### Working for a better future



## REPORT

Assess ment of Ready Biodegradability b y

Respirometry

HLS s tudy number: CEF0011

Version ID: Audited Draft 2 Report

Issue date: 12 September 2013

Page 1 of 22Huntingdon Life Sciences

CEF0011



# Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK

2Huntingdon Life Sciences

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# T

able of Contents

Sponsor and Test Facility Details

................................ ................................ .......................... 2

Compliance with Good Laboratory Practice

................................ ................................ .......... 4

Quality Assurance Statement

................................ ................................ ................................ 5

Contributing Scientists

................................ ................................ ................................ .......... 6

Summary

................................ ................................ ................................ .............................. 7

1. Introduction

................................ ................................ ................................ .................. 8

2. Test Substance

................................ ................................ ................................ ............ 9

3. Experimental Procedure

................................ ................................ ............................. 10

3.1 Respirometry test

................................ ................................ ............................ 10

3.1.1 Reagents

................................ ................................ ............................. 10

3.1.2 Inoculum

................................ ................................ ............................. 10

3.1.3 Test design

................................ ................................ ......................... 10

3.2 Validity criteria

................................ ................................ ................................ 12

3.3 Protocol deviations

................................ ................................ ......................... 12

4. Calculation of Results

................................ ................................ ................................ . 13

4.1 Calculation of theoretical oxygen demand (ThOD)

................................ .......... 13

4.2 Biochemical oxygen demand (BOD) and percentage degradation

.................. 14

4.3 Maintenance of Records

................................ ................................ ................. 14

5. Results

................................ ................................ ................................ ....................... 15

5.1 Calculation of theoretical oxygen demand (ThOD)

................................ .......... 15

5.2 Respirometry test

................................ ................................ ............................ 15

6. Conclusion

................................ ................................ ................................ ................. 16



# List of Figures

Figure 1 Percentage degradation of the test and reference substances

..................... 17



# List of Tables

Table 1 Respirometry test

- cumulative consumption and bl ank corrected values of

oxygen demands

................................ ................................ .......................... 18

Table 2 Cumulative consumption and blank corrected values of oxygen demands in

ATU treated mixtures

................................ ................................ ................... 19

Table 3 Respirometry test

– biodegradation of the t est and reference substances

... 20



# List of Appendices

Appendix 1 Preparation of mineral salts medium

................................ ............................ 21

Appendix 2 Compliance statement

................................ ................................ ................. 22

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# Compliance with Good Laboratory Practice

: Assessment of Ready Biodegradability by Respirometry

The study described in this report was conducted in compliance with the following Good

Laboratory Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No.

3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997),

ENV/MC/CHEM (98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No

L 50/44).

These principles of Good Laboratory Practice are accepted by the regulatory authorities of

the United States of America a nd Japan on the basis of intergovernmental agreements.

In line with normal practice in this type of study, an assessment of the stability or

homogeneity of the test substance formulation and achieved concentration was not

conducted.

Jordan Davies BS c MSc

Study Director

Huntingdon Life Sciences Date

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# Quality Assurance Statement

: A ssessment of Ready Biodegradability by Respirometry

The following inspections and audits have been carried out in relation to this study:

Study Phase Date(s) of In spection Date of Reporting to Study

Director and Management

Protocol Audit 03 Jun 2013 03 Jun 2013

Report Audit 27 Aug 2013 27 Aug 2013

Process based inspections : At or about the time this study was in progress inspections of

procedures employed on this type of study were carried out. These were conducted and

reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reporting to

Management

Environmental measurements &

monitoring/Ammo nia analysis 07 Mar 2013 07 Mar 2013

Set

- up of test or reference

compound/inoculum mixture 04 Jul 2013 04 Jul 2013

Study management & conduct 25 Jul 2013 25 Jul 2013

In addition, an inspection of the facility where this study was conducted was carried out on an

annual basis. These inspections were reported to Company Management.

........................................................................................ ...................... Tom Pullinger MRQA Date

Group Manager

Departme nt of Quality Assurance

Huntingdon Life Sciences Ltd

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# Contributing Scientists

: A ssessment of Ready Biodegradability by Respirometry



## Study management

Aquatic Ecotoxicology and Biodegradation

Jordan Davies BSc MSc

Study Director

6Huntingdon Life Sciences

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# Summary

The rea dy biodegradability of was assessed in accordance with Directive (EC)

440/2008, C.4-D, ‘Determination of Ready Biodegradability, Manometric respirometry’ and

OECD Procedure 301F ‘Ready Biodegradability, Manometric Respirometry Test’, adopted

17 July 1992.

was added to two bottles containing mineral salts medium inoculated with

activated sludge (30 mg solids/L) to give a nominal test concentration of 50 mgO 2 

### /L. Two

control cultures contained inoculated mineral salts medium alone. Two cultures contained

inoculated mineral salts medium plus the reference substance sodium benzoate (50 mgO 2 

### /L)

of which one also contained (50 mgO 2 

### /L) in order to assess the potential

inhibitory effects of the test substance on the microbial inoculum. As th e test substance

contained nitrogen, two further mixtures (one blank-control and one mixture containing the

test substance) contained an inhibitor of nitrification, allylthiourea (ATU) at 11.6 mg/L. The

results of the additional cultures were used to consi der the impact of nitrification in the test.

The test system comprised of an automated system for oxygen (O 2 

### ) generation and the

cultures were stirred and held in a thermostatically -controlled water bath.

The blank- corrected oxygen demanded by the culture containing the reference substance had

achieved 15.02 mgO 2 

### /500 mL or 60% of the ThOD (25 mgO 2 

### /500 mL) after 2 days of

incubation, and 27.6 mgO 2 

### /500 mL or 111% by Day 28. Comparing the ATU control with

the blank controls, suggests that nitrification had occurred, which is implicated in causing the

conversion of nitrogen naturally present in the cultures during the decline phase of the

degradation curve, as cells begin to lyse and nutrient turnover processes occur when the

sodium benzoate is exhausted from the culture. This may be why the % degradation at the

end of the study in the refer ence culture was higher than expected. In the presence of

, degradation of sodium benzoate had achieved 64 % by Day 3 . Cumulative levels

of oxygen consumption by the controls after 28 days (18.03 and 21.38 mgO 2 

### /500 mL,

equivalent to 36.06 and 42.76 mgO 2 

### /L) were considered to be acceptable for this assay

system. These results confirm that was not inhibitory to the activity of the

microbial inoculum and that the test was valid.

Oxygen consumption by the control mixture containing Allylthiourea (ATU) was equivalent

to 20.24 mgO 2 

### /500 mL on Day 28 of the test. T he test substance had achieved 81%

degradation in the presence of ATU by 28 days.

Mean oxygen consumption in mixtures containing was equivalent to 24 % of the

theoretical value (25 mgO 2 

### /500 mL) after approximately 2 days, 63 % after approximately 26

days and 67% at the end of the test (Day 28). This pattern of degradation indicates that the

microbial po pulation had adapted to the degradation products following the primary

(hydrolytic) degradation of the test material.

Substances are considered to be readily biodegradable in this type of test if oxygen

consumption is equal to or greater than 60% of the Th OD of the test mixtures within ten days

of the consumption achieving 10%. Therefore, was not considered to be readily

biodegradable under the conditions of this test, although 60% pass level was achieved by the

Day 28.

7Huntingdon Life Sciences

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# 1. Introduction

The objective of this study was to assess the biodegradability of . It was

conducted in accordance with Directive (EC) 440/2008, C.4-D, ‘Determination of Ready

Biodegradability, Manometric respirometry’ and OECD Procedure 301F ‘Ready

Biodegradability, Manometric Respirometry Test’, adopted 17 July 1992. The test system

was chosen on the basis that it was the most suitable OECD 301 method for this type of test

substance.

The parameter determined in this test is the cumulative amount of oxygen consumed by

mixtures containing , mineral salts medium, and an inoculum of activated sludge

after 28 days of incubation at temperatures nominally in the range 22 ± 2 ° C.

The protocol was approved by Huntingdon Life Sciences Management and the Study

Director on the 4 June 2013 and by the Sponsor on 5 June 2013.

The experimental start and completion dates were 19 June 2013 and 01 August 2013,

respectively.

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# 2. Test Substance

Name :

Chemical name :

CAS No. :

Physical stat e: White crystalline powder

Batch number: 12342

Purity : To be included in the raw data when available

Storage : Room temperature in the dark

Expiry Date : To be included in the raw data when available

Molecular Formula : C 26 

### H 36 

### BNO 6



### Molecular Weight : 469.38

Sample received: 4 March 2013

9Huntingdon Life Sciences

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# 3. Experimental Procedure

3.1 Respirometry test

3.1.1 Reagents

The following AR grade reagents were used:

Sodium benzoate (for preparation of the reference substance stock solution).

Copper sulfate pentahy drate (for preparation of the 1 M electrolyte solution).

Potassium hydroxide pellet s (for the preparation of the 2 M CO 2 

### absorber solution).

3.1.2 Inoculum

A sample of activated sludge was obtained from Worlingworth sewage treatment works

(Suffolk, UK), which treats predominantly domestic waste. At the time of collection, the

sludge was sieved (1 mm 2 

### ) then transported to the laboratory and left to stand for

approximately 50 minutes to allow the sewage solids to settle. A portion of the supernatant

was removed and the sludge aerated until required.

The concentration of suspended solids in a homogenised sample was determined before the

start of the test. Aliquots (10 mL) of the sludge were filtered through dried and preweighed

Whatman GF/C filters, which were then dried again at approximat ely 105 ° C for one hour,

allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS)

content of the sludge was then determined and the volume required to give a solids level of

30 mg/L in test cultures was calculated. This was added to bottles one day before the

addition of test material to allow a period of ageing.

3.1.3 Test design

In this test, a Co

- ordinated Environmental Services (CES) Ltd automated respirometer and

associated software was used to monitor the cumulative amount of oxygen consumed by the

mixtures.

One day before the addition of test material , a volume ( 5 litres) of mineral salts medium

(MSM, see Appendix 1 ) was prepared and the pH determined and adjusted to 7.43 with 5M

HCl. Appropri ate volumes of the MSM were then added to eight amber glass culture bottles

(500 mL) and inoculated with activated sludge. Magnetic stirrer bars were added to the

cultures and each bottle was fitted with an electrolytic cell assembly (containing the

elect rolyte, 1M copper sulfate solution, and the CO 2 

### absorber, 5 mL of 2M potassium

hydroxide) and connected to the respirometer. A magnetic stirrer was set to give a vortex in

each test mixture and the instrument was initiated.

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### The following schedule describes the contents of the bottles used in the study:

Identity No. of bottles Contents

Blank

– control 2 Inoculated MSM alone

Reference 1 Inoculated MSM + sodium benzoate (25 mgO 2 /500 mL)

Test 2 in inoculated MSM (25 mgO 2 /500 mL)

Inhibition assay 1 (25 mgO 2 /500 mL) + sodium benzoate

(25 mgO 2 /500 mL) in inoculated MSM

ATU control 1 Inoculated MSM + ATU (11.6 mg/L)

ATU test 1 in inoculated MSM (25 mgO 2 /500 mL) + ATU

(11.6 mg/L)



### The test substance (117 mg) was weighed into a volumetric flask (50 mL) containing acetone

( ca . 30 mL), shaken and made up to volume with acetone. Aliquots (5 mL) of this solution

were added to the empty bottles designated to contain the test substance, after which the

acetone w as evaporated off with nitrogen. The bottles were left to stand for an hour and

further flushed with nitrogen. Inoculated media was added to the volume in each bottle to

give a nominal concentration of test substance with an oxygen demand of 50 mgO 2 

### /L.

I n order to ensure similarity in the preparation of all mixtures, the control and reference

bottles were also treated with acetone in an identical manner as described for the test groups

above.

Sodium benzoate (20 mL) was added as an aqueous stock solution (0.750 g/L) in MSM to the

reference and inhibition assay cultures to give a final nominal concentration of

25 mgO 2 

### /500 mL or 50 mgO 2 

### /L.

A stock solution of allylthiourea (ATU) was prepared in MSM at a nominal concentration of

1.16 g/L. An aliquot (5 mL) of this stock solution was added to one culture containing

inoculated MSM alone and to one containing the test substance.

The pH of the cultures was measured and no adjustment was necessary.

The CO 2 

### absorber was replaced in each cell (to ensure the maximu m adsorption capacity in

the test) and the cultures were sealed and returned to the water bath to equilibrate. The cells

were connected to the computer

- controlled system and the test was initiated.

A record of the cumulative oxygen demand made by each cel l was printed at, typically,

hourly intervals.

The correct operation of the magnetic stirrers and the temperature of the water bath were

recorded at appropriate intervals during the test.

The nominal temperature range for the test was 22 ± 2oC.

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### On Day 6, t he inhibition assay mixture was terminated and the pH measured.

On Day 28

, the test was halted and the pH of each mixture was measured.



## 3.2 Validity criteria

• Biodegradation of the reference substance alone should be ≥60% by Day 14.

• Oxygen consumption by the c ontrols should be, typically, 20 to 30 mgO 2 

### /L but

should not exceed 60 mgO 2 

### /L.

• Biodegradation of the reference substance in the presence of the test substance

(inhibition assay) should not be <25% on Day 14.

• The difference between extremes of replicate val ues for the removal of the test

substance at the plateau, at the end of the test or at the end of the 10

- day window, as

appropriate, should be ≤20%. This only applies where the test substance is

considered to be readily biodegradable.



## 3.3 Protocol d eviations

The re were no protocol deviations.

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# 4. Calculation of Results

4.1 Calculation of theoretical oxygen demand (ThOD)

The ThOD value s of was calculated from its empirical formula and molecular

weight (469.38) in the following way:

ThOD ammonia:

ThOD = 16 [2c + 1⁄2(h

- 3n) - o] mgO 2 

### /mg

MW

ThOD Nitrite:

ThOD = 16 [2c + 1⁄2(h) + 3/2n - o] mgO 2 

### /mg

MW

ThOD Nitrate:

ThOD = 16 [2c + 1⁄2(h) + 5/2n - o] mgO 2 

### /mg

MW

where MW is the molecular weight.

The ThOD value of sodium benzoate was calculated from its empirical formula and

molecular weight in the following way:

ThOD (mgO 2 

### /mg) = 16 [2c + 1⁄2(h) + 1⁄2na - o] mgO 2 

### /mg

MW

Where

c = carbon, h = hydrogen, o = oxygen, na = sodium

MW is the molecular weight.

These equations assume that carbon is mineralised to CO 2 

### , hydrogen to H 2 

### O, and sodium to

Na 2 

### O. Nitrogen is presume d to be eliminated as ammonia, nitrite and nitrate.

13Huntingdon Life Sciences

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## 4.2 Biochemical oxygen demand (BOD) and percentage

degradation

The amounts of oxygen consumed in biotic mixtures containing the reference or test

substances in the respirometer test were calculated in the following way:

BOD (n) 

### = uptake by the test mixture (mgO 2 

### /culture) - mean uptake by blank controls

(mgO 2 

### /culture)

The percentage degradation of the reference or test substances at daily intervals during the

test were calculated as a percentage of their ThO D values in the following way:

% degradation (n) 

### = BOD (n) 

### (mgO 2 

### ) x 100

ThOD/(mgO 2 

### ) of mixture

The ThOD of the reference or test mixture was calculated by multiplying the total weight

(mg) added to the test vessel by the ThOD of the substance.

In mixtures containing sodium benzoate and the apparent BOD of sodium

benzoate was calculated by subtracting the mean oxygen consumption in control vessels and

consumption attributable to the test substance from that of the mixture containing sodium

benzoate plus the test substance. The resulting BOD was expressed as a percentage of the

ThOD of sodium benzoate.



## 4.3 Maintenance of R ecords

This report has been compiled from original data, which will be stored, together wit h a copy

of the final report, a copy of the protocol and amendments in Huntingdon Life Sciences’

Archives, Huntingdon Life Sciences Ltd, Eye, Suffolk, UK.

Records will be retained for a minimum period of at least one year from the date of issue of

the final report. At the end of the one year retention period the Sponsor will be contacted and

advice sought on the future requirements. Under no circumstances will any item be discarded

without the Sponsor’s knowledge.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study for

twenty years, and a copy of the final report in its archives indefinitely.

14Huntingdon Life Sciences

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# 5. Results

5.1 Calculation of theoretical oxygen demand (ThOD)

The ThOD values of the test substance and sodium benzoate were calculat ed from their

empirical formula to be:

: C 26 

### H 36 

### BNO 6 

### MW = 469.4 ThOD ammonia: 2.13 mgO 2 

### /mg

ThOD nitrite: 2.23 mgO 2 

### /mg

ThOD nitrate: 2.27 mgO 2 

### /mg

Sodium benzoate: C 7 

### H 5 

### O 2 

### Na MW = 144 ThOD: 1.67 mgO 2 

### /mg

The theoretical oxygen content of the mixtures containing the test substance alone and

sodium benzoate were calculated to be 25 mgO 2 

### /500 ml or 50 mgO 2 

### /L.



## 5.2 Respirometry test

The cumulative amounts of oxygen consumed by each mixture and the blank controlcorrected cumulative oxygen consumption are given in Table 1 (Test, reference and

inhibition) and Table 2 (ATU mixture) . The percentage biodegradation is given in Table 3

. The extent of biodegradation of the test and reference substances is illustrated in Figure 1

.

The blank- corrected oxygen demanded by the culture containing the reference substance had

achieved 15.02 mgO 2 

### /500 mL or 60% of the ThOD (25 mgO 2 

### /500 mL) after 2 days of

incubation, and 27.6 mgO 2 

### /500 mL or 111% by Day 28. In the presence of ,

degradation of sodium benzoate had achieved 64% by Day 3. Cumulative levels of oxygen

consumption by the controls after 28 days (18.03 and 21.38 mgO 2 

### /500 mL, equivalent to

36.06 and 42.76 mgO 2 

### /L) were considered to be acceptable for this assay system. These

results confirm that was not inhibitory to the activity of the microbial inoculum

and that the test was valid.

Oxygen consumption by the control mixture containing Allylthiourea (ATU) was equivalent

to 20.24 mgO 2 

### /500 mL on Day 28 of the test. The test substance had achieved 81%

degradation in the presence of ATU by 28 days.

Mean oxygen consumption in mixtures containing SABoTBA was equivalent to 24% of the

theoretical value (25 mgO 2 

### /500 mL) after approximately 2 days, 63% after approximately 26

days and 67% at the end of the test (Day 28).

Substances are considered to be readily biodegradable in this type of test if oxygen

consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days

of the consumption achieving 10%. Therefore, was not considered to be readily

biodegradable under the conditions of this test.

The pH of test mixtures ranged between 7.36 and 7.41 at the start of the test and 7.38 and

7.58 at the end.

The temperature of the water bath ranged from 21.1 to 23.6 ° C.

15Huntingdon Life Sciences

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# 6. Conclusion

Mean oxygen consumption in mixtures containing was equivalent to 24% of the

theoretical value (25 mgO 2 

### /500 mL) after approximately 2 days, 63% after approximately 26

days and 67% at the end of the test (Day 28). This pattern of degradation indicates that the

microbial population had adapted to the degradation products following the primary

(hydrolytic) degradation of the test material

.

Substances are considered to be readily biodegradable in this type of test if oxygen

consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days

of the consumption achieving 10%. Therefore, was not considered to be readily

biodegradable under the conditions of this test.

The results obtained for the rate of degradation of sodium benzoate (60 % of its ThOD after 2

days) and for the cumulative amount of oxygen consumed by the control mixtures (36.06 and

42.76 mgO 2 /L ) fulfilled the validity criteria for this test. Comparing the ATU control with

the blank controls, suggests that nitrification had occurred, which is implicated in causing the

conversion of nitrogen naturally present in the cultures during the decline ph ase of the

degradation curve, as cells begin to lyse and nutrient turnover processes occur when the

sodium benzoate is exhausted from the culture. This may be why the % degradation at the

end of the study in the reference culture was higher than expected.

In the presence of the degradation of sodium benzoate achieved 64 % after 3 days

indicating that the test substance was not inhibitory to the microbial inoculum.

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### Figure 1 Percentage degradation of the test and reference sub stances

17Huntingdon Life Sciences: CEF0011



### Table 1 Respirometry test

- cumulative consumption and blank corrected values of oxygen demands

Day Control Sodium Benzoate

(50 mgO 2 / L) ( 50 mgO 2 /L) Sodium benzoate

(50 mgO 2 /L) in inhi bition mixture

Cumulative oxygen demands

(mgO 2 ) Cumulative

oxygen

demands

(mgO 2 ) Blank

corrected

(mgO 2 ) Cumulative oxygen demands

(mgO 2 ) Blank corrected

(mgO 2 ) Cumulative

oxygen

demands

(mgO 2 ) Blank corrected

(mgO 2 )

Culture 1 Culture 2 Culture 1 Cult ure 2 Culture 1 Culture 2

1 2.18 1.89 14.41 12.38 2.06 3.24 0.02 1.21 14.45 11.80

2 3.83 3.54 18.70 15.02 9.53 9.78 5.85 6.10 23.71 14.06

3 5.18 4.93 21.99 16.94 11.07 11.28 6.02 6.23 27.17 16.00

4 6.41 6.24 25.09 18.77 12.55 12.64 6.23 6.32

- -

5 7 .35 7.39 27.44 20.07 13.80 13.72 6.43 6.35

- -

6 8.14 8.43 29.27 20.99 14.91 14.80 6.63 6.52

- -

7 8.80 9.30 30.67 21.62 15.82 15.61 6.77 6.56

- -

8 9.41 10.16 31.88 22.10 16.72 16.43 6.94 6.65

- -

9 9.91 10.91 32.90 22.49 17.63 17.05 7.22 6.64

- -

10 10.51 11.68 33.98 22.89 18.55 17.72 7.46 6.63

- -

11 10.99 12.41 35.02 23.32 19.42 18.36 7.72 6.66

- -

12 11.51 13.07 35.98 23.69 20.13 18.92 7.84 6.63

- -

13 11.91 13.68 36.88 24.09 20.82 19.34 8.03 6.55

- -

14 12.37 14.30 37.77 24.44 21.49 19.86 8.1 6 6.53

- -

15 12.82 14.99 38.71 24.81 22.19 20.47 8.29 6.57

- -

16 13.36 15.63 39.62 25.13 22.92 21.09 8.43 6.60

- -

17 13.72 16.07 40.31 25.42 23.40 21.51 8.51 6.62

- -

18 14.11 16.55 41.02 25.69 23.96 22.09 8.63 6.76

- -

19 14.55 17.11 41.79 25.96 2 4.71 22.96 8.88 7.13

- -

20 14.97 17.65 42.50 26.19 25.40 24.09 9.09 7.78

- -

21 15.43 18.20 43.21 26.40 26.17 25.86 9.36 9.05

- -

22 15.78 18.61 43.79 26.60 27.13 29.19 9.94 12.00

- -

23 16.20 19.11 44.44 26.79 28.15 32.29 10.50 14.64

- -

24 16.57 19 .65 45.12 27.01 29.71 33.34 11.60 15.23

- -

25 16.99 20.13 45.73 27.17 31.63 34.13 13.07 15.57

- -

26 17.34 20.55 46.31 27.37 34.42 34.84 15.48 15.90

- -

27 17.70 21.01 46.85 27.50 36.38 35.52 17.03 16.17

- -

28 18.03 21.38 47.35 27.65 36.90 36.11 17.2 0 16.41

- - The data presented were calculated using unrounded values stored in the computer database. Minor numerical differences may b e observed if rounded values are used to calculate the data.

This minor discrepancy is not considered to be significa nt.

18Huntingdon Life Sciences: CEF0011



### Table 2 Cumulative consumption and blank corrected values of oxygen

demands in ATU treated mixtures

Day Control (50 mgO 2 /L)

Cumulative oxygen

demands

(mgO 2 ) Cumulative oxygen

demands

(mgO 2 ) Blank corrected

(mgO 2 )

1 1.31 2.58 1.27

2 2.41 9.05 6.64

3 3.33 10.39 7.06

4 4.26 11.68 7.42

5 4.97 12.78 7.81

6 5.58 13.84 8.26

7 5.99 14.78 8.79

8 6.49 15.66 9.17

9 6.97 16.34 9.37

10 7.68 17.09 9.41

11 8.18 17.78 9.60

12 8.70 18.43 9.73

13 9.16 19.01 9.85

14 9.64 19.59 9.95

15 10.05 20.15 10.10

16 10.66 20.86 10.20

17 11.01 21.32 10.31

18 11.43 21.88 10.45

19 11.93 22.53 10.60

20 12.41 23.21 10.80

21 12.91 24.01 11.10

22 13.32 25.01 11.69

23 13.76 26.21 12.45

24 14.22 28.21 13.99

25 14.66 31.13 16. 47

26 15.05 34.27 19.22

27 15.43 35.25 19.82

28 15.76 36.00 20.24

The data presented were calculated using unrounded values stored in the

computer database. Minor numerical differences may be observed if

rounded values are used to calculate the data. This minor discrepancy is not

considered to be significant.

19Huntingdon Life Sciences: CEF0011



### Table 3 Respirometry test – b iodegradation of the test and reference

substances

Day % Biodegradation

(50 mgO 2 /L) (50 mgO 2 /L)

in the presence

of ATU Sodium

Benzoate

(50 mgO 2 /L) Sodium benzoate

(50 mgO 2 /L)

in the inhibition

mixture Test 1 Test 2 Mean

1 0 5 2 5 50 47

2 23 24 24 27 60 56

3 24 25 24 28 68 64

4 25 25 25 30 75

-

5 26 25 26 31 80

-

6 27 26 26 33 84

-

7 27 26 27 35 86

-

8 28 27 27 37 88

-

9 29 27 28 37 90

-

10 30 27 28 38 92

-

11 31 27 29 38 93

-

12 31 27 29 39 95

-

13 32 26 29 39 96

-

14 33 26 29 40 98

-

15 33 26 30 40 99

-

16 34 26 30 41 101

-

17 34 26 30 41 102

-

18 35 27 31 42 103

-

19 36 29 32 42 104

-

20 36 31 34 43 105

-

21 37 36 37 44 106

-

22 40 48 44 47 106

-

23 42 59 50 50 107

-

24 46 61 54 56 108

-

25 52 62 57 66 109

-

26 62 64 63 77 109

-

27 68 65 66 79 110

-

28 69 66 67 81 111

-

The data presented were calculated using unrounded values stored in the computer database.

Minor numerical differences may be observed if rounded values are used to calculate the data.

This minor discrepancy is not considered to be significant

.

20Huntingdon Life Sciences: CEF0011



### Appendix 1 Preparation of mi neral salts medium

Dilution water

The dilution water used to prepare stock and test solutions of mineral salts medium was tap

water that had been softened and treated by reverse osmosis and then purified; nominal

resistivity, ≥ 18 MegOhm.cm. This water co mplies with the relevant standards (British

Standard (BS) 3978, 1987; American Society for Testing and Materials (ASTM) D 1193

- 06)

for classification as Grade 1 or Type 1 water for laboratory use.

Mineral salts medium

The mineral salts medium (MSM) for the test was prepared by establishing 10 mL of stock

solution 1 and 1 mL of solutions 2, 3 and 4 in each litre of water required for the test.

Stock solution 1 g/L

KH 2 

### PO 4 

### (potassium dihydrogen phosphate) 8.50

K 2 

### HPO 4 

### (dipotassium hydrogen phosphate) 21.75

N a 2 

### HPO 4 

### .2H 2 

### O (disodium monohydrogen

phosphate dihydrate) 33.40

NH 4 

### Cl (ammonium chloride) 0.50

Stock solution 2

MgSO 4 

### .7H 2 

### O (magnesium sulphate

heptahydrate) 22.50

Stock solution 3

CaCl 2 

### .2H 2 

### O (calcium chloride dihydrate) 36.40

Stock solution 4

FeCl 3 

### .6H 2 

### O (iron (III) chloride hexahydrate) 0.25

Minor variations in the weights taken are not considered to be significant.

21Huntingdon Life Sciences: CEF0011



### Appendix 2 Compliance statement

22