Good Laboratory Practice Assignment 2017

(A0301)
April 3, 2017

GOOD LABORATORY PRACTICE FINAL ASSIGNMENT

M0106

By
1. Dawit Gebremichael(CHIR)
2. Gidey Gebremeskel (CHIR)
 Good Laboratory Practice Assignment (A0301) 2017

April 3, 2017

FINAL REPORT

FISH ACUTE TOXICITY TEST OF OXYTETRACYCLINE

Report distribution

Type Recipient Location Date of report Copie

s

Orginal Sponsor London, united kingdom March 31, 2017 1

First copy Study director Faro, portugal March 31, 2017 1

Second Study personnel Faro, portugal March 31, 2017 1

copy

Third copy Quality assurance unit Faro, portugal March 31, 2017 1
 Good Laboratory Practice Assignment (A0301) 2017

Report Comprising: Coversheet plus pages 2 to 22

GENERAL INFORMATION

Study Title: Fish Acute Toxicity Test of Oxytetracycline.

Study Code: CHIR-001

SPONSOR
Charles Lobster
The European Chemical Company (ECC)
356 Octopus Street
London EC4M 9AJ
United Kingdom
Telf: 777669988
e-mail: clobster@ecc.com

LABORATORY
Erasmus Mundus Master in chemical innovation and regulation (CHIR)
Laboratory Manager e-mail:
dawit.g.ezana@gmail.com
Dawit Gebremichael Study Director
Penha 16 Dawit Gebremichael
Quality Assurance Unit
8002, Faro, Portugal Penha 16
Gidey Gebremeskel
Telf: 920147846 8002, Faro, Portugal
Penha 48
e-mail: Telf: 920147846
dawit.g.ezana@gmail.com 208, Faro, Portugal

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 Good Laboratory Practice Assignment (A0301) 2017

Telf: 967467920 e-mail: ghidena12@gmail.com

STATEMENT

Sponsor: Charles Lobster, European Chemical Company (ECC)

Study title: Fish acute toxicity test of Oxytetracycline
Study code: CHIR-001

I, undersigned, hereby declare that this report provides correct English translation and version of final report
(study number CHIR-001, issued on March31, 2017)

Study director: Dawit Gebremichael

Signature:
Date: March31, 2017

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 Good Laboratory Practice Assignment (A0301) 2017

GOOD LABORATORY PRACTICE (GLP) STATEMENT

Sponsor: Charles Lobster, European Chemical Company (ECC)

Study title: Fish acute toxicity test of Oxytetracycline
Study code: CHIR-001

The study described in this report was conducted in compliance with the following GLP principles
1. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring Number 1,
1997

This report relates specifically to the sample(s) tested in so far as that the sample(s) is truly representative of
the sample source as supplied.

Study director: Dawit Gebremichael

Signature:
Date: March31, 2017

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 Good Laboratory Practice Assignment (A0301) 2017

QUALITY ASSURANCE UNIT STATEMENT

Sponsor: Charles Lobster, European Chemical Company (ECC)

Study title: Fish acute toxicity test of Oxytetracycline
Study code: CHIR-001

The study was conducted as per the requirement of OECD Series on Principles of Good Laboratory Practice
and Compliance Monitoring Number 1, 1997. I assure that the final report accurately describes the test
methods and procedures, and that the reported results accurately reflect the raw data of the study. The
inspections and audit of this study were carried out and the results were reported to the study director and
the test facility management by quality assurance unit as follows.

Item of inspection/audit Date of inspection/audit Date of report to study director

protocol draft February15, 2017 February15, 2017
Protocol February17, 2017 February17, 2017
First protocol amendment February22, 2017 February23, 2017
Preparation of stock solution February 28, 2017 February 28, 2017
Analysis of test water March 05, 2017 March 05, 2017
Start of exposure and after exposure March 10, 2017 March 11, 2017
March 15, 2017 March 20, 2017
Raw data and final draft report March 27, 2017 March 30, 2017
Final report March 31, 2017 March 31, 2017

Head of quality assurance unit: Gidey Gebremeskel

Signature:
Date: March31, 2017

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TABLE OF CONTENTS
1. STUDY SCHEDULE.......................................................................................................- 8 -
2. STUDY OBJECTIVE......................................................................................................- 8 -
3. GUIDELINES..................................................................................................................- 8 -
4. TEST AND REFERENCE ITEM....................................................................................- 8 -
5. TEST SYSTEM...............................................................................................................- 9 -
6. METHOD......................................................................................................................- 10 -
6.1. General method description....................................................................................- 10 -
6.2. Water......................................................................................................................- 10 -
6.3. Test item solutions preparation...............................................................................- 10 -
6.4. Range-finding and definitive test...........................................................................- 10 -
6.5. Observations...........................................................................................................- 11 -
6.6. Determination of Test item Concentration.............................................................- 11 -
6.7. Validity of the Test.................................................................................................- 12 -
7. RESULTS......................................................................................................................- 12 -
8. REPORT SUMMERY...................................................................................................- 16 -
9. RECORDS.....................................................................................................................- 16 -
10. REFERENCES...........................................................................................................- 16 -

LIST OF TABLES, FIGURES AND ANNEX TABLES

Table 1.Standard and test result of the chemical......................................................................- 8 -
Table 2 Experimental design used in zebra fish 96-h exposure tests.....................................- 11 -
Table 3. Observation parameters and time frequencies.........................................................- 11 -
Table 4. Acceptable working range of Oxytetracycline % recovery in test water..................- 12 -
Table 5. Summary of percent of mortality at different concentrations and exposure time....- 13 -

Figure 1.structure of Oxytetracycline -9-

Figure 2. Summary of percent of mortality at different concentrations and exposure time - 14 -
Figure 3. Cumulative percent mortality vs. exposure time - 14 -
Figure 4. Calibration graph of HPLC-UV analysis of Oxytetracycline in a test item - 15 -

Annex table 1. Range finding test - 17 -

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Annex table 2. Definitive test - 18 -

Annex table 3. Concentration of test conditions before and after renewal - 19 -
Annex table 4. Measured concentrations of test item in a test solution before and after renewal- 20 -
Annex table 5.Calibration working standards and analysis peak area value - 20 -
Annex table 6. Method recovery determination by standard addition (spiking) method 21

1. STUDY SCHEDULE

Experimental phase starting date February 23, 2017

Experimental phase ending date March 30, 2017

2. STUDY OBJECTIVE

The aim of this study was to determine the LC50 of Oxytetracycline, i.e., the concentration of product that
kills the 50% of the fishes during a 96 hours exposure time.
3. GUIDELINES

The study was conducted according to the OECD 203 guideline “Fish Acute Toxicity Test”.

4. TEST AND REFERENCE ITEM

Product name: Oxytetracycline

CAS Number: 2068-46-0 Certificate No: 20110608
Batch No: X24-2016 Report date: 2017-02-25
Manufacturing date: 2016-08-25 Expiry date: 2020-08-24
Table 1.Standard and test result of the chemical

ASSAY ITEM STANDARD TEST DATA

Description A yellow ,crystalline powder A yellow, crystalline powder
Identification Positive Positive
PH 4.5-7.5 5.6
Absorbance 290-310 298
Light-absorbing impurities 430nm A≤0.25 0.11
Related substances ----- ---
Heavy metal ≤50mg/L 50 mg/L

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Water 6.0-9.0% 7.5%

Sulphated ash ≤0.5% 0.13%
Assay 95.0 -102.0% 98.1%

Oxytetracycline Hydrochloride is an antibiotic isolated from Streptomyces rimosus, a bacteriostatic

antibiotic that inhibits protein synthesis.

Figure 1.structure of Oxytetracycline

Test item was provided to the Study Director by the Sponsor together with a certificate of analysis. Test item
was stored at the laboratory according to environmental conditions established in the MSDS. A sample of
the test item will be retained at the laboratory until its expiry date. As reference item, Oxytetracycline
O5875 Sigma Oxytetracycline h
hydrochloride ≥95% (HPLC), crystalline was used.
.
5. TEST SYSTEM

In this study Zebra-fish (Brachydanio rerio) of an average weight and length of 350±5 mg and 2 ± 0.2 cm,
respectively, was used as a test system. Fish was acquired from an adequate supplier and held at their arrival
in a 200L glass tank (100x50x40cm) in the laboratory for 12 days before using for testing, under the
following environmental conditions:
 Light: 12 hours photoperiod daily
 Temperature: 23 ±2 oC
 Oxygen concentration: more than 80% of air saturation value
 pH: between 7.5 and 8.5
 Feeding: daily until 24 hours before the test is started.

Fish were stored in a glass big tank and, after a 48-hour settling-in period, mortality was recorded daily. The
batch was accepted due to the reason that mortalities were under 5% of population after 10 days. The test
was conducted in 5L plastic tanks (33X12X13cm) under the same environmental conditions.

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6. METHOD

6.1. General method description

SOP TOX-EXP-33 “Fish Toxicity Test” was followed. All the apparatus used in this study, as well as their
associated SOPs, were recorded in a table attached to the study notebook specifically designed for this study.
Fish were exposed to the test item for a 96-hour period. Observations and mortalities were recorded at 1, 6,
24, 48, 72 and 96 hours in order to calculate LC50. Each test tank contained 4.5L of water and 10 fish. There
were 6 tanks in total (5 test concentrations plus control). The test was conducted under semi-static
conditions, renovating of 40% of the total volume every 24 hours.

6.2. Water

Reconstituted water prepared according to 203 OECD Guideline was used to fill the tanks and prepare the
test item solutions. A SOP TOX-REG-02 was followed.

6.3. Test item solutions preparation

A stock solution of test item at 1000 mg/L of active ingredient was prepared with reconstituted water. The
different test item concentrations were prepared by dilution of stock solution with reconstituted water. pH
was measured and adjusted to be between 7.5 and 8.5 before pouring the test item solution into the tank.

6.4. Range-finding and definitive test

The test was started with a range-finding, in which 5 concentrations of test item (100, 10, 1, 0.1 and 0.01
mg/L) and a negative control was tested. This test helped to determine more precisely the final
concentrations of test item tested. Based on the results of preliminary fish mortality study, a definitive test
with 5 concentrations placed between the 0 and 100% of mortality was conducted. The final concentrations
were recorded in the raw data and the final report.

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Table 2 Experimental design used in zebra fish 96-h exposure tests

Acute toxicity tests Oxytetracycline concentration (mg/L)

Range finding 0, 0.01, 0.1, 1, 10, and 100
Definitive 0, 7, 15, 30, 70, and 100

6.5. Observations

Table 3. Observation parameters and time frequencies

Determinations Settling in 0hr 6h 24h 48h 72h 96h

Temperature X X X X X X
pH X X X X X
Oxygen X X X X X
Mortality X X X X X X X
Light intensity X X
Abnormality X X X X X X

Measurements were done one hour after daily water renovation and recorded in the study notebook. Fish
were observed at 1, 6, 24, 48, 72 and 96 hours after the starting of the test, recording mortality and any
abnormality in their behavior (Annex table 1).

6.6. Determination of Test item Concentration

Water samples were obtained from a central point of the tank, after stirring. Duplicate samples were
obtained at the beginning and end of the test, and before and after each water renovation for each one of the
concentrations and control (Annex table 4). Water samples were labeled and stored at -30 oC until their
analysis.

Oxytetracycline levels in water were measured by an HPLC-UV validated method and according to SOP
TOX-ANL-24. The actual Oxytetracycline concentration was determined by interpolation of
chromatographic area in a calibration curve that was prepared by standard addition of Oxytetracycline

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standard. The calibration curves were considered acceptable if the % of recovery fulfils the following
criteria:

Table 4. Acceptable working range of Oxytetracycline % recovery in test water

Sample concentration Percent of recovery

0.1 - 10 mg/L -20-10%
10-100 mg/L -10-7%

Daily quality controls (QC) were prepared from a different standard solution that the one used for the
calibration curve. Two concentrations were used, one of them close to the limit of quantification (LOQ).
Quality controls represented at least 5% of the total samples of each analytical series, and the results were
accepted if at least the 60% of the QC concentrations had less than 20% of deviation from the nominal
concentration.
The final results are reported as concentration of Oxytetracicline in water in mg/L.

6.7. Validity of the Test

The test was considered as valid with the following requirements:

 The mortality in the controls did not exceed 10%.
 Conditions were maintained constant during the entire test.
 The dissolved oxygen concentration was above 60% of the air saturation during the entire test.
 The test item concentration in the tanks was at least 80% of the nominal concentration throughout the
test.

7. RESULTS
The temperature of the test system was maintained between 21 and 24 oc with an average of 22.5 oc. The
dissolved oxygen was measured every 24 hours in all of the test concentrations and the control tank. The
level of dissolved oxygen of the test system was maintained above 80%. The duration for the study was 96
hrs. It was found that the mortalities were observed after 12 hours, majority of which occurred at the end of
96 hours.

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The maximum concentration of Oxytetracycline causing no mortality was below 10 mg/L (Annex table1) in
the range finding test so that 7 mg/L is selected as the maximum concentration at which no mortality is
observed and put in the definitive test as minimum concentration (Annex table 2).

The minimum concentration causing 100% mortality is greater than 100 mg/L due to target of the study that
it is to determine LC50, absolute mortality was not observed with the tested concentrations. Five test item
concentrations were used in the definitive test along with the negative control. There were no mortality
recorded in the control and the 7 mg/L tanks (Table 5). However, the tank with 15 mg/L concentration
showed a cumulative percent mortality of 15% at the end of 96 hrs. 10% and 30% mortality rate has been
observed in a 30 mg/L tank at 72 hours and 96 hours, respectively.

Table 5. Summary of percent of mortality at different concentrations and exposure time

Concentration (mg/L) Comulative mortality (%)

1h 6h 24h 48h 72h 96h
Control 0 0 0 0 0 0
7 0 0 0 0 0 0
15 0 0 0 0 0 15
30 0 0 0 0 10 30
70 0 0 0 10 30 50
100 10 20 30 50 60

At 70 mg/L test concentration, a cumulative percent mortality of 10%, 30%, and 50% were observed at the
end of 48 hrs, 72 hrs, and 96 hrs, respectively. Finally, the cumulative percent mortality rates in 100 mg/L
tank were found to be 20%, 30%, 50%, and 60% at the end of 24 hrs, 48 hrs, 72 hrs, and 96 hrs,
respectively. Percent mortality of fish was taken as indicators of toxicity of the test item. Calculation of
mortality percentage was done using the following equation:
100∗(Lc−L)
Mortality =
Lc
Where, Lc and Lt are the number of live fish in the control and the treated samples, respectively. Percent
mortality was regressed versus concentration and converted to a log scale to calculate the LC50 values.
Moreover, mortality percent versus exposure concentration correlation is done (Figure 2).

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70

60
f(x) = 19.7709097567132 ln(x) − 33.6477407088108
50 R² = 0.99033521209614

40
%Mortality

30

20

10

0
0 20 40 60 80 100 120
concentration

Figure 2. Summary of percent of mortality at different concentrations and exposure time

Regression line with logarithmic equation was fitted to interpolate the LC50 and calculated LC50 result is
68.3 mg/L.
Mortality ( % )=19.771∗ln ( [ Oxytetracycline ] ) −33.648

Water samples were taken after stirring and homogenizing thoroughly. Triplicate samples were obtained at
the beginning and end of the test, and before and after each water renovation for each one of the
concentrations and control (Annex table 4) resulting with recovery of 87.9 to 99.5% which is in the
acceptable range (Table 4). A time response relationship of Oxytetrcycline concentrations on fish is shown
in Figure3 that demonstrates a progressive relationship between mortality percentage and length of
exposure. Linear regression analysis indicated a strong positive association with a high value of R 2.
120

100
Cumulative %Mortality

80 f(x) = 1.25315985130112 x + 1.48327137546468

R² = 0.984869759339766
60

40

20

0
0 10 20 30 40 50 60 70 80
Exposure time to Oxytetracycline (h)

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Figure 3. Cumulative percent mortality vs. exposure time

Moreover, at the maximum concentration of Oxytetrcycline, abnormalities such as hyperactivity, edema, and
loss of equilibrium were observed after 1hour exposure time (Annex table1).

The calibration graph was linear over the range of 0.01 to 100 mg/L of test item. The equation from the
curve was: y = 5.02x - 0.0024, where y is the peak area found through HPLC-UV analysis and x is the
concentration of Oxytetracycline in the test water samples (Figure 4). Calibration curves had a correlation
coefficient of R2 =0.999(Annex table 5). Limit of detection and limit of quantification was determined by
analyzing control sample 10 times resulting with standard deviation value of 0.0013 so that taking three
times and ten times of this standard deviation to get lower limit of detection and quantification 0.004 and
0.010 mg/L, respectively.

700.0
600.0
f(x) = 5.02002689070342 x − 0.00238808775819166
500.0 R² = 0.999999999813126
Peak area

400.0
300.0
200.0
100.0
0.0
0 20 40 60 80 100 120 140
concetration(mg/L)

Figure 4. Calibration graph of HPLC-UV analysis of Oxytetracycline in a test item

Method recovery was done by spike method (standard addition method) and calculated
spiked sample peak area−unspiked sample peak area
as; Recovery= ∗100
spiking conentration peak area
Accordingly, the percent recoveries of the test item were calculated for each tank, and they were found to
range from 93.9– 106.7 % (Annex Table 6). Hence, percent of recovery for each test is within the acceptable
range. Moreover, routine quality controls (QC) were conducted by using standard concentrations of 0.1 and
40 mg/L. The results showed that all the QC concentrations had less than 20% of deviation from the nominal
concentration.

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8. REPORT SUMMERY

Generally, all the working conditions were maintained within their corresponding acceptable limits at the
staring, before renewal, after renewal and at the end of observation (Annex table 3). Mortality was observed
at 12 hours post exposure. Absolute mortality was not observed at the maximum test item concentration of
100 mg/L. The LC50 was determined as 68.3 mg/L and all recoveries and quality parameters are in the
acceptable range showing that the determined LC50 value is acceptable.

9. RECORDS

All the documentation related to the Study (Protocol, Raw Data and Final Report) will be kept in the GLP
Archive of CHIR laboratory for 5 years. After this period, CHIR laboratory will not be responsible for any
loss or malfunction of the archive. However, the sponsor may request for special handling at his own cost of
archiving, as per the fees requested by CHIR laboratory.

10. REFERENCES
1. OECD GUIDELINE FOR TESTING OF CHEMICALS 203 “Fish Acute Toxicity Test, adopted on
17th July 1992.
2. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring Number 1, 1997
3. OECD principles of good laboratory practice (November 26, 1997, ENV/MC/CHEM (98)17

SOP LIST
1. TOX-EXP-33: Fish Toxicity Test
2. TOX-ANL-24: Analysis of Oxytetracycline by HPLC-UV
3. TOX-CAL-05: LC50 Calculating by Probit Analysis Software
4. TOX-DOC-03: Study Plan Amendments and Deviations
5. TOX-DOC- 04: Final Reports

Annex table 1. Range finding test

Conc (mg/L) Determinations Settling in 0h 1h 6h 24h 48h 72h 96h

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0 Temp 22 22 - - 23 23 23 22
pH - 8.0 - - 8.1 8.1 7.9 7.9
light X X - -
oxygen(mg/L) - 7.2 - - 7.1 7.3 7.6 7.7
mortality N N N N N N N N
abnormality N N N N N N N
0.01 Temp 21 22 - - 22 23 22 21
pH - 7.9 - - 8.0 7.9 7.8 7.7
light X X - -
oxygen(mg/L) - 7.2 - - 7.1 7.3 7.6 7.8
mortality N N N N N N N N
abnormality N N N N N N N
0.1 Temp 22 23 - - 24 23 22 21
pH - 7.9 - - 8.0 7.9 7.8 7.7
light X X - -
oxygen(mg/L) - 7.2 - - 7.1 7.3 7.6 7.9
mortality N N N N N N N N
abnormality N N N N N N N
1 Temp 22 24 - - 23 23 21 22
pH - 7.9 - - 8.1 8.0 7.9 7.7
light X X - -
oxygen(mg/L) - 7.2 - - 7.1 7.3 7.6 7.9
mortality N N N N N N N N
abnormality N N N N N N N
10 Temp 22 24 - - 22 23 21 22
pH - 7.9 - - 8.1 8.0 7.9 7.7
light X X - -
oxygen(mg/L) - 8.0 - - 7.7 7.3 7.6 7.9
mortality N N N N N N N 1
abnormality N N N N N 1 2
100 Temp 22 21 - - 23 24 23 22
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7.9
pH - 7.9 - - 8.1 8.0 7.8
light X X - - 7.6
oxygen(mg/L) - 7.2 - - 7.1 7.3 5 7.9
mortality N N N 1 2 3 9 7
abnormality N 1 2 3 6 10
1. Environmental conditions (Temp 23 ±2 oC, Light 12 hours photoperiod daily, Oxygen (mg/l) <8.0, pH 7.5- 8.5 the
empty once are at which observations were not conducted.
2. Mortality and abnormality at 0, 1, 6, 24, 48, 72 and 96 hours varying concentration from 0 to 100 mg/l. note that N
stands no mortality and abnormality has observed at that specific conditions
3. All readings are mean of triplicates
Annex table 2. Definitive test
Conc Determination Settling in 0 hr 1 hr 6 hr 24 hr 48 hr 72 hr 96 hr
(mg/L)
0 Temp 21 22 23 23 23 22
pH 7.9 8.1 8 7.9 7.8
light X X
oxygen(mg/L) - 7.2 7.1 7.6 7.6 7.7
Mortality N N N N N N N N
Abnormality - N N N N N N N
7 Temp 22 23 24 23 22 21
pH 7.9 8.0 7.9 7.8 7.7
light X X - - - -
oxygen(mg/L) - 7.2 7.1 7.3 7.6 7.9
Mortality N N N N N N N N
Abnormality - N N N N N N N
15 Temp 22 22 23 24 22 21
pH 7.9 8.1 8 7.8 7.9
light X X - - - -
oxygen(mg/L) - 7.6 7.3 7.5 7.6 7.7
Mortality N N N N N 2
Abnormality - N N 1 3 3
30 Temp 22 23 22 21 24 21
pH 80 7.9 8 7.9 7.8
light X X
oxygen(mg/L) - 7.2 7.3 7.6 7.6 7.7
Mortality N N N N N N 1 4
Abnormality - N N N N 1 4 5
70 Temp 22 24 22 21 24 21
pH 80 7.8 8.1 7.9 7.8
light X X
oxygen(mg/L) - 7.2 7.3 7.5 7.6 7.7
Mortality N N N N 1 2 4 6
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Abnormality - N N 1 2 5 7 8
100 Temp 22 23 22 21 24 21
pH 80 7.9 8 7.9 7.8
light X X
oxygen(mg/L) - 7.2 7.3 7.6 7.6 7.7
Mortality N N N 1 2 3 5 7
Abnormality - N 1 2 3 6 9 10
1. Environmental conditions (Temp 23 ±2 oC, Light 12 hours photoperiod daily, Oxygen (mg/l)<8.0, pH 7.5 - 8.5.
2. Mortality and abnormality at 0, 1, 6, 24, 48, 72 and 96 hours varying concentration from 5 to 100 mg/l. note that N stands to
no mortality and abnormality has observed at that specific conditions
3. All readings are mean of triplicates
Annex table 3. Concentration of test conditions before and after renewal

Conc Condition 0h 24h 48h 72h 96h

(mg/L)
at the BR AR BR AR BR AR at the
start end
0 Temp 22.0 23.0 24.0 23.0 24.0 23.0 24.0 22.0
pH 8.0 8.1 8.4 8.1 8.3 7.9 8.0 7.9
oxygen(mg/L) 7.2 7.1 7.3 7.4 7.8 7.5 7.9 7.6
7 Temp 22.0 22.0 24.0 23.0 24.0 22.0 23.0 21.0
pH 7.9 8.0 7.9 7.9 8.1 7.8 8.0 7.7
oxygen(mg/L) 7.2 7.1 7.3 7.3 7.4 7.6 7.9 7.8
15 Temp 23.0 23.0 24.0 23.0 24.0 22.0 23.0 21.0
pH 7.9 8.0 8.1 7.9 8.0 7.8 7.9 7.7
oxygen(mg/L) 7.2 7.1 7.3 7.3 7.4 7.6 7.9 7.8
30 Temp 24.0 23.0 24.0 23.0 24.0 21.0 23.0 22.0
pH 7.9 8.1 8.2 8.0 8.2 7.9 8.2 7.7
oxygen(mg/L) 7.2 7.1 7.3 7.3 7.4 7.6 7.8 7.9
70 Temp 24.0 22.0 23.0 23.0 24.0 21.0 24.0 22.0
pH 7.9 8.1 8.2 8.0 8.2 7.9 8.2 7.7
oxygen(mg/L) 8.0 7.7 7.9 7.3 7.9 7.6 7.9 7.9
100 Temp 21.0 23.0 24.0 24.0 24.0 23.0 24.0 22.0
pH 7.9 8.1 8.2 8.0 8.2 7.9 8.2 7.8
oxygen(mg/L) 7.2 7.1 7.3 7.3 7.5 7.6 7.8 7.9

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Note: BR and AR stand for before renewal and after renewal

Annex table 4. Measured concentrations of test item in a test solution before and after renewal

0h 24h 48h 72h 96h

Conc
(mg/L)
at the start BR AR BR AR BR AR at the %R
end
0 ND ND ND ND ND ND ND ND NA
7 5.12 4.5 4.9 4.6 4.7 4.5 4.6 4.5 87.9
15 12.8 11.6 11.8 11.5 11.7 11.4 11.6 11.3 88.3
30 31 30.5 31.5 30 30.7 29.5 30.5 29.9 96.3
70 71 70 70.5 71 69 70 71 70.6 99.4
100 100 98 97 99 100 100.5 98.5 99.5 99.5
1. Note: BR and AR stand for before renewal and after renewal and ND and NA are for not detected and not analyzed
2. ND stands to not detectable and is a value below 0.010 which is the quantification limit of the method
3. All readings are mean of triplicates

Annex table 5.Calibration working standards and analysis peak area value

Replicates concentration peak area

5 0 0.0
5 0.03 0.1
5 3 15.1
5 30 150.6
5 60 301.2
5 120 602.4

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Annex table 6. Method recovery determination by standard addition (spiking) method

Exposure Sample
Replicates time Conc(mg/L) unspiked p. area spiking conc spiking p. area spiked p. area
3 0 0 0.0 5 48 50
3 24 0 0.0 5 49 50
3 48 0 0.0 5 50 51
3 72 0 0.0 5 49 51
3 96 0 0.0 5 48 52
AVERAGE 0 48.8 50.8
STDEV 0.0 0.8 0.8
%R 104.1
3 0 7 12.5 5 48 63.0
3 24 7 12.5 5 49 64.0
3 48 7 13.0 5 50 63.5
3 72 7 13.0 5 49 63.0
3 96 7 12.5 5 48 62.0
AVERAGE 12.7 48.8 63.1
STDEV 0.3 0.8 0.7
%R 103.3
3 0 15 30.0 5 48 80
3 24 15 30.0 5 49 80
3 48 15 29.0 5 50 81
3 72 15 30.0 5 49 82
3 96 15 29.0 5 48 82
AVERAGE 29.6 48.8 81
STDEV 0.5 0.8 1.0
%R 105.3

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 Good Laboratory Practice Assignment (A0301)

3 0 30 75.0 5 48 125
3 24 30 75.0 5 49 125
3 48 30 76.0 5 50 126
3 72 30 77.0 5 49 127
3 96 30 76.0 5 48 126
AVERAGE 75.8 48.8 125.8
STDEV 0.8 0.8 0.8
%R 102.5
3 0 70 175.0 50 136 300
3 24 70 174.0 50 138 301
3 48 70 172.0 50 135 301
3 72 70 175.0 50 134 302
3 96 70 175.0 50 134 303
AVERAGE 174.2 135.4 301.4
STDEV 1.3 1.7 1.1
%R 93.9
3 0 100 250.0 50 136 400
3 24 100 249.0 50 138 394
3 48 100 250.0 50 135 401
3 72 100 254.0 50 134 400
3 96 100 250.0 50 134 405
AVERAGE 250.6 135.4 400
STDEV 1.9 1.7 3.9
%R 106.5
Note: This spiking technique is done to determine the method suitability with spiking concentration of 5 and 50mg/L

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