Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-28 to 2010-11-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
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:
- Name as cited in study report: POPDA
- color: clear, colorless (sponsor identification : yellowish liquid)
- Lot/Batch nulber: LL100365
- Purity: 94%
- Impurity: polypropylene glycol (approximately 6%)
- Date received: 2010-09-21
- Storage: in the dark
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 29 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 - <= 28 d
Initial conc.:
17.6 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 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, Shimadzu TOC-VCPH 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 680¿C 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 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Evalaution of data:
Calculation of carbon content
The theoretical amount of the carbon present in the test item, POPDA (C9H22O2N2), was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the test item multiplied by 100%.Thus for a 10 mg C/l test concentration (a total of 70.4 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.

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).
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). A further 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.

The results obtained from the samples taken for DOC analysis from the preliminary investigational work 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 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 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 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:
0
Sampling time:
28 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 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 with the exception of control replicates R1 and R2. 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.
Results with reference substance:
Sodium benzoate attained 62% degradation after 14 days and 94% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Sodium benzoate attained 100% and 99% degradation respectively 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

1.98

0.35

1.87

0.35

2.10

0.35

1.63

0.35

0.47

0.35

0.35

0.35

0.35

0.35

2

10.90

-

9.86

-

29.00

-

33.87

-

10.09

-

9.86

-

30.39

-

6

24.45

-

22.60

-

51.09

-

55.71

-

21.34

-

22.03

-

46.36

-

8

26.72

-

25.69

-

52.06

-

55.84

-

22.59

-

23.62

-

48.85

-

10

31.35

-

29.41

-

47.08

-

56.20

-

24.05

-

26.79

-

51.87

-

14

38.19

-

37.40

-

62.22*

-

62.90

-

25.16

-

30.26

-

58.03

-

21

38.19

-

37.74*

-

75.49

-

73.01

-

33.80

-

33.35

-

-

-

28

46.14

-

45.47

-

78.73

-

77.84

-

41.55

-

39.87

-

-

-

29

44.42

2.09

43.98

2.44

81.94*

2.44

81.39*

2.44

43.09

2.44

44.98

2.67

-

-


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

53

0

25

6

75

0

29

8

69

0

28

10

53

0

27

14

62

0

25

21

91

0

-

28

81

0

-

29*

94

0

-

 


-= 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

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

10.05

0.14

1

Sodium Benzoate

10 mg C/l R2

10.06

-0.01

0

Test Item

10 mg C/l R1

9.51

-0.16

0

Test Item

10 mg C/l R2

9.75

-0.30

0

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

19.91

0.04

0


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 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

9.92

99

      <control

0

100

Sodium Benzoate

10 mg C/l R2

10.07

101

0.13

1

99

Test Item

10 mg C/l R1

9.67

97

9.17

95

5

Test Item

10 mg C/l R2

10.05

101

9.32

93

7


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

7.7

Sodium Benzoate

10 mg C/l R2

7.7

Test Item

10 mg C/l R1

7.8

Test Item

10 mg C/l R2

7.8


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:
under test conditions no biodegradation observed
Conclusions:
The test item attained 0% degradation after 28 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 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 0% degradation after 28 days.

Conclusion.

The test item attained 0% degradation after 28 days.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2005-07-28 to 2006-01-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
no data on purity or stability of test substance (responsibility of Test Sponsor)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
No data on purity or stability of test substance.
GLP compliance:
yes
Remarks:
Statement, no certificate
Specific details on test material used for the study:
- Name as cited in study report: Jeffamine D-230
- Substance type: Active
- Color: colorless
- Lot/Batch number: 4W512
- Date receipt 2005-07-05 727.9g; 2005-07-13: 750.6 g
- Mean carbon content: 56.73%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from Brazos River Authority Sewage Treatment Plant, Sugar Land, Texas was collected Aug 05, 2005. The sample was aerated during transport and immediately placed under aeration upon arrival in the laboratory.
Duration of test (contact time):
31 d
Initial conc.:
> 10 - < 20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Inocculated nutrient mineral medium
- Test temperature: 19 deg C
- pH:7.4 +/- 0.5
- pH adjusted: yes
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 3 L flasks
- Number of culture flasks/concentration: 8 (2 reference,2 blank, 2 toxicity reference, 2 test substance)
- Method used to create aerobic conditions: aeration and continual stirring with magnetic stirrers.
- Measuring equipment: pH meter, titration apparatus
- Test performed in closed vessels due to significant volatility of test substance: no-closed system to allow for CO2 free air to be pumped through the test system.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: 3 CO2 absorbing bottles, each containing 100mL 0.0125 M Ba(OH)2, connected in series. Solution adjusted to 7.4 +/- 0.5 with HCl or NaOH.

SAMPLING
- Sampling frequency: Day 3, 4, 5, 7, 9, 13, 14, 15, 24, 27, 28, 29 and 30.
- Sampling method: The collection bottle nearest the test flask was removed and replaced. The solution was titrated with 0.05 N HCl to determine the amount of CO2 evolved, and % ThCO2.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 500 mL mineral media only.
- Other: 500 mL Reference Toxicant-sodium acetate in mineral media.

Reference substance:
acetic acid, sodium salt
Remarks:
Fisher Lot 980374
Test performance:
Test was considered valid.
Key result
Parameter:
other: ThCO2
Value:
9.83
Sampling time:
28 d
Results with reference substance:
Sodium acetate yielded 72.15% ThCO2 at 15 days. The test is considered valid.
Validity criteria fulfilled:
yes
Remarks:
Reference substance yielded >70% ThCO2 in < 28 days.
Interpretation of results:
not readily biodegradable
Conclusions:
The test material yielded a maximum average biodegradation of 9.83% ThCO2 in 28 days. Therefore, the test substance was not considered to be readily biodegradable.

Description of key information

The biodegradability of the substance was determined using the CO2 evolution method (Clarke, 2010; Stillmeadow Inc., 2006). The substance cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Two biodegradation tests are available (Clarke N., 2010 and Stillmeadow Inc., 2006) measuring the biodegradability of the substance following the OECD 301B guideline. The biodegradation of the substance exposed to a microbial inoculum in aerobic, mineral salts medium at an initial test concentration of 10 mg Carbon/L was measured. The inoculum was activated sewage sludge (predominantly domestic). The percentage of theoretical CO2 values (or percentage of biodegradation) was 0% and 9.8% respectively after day 28. Therefore, the substance cannot be considered to be readily biodegradable under the strict conditions of OECD Guideline 301B.