Working for a better future

REPORT 

 Assessment of Ready Biodegradability by 

Respirometry

HLS study number: CEF0011

Version ID: Audited Draft 2 Report

Issue date: 12 September 2013

Page 1 of 22
Huntingdon Life Sciences 

CEF0011

Sponsor and Test Facility Details 

Sponsor

 

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK

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CEF0011

Table 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 blank 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 test 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 and 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 BSc MSc

Study Director

Huntingdon Life Sciences 

Date

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Quality Assurance Statement

: Assessment of Ready Biodegradability by Respirometry

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

Study Phase Date(s) of Inspection 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/Ammonia 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

Department of Quality Assurance

Huntingdon Life Sciences Ltd

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Contributing Scientists

: Assessment of Ready Biodegradability by Respirometry

Study management 

Aquatic Ecotoxicology and Biodegradation

Jordan Davies BSc MSc

Study Director

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Summary

The ready 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 mgO2/L. Two 

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

inoculated mineral salts medium plus the reference substance sodium benzoate (50 mgO2/L) 

of which one also contained (50 mgO2/L) in order to assess the potential 

inhibitory effects of the test substance on the microbial inoculum. As the 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 consider the impact of nitrification in the test. 

The test system comprised of an automated system for oxygen (O2) 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 mgO2/500 mL or 60% of the ThOD (25 mgO2/500 mL) after 2 days of 

incubation, and 27.6 mgO2/500 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 reference 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 mgO2/500 mL, 

equivalent to 36.06 and 42.76 mgO2/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 mgO2/500 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 was equivalent to 24% of the 

theoretical value (25 mgO2/500 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, although 60% pass level was achieved by the 

Day 28. 

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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 state: 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: C26H36BNO6

Molecular Weight: 469.38 

Sample received: 4 March 2013 

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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 pentahydrate (for preparation of the 1M electrolyte solution).

Potassium hydroxide pellets (for the preparation of the 2M CO2 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 mm2) 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 approximately 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. Appropriate 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 

electrolyte, 1M copper sulfate solution, and the CO2 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 mgO2/500 mL)

Test 2 in inoculated MSM (25 mgO2/500 mL)

Inhibition assay 1 (25 mgO2/500 mL) + sodium benzoate 

(25 mgO2/500 mL) in inoculated MSM

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

ATU test 1 in inoculated MSM (25 mgO2/500 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 was 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 mgO2/L.

In 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 mgO2/500 mL or 50 mgO2/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 CO2 absorber was replaced in each cell (to ensure the maximum 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 cell 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, the 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 controls should be, typically, 20 to 30 mgO2/L but 

should not exceed 60 mgO2/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 values 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 deviations

There were no protocol deviations.

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4. Calculation of Results 

4.1 Calculation of theoretical oxygen demand (ThOD)

The ThOD values 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] mgO2/mg

 MW

ThOD Nitrite:

ThOD = 16 [2c + 1⁄2(h) + 3/2n - o] mgO2/mg

 MW

ThOD Nitrate:

ThOD = 16 [2c + 1⁄2(h) + 5/2n - o] mgO2/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 (mgO2/mg) = 16 [2c + 1⁄2(h) + 1⁄2na - o] mgO2/mg

 MW 

Where

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

MW is the molecular weight.

These equations assume that carbon is mineralised to CO2, hydrogen to H2O, and sodium to 

Na2O. Nitrogen is presumed to be eliminated as ammonia, nitrite and nitrate.

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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 (mgO2/culture) - mean uptake by blank controls 

(mgO2/culture)

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

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

% degradation(n) = BOD(n) (mgO2) x 100 

 ThOD/(mgO2) 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 Records

This report has been compiled from original data, which will be stored, together with 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. 

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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 calculated from their 

empirical formula to be:

: C26H36BNO6 MW = 469.4 ThOD ammonia: 2.13 mgO2/mg

 ThOD nitrite: 2.23 mgO2/mg

 ThOD nitrate: 2.27 mgO2/mg

Sodium benzoate: C7H5O2Na MW = 144 ThOD: 1.67 mgO2/mg

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

sodium benzoate were calculated to be 25 mgO2/500 ml or 50 mgO2/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 mgO2/500 mL or 60% of the ThOD (25 mgO2/500 mL) after 2 days of 

incubation, and 27.6 mgO2/500 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 mgO2/500 mL, equivalent to 

36.06 and 42.76 mgO2/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 mgO2/500 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 mgO2/500 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.

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6. Conclusion

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

theoretical value (25 mgO2/500 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 mgO2/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 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 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 substances

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Table 1 Respirometry test - cumulative consumption and blank corrected values of oxygen demands 

Day

Control Sodium Benzoate

(50 mgO2/L) (50 mgO2/L) Sodium benzoate(50 mgO2/L) in inhibition mixture

Cumulative oxygen demands

(mgO2) 

Cumulative 

oxygen 

demands

(mgO2)

Blank 

corrected

(mgO2) 

Cumulative oxygen demands

(mgO2) 

Blank corrected

(mgO2) 

Cumulative 

oxygen 

demands

(mgO2)

Blank corrected

(mgO2) 

Culture 1 Culture 2 Culture 1 Culture 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 8.16 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 24.71 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 17.20 16.41 - -

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.

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Table 2 Cumulative consumption and blank corrected values of oxygen 

demands in ATU treated mixtures

Day

Control (50 mgO2/L)

Cumulative oxygen 

demands

(mgO2)

Cumulative oxygen 

demands

(mgO2)

Blank corrected

(mgO2) 

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.

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Table 3 Respirometry test – biodegradation of the test and reference 

substances

Day

% Biodegradation

(50 mgO2/L) (50 mgO2/L)

in the presence 

of ATU

Sodium 

Benzoate

(50 mgO2/L)

Sodium benzoate

(50 mgO2/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.

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Appendix 1 Preparation of mineral 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 complies 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

KH2PO4 (potassium dihydrogen phosphate) 8.50

K2HPO4 (dipotassium hydrogen phosphate) 21.75

Na2HPO4.2H2O (disodium monohydrogen 

phosphate dihydrate) 33.40

NH4Cl (ammonium chloride) 0.50

Stock solution 2

MgSO4.7H2O (magnesium sulphate 

heptahydrate) 22.50

Stock solution 3

CaCl2.2H2O (calcium chloride dihydrate) 36.40

Stock solution 4

FeCl3.6H2O (iron (III) chloride hexahydrate) 0.25

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

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Appendix 2 Compliance statement

22