Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-11-2011 to 16-02-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

GLP compliance:

yes

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): Cambridge Wastewater Treatment Facility, Cambridge, Maryland, USA.
- Laboratory culture: No
- Method of cultivation: None
- Storage conditions: Sieved using a 2 mm screen and aerated until adjustment.
- Storage length: 6 days (collected Dec 08, 2011)
- Preparation of inoculum for exposure: Diluted in test medium to approximately 30 mg total suspended solids/L and aerated with CO2-free air until adjustment. The inoculated medium used in the test was diluted to 4 mg total suspended solids/L and aerated for 30 minutes.
- Concentration of sludge: 0.5 mL
- Initial cell/biomass concentration: 2.8 x 102 CFU/mL
- Pretreatment: No
- Water filtered: no

Duration of test (contact time):

35 d

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Aliquots of inoculated test medium were dispensed into replicate bottles to give a headspace to liquid ratio of approximately 1:2 (e.g. 107 mL in each 160 mL bottle). The blank control chambers contained only inoculated medium and were not dosed with a carbon source. Reference group test chambers were dosed with sufficient reference substance necessary to deliver 20 mg C/L. Treatment group test chambers were dosed with sufficient test substance necessary to deliver approximately 20 mg C/L..
- Additional substrate: None
- Test temperature: 19.5 to 21.3°C
- pH: not specified
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: No

TEST SYSTEM
- Culturing apparatus: Glass serum bottles with nominal volume of 160 mL, sealed with butyl rubber septa and crimp caps
- Number of culture flasks/concentration: 27
- Method used to create aerobic conditions: aerated test medium
- Measuring equipment: Shimadzu Model TOC-VCSH carbon analyzer.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No


SAMPLING
- Sampling frequency: Once a week.
- Sample storage before analysis: No

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

Reference substance:

other: 1-octanol

Preliminary study:

Not applicable

Test performance:

The viability of the inoculum and validity of the test were supported by the results of the reference substance, 1-octanol, from which an average of 94.1% of theoretical IC was evolved An average percent biodegradation of greater than 60% was achieved by Day 7, thereby fulfilling the criteria for a valid test by reaching the pass level by Day 28.

Parameter:

% degradation (inorg. C analysis)

Remarks:

average cumulative percent biodegradation

Value:

>= 73.1

Sampling time:

35 d

Remarks on result:

other: test item is not readily biodegradable because ≥ 60% ThIC was not achieved within a 10-day window of reaching 10 % ThIC

Remarks:

test item is rapidly degradable since the pass level of 60% ThIC was achieved within 28 days

Details on results:

Result tables attached.
The control chambers evolved an average of 2.1 mg IC/L by Day 28 (Table 2). The amount of IC evolved by the control chambers was < 15% of the organic carbon added initially as the test substance (20 mg C/L), which is considered the acceptable limit for this study.
The viability of the inoculum and validity of the test were supported by the results of the reference substance, 1-octanol, from which an average of 94.1% of theoretical IC was evolved (Table 4). An average percent biodegradation of greater than 60% was achieved by Day 7, thereby fulfilling the criteria for a valid test by reaching the pass level by Day 28. The average cumulative percent biodegradation for test material was 73.1% (Table 4). The test material may not be considered readily biodegradable under aerobic conditions since the pass level of 60% ThIC was not achieved within a 10-day window of reaching 10% ThIC. However, as a UVCB, the test substance may be considered rapidly biodegradable since the pass level of 60% of ThIC was achieved within 28 days.

See attached document.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

As a UVCB, the test item may be considered rapidly degradable since the pass level of 60 % ThIC was achieved within 28 days

Executive summary:

Test Guideline

OECD Guideline 310

Method and materials

In the Headspace Test, inoculated test medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and sealed. CO2 evolution from the ultimate aerobic biodegradation of the test substance is determined by measuring the inorganic carbon (IC) produced in the test bottles over that produced in the blank control bottles. The amount of IC produced by the test substance (corrected for that evolved by the controls and the amount added by basification, where applicable) is expressed as a percentage of the theoretical amount of IC (ThIC) that could have been produced if complete biodegradation of the test substance occurred. The test contained a control group, a reference group and a treatment group. Each group contained twenty-seven replicate test chambers. The control chambers were used to measure the background IC production of the inoculum medium and were not dosed with a carbon source. The reference chambers were dosed with 1-octanol, a substance known to be biodegradable, at a nominal concentration of 20 mg C/L. The treatment group test chambers were used to evaluate the test item at a nominal concentration of approximately 20 mg C/L.

Results

The results at the end of the 35-day test period indicated that the activated sludge inoculum was active, degrading the reference substance 94.1 %. The average cumulative percent biodegradation for the test item was 73.1 %. The test item may not be considered readily biodegradable under aerobic conditions since biodegradation ≥ 60 % ThIC was not achieved within a 10-day window of reaching 10 % ThIC. However, as a UVCB, the test item may be considered rapidly degradable since the pass level of 60 % of ThIC was achieved within 28 days.

Conclusion

The test substance is rapidly degradable under the conditions of the study.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 1998-04-01 to 1998-11-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

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

Principles of method if other than guideline:

not applicable.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
none

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was collected from the Prospect Bay Wasterwater Treatment Facility.
- Method of cultivation: The sludge was sieved using a 2 mm screen and then aerated for approximately 4 hours. The sludge was homogenized in a blender at medium speed for approximately 2 minutes and then was allowed to settle for approximately 30 minutes. The supernatant was used as the inoculum for this study and was used the same day that it was prepared. A total suspended solids measurement and standard plate count were performed on the inoculum using procedures based on Standard Methods. The plates were incubated at 20 +/- 3°C for ca. 48 h, and then the number of colony forming units was enumerated.
- Concentration of sludge: 78.6 mg/L
- Initial cell/biomass concentration: 0.786 mg/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

10 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
- 2943 mL of nanopure water
- 3 mL of ammonium sulfate solution
- 3 mL calcium chloride solution
- 12 mL of ferric chloride solution
- 3 mL of magnesium sulfate solution
- 6 mL of phosphate buffer
- 30 mL of activated sludge inoculum.
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 18-20°C
- pH: 4.3-6.5
- pH adjusted: no data
- Aeration of dilution water: yes
- Suspended solids concentration:
- Continuous darkness: no data
- Other: none

TEST SYSTEM
- Culturing apparatus: ambered 4-L bottles.
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: the air entrering the chambers was passed through Drierite to remove ambient moisture and then through Ascarite to produce CO2 free air.
- Measuring equipment: Shimadzu TOC-5000 carbon analyzer.
- Details of trap for CO2 and volatile organics if used: bottles contained ca. 100 mL of 0.5 N KOH and the amount of CO2 detected in the KOH solution was substracted from the CO2 in the blank control traps to determine the amount of CO2 produced by the inoculum in the blank control.
- Other: none.

SAMPLING
- Sampling frequency: 3-4 days
- Sampling method: The CO2 trap nearest the chamber was removed and analyzed for inorganic carbon. The 2 remaining traps were placed one position closer to the test chamber and a new trap was placed on the end of the series.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes
- Other: none

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

57

St. dev.:

9.1

Sampling time:

28 d

Results with reference substance:

Percentage of Theoretical carbon dioxide : 99.3 +/- 4.3 %

Tables are presented in attached document (cf. Tables 331E-111.pdf)

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which 99.3% of theretical CO2 was evolved. An average percent biodegradation of greater than 60 % was achieved within 7 days, thereby fulfilling the criteria for a valid test by reaching the pass level by day 14. The average cumulative percent of theoretical carbon dioxide produced by the test substance at 10 mg C/L was 57.0 %. The test item may not be considered readily bioldegradable under the conditions tested since the pass level of 60% TCO2 was not achieved.

Executive summary:

Test Guidance

The study was conducted according to the procedures outlined in the protocol "Ready biodegradability by the carbon dioxide evolution test method". The protocol was based on the procedures specified in the OECD guideline for testing of chemicals, Methods 301B, and EEC Method C.4 -C.

Results

The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which 99.3 % of theretical CO2 was evolved. An average percent biodegradation of greater than 60 % was achieved within 7 days, thereby fulfilling the criteria for a valid test by reaching the pass level by day 14. The average cumulative percent of theoretical carbon dioxide produced by the test substance at 10 mg C/L was 57.0 %.

Conclusions

Evidence of ready biodegradability in a carbon dioxide evolution test is 60 % TCO2 within the 28-d test period. In addition, the pass value must be reached within 10 days of achieving 10 % TCO2. The test item may not be considered readily biodegradable under the conditions tested since the pass level of 60 % TCO2 was not achieved. However since tests of ready biodegradability provide limited opportunity for biodegradation to occur, a negative result does not mean that the test substance will not be biodegraded under relevant environmental conditions.

Description of key information

The test item cannot be declared readily biodegradable but was determined to be rapidly degradable. At the end of the 35-day test period in the key study, the activated sludge inoculum was active, degrading the reference substance 94.1 % and, as a UVCB, the test item may be considered rapidly biodegradable since the pass level of 60% of ThIC was achieved within 28 days (OECD 310).

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater

Additional information

Key study

Test Guideline

OECD Guideline 310

Method and materials

In the key Headspace Test, inoculated test medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and sealed. CO2 evolution from the ultimate aerobic biodegradation of the test substance is determined by measuring the inorganic carbon (IC) produced in the test bottles over that produced in the blank control bottles. The amount of IC produced by the test substance (corrected for that evolved by the controls and the amount added by basification, where applicable) is expressed as a percentage of the theoretical amount of IC (ThIC) that could have been produced if complete biodegradation of the test substance occurred. The test contained a control group, a reference group and a treatment group. Each group contained twenty-seven replicate test chambers. The control chambers were used to measure the background IC production of the inoculum medium and were not dosed with a carbon source. The reference chambers were dosed with 1-octanol, a substance known to be biodegradable, at a nominal concentration of 20 mg C/L. The treatment group test chambers were used to evaluate the test item at a nominal concentration of approximately 20 mg C/L.

Results

The results at the end of the 35-day test period indicated that the activated sludge inoculum was active, degrading the reference substance 94.1%. The average cumulative percent biodegradation for the test item was 73.1%. Biodegradation ≥ 60% ThIC within a 10-day window of reaching 10% ThIC in this test demonstrates that a test substance is readily biodegradable under aerobic conditions. The average cumulative percent biodegradation for the test item was 73.1%.

Conclusion

The test item may not be considered readily biodegradable under aerobic conditions since biodegradation ≥ 60% ThIC was not achieved within a 10-day window of reaching 10% ThIC. However, as a UVCB, the test item may be considered rapidly biodegradable since the pass level of 60% of ThIC was achieved within 28 days.

Supporting study

Test Guidance

The supporting study was conducted according to the procedures outlined in the protocol "Ready biodegradability by the carbon dioxide evolution test method". The protocol was based on the procedures specified in the OECD guideline for testing of chemicals, Methods 301B, and EEC Method C.4 -C.

Results

The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which 99.3 % of theretical CO2 was evolved. An average percent biodegradation of greater than 60 % was achieved within 7 days, thereby fulfilling the criteria for a valid test by reaching the pass level by day 14. The average cumulative percent of theoretical carbon dioxide produced by the test substance at 10 mg C/L was 57.0 %.

Conclusions

Evidence of ready biodegradability in a carbon dioxide evolution test is 60 % TCO2 within the 28-d test period. In addition, the pass value must be reached within 10 days of achieving 10 % TCO2. The test item may not be considered readily biodegradable under the conditions tested since the pass level of 60 % TCO2 was not achieved. However since tests of ready biodegradability provide limited opportunity for biodegradation to occur, a negative result does not mean that the test substance will not be biodegraded under relevant environmental conditions.