96Hrs Acute Toxicity Testing of a Leak Detector, LD/TT/2021/002 against

Freshwater Fish - Oreochromis niloticus and Brackish water Shrimp -

Palaemonetes africanus under Nigerian Condition

Prepared for

DEPARTMENT OF PETROLEUM RESOURCES,

7, Kofo Abayomi Street,
Victoria Island, Lagos

DPR’s new address is:

7 Sylvester Ugo Crescent, Off Awolowo Way,
Jabi District, Abuja

By

ETINA ENVIRONMENTAL SERVICES LIMITED

34, Anite Crescent, Off Okota Road,
Isolo, Lagos.

FEBRUARY, 2021

1 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 TABLE OF CONTENT

Page

Executive Summary.................................................................................................................................3
List of Tables...........................................................................................................................................4
List of Plates............................................................................................................................................5
List of Abbreviations and Acronyms…...................................................................................................6
List of Preparers.......................................................................................................................................7
Acknowledgment.....................................................................................................................................8

CHAPTER ONE
1.0 INTRODUCTION......................................................................................................................9
1.1 Background
1.2 Aim and Objectives
1.3 Standards and Compliance

CHAPTER TWO
2.0 MATERIALS AND METHOD................................................................................................13
2.1 Description and Acclimatization of organisms
2.2 Collection and Transportation
2.3 Equipment and Materials
2.4 Physicochemical Analysis of LD/TT/2021/002 and Test Habitat
2.5 Methodology for the 96hour Acute Toxicity Bioassay
2.6 Quality Assurance/Quality Control

CHAPTER THREE
3.0 RESULTS.................................................................................................................................18 15
3.1 Physicochemical Analysis
3.2 Range Finding Test
3.3 Bioassay Result of LD/TT/2021/002
3.4 LC50 Values and Toxicity Factor

CHAPTER FOUR
4.0 DISCUSSIONS.........................................................................................................................22 18
4.1 Acute Toxicity of LD/TT/2021/002

CHAPTER FIVE
5.0 CONCLUSION.........................................................................................................................23 19
5.1 Recommendation
References
Appendices
Standards and compliance not in the report (omitted)
Page numbers should be given, References and Appendices are not part of Chapter Five

2 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 EXECUTIVE SUMMARY

The test chemical, LD/TT/2021/002 is a pinkish powder with a density of 10mg/L. It has an
unpleasant odour. The pH is 6.96, salinity 3.84 ppt, conductivity 7740 µScm-1, COD
253mg/L, BOD 2.85mg/L, Alkalinity 18.75mg/L and Total Dissolved Solid 54.50mg/L.

The 96hLC50 of LD/TT/2021/002 on exposure to Palaemonetes africanus and Oreochromis

niloticus were 169.9 mg/L and 201.5mg/L respectively. On the other hand, the 96h LC 50 of
the reference chemical; KCL when tested against P. africanus and O. niloticus were 86.92
mg/L and 341.74mg/L respectively. The Leak Detector LD/TT/2021/002 was practically
non-toxic (LC50 100 - ≤ 1000) to both the brackish water shrimps and the freshwater fish.
While, the reference chemical; KCL was slightly toxic (LC50 10 - ≤ 100) to brackish water
shrimps and practically non-toxic (LC50 100 - ≤ 1000) to freshwater fish.

Furthermore, the Toxicity Factor for P. africanus was 2.3 while that of O. niloticus was 0.5.
These values show the toxicity of LD/TT/2021/002 with reference to KCL, indicating that
the reference chemical KCL is more toxic than the test chemical LD/TT/2021/002 (TF>1)
for brackish water media and less toxic than the test chemical LD/TT/2021/002 (TF<1) for
fresh water media. The result also indicated that O. niloticus was less sensitive to the test
chemical than P. africanus. This may be attributed to the physiochemical constituents of the
receiving environment, difference in the behaviour and morphology of the organisms and
their metabolic activities.

The Leak Detector Dye, LD/TT/2021/002 is hence recommended as safe for use in the
Nigerian environment at Maximum Allowable Toxicant Concentration (MATC) of
20.15mg/L for P. africanus and 16.99mg/L for O. niloticus.

Zoological names should be written in italics

KCL should be written as KCl

3 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 LIST OF TABLES

Table Page

Table 1: Test concentrations of LD/TT/2021/002 used for 96hour LC50 Bioassay 15

Table 2: Test concentrations of (KCL) used for 96hour LC50 Bioassay 15
Table 3: GESAMP rating/classification of acute toxicity to aquatic organisms by
chemicals/substances 16
Table 4: Physico-chemical characteristics of Test Chemical and Habitat 18
Table 5: Range finding tests of LD/TT/2021/002 against P. africanus 19
Table 6: Range finding tests of LD/TT/2021/002 against O. niloticus 19
Table 7: 96h. Acute toxicity tests of LD/TT/2021/002 against P. africanus 20
Table 8: 96h. Acute toxicity tests of LD/TT/2021/002 against O. niloticus 20
Table 9: 96h Acute toxicity tests of KCL against P. africanus 20
Table 10: 96h Acute toxicity tests of KCL against O. niloticus 21
Table 11: 96h. LC50 values for P. africanus and O. niloticus 21

Tables 5 and 6 are not in the body of the report

The page numbers do not match the actual pages where the tables are located
KCL should be written as KCl

4 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 LIST OF PLATES

Plates Page

Plate 1: Bioassay set up for LD/TT/2021/002 against P. africanus and O. niloticus 14

Plate 2: Well-maintained organisms in large stock tanks 14

Plate 3: Measurement of the length of O. niloticus 14

Plate 4: Measurement of the length of P. africanus 14

Plates are on Page 11, not page 14

5 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 LIST OF ABBREVIATIONS AND ACRONYMS

APHA American Public Health Association

API American Petroleum Institute
ASTM American Society for Testing and Materials
BOD Biochemical Oxygen demand
cm Centimeter
DPR Department of Petroleum Resources
EGASPIN Environmental Guidelines and Standards for the Petroleum Industry in Nigeria
0
C Degree Celcius
g. Gram me
h Hour
KCL Potassium Chloride
L. Litre
LC50 Median Lethal Concentration
LT50 Median Lethal Time
MATC Maximum Allowable Toxicant Concentration
mg Milligramme
mg/L Milligramme per Litre
ml Millilitre
ml/L Millilitre per Litre
mm Millimetre
ND Not Detected
QA/QC Quality Assurance/Quality Control
ppt, ‰ Part Per Thousand
TF Toxicity Factor
µScm-1 Micro Siemens per Centimetre
SPSS Statistical Package for the Social Sciences
TDS Total Dissolved Solids

BOD5

You have Hour written as Hr, Hrs in some other parts of the report (not only h)

You have centimeter and litre (mixture of US and UK styles)

6 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 LIST OF PREPARERS

This report was prepared through the contributions of the following:

Name Designation

1. Prof. Adebayo Otitoloju Head, Technical Advisory and Coordination

2. Mr Joseph Anite Head, Business Development

3. Mr. Eniola Olowu Head, Project and Research.

4. DR Samuel A. Head, Health and Safety

5. Mr. Franklyn Nnorom Executive, Project and Research.

6. Mr. Benedict Begusa Consulting Analyst.

See submitted report for updated list

7 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 ACKNOWLEDGMENT

We are grateful to the Department of Petroleum Resources (DPR) for recognizing and
approving our laboratory for the purpose of carrying out Aquatic Toxicity Bioassay of
chemicals to be used in the Oil industry in Nigeria. We are particularly grateful and
thankful for the confidence the following staff of the Department have for our ability.

a. Mr O.S Bajomo
b. Mr A.N Kamba
c. Mr. Illo Mohammed
d. Mr. Shehu
e. Mr. Idris Ahmed

We acknowledge with gratitude Dr. Yakubu of NIOMR for supplying our laboratory with
sufficient healthy frys of Oreochromis niloticus from their Research Center (hatchery) and
Mr. O. Matthew and his team for painstakingly collecting the juveniles of the shrimp
Palaemonetes africanus from mangrove creeks in the Lagos Lagoon at Ikorodu area.

8 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 CHAPTER ONE

1.0 INTRODUCTION

There are numerous chemicals used in oil and gas production. Leak Detector is a tracer dye
specially developed for detecting leaks from closed-loop system that are used in petroleum
production and/or transportation. The dye is formulated in a manner that enables rapid and
effective detection with an optimal dosage.

In ecotoxicology, toxicity tests are great tools used in deriving environmental quality
standards, and it’s used to predict the toxicity of chemicals upon which, regulatory agencies
make decision on chemicals usage in the environment.

The toxicity of many chemicals is unknown even when they can be detected. Different
chemicals combined together may have unknown additive or synergistic effects even when the
toxicity of each individual chemical is well-known (Randall M., 1999; Ogbonna and
Amajuoyi, 2009). Bioassay is the use of living organism to determine the presence, amount,
changes and effects of pollutants on life forms in the environment (Don-Pedro, 2009). The
combination of bioassay techniques as well as the analysis of the physico-chemical parameters
of the chemical and test habitat gives better inferences.

1.2 Aim and Objectives

The aim of this study is to determine the toxicity of the Leak Detector (LD/TT/2021/002) when
used both in the freshwater and brackish water environments in Nigeria. The objectives
include:

i. To determine the 96hr LC50 for the toxicant/chemical used against the freshwater and
brackish water organisms.
ii. To evaluate the toxicity factors for freshwater and brackish water organisms.
iii. To recommend the chemical for use in the Nigerian environment.

9 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 Description and Acclimatization of Organism

Juvenile of Palaemonetes africanus having an average length of 17mm and weight of 0.18g as
well as fry of Oreochromis niloticus having average length of 18mm and weight of 1.7g were
obtained. These were acclimatized for a minimum of ten days in large stock tanks at a
temperature of 26±20C, 12-hour daylight/12-hour darkness, and continuous aeration. The
organisms were fed once daily with commercial feed and the stock tank was renewed daily
along with constant removal of dead organisms to maintain a clean habitat.

2.2 Collection and Transportation

The test chemical is a Leak Detector, LD/TT/2021/002 and this was obtained from the DPR,
Lagos office, 7, Kofo Abayomi Street, Victoria Island, Lagos while the recommended
reference chemical is Potassium Chloride (KCL). This was prepared under strict QA/QC in our
Laboratory.

2.3 Equipment and materials

The equipment and materials used in this study includes:
i. Refrigerator (temp maintained at 1-60C)
ii. Stock tanks (5L and 20L)
iii. Aerators
iv. 50ml glass density bottle
v. Analytical balance
vi. 25L Plastic kegs
vii. 100L Plastic containers
viii. 1-gallon, 2-gallon or 3.5 gallon mixing containers (plastic buckets)
ix. Mesh
x. Glass beakers (1L and 500ml)
xi. Measuring cylinder (2000ml, 1000ml etc)
xii. Disposable syringe (0.lml intervals)
xiii. Multi-testing water quality meter (pH, EC, Temp, TDS)
xiv. Dissolved Oxygen (DO) meter

10 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 xv. Salinity meter
xvi. Alkalinity meter
xvii. High-shear meter
xviii. Magnetic stir bar
xix. Magnetic stir plate
xx. De-ionized water
xxi. Habitat water (diluent)
xxii. 6N Hydrochloric acid (HCl)
xxiii. Sodium Hydroxide (NaOH) solution
xxiv. pH buffer solution
xxv. Potassium Chloride (KCL)-Standard Reference Toxicant (SRT)
xxvi. Computer with SPSS 20 application

3. 4.

Plate 1 showing the bioassay set for LD/TT/2021/002 using P. africanus and O. niloticus as test organisms.
Plate 2 showing well aerated stock tanks for maintaining the test organisms. Plate 3 and 4 shows the
determination of the average length of both and O. niloticus and P. africanus using a ruler.

These plate images are no longer being used. Please see submitted report for new plate
images

11 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

2.4 Physio-chemical Analysis of LD/TT/2021/002 and the Test habitat

The physio-chemical parameters of the Leak Detector and test habitat were analyzed according
to internationally acceptable methods such as APHA and ASTM as recommended by DPR
(EGASPIN, 2018). These parameters include colour, odour, pH, temperature, electrical
conductivity, salinity, alkalinity, total dissolved solids, total suspended solids, chemical and
biological oxygen demand.

2.5 Methodology for the 96-hour Acute Toxicity Bioassay

The acute toxicity bioassay method followed in this study is the semi-static renewal test
procedure. The 96-hour bioassay began with a range finding test to determine the least
concentration that gave no effect as well as the concentration that gave 100% mortality. The
various concentrations examined for both P. africanus and O. niloticus ranged between
50mg/L and 900mg/L using the test and reference chemicals. Having determined a suitable
range of the concentrations, the 96-hr LC50 bioassay was setup with the following in Tables 1-
2.

Table 1: Test concentration of LD/TT/2021/002 used for 96-hour LC50 Bioassay

Test organism Test concentration (mg/L) of LD/TT/2021/002

P. africanus 70 140 280 560 900 Control

O. niloticus 50 100 200 400 800 Control

Table 2: Test concentration Reference Chemical (KCL) used for 96-hour LC50 Bioassay

Test organism Test concentration (mg/L) of KCL

P. africanus 26.91 52.19 104.37 208.73 372.75 Control

O. niloticus 52.19 104.37 208.73 417.45 834.90 Control

12 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

Each bioassay concentration was duplicated and contained ten (10) randomly selected
organisms. Agitation of the bioassay was done with a glass stirrer rod intermittently for about
five minutes each, three times daily and organisms were regularly observed.

2.5.1 Bioassay calculations

a. Density = Mass
Volume

b. LC50 = Concentration of Toxicant that would cause 50% mortality of

test organisms.

c. Toxicity factor = 96hr LC50 of Test Chemical

96hr LC50 of Reference Compound

Mortality in the bioassay was recorded at intervals of 24, 48, 72 and 96hours. The results
obtained were analyzed to determine the LC50 values for fresh and brackish water organisms.
These values were obtained using the statistical application, SPSS 20. In addition, the toxicity
factors were determined and in accordance with the GESAMP toxicity rating/classification
(Table 4), the chemical was classified.

Table 3: GESAMP rating/classification of acute toxicity to aquatic organisms of

chemicals/substances

Toxicity Rating Acute Toxicity (LC50)

Non-toxic >1000
Practically non-toxic 100- ≤ 1000
Slightly toxic 10 - ≤ 100
Moderately toxic 1.0 - ≤ 10
Highly toxic 0.10 - ≤ 1.0
Very highly toxic 0.01 - ≤ 0.10
Extremely toxic < 0.01

The referenced table is Table 3, not Table 4

13 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

2.6 Quality Assurance/Quality Control
The integrity of physico-chemical analysis and bioassay is significantly dependent on the
quality control and assurance implemented on the system. To this end, internationally accepted
methodology such as those of APHA, ASTM, and others prescribed by DPR were adopted.
Specific mechanisms for QA/QC include regular calibration of laboratory instruments. Sample
blanks were taken and analyzed for each set of samples and the laboratory was usually kept
clean. All apparatus, sample containers and glass wares were thoroughly cleaned using
standard prescribed methods. The glass tanks for toxicity testing as well as toxicity testing area
were well labeled and all data were logged in appropriately into the bioassay log book. The test
organisms were of about the same length and size and were randomly selected into the glass
tanks.

Etina Environmental Services Limited understands that there are chances of introducing errors
at every stage of data processing and handling; hence the research team ensured that error in
data handling during data entry and retrieval were always rectified. To reduce such errors,
direct electronic recording and data transfer processes were used. In cases where data are
handled or manually transcribed, they were normally checked against the original records.
Another common problem is loss of data due to accidental erasure of computer files. To avoid
data loss, back up files were created for every set of data and were regularly updated.

14 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 CHAPTER THREE

3.0 RESULTS

3.1 Physico-chemical Analysis

The physico-chemical analysis of the fresh water, Lagos lagoon water and the test chemical,
LD/TT/2021/002 yielded the result below (Table 4).

Table 4: Physico-chemical characteristics of Test Chemical and Habitat

S/ Physico-Chemical Parameter Level Detected
N
Freshwater Brackish LD/TT/2021/002
3.2
water
1 Range
ColourFinding Test Clear Light brown Pink
2 Odour ND ND Unpleasant
3 Ph 6.82 7.12 6.96
4 Temperature, (0C) 26.0 26.0 27.3
5 Alkalinity (mg/L) 7.5 18 18.75
6 Salinity (‰) 0.01 0.45 3.84
7 Conductivity, (µScm-1) 150 550 7740
8 Chemical Oxygen Demand, 16.2 253
8.2
(mg/L)
9 Biological Oxygen 2.9 5.2 2.85
Demand,(mg/L)
10 Dissolved Oxygen, (mg/L) 5.9 7.5 *
11 TDS, (ppm) 80 2120 54.50
12 Density (mg/cm3) 1047.2 1067.6 10

Initial range finding tests were conducted to establish suitable concentration range of toxicants
to be administered to the test organisms in the definitive tests. Six concentrations including
control were chosen for range finding. Test was run for 48 hours using 10 organisms per test
concentration.

3.3 Bioassay Result of LD/TT/2021/002

15 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

The 96-hour bioassay for using P. africanus showed total mortality at concentration higher
than 900 mg/L and no mortality at concentration below 70mg/L. On the other hand, the 96-
hour bioassay for using O. niloticus showed total mortality at concentration higher than
800mg/L and no mortality at concentration below 50mg/L.

The results of the 96-hour acute toxicity bioassay conducted on the Leak Detector,
LD/TT/2021/002 and the reference compound, KCL are presented in Tables 8-11 below
Tables below is 7-11
After Table 4, next table should have been labeled 5 and onwards

Table 7: 96h. Acute toxicity tests of LD/TT/2021/002 against P. africanus

Test Medium Number of No. of deaths from 24h to 96h of exposure
Concentrations Organisms
(mg/L) N 24h. % 48h. % 72h. % 96h. %
Control, 0.0 20 0 0.0 0 0.0 0 0.0 0 0.0
70 20 0 0 1 5 1 5 3 15
140 20 1 5 3 15 7 35 8 40
280 20 4 20 5 25 9 45 12 60
560 20 7 35 11 55 14 70 16 80
900 20 10 50 14 70 17 85 19 95

Table 8: 96h. Acute toxicity tests of LD/TT/2021/002 against O. niloticus

Test Medium Number of No. of deaths from 24h to 96h of exposure
Concentrations Organisms
(mg/L) N 24h. % 48h. % 72h. % 96h. %
Control, 0.0 20 0 0.0 0 0.0 0 0.0 0 0.0
50 20 0 0 1 5 2 10 3 15
100 20 1 5 3 15 5 25 7 35
200 20 3 15 5 25 8 40 11 55
400 20 7 35 10 50 13 65 15 75
800 20 9 45 12 60 16 80 18 90

Table 9: 96h. Acute toxicity tests of KCL against P. africanus

16 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

Test Medium Number of No. of deaths from 24h to 96h of exposure
Concentrations Organisms
(mg/L) N 24h. % 48h. % 72h. % 96h. %
Control, 0.0 20 0 0.0 0 0.0 0 0.0 0 0.0
26.91 20 0 0.0 1 5.0 3 15.0 3 15.0
52.19 20 2 10.0 5 25.0 7 35.0 8 40.0
104.37 20 3 15.0 6 30.0 10 50.0 12 60.0
208.74 20 5 25.0 7 35.0 13 65.0 15 75.0
372.75 20 9 45.0 12 60.0 14 70.0 19 95.0

Table 10: 96h. Acute toxicity tests of KCL against O. niloticus

Test Medium Number of No. of deaths from 24h to 96h of exposure
Concentrations Organisms
(mg/L) N 24h. % 48h. % 72h. % 96h. %
Control, 0.0 20 0 0.0 0 0.0 0 0.0 0 0.0
52.19 20 1 5.0 1 5.0 2 10.0 2 10.0
104.37 20 1 5.0 2 10.0 4 20.0 7 35.0
208.74 20 3 15.0 5 25.0 6 30.0 9 45.0
417.48 20 4 20.0 7 35.0 13 65.0 14 70.0
834.90 20 7 35.0 10 50.0 15 75.0 18 90.0

3.4 LC50 values and Toxicity factor

The LC50 values obtained for the P. africanus and O. niloticus on exposure to the
LD/TT/2021/002 and standard reference toxicant KCL are shown in Table 12 below.
Furthermore, the Toxicity Factor, TF for LD/TT/2021/002against P. africanus was 2.3 and 0.5
for O. niloticus.

Table 11: 96h. LC50 values for P. africanus and O. niloticus

Chemicals LC 50 (mg/L)
P. africanus O. niloticus
TT/2021/ 201.5 169.9
KCL 86.92 341.74

There is no Table 12
CHAPTER FOUR

17 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

4.0 DISCUSSION

4.1 Acute Toxicity of LD/TT/2021/002

This work was undertaken to investigate the dose-time effect of LD/TT/2021/002 on

organisms of fresh water and brackish water habitats. The species used for the acute toxicity
were freshwater fish, Oreochromis niloticus (fry) and brackish water shrimp, Palaemonetes
africanus as recommended in EGASPIN, 2018.

From the result, the 96hLC50 of the LD/TT/2021/002 on exposure to the shrimp was
201.5mg/L while it was 169.9mg/L for fish. On the other hand, the 96h LC 50 of the reference
chemical; KCL when tested against the P. africanus and O. niloticus were 86.92mg/L and
341.74mg/L respectively. According to the GESAMP (2002) toxicity rating, the chemical
alone is practically non-toxic (LC50 100 - ≤ 1000mg/l) to both the brackish water shrimps
and fresh water fish.

The Toxicity Factors (TF) were obtained based on LC50 of the test chemical,
LD/TT/2021/002 and reference chemical, KCL on exposure to Palaemonetes africanus and
Oreochromis niloticus. The TF of Palaemonetes africanus was found to be 2.3 while that of
Oreochromis niloticus was 0.5. The values of the toxicity factors showed the toxicity of the
LD/TT/2021/002 with reference to KCL. Hence, LD/TT/2021/002 was found to be less toxic
than the reference chemical (KCL) in brackish water (TF˃1) and more toxic than the
reference chemical (KCL) in freshwater (TF<1).

Furthermore, the result indicated that P. africanus was less sensitive to the test chemical,
LD/TT/2021/002 than O. niloticus. This may be attributed to the physiochemical
constituents of the receiving environment, differential behavior and morphology of
organisms and their metabolic activities.

CHAPTER FIVE

18 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

5.0 CONCLUSION

The physicochemical constituents of the chemical and test habitat played a major role in
determining the mortality rate of the aquatic organisms. P. africanus was less sensitive to the
LD/TT/2021/002 compared to O. niloticus.  The bioassay revealed that both organisms
responded in dose and time dependent manners, in such a way that the higher the
concentration of the chemical, LD/TT/2021/002, the higher the % mortality recorded and as
well the longer the duration of exposure, the higher the % mortality.  

This work will make for a better understanding of the concept of basic methods of chemical
toxicity testing and as well validate the age long theory that “the most important factor that
determines the effect of any substance is the dose – time relationship. Furthermore,
combination of chemicals will interact to exhibit antagonistic, additive or synergistic effects
leading to increase or decrease in their toxicity.  

5.1 Recommendation
The LD/TT/2021/002 was practically non-toxic to both the brackish water organism and
freshwater organism (100-≤1000mg/L) (GESAMP, 2002). The application factor advised for
the use of industrial chemicals and pesticides is between 0.01- 0.1mg/L.

Hence, we recommend the Chemical; LD/TT/2021/002 as safe for use in the Nigerian
environment based on the LC50 values at a Maximum Allowable Toxicant Concentration of
20.15mg/L and 16.99mg/L respectively for P. africanus and O. niloticus. At these
concentrations, strict compliance should be mandated as well as periodic in situ bio-
monitoring studies to identify possible impact on the environment due to its use.

In a sentence without figures, its more appropriate to write percentage in words, rather than its symbol (%)

19 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 REFERENCES

American Public Health Association (1998). Standard Methods for the Examination of Water
and Wastewater 20th Edition Edited by Andrew D. Greenberg et al.

Dede, E. B; Dogara, F. M. (2004). The Acute toxicological effect of Gamalin 20 (Gamma

Isomer of Hexachlorocyclohexane) on the Lung and Pancrease of Guinea Pig. J. Appl. Sci. &
Env. Mgt.  8 (1) 33 - 35

Department of Petroleum Resources (2002). Environmental Guidelines & Standards for the
Petroleum Industry in Nigeria, pp 281-291

Duffs, J. H. (1980).  Environmental Toxicology. Resources and Environmental Science

Series.   Edward Arnold Limited, London pp1-19. 

Finney, D. J. (1978). Statistical Methods in Biological Assay 3rd Edition. Charles Griffin,
London.

Franklin, F. L. (1973). Assessing the Toxicity of Industrial Wastes, with Particular Reference
to Variations in Sensitivity of Test Animals / Fish. Rep MAFF Direct Fish Res., Lowerstoft.

Joint Group of Experts on Scientific Aspects of Marine Environmental Protection (GESAMP)

(2002). The Revised GESAMP Hazard Evaluation Procedure for Chemical Substances Carried
by Ships. GESAMP Report and Studies No. 64: 493 (02)

Ogbonna, F. Joel; Amajuoyi, Chinomso A. (2009). Synergistic Effect of Test Chemical with
Crude oil Contaminated Aquatic Habitat, J.NES, 2009, Volume 5 Number three, Nigeria.

Okpokwasili, G. C; Odokuma, L. O. (1994). Toxicity of some Nigerian Crude Oils, Bulletin of

Environmental Contamination and Toxicology 52: 388-399.

Randall M. (1999). Whole Toxicity Testing and Limits, Department of Ecology, Water Quality
Program, Sate of Washington, pp 173-205 WAC.

20 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 APPENDICES

A. Density Calculation
Density of LD/TT/2021/002
Volume of density bottle = 100ml
Mass of Chemical = 1g
Density = 2g ÷ 100ml = 0.01g/ml = 10mg/L

Density of Fresh Water

Weight of density bottle = 13.64g
Weight of chemical and bottle =66.00g
Volume of density bottle = 50ml
Mass of Chemical = 66.00-13.64 = 52.36g
Density =52.36 ÷50ml = 1.0472g/ml = 1047.2mg/cm3 or mg/L

Density of Brackish Water

Weight of density bottle = 13.64g
Weight of chemical and bottle = 67.02g
Volume of density bottle = 50ml
Mass of Chemical = 67.02-13.64 = 53.38g
Density = 53.38g÷50ml = 1.0676g/ml = 1067.6mg/cm3 or mg/L

B. Toxicity Factor
Toxicity Factor for P. africanus
The 96h LC50 of KCL against P. africanus = 86.92mg/L
The 96h LC50 of LD/TT/2021/002 against P. africanus = 201.45mg/L.
96 h . LC 50 LD /TT /2021/002
Toxicity Factor, TF = 96 h . LC 50 of KCL

201.45
¿
86.92
= 2.3

Toxicity Factor for O. niloticus

The 96h LC50 of KCL against O. niloticus = 341.74mg/L
The 96h LC50 of LD/TT/2021/002 against P. africanus = 169.92mg/L.
96 h . LC 50 LD /TT /2021/002
Toxicity Factor, TF = = 96 h . LC 50 of KCL

21 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 169.92
¿
341.74
= 0.5

22 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

 Confidence Limits and Probit Response Curves for P. africanus and O. niloticus

1. LD/TT/2021/002 on P. africanus

Confidence Limits

Probability 95% Confidence Limits for Concentration 95% Confidence Limits for log(Concentration)a

Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

.050 35.746 11.715 62.701 1.553 1.069 1.797

PROBIT .500 201.451 141.920 272.107 2.304 2.152 2.435

.950 1135.302 699.179 2903.823 3.055 2.845 3.463

a. Logarithm base = 10.

23 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

2. LD/TT/2021/002 on O. niloticus

Confidence Limits

Probability 95% Confidence Limits for Concentration 95% Confidence Limits for log(Concentration)a

Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

.050 22.705 5.943 43.287 1.356 .774 1.636

PROBIT .500 169.919 116.054 240.942 2.230 2.065 2.382

.950 1271.651 698.911 4348.687 3.104 2.844 3.638

a. Logarithm base = 10.

24 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

3. KCL on O. niloticus

Confidence Limits

95% Confidence Limits for VAR00003 95% Confidence Limits for log(VAR00003)a
Probabil
ity Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

PROBIT .050 29.829 8.926 54.055 1.475 .951 1.733

.500 341.735 173.262 353.687 2.383 2.239 2.549

.950 1455.625 802.393 4876.864 3.163 2.904 3.688

a. Logarithm base = 10.

25 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002

4. KCL on P. africanus

Confidence Limits

95% Confidence Limits for VAR00003 95% Confidence Limits for log(VAR00003)a

Probability Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound

PROBIT .050 15.721 4.888 27.957 1.196 .689 1.446

.500 86.916 73.299 144.338 2.012 1.865 2.159

.950 673.727 379.487 2158.465 2.828 2.579 3.334

a. Logarithm base = 10.

On arrangements, you had P. africanus, then O. niloticus for confidence limits and
probits for the test chemical. For the reference chemical (KCl), you now had O. niloticus
before P. africanus. Let your arrangements be consistent. Either P. africanus always
comes first or vice versa.

26 | Acute Toxicity Testing of Leak Detector - LD/TT/2021/002