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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
January 2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE

EPI Suite v4.1

2. MODEL (incl. version number)

BIOWIN v4.10

The sub-models in BIOWIN v4.10 used for this QSAR analysis, are:
- BIOWIN3
- BIOWIN5


3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

BIOWIN3 and BIOWIN5 only require a chemical structure as SMILES as input. All SMILES codes are available in the attached QPRF.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.

5. APPLICABILITY DOMAIN
All constituents and biotransformation products fell within the molecular weight domain of BIOWIN3 and BIOWIN5, but some did not completely fall within their structural applicability domain. Compounds that did not completely fall within the structural domain of BIOWIN3, did fall within the structural domain of BIOWIN5, and vice versa. As those compounds were therefore always within the domain of one of the two models, this was not expected to affect the results or the suitability to assess the biodegradability of those compounds. More details on this assessment can be found in the attached QPRF.


6. ADEQUACY OF THE RESULT
The main purpose of this QSAR analysis, was to assess the ready biodegradation potential of the predicted biodegradation products of Amine C8 (CAS 68909-77-3) for use in the PBT/vPvB assessment. Overall, the predictions can be considered appropriate for this use.
Reason / purpose for cross-reference:
other: EAWAG-BBD PPS prediction of the biodegradation products of Amine C8 (CAS 68909-77-3) used in this QSAR analysis to estimate the biodegradation potential.
Principles of method if other than guideline:
Estimation of the ready biodegradation in water using the Ready Biodegradability prediction in BIOWIN v4.10, as based on its sub-models BIOWIN3 and BIOWIN5.
GLP compliance:
no
Specific details on test material used for the study:
Ready biodegradability predictions were made for the biodegradation products identified with the EAWAG-BBD Pathway Prediction System.
Oxygen conditions:
aerobic

The following predictions for ready biodegradation were made with the BIOWIN v4.10 model: 

Name BIOWIN v4.10 Ready biodegradability prediction
Ethyldiglycolamine (EDGA)  YES
2-morpholinoethylamine   NO
morpholin-3-one (M-3-ONE)   NO
4-[(2-hydroxyethoxy)ethyl] morpholine (HEEM/MEE)  NO
4-[2-amino(2-ethoxyethyl)]Morpholine (AEEM)  NO
Bis 2-(2-aminoEthoxyethanol (bis-DGA)  YES
Bis-Amino Ethoxyethylmorpholine (Bis-AEEM)  NO
4,4'-(ethane-1,2-diyl)bismorpholine (DMORE/DME)   NO
2-(2-aminoethoxy)acetaldehyde  YES
2-(2-aminoethoxy)acetate   YES
2-(2-oxoethoxy)acetate   YES
diglycolate  YES
2-(2-hydroxyethoxy)acetaldehyde  YES
2-Hydroxyethoxyacetate    YES
2-[2-(ethylamino)ethoxy]acetaldehyde  YES
2-[2-(ethylamino)ethoxy]acetate  YES
2-(morpholin-4-yl)acetaldehyde  NO
2-(morpholin-4-yl)acetate  YES
2-{[2-(2-oxoethoxy)ethyl]amino}acetate  YES
2-{[2-(carboxylatomethoxy)ethyl]amino}acetate  YES
2-{2-[(2-aminoethyl)amino]ethoxy}acetaldehyde  YES
2-{2-[(2-aminoethyl)amino]ethoxy}acetate   YES
2-{2-[(2-oxoethyl)amino]ethoxy}acetate  YES
2-[2-(morpholin-4-yl)ethoxy]acetaldehyde  NO
2-[2-(morpholin-4-yl)ethoxy]acetate  YES
2-(2-{[2-(2-oxoethoxy)ethyl]amino}ethoxy)acetate  YES
2-(2-{[2-(carboxylatomethoxy)ethyl]amino}ethoxy)acetate  YES
2-(2-{[2-(2-hydroxyethoxy)ethyl]amino}ethoxy)acetaldehyde  YES
2-(2-{[2-(2-hydroxyethoxy)ethyl]amino}ethoxy)acetate  YES
2-(2-{[2-(2-aminoethoxy)ethyl]amino}ethoxy)acetaldehyde  YES
2-(2-{[2-(2-aminoethoxy)ethyl]amino}ethoxy)acetate  YES
2-(2-aminoethoxy)ethan-1-amine  NO
2-[2-({2-[2-(morpholin-4-yl)ethoxy]ethyl}amino)ethoxy]acetaldehyde  NO
2-[2-({2-[2-(morpholin-4-yl)ethoxy]ethyl}amino)ethoxy]acetate  NO
17-(morpholin-4-yl)-3,9,15-trioxa-6,12-diazaheptadecanal  NO
17-(morpholin-4-yl)-3,9,15-trioxa-6,12-diazaheptadecanoate  NO
2-[2-({2-[2-(morpholin-4-yl)ethoxy]ethyl}amino)ethoxy]ethan-1-ol  NO
17-hydroxy-3,9,15-trioxa-6,12-diazaheptadecanal  YES
17-hydroxy-3,9,15-trioxa-6,12-diazaheptadecanoate  YES
17-oxo-3,9,15-trioxa-6,12-diazaheptadecanoate  YES
3,9,15-trioxa-6,12-diazaheptadecanedioate  YES
2-(2-{[2-(2-aminoethoxy)ethyl]amino}ethoxy)ethanol  YES
23-oxo-3,9,15,21-tetraoxa-6,12,18-triazatricosanoate  YES
3,9,15,21-tetraoxa-6,12,18-triazatricosanedioate  YES
17-amino-3,9,15-trioxa-6,12-diazaheptadecanoate   YES
bis[2-(2-aminoethoxy)ethyl]amine  NO
2-(2-{[2-(morpholin-4-yl)ethyl]amino}ethoxy)acetaldehyde  NO
2-(2-{[2-(morpholin-4-yl)ethyl]amino}ethoxy)acetate  NO
14-oxo-3,12-dioxa-6,9-diazatetradecanoate  YES
3,12-dioxa-6,9-diazatetradecanedioate  YES
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-06 - 2010-11-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Sponsor's identification: Amine C8
Description: dark brown liquid
Batch number: T7-271109
Purity: 99.65%
Date received: 21 September 2010
Expiry date: 31 December 2011
Storage conditions: room temperature in the dark

A Certificate of Analysis for the test item is given in Appendix 1, attached in overall remarks and attachments.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 5 October 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Concentration of sludge: The suspended solids concentration was equal to 2.9 g/l prior to use.


* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven
Duration of test (contact time):
28 d
Initial conc.:
18.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Culture medium
The culture medium used in this study (see Appendix 2 attached in overall remarks and attachments section) was that recommended in the OECD Guidelines.

Preparation of test system
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test item, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test item plus the reference item in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21DegC, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 ml of culture medium and 31.0 ml of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

SAMPLING
-CO2 analysis:
Samples (2 ml) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.
The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately.
On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser, a Shimadzu TOC-VCSH TOC analyser and a Shimadzu TOC-VCPH TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.


Dissolved Organic carbon (DOC) analysis:
Samples (30 ml) were removed from all culture vessels on Day 0 and from the control, reference and test item culture vessels on Day 28 and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-VCSH TOC analyser. Samples (50 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680DegC using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.


pH Measurement:

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Evalaution of data:
Determination of carbon content
The test item contains 54.10% carbon (data obtained from 3 replicate 100 mg/l stock solutions in Harlan Laboratories Ltd. UK Project Number: 41003980) and so for a concentration of 10 mg C/l (a total of 55.5 mg of test item in 3 litres) the total organic carbon present was 30 mg C.

The theoretical amount of the carbon present in the reference item, sodium benazoate, (C6H5COONa), was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the reference item multiplied by 100%.Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.

CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An aliquot (55.5 ml) of the test item stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 18.5 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.


Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values,
The values of Replicates R1 and R2 are meaned for the control, test and reference items before substitution in the equation.
%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test chemical multiplied by 100%. The percentage degradation from the results of the DOC analysis, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Equation: % degradation=[1-mg DOC in test flask on day 28/mg DOC in test flask on day 0] x 100%.

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total Co2 evolution = mgIC in control x 100/%C of Co2 x 1/test volume.

Validation criteria:
The results of the degradation test are considered valid if in the same test the reference item yields equal to or greater than 60% degradation by Day 14.
The test item may be considered to be readily biodegradable if equal to or greater than 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain equalt to or greater than 25% degradation by Day 14 for the test item to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/l medium.
The IC content of the test item suspension in the mineral medium at the beginning of the test should be < 5% of the TC.


Reference substance:
other: sodium benzoate
Preliminary study:
Preliminary Investigational Work
During the study, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus, Preliminary Investigation Work was conducted under Harlan Laboratories ltd. UK Project Number: 41003980 in order to investigate this.
The results obtained from the samples taken for DOC analysis from the preliminary investigational work conducted under Harlan Laboratories ltd. Project Number: 41003980) indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. However due to a shortage of 0.45 µm AcroCap filters, for the purpose of the study, the samples taken for DOC analysis were centrifuged to remove the suspended solids present without the loss of any test item.
Test performance:
Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1 (see any other infromation on results including tables section). Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 (see any other information on results including tables section) and the biodegradation curves are presented in Figure 1 (attached in overall remarks and attachments section). Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3 (see any other information on results including tables section), and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4 (see any other information on results including tables section. The pH values of the test preparations on Day 28 are given in Table 5 (see any other information on results including tables section). Observations made on the test vessels are given in Table 6 (see any other information on results including tables section).
The total CO2 evolution in the control vessels on Day 28 was 21.89 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3 in any other information on results including tables section) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
21
Sampling time:
28 d
Key result
Parameter:
% degradation (DOC removal)
Value:
25
Sampling time:
28 d
Remarks on result:
other: degradation value calculated from a mean of 2 replicate values
Details on results:
The total CO2 evolution in the control vessels on Day 28 was 21.89 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The toxicity control attained 49% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:
Sodium benzoate attained 91% degradation after 14 days and 102% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Sodium benzoate attained 100% degradation for Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis.

Table1              Inorganic Carbon Values on Each Analysis Occasion

Day

Control (mg IC)

Sodium Benzoate
(mg IC)

Test Item (mg IC)

Test Item
plus Sodium Benzoate Toxicity Control
(mg IC)

R1

R2

R1

R2

R1

R2

R1

Abs1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

0

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

2

6.03

-

6.03

-

23.55

-

22.97

-

6.61

-

4.64

-

18.68

-

6

10.61

-

11.76

-

33.45

-

33.22

-

13.15

-

10.73

-

31.37

-

8

12.04

-

14.45

-

36.46

-

36.23

-

15.37

-

13.07

-

36.81

-

10

13.23

-

16.99

-

39.10

-

37.85

-

16.42

-

14.59

-

37.96

-

14

14.28

-

17.00

-

41.14

-

44.54

-

20.06

-

17.68

-

44.99

-

21

14.08

-

17.46

-

41.12

-

44.95

-

21.97

-

18.70

-

-

-

28

16.80

-

19.04*

-

48.27

-

51.52

-

26.32

-

24.08

-

-

-

29

18.37

1.74

22.49

1.28

49.21

1.74

52.44

1.39

26.39

1.74

26.61

1.74

-

-


R1¿ R2= Replicates 1 and 2

Abs= CO2absorber vessel

- = No value determined

*Duplicate sample analysed as the original sample result was deemed anomalous

Table2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

57

0

21

6

74

3

34

8

77

3

39

10

78

1

38

14

91

11

49

21

91

15

-

28

107

24

-

29*

102

21

-


-= No degradation result obtained due to toxicity control being terminated after 14 days.

*Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Table 3              Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon*

(mg/l)

Inorganic Carbon*

(mg/l)

IC Content (% of TC)

Sodium Benzoate

10 mg C/lR1

9.38

-0.01

0

Sodium Benzoate

10 mg C/l R2

9.26

-0.26

0

Test Item

10 mg C/l R1

9.79

-0.12

0

Test Item

10 mg C/l R2

8.72

-0.17

0

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

18.82

0.70

4


R1¿ R2= Replicates 1 and 2

*Corrected for control values. Negative values are due toasured concentrations being less than control values

Table 4              Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

Test Vessel

DOC*Concentration

Day 0

Day 28

mg C/l

% of Nominal Carbon Content

mg C/l

% of Initial Carbon Concentration

% Degradation

Sodium Benzoate

10 mg C/l R1

9.39

94

0.02

0

100

Sodium Benzoate

10 mg C/l R2

9.51

95

0.00

0

100

Test Item

10 mg C/l R1

9.91

99

8.01

81

19

Test Item

10 mg C/l R2

8.89

89

6.16

69

31


R1¿ R2= Replicates 1 and 2

*Corrected for control values.

Table5              pH Values of the Test Preparations on Day 28

Test Vessel

pH

ControlR1

7.7

Control R2

7.7

Sodium Benzoate

10 mg C/l R1

7.8

Sodium Benzoate

10 mg C/l R2

7.8

Test Item

10 mg C/l R1

7.8

Test Item

10 mg C/l R2

7.7


R1¿ R2= Replicates 1 and 2

Table6              Observations on the Test Preparations Throughout the Test Period

 

Test Vessel

Observations on Test Preparations

Day 0

Day 5

Day 12

Day 19

Day 26

Control

R1

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

 

R2

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Reference Item

R1

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

 

R2

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Test Item

R1

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

 

R2

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Toxicity Control

 

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

-

-


R1¿ R2= Replicates 1 and 2

-= No observations made due to toxicity control being terminated after 14 days

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 21% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

Methods.

The test item, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.

The test item attained 21% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Conclusion.

The test item attained 21% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-28 - 2010-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECHA Reach Guidance Document Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance
Deviations:
yes
Remarks:
The test duration may be extended up to 60 days dependent on the level of biodegradation observed. The test volume employed was increased from 3 litres to 4 litres.The concentration of inoculum used was increased from 30 to 60 mg suspended solids/l
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Sponsor's identification: Amine C8
Description: dark brown liquid
Batch number: T7-271109
Purity: 99.65%
Date received: 21 September 2010
Expiry date: 31 December 2011
Storage conditions: room temperature in the dark



A Certificate of Analysis is given in Appendix 1 (attached in overall remarks and attachments section).
Oxygen conditions:
aerobic
Inoculum or test system:
other: The study used a composite microbial inoculum, derived from soil and a wastewater treatment facility.
Details on inoculum:
Test Species
The study used a composite microbial inoculum, derived from soil and a wastewater treatment facility.
A mixed population of activated sewage sludge micro-organisms was obtained on
5 October 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
A mixed population of soil micro-organisms was collected from Allestree Park, Derby, Derbyshire on 28 September 2010.
The soil surface was cleared of leaf litter and a sample collected from a depth of up to 20 cm below the soil surface. The soil sample was transported to the laboratory in a loosely tied black polythene bag. Once in the laboratory stones, plant remains and invertebrates were removed from the soil prior to sieving through a 2 mm mesh and then storing at approximately 4°C in a loosely tied black polythene bag until use.

Preparation of inoculum
The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. To one litre of washed activated sewage sludge, 1.0 g of soil was added prior to determination of the suspended solids level of the activated sewage sludge/soil being carried out by filtering a sample (100 ml) of the washed activated sewage sludge/soil by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.3 g/l prior to use. The pH of the inoculum was pH 7.4.
* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven
Duration of test (contact time):
0 - 42 d
Initial conc.:
18.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Culture medium
The culture medium used in this study (see Appendix 3 attached in overall remarks and attachments section) was that recommended in the OECD Guidelines.

Preparation of test system
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 4 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test item, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test item plus the reference item in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 60 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21degC, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 3400 ml of culture medium and 72.7 ml of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 4 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.



Sampling and analysis
CO2 analysis
Samples (2 ml) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28, 35, 42 and 43 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 43 for the control, reference and test item and on Day 0 for the toxicity control.
The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28, 35, 42 and 43 were analysed for CO2 immediately.
On Day 42, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (30 ml) were removed from all culture vessels on Day 0 and from the control, reference and test item culture vessels on Day 28 and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-VCPH TOC analyser. Samples (50 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680DegC using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

pH measurements
The pH of the test preparations was determined on Day 42, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Evalaution of data:
Determination of carbon content
In order to calculate the percentage carbon content of the test item, dissolved organic carbon analysis was performed on three replicate 100 mg/l stock solutions. (see preliminary study section and Appendix 1 attached in overall remarks and attachments section)
Thus for a concentration of 10 mg C/l (a total of 74 mg of test item in 4 litres) the total organic carbon present was 40 mg C.


The theoretical amount of the carbon present in the reference item, sodium benazoate, (C6H5COONa), was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the reference item multiplied by 100%.Thus for a 10 mg C/l test concentration (a total of 68.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 40 mg C.

Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values,
The values of Replicates R1 and R2 are meaned for the control, test and reference items before substitution in the equation.
%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test chemical multiplied by 100%. The percentage degradation from the results of the DOC analysis, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Equation: % degradation=[1-mg DOC in test flask on day 42/mg DOC in test flask on day 0] x 100%.

Validation criteria
The results of the degradation test are considered valid if in the same test the reference item yields equal to or greater than 60% degradation by Day 14.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
The toxicity control (test item and sodium benzoate) should attain equalt to or greater than 25% degradation by Day 14 for the test item to be considered as non-inhibitory.The IC content of the test item suspension in the mineral medium at the beginning of the test should be < 5% of the TC.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Preliminary Investigational Work
During the study, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analysed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-5050A analyser (see Appendix 2 attached in overall remarks and attachments section).
An amount of test item (100 mg) was dissolved in culture medium (1000 ml) to give a 100 mg/l stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a Gelman 0.45 µm AcroCap filter (discarding the initial 5 ml to pre-condition the filter). Two further amounts of test item (100 mg) were separately dissolved in culture medium (1000 ml) to give two separate 100 mg/l stock solution. One untreated sample from each stock was taken for DOC analysis. An additional amount of test item (100 mg) was dissolved in culture medium and inoculated at a concentration of 30 mg suspended solids (ss)/l prior to adjusting to a final volume of 1000 ml. Two samples were taken for DOC analysis; one after filtration through a Gelman 0.45 µm AcroCap filter (discarding the initial 5 ml to pre-condition the filter) and the other after centrifugation at 3500 rpm for 15 minutes. Control samples were prepared by inoculating culture medium (1000 ml) at a suspended solids level of 30 mg ss/l and then filtering or centrifuging as per the test item samples.
Test performance:
Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1(see any other information on results including tables section). Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2(see any other information on results including tables section) and the biodegradation curves are presented in Figure 1(attached in overall remarks and attachments section). Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3(see any other information on results including tables section), and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 42 are given in Table 4(see any other information on results including tables section). The pH values of the test preparations on Day 28 are given in Table 5(see any other information on results including tables section). Observations made on the test vessels throughout the study period are given in Table 6(see any other information on results including tables section).
Key result
Parameter:
% degradation (CO2 evolution)
Value:
15
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
18
Sampling time:
42 d
Details on results:
The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3 in any other information on results including tables section) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guideline.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guideline.
Acidification of the test vessels on Day 42 followed by the final analyses on Day 43 was conducted according to the methods specified in the Test Guideline. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 43 samples originated from dissolved CO2 that was present in the test vessels on Day 42 and hence the biodegradation value calculated from the Day 43 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 43 showed an increase in all replicate vessels. Inorganic carbon analysis of the samples from the second absorber vessels on Day 43 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
Results with reference substance:
Sodium benzoate attained 68% degradation after 14 days, 82% degradation after 28 days and 96% degradation after 42 days thereby confirming the suitability of the inoculum and test conditions.
Sodium benzoate attained 100% and 98% degradation respectively for Replicates R1 and R2 calculated from the results of the DOC analyses.

Table1              Inorganic Carbon Values on Each Analysis Occasion

Day

Control (mg IC)

Sodium Benzoate
(mg IC)

Test Item (mg IC)

Test Item
plus Sodium Benzoate Toxicity Control
(mg IC)

R1

R2

R1

R2

R1

R2

R1

Abs1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

0

0.35

0.35

0.35

0.35

0.35

 0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

0.35

2

8.47

-

9.51

-

29.69

-

29.12

-

8.93

-

9.40

-

27.84

-

6

21.45

-

24.80

-

48.09

-

52.01

-

18.22

-

25.03

-

51.09

-

8

24.42

-

27.75

-

47.93

-

59.86

-

21.33

-

28.09

-

52.52

-

10

27.36

-

32.94

-

50.04

-

55.98

-

22.46

-

31.01

-

51.53

-

14

34.57

-

39.21

-

64.60

-

63.35

-

29.24

-

37.85

-

60.29

-

21

36.28

-

42.02

-

67.37

-

68.39

-

42.36

-

43.26

-

-

-

28

40.32

-

46.93*

-

75.26

-

77.95

-

49.39

-

49.95

-

-

-

35

45.31

-

54.44

-

84.17

-

87.95

-

54.55

-

54.55

-

-

-

42

45.37*

-

54.78

-

87.87*

-

80.23

-

56.55*

-

52.90

-

-

-

43

47.08*

2.09

56.10*

1.85

92.62

2.09

87.01*

2.09

57.75

1.97

59.84

2.09

-

-


R1¿ R2= Replicates 1 and 2

Abs= CO2absorber vessels

- = No value determined

* Duplicate sample analysed as the original sample result was deemed anomalous

Table2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

51

0

24

6

67

0

35

8

70

0

33

10

57

0

27

14

68

0

29

21

72

9

-

28

82

15

-

35

90

12

-

42*

85

12

 

43

96

18

-


-= No degradation result obtained due to toxicity control being terminated after 14 days.

*Day 43 values corrected to include any carry-over of CO2detected in Absorber 2

Table3              Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon*

(mg/l)

Inorganic Carbon*

(mg/l)

IC Content (% of TC)

Sodium Benzoate

10 mg C/lR1

9.65

0.06

1

Sodium Benzoate

10 mg C/l R2

9.95

0.23

2

Test Item

10 mg C/l R1

9.91

0.19

2

Test Item

10 mg C/l R2

9.87

-0.01

0

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

19.97

0.27

1


R1¿ R2= Replicates 1 and 2

*Corrected for control values. Negative values are due toasured concentrations being less than control values

 

Table4              Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and42

Test Vessel

DOC*Concentration

Day 0

Day 42

mg C/l

% of Nominal Carbon Content

mg C/l

% of Initial Carbon Concentration

% Degradation

Sodium Benzoate

10 mg C/l R1

9.59

96

-0.12

-1

100

Sodium Benzoate

10 mg C/l R2

9.72

97

0.23

2

98

Test Item

10 mg C/l R1

9.72

97

4.53

47

53

Test Item

10 mg C/l R2

9.88

99

6.56

64

36


R1¿ R2= Replicates 1 and 2

*Corrected for control values.

Table5              pH Values of the Test Preparations on Day42

Test Vessel

pH

ControlR1

7.8

Control R2

7.7

Sodium Benzoate

10 mg C/l R1

7.8

Sodium Benzoate

10 mg C/l R2

7.7

Test Item

10 mg C/l R1

7.7

Test Item

10 mg C/l R2

7.7


R1¿ R2= Replicates 1 and 2

Table6              Observations on the Test Preparations Throughout the Test Period

 

Test Vessel

Observations on Test Preparations

Day 0

Day 5

Day 12

Day 19

Day 26

Control

R1

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

 

R2

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Reference Item

R1

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

 

R2

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Test Item

R1

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

 

R2

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Toxicity Control

 

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

-

-


 

Test Vessel

Observations on Test Preparations

Day 33

Day 40

Control

R1

Light brown dispersion

Light brown dispersion

 

R2

Light brown dispersion

Light brown dispersion

Reference Item

R1

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

 

R2

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Test Item

R1

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

 

R2

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible


R1¿ R2= Replicates 1 and 2

-= No observations made due to toxicity control being terminated after 14 days

 


Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 15% degradation after 28 days and 18% degradation after 42 days.
Executive summary:

Introduction.

A study was performed to assess the biodegradability of the test item in an aerobic aqueous medium. The test design was based on the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

However following the recommendations of the ECHA Reach Guidance Document : "Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance", the following modifications to a standard OECD 301B test were made:

Test duration: The test duration may be extended up to 60 days dependent on the level of biodegradation observed.

Testing in larger vessels: The test volume employed was increased from 3 litres to 4 litres.

Increasing the biomass: The concentration of inoculum used was increased from 30 mg suspended solids (ss)/l to 60 mg ss/l.

Methods.

The test item, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge and soil micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 42 days.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.

The test item attained 15% degradation after 28 days and 18% degradation after 42 days.

Conclusion.

The test item attained 15% degradation after 28 days and 18% degradation after 42 days.

Description of key information

Two key studies are available and used to conclude on the ready biodegradability of the substance in an aerobic aqueous medium. Both studies are given a Klimisch score of 1 and were conducted under GLP. The first study (Clarke, 2010 - report 41003975) is carried out according to the OECD guideline 301B (CO2 evolution test), EC Method C.4-C. After 28 days, the observed biodegradation was 21% and the test substance is regarded as not readily biodegradable. The second study (Clarke, 2010 - report 41003980) is an enhanced biodegradation test carried out according to the OECD guideline 301B (CO2 evolution test), EC Method C.4-C.  After 28 days, the observed biodegradation was 15%, and after 42 days, the observed biodegradation was 18%. The test substance is regarded as not readily biodegradable.

 

Additionally, the relevant degradation products of Amine C8 were identified by means of the EAWAG-BBD Pathway Prediction System model. The biodegradability of each of these degradation products was predicted using the QSAR model BIOWIN available in the EPI Suite software. 42 degradation products were identified, of which 11 were predicted to be not readily biodegradable. The remaining 31 degradation products were predicted to be readily biodegradable. 

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

In the first study  (Clarke, 2010 - report 41003975), the ready biodegradability of the substance was determined in a CO2 evolution test (OECD guideline 301B, EC Method C.4-C). After 28 days, the observed biodegradation was 21%. The toxicity control and positive control were both valid. The test substance is regarded as not readily biodegradable.

 

In the second study (Clarke, 2010 - report 41003980), the ready biodegradability of the substance was tested in an enhanced CO2 evolution test, modified with an extended duration, an increased test volume from 3 liters to 4 liters and an increased concentration of inoculum from 30 to 60 mg suspended solids/l. After 28 days, the observed biodegradation was 15%, and after 42 days, the observed biodegradation was 18%. The toxicity control and positive control were both valid. The test substance is regarded as not readily biodegradable.