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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19 Jul -17 Aug 2005
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)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)
Remarks:
2003-02-13, The Department of Health of the Government of the United Kingdom
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of activated sludge micro-organisms was obtained on 18 Jul 2005 from the aeration stage of Seven Trent Water sewage treatment plant at Loughborough Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: washed three times by settlement and resuspension in culture medium, continuous aeration at 21°C
- Concentration of sludge: 1.8 g/L
Duration of test (contact time):
29 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test vessels: 5 L glass culture vessels each containing 3 L of solution
- Composition of medium: according to OECD Guideline
- Suspended solids concentration: 30 mg/L
- Temperature: 21°C
- Method used to create aerobic conditions: Test vessels were sealed and CO2-free air bubbled through the solution at a rate of approx. 40 mL/min, stirred with magnetic stirrer
- CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05M NaOH
- pH: 7.4-7.5
- pH adjusted: yes
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Measuring equipment: WTW pH/Oxi 340I pH and dissolved oxygen meter

SAMPLING
- Sampling frequency: Day, 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28, 29
- Sample storage before analysis: at -20°C on day 12 and 18, other samples measured immediately

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Toxicity control: 1


Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
100
Sampling time:
28 d
Details on results:
10-day window fulfilled. The test substance is readily biodegradable.
The toxicity control achieved 103% degradation after 28 days.
Results with reference substance:
30 mg/L sodium benzoate were degraded to 92% after 28 days;
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: sewage treatment plant Hochdahl treating predominantly domestic sewage
- Preparation of inoculum for exposure: the effluent was filtered through a coarse paper, the first 200 mL were discarded
- Concentration of sludge: 1 drop effluent/L
Duration of test (contact time):
30 d
Initial conc.:
2 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Deionized water O2 concentration: 8-9 mg/L

TEST SYSTEM
- Culturing apparatus: completely filled closed bottles


CONTROL AND BLANK SYSTEM
no details provided
Reference substance:
other: dodecyl ester sodium salt (CAS 151-21-3) 2 mg/L
Parameter:
% degradation (O2 consumption)
Value:
69
Sampling time:
30 d
Details on results:
Under the described test conditions 69% of the test substance was degraded within the 30-day test period. Since the pass level (60% ThOD) and the 14-day window were reached the substance is considered to be readily biodegradable.

Table 1: Biodegradation of the test and reference substance:

Day % Degradation
Test substance [2 mg/L] Reference substance [2 mg/L]
5 45 70
15 63 99-106
30 69 96
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Apr - 10 May 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
According to EPA GLP Standards (40 CFR, Part 792)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from Sycamore Sewage Treatment Plant, Cincinnati, Ohio. This STP receives primarily domestic wastewater
- Storage conditions: Sludge was aerated to prevent settling of solids until ready to use
- Storage length: The inoculum was used on the same day of preparation
- Preparation of inoculum for exposure: The sludge was homogenized (in a blender for 2 minutes at medium speed) and allowed to settle in beaker for approx. 30 minutes and supernatant was carefully decanted. Sufficient volumes of the mixed liquor was collected and treated at one time to provide enough inoculum for all test bottles. Microbial plate counts were also performed on the inoculum. For microbial plate counts, serial dilutions using the nutrient broth (5 g of pancreatic digest of gelatin and 3 g of beef extract in 1L of bottle) were performed on the inoculum and then plated onto trypticase soy agar plates (agar contained 15 g of pancreatic digest of casein, 5 g of papaic digest of soybean meal, 5 g of sodium chloride and 15 grams of agar in 1L of water). The plates were incubated at room temperature for 48 h.
- Concentration of sludge: 1% v/v (10 mL/L).
- Initial cell/biomass concentration: 2.3 x 10(5) CFU/mL (average plate count conducted in duplicates)
- Water filtered: Not applicable

Duration of test (contact time):
35 d
Initial conc.:
20 mg/L
Based on:
act. ingr.
Initial conc.:
11 mg/L
Based on:
other: TOC (measured)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The test medium was modified biochemical oxygen demand (BOD) water similar to OECD 301 B media except the addition of 1 mL of 4% (w/v) solution of (NH4)2(SO4)
- Additional substrate: No
- Solubilising agent: None used
- Test temperature: Room temperature
- pH: No data
- pH adjusted: Not reported
- Aeration of dilution water: Test medium (BOD water) was added to each test bottle and connected to the CO2-free apparatus. The test bottles were placed on a rotary shaker at 145±10 rpms at room temperature and aerated overnight.
- Suspended solids concentration: Not reported
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: Clear handled Jugs (1 gallon) with stopper containing 2 L final combined volume of test medium, test substance, and inoculum as follows:
1950 ml test medium
40 ml stock solution of test substance
10 ml inoculum
2000 ml Total
- Number of culture flasks/concentration: Two each for test, inoculum blank and procedure control.
- Method used to create aerobic conditions: By continuous aeration with CO2-free air (flow was controlled using flow meters).
- Measuring equipment: Carbon dioxide measuring apparatus consisted of trapping train of three french square jars filled with 100 mL of 0.024 N Ba(OH)2 and were connected in series to the exit air line of each test bottle.
- Test performed in closed vessels: No
- Details of trap for CO2 and volatile organics if used: Three french square jars which were filled with 100 mL of 0.024 N Ba(OH)2 and were connected in series to the exit air line of each test bottle. Periodically the traps nearest to the test bottles were removed for titration and the remaining two traps were moved one place closer to the respective test bottles and a new trap was added to the end of the series. With every change of traps, an extra bottle containing 100 mL of Ba(OH)2 solution was included. This was titrated as the "initial" to determine the amount of CO2 produced in the control (blank). The entire content of each of the Ba(OH)2 traps and "initial" solution were titrated with 0.05 N HCl to end point equivalence.
- Other: The CO2 produced in each bottle was reacted with 0.024N Ba(OH)2 in the traps and precipitated as BaCO3. The amount ofCO2 produced was determined by titrating the remaining Ba(OH)2 in the traps with 0.05N standard HCI. After 35 d the study was terminated by acidifying the flasks with 1 mL conc. HCl and final titration was performed after overnight aeration.

SAMPLING
- Sampling frequency: On Day 0, 3, 5, 9, 15, 17, 23, 29, 35 and 36
- Sampling method: The first base trap in the trapping train was removed, and titrated. The remaining 2 traps were moved forward, and a new trap added to the end of series
- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: No
- Reference control: Yes

STATISTICAL METHODS: Not reported
Reference substance:
other: glucose (20 mg/L)
Preliminary study:
Not applicable
Test performance:
No data
Parameter:
% degradation (CO2 evolution)
Value:
21.7
Sampling time:
3 d
Remarks on result:
other: Mean value of 2 replicates, indicating start of 10 d window criterion
Parameter:
% degradation (CO2 evolution)
Value:
60.7
Sampling time:
10 d
Remarks on result:
other: Mean value of 2 replicates, met the 10 day window. (Straight line interpolation between measurements on days 9 and 15.)
Parameter:
% degradation (CO2 evolution)
Value:
70.4
Sampling time:
15 d
Remarks on result:
other: Mean value of 2 replicates
Parameter:
% degradation (CO2 evolution)
Value:
79.1
Sampling time:
29 d
Remarks on result:
other: Mean value of 2 replicates, indicating final percentage of degradation
Details on results:
- Final SOC (in mg/L) of test substance was as found to be <0.2 and 0.3 for replicate 1 and 2 respectively.
- For details on biodegradation results, please refer to 'Table 1' in the ‘Any other information on results incl. tables’.
-Final SOC (in mg/L) was found to be <0.2 in both blanks.
Results with reference substance:
- The Reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window. Pass level of >60% reached at Day 9 in both replicates. (Mean values of all both replicates on Day 3 = 37.9%, Day 9 = 69.4% and Day 29 = 89.4% TCO2)
For details on individual replicate results, please refer to 'Table 2' in the ‘Any other information on results incl. tables’
- Final mg Soluble Organic Carbon (SOC)/L were found to be 0.4 and < 0.2 mg/L in replicate 1 and 2 respectively.

Table 1: Ready Biodegradation Test of C14 alkyl sulfate- sodium (study #35564)

% of TCO2

Days

Replicate 1

Replicate 2

0

0

0

3

21.45

22.04

5

38.32

36.75

9

58.74

58.73

15

69.37

71.6

17

70.5

73.63

23

78.48

76.65

29

79.87

78.28

35

78.34

77.63

36

78.36

79.99

Table 2: Ready Biodegradation Test of glucose (reference substance)

% of TCO2

Days

Replicate 1

Replicate 2

0

0

0

3

39.76

35.95

5

55.89

60.14

9

63.71

75.08

15

75.59

90.2

17

78.67

91.85

23

80.11

96.08

29

81.34

97.47

35

79.87

99.12

36

80.59

97.16

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Sodium tetradecyl sulfate (C14-alkyl sulfate) is readily biodegradable.
Executive summary:

A ready biodegradation test, following OECD 301B guideline, was conducted on sodium tetradecyl sulfate (C14-alkyl sulfate; C14AS). The test concentration was 20 mg active/L, and the inoculum was 1% v/v. The test treatments, inoculum blank and the reference were measured in duplicate.  The deviation between replicate treatments was < 20% (validity criteria of the guideline).

Greater than 10% biodegradation of the test material was reached at Day 3 and > 60% biodegradation was reached at Day 10 in all replicates.  At Day 3, the average percent biodegradation was 21.8, and at Day 10, the average percent biodegradation was 60.7.  The final level of biodegradation on Day 29 was 79.8%. Based on these results, C14AS is readily biodegradable.

The reference material, glucose reached > 10% biodegradation at Day 3 and > 60% at Day 9 in all replicates. Hence, it met the validity criteria established in the guideline for a reference material. 

This ready biodegradation test is classified as acceptable, and satisfies the guideline requirements for the OECD 301B.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Parameter:
% degradation (O2 consumption)
Value:
69
Sampling time:
30 d
Remarks on result:
other: Source, key, 1120-01-0, 2001
Parameter:
% degradation (CO2 evolution)
Value:
79.1
Sampling time:
29 d
Remarks on result:
other: Source, key, 1191-50-0, 1995

Description of key information

Ready biodegradable according to OECD criteria.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Since no reliable biodegradability tests are available for C14-16AS Na (CAS 91648-54-3), in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across to another structurally related category members was conducted. Read-across data from C14AS Na (CAS 1191-50-0), C16AS Na (CAS 1120-01-0), and C12-16 (CAS 73296-89-6) were used.

One ready biodegradability study is available for C16AS Na (CAS 1120-01-0). In the non-GLP supporting study according to OECD 301 D guideline (BASF, 2001) effluent of a municipal sewage treatment plant was used as inoculum. The initial concentration of the test substance was 2 mg a.s./L. Biodegradation based on O2 consumption reached 69% after 30 days. Since the pass level was reached, C16AS Na (CAS 1120-01-0) is considered to be readily biodegradable according to the OECD conditions.

One GLP ready biodegradation test is available for C14AS Na (CAS 1191-50-0; P&G, 1995). This study was performed according to OECD 301 B guideline using activated, domestic, non-adapted sludge as inoculum (10 mg sludge/L). The initial concentration of the test substance 11 mg/L as TOC. Biodegradation measured as CO2 evolution was greater than 60% after 10 days. The final level of biodegradation after 28 days was 79.8%.

Moreover, the biodegradability of C12-16AS Na (CAS 7329-89-6) was investigated in a GLP guideline study according to OECD 301 B (KAO, 2005). Domestic, activated and non-adapted sludge was used as inoculum. After 28 days of exposure the biodegradation measured as CO2 evolution reached 100%; the substance is therefore considered as readily biodegradable.

Due to structural and property similarities with the tested category members it can be concluded that the above results apply also to C14-16AS Na (CAS 91648-54-3). The substance can be considered as readily biodegradable according to OECD criteria.