to
pollution stress and tolerance in LRK and IRK products in
various aquatic ecosystems becomes imperative. This study
was, therefore, designed to assess the tolerance and toxicity
levels of Nitrobacter in triaquatic microcosms incorporated
with LRK and IRK products in Nigeria.
MATERIALS AND METHODS
Source and collection of samples
Three (3) water samples from various sources were
collected with one (1L) litre of sterile plastic container and
used as the tri-aquatic microcosms. They are Marine water
(MW) from Bonny River in Bonny, freshwater (FW) from
Muu Bagia Biara, Gokana LGA and Brackish water (BW)
from Eagle Island, Nkpolu-Oroworukwo, Port Harcourt City
LGA all in Rivers State.
Pollutant/toxicant samples
Local refined kerosene (LRK) was purchased at Nembe
waterfront, Creek Road whereas Industrial refined
kerosene (IRK) was purchased at NNPC Filling Station, all in
Port Harcourt, Rivers State, Nigeria.
Test Bacterium
The test bacterium, Nitrobacter species isolated from the
river water sample was obtained from a stock culture at the
Department of Microbiology, Rivers State University from
previous work using Winogradsky agar medium (Odokuma
and Nrior, 2015). The compositions of this medium were:
KNO2 (0.1g), Na2CO3 (1.0g), NaCl (0.5g), FeSO4.7H2O (0.4g),
Agar agar (15.0g), Distilled water (1000ml). The
Winogradsky agar medium was autoclaved at 121 for
15minutes and aseptically transferred to sterile Petridshes
after cooling to about 40°C. The Petridishes were then
inoculated with the river water and incubated aerobically
for 4days at room temperature (30± 2°C). Cultural and
morphological characteristics revealed; grayish, mucoid,
flat colonies and Gram’s reaction of the colonies revealed
pear shaped, and other biochemical tests for identification
of Nitrobacter were carried out as earlier reported (Colwell
and Zambuski, 1972; Okpokwasili and Odokuma, 1996).
The colonies were aseptically streaked on fresh
Winogradsky agar and incubated for 2 days at 30±2°C.
Furthermore, the grayish, mucoid and flat colonies were
aseptically transferred from the Petridishes into 200ml
Erlenmeyer flasks containing the growth medium and
incubated for 24hours at room temperature.
A solution of 0.2mg sodium nitrite (NaNO2) per litre was
autoclaved at 121°C for 15minutes and reconstituted
30±2°C with 100ml distilled water which in turn was
transferred aseptically into 250ml Erlenmeyer flasks, this
served as diluents for the effective determinations of
subculture on Winogradsky agar.
Toxicity Test Procedure for Nitrobacter species
The acute toxicity bioassays were determined for a
duration of 24hours as described in the guidelines (APHA,
1992; DPR, 2002 (formally NNPC Inspectorate Division).
The test was carried out in separate test tubes containing
appropriate volume of filtered waters; FW, BW and MW
from the organism’s habitat. For each of the experimental
set up, a toxicant in percentage (%) concentrations of 50,
25, 12.5, 6.5 and 3.25% were added into tubes later
inoculated with test organism and loosely plugged with
cotton wool and repeated for the other toxicant (Table 1).
Aliquots (0.1ml) of each concentrations of the effluent was

Nrio et al. 201
Table 1: Toxicity test set-up using industrial and local refined kerosene on Nitrobacter sp. in Freshwater (FW), Brackish water (BW) and Marine
Water (MW)
Industrial Refined Kerosene (IRK)
Local Refined Kerosene (LRK)
Microcosm
Setup Label
Concentrati
on
Volume of
Toxicant
Volume of
Diluent
Volume of
Test
Organism
Microcosm
Setup Label
Concent
ration
Volume of
Toxicant
Volume of
Diluent
Volume of
Test
Organism
1
Control (0%)
0.0ml IRK
10ml FW
1ml
19
Control (0%)
0.0ml LRK
10ml FW
1ml
2
3.25%
0.3ml IRK
9.7ml FW
1ml
20
3.25%
0.3ml LRK
9.7ml FW
1ml
3
6.5%
0.7ml IRK
9.3ml FW
1ml
21
6.5%
0.7ml LRK
9.3ml FW
1ml
4
12.5%
1.3ml IRK
8.7ml FW
1ml
22
12.5%
1.3ml LRK
8.7ml FW
1ml
5
25%
2.5ml IRK
7.5ml FW
1ml
23
25%
2.5ml LRK
7.5ml FW
1ml
6
50%
5.0ml IRK
5.0ml FW
1ml
24
50%
5.0ml LRK
5.0ml FW
1ml
7
Control (0%)
0.0ml IRK
10ml BW
1ml
25
Control (0%)
0.0ml LRK
10ml BW
1ml
8
3.25%
0.3ml IRK
9.7ml BW
1ml
26
3.25%
0.3ml LRK
9.7ml BW
1ml
9
6.5%
0.7ml IRK
9.3ml BW
1ml
27
6.5%
0.7ml LRK
9.3ml BW
1ml
10
12.5%
1.3ml IRK
8.7ml BW
1ml
28
12.5%
1.3ml LRK
8.7ml BW
1ml
11
25%
2.5ml IRK
7.5ml BW
1ml
29
25%
2.5ml LRK
7.5ml BW
1ml
12
50%
5.0ml IRK
5.0ml BW
1ml
30
50%
5.0ml LRK
5.0ml BW
1ml
13
Control (0%)
0.0ml IRK
10ml MW
1ml
31
Control (0%)
0.0ml LRK
10ml MW
1ml
14
3.25%
0.3ml IRK
9.7ml MW
1ml
32
3.25%
0.3ml LRK
9.7ml MW
1ml
15
6.5%
0.7ml IRK
9.3ml MW
1ml
33
6.5%
0.7ml LRK
9.3ml MW
1ml
16
12.5%
1.3ml IRK
8.7ml MW
1ml
34
12.5%
1.3ml LRK
8.7ml MW
1ml
17
25%
2.5ml IRK
7.5ml MW
1ml
35
25%
2.5ml LRK
7.5ml MW
1ml
18
50%
5.0ml IRK
5.0ml MW
1ml
36
50%
5.0ml LRK
5.0ml MW
1ml
plated out after 4, 8, 12 and 24hours onto Winogradsky
agar and incubated for 4days. Plates were then counted as
colony forming unit per millilitre (CFU/ml).
The Percentage Log Survival of Nitrobacter in Kerosene
The percentage log survival of the bacterial isolates in the
kerosene effluent used in the study was calculated using the
formula adopted by (Williamson and Johnson 1981; Nrior
and Obire, 2015). The percentage log survival of the
bacterial isolates in the effluent was calculated by obtaining
the log of the count in each toxicant concentrations (Log C),
divided by the log of the count in the zero toxicant
concentration ( Log c) and multiplying by 100. Thus:
% log survival = Log C ×100
Log c
The Percentage Log Mortality of Nitrobacter in
Kerosene
The formula for the calculation of percentage (%) mortality
was adopted from (APHA, 1992). The percentage (%) log
mortality was done by using the percentage (%) log
survival in zero toxicant concentration to subtract the
percentage (%) log survival. Thus: percentage (%) log
mortality = % log survival in zero toxicant concentration
(100) - percentage (%) log survival in test concentrations.
Statistical Analysis
The results 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
mortality of the test bacterium and toxicants, kerosene. The
median lethal concentrations of toxicants with respect to
bacterium with respect were calculated using regression
analysis.
RESULTS AND DISCUSSION
The Log Mortality of Nitrobacter with LRK and IRK at
different (%) concentrations; 0, 3.25, 6.5, 12.5, 25 and 50 in
triaquatic microcosms ((Freshwater, brackish and marine
water) exposed for periods of; 0, 4, 8, 12 and 24h are shown
in Figures 1-3. There is a constant decrease in the log
survival of the test species, Nitrobacter sp. which reflected
in the increase in mortality rate from 0-24hours. FW, BW
and MW samples were used in the study as a specimen to
assess the probable toxic level of LRK and IRK on
Nitrobacter which is a key environmental pollution monitor
in aquatic ecosystem.
The Lethal toxicity of IRK and LRK results also indicated
that the increased kerosene concentrations resulted in high
mortality rate of Nitrobacter which is suggestive of adverse

Int. Res. J. Public Environ. Health 202
Figure 1: Percentage (%) log mortality of Nitrobacter with IRK and LRK in freshwater (FW)
Figure 2: Percentage (%) log mortality of Nitrobacter with IRK and LRK in Brackish water (BW)
Figure 3: Percentage (%) log mortality of Nitrobacter with IRK and LRK in Marine water (MW)
negative effect on growth and survival of the species as
earlier reported (Kobeticova et al., 2012). On the other
hand, earlier Laboratory investigations have shown that
nitrifying bacteria could utilize kerosene and other
petroleum products as carbon source which may vary both
in rates of utilization and growth profile (Eze et al.,
2013a,b). Furthermore, the influence of salinity on the
sensitivity of Nitrobacter to various microcosms was

Figure 4A: Percentage Median Lethal Concentration (LC50)
of IRK and LRK on Nitrobacter in tri-aquatic ecosystem.
Figure 4B: Percentage Toxicant effect of IRK and LRK on
Nitrobacter in tri-aquatic ecosystem
Figure 4C: The Summation of Percentage Median Lethal
Concentration (∑%LC50) of IRK and LRK in the three
aquatic ecosystem combined
Nrio et al. 203
which corroborates earlier work reported (Wemedo and
Nrior, 2017).
The percentage median Lethal Concentration (%LC50) of
microcosms, toxicant effect and toxicant ratio of Nitrobacter
exposed for periods of 0, 4, 8, 12 and 24h at different (%)
concentrations of; 0, 3.25, 6.5, 12.5, 25 and 50 in LRK and
IRK are represented in Figures 4A-C. The results indicated
that IRK was more toxic than LRK (Figure 4A). Comparative
values of the various microcosms were in the order (noting;
the lower the %LC50 the more toxic the test toxicant):
Nitrobacter with IRK in FW (34.41%) < Nitrobacter with
IRK with MW (37.89%) <. Nitrobacter with LRK in FW
(39.43%) < Nitrobacter with IRK in BW (40.99%MW
(41.56%), Nitrobacter with LRK in BW (45.35%)
respectively (Figure 4A).
This study also demonstrated that the level of
purification and concentrations of un volatilized substances
in the Kerosene may be toxic and affect the test bacteria
which confirms similar observation on substances in
electronics (Hermann and Urbach, 2000). Some advantages
observed in the use of bacterial bioassay organism include;
low cost, small space, simplicity and rapidity. The use of
Nitrobacter sp. mortality rate to express as Median Lethal
Concentration (LC50) in this study as indices to monitor
toxicity has earlier been reported (Odokuma and Nrior,
2015).
Toxicity seems to be affected by the salinity of the
medium, as the kerosene shows to be more toxic in
freshwater (LC50 31%) > Marine water (33%) and least in
Brackish water (36%); noting that the lower the LC50 the
more toxic the toxicant (Figure 4B). This may be attributed
to chemical reactions with the compounds in Kerosene and
the salt likely to be present in the waters, particularly in
BW. The analytical summation of Percentage LC50 in the
three aquatic microcosms combined revealed that IRK
(∑LC50 37.6±3.29%) was more toxic to the test bacteria,
Nitrobacter sp. than in LRK (∑LC50 42.11±3.0%) (Figure
4C). Comparative evaluation of the toxicity strength gap
between the two toxicants shows a significant gap between
the toxicants. This also, may be due to combined effect of
Kerosene and the salinity in these waters. The marked
decrease in the number of Nitrobacter sp. as the Kerosene
concentration increases, suggest that components present
in the kerosene is highly toxic to Nitrobacter and may
interfere with the nitrogen cycle in the environment (Nrior
and Gboto, 2017). Futhermore, the inability of the organism
to thrive well at kerosene concentration above 1% (v/v)
may be due to enzyme inactivation at increased
concentrations. Such adverse effects may also be attributed
to sudden exposure of the organism to hostile xenotic
microenvironment which obstructed gradual recovery
within study period.
Conclusion
This study revealed that different concentrations of
kerosene had profound negative effects on growth and

Int. Res. J. Public Environ. Health 204
survivability (i.e., high mortality rate) of Nitrobacter
species. Toxicity results indicated that the sensitivity of
Nitrobacter species was a function of both the contact time
and concentrations, and also reflected lethal effects of the
pollutants/toxicants (kerosene). This study also,
demonstrated that IRK + FW microcosm was the most toxic
(LC50) and least in BW microcosms.
Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this manuscript.
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