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EC number: 946-364-2 | CAS number: -
Biodegradation in water: screening tests
Administrative data
Link to relevant study record(s)
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-04-14 - 2015-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guidline study in compliance with GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
- Deviations:
- yes
- Remarks:
- On Day 19 of the study the temperature in a vessel containing water which was incubated under the same conditions as the test vessels was recorded as being 24. 7 °C which is outside the temperature range of 22 ± 2 °C.
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
- Deviations:
- yes
- Remarks:
- On Day 19 of the study the temperature in a vessel containing water which was incubated under the same conditions as the test vessels was recorded as being 24. 7 °C which is outside the temperature range of 22 ± 2 °C.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 835.3110 (Ready Biodegradability)
- Deviations:
- yes
- Remarks:
- On Day 19 of the study the temperature in a vessel containing water which was incubated under the same conditions as the test vessels was recorded as being 24. 7 °C which outside the temperature range of 22 ± 2 °C.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic (adaptation not specified)
- Details on inoculum:
- The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any 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. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (50 mL) of the washed activated sewage sludge by suction through preweighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.6 g/L prior to use.
- Duration of test (contact time):
- >= 29 d
- Initial conc.:
- >= 14.2 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 to 25 °C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 25.0 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter and the pH values was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with C02 free air.
The test vessels were sealed and C02-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb ) granules.
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
Samples (2 mL) were taken from the first C02 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
All samples were analyzed for IC immediately.
On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analyzed for IC using a Tekmar-Dohrmann Apollo 9000 TOC analyzer or a Shimadzu TOC-VcsH TOC analyzer. Samples (300 or 50 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.
IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
The samples were analyzed for IC and TC using a Shimadzu TOC-V cPH TOC Analyzer. Samples (50 μL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.
The test item consists of fourteen components which contribute to the total carbon content. The amount of carbon present in each component was calculated using the following equation and corrected for the amount of each component in the test item.
((No of C atoms * mol wt of C) / (mol wt of test item)) * 100%
Therefore for a concentration of 10 mg C/L (a total of 42.6 mg of test item in 3 litres) the total organic carbon present was 30 mg C.
The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:
((No of C atoms * mol wt of C) / (mol wt of sodium benzoate)) * 100
((7 * 12.011) / (144.11)) * 100 = 58.34%
Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.
The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference items before substitution into the following equation:
%THCO2 (=%biodegradation#) = ((mg IC in test flask - mg IC in control flask) / (mg TOC added as test chemical)) * 100
# = The conversion factor for carbon to carbon dioxide is 3.67
The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control * (100 / (%C of CO2)) * (1 / (test volume))
= mg IC in control * (100 / 27.29) * (1 / 3)
The results of the degradation test are considered valid if in the same test the reference item yields >= 60% degradation by Day 14.
The test item may be considered to be readily biodegradable if >= 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain >=25% degradation by Day 14 for the test item to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-Day window, as appropriate, is less than 20%.
The total C02 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.
The IC content of the test item suspension in the mineral medium at the beginning of the test should be <5% of the TC. - Reference substance:
- benzoic acid, sodium salt
- Preliminary study:
- not applicable
- Test performance:
- Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0 and 28 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.
- Key result
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 94
- Sampling time:
- 28 d
- Details on results:
- Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved C02 present in the test vessels. Therefore any additional C02 detected in the Day 29 samples originated from dissolved C02 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of test item replicates 1 and 2 and the toxicity control. This decrease was considered to be due to sampling/analytical variation.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of C02 into the second absorber vessels occurred.
The test item attained 94% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of thebiodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 83% biodegradation after 14 days and 84% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 97% biodegradation after 14 days and 102% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation. - Validity criteria fulfilled:
- yes
- Remarks:
- in accordance with the validation criterions given in the OECD Test Guidelines
- Interpretation of results:
- readily biodegradable
- Conclusions:
- The test item attained 94% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- Executive summary:
The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).
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 temperatures of between 20 to 25 °C for 28 days.
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.
The test item attained 94% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2004-11-22 - 2001-12-31
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guidline study in compliance with GLP
- 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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 835.3110 (Ready Biodegradability)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic (adaptation not specified)
- Details on inoculum:
- The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium and then was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a washed sample (100 ml) of the activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel . The filter paper was then dried in an oven at approximately 105°C
for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids was equal to 2.7 g/L prior to use.
* previously rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven - Duration of test (contact time):
- >= 29 d
- Initial conc.:
- >= 14.2 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbo/L.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at 21°C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 33.3 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing selfindicating soda lime (Carbosorb® )granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.
Samples (2 ml) were taken from the first C02 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18,20,23,27,28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0, 1, 2, 3, 6, 8, 12, 14, 16, 20, 23, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 10 and 18 were stored at approximately -20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that degradation of the test material did not meet the 10-Day window validation criterion given in the OECD 'Guidelines and therefore additional analyses were considered to be unnecessary.
On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analysed for C02 using a Tekmar-Dohann Apollo 9000 TOC analyser, and the Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 pl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to C02 by orthophosphoric acid using zero grade air or nitrogen (oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2C03). Each analysis was carried out in triplicate.
Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon/Total Carbon (IC/TC) ratio in the test media. The samples were filtered through Gelman 0.45 pm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels and filtered through Gelman 0.45 pm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 mycroL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pWOxi 3401 pH and dissolved oxygen meter.
The test material contains 70.66% carbon and so for a concentration of 10 mg C/L (a total of 42.6 mg) the total organic carbon present was 30 mg C.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6H5COONa) was calculated as follows:
((No of C atoms * mol wt of C) / (mol wt of sodium benzoate)) * 100 % = ((7 * 12.011) / (144.11)) * 100 = 58.34 %
Thus for a 10 mg C/L test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values given in Table 1 in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.
% ThCO2 (= % degradation) = ((mg IC in test flask - mg IC in control) / (mg TOC as test material)) * 100 %
The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.
Percentage degradation = [1 - ((mg DOC in test flask on Day 28) / (mg DOC in test flask on Day 0))] * 100 %
The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.
Total CO2 evolution = mg IC in control * ((100) / (% C of CO2)) * ((1) / (test volume))
= mg IC in control * ((100) / (27.29)) * 1/3
The results of the degradation test are considered valid if in the same test the standard material yields >= 60% degradation by Day 14.
The test material may be considered to be readily biodegradable if >= 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain >= 25% degradation by Day 14 for the test material to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
The total C02 evolution in the control vessels at the end of the test should not normally exceed 40 mg/L medium.
The IC content of the test material suspension in the mineral medium at the beginning of the test should be < 5% of the TC. - Reference substance:
- benzoic acid, sodium salt
- Preliminary study:
- not applicable
- Test performance:
- Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 94
- Sampling time:
- 28 d
- Details on results:
- The total CO2 evolution in the control vessels on Day 28 was 21.77 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
he IC/TC ratio of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% and hence satisfied the validation criterion given in the 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 criterion given in the OECD Test Guidelines.
The test material attained 94% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test material cannot be considered to be readily biodegradable as the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. However, the test material has exhibited the potential for rapid degradation. In terms of the classification and labelling requirements (EU Directive for Dangerous Substances, L 1 1 OA) the test material may be considered as readily biodegradable as evidence of >70% degradation has been shown over a 28-Day period in a standard biodegradation study.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of standard material replicate R2 and test material replicate R2. These increases were considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28 and coupled with the decrease in inorganic carbon within test material replicate R2 resulted in an increase in the percentage degradation value for the test material from 94% on Day 28 to 95% on Day 29.
The toxicity control attained 86% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The increase in inorganic carbon in the control first absorber vessels on Day 29 resulted in a decrease in the percentage degradation value for the toxicity control from 86% on Day 28 to 85% on Day 29. The decrease in degradation obtained for the toxicity control on Day 29 was considered to be due to the increases in inorganic carbon within the replicate control vessels being greater than those within the toxicity control vessel.
Sodium benzoate attained 106% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the control first absorber vessels on Day 29 coupled with the decrease in inorganic carbon within standard material replicate R2 resulted in a decrease in the percentage degradation value for the standard material from 106% on Day 28 to 103% on Day 29. The decrease in degradation obtained for the standard material on Day 29 was considered to be due to the increases in inorganic carbon within the replicate control vessels and standard material replicate R1 being less than the decrease in inorganic carbon within the standard material replicate R2. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of C02 into the second absorber vessels occurred.
Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 102% and 100% respectively for Replicates R1 and R2. Degradation values in excess of 100% were considered to be due to sampling/analytical variation. Observations made throughout the test period (see Table 6), showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were cloudy light brown dispersions with no undissolved test material visible. The content of the toxicity control vessel was a cloudy light brown dispersion With no undissolved standard material or test material visible.
6. - Validity criteria fulfilled:
- yes
- Interpretation of results:
- readily biodegradable, but failing 10-day window
- Conclusions:
- The test material attained 94% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test material cannot be considered to be readily biodegradable as the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. However, the test material has exhibited the potential for rapid degradation. In terms of the classification and labelling requirements (EU Directive for Dangerous Substances, L11 OA) the test material may be considered as readily biodegradable as evidence of >70% degradation has been shown over a 28-Day period in a standard biodegradation study.
- Executive summary:
The study was performed to assess the ready biodegrdability of the test material in an aerobic aqueous medium. The method that described in the OECD Guidelines for Testing of Chemicals (1992) No 301 B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3 110 Paragraph (m).
The test material, at a concentration of 10 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21°C for 28 days.
The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.
The test material attained 94% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test material cannot be considered to be readily biodegradable as the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. However, the test material has exhibited the potential for rapid degradation. In terms of the classification and labelling requirements (EU Directive for Dangerous Substances, L 110A) the test material may be considered as readily biodegradable as evidence of >70% degradation has been shown over a 28-Day period in a standard biodegradation study.
Referenceopen allclose all
Table 1: Inorganic Carbon Values on Each Analysis Occasion (attached)
Table 2: Percentage Biodegradation Values
Day |
% Biodegradation |
|||||
Procedure Control |
Test Item |
Toxicity Control |
||||
0 |
0 |
0 |
0 |
|||
2 |
65 |
18 |
42 |
|||
6 |
92 |
19 |
74 |
|||
8 |
80 |
38 |
70 |
|||
10 |
97 |
52 |
70 |
|||
14 |
97 |
69 |
83 |
|||
21 |
103 |
83 |
83 |
|||
28 |
105 |
103 |
88 |
|||
29* |
102 |
94 |
84 |
|||
* Day 29 values corrected to include any carry-over of CO2detected in Absorber 2 |
||||||
Table 3: Total and Inorganic Carbon Values in the Culture Vessels on Day 0
Test vessel |
Total Carbon* (mg/L) |
Inorganic Carbon* (mg/L) |
IC Content (% of TC) |
Test Item 10 mg C/L R1 |
10.50** |
-0.24 |
0 |
Test Item 10 mg C/L R2 |
9.70** |
-0.40 |
0 |
R1- R2 |
= |
Replicates 1 and 2 |
|
* |
= |
Corrected for control values. Negative values are due to measured concentration being less than control values |
|
** |
= |
Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item |
|
Table 4: pH Values of the Test Preparations on Days 0 and 28
Test Vessel |
pH |
||
| Day 0 Pre-Adjustment | Day 0 Post-Adjustment | Day 28 | |
| Inoculum Control R1 | 7.7 | 7.5 | 7.5 |
| Inoculum Control R2 | 7.7 | 7.6 | 7.5 |
| Procedure Control R1 | 7.7 | 7.5 | 7.6 |
| Procedure Control R2 | 7.7 | 7.5 | 7.4 |
| Test Item R1 | 7.7 | 7.5 | 7.4 |
| Test Item R2 | 7.7 | 7.5 | 7.4 |
| Toxicity Control | 7.7 | 7.5 | 7.4 |
| R1- R2 | = | Replicates 1 and 2 |
|
Table 5: Observation on the Test Preparations Throughout the Test Period
| Test Vessel | Observations on Test Preparations | |||||
| Day 0 | Day 6 | Day 13 | Day 20 | Day 27 | ||
| Inoculum Control | R1 | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion |
| R2 | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion | |
| Procedure Control | R1 | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible |
| R2 | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | Light brown dispersion, no undissolved reference item visible | |
| Test Item | R1 | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible |
| R2 | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | Light brown cloudy dispersion, no undissolved test item visible | |
| Toxicity Control | Light brown cloudy dispersion, no undissolved test or reference item visible | Light brown cloudy dispersion, no undissolved test or reference item visible | Light brown cloudy dispersion, no undissolved test or reference item visible | Light brown cloudy dispersion, no undissolved test or reference item visible | Light brown cloudy dispersion, no undissolved test or reference item visible | |
Figure 1: picture-file attached
Table 1: Inorganic carbon values on each analysis occasion (attached)
Table 2: Percentage Biodegradation Values
| Day | % Degradation Sodium Benzoate |
% Degradation Test Material |
% Degradation Test Material plus Sodium Benzoate Toxicity Control |
|
| 0 | 0 | 0 | 0 | |
| 1 | 29 | 10 | 20 | |
| 2 | 59 | 27 | 30 | |
| 3 | 59 | 33 | 33 | |
| 6 | 60 | 33 | 34 | |
| 8 | 63 | 49 | 42 | |
| 12 | 86 | 52 | 43 | |
| 14 | 88 | 67 | 54 | |
| 16 | 92 | 73 | 68 | |
| 20 | 95 | 83 | 69 | |
| 23 | 101 | 89 | 82 | |
| 27 | 102 | 89 | 88 | |
| 28 | 106 | 94 | 86 | |
| 29* | 103 | 95 | 85 | |
| * Day 29 values corrected to include any carry-over of CO2detected in Absorber 2 | ||||
Table 3: Total and inorganic carbon values in the culture vessels on Day 0:
| Test vessel | Total Carbon* (mg/L) | Inorganic Carbon* (mg/L) | IC/TC Ratio (%) |
| Sodium Benzoate 10mgC/L R1 | 9.52 | 0.25 | 3 |
| Sodium Benzoate 10mgC/L R2 | 9.45 | 0.22 | 2 |
| Test Material 10 mg C/L R1 | 10.24** | 0.15 | 1 |
| Test Material 10mgC/L R2 | 10.10** | 0.09 | 1 |
| Test Material plus Sodium Benzoate Toxicity Control 20 mg C/L | 19.64** | -0.10 | 0 |
| R1 - R2 = Replicates 1 and 2 | |||
| * Corrected for control values | |||
| ** Total carbon value given is the sum of the TC value optained from analysis and the nominal TC contribution of the test material and sodium benzoate where applicable | |||
Table 4: Dissolved orgnic carbon (DOC) values in the culture vessels on Days 0 and 28
| DOC* Concentration | |||||
| Test Vessel | Day 0 | Day 28 | |||
| mg C/L | % of Nominal Carbon Content | mg C/L | % of Initial Carbon Concentration | % Degradation | |
| Sodium Benzoate 10 mg C/L R1 | 9.26 | 93 | -0.19 | -2 | 102 |
| Sodium Benzoate 10mg C/L R2 | 9.22 | 92 | -0.01 | 0 | 100 |
| R1 - R2 = Replicates 1 and 2 | |||||
| * Corrected for control values. Negative values are due to measured concentration being less than control values | |||||
Table 5: pH values of the test preparations on Day 28
| Test Vessel | pH |
| Control R1 | 7.6 |
| Control R2 | 7.5 |
| Sodium Benzoate 10 mg C/L R1 | 7.5 |
| Sodium Benzoate 10 mg C/L R2 | 7.6 |
| Test Material 10 mg C/L R1 | 7.6 |
| Test Material 10 mg C/L R2 | 7.6 |
| Test Material plus Sodium Benzoate Toxicity Control 20 mg C/L | 7.6 |
| R1- R2= Replicates 1 and 2 | |
Table 6: Observation on the test preparations throughout the test period
| Test Vessel | Observations on Test Preparations | |||||
| Day 0 | Day 5 | Day 12 | Day 19 | Day 26 | ||
| Control | R1 | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion |
| R2 | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion | Light brown dispersion | |
| Standard Material | R1 | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible |
| R2 | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | Light brown dispersion, no undissolved standard material visible | |
| Test Material | R1 | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible |
| R2 | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | Cloudy light brown dispersion with no undissolved test material visible | |
| Toxicity Control | Cloudy light brown dispersion with no undissolved standard or test material visible | Cloudy light brown dispersion with no undissolved standard or test material visible | Cloudy light brown dispersion with no undissolved standard or test material visible | Cloudy light brown dispersion with no undissolved standard or test material visible | Cloudy light brown dispersion with no undissolved standard or test material visible | |
| R1- R2= Replicates 1 and 2 | ||||||
Description of key information
The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).
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 temperatures of between 20 to 25 °C for 28 days.
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.
The test item attained 94% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Key value for chemical safety assessment
- Biodegradation in water:
- readily biodegradable
Additional information
For Supporting study:
The study was performed to assess the ready biodegrdability of the test material in an aerobic aqueous medium. The method that described in the OECD Guidelines for Testing of Chemicals (1992) No 301 B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3 110 Paragraph (m).
The test material, at a concentration of 10 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21°C for 28 days.
The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.
The test material attained 94% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No 301B the test material cannot be considered to be readily biodegradable as the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. However, the test material has exhibited the potential for rapid degradation. In terms of the classification and labelling requirements (EU Directive for Dangerous Substances, L 110A) the test material may be considered as readily biodegradable as evidence of >70% degradation has been shown over a 28-Day period in a standard biodegradation study.
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