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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:
2014-04-08 to 2014-05-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
Test temperature fell below 22 ± 2°C on three occasions. As this was a short period and the temperature consistent throughout the rest of the study period it was not considered to have an adverse effect.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
see above
Principles of method if other than guideline:
Following the recommendations of ISO 1995 for organic substances with low water solubility, the test item was dissolved in an auxiliary solvent prior to being absorbed onto filter paper and subsequent dispersal in the test media using high shear mixing to break up the filter paper. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
GLP compliance:
yes (incl. certificate)
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): obtained on 7 April 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Preparation of inoculum for exposure: The sample waswashed twice by settlement and resuspension in mineral medium to remove excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection.
- Suspended solids concentration was equal to 2.9 g/L prior to use.
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper
Duration of test (contact time):
>= 28 d
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Prepared to OECD Guideline
- Solubilising agent (type and concentration if used): The test item was dissolved in an auxillary solvent prior to adsorption onto filter paper and then added to the test medium using high shear mixing.
An amount of test item (792 mg) was dissolved in 10 mL of acetone to give a 792 mg/10 mL solvent stock solution. An aliquot (500 µL) of this solvent stock solution was dispersed onto filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed into approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to innoculated mineral medium. The volume was then adjusted to 3 litres to give a final concentration of 13.2 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
- Test temperature: 2°1C
- pH: 7.5 - 7.6
- pH adjusted: no
- Aeration of dilution water: No
- Suspended solids concentration: final: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2 free air bubbled through solution at a rate of 30 to 100 mL/min
- Measuring equipment: IC analysis using a Tekmar=Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser and a Shimadzu TOC-VCPH analyser.
- Test performed in closed vessels due to significant volatility of test substance: No
- Details of trap for CO2 and volatile organics if used: Two 500 mL Dreschel bottles containing 350 mL of 0.05M NaOH. CO2 absorbing solutions prepared using purified de-gassed water.

SAMPLING
- Sampling frequency: Days 0, 2, 6, 8, 10, 14, 21, 28 and 29.
- Sampling method: 2mL removed from first CO2 absorber vessel
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate vessels consistingof inoculated mineral medium plus a filter paper
- Abiotic sterile control: No
- Toxicity control: Test item plus sodium benzoate to give a final concentration of 20 mg carbon/L
- Other: Sodium benzoate in duplicate plus filter paper to give a final concentration of 10 mg carbon/L
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable. The test item is an unstable UVCB substance in aqueous conditions and hydroyses rapidly (less than 30 minutes). The water solubility of the test item is therefore equivalent to the water solubility of the two main degradation products boric acid and 2-propyl heptan-1-ol. Both degradation products have been registered under REACH. Boric acid is an inorganic substance with high water solubility and will not undergo biodegradation. 2-propyl heptan-1-ol is a poorly soluble substance (> 100 mg/L) and is readily biodegradable with over 90% degradation in 28-days.
Test performance:
The total CO2 evolution on the inoculum control vessels on Day 28 was 33.38 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given inthe oECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation croterion given in the OECD Test Guidelines.
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
40
Sampling time:
8 d
Parameter:
% degradation (CO2 evolution)
Value:
50
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
66
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
74
Sampling time:
29 d
Remarks on result:
other: Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2
Details on results:
The results of the inorganic carbon analysis of samples from the first absorber vessels om Day 29 showed an increase in all replicate vessels with the exception of inoculum control R2 and procedure control R2.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over into the second absorber vessels occurred.
The test item attained 74% biodegradation after 28 days. As the test material is a UVCB the 10 -day window validation criterion are not applicable. Therefore, the test material can be considered to be readily biodegradable.The organic degradation product derived from hydrolysis of the test item is readily biodegradable.
The toxicity control attained 69% biodegradation after 14 days and 77% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatement micro-organisms in the test.
Sodium benzoate attained 82% biodegradation after 14 days and 87% biodegradation after 28 days confirming the suitability of the inoculum and test conditions.
Results with reference substance:
Sodium benzoate attained 82% biodegradation after 14 days and 87% after 28 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 74% biodegradation after 28 days. The test item is readily biodegradable
Executive summary:

Guidance

OECD Guideline No. 301B and EC Method C.4-C : CO2 Evolution test

Method

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of ISO 1995, the test item was dissolved in an auxillary solvent prior to being absorbed onto filter paper and subsequent dispersal in the test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 74% biodegradation after 28 days. As the test material is a UVCB the 10 -day window validation criterion is not applicable. Therefore, the test material can be considered to be readily biodegradable.The organic degradation product derived from hydrolysis of the test item is readily biodegradable.

The test item is an unstable UVCB substance in aqueous conditions and hydroyses rapidly (less than 30 minutes). The water solubility of the test item is therefore equivalent to the water solubility of the two main degradation products boric acid and 2-propyl heptan-1-ol. Both degradation products have been registered under REACH. Boric acid is an inorganic substance with high water solubility and will not undergo biodegradation. 2-propyl heptan-1-ol is a poorly soluble substance (> 100 mg/L) and is readily biodegradable with over 90% degradation in 28-days.

The toxicity control reached 69% biodegradation after 14 days and 77% biodegradation after 28 days.

Sodium benzoate attained 82% biodegradation after 14 days and 87% after 28 days.

Conclusion

The test item attained 74% biodegradation after 28 days. The test item is readily biodegradable

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 January 2015 to 13 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
yes
Remarks:
change to stock solution concentration with no impact on validity of study (see below)
Qualifier:
according to
Guideline:
EU Method C.4-F (Determination of the "Ready" Biodegradability - MITI Test)
Deviations:
yes
Remarks:
change to stock solution concentration with no impact on validity of study (see below)
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004
Deviations:
yes
Remarks:
change to stock solution concentration with no impact on validity of study (see below)
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Degradation and Accumulation [M]. 2nd edition. Beijing: China Environment Press. 2013: 38-43.
Deviations:
yes
Remarks:
change to stock solution concentration with no impact on validity of study (see below)
Qualifier:
according to
Guideline:
other: GB/T 21802-2008. Chemical Ready Biodegradation-Modified MITI Test (I) [S]. Beijing: SAC, 2008.
Deviations:
yes
Remarks:
change to stock solution concentration with no impact on validity of study (see below)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Activated sludge, surface soil and surface water were sampled from ten sites distributed in four districts throughout Nanjing city, such as Chengdong, Chengbei, Jiangxinzhou and Jiangning.
- Sludge, soil and water (1 L) were collected and mixed thoroughly together.
- After removing floating matter, the mixture was allowed to stand and then the supernatant was filtrated through filter paper.
- The supernatant was then adjusted to pH 7.0 with sodium hydroxide or phosphoric acid.
- Finally an appropriate volume of the filtered supernatant was transferred to a fill-and-draw activated sludge vessel and aerated for about 23.5 hours (Batch No IN201411051).
- Thirty minutes after stopping the aeration, about one third of the whole volume of supernatant was discarded. - An equal volume of the solution (pH 7.0) containing 0.1 % each of glucose, peptone and potassium orthophosphate was then added into the settled material and aerated again. This procedure was repeated once per day for one month.
- Before use the mixture was allowed to stand, and the supernatant was removed.
- A small quantity of sludge was taken to be centrifuged (2500 r/minx 10 min) and then weighed.
- The sludge was then dried in the oven and weighed again in order to calculate the content of dry sludge (11 .0%).
- Centrifuged sludge (8.90 g ) was diluted 1 L with basal culture medium (BSM) to give activated sludge suspension with a concentration of 1000 mg/L (dry weight) (Batch No 120150109).
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
OVERVIEW
- Five groups of "abiotic", "procedure control", "blank control", 'toxicity control' and "test" were
set up simultaneously.
- The "abiotic" contained mineral salts medium and a measured amount of test substance in order to determine whether there was any change in the test chemical during the testing period.
- The "procedure control" contained inoculated mineral salts medium and a measured amount of a reference substance for validating the test result.
- The "blank control" contained only inoculated mineral salts medium.
- The "test" vessels contained inoculated mineral salts medium and a measured amount of the test substance.
- The "toxicity control" contained inoculated mineral salts medium and a measured amount of a reference substance and test substance for check the inhibition effect on the inoculum.
- The progress of degradation was followed by the determination BOD in the "test" and "blank control".
- Degradation was expressed as the ratio of the biochemical oxygen demand (BOD) and the theoretical oxygen demand (ThOD) in order to evaluate the ready biodegradability of chemical substance.
- The ready degradation rate was also expressed as percentage of initial concentration of test substance, where the residue analysis of the test substance was performed at the end (28 d) of the test.
- Substances are considered to be "ready biodegradable" if the ready degradation rate is equal to or greater than 60% during the 28-day test period.

PREPARATION OF TEST MEDIUM
- The Based Salt Medium (BSM) was prepared by adding 3 mL of stock solutions A, B, C and D prepared in pre-aerated deionised water to 1 L of pure water. A total of 3L of BSM was prepared (Batch BSM20150109)
- Composition of the stock solutions used to prepare BSM are shown in the table below.

PREPARATION OF TEST SUBSTANCE SOLUTIONS
- Test substance (5.0012 g) was dissolved in acetonitrile in a 50 mL volumetric flask to give a stock solution of 100000 mg/L.
- Sodium benzoate (1.0011 g) was dissolved in BSM in a 1L volumetric flask to give a stock solution of 1001 mg/L.

TEST CONDITIONS
- An appropriate amount of glass filter paper was added to vessels numbered 1, 2, 3 and 4.
- Stock solution (300 µL) was dispersed onto the filter paper in vessel 1 (abiotic vessel) to give a test substance concentration of 100 mg/L.
- Stock solution (90 µL) was dispersed onto the filter paper in vessels 2, 3 and 4.
- Solvent was evaporated to dryness for approximately 15 minutes.
- Acetonitrile (90 µL) was added to a vessel numbered 6 and solvent was allowed to evaporate to dryness for approximately 15 minutes.
- Deionised water (300 mL) was added to vessel 1.
- BSM (200 mL) was added to vessels 2 and 3.
- Mixing with the aid of high shear took place for 10 minutes prior to addition of inoculum.
- Activated sludge suspension (9 mL) was added to vessels 2 and 3 (test vessels) and the volume adjusted to 300 mL with BSM. Concentration of activated sludge was 30 mg/L and concentration of test substance was 30 mg/L.
- Vessel 4 (toxicity control) was prepared by adding reference substance stock solution (30 mL) plus activated sludge suspension (9 mL) and adjusting the volume to 300 mL with BSM. Concentration of activated sludge was 30 mg/L, concentration of test substance was 30 mg/L and concentration of reference substance was 100 mg/L.
- Vessel 5 (procedure control) was prepared by adding reference substance stock solution (30 mL) plus activated sludge suspension (9 mL) and adjusting the volume to 300 mL with BSM. Concentration of activated sludge was 30 mg/L and concentration of reference substance was 100 mg/L.
- Vessel 6 (blank control) was prepared by adding activated sludge suspension (9 mL) and adjusting the volume to 300 mL with BSM. Concentration of activated sludge was 30 mg/L.
- The equipment was then checked to ensure it was airtight, stirring was started, and measurement of oxygen uptake in conditions of darkness was begun.
- Temperature plus operation of the stirrer and recorder were checked daily.
- Any changes in the colour of the vessel contents were recorded.
- Biological Oxygen Demand (BOD) was recorded for the six vessels.
- Concentration of the test substance in the six vessels was analysed after 28 days.

TEST VALIDITY
1. Biodegradation of the reference substance > 40% on day 7 and 65% on day 14 day.
2. The pH value is inside the range 6.0 to 8.5.
3. Oxygen uptake of the inoculum blank should not be greater than 60 mg O2/L in 28 days.
4. The difference of extremes of replicate values of the removal of the test substance during 28-day test is less than 20%.
5. The oxygen consumption by the test substance is greater than 60% of inoculum blank.

CALCULATION OF THEORETICAL OXYGEN DEMAND
- Theoretical oxygen demand was calculated as shown in the attached equation.
- Carbon was assumed to be mineralised to CO2, hydrogen to H2O, Cl to HCl, S to SO3 and Na to Na2O3.
- Halogens were presumed to be eliminated as hydrogen halides and nitrogen as ammonia.
- The ThOD(NH3) of the test substance was calculated to be 2.98 mg O2/mg and the ThOD(NH3) of sodium benzoate was calculated to be 1.67 mg O2/mg.

CALCULATION OF PERCENTAGE BIODEGRADABILITY
- Percentage biodegradation was calculated using the equation % degradation = [(BOD x V – B x V) / (ThoD x V x C) x 100
- Where BOD = Biological oxygen demand of the test compound measured on the BOD curve (mg/L); B = Oxygen consumption of basal culture medium to which the inoculum was added measured on the BOD curve (mg/L); V = Test volume (mL); C = Concentration of test substance in test vessel (mg/L); ThOD = Theoretical oxygen demand (theoretical, mg) required when the test compound is completely oxidised (mg/mg).
- Percentage degradation from the results of chemical analysis was calculated using the equation % degradation = [(Sb / Cb) – (Sa / Ca) / (Sb / Cb) x 100]
- Where Sa = Concentration in the "test vessel" after completion of the biodegradability test (mg/L); Sb = Concentration in the "abiotic control" after completion of the biodegradability test (mg/L); Ca = Concentration of the test substance added in the "test vessel" at the start of the test (mg/L); Cb = Concentration of the test substance added in the "abiotic control" at the start of the test (mg/L).
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
28 d
Parameter:
% degradation (TOC removal)
Value:
71.3
Sampling time:
28 d
Details on results:
WORK CURVE
- A series of standard solutions with concentration at 0.50, 1.00, 2.00, 5.00, 10.0, 20.0 and 50.0 mg/L were measured under the TOC conditions mentioned above.
- A linear regression equation was obtained with the peak area versus standard solution concentration (A=178.05c+229.4) where A = peak square (AU) and c = TOC concentration (mg/L) (see Figure 2, attached).
- Results showed that linearity for a concentration range of 0.50 mg/L to 50 mg/L was good (r2 = 0.999).

RECOVERY TEST
- Results of the recovery test are shown in Table 4 (attached).
- Mean recovery rate was 95.7 % for the spiked concentration of 10 mg/L and 84.5 % for the spiked concentration of 100 mg/L.
- Relative standard deviations were 5.28 % to 6.46 %.

DETECTION LIMIT
- The minimum detection amount of TOC analyser was 0.50 mg/L TOC.
- The minimum detection concentration for water samples was 0.67 mg/L test substance.

READY BIODEGRADATION OF TEST SUBSTANCE
- During the test, the temperature was kept at 24.7°C to 25 .2 °C and pH was kept at 6.83 - 7.40.
- The total oxygen uptake in the inoculum blank was 33.4 mg O2/L at the end of the test.
- Biodegradation of the reference substance, sodium benzoate, reached 71.8% at 7 days {> 40%), and 75.5% at 14 days {> 60%).
- The difference of replicate values for inoculum blank and test, during the test, were both less than 20%.
- Biodegradation of inhibition control was 72.2% at 14 days (>25%), indicating that there was no
inhibition effect to inoculum.
- The oxygen consumption by the test substance was greater than 60% of the inoculum blank. Thus, the test was considered valid.
- The BOD results showed that biodegradation of the test substance was 63.0% after 28 days.
- Based on the residue analysis, biodegradation of the test substance was 71.3% during the testing period.
Results with reference substance:
Biodegradation of the reference substance, sodium benzoate, reached 71.8% at 7 days {> 40%), and 75.5% at 14 days {> 60%).
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The BOD results showed that biodegradation of the test substance was 63.0 % after 28 days. Based on residue analysis, biodegradation of the test substance was 71.3 % during the test period.
Executive summary:

GUIDELINE

Ready biodegradation of the test item was investigated according to HJ/T 153-2004 “The guidelines for the testing of chemicals, Degradation and Accumulation” (2ndedition) (Beijing: China Environment Press, 2013) and procedure 301 C of the Guidelines for Testing of Chemicals “Modified MITI Test (I) (OECD 1992).

 

METHODS

Test solutions were prepared in inorganic salts medium inoculated with a number of microorganisms collected from ten places in Nanjing city. Test substance and microorganisms not adapted to the test item were added to aerobic aqueous medium in BOD bottles. The Biological Oxygen Demand (BOD) and residual chemicals in the BOD bottles were measured over a 28-day period.

 

RESULTS

During the test, the temperature was kept at 24.7 to 25.2 °C and pH was kept at 6.83 to 7.40. Total oxygen uptake in the inoculum blank was 33.4 mg O2/L at the end of the test. Biodegradation of the reference substance (sodium benzoate) reached 71.8 % at 7 days and 75.5 % at 14 days. The difference between replicate values for inoculum blank and test item were both less than 20 %. Biodegradation of the inhibition control was 72.2 % at 14 days indicating there was no inhibitory effect on the inoculum. Oxygen consumption by the test substance was greater than 60 % of inoculum blank. The test was considered valid.

 

CONCLUSION

The BOD results showed that biodegradation of the test substance was 63.0 % after 28 days. Based on residue analysis, biodegradation of the test substance was 71.3 % during the test period.

Description of key information

In the key study, the test item attained 74% biodegradation after 28 days (OECD 301 B and EU Method C.4 -C). In a supporting study, the  BOD results showed that biodegradation of the test substance was 63.0 % after 28 days. Based on residue analysis, biodegradation of the test substance was 71.3 % during the test period (HJ/T 153 -2004, OECD 301 C and EU Method C.4 -F). The test item is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Key study

GUIDELINE

In a study performed to OECD Guideline No. 301 B and EC Method C.4-C: CO2Evolution test the test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21 °C for 28 days.

METHODS

Following the recommendations of ISO 1995, the test item was dissolved in an auxiliary solvent prior to being absorbed onto filter paper and subsequent dispersal in the test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item sodium benzoate, together with a toxicity control were used for validation purposes.

RESULTS

The test item is an unstable UVCB substance in aqueous conditions and hydroyses rapidly (less than 30 minutes). The water solubility of the test item is therefore equivalent to the water solubility of the two main degradation products boric acid and 2-propyl heptan-1-ol. Both degradation products have been registered under REACH. Boric acid is an inorganic substance with high water solubility and will not undergo biodegradation. 2-propyl heptan-1-ol is a poorly soluble substance (> 100 mg/L) and is readily biodegradable with over 90% degradation in 28-days.

CONCLUSION

The test item attained 74% biodegradation after 28 days. As the test material is a UVCB the 10 -day window validation criterion are not applicable. Therefore, the test material can be considered to be readily biodegradable. The organic degradation product derived from hydrolysis of the test item is readily biodegradable.

 

Supporting study

GUIDELINE

Ready biodegradation of the test item was investigated according to HJ/T 153-2004 “The guidelines for the testing of chemicals, Degradation and Accumulation” (2ndedition) (Beijing: China Environment Press, 2013) and procedure 301 C of the Guidelines for Testing of Chemicals “Modified MITI Test (I) (OECD 1992).

 

METHODS

Test solutions were prepared in inorganic salts medium inoculated with a number of microorganisms collected from ten places in Nanjing city. Test substance and microorganisms not adapted to the test item were added to aerobic aqueous medium in BOD bottles. The Biological Oxygen Demand (BOD) and residual chemicals in the BOD bottles were measured over a 28-day period.

 

RESULTS

During the test, the temperature was kept at 24.7 to 25.2 °C and pH was kept at 6.83 to 7.40. Total oxygen uptake in the inoculum blank was 33.4 mg O2/L at the end of the test. Biodegradation of the reference substance (sodium benzoate) reached 71.8 % at 7 days and 75.5 % at 14 days. The difference between replicate values for inoculum blank and test item were both less than 20 %. Biodegradation of the inhibition control was 72.2 % at 14 days indicating there was no inhibitory effect on the inoculum. Oxygen consumption by the test substance was greater than 60 % of inoculum blank. The test was considered valid.

 

CONCLUSION

The BOD results showed that biodegradation of the test substance was 63.0 % after 28 days. Based on residue analysis, biodegradation of the test substance was 71.3 % during the test period.