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Environmental fate & pathways

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:
other: Limited study summary, no guideline and no GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The activated sludge test was based on the semicontinuous activated sludge test of the soap and Detergent Association (1965) and Zahn and Huber (1975). The concentration of test substance was 50 and 100 ppm. During 19 days treatment, the sample was taken at 30 min, 3, 6, and 24 h, and on days 2, 3, 4, 6, 8, 10, 13, 16, and 19. DOC removal was measured.
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): Activated sludge is collected from a sewage treatment plant that principally treats domestic waste.
- Laboratory culture: Following a period of adaptation to gradually increasing test substance concentrations, an activated sludge sample is exposed to a mineral salts medium plus test substance.
- Concentration of sludge: The suspended solids content of the sludge is determined and adjusted to 2500 ppm by adding tap water or by decanting supernatant.

No additional data.
Duration of test (contact time):
19 d
Initial conc.:
50 other: ppm
Based on:
DOC
Initial conc.:
100 other: ppm
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- pH: 6.5-8.0
- pH adjusted: yes
- Aeration of dilution water: Aeration is continued throughout the experiment.
- Suspended solids concentration: 500 ppm

TEST SYSTEM
- Culturing apparatus: The reaction unit consists of a 4-L capacity percolator fitted with an aeration stone at the narrow end. Each percolator is suspended vertically in a ring stand, and clean air is provided to the aeration stone, which is sealed to make it water- tight.
- Measuring equipment: Technicon Autoanalyzer or Beckman Infrared Analyzer Model IR-315.

SAMPLING
- Sampling frequency: Samples are collected at 30 min, 3, 6, and 24 h, and on days 2, 3, 4, 6, 8, 10, 13, 16, and 19.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

No additional data.
Reference substance:
not specified
Test performance:
The concentration of test substance was 50 and 100 ppm. During 19 days treatment, the sample was taken at 30 min, 3, 6, and 24 h, and on days 2, 3, 4, 6, 8, 10, 13, 16, and 19. DOC removal was measured.
Parameter:
% degradation (DOC removal)
Value:
ca. 100
Sampling time:
19 d
Remarks on result:
other: 50 ppm
Parameter:
% degradation (DOC removal)
Value:
> 100 - < 110
Sampling time:
19 d
Remarks on result:
other: 100 ppm
Details on results:
Acclimation to the activated sludge cultures required the minimum of 3 days, and >95% degradation occurred within the first 30 min of the test.
Results with reference substance:
No information provided.
Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
The DOC removal rate of the test substance can reach more than 100% in the condition of the test. In conclusion, the test substance is completely biodegradable under the condition of the test.
Executive summary:

The activated sludge test was based on the semicontinuous activated sludge test of the soap and Detergent Association (1965) and Zahn and Huber (1975). The concentration of test substance was 50 and 100 ppm. During 19 days treatment, the sample was taken at 30 min, 3, 6, and 24 h, and on days 2, 3, 4, 6, 8, 10, 13, 16, and 19. DOC removal was measured. The DOC removal rate of the test substance can reach more than 100% in the condition of the test. In conclusion, the test substance is completely biodegradable under the condition of the test.

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:
other: Limited study summary, no guideline and no GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Biochemical Oxygen Demand (BOD) test was made according to the standard BOD test method. The test substance in BOD bottles in concentrations of 1, 2, 4, 8, and 16 ppm and the control is mixture of 50% glucose:50% glutamic acid, run in concentrations of 21/2 or 5 ppm. In 28 days of test, the DO content of each bottle is measured on days 1, 3, 6, 9, 13, 15, 21 and 28 or some similar schedule.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent/soil microbial culture, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): secondary effluent/soil microbial culture that has been acclimated to 10 ppm concentrations of the test substance for 2 weeks
- Preparation of inoculum for exposure: Adaptive transfers are made every 48 to 72 h during the acclimation period, resulting in about 4 transfers.

No additional data.
Duration of test (contact time):
28 d
Initial conc.:
1 other: ppm
Based on:
test mat.
Initial conc.:
2 other: ppm
Based on:
test mat.
Initial conc.:
4 other: ppm
Based on:
test mat.
Initial conc.:
8 other: ppm
Based on:
test mat.
Initial conc.:
16 other: ppm
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Test temperature: room temperature
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Sealed BOD bottles
- Number of culture flasks/concentration: 2
- Measuring equipment: Orion Model 97-08 DO specific ion electrode ion conjunction with an Orion Model 701A pH/mV meter

SAMPLING
- Sampling frequency: The DO content of each bottle is measured on days 1, 3, 6, 9, 13, 15, 21 and 28 or some similar schedule.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum and dilution water but no organic chemical.
- Toxicity control: A mixture of 50% glucose:50% glutamic acid, run in concentrations of 21/2 or 5 ppm of each.


No additional data.
Test performance:
The test substance in concentrations of 1, 2, 4, 8, and 16 ppm and the control is mixture of 50% glucose: 50% glutamic acid, run in concentrations of 21/2 or 5 ppm. The DO content of each bottle is measured on days 1, 3, 6, 9, 13, 15, 21 and 28 or some similar schedule.
Parameter:
% degradation (O2 consumption)
Value:
> 60
Sampling time:
28 d
Details on results:
Quantitative biodegradation of test substance started immediately and occurred in most cases within the first 3 days of the experiment. The test substance with higher concentration has more O2 consumption. The O2 consumption curve against time have been showed in Fig 1 (see attached document).
Parameter:
BOD5
Value:
> 0.5 - < 5 g O2/g test mat.
Results with reference substance:
No information provided.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
The test substance degraded rapidly within the first 3 days, the higher concentration the more O2 consumption in the condition of the test.
Executive summary:

The Biochemical Oxygen Demand (BOD) test was made according to the standard BOD test method. The test substance in BOD bottles in concentrations of 1, 2, 4, 8, and 16 ppm and the control is mixture of 50% glucose:50% glutamic acid, run in concentrations of 21/2 or 5 ppm. In 28 days of test, the DO content of each bottle is measured on days 1, 3, 6, 9, 13, 15, 21 and 28 or some similar schedule.

The test substance degraded rapidly within the first 3 days, the higher concentration the more O2 consumption in the condition of the test.

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:
other: Study run to a method comparable with current guidelines, no GLP available at the time.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
The OECD SIDS dossier reports that this test was equivalent to their 301B guideline.
Deviations:
not specified
Principles of method if other than guideline:
The CO2 Evolution test was made based on the method of Sturm (1973). The concentration of test substance was 10 mg/l and glucose was used as control. During 28 days treatment, the proximal absorber is analyzed on days 1, 2, 5, 7, 9, 12, 15, 21 and 28 or some similar schedule. CO2 trapped as barium carbonate is analyzed.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
not specified
Details on inoculum:
- Preparation of inoculum for exposure: Adaptive transfers are made every 48 to 72 h during the acclimation period, resulting in about 4 transfers. On the 13th day of acclimation, equal volume from each shake flask culture, including test and reference compounds and blanks, are combined to form a composite inoculums culture.

No additional data.
Duration of test (contact time):
28 d
Initial conc.:
10 other: ppm
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: Room temperature
- Continuous darkness: yes
- Other: CO2-free air

TEST SYSTEM
- Culturing apparatus: 4-L capacity glass jug.
- Details of trap for CO2 and volatile organics if used: CO2- free air is passed through a rotameter, a drying tube, and a CO2 check arranged in a series. CO2 trapped as barium carbonate is analyzed by titrating the Ba(OH)2 remaining in solution to the phenolphthalein endpoint with 0.1 N HCl.

SAMPLING
- Sampling frequency: The proximal absorber is analyzed on days 1, 2, 5, 7, 9, 12, 15, 21 and 28 or some similar schedule.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: Glucose as reference compound.

No additional data.

Reference substance:
other: glucose
Test performance:
The CO2 Evolution test was made based the concentration 10 ppm of test substance and glucose as control. The proximal absorber is analyzed on days 1, 2, 5, 7, 9, 12, 15, 21 and 28 and CO2 trapped as barium carbonate is analyzed.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
95
Sampling time:
28 d
Details on results:
CO2 Evolution test where about 60% of the compound was mineralized in 7 days and 95% in 28 days.
Results with reference substance:
No information provided.
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
The OECD reported that this study was equivalent to the 301B (Sturm method), measuring CO2 evolution. In 7 days 60%of the applied substance was mineralized and 95% was mineralized in 28 days. Therefore, meeting the criteria for ready biodegradability.
Executive summary:

The OECD reported that this study was equivalent to the OECD 301B (Sturm method), measuring CO2 evolution. The CO2 Evolution test was made based on the method of Sturm (1973). The concentration of test substance was 10 ppm and glucose was used as control. During 28 days treatment, the proximal absorber is analyzed on days 1, 2, 5, 7, 9, 12, 15, 21 and 28 or some similar schedule. CO2 trapped as barium carbonate is analyzed. In 7 days 60%of the applied substance was mineralized and 95% was mineralized in 28 days. Therefore, meeting the criteria for ready biodegradability.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited study summary, no guideline and no GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Gledhill test combines elements of both the CO2 Evolution test and Shake Flask test. The concentration of test substance was 20 ppm and glucose was used as a control. During 21 days treatment the sample was taken at days 3, 7, 14, 21, and 28. The CO2 evolution and loss of DOC were measured.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): mixture of raw sewage/soil
- Method of cultivation: Inoculum hat has been acclimated to 10 ppm concentrations for 2 weeks and 3 adaptive transfers were made during this period. At the end of the accilimation period, inocula for test substance and blanks are pooled to form a composite inocula. This is then combined with freshly settled raw sewage.

No additional data.
Duration of test (contact time):
21 d
Initial conc.:
20 other: ppm
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
Shake Flask Test
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
CO2 Evolution Test
Details on study design:
TEST CONDITIONS
- Test temperature: room temperature
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: An open container suspendent in a 2-L capacity Erlenmeyer flask .
- Measuring equipment: Technicon Autoanalyzer or Beckman Infrared Analyzer Model IR-315.
- Details of trap for CO2 and volatile organics if used: CO2 trapped as barium carbonate is analyzed by titrating the Ba(OH)2 remaining in solution to the phenolphthalein endpoint with 0.1 N HCl.

SAMPLING
- Sampling frequency: Sample at days 3, 7, 14, 21, and 28.
- Sampling method: Base from the center well is removed for analysis, and small aliquots are removed from the basal medium for DOC analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blank receiving inoculated media and no test substance.
- Abiotic sterile control: Sterile controls containing inoculated medium, test substance and 50 mg HgCl2/L.
- Toxicity control: Glucose as reference compound.

No additional data.
Reference substance:
other: Glucose
Test performance:
The concentration of test substance was 20 ppm and glucose was used as a control. During 21 days treatment The sample was taken at days 3, 7, 14, 21, and 28. The CO2 evolution and loss of DOC were measured.
Parameter:
% degradation (DOC removal)
Value:
ca. 100
Sampling time:
21 d
Remarks on result:
other: Shake Flask Test
Parameter:
% degradation (CO2 evolution)
Value:
ca. 100
Sampling time:
21 d
Remarks on result:
other: CO2 Evolution Test
Details on results:
Quantitative biodegradation of test substance started immediately and occurred in most cases within the first 3 days of the experiment. Test substance was not abiotically mineralized to CO2.
Results with reference substance:
No information provided.
Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
The test substance is completely biodegradable. Although the test substance can nonbiologically degraded, was not abiotically mineralized to CO2.
Executive summary:

The Gledhill test combine elements of both the CO2 Evolution test and Shake Flask test. he concentration of test substance was 20 ppm and glucose was used as a control. During 21 days treatment The sample was taken at days 3, 7, 14, 21, and 28. The CO2 evolution and loss of DOC were measured.

The test substance is completely biodegradable. Although the test substance can nonbiologically degraded, was not abiotically mineralized to CO2.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines, no GLP available at the time.
Qualifier:
according to
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Deviations:
yes
Remarks:
exposure period was shorten to 21 days
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary sewage and garden soil
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A culture from secondary sewage and garden soil
- Preparation of inoculum for exposure: Adaptive transfers are made every 48 to 72 h during the acclimation period, resulting in about 4 transfers.
- Method of cultivation: Microorganisms are inoculated into flasks containing dilute nutrient soil.

No additional data.
Duration of test (contact time):
21 d
Initial conc.:
20 other: ppm
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST SYSTEM
- Culturing apparatus: Shake flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration is accomplished by continuous shaking of the flask.
- Measuring equipment: millipore membrane, technician Autoanalyzer or Beckman Infrared Analyzer Model IR-315

SAMPLING
- Sampling frequency: Samples for DOC analysis are collected on days 1, 2, 3, 4, 7, 10, 15, and 21, or some similar schedule.
- Other: Samples were immediately filtered through prewashed in-line 0.22 μm disposable. Millipore membranes and preserved by acidifying to pH 2 or less with concentrated H2SO4.

CONTROL AND BLANK SYSTEM
- Toxicity control: A 20 ppm concentration (as OC) of LAS was used as a reference control.

No additional data.
Reference substance:
other: linear alkylate sulfonate (LAS)
Test performance:
In the shake flask test, the concentration of test substance was 20 ppm and LAS was used as a control. During 21 days treatment, samples for DOC are collected on days 1, 2, 3, 4, 7, 10, 15, and 21, or some similar schedule.
Parameter:
% degradation (DOC removal)
Value:
ca. 100
Sampling time:
21 d
Details on results:
The DOC removal rate of the test substance was almost 100% at viable condition after 1 day, while the rate was almost 80 % at sterile condition at the 21 days.
Results with reference substance:
No information provided
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance is completely biodegradable under the condition of the test.
Executive summary:

The shake flask test was made according to Modified of OECD Screening Test 301E. The concentration of test substance was 20 ppm and LAS was used as a control of 20 ppm. During 21 days treatment, samples are collected on days 1, 2, 3, 4, 7, 10, 15, and 21, or some similar schedule and DOC was measured.

The test substance is completely biodegradable under the conditions of the test.

Description of key information

Means and Anderson (1981) have investigated the test substance in assays equivalent to the OECD 301B (Sturm test) and the 301E (CO2evolution test) and further studies including an activated sludge test, BOD test and Gledhill test.

The OECD concluded that the substance met the criteria for readily biodegradable in the equivalent 301B and 301E studies. The other three studies showed high levels of degradation under different test conditions.

In addition to the Means and Anderson (1981) there are several studies available which indicate varying levels of biodegradation under aerobic and anaerobic conditions.

The substance has therefore been classified as “readily biodegradable” based on the data from the equivalent 301B and 301E studies.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Although no studies are available that are performed and reported according to the guideline OECD 301, the studies mentioned above are very similar to guideline studies and are considered to show readily biodegradability of the test substance in a weight of evidence approach.

The test substance is considered to be readily biodegradable.