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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP-guideline study with acceptable restrictions: The guideline requires 50 – 100 mg ThOD/L of test substance and 30 mg suspended solids/L of inoculum. In the test, only 30 mg ThOD/L test substance were used without adjustment of the amount of inoculum.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted in 1992
Deviations:
yes
Remarks:
Guideline requirements: 50 – 100 mg ThOD/L of test substance and 30 mg suspended solids/L of inoculum. Test conditions: 30 mg ThOD/L test substance without adjustment of inoculum.
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
adopted in 2008
Deviations:
yes
Remarks:
Guideline requirements: 50 – 100 mg ThOD/L of test substance and 30 mg suspended solids/L of inoculum. Test conditions: 30 mg ThOD/L test substance without adjustment of inoculum.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
adopted in 1998
Deviations:
yes
Remarks:
Guideline requirements: 50 – 100 mg ThOD/L of test substance and 30 mg suspended solids/L of inoculum. Test conditions: 30 mg ThOD/L test substance without adjustment of inoculum.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch bioreactor at the Midland Municipal Wastewater Treatment Plant (Midland MI, USA) on 30 Mar 2011. This facility treats an excess of 11 million litres of wastewater per day, of which > 90% is from domestic sources.
- Storage conditions: The activated sludge was continuously aerated until use.
- Storage length: one day
- Preparation of inoculum for exposure: Prior to use, the activated sludge was screened through 500 µm nylon mesh and briefly homogenized in a Waring blender (Waring Products Inc., Torrington CT, USA).
- Concentration of sludge mixed liquor suspended solids: 1267 mg/L
Duration of test (contact time):
28 d
Initial conc.:
30.1 mg/L
Based on:
other: ThOD
Initial conc.:
17.1 mg/L
Based on:
test mat.
Initial conc.:
8.81 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Defined mineral medium was prepared as specified in the OECD guideline.
- Test temperature: 22.5 - 23.5 °C
- pH: 6.67 - 7.46
- pH adjusted: yes
- Suspended solids concentration: 30 mg/L test solution
- Continuous darkness: yes
- Other: The medium was filter sterilised (0.2 µm).

TEST SYSTEM
- Culturing apparatus: Specially designed 1 L glass Pyrex bottles (10 cm wide, 23 cm high), each containing 500 mL of the inoculated mineral medium. The vessels are fitted with 20 x 105 mm glass side baffles to facilitate complete mixing and aeration.
- Number of culture flasks/concentration: 2
- Measuring equipment: Oxygen and CO2 were measured using the Columbus Micro-Oxymax automated respirometry system with a paramagnetic oxygen sensor and a non-dispersive infrared CO2 detector.
- Other: Continuous stirring with magnetic stir bars at 150 rpm was applied.

SAMPLING
- Sampling frequency: Concentrations of oxygen and CO2 in the headspace of each reaction vessel were recorded at 6 h intervals over the entire 28 d test period.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 vessels
- Abiotic sterile control: A single abiotic control mixture was prepared by adding HgCl2 (approx. 250 mg/L) to inoculated mineral medium containing the test substance. This abiotic control was used to determine the amount of O2 consumption, CO2 evolution, and DOC removal recorded in the test mixtures which could be attributed to abiotic reactions.
- Toxicity control: A single toxicity control reaction mixture was similarly prepared by combining the test substance (30 mg ThOD/mL) and 100 mg/L aniline in the inoculated mineral medium. The rate and extent of aniline degradation in this reaction mixture was used to assess whether the test substance was inhibitory to the sludge inoculum.
- Positive control with aniline: 2 vessels

STATISTICAL METHODS: Descriptive statistics (mean, standard deviation) were used where applicable.
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
3.4
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
7.9
Sampling time:
28 d
Details on results:
- No biodegradation of the test substance was evident based on measurement of BOD over 28 days.
- The onset and rate of biodegradation in the toxicity control mixture closely matched that in the positive controls indicating that the test substance at 17.1 mg/L was not toxic to the sludge inoculum.
- The abiotic control exhibited no O2 consumption or CO2 production.
- The cumulative oxygen consumption and CO2 production in the test mixtures were less than that in the inoculum blanks. This result suggests that the test substance was at least partially inhibitory to the inoculum under the conditions of the test.
Results with reference substance:
The reference substance attained > 60% degradation within 8.4 days. After 28 days, biodegradation reached 109.2%, 73.3% and 97.4% based on O2 consumption, CO2 evolution and DOC removal, respectively.
Interpretation of results:
under test conditions no biodegradation observed
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted in 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
adopted in 1998
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The inoculum consisted of secondary effluent, collected from the Midland Municipal Wastewater Treatment Plant (Midland MI, USA). This facility treats an excess of 3 million gallons of wastewater per day, of which > 90% is from domestic sources.
- Storage length: The effluent was collected on the day before test start.
- Preparation of inoculum for exposure: The pH of the inoculated mineral medium was measured and adjusted to 7.4 ± 0.2 using NaOH or HCl solutions. Prior to the initiation of the test the inoculated mineral medium was sparged with ambient laboratory air for at least 30 minutes, and then allowed to stand for at least 20 h under slow stirring. The concentration of dissolved oxygen in the equilibrated, inoculated mineral medium was measured and recorded to ensure that it is approximately 8.5 mg/L prior to initiating the test.
- Effluent filtered: yes
- Type and size of filter used: Whatman 114V
Duration of test (contact time):
28 d
Initial conc.:
1.98 mg/L
Based on:
test mat.
Initial conc.:
3.96 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The defined mineral medium specified by OECD 301D wa prepared by dissolving appropriate volumes of concentrated mineral stock solutions in deionised water.
- Solubilising agent (type and concentration if used): Silica gel (35-60 mesh) was fired in a muffle furnace (550 °C) to remove any trace organic contaminants and was added to all reaction mixtures at 2 g/BOD bottle.
- Test temperature: 22.3 ± 0.4 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 300 mL glass BOD bottles with magnetic stirr bars were filled to approx. 85% capacity with the inoculated mineral medium and allowed to mix overnight with open tops to equilibrate the mixture. The bottles were then sealed with ground glass stoppers and plastic covers.
- Number of culture flasks/concentration: 2 per concentration for each test group with 1.98 or 3.96 mg/L test substance.
- Measuring equipment: Orion model 97-08 dissolved oxygen electrode or Hach luminescent dissolved oxygen probe. The concentrations of dissolved nitrate and nitrite were determined using an ion chromatographic method based on EPA method 300.0.

SAMPLING
- Sampling frequency: on day 0, 7, 14, 21 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 10 bottles
- Toxicity control: yes, 10 bottles with each 4 mg/L sodium benzoate and 2 or 4 mg/L test substance
- Positive control: yes, 10 bottles
- Other: Replicate reaction mixtures of the inoculum blanks, 4mg/L test solution, and positive controls containing no silica gel were prepared for analysis on days 0, 14, and 28. These samples were used to assess the effect of adding silica gel to BOD reaction vessels.

STATISTICAL METHODS: Descriptive statistics (mean, standard deviation) were used where applicable.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
< 6
Sampling time:
28 d
Details on results:
- Nitrite and nitrate concentrations were below the limit of quantitation (1.0 mg/L). Therefore, results were not corrected for nitrification.
- No degradation was observed for the test substance concentration of 3.96 mg/L.
- The toxicity controls attained 27.5 and 24% degradation, respectively.
Results with reference substance:
The reference substance attained approx. 50% degradation after 7 days and 57% degradation after 14 days.

Summary of biodegradation based on oxygen consumption. Percent biodegradation values are reported as average value ± 1 standard deviation. (a) Calculation based on the COD or ThOD of both the test and reference material. (b) Calculation based on the ThOD of the reference material only.

Table 1: % Biodegradation of 1.98 mg/L Test Chemical

Incubation Time (Days)

Test Chemical

Positive Controls

Toxicity Controls (a)

Toxicity Controls (b)

0

_

_

_

_

7

0.43 ± 0.2

50.23

33.56

50.09

14

Not detected

57.19

29.44

50.16

21

Not detected

51.72

28.02

47.74

28

2.60 ± 5.1

48.17

27.48

46.82

 

Table 2: % Biodegradation of 3.96 mg/L Test Chemical

Incubation Time (Days)

Test Chemical

Positive Controls

Toxicity Controls (a)

Toxicity Controls (b)

0

_

_

_

_

7

0.87 ± 1.43

50.23

25.92

51.44

14

0.79 ± 6.94

57.19

28.03

55.63

21

Not detected

51.72

24.34

48.31

28

Not detected

48.17

27.28

54.13

 

 

Interpretation of results:
under test conditions no biodegradation observed
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Mar - 9 Apr 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: DIN EN 29888 Part 25
GLP compliance:
not specified
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): sewage plant Brühl, Germany
- Preparation of inoculum for exposure: centrifuged and washed multiple times
- dry solids: 25 g/L
- Concentration of sludge: 1g dry activated sludge per test batch
Duration of test (contact time):
7 d
Initial conc.:
0.5 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: COD
Details on study design:
TEST CONDITIONS
- Test temperature: 20 ± 2 °C
- Suspended solids concentration: 25 g/L

TEST SYSTEM
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: 0 h, 3 h, 24 h, 7 d

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
Reference substance:
ethylene glycol
Parameter:
other: COD
Value:
> 90
Sampling time:
7 d

Table 1: Measurements, mg COD/L after x hours or x days

 

0 hours

3 hours

24 hours

7 days

Dummy value

3

9

27

24

Control

660

609

390

40

Test 1

860

840

825

90

Test 2

880

845

830

100

Poisoned Batch

870

845

860

860

Inhib. Test

1540

1410

1100

110

Table 2: Total elimination in % after x hours or x days

 

3 hours*

24 hours

7 days

Control

9

40

>90

Test 1

<5

<5

>90

Test 2

5

<5

>90

Average

<5

<5

>90

Poisoned Batch

<5

<5

<5

Inhib. Test

9

23

>90

* reference value at hour 0

Description of key information

Not readily biodegradable: 3.4% (CO2) in 28 days, 7.9% (DOC) in 28 days (OECD 301 F); <6% (O2) in 28 days (OECD 301 D)
Inherently biodegradable: >90% (COD) in 7 days (DIN EN 29888 Part 25)

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

The biodegradability of 2-amino-2-ethyl-1,3-propanediol (AEPD) was investigated in two ready biodegradability studies and one inherent biodegradability study. All three tests are regarded as key studies. The first ready biodegradability study was conducted following OECD guideline 301 F and GLP (Gonsior and Hales, 2011). Activated sludge was used as inoculum. The second ready biodegradability study followed OECD guideline 301 D and GLP, with domestic sewage as inoculum. In both ready biodegradability studies biodegradation remained below 10% during the 28-day test period. The inherent biodegradability test was performed following the national guideline DIN EN 29888 Part 25 (Hornscheidt et al., 1996). Activated sludge from a domestic sewage treatment plant was used as inoculum, and over 90% biodegradation was reached during the 7-day test period. Based on the available biodegradability data, AEPD is considered rapidly biodegradable, but not readily biodegradable according to OECD criteria.