South Asian Journal of Research in Microbiology

6(2): 10-23, 2020; Article no.SAJRM.55886

ISSN: 2582-1989

Toxic Effect of Refinery Industrial Effluent Using

Three Toxicity Bioassays
Samson Ogagaoghene Egurefa1, Micheal Uchenna Orji1
and Bright Obidinma Uba2*
1
Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, P.M.B. 5025, Awka,
Anambra State, Nigeria.
2
Department of Microbiology, Chukwuemeka Odumegwu Ojukwu University, P.M.B.02, Uli,
Anambra State, Nigeria.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors SOE, MUO and BOU designed
the study. Author BOU performed the statistical analysis and wrote the protocol. Authors SOE and
BOU wrote the first draft of the manuscript while authors SOE and MUO managed the analyses of the
study. All authors managed the literature searches, read and approved the final manuscript.

Article Information

DOI: 10.9734/SAJRM/2020/v6i230145
Editor(s):
(1) Dr. Chamari Hettiarachchi, University of Colombo, Sri Lanka.
Reviewers:
(1) Raúl Gutiérrez-Lucas, Universidad Autónoma Metropolitana, México.
(2) Sajid Farid, Pakistan.
(3) Randa Mohammed Osman, National Research Centre, Egypt.
Complete Peer review History: http://www.sdiarticle4.com/review-history/55886

Received 13 February 2020

Accepted 18 April 2020
Original Research Article
Published 20 May 2020

ABSTRACT
Aims: To determine the toxic effect of refinery industrial effluents using three toxicity bioassays.
Study Design: Five treatments and the controls designs were set up in triplicates containing
6.25%,12.5%, 25%, 50%, 100% and 0% of the industrial effluents and incubated at 24°C for 0 - 96
h. The five treatments and control set ups designated as PH, Warri and Control (Without effluent)
were used to determine the toxic effect of industrial effluents.
Place and Duration of Study: Department of Microbiology, Faculty of Natural Sciences,
Chukwuemeka Odumegwu Ojukwu University, Uli Nigeria between September, 2019 and
December, 2019.
Methodology: A laboratory scale study was carried on two composite samples of the produced
water samples from the two studied areas using physicochemical analyses, microalgal toxicity test,
mollusk toxicity and Zea mays test.
Results: The results revealed that Port Harcourt refinery effluent contains higher quantities of
physicochemical parameters than the Warri effluent sample. Warri sample had the most harmful
_____________________________________________________________________________________________________

*Corresponding author: Email: ubabright4real@yahoo.com;

 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

effects on Selenastrum capricornutum, Lymnaea stagnalis and Zea mays, with ErC50 values of
47.62%, LC50 of 51.86% and EC50 of-32.68%, respectively. Inhibition (%) and mortality (%) of all
species used were found to be concentration dependent with a significant (P < 0.05) strong
positive correlation at increasing concentrations of industrial effluents.
Conclusion: Thus, these raw industrial effluents from Port Harcourt and Warri refineries are toxic
and induced growth inhibition, mortality and phytotoxicity and adequate measures should be taken
by these industries to minimize their negative environmental impacts.

Keywords: Industrial effluents; growth inhibition; mortality; phytotoxicity and bioassay.

1. INTRODUCTION ecological quality of natural and man-made

environments. A sufficiently broad range of
Produced water (PW) is the waste usually freshwater toxicity bioassays have been devised
generated in largest volume during production of and are to date used in global practice. The
oil and gas from offshore oil and gas wells [1]. applicability of toxicity bioassays as efficient
Most PW is fossil water (formation water) that analytical tools should be supported by their
has accumulated over millions of years with fossil standardization and validation [5].
fuels in geologic formations deep in the earth.
PW also may contain some surface water that Algae are often included among the species
has been injected into the formation for used in biotest batteries for hazard assessment
enhanced oil recovery. PW reaches the surface of chemically contaminated wastes and
from natural oil seeps worldwide and during leachates. As primary producers, changes in the
production of oil and gas from a well [1]. The structure and productivity of the algal community
chemical characteristics of PW are different for may induce direct structural changes in the rest
each production platform or formation from which of the ecosystem and/or indirectly affect the
the oil is extracted. It is typically highly saline and ecosystem by affecting water quality. It is
contains elevated levels of heavy metals, therefore crucial to assess the toxicity of
hydrocarbons (including polycyclic aromatic chemicals to algae as the pollution is likely to end
hydrocarbons (PAHs)), alkylphenols, ammonia up in water bodies via industrial or household
and radionuclides compared to the receiving waste [6]. Selenastrum capricornutum was
environment [2,3]. selected in this study because it is a model
organism recommended by ISO [7].
In Nigeria, the Department of Petroleum Among aquatic organisms used in
Resources (DPR), a Regulatory body of the Oil ecotoxicological studies, invertebrates have been
and Gas Industry in Nigeria has stipulated employed due to their importance in trophic
guidelines and standards for the management chains and greater sensitivity response to
and discharge of Produced water and has set chemical pollutants. Although, mollusks are the
limits within which waste water generated from second largest group in kingdom Animalia, they
the activities of the petroleum industry in Nigeria have not been considered in environmental risk
must meet. This is prior to its discharge into the assessment so far, mainly due to the lack of
aquatic ecosystem (brackish and saline water). standardized protocols. In this sense,
In an endeavour to operate within these gastropods, the most abundant mollusks, have
stipulated regulatory limits, most oil companies been successfully used as pollution indicators by
treat their wastewater before they are discharged different compound classes, such as, metals,
into the environment. Nevertheless, studies have pesticides and polluted waste water [8]. The use
revealed that some of the treated produced water of Lymnaea stagnalis which are fresh water
do not meet the DPR limits with respect to some snails by several researchers have demonstrated
of the parameters, before being discharged into that this species is a good model for laboratory
the surrounding [4]. and monitoring environmental studies, very
common, readily available, cheap and globally
Biotesting for environmental samples is integral distributed especially here in Nigeria [9–13].
to environmental regulations in many countries,
including Nigeria. Lab testing, involving Among the bioassays developed for detection of
application of ‘short-term bioassays’ gives results mutagenicity, genotoxicity, cytotoxicity,
that complement bioindication research data and phytotoxicity and clastogenicity due to
improve credibility of biodiagnostics of the environmental pollutants, plant systems have

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proven to be sensitive, cheap, and effective. effluents samples were prepared by centrifuging
Plant bioassays, which are mostly sensitive for and filtering the suspensions through glass fiber
the detection of phytotoxicity, may provide a filters (d = 0.45 μm) using membrane filtration
warning of environmental hazards in the water technique prior to the physico – chemical and
[14]. Zea mays test has demonstrated that toxicological assays [6,18–20].
germination, root elongation and shoot length are
the most authoritative parameters that indicate 2.3 Physicochemical Analysis
changes in environmental quality [15–17].
The following analysis were carried out on the
Several studies and literatures abound on the samples: temperature, pH, conductivity and total
effects of refinery industrial effluents on dissolved solids, total suspended solids, total
microorganisms, invertebrates, plants and solids, turbidity, total hardness, total chloride,
animals but there is paucity of information nitrate, phosphate, sulphate, total alkalinity,
regarding their toxicities on Selenastrum dissolved oxygen, biochemical oxygen demand
capricornutum, Lymnaea stagnalis and Zea mays (BOD5)and chemical oxygen demand, oil and
especially in our country Nigeria and hence, grease content according to the method of APHA
necessitated this study. So, this is the first study [21]. Also, oil and grease content, total petroleum
to be carried to the best of our knowledge here in hydrocarbon (TPH), total hydrocarbon content
our country Nigeria. This study was undertaken (THC) and total phenol were determined by
to determine the toxic effects of refinery industrial adopting the methods of Kiepper [22], Akpveta et
effluents on Selenastrum capricornutum, al. [23], Nwineewii and Azuonwo [24] and
Lymnaea stagnalis and Zea mays using a battery Leouifoudi et al. [25]. The heavy metals such as
of bioassays. iron, mercury, copper, chromium and lead) were
determined by atomic absorption spectrometry
2. MATERIALS AND METHODS following the method of APHA [21].

2.1 Description of the Studied Area 2.4 Microbiological Analysis

The studied areas were Port Harcourt Refining 2.4.1 Determination of total cultural
Company (PHRC) located in Alesa-Eleme, in heterotrophic bacterial and fungal
Eleme Local Government Area of Rivers State count (TCHBC/TCHFC)
with coordinates 4o 44’N and 6o 49’E and Warri
Refinery and Petrochemical Company (WRPC) Total heterotrophic bacterial and fungal counts
located in Ubeji Community in Warri, Warri South for each effluent samples were enumerated
Local Government Area of Delta State with using spread plate method as described by
o o
coordinates 5 33’N and 5 42’E, respectively. Willey et al. [26]. An aliquant (0.1 mL) of the
4
dilution of 10 were aseptically transferred unto
2.2 Sample Collection and Preparation properly dried Nutrient Agar and Sabouraud
Dextrose Agar plates containing antibiotic
Ten random samples of the refinery industrial (tetracycline) to inhibit bacterial growth in
effluents (produced water) were collected from duplicate, spread evenly using bent glass rod
the ten (10) designated points of the two and incubate at 28°C for 24 h and at 28°C for 3
sampling sites. The samplings were done once in days, respectively. After incubation, the bacterial
each of the two sampling sites in August, 2019. and fungal colonies that grew on the plates were
The samples were mixed together to obtain a counted and average counts were taken. The
total of two representative samples which were colony forming unit for the THBC of effluent
used for the analysis. The water samples were samples were then calculated using the formula;
collected at the air-water interface by hand THFC (CFU/mL) = Number of Colonies x Dilution
dipping of the sterile and clean 2 L plastic 4
factor (10 ) x volume plated (0.1 mL) [27].
containers. The containers were aseptically
rinsed with the samples thrice before collection. 2.4.2 Determination of hydrocarbon utilizing
All the representative sample containers were bacterial and fungal count
labelled with sample type, date, time, and place (HUBC/HUFC)
of collection. They were placed into a sterile
polythene bags in ice packed coolers and then The vapour phase method was used to
transported to the Microbiology Laboratory, determine the HUBC and HUFC on mineral salt
Chukwuemeka Odumegwu Ojukwu University, agar containing 0.04 g MgSO4. 7H2O, 0.03 g
Uli Campus, Nigeria. The refinery industrial KCl, 0.09 g KH2PO4, 0.04 g NaNO3, 0.13 g

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

K2HPO4, 2.0 g NaCl, 15 g of agar powder, 100 D3165, West Germany) at 23 ± 2°C under
mL of distilled water amended with 0.1 g of continuous white fluorescent lamps. After the 3
nystatin to inhibit the growth of fungi and days of incubation, the suspension was placed in
tetracycline to inhibit bacteria [27]. Then, it was the spectrophotometer and the optical density
sterilized by autoclaving at 121°C and 15 psi for (OD) was read at 700 nm after 10 seconds. The
15 min and allowed to cool to about 45°C. The number of alga corresponding with OD was
already prepared medium was poured into Petri interpolated from the optical density/algal number
dishes and allowed to gel, then 0.1 mL of the (OD/N) standard curve and was used to
inocula was spread on plates with rod determine the dilution factor needed to reach an
aseptically. Filter paper (Whatman No 1) was optical density equal to OD2, corresponding to
4
saturated with crude oil and the crude oil an algal density of 5 x 10 cells/mL. The algal
impregnated papers were aseptically placed onto suspension from the flask was transferred into a
the Petri covers. The crude oil saturated filter 100 mL sterile flask and the volume of algal
papers supply diesel by vapour - phase transfer growth medium needed to make up a 5.104 algal
to the inocula. The plates were incubated by cells/mL suspension was added and the flask
inversion for 7 to 10 days at 28°C. Plates yielding shaken thoroughly to distribute the algae evenly.
30 to 300 colonies were enumerated from
triplicates and mean values were recorded and 2.5.1.3 Choice of test sample concentration and
calculated in CFU mL-1. preparation

2.5 Toxicity Bioassay The microalga cells were exposed to

concentrations of the test sample in a geometric
2.5.1 Algal growth inhibition test with series with a ratio not exceeding 2.0 as follows:
unicellular green algae 0%, 6.25%, 12.5%, 25%, 50%, and 100%. The
Port Harcourt and Warri samples were prepared
The algal growth inhibition test was carried out as previously described in 3.2 above after which
according to the methods of ISO [7] with little nutrient stock solutions 1, 2, 3, and 4 were added
modifications. to the samples as prepared in accordance with
3.5.1.1. A quality control test was carried out with
2.5.1.1 Preparation of growth medium the reference chemical potassium dichromate
(K2Cr2O7) having toxicant dilution series: 1.0
The growth medium was prepared by adding an mg/L, 10 mg/L, 18 mg/L, 32 mg/L, and 56 mg/L.
appropriate volume of the nutrient stock solutions
to water. Then, add to approximately 500 mL of 2.5.1.4 Preparation of test and control batches
water, 10 mL of stock solution 1 (NH4Cl,
MgCl2.6H2O, MgSO4.7H2O and KH2PO4); 1 mL of
The test batches were prepared by mixing the
stock solution 2 (FeCl3.6H2O, Na2EDTA.2H2O); 1
appropriate volumes of test sample, growth
mL of stock solution 3 (H3BO3, MnCl2.4H2O,
medium and inoculum in all the test vessels.
ZnCl2,CoCl2.6H2O, CuCl2.2H2O, Na2MoO4.2H2O)
Three replicate batches for each test sample
; 1 mL of stock solution 4 (NaHCO3) and finally
concentration and six replicates control batches
made up to 1000 mL with water. Before use, the
were prepared by adding the appropriate volume
medium was equilibrated overnight, adjusted to
of inoculum to growth medium. The pH of a
8.1 ± 0.2 using 1 mol/L sodium hydroxide
replicate batch at each test concentration and in
solution and finally buffered with hydrogen
one control replicate were measured.
carbonate.

2.5.1.2 Preparation of pre-culture and inoculum 2.5.1.5 Incubation and measurement

One of the two tubes containing the microalga The test vessels were covered with lids to avoid
(Selenastrum capricornutum) inoculum was airborne contamination and to reduce water
taken, shaken vigorously and the content was evaporation and then incubated at 23 ± 2℃
poured into 100 mL flask.The (same) tube was under aeration and continuous white fluorescent
rinsed twice with 7.5 mL algal growth medium lamps for 72 h. The cell density in each test
and the content was transferred into the flask to batch (including the controls) were measured at
ensure the total transfer of the microalga 0, 24, 48 and 72 h by mixing the test batches
inoculum. The flask was closed with the lid and prior measurement. At the end of the test, the pH
incubated for 3 days in an incubator (Kottermann of the samples of at least one replicate batch at

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each test sample concentration and one control the methods described by Sheir [28] and Atlia
replicate were measured. and Grosell [13].

2.5.1.6 Calculation 2.5.2.2 Adult snail exposure assay

2.5.1.6.1 Plotting of growth curve Static-renewal acute toxicity assays were
The cell density measurements for each test performed with adult snails (Lymnaea stagnalis)
batch was tabulated according to the at least four months old and with shell diameter
concentration of the test sample and the duration of 20 mm were not fed 24 h before test start. The
of measurement. Growth curve for each test mortality before assay start did not exceed 10
concentration and control was plotted as a graph percent. The animals were exposed to five Port
of the logarithm of the mean cell density against Harcourt and Warri effluents concentrations of
time. The mean growth rate, specific growth rate 6.25, 12.5, 25, 50 and 100% and a negative
and growth inhibition was calculated using the control group that was exposed to only tap water
formula: under the same experimental conditions. The
chosen lethal concentrations were determined by
previous work done by Tallarico et al. [8].
Exposures were performed for 96 h in triplicate
for a total of 10 snails per treatment. After 24 h of
exposure, snails were washed and observed
where: t0 is the time of test start; tL is the time of daily (24, 48, 72 and 96 h). Mortality and
test termination or the time of the last abnormal responses, as retraction or extending
measurement within the exponential growth of body and release of mucus and hemo- lymph,
period in the control; No is the nominal initial cell were recorded at each observed time. The
density; NL is the measured cell density at time median lethal concentration (LC50) of the
tL. Alternatively, it was also calculated from the samples was thus determined.
slope of the regression line.
2.5.3 Zea mays test

The modified guidelines for the testing of

effluents on seeds of terrestrial plants according
Where: Iμi is the percentage inhibition (growth to the Organization for Economic Cooperation
rate) for test concentration I; μi is the mean Development (OECD) [29] were used for this
growth rate for test concentration i; μc is the study. First, floatation method was adopted to
mean growth rate for control c. The effective determine the viability of the seeds. The seeds
concentration (ErCx) was determined by that sink represent the viable seeds while the
tabulating and plotting the normalized inhibition seeds that float represent seeds the non – visible
(Iμi) against the test concentration on the seeds. Surface sterilization of the viable seeds
logarithmic scale. was then performed by immersing the seeds into
2.5.2 Mollusc toxicity test 70% ethanol for 5 minutes to reduce
contamination and finally washed thoroughly with
2.5.2.1 Laboratory animal water. Ten maize seedlings were placed in Petri
dishes lined with tissue paper containing 30 mL
The freshwater snails, (Lymnaea stagnalis) were each of the effluent (Port Harcourt and Warri) in
collected from Omor River in Ayamelum Local different concentrations (6.25%, 12.5%, 25%,
Governement Area of Anambra State, Nigeria 50% and 100%), while the Petri dishes lined with
(body weight = 23.73 ± 0.21 g) and transported tissue paper containing 30 mL of distilled water
in field water to the laboratory. The snails were served as the control. The investigation of each
kept after two weeks collection for adaptation in concentration including the control was carried
aerated and filtered tap water contained in 5 L out in triplicates. The Petri dishes were covered
per six 10 L aquarium containers (dimension: 43 with lids to prevent evaporation. The seeds were
cm X 32 cm x 25 cm). The snails were planted under room temperature for seven (7)
maintained under laboratory conditions (BOD = days, after which the seed germination (%),
11.30 mg/L, temperature = 28.30°C, pH = 7.90) shoot length (cm), root length (cm), relative root
with photoperiod 12 h light: 12 h dark. The length percentage, germination index (%) and
culture was fed fresh carrots ad libitum and a vigour index were measured and calculated [30].
balanced diet before toxicity studies according to The shoot length measurement was taken from

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

the base to the apical leaf of the plant using a 3. RESULTS

transparent plastic ruler; while the root length
was also measured by the same procedure after 3.1 Physicochemical Profile
it was harvested and carefully washed with
distilled water. The result of the physicochemical qualities of the
refinery produced discharge water is presented
2.6 Analysis of Data in Table 1. From the result, the Port Harcourt
(PH) produced water had the highest values of
The data obtained in this study were subjected to conductivity, TDS, turbidity, TS, total Chloride,
descriptive statistics, one - or two-way ANOVA nitrate, temperature, phosphate, sulphate, DO,
with Dunnet multiple comparison, paired two BOD, COD, oil and grease, TPH, THC, TCHBC,
tailed T – test and Pearson coefficient of TCHFC, copper, mercury, lead and phenol while
variation among the samples, controls and Warri sample had the highest values of pH, total
concentrations at 95% confidence intervals such hardness, total alkalinity, TCHB, and iron. Both
that values lower than 0.05 probability level are samples had too low to count (TLTC) in their
considered statistically significant (P < 0.05). hydrocarbon utilizing bacteria count (HUBC) and

Table 1. Physicochemical qualities of the refinery produced discharge water

Parameters PH sample Warri sample DPR standard

PH 5.30 6.30 6.50 - 8.50
Conductivity (µS/cm) 460.00 350.00 -
Temperature (℃) 28.90 28.60 30.00
Turbidity (NTU) 1898.00 193.25 15.00
TDS (mg/L) 345.00 262.50 5000.00
TSS (mg/L) 39.50 42.70 50.00
TS (mg/L) 384.50 304.70 -
Total hardness (mg/L) 420.00 465.00 -
Total Chloride (mg/L) 97.63 44.40 2000.00
Nitrate (mg/L) 26.96 10.02 -
Phosphate (mg/L) 3.91 3.69 -
Sulphate (mg/mL) 617.40 411.60 200.00
Total alkaline (mg/L) 340.00 490.00 -
DO (mg/L) 16.20 12.80 -
BOD (mg/L) 262.00 22.00 125.00
COD (mg/L) 393.00 33.00 125.00
Oil and grease (mg/L) 51,900.00 7,900.00 40.00
TPH (mg/L) 18,270.00 17,590.00 -
THC (mg/L) 11,390.00 9,296.00 40.00
Phenol (GAE) 594.52 441.85 -
5 5 -
TCHBC (CFU/mL) 4.1x10 9.1X10
TCHFC(CFU/mL) 3.2x105 TLTC -
HUBC (CFU/mL) TLTC TLTC -
HUFC (CFU/mL) TLTC TLTC -
Iron (ppm) 3.13 3.77 1.00
Chromium (ppm) 0.07 0.03 0.50
Mercury (ppm) 0.03 0.02 0.00
Lead (ppm) 0.68 0.79 -
Copper (ppm) 0.98 0.94 -
Key: PH = Port Harcourt, TDS=Total dissolved solid, TSS = Total suspended solid, TS=Total solid,
DO=Dissolved oxygen, BOD=Biochemical oxygen demand, COD=Chemical oxygen demand, TPH=Total
petroleum hydrocarbon, THC=Total hydrocarbon content, TCHBC = Total culturable heterotrophic bacterial
count, TCHFC = Total culturable heterotrophic fungal count, GAE= Gallic acid equivalent, ppm= Part per million,
CFU/ml=Colony forming unit per milliliter, mg/L=Milligram per liter, µS/cm= micro Siemen per centimeter,
°C = Degree centigrade, NTU=Nephelo transmitting unit, HUBC= Hydrocarbon utilizing bacterial count,
HUFC = Hydrocarbon utilizing fungal count, DPR = Department of Petroleum Resources, TLTC = Too low to
count

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utilizing fungi count (HUFC) while total culturable optimum mortality rate at all the concentrations
heterotrophic fungi count (TCHFC) in Warri with LC50 value of 51.86% after 72 h exposure.
sample is TLTC. Also, the toxic effect of refinery discharged
effluents to Lymneae stagnalis after 72 h
3.2 Acute Toxicity Profile exposure is shown in Fig. 3. From the result,
Warri sample had higher percentage mortality at
3.2.1 Algal toxicity studies all the concentration while PH sample recorded
optimum toxicity at increase concentrations (0 –
The results of the algal growth inhibition by
100%).
refinery discharge effluents and potassium
dichromate as well as 72 h effective growth rate
3.2.3 Zea mays toxicity profile
concentration of refinery discharge effluents and
potassium dichromate on Selenastrum
The results of the growth features of Zea mays
capricornutum are shown in Figs. 1 and 2. From
seedlings exposed to Port Harcourt and Warri
the results, 100% concentration of Warri sample
refinery discharged effluent are presented in
had the highest percentage (%) inhibition
Tables 4 and 5. From the results, 100% and
(65.84%) while PH sample had the lowest
6.25% PH effluent sample had the lowest and
percentage (%) inhibition (65.85%). Also, PH and
highest GI and VI values of 13.11%, 86.94%,
Warri samples had the highest and lowest ErC10,
1.10 and 12.53, respectively. Similarly, 100 %
ErC50, ErC90 values of 10.53%, 52.58%, 94.74%
and 6.25% Warri effluent sample had the lowest
and 9.52%, 47.62%, 85.71%, respectively.
and highest GI and VI values of 7.55%, 76.85%,
3.2.2 Mollusc toxicity profile 0.83 and 10.53, respectively. The result of the
toxicity threshold concentrations of refinery
The toxic responses of Lymnaea stagnalis to PH discharged effluents on Zea mays seedlings is
and Warri samples refinery discharged effluents shown in Fig. 4. From the Figure, Warri and PH
are shown in Tables 2 and 3. From the results, samples had the highest and lowest EC10, EC50
there was a higher mortality rate in PH sample at and EC90 values of - 80.40%, - 32.68%, 15.04%
100 % and 50 % concentrations with LC50 value and - 87.54%, - 37.56% and 12.43%,
of 85.47 % while Warri sample recorded an respectively.

Fig. 1. Algal growth inhibition by refinery discharge effluents and potassium dichromate

Table 2. Toxic response of Lymnaea stagnalis to Port Harcourt refinery discharged effluent

Concentration Mortality rate Total (%) Mean ± S.D LC50 (%) (CL)
(%) 0h 24 h 48 h 72 h 96 h
100 0 3 3 4 4 14 (46.7) 3.5±0.27
50 0 2 3 3 4 12 (40.0) 3±0.50
25 0 1 2 2 4 9 (30.0) 2.25±51.11 85.47 (-50.3 - 29)
12.5 0 1 2 2 2 7 (23.3) 1.75±1.36
6.25 0 0 1 1 2 4 (13.3) 1±1.92
0 0 0 0 0 1 1 (3.33) 0.25±0.00

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

Fig. 2. 72 h effective growth rate concentration of refinery discharge effluents and potassium
dichromate on Selenastrum capricornutum

Table 3. Toxic response of Lymnaea stagnalis to Warri refinery discharge effluent

Concentration Mortality rate Total Mean ± SD LC50 (%) (CL)

(%) 0h 24 h 48 h 72 h 96 h (%)
100 0 1 1 9 9 20 (66.7) 5 ± 1.79
50 0 1 1 9 9 20 (66.7) 5 ± 1.79 51.86 (- 624 - 136)
25 0 1 0 9 10 20 (66.7) 5 ± 1.69
12.5 0 0 0 10 10 20 (66.7) 5 ± 1.58
6.25 0 0 0 8 8 16 (53.3) 4 ± 1.06
0 0 0 0 0 1 1 (3.3) 0.25 ± 0.00

90
80 y = 0.3391x + 42.955
R² = 0.2528 Warri
70
60
y = 0.3874x + 13.595 PH
50
Mortality (%)

R² = 0.8014
40 Linear ( Warri)
30
20 Linear (PH)
10
0
0 20 40 60 80 100 120
Concentration (%)

Fig. 3. Toxic effect of refinery discharge effluents to Lymnae stagnalis after 72 h of exposure

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

Table 4. Growth features of Zea mays seedlings exposed to Port Harcourt refinery discharged
effluent

Concentration (%)
Control 6.25 12.5 25 50 100
Number of seed germinated 9 8 6 5 3 2
Root length (cm) 5.00 4.94 4.30 4.01 3.50 2.95
Shoot length (cm) 10.00 9.30 7.00 6.00 3.7 2.00
% relative seed germination - 88.00 66.67 55.56 33.33 22.22
% relative root growth - 98.80 86.00 80.20 70.00 59.00
Germination Index (GI) - 86.94 57.34 44.56 23.33 13.11
Vigour index (VI) - 12.53 7.53 5.56 2.40 1.10

Table 5. Growth features of Zea mays seedlings exposed to Warri sample refinery discharged
effluent

Concentration (%)
Control 6.25 12.5 25 50 100
Number of seed germinated 9 7 6 4 2 1
Root length (cm) 5.00 4.94 4.70 4.20 3.95 3.40
Shoot length (cm) 10.00 8.60 6.30 6.30 4.70 4.07
% relative seed germination - 77.78 66.67 44.44 22.22 11.11
% relative root growth - 98.80 94.00 84.00 79.00 68.00
Germination Index (GI) - 76.85 62.67 37.33 17.55 7.55
Vigour Index (VI) - 10.53 7.53 7.33 1.92 0.83

pH sample Warri sample

12.43 15.04

EC10 EC50 EC90

-37.56 -32.68

-80.4
-87.54

Fig. 4. Toxicity threshold concentrations of refinery discharged effluents on Zea mays

seedlings

4. DISCUSSION processes. This research is aimed at

ascertaining the degree of toxicity of the effluents
Many industries particularly refinery industries in on Selenastrum capricornutum, Lymnaea
Port Harcourt and Warri areas of Niger Delta stagnalis and Zea mays and to suggest ways to
produce effluents during production processes. possibly curb the menace. From Table 1, the
These effluents are discharged most times physicochemical analysis revealed that the
indiscriminately into the aquatic and terrestrial samples are moderately acidic with a moderate
ecosystems without adequate detoxification temperature. This could be attributed to a high

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

content of acidic salts and compounds in the mercury and phenol, while Warri sample had a
samples. The presence of these soluble organic higher TSS, total hardness, total alkalinity,
salts and ions also made the samples to have TCHB, iron, and lead. From these results, it is
high conductivity values. The concentration of obvious that the two samples are highly
DO is low and could results to respiratory contaminated even though PH sample proved to
malfunction in aquatic organisms due to stress be more contaminated than the Warri sample.
caused by the effluents. The high BOD and COD These chemicals such as chloride, phosphate,
levels showed that a high biological activities and nitrates and sulphates can result to pollution and
chemical oxidation occurred in the samples make water hazardous for drinking, entertaining
resulting to decomposition of organic events and fishing activities when they are in
compounds. The BOD determines only the high quantity [35]. They also encourage the
decomposable organics and needs a reasonably growth of bacteria and substantial algae blooms
long period of time to obtain desired results. [33]. AIso, Okereke, and Nnoli [36] reported that
Also, the COD test processes the oxygen high content of organic and inorganic phosphates
correspondent of the biological substances in results to eutrophication and either
waste water that can be oxidized chemically. The disappearance or shallowness of some surface
COD will always be greater than the BOD, water bodies. It is imperative to note that the
because the COD checks materials that are both samples exceeded most of the DPR standard
chemically and biologically oxidized. The ratio of limits for produced wastewater discharge which
COD: BOD gives a vital guide to the amount of means that the environment where these
organic material present in waste water [31,32]. effluents are discharged is unsafe for inhabitation
Okereke et al. [33] also reported that the dissolve of living organisms. The physicochemical results
oxygen is beneficial in aerobic respiration of of this study revealed that the effluents contain
organisms which supports the observation made chemicals mainly heavy metals and
in this research work. The total hardness showed hydrocarbons which are capable of altering the
2+
high presence of magnesium ion (Mg ) and normal concentration of a given habitat.
2+)
calcium ion (Ca . The samples also contain Considering the standard limits for produced
linear and branched chains of aromatic and water by the Department of Petroleum
aliphatic hydrocarbon fragments as showed in Resources (DPR) [37], the physicochemical
the THC and TPH. The samples contain high oil qualities of both samples (PH/Warri) vary
and grease as well as phenol content which may tremendously but similar results were recorded
be attributed to the high THC, TPH and phenolic by Ajuzieogu et al. [18]. The following
compounds used and produced during refining parameters have their contents greater than the
processes. This increase had led to the selection DPR standard limits: oil and grease, BOD, COD,
of bacteria and fungi (though low counts) that turbidity and THC while TDS, TSS, total chloride,
can utilize or metabolize the hydrocarbons as and chromium. From the result, the BOD and
carbon and energy sources known as COD of Warri sample are lower than the DPR
hydrocarbon utilizers. The findings are similar to standard limits while that of PH is higher.
the works published by Okoro [34] and Ajuzieogu
et al. [18] who both reported that the low The results in Figs. 1 and 2 revealed that there
microbial counts revealed that there is paucity of was an exponential decrease in the algal cell
nutrients in oil produced water thereby counts which intensified as the time of exposure
supporting low population counts. The turbidity of increases (24 - 72 h). The decrease in the cell
the samples is high which shows that there is a number of algae when exposed to the samples
high rate of impurities and dissolved substances. showed that an unfavorable condition which
This is capable of increasing the BOD and COD decreased the rate of metabolism had been
of the samples as revealed above. The high TDS created. The cells were unable to withstand the
values of both samples correlated with the dynamic nature of the effluents which eventually
conductivity findings above as TDS is a led to a distinct reduction in proliferation. This
surrogate value of salinity and conductivity. shows that the both samples (Warri / PH) sample
There were lower values of total chloride, nitrate, had a high hydrocarbon and heavy metal
phosphate, sulphate, total alkalinity but higher contents even though PH sample was a bit
values of the heavy metals analyzed. higher. The specific growth rate decreases as the
Comparatively, PH sample had a higher turbidity, concentration of the samples increases which
conductivity, acidity, TDS, TS, total chloride, means that there is a decrease in cell density per
nitrate, phosphate, sulphate, DO, BOD, COD, oil time. This can be expressed in reciprocal days
-1
and grease, TPH, THC, copper, chromium, (day ) [7]. The average growth rate (3.61),

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

2
coefficient of variation (4.7 %) and pH (9.10) of samples and control with R value of 0.207. The
our control replicates met the validity criteria of likely reason behind this could be as a result of
International Standard Organization [7] which the presence of heavy metals especially
states that the average growth rate, variation chromium present in the both samples.
coefficient (CV) and pH in the control replicates Hexavalent chromium has been widely examined
-1
shall be at least 1.4 d , not more than 5% and for toxicity and used as reference chemical
1.5 relative to the pH of the growth medium. This compound in ecotoxicological studies to different
growth rate corresponds to an increase in cell aquatic organisms such as fresh water snails.
density by a factor 67 in 72 h. The results further Prato et al. [41] reported that mercury was more
showed that Warri sample proved to be the most toxic to Gammarus aequicauda, Corophium
harmful effluent (ErC50 47.62%) while other insidiosum, Idoteabaltica, Sphaeromaserratum,
samples also had a harmful effects on the growth and Mytilus galloprovincialis than copper and
rate of the algal species according to GESAMP cadmium. Tallarico et al. [8] reported that
[38] and CSP [39] toxicity classification system WWWTP water samples showed acute toxicity to
which states that: highly toxic - LC50/EC50< 1 B. glabata adult snails in the samplings II and III;
mg/L, toxic- 1 mg/L < LC50/EC50 ≤ 1 mg/L, samplings II were toxic to snails (LC50 = 41.25 %)
harmful/hazardous for aquatic environment - 10 and sampling III was slightly toxic (LC50 =
mg/L < LC50/EC50 ≤ 100 mg/L, very low toxic – 84.16%) and no toxicity was observed in the
non – toxic - LC50/EC50 > 100 mg/L. There were sampling IV.
statistically significant differences detected (P <
2
0.05) among the samples and control with R Several researchers have demonstrated that
value of 0.992. Similar observation was made by germination, root elongation and shoot length are
Yamagishi et al. [40] who reported that the the most authoritative parameters that indicate
growth of a green alga decreases in the changes in environmental quality [42–44] and the
presence of a toxic substance in high results in Tables 4 and 5 showed that there was
concentration but Prato et al. [41] discovered that general and remarkable decrease in all the
some aquatic organisms such as Sphaeroma growth features of the Zea mays seedlings
serratum can tolerate some toxicants (Cu, Cd exposed to both effluents. The inhibition of root
and Hg). and shoot growth was concentration dependent
and statistically significant (P < 0.05) at the
Further study was carried out to ascertain the tested concentrations of PH and Warri samples
environmental effects of the effluents using in comparison with their controls with R2 values
Lymnaea stagnalis (freshwater snail) as of 0.6256 and 0.7067, respectively. The reason
bioindicators for water monitoring. The for these decreases and differences could be
standardization and justification of this native due to the accumulations and magnifications of
species for ecotoxicological studies are heavy metals and hydrocarbon components
applicable mainly for West Africans where present in the samples by the root cells of these
dogmas and protocols concerning the bio – indicators which further affect the growth
environments are still quite primitive compared to and structures of the seed embryos negatively.
other countries. The results in Tables 2 and 3 The result in Fig. 4 revealed that Warri sample
depict several changes which occurred in exhibited the most growth inhibitory effect on
movement, retraction, production of mucus, and 50% population of Z. mays with EC50 value of -
mortality. These features increased with time and 32.68%. Using two tailed paired T-TEST, there
concentration in Warri sample while PH sample was no significant difference (P > 0.05) between
recorded a moderate mortality rate at every time PH and Warri samples revealing that both
and concentration. When the molluscs were samples had equal and significant harmful
supplied with a vitamin source, similar mortality effects on the root and shoot growths of the Zea
was recorded. This showed that the refinery mays seedlings. Our result was validated by the
industrial effluents contained a high hydrocarbon research work of Odutayo et al. [44] who
content which cannot be metabolized by the reported that rate of growth and length of roots
molluscs. The result in Fig. 3 demonstrated that differed according to effluent concentrations of
the Warri sample exhibited the most harmful industrial effluent with the lowest shoot and root
effect on 50% population of Lymnaea stagnalis growth occurring at 100% concentration and the
with LC50 value of 51.86% in accordance with the highest growth seen at the control. A similar
GESAMP [38] and CSP [39] acute toxicity study was been conducted by Gvozdenac et al.
criterion. Statistically, there was no significant [45] where germination, root and shoot length of
difference detected (P > 0.05) among the selected plants were used as indicators of water

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 Egurefa et al.; SAJRM, 6(2): 10-23, 2020; Article no.SAJRM.55886

quality and also by Orhue et al. [46] where they and phenols to algae Pseudokirchneriella
studied the effect of brewery effluents on the subcapitata and bacteria Vibrio fischeri:
growth of maize crop. Comparison with published data and
QSARs. Chem. 2011;84(10):1310–1320.
5. CONCLUSION 7. ISO. Water quality- Fresh water algal
growth inhibition test with unicellular green
This research study has shown that untreated algae, ISO (2012) 8692:2012 (E),
industrial effluents from refiners are highly toxic Case postale 56.CH-1211. Geneva,
to plants, invertebrates and microorganisms due Switzerland: International Standard
to their high hydrocarbon, heavy metals and Organization; 2012.
other pollutant contents. The toxicity values > 1 8. Tallarico LD, Borrely S, Hamada N,
EC50/ECr50/LC50 < 100 obtained in this study Grazeffe VS, Ohlweiler FP, Okazaki K,
are in line with other toxicity data on untreated Granatelli AT, Pereira IW, Pereira CA and
refinery industrial effluents. Also, appropriate Nakano E. Developmental toxicity, acute
care should be employed because effect toxicity and mutagenicity testing in
observed particularly in Zea mays can also occur freshwater snails Biomphalaria glabrata
in plants when exposed to the effluents. (Mollusca: Gastropoda) exposed to
chromium and water samples. Ecotox
COMPETING INTERESTS Environ Saf. 2014;110:208–215.
9. Pyatt AJ, Pyatt FB, Pentreath VW. Lead
Authors have declared that no competing toxicity, locomotion, and feedingin the
interests exist. freshwater snail, Lymnaeastagnalis (L.).
Invertebr. Neurosci. 2002;4:135–140.
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