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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
Study period:
04 November - 03 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No: CNAA053604
Storage: At room temperature, ca. 20°C
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source: Sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant.
- Preparation of inoculum for exposure: The sludge was pre-conditioned for 7 days (aerated but not fed) to reduce the amount of O2 consumed by the blank controls. Therefore, the sludge was washed twice with tap water, by 2 centrifugation steps.
- Pretreatment: The activated sludge was used after sampling from the treatment plant without adaptation.
- Concentration of sludge: 30 mg/L dry matter in the final mixture.
Duration of test (contact time):
28 d
Initial conc.:
40.8 mg/L
Based on:
other: nominal test item concentration based on 98.8 mg ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of test medium: Aerobic mineral salts medium prepared with ultrapure water (conductivity: <1.5 µS/cm; DOC: <0.5 mg/L)
* mineral stock solution A (10mL/L): 8.5 g/L KH2PO4, 28.49 g/L K2HPO4.3H2O, 33.4 g/L Na2HPO4.2H2O, 0.5 g/L NH4Cl, pH 7.4
* mineral stock solution B (1 mL/L): 36.4 g/L CaCl2.2H2O
* mineral stock solution C (1 mL/L): 22.5 g/L MgSO4.7H2O
* mineral stock solution D (1 mL/L): 0.25 g/L FeCl3.6H2O
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium
After centrifugation, the sludge was suspended in test medium, at about 1 g/L dry matter. Before the test, this suspension was diluted down to 60 mg/L dry matter, i.e. twice the final concentration, since this suspension was diluted 1:1 (v:v) afterwards.

The test item was applied by direct addition into the test vessels, so as to give a final test concentration of about 100 mg ThOD/l.
The reference item was dissolved in the test medium at a concentration twice of the final concentration to be achieved for the test. This stock solution was diluted with the sludge suspension 1:1 (v:v) to give a final test concentration of about 100 mg ThOD/l.

- Test temperature: 22±2°C, controlled at ± 1°C, in a thermostat cabinet
- pH: 7.2±0.2°C (measured prior to testing and if necessary adjusted with NaOH or HCl (except in flask C; and in flask T and X only if the test item is soluble).
- Continuous darkness: yes, test bottles were in a thermostat cabinet.

TEST SYSTEM
- Test flasks: 510 mL glass bottles (tightly closed with manometric BOD measuring devices) containing a total volume of test solution of 200 mL. The bottles were equipped with stirring rods and butyl rubber quivers which contain 2 pellets of sodium hydroxide each to absorb the produced CO2 from the head space.
- Test performed in duplicate (two test flasks)

CONTROL AND BLANK SYSTEM
B: Inoculum blank (two replicates)
R: Procedure control (two replicates): 60.1 mg/L Sodium benzoate (100 mg ThOD/L)
C: Abiotic sterile control (one replicate): 40.0 mg/L test item (97.0 mg ThOD/L)
X: Toxicity control (one replicate): 41.5 mg/L test item and 60.1 mg/L reference item (total 201 mg oxygen demand/L)

SAMPLING
The test vessels were stirred by an inductive stirring system for a maximum test period of 28 days. During the test the O2 uptake was continuously measured with a manometric BOD measuring device. Temperature was recorded with a data logger.
At the end of the test, the pH was measured in all flasks except the abiotic sterile control (C).
Total elimination was determined in each test vessel (test suspension, blank and procedure control) based on the theoretical carbon content and the analysis of the dissolved organic carbon (DOC) concentrations at the end of the test.

STATISTICAL METHODS:
Values of % degradation were calculated for each test flask and day. The arithmetic mean of % degradation in each test flask on each day was calculated.
Reference substance:
other: Sodium benzoate
Remarks:
CAS 532-32-1
Test performance:
The procedure control sodium benzoate reached 96% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.
Key result
Parameter:
% degradation (O2 consumption)
Value:
67
Sampling time:
28 d
Remarks on result:
other: test flask 1 after 28 d: 69.9% test flask 2 after 28 d: 63.7%
Details on results:
The biodegradability of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-secbutylphthalimide based on O2 consumption was calculated to be 67% after 28 days as compared to the theoretical oxygen demand (ThOD). The biodegradation curve showed three plateaus at about 4%, 37% and 65% after about 2, 8 and 23 days respectively, which are expected to correspond to the sequential degradation of the three components of the test item.
The biodegradation of the test item reached 57% at the end of the 10-d window. Biodegradation of the test item was observed without any lag-phase.

The respective concentrations of organic carbon at the beginning (theoretical value based on the test item’s carbon content and the test concentration, as mg TOC/L) and at the end of the test (mean measured value of the two replicates, as mg DOC/L) were, respectively:
- test units: 28.8 and 2.65 (mean of two replicates)
- blank control: <0.5 and 1.02 (mean of two replicates)
- procedure control: 35.0 (theoretical value calculated based on the carbon content and the test concentration) and 1.27 (mean of two replicates)

The total elimination calculated based on dissolved organic carbon (DOC) measurements reached 94% for the test item and 99% for sodium benzoate, respectively. This data is in line with the degradation calculated based on O2 consumption.
Results with reference substance:
The procedure control sodium benzoate reached 96% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

Toxicity control:

At the applied initial test concentration of 41.5 mg/L the test item was not judged to have any inhibitory effect on the microbial population, since the biodegradation of the mixture (test item + reference item sodium benzoate) exceeded 25% within 14 days.

Abiotic steril control:

The test item was not abiotically degraded (by processes using O2) during the whole test period of 28 days in the absence of microorganisms as confirmed by the lack of oxygen consumption.

Remark on 10 -day window

The substance did not reach the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window (but it was reached within 28 days).

According to the OECD Guidelines for testing of chemicals, Section 3 Part I: Principles and strategies related to the testing of degradation of organic chemicals" (OECD, 2006b), the 10-day window need not be applied if the test is carried out on a mixture of structurally similar constituents and if a sequential biodegradation of the individual constituents is taking place. As a sequential biodegradation of the individual constituents was observed and the pass level was reached within 28 days of incubation during the performed OECD 301F test, it can be concluded that reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide is readily biodegradable.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
A 28-d ready biodegradability test (OECD 301F, Manometric Respirometry Test) using activated sludge of a municipal sewage treatment plant indicated that the reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide reached a biodegradation of 67%. As a sequential biodegradation of the individual structurally similar constituents was observed, the 10-day window is not applicable for this substance and it can be considered as readily biodegradable.

Executive summary:

The biodegradability of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-secbutylphthalimide exposed to microorganisms derived from activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions, following the test guideline OECD 301 F.

The biodegradability of the test item based on O2 consumption was calculated to be 67% after 28 days as compared to the theoretical oxygen demand (ThOD). The biodegradation curve showed three plateaus at about 4%, 37% and 65% after about 2, 8 and 23 days respectively, which are expected to correspond to the sequential degradation of the three components of the test item.

The biodegradation of the test item reached 57% at the end of the 10-d window.

Biodegradation of the test item occurred without any lag phase.

The procedure control sodium benzoate reached 96% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

The total elimination calculated based on dissolved organic carbon (DOC) measurements reached 94% for the test item and 99% for sodium benzoate, respectively. This data is in line with the degradation calculated based on O2 consumption.

Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide did not reach the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window. However, the pass level was reached within 28 days of incubation.

According to the OECD Guidelines for testing of chemicals, Section 3 Part I: Principles and strategies related to the testing of degradation of organic chemicals" (OECD, 2006b), the 10-day window need not be applied if the test is carried out on a mixture of structurally similar constituents and if a sequential biodegradation of the individual constituents is taking place. As a sequential biodegradation of the individual constituents was observed and the pass level was reached within 28 days of incubation during the performed OECD 301F test, it can be concluded that reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide is readily biodegradable.

All validity criteria were fulfilled.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 August 1990 to 13 September 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study follows Modified MITI (II) guideline, but the report is without detailed documentation. The inherent biodegradability study can be used as supportive evidence.
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
not specified
GLP compliance:
no
Specific details on test material used for the study:
Description: Straw coloured liquid
Storage: In original container at room temperature.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The aeration stage of Severn Trent PLC Sewage Treatment Plant, Belper, Derbyshire.
- Preparation of inoculum for exposure: The activated sludge was maintained at a temperature of 21+/- 1°C for 24 hours. An aliquot of the sludge was added 10 L of diluted water to give a suspended solids level of 111.35 mg/l (100 mg SS/L on dilution for the test) and aerated for seven days prior to the start of the test to reduce the oxygen demand of the control.
- Usage rate: equivalent to 100 mg dry weight/L
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
measured by direct manometer reading
Details on study design:
TEST CONDITIONS
- Composition of medium: water
- Additional substrate: no
- Test temperature: 25 +/- 1 °C
- Agitation by magnetic stirrer
- Suspended solids concentration: 111.35 mg/l
- Continuous darkness: yes
Reference substance:
aniline
Remarks:
100 mg/l
Test performance:
The reference substance aniline attained 66% biodegradation after 7 days and 70% after 14 days thereby confirming the suitability of the inoculum and culture conditions. Total Organic Carbon (TOC) analysis showed that aniline attained 99% degradation after 28 days.
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
calculated by comparison of the oxygen uptake with the Chemical Oxygen Demand (COD)
Value:
> 75 - < 77
Sampling time:
28 d
Key result
Parameter:
% degradation (TOC removal)
Value:
100
Sampling time:
28 d

The COD was determined to be 2.66 mg O2/mg.

Validity criteria fulfilled:
not specified
Remarks:
The reference substance aniline attained 66% biodegradation after 7 days and 70% after 14 days thereby confirming the suitability of the inoculum and culture conditions.
Interpretation of results:
inherently biodegradable
Conclusions:
The test item attained 75-77% degradation within 28 days calculated from oxygen uptake and so can, therefore, be considered as inherently biodegradable under the strict terms and conditions of the OECD Guidelines. Total Organic Carbon (TOC) analysis showed that the test item attained 100% degradation within 28 days.
Executive summary:

In this study on inherent biodegeradability according to Modified MITI II, the test item attained 75-77% degradation within 28 days calculated from oxygen uptake and so can, therefore, be considered as inherently biodegradable under the strict terms and conditions of the OECD Guidelines. Total Organic Carbon (TOC) analysis showed that the test item attained 100% degradation within 28 days.

Description of key information

A GLP study according to OECD 301F was conducted with the substance "Reaction mass of N-butylphthalimide and N-propylphthalimide and N-secbutylphthalimide" (ACH, 2017). The study is reliable without restriction and was rated as Klimisch 1.

The biodegradability of the test item based on O2 consumption was calculated to be 67% after 28 days as compared to the theoretical oxygen demand (ThOD). The biodegradation curve showed three plateaus at about 4%, 37% and 65% after about 2, 8 and 23 days respectively, which are expected to correspond to the sequential degradation of the three components of the test item.

The biodegradation of the test item reached 57% at the end of the 10-d window.

Biodegradation of the test item occurred without any lag phase.

The procedure control sodium benzoate reached 96% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

The total elimination calculated based on dissolved organic carbon (DOC) measurements reached 94% for the test item and 99% for sodium benzoate, respectively. This data is in line with the degradation calculated based on O2 consumption.

Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide did not reach the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window. However, the pass level was reached within 28 days of incubation.

All validity criteria were fulfilled.

In the OECD 301F study (ACH, 2017), the substance is readily biodegradable, but fails the 10 d window.

According to the OECD Guidelines for testing of chemicals, Section 3 Part I: Principles and strategies related to the testing of degradation of organic chemicals" (OECD, 2006b), the 10-day window need not be applied if the test is carried out on a mixture of structurally similar constituents and if a sequential biodegradation of the individual constituents is taking place. As a sequential biodegradation of the individual constituents was observed and the pass level was reached within 28 days of incubation during the performed OECD 301F test, it can be concluded that reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information