Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 September 2009 to 9 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3100 (Aerobic Aquatic Biodegradation)
Deviations:
no
GLP compliance:
yes
Remarks:
Date of GLP inspection: 19-08-2008 Date of Signature on GLP certificate: 04-03-2009
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of sewage treatment micro-organisms was obtained on 5 October 2009 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture: Not recorded

- Method of cultivation: Not recorded

- Storage conditions: Not recorded

- Storage length: Not recorded

- Preparation of inoculum for exposure: The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1ºC prior to use.

- Pretreatment: Not recorded

- Concentration of sludge: 1 % V/V

- Initial cell/biomass concentration: not recorded

- Water filtered: yes

- Type and size of filter used, if any: filtered through coarse filter paper
Duration of test (contact time):
35 d
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
Results of DOC analyses on vessels containing test material on Days 0 and 35 given in Table 2. These results cannot be used to calculate the degradation of test material based on DOC analysis due to insoluble nature of test material in water
Details on study design:
METHODS
Preliminary Work
The test material was a poorly water soluble, volatile liquid and hence following the recommendations of the International Standards Organisation (ISO 1995) for dealing with such compounds, for the purpose of the biodegradation test the test material was added directly to the test vessels using a high precision gas tight syringe. Preliminary work conducted showed that a volume of 5.5 µL of test material injected using a gas tight syringe gave a measured weight of 5 mg, mean of 15 separate weighings.

Experimental Preparation
For the purpose of the test, the test material was dispersed directly in culture medium. An aliquot (5.5 µL) of test material was injected under the surface of inoculated culture medium (495 mL culture medium plus 5 mL inoculum) to give the required test concentration of 10 mg/L. A reduced test concentration of 10 mg/L was selected for use in the study at the request of the Sponsor. Analysis of the concentration, homogeneity and stability of the test material in the test preparations were not appropriate to the Test Guideline, however at the request of the Sponsor chemical and DOC analysis were performed (Appendix 2).

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1ºC prior to use.

Culture Medium
The culture medium used in this study (see Appendix 3) was that recommended in the OECD Guidelines.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Five replicate bottles containing inoculated culture medium to act as the control.
b) Three replicate bottles containing inoculated culture medium and the standard material, aniline, at a concentration of 100 mg/L.
c) Five replicate bottles containing inoculated culture medium and the test material at a concentration of 10 mg/L.
d) Three replicate bottles containing inoculated culture medium, the standard material, aniline, at a concentration of 100 mg/L and the test material at a concentration of 10 mg/L to act as toxicity control vessels.
e) Four replicate bottles containing deionised reverse osmosis water and the test material at a concentration of 10 mg/L.
Vessels a - d were inoculated with the prepared inoculum at a rate of 1% v/v. On Day 0, one control, one standard material, one test material, one toxicity control and one abiotic control vessel were sampled for pH and DOC analysis. Additionally, on Day 0, one control, one test material and one abiotic control vessel were sampled for compound specific analysis. The pH of all remaining control, test material, standard material, toxicity control and abiotic control vessels was measured prior to the vessels being placed in the CES Multi-channel Aerobic Respirometer. The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer. As biodegradation progressed, the micro-organisms converted oxygen to carbon dioxide which was absorbed into the ethanolamine (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggered the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolysing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated PC. The test was conducted in diffuse light at a temperature of 21 ± 1ºC. On Day 35, two control vessels, one standard material, two test material, one toxicity control and one abiotic control vessel that were considered to have given the most consistent BOD values over the study period were selected for pH determinations and/or compound specific analysis. Additionally DOC analysis was performed on two control vessels, standard material and toxicity control vessel. The remaining test material and abiotic control vessels also had DOC analysis performed on them. The remaining vessels which were not sampled were discarded and not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

Observations and Physico-chemical Measurements
The daily BOD values for the control, test and standard materials, the toxicity control and the abiotic control are given in Appendix 4. The temperature of the water bath was recorded daily. The pH of the relevant control, test and standard material vessels, toxicity control and abiotic control was recorded on Days 0 and 35.

Analyses

pH Measurements
In order to confirm whether the pH of the test preparations were changed, the pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter on Days 0 and 35.

Dissolved Organic Carbon (DOC) Analysis
On Day 0, DOC analyses were carried out on one control, standard material, test material, toxicity control and abiotic control vessel. On Day 35, DOC analyses were carried out on two control, one standard material, one test material, one toxicity control vessel and one abiotic control vessel. The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total Carbon analysis was carried out at 680ºC using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involved conversion of the sample by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) prepared in deionised water purified by reverse osmosis. Each analysis was carried out in triplicate.

Compound Specific Analyses
Compound specific analyses were carried out on Days 0 and 35 from the test vessels with inoculum and deionised water. The method of analysis, recoveries and test preparation analysis are described in Appendix 2.

Evaluation of Data
Please see attached evaluation of data.

Statistical Analysis
Statistical analysis of the Day 35 BOD values for the control and test material vessels was performed using a Students t-test. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validation Criteria
The BOD of the inoculated culture medium (control) is normally 20 to 30 mg O2/L and should not exceed 60 mg O2/L after 28 days. Values higher than 60 mg O2/L require critical examination of the data and the experimental technique. If the pH of the inoculated test vessels after 28 days is outside the range 6.0 to 8.5 and the degradation rate of the test material is less than 60%, the test should be repeated at a lower test concentration.

The test was considered valid if the difference between extremes of replicate BOD values at the plateau or at the end of the test is less than 20%. The toxicity control (test material and aniline) should attain ≥ 25% degradation by Day 14 of the study for the test material to be considered as non-inhibitory. The percentage degradation of aniline calculated from oxygen consumption values must be ≥60% after 14 days. Test materials giving a result of greater than 60% degradation after 28 days calculated from the oxygen consumption values may be regarded as readily biodegradable. This level of degradation must be reached within 10 days of the degradation rate exceeding 10% (the 10-Day window).
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
35 d
Remarks on result:
other: No significant biodegradation observed.
Details on results:
Please see first "Any other information on results" section
Results with reference substance:
Aniline attained 75% degradation after 14 days and 80% degradation after 35 days thereby confirming the suitability of the inoculum and test conditions.
Aniline attained 97% degradation after 35 days, calculated from the results of the DOC analyses performed on Days 0 and 35. The degradation rate calculated from the results of the DOC analyses was higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the aniline into the microbial biomass prior to degradation, and hence oxygen consumption, occurring.

The BOD values and the degree of degradation calculated from the oxygen consumption data for the control, test and standard materials, toxicity control and abiotic control are given in Table 1. The daily BOD values and percentage degradation values are given in attached Appendices 4 and 5. The calculated Theoretical Oxygen Demand values for the test and standard materials are given in attached Appendix 6. For BOD curves and biodegradation curves are given in attached Figures 2 and 3 respectively.

The results of the DOC analyses are summarised in Table 2. Daily temperature readings are given in Table 3 and pH values of each individual vessel are given in Table 4.

 

Table1              BOD and Biodegradation Values from Days 0 to35

Sample Description

ThOD

(mg O2/L)

Day 7

Day 14

Day 28

Day 35

BOD

(mg O2/L)

Degradation (%)

BOD

(mg O2/L)

Degradation (%)

BOD

(mg O2/L)

Degradation (%)

Mean Biodegradation (%)

BOD

(mg O2/l)

Degradation (%)

Mean Biodegradation (%)

Control

R1

-

6.46

-

12.04

-

25.70

-

-

43.36

-

-

R2

-

6.74

-

13.36

-

28.08

-

-

52.32

-

Aniline

 

309

181.18

56

243.24

75

281.92

83

-

296.34

80

-

Test Material

R1

30.3

8.20

5

15.70

10

29.16

7

6

34.90

-43

-43

R2

30.3

7.46

3

14.92

7

28.54

5

34.94

-43

Toxicity Control

 

339.3

6.50

0

115.06

30

229.28

60

-

246.44

59

-

Abiotic Control

 

30.3

0.70

-

2.38

-

5.40

-

-

6.20

-

-


 

Table 2              Dissolved Organic Carbon and Biodegradation Values

Sample Description

Theoretical Carbon Content

(mg C/l)

Day 0*

Day 35

DOC (mg C/L)

DOC (mg C/L)

Measured

Corrected for Control Value

% Theoretical Carbon Content

Measured

Corrected for Mean Control Values

Degradation (%)

Control

R1

-

<LOQ

-

-

<LOQ

-

-

R2

-

-

-

-

1.78

-

-

Aniline

 

77.38

78.08

78.08

101

2.91

2.02

97

Test Material

R1

8.98

1.17

1.17

NA***

-

-

-

R2

8.98

-

-

-

-

-

-

R3

8.98

-

-

-

3.33

2.44

NA***

Toxicity Control

86.36

77.40

77.40

NA***

5.07

4.18

NA***

Abiotic Control

8.98

<LOQ

<LOQ

NA***

6.89

6.89

NA***

RO Water

-

1.94

-

-

-

-

-

Aniline Stock Solution**

77.38

77.85

75.91

98

-

-

-

***R1


Table 3              Daily Temperature Readings of the Water Bath

Day

Temperature (ºC)

1

21.8

2

21.9

3

22.4

4

22.2

5

22.2

6

22.2

7

22.4

8

22.4

9

22.3

10

22.4

11

22.4

12

22.3

13

22.2

14

22.3

15

22.2

16

22.3

17

22.4

18

22.4

19

22.4

20

22.4

21

22.4

22

22.4

23

22.4

24

22.3

25

22.4

26

22.3

27

22.4

28

22.4

29

22.4

30

22.4

31

22.4

32

22.4

33

22.4

34

22.4

35

22.4


Table 4              pH Values of the Control, Test Material, Standard Material, Toxicity Control and Abiotic Control Preparations

Test Vessel

pH Value

Day 0

Day 35

Control

R1

7.8

7.5

R2

7.8

7.5

Aniline

 

7.8

8.0

Test Material

R1

7.9

8.0

R2

7.8

8.1

R3

7.8

8.2

Toxicity Control

 

7.8

8.3

Abiotic Control

R1

8.5

9.2

R2

8.5

9.2



R1– R2= Replicates 1 and 2

 LOQ = Limit of quantitaiton (assessed down to 1.0 mg C/L)

*Day 0 DOC values represent the analysis of the single replicate vessel sacrificed at Day 0 for DOC determination

**Stock Solution analyses represent the analysis of the initial stock solution diluted to a concentration of 100 mg/L prior to analysis

*** Not Applicable due to the insoluble nature of the test material in water

R1- R2= Replicates 1 and 2

R1- R2= Replicates 1 and 2

 

The mean BOD of the inoculated culture medium (control) was 26.89 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

 

As this test ran for 35 days it was not possible to determine pH values after 28 days. However after 35 days, although the degradation rate of the test material was below 60%, the pH values ranged from 8.0 – 8.3 so were inside the range of 6.0 – 8.5 for the 28-Day validation criteria.

The difference between extremes of replicate BOD values at the end of the test was <20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

 

 

 

Percentage Degradation Values

The test material attained no significant degradation calculated from oxygen consumption values after 35 days. The test material therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F. 

The slight degradation of the test material shown between Days 27 and 35 was considered to be due to variation in the BOD values between the control and test material vessels between this time.  As a result of the low test concentration (10 mg/l) employed for the test, these small variations in measured BOD values lead to relatively large variations in the calculated biodegradation rates.

There was no statistically significant difference (P≥0.05) between the control and test material BOD values on Day 35. The test material was therefore considered not to have had an inhibitory effect on the sewage treatment micro-organisms used in the test and this was confirmed by the toxicity control results whereby the toxicity control attained 30% degradation after 14 days and 59% degradation after 35 days.

Aniline attained 75% degradation after 14 days and 80% degradation after 35 days thereby confirming the suitability of the inoculum and test conditions.

Aniline attained 97% degradation after 35 days, calculated from the results of the DOC analyses performed on Days 0 and 35. The degradation rate calculated from the results of the DOC analyses was higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the aniline into the microbial biomass prior to degradation, and hence oxygen consumption, occurring.

The results of the DOC analyses performed on the vessels containing the test material on Days 0 and 35 are given in Table 2. These results cannot be used to calculate the degradation of the test material based on DOC analysis due to the insoluble nature of the test material in water.

 

Compound Specific Analyses

The compound specific analyses conducted on Day 0 (see Appendix 2) showed the measured concentration in the test material plus inoculum vessel to be 83% of the nominal value.  In the test material in deionised water vessel (abiotic control) the measured concentration was 85% of the nominal value.  Examination of the chromatograms from Day 0 of the test period showed four peaks attributable to the test material in both the vessels containing the test material.

The compound specific analyses conducted on Day 35 showed measured concentrations in the test material plus inoculum vessels to range from 32% to 34% of nominal values with four peaks attributable to the test material (see Appendix 2).  In the test material in deionised water vessel (abiotic control) the measured concentration was 55% of the nominal value with four peaks attributable to the test material.  

Although the results from chemical analysis on Day 35 indicated that there was an approximate 65% loss of test material from the inoculated test material vessels, this loss was considered not to be due to biological degradation. The test material in deionised water vessel also showed by a 45% loss of test material over the test period.  This loss of the test material was considered to be due to the volatile nature of the test material resulting in the loss of test material into the headspace of the sealed vessels and possibly the adsorption of the test material to the inoculum in the inoculated test vessels. The oxygen consumption values from the inoculated test material vessels confirmed no biological degradation of the test material occurred over the test period.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material showed no significant degradation calculated from oxygen consumption values after 35 days. The test material therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.
Executive summary:

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (q)).

At the request of the Sponsor, the test material, at a reduced test concentration of 10 mg/L, was exposed to sewage treatment micro-organisms with culture medium in sealed culture vessels in diffuse light at 21 ± 1ºC for 35 days. The degradation of the test material was assessed by the measurement of daily oxygen consumption values and compound specific analysis on Days 0 to 35. At the request of the Sponsor, Dissolved Organic Carbon (DOC) analysis was also performed on Days 0 and 35. Control solutions with inoculum and the standard material, aniline, together with a toxicity control were used for validation purposes.

 

The test material showed no significant degradation calculated from oxygen consumption values after 35 days. The test material therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F. The results obtained from the toxicity control vessel confirmed that, at the concentration of 10 mg/L, the test material did not exert any inhibitory effects. The compound specific analyses conducted on Day 0 showed the measured concentration in the test material plus inoculum vessel to be 83% of the nominal value.  In the test material in deionised water vessel (abiotic control) the measured concentration was 85% of the nominal value.  Examination of the chromatograms from Day 0 of the test period showed four peaks attributable to the test material in both the vessels containing the test material. The compound specific analyses conducted on Day 35 showed measured concentrations in the test material plus inoculum vessels to range from 32% to 34% of nominal values with four peaks attributable to the test material.  In the test material in deionised water vessel (abiotic control) the measured concentration was 55% of the nominal value with four peaks attributable to the test material.  

Although the results from chemical analysis on Day 35 indicated that there was an approximate 65% loss of test material from the inoculated test material vessels, this loss was considered not to be due to biological degradation. The test material in deionised water vessel also showed a 45% loss of test material over the test period.  This loss of the test material was considered to be due to the volatile nature of the test material resulting in the loss of test material into the headspace of the sealed vessels and possibly the adsorption of the test material to the inoculum in the inoculated test vessels. The oxygen consumption values from the inoculated test material vessels confirmed no biological degradation of the test material occurred over the test period.

The test material showed no significant degradation calculated from oxygen consumption values after 35 days. The test material therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.

Description of key information

Biodegradation screening tests indicate little or no biodegradation of the components of this substance. 

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The components of DVB-55, -65, and -HP have exhibited little or no biodegradation in screening tests of ready biodegradability. However, this does not necessarily indicate the substances to be persistent in the environment. The testing of biodegradation potential of the DVB substances is confounded by their volatility and potential for toxicity/inhibition of the microbial inocula using the relatively high concentrations used in these tests. Similar substances, such as ethylbenzene, xylenes, styrene, etc. have been shown to be biodegradable in water and soil; and the pathways for their aerobic biodegradation have been elucidated. Therefore, these substances are expected to be ultimately biodegradable in the environment.