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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:
- 9 November 2006 - 7 December 2006
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds ill aqueous medium - Method by analysis of inorganic carbon in sealed vessels (C02 headspace test)"
- Deviations:
- no
- GLP compliance:
- yes
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, non-adapted
- Details on inoculum:
- - Source of inoculum/activated sludge: Severn trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately I hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the
supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until use.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 20 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- TEST SPECIES:
A mixed population of sewage sludge micro-organisms was obtained on 9 November 2006 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
TEST SYSTEM
The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:
a) A control consisting of inoculated culture medium, plus 100 mg silica gel, 11 replicate vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of20 mg carbon/l, 11 replicate vessels.
c) The test material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of mg carbon/I, 7 replicate vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 7 replicate vessels.
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.
Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 mI/l.
Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20± 1°C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).
Data from the control and standard material vessels was shared with similar concurrent studies.
SAMPLING
The slight variation in the degradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the nature of the study design (individual replicates sacrificed on each sampling occasion), the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected.
Dissolved Organic Carbon (DOC) analyses
Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R2. The degradation rates for the standard material were similar to those determined by IC analyses.
CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/I, equivalent to a total of20 mg carbon/I.
EVALUATION OF DATA
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material was determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:
Carbon: 85.44% w/w
Hydrogen: 15.01% w/w
Nitrogen: 0.09% w/w
Thus for a test concentration of 20 mg C/I (a total of2.50 mg) the total organic carbon present was 2.14mgC.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6H6COONa) was calculated as follows:
[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%
Thus for a 20 mg C/I test concentration (34.3 mg/I) the total organic carbon present in each test vessel was 2.14mgC.
Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.
The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.
The toxicity control should attain ≥ 25% degradation by Day 14 for the test material to be considered as noninhibitory.
The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test material. - Reference substance:
- other: Sodium benzoate
- Preliminary study:
- Preliminary work conducted showed that a volume of 3.2 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE PIN 001 100 5FX) gave a measured weight of 2.51 mg, mean of 15 separate weighings.
- Test performance:
- The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 43
- Sampling time:
- 28 d
- Details on results:
- The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards Organisation (ISO 1996) and in the published lilerature (Handley et ai, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.
The test material attained 43% degradation after 28 days.
The toxicity control attained 60% degradation after 14 days and 66% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that
the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring. - Results with reference substance:
- Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test material attained 43% degradation after 28 days.The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (3 cSt) is not readily biodegradable.
- Executive summary:
An ISO 14593 study with GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (3 cSt) has been conducted.
The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking.
A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance.
Assessment of biodegradation was made by CO2 analysis only, based on 2-weekly samplings. At the end of the 28 d test period, 43% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes.
Conclusion: The test material attained 43% degradation after 28 days. The study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (3 cSt) is not readily biodegradable.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-01-14 to 2013-02-15
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
- GLP compliance:
- yes (incl. QA statement)
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic, non-adapted
- Details on inoculum:
- A mixed population of activated sludge micro-organisms was obtained on 14 January 2013 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Preparation of inoculum:
The activated sewage sludge sample was washed three times 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 them 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 (100 mL) of the washed activated sewage sludge by suction through pre-weighted GF/A filter paper using a Buchner funnel. Filtration was continued for a further three minutes after rinsing the filter three successive timeswith 10 mL of deionised 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 3.0 g/L prior to use.
Abiotic samples were prepared by the autoclaving an aliquot of inoculum prior to addition to the test vessels. Additionally an aliquot (20 mL) of a 10 g/L aqueous solution of sodium azide was added to the test vessels to maintain sterility through the duration of the incubation period. - Duration of test (contact time):
- 29 d
- Initial conc.:
- 10 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- Preparation of test system
The following test preparations were prepared and inoculated in a 5 litre test culture each containing 3 litres of solution
a) an inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter paper to give a final concentration of 10 mg carbon/L
c) The test item on a filter paper, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item on a filter paper, in duplicate, in inoculated mineral medium plus an aliquot (20 mL) of a 10 g/L aqueous solution of sodium azide to give a final concentration of 10 mg carbon/L.
A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels. 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±1°C in darkness. - Reference substance:
- benzoic acid, sodium salt
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 65
- Sampling time:
- 28 d
- Details on results:
- The biotic test item attained 65% after 28 days and 70% degradation after 29 days. The abiotic test item attained 0% degradation after 28 days.
Chemical analysis of the biotic test preparations at Day 29 showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 5.3 mg/l/ The abiotic test sample had a concentration of 48% of nominal.
Analysis of the test preparations of the abiotic test vessels at Day 29 showed the losses from the test system to be via several mechanisms, not solely via biological pathways. Losses from the system were considered to be mainly via volatisation due to the chemical nature of many of the constituents of the test item but may also be due to losses due to formation of non-extractable residues. - Results with reference substance:
- Sodium benzoate attained 70% degradation after 14 days and 81% degradation after 28 days.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- readily biodegradable
- Conclusions:
- The test item attained 65% degradation in 28 days (CO2 evolution) in a study conducted in accordance with OECD 301B.
- Executive summary:
A study was performed to assess the ready biodegradability of the test material GTL base oil [‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (3 cSt) in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "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 835.3110 Paragraph (m).’
The test material GTL base oil (3 cSt) attained 65% degradation after 28 days and therefore cannot 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:
- key study
- Study period:
- Study initiation: March 24, 2009; Study completion: August 7, 2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
- GLP compliance:
- yes (incl. QA statement)
- 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):
Chemicals Evaluation and Research Institute, Japan
- Concentration of sludge: Mixed liquor suspended solid (MLSS): 3300 mg/L - Duration of test (contact time):
- 28 d
- Initial conc.:
- 100 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- other: The biological oxygen demand (BOD) was measured over a 28-day period. After this period, amounts of the dissolved organic carbon (DOC) and residual test susbtance were measured. The biodegradability of the test substance was evaluated from these results.
- Details on study design:
- Study conditions:
1) Temperature: 25 ± 1ºC
2) Exposure period: 28 days
3) Stirring: Continuous stirring with magnetic stirrer
4) Test volume: 300 mL
5) Concentration: test substance (bottles 3-6): 100 mg/l
Aniline (bottle 1) : 100 mg/l
Activated sludge (bottles 1-5): 30 mg/l
Test bottle contents:
Bottle 1: Activity control (aniline + activated sludge + basal medium)
29.5 μL (30.0 mg) of aniline was added to the basal medium, and then 2.7 ml of activated sludge was added.
Bottle 2: Inoculum blank (activated sludge + basal medium)
2.7 ml of activated sludge was added to the basal medium.
Bottles 3, 4, 5: Test suspensions (test substance + activated sludge + basal medium)
30.0 mg of the test substance was added to the basal medium then 2.7 ml activated sludge was added.
Bottle 6: Abiotic control (test substance + purified water)
30.0 mg of the test substance was added to 300 ml of purified water. - Reference substance:
- aniline
- Test performance:
- There was no specific factor which might have affected the reliability of the test results.
The test was judged valid since the measurement results fulfilled all of the validity criteria of the guideline. - Parameter:
- other: BOD
- Value:
- 52
- Sampling time:
- 28 d
- Parameter:
- % degradation (test mat. analysis)
- Value:
- 47
- Sampling time:
- 28 d
- Details on results:
- Observation of the test solutions after exposure period:
The color of the test solutions and growth of the sludge were observed in contrast with the control (bottle 2).
The test solutions in bottle 1 and 5 were white, solutions in bottles 3, 4 and 6 were slightly white.
Growth of the sludge was observed in bottle 1, 3, 4 and 5.
pH measurement:
After 28 days of exposure, pH values were 6.9, 7.0, 7.0, 8.2 for bottles 3, 4, 5 and 6, respectively.
Degradability based on the BOD:
The BOD (maximum theoretical value = 103.7 mg) in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 49.2, 57.0 and 56.3 mg respectively, and the BOD in bottle 6 was 0.7 mg.
The degrability based on the BOD measurement was calculated to be 47, 55 and 54% for bottles 3, 4 and 5 respectively.
Degradability based on the DOC:
The DOC (initial amount 25.8 mg) in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 0.1, 0.0 and -0.2 mg respectively, and the DOC in bottle 6 was 1.7 mg.
The degradability based on the DOC was not calculated since the test substance was insoluble in water and the DOC in bottle 6 is less than 90% of initial amount.
Degradability based on the residual test substance amount:
The amounts of residual test substance (Initial amount = 30.0 mg) were 16.6, 14.9 and 15.3 mg in bottles 3, 4 and 5 respectively and 29.3 mg in bottle 6.
The degradabilities based on the residual test substance amount were calculated to be 43, 49 and 48% for bottles 3, 4, and 5, respectively.
From the degradability results based on the BOD (av. 52%) and the residual test substance amount (av. 47%), it is concluded that the test substance is not readily biodegradable under the conditions of this test.
Result tables attached as attached background material. - Results with reference substance:
- The aniline control (Bottle 1) attained 73.0% degradability after 28 days.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test substance is not readily degradable. No transformed product was formed.
- Executive summary:
A ready biodegradation test of GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] has been conducted, using the Japanese methodology for new substance notification, i.e. OECD 301C MITI (I) methodology. The method for introduction of the test substance into the test system was not described.
The test substance concentration was 100 mg/l. There was no indication of steps being taken to minimise or prevent evaporation during test substance loading or during the test. The inoculum was a mixed liquor obtained from CERI, Japan; in accordance with the guideline the sludge should be non-adapted and whilst this is not explicitly stated, it is reasonable to assume that such an important criterion would have been met. The concentration of inoculum in the test system was 30 mg suspended solids/l.
Assessment of biodegradation in both tests was made by BOD, analysed by closed system oxygen consumption analysis, supported by residual test substance analysis by gel permeation chromatography.
DOC measurements were made but degradation was not assessed on this basis due to poor recoveries in the abiotic control, possibly due to the very low solubility of the test substance.
At the end of the 28 d test period, the following results are reported: 47% - 55% degradation (based on BOD); 43% - 49% degradation (based on residual test substance).
The kinetics appear to show that the degradation proceeded smoothly without significant plateaux in the curve. The study was conducted in accordance with GLP and is considered to be reliable, though the results are not necessarily meaningful for exposure assessment purposes.
The substance GTL Base oil ['Distillates (Fischer-Tropsch), C18-50-branched, cyclic and linear'] was not readily biodegradable in this study.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 9 November 2006 - 7 December 2006
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds ill aqueous medium - Method by analysis of inorganic carbon in sealed vessels (C02 headspace test)"
- Deviations:
- no
- GLP compliance:
- yes
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, non-adapted
- Details on inoculum:
- - Source of inoculum/activated sludge: Severn trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately I hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the
supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until use.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 20 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- TEST SPECIES:
A mixed population of sewage sludge micro-organisms was obtained on 9 November 2006 from the secondary treatment stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
TEST SYSTEM
The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:
a) A control consisting of inoculated culture medium, plus 100 mg silica gel, 11 replicate vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of20 mg carbon/l, 11 replicate vessels.
c) The test material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of mg carbon/I, 7 replicate vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 7 replicate vessels.
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.
Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 mI/l.
Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20± 1°C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).
Data from the control and standard material vessels was shared with similar concurrent studies.
SAMPLING
The slight variation in the degradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the nature of the study design (individual replicates sacrificed on each sampling occasion), the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected.
Dissolved Organic Carbon (DOC) analyses
Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R2. The degradation rates for the standard material were similar to those determined by IC analyses.
CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/I, equivalent to a total of20 mg carbon/I.
EVALUATION OF DATA
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material were determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:
Carbon: 85.44% w/w
Hydrogen: 15.01% w/w
Nitrogen: 0.09% w/w
Thus for a test concentration of 20 mg C/I (a total of2.50 mg) the total organic carbon present was 2.14mgC.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6H,COONa) was calculated as follows:
[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%
Thus for a 20 mg C/I test concentration (34.3 mg/I) the total organic carbon present in each test vessel was 2.14mgC.
Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.
The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.
The toxicity control should attain ≥ 25% degradation by Day 14 for the test material to be considered as noninhibitory.
The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test material. - Reference substance:
- other: Sodium benzoate
- Preliminary study:
- Preliminary work conducted showed that a volume of 3.2 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE PIN 001 100 5FX) gave a measured weight of 2.51 mg, mean of 15 separate weighings.
- Test performance:
- The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 24
- Sampling time:
- 28 d
- Details on results:
- The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% of the organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.
The test material attained 24% degradation after 28 days.
The toxicity control attained 56% degradation after 14 days and 58% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that
the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring. - Results with reference substance:
- Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test material attained 24% degradation after 28 days; the study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) is not readily biodegradable.
- Executive summary:
An ISO 14593 study with the substance GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (4 cSt) has been conducted. The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking.
A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance. Assessment of biodegradation was made by CO2 analysis only, based on 2-weekly samplings.
At the end of the 28 d test period, 24% degradation was reported. It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes.
Conclusion: The test material attained 24% degradation after 28 days. The study demonstrates that GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (4 cSt) is not readily biodegradable.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 9 November 2006 - 7 December 2006
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds ill aqueous medium - Method by analysis of inorganic carbon in sealed vessels (C02 headspace test)"
- Deviations:
- no
- GLP compliance:
- yes
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, non-adapted
- Details on inoculum:
- - Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure:
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded). In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air for approximately I hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.6 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2.2 litres) of the
supernatant for use in the study. The supernatant was maintained on aeration using CO2-free air until use.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 20 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- TEST SPECIES:
A mixed population of sewage sludge micro-organisms was obtained on 9 November 2006 from the secondary treatment stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
TEST SYSTEM
The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:
a) A control consisting of inoculated culture medium, plus 100 mg silica gel, 11 replicate vessels.
b) The standard material (sodium benzoate) in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of20 mg carbon/l, 11 replicate vessels.
c) The test material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of mg carbon/I, 7 replicate vessels.
d) The test material plus the standard material in inoculated culture medium, plus 100 mg silica gel, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 7 replicate vessels.
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.
Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 mI/l.
Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel (one vessel for analysis on Day 0, one vessel for analysis on Day 14 and five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20± 1°C in darkness with constant shaking at approximately 150 rpm (INFORS TR-225 orbital platform shaker).
Data from the control and standard material vessels was shared with similar concurrent studies.
SAMPLING
The slight variation in the degradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the nature of the study design (individual replicates sacrificed on each sampling occasion), the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected.
Dissolved Organic Carbon (DOC) analyses
Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the replicate standard material vessels attained 99% degradation for Replicates R1 and R2. The degradation rates for the standard material were similar to those determined by IC analyses.
CONTROL AND BLANK SYSTEM
- Toxicity control:
23.4 mg test material/l plus 34.3 mg sodium benzoate/I, equivalent to a total of20 mg carbon/I.
EVALUATION OF DATA
Calculation of carbon content
The carbon, hydrogen and nitrogen content of the test material was determined by elemental analysis performed by Warwick Analytical Services. The following results were obtained:
Carbon: 85.44% w/w
Hydrogen: 15.01% w/w
Nitrogen: 0.09% w/w
Thus for a test concentration of 20 mg C/I (a total of2.50 mg) the total organic carbon present was 2.14mgC.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6H,COONa) was calculated as follows:
[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%
Thus for a 20 mg C/I test concentration (34.3 mg/I) the total organic carbon present in each test vessel was 2.14mgC.
Validation criteria:
Test materials giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.
The test is considered valid if the standard material degradation rate is ≥ 60% by Day 14.
The toxicity control should attain ≥ 25% degradation by Day 14 for the test material to be considered as noninhibitory.
The TIC produced from the control bottles at the end of the test should be ≤15% of the TOC added initially as test material. - Reference substance:
- other: Sodium benzoate
- Preliminary study:
- Preliminary work conducted showed that a volume of 3.2 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE PIN 001 100 5FX) gave a measured weight of 2.51 mg, mean of 15 separate weighings.
- Test performance:
- The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 29
- Sampling time:
- 28 d
- Details on results:
- The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 1% ofthe organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
The test material was a poorly water soluble liquid and hence following the recommendations of the International Standards Organisation (ISO 1996) and in the published lilerature (Handley et ai, 2002), for the purpose of the study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium. Silica gel was used to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.
The test material attained 29% degradation after 28 days.
The toxicity control attained 49% degradation after 14 days and 57% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that
the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring. - Results with reference substance:
- Sodium benzoate attained 69% degradation after 14 days and 70% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Dissolved Organic Carbon (DOC) analyses conducted on samples taken from the standard material vessels on Days 0 and 28 showed that the standard material vessel attained 100% degradation. The degradation rate for the standard material was higher than that determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO, evolution occurring.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test material attained 29% degradation after 28 days; the study demonstrates that 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (4 cSt) is not readily biodegradable.
- Executive summary:
An ISO 14593 study with the substance GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (4 cSt) has been conducted.
The test substance was loaded into the test system by being coated onto granular silica gel. The test substance concentration was 20 mg C/l. The inoculum was non-adapted activated sludge sampled from a municipal waste water treatment plant treating predominantly domestic sewage. The concentration of inoculum in the test system was 100 ml effluent/l. The test was conducted in darkness at 20°C with constant shaking.
A toxicity control was included and the study determined, as far as possible, the absence of inhibition of microorganisms by the test substance.
Assessment of biodegradation was made by CO2 analysis only, based on 2-weekly samplings.
At the end of the 28 d test period, 29% degradation was reported.
It is not possible to interpret the results further than this or discriminate between constituents. The validation criteria were met. The study is considered to be reliable (K2), though the results are not necessarily meaningful for exposure assessment purposes.
Conclusion:
The test material attained 29% degradation after 28 days. The study demonstrates that GTL Base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (4 cSt) is not readily biodegradable.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The definitive study was conducted between 20 January 2004 and 18 February 2004 and the last date that data was acquired for the study was 19 February 2004.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of Inspection: 2nd December 2002 Date of signature: 13 February 2003
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, non-adapted
- Details on inoculum:
- - Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a
temperature of 21°C and was used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a 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 I hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
The suspended solids were equal to 3.3 g/l prior to use.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 10.1 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on 19 January 2004 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage
TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 Iitres of solution:
a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10.1 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20.1 mg carbon/l to act as a toxicity control (one vessel only).
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/1. The study 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 27.3 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 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 de-gassed water.
SAMPLING
-CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0 from the first absorber vessels and the samples taken on Days 1, 2, 3, 6, 8, 10, 14, 16, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Day 0 from the second absorber vessels were stored at approximately -20 °C prior to analysis. The samples taken on Days 12 and 18 were also stored at approximately 20 °C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed degradation did not attain 60% 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 CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and an Ionies 1555B TOC analyser. Samples (300 or 40 µl) 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 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.
Dissolved Organic carbon (DOC) analysis:
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 content in the test media. The samples were filtered through Gelman 0.45 µm 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 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (27 or 13 µl) 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
pH Measurement:
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter and dissolved oxygen meter.
CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (35.4 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No.101K3700) prior to dispersal in approximately 100 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 10 minutes. The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 3 litres to give a final concentration of 11.8 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20.1 mg carbon/l.
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 = mgIC in control x (100/%C of Co2) x (1/test volume).
EVALUATION OF DATA
Calculation of carbon content
The test material contains 85.50% carbon and so for a concentration of 10.1 mg C/I (a total of35.4 mg) the total organic carbon present was 30.3 mg C.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6HSCOONa) was calculated as follows:
[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%
Thus for a 10 mg C/I test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.
Validation criteria:
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 material 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 CO2 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:
- other: Sodium benzoate
- Preliminary study:
- At the request of the Sponsor and in view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organization (ISO 1996) and in the published literature (Handley et ai, 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels.
Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation. - Test performance:
- The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material 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 in the OECD Test Guidelines.
At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material 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 in the OECD Test Guidelines. - Parameter:
- % degradation (CO2 evolution)
- Value:
- 46
- Sampling time:
- 28 d
- Details on results:
- The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material 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 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.
At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.
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. 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 resulted in an increase in the percentage degradation value for the test material from 44% on Day 28 to 46% on Day 29.
The addition of the acid causes all biological activity to stop, and so the test material attained 46% degradation after 28 days as CO2 was present in solution on Day 28 but was not released until after the addition of the acid. The test material, therefore, cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301H.
Variation in degradation rates for the test material on different sampling days was considered to be due to variation in the CO2 production rates between the replicate control and replicate test vessels.
The toxicity control attained 58% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 47% on Day 28 to 58% on Day 29.
Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation values for the standard material from 69% on Day 28 to 71% on Day 29.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 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) gave percentage degradation values of 101% and 91% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analysis were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions of inoculum and silica gel with no undissolved standard material visible. The test material vessels were cloudy light brown dispersions of the test material, inoculum and silica gel. The toxicity control vessel was a cloudy light brown dispersion of test material, inoculum and silica gel with no undissolved standard material. - Results with reference substance:
- Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the standard material from 69% o Day 28 to 71% on Day 29.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test material attained 46% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- Executive summary:
Introduction: A study was performed to assess the ready biodegradability of the test material GTL base oil [‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] (4 cSt) in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "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 835.3110 Paragraph (m).’
Method: The test material, at a concentration of 10.1 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed vessels in the dark at a temperature of 21 °C for 28 days.
Following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium i n order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.
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.
Results & Conclusion: The test material attained 46% degradation after 28 days and therefore cannot 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:
- key study
- Study period:
- The definitive study was conducted between 20 January 2004 and 18 February 2004 and the last date that data was acquired for the study was 19 February 2004.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of Inspection: 2nd December 2002 Date of signature: 13 February 2003
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, non-adapted
- Details on inoculum:
- - Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a
temperature of 21°C and was used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a 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 I hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
The suspended solids were equal to 3.3 g/l prior to use.
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 10.1 mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on 19 January 2004 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage
TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 Iitres of solution:
a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10.1 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20.1 mg carbon/l to act as a toxicity control (one vessel only).
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/1. The study 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 27.3 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 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 de-gassed water.
SAMPLING
-CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0 from the first absorber vessels and the samples taken on Days 1, 2, 3, 6, 8, 10, 14, 16, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Day 0 from the second absorber vessels were stored at approximately -20 °C prior to analysis. The samples taken on Days 12 and 18 were also stored at approximately 20 °C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed degradation did not attain 60% 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 CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and an Ionies 1555B TOC analyser. Samples (300 or 40 µl) 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 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.
Dissolved Organic carbon (DOC) analysis:
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 content in the test media. The samples were filtered through Gelman 0.45 µm 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 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (27 or 13 µl) 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
pH Measurement:
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter and dissolved oxygen meter.
CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (35.4 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No.101K3700) prior to dispersal in approximately 100 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 10 minutes. The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 3 litres to give a final concentration of 11.8 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20.1 mg carbon/l.
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 = mgIC in control x (100/%C of Co2) x (1/test volume).
EVALUATION OF DATA
Calculation of carbon content
The test material contains 85.50% carbon and so for a concentration of 10.1 mg C/I (a total of 35.4 mg) the total organic carbon present was 30.3 mg C.
The theoretical amount of carbon present in the standard material, sodium benzoate (C6HSCOONa) was calculated as follows:
[(No. of C atoms x mol wt of C)/(mol wt of sodium benzoate)] x 100% = [(7 x 12.011)/144.11)] x 100% = 58.34%
Thus for a 10 mg C/I test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.
Validation criteria:
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 CO2 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:
- other: Sodium benzoate
- Preliminary study:
- At the request of the Sponsor and in view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels.
Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation. - Test performance:
- The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC/TC ratio of the test material suspension in the mineral medium at the start of the test was below 5% and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for C02 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. - Parameter:
- % degradation (CO2 evolution)
- Value:
- 11
- Sampling time:
- 28 d
- Details on results:
- The total CO2 evolution in the control vessels on Day 28 was 35.06 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material 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 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.
At the request of the Sponsor and following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms.
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. These increases were considered to be due to C02 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in an increase in the percentage degradation value for the test material from 48% on Day 28 to 55% on Day 29.
The addition of the acid causes all biological activity to stop, and so the test material attained 55% degradation after 28 days as CO2 was present in solution on Day 28.
The increase in inorganic carbon in the test material Replicate R2 first absorber vessel resulted in an increase in the percentage degradation value for the test material from 10% on Day 28 to 11% on Day 29.
Variation in degradation rates for the test material on different sampling days was considered to be due to variation in the CO2 production rates between the replicate control and replicate test vessels.
The toxicity control attained 55% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 47% on Day 28 to 55% on Day 29.
Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the standard material from 69% on Day 28 to 71% on Day 29.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 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) gave percentage degradation values of 101% and 91% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analysis were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions of inoculum and silica gel with no undissolved standard material visible. The test material vessels were cloudy light brown dispersions of the test material, inoculum and silica gel. The toxicity control vessel was a cloudy light brown dispersion of test material, inoculum and silica gel with no undissolved standard material. - Results with reference substance:
- Sodium benzoate attained 71% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the standard material Replicate R1 first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the standard material from 69% on Day 28 to 71% on Day 29.
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test material attained 11% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- Executive summary:
Introduction: A study was performed to assess the ready biodegradability of the test material GTL Base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’] (8 cSt) in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "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 835.3110 Paragraph (m)).
Method: The test material, at a concentration of 10.1 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed vessels in the dark at a temperature of 21 °C for 28 days.
Following the recommendations of the International Standards Organization (ISO 1996) and in the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.
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.
Results & Conclusion: The test material attained 11% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Referenceopen allclose all
Table 1: Percentage biodegradation values
Day |
% Biodegradation |
||
Sodium benzoate Procedure control |
Test item |
Abiotic test item |
|
0 |
0 |
0 |
0 |
2 |
41 |
3 |
0 |
6 |
53 |
4 |
0 |
8 |
54 |
8 |
0 |
10 |
61 |
17 |
0 |
14 |
70 |
40 |
0 |
21 |
79 |
58 |
0 |
28 |
81 |
65 |
0 |
29* |
79 |
70 |
0 |
*Day 29 value was corrected to include any carry over of CO2detected in Absorber 2
From the degradability results based on the BOD (av. 52%) and the residual test substance amount (av. 47%), it is concluded that the test substance is not readily biodegradable under the conditions of this test.
To confirm existence of transformed product, HPLC with reverse-phase column analysis and LCIMS analysis were performed. Neither new peak on chromatogram nor molecular-related ion on Mass spectrum were detected. It is concluded that the test substance was not transformed structurally under the conditions of this test.
Percentage Biodegradation value:
Day |
% Degradation Sodium Benzoate |
% Degradation Test Material |
% Degradation Test Material plus Sodium Benzoate Toxicity Control |
0 |
0 |
0 |
0 |
1 |
22 |
4 |
1 |
2 |
29 |
2 |
0 |
3 |
43 |
1 |
0 |
6 |
50 |
7 |
35 |
8 |
54 |
10 |
35 |
10 |
58 |
13 |
37 |
14 |
62 |
23 |
37 |
16 |
63 |
28 |
50 |
20 |
67 |
36 |
53 |
22 |
66 |
35 |
53 |
24 |
67 |
41 |
56 |
27 |
69 |
45 |
50 |
| 28 | 69 | 44 | 47 |
| 29* | 71 | 46 | 58 |
* Day 29 values corrected to include any carry-over of CO2detected in Absorber 2
Percentage Biodegradation value:
Day |
% Degradation Sodium Benzoate |
% Degradation Test Material |
% Degradation Test Material plus Sodium Benzoate Toxicity Control |
0 |
0 |
0 |
0 |
1 |
22 |
2 |
9 |
2 |
29 |
2 |
14 |
3 |
43 |
0 |
27 |
6 |
50 |
0 |
33 |
8 |
54 |
2 |
34 |
10 |
58 |
0 |
35 |
14 |
62 |
13 |
41 |
16 |
63 |
13 |
41 |
20 |
67 |
9 |
43 |
22 |
66 |
5 |
48 |
24 |
67 |
12 |
48 |
27 |
69 |
16 |
49 |
| 28 | 69 | 10 | 47 |
| 29* | 71 | 11 | 55 |
* Day 29 values corrected to include any carry-over of CO2detected in Absorber 2
Description of key information
- experimental results of biodegradability studies of the closely related test item GTL base oil (CAS 848301-69-9, EC 482-220-0) with different viscosities (3, 4 and 8 cSt at 40°C), and consisting of linear and branched alkanes (paraffins) with a small amount of cycloalkanes in the carbon range from C18 to C50, ranged from 11 to 65 % after 28 days; main factors influencing the results include the guideline/method followed, the dosing method and the variable composition of the complex, hydrophobic test item; it is assumed that the extent of biodegradation observed in these (standard) tests was reduced due to lack of bioavailability and abiotic losses;
- the available measured data for GTL base oil in biodegradation studies, covering the low molecular fraction of the registration substance up to C50 are consistent with the expectation that rate of biodegradation decreases with increasing carbon number range; accordingly, individual components with lower carbon numbers may be susceptible to biodegradation, which is also indicated by the degradation behaviour of the lowest viscosity product GTL Base Oil 3 (covering the low molecular weight fraction of the registration substance up to C30);
- based on the test results for GTL base oil with different viscosities it is concluded that the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' containing higher molecular weight constituents (C25-150, about 30-55 % >C50) is not expected to be readily biodegradable, but is likely to be inherently biodegradable.
Key value for chemical safety assessment
- Biodegradation in water:
- inherently biodegradable, not fulfilling specific criteria
Additional information
No measured biodegradability data are available for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' itself.
However, reliable data of the closely related substance GTL base oil (CAS 848301-69-9, EC 482-220-0) - consisting of linear and branched alkanes (paraffins) with a small amount of cycloalkanes in the carbon range from C18 to C50 - can be used to address the endpoint, which is appropriate to draw conclusions on the degradation behaviour of the registration substance.
A large number of ready biodegradability tests have been carried out for GTL base oil [with different viscosities including the three grades relevant for EU supply, 3, 4 and 8 cSt (at 40°C)]. The results of these biodegradability studies ranged from 11-65% after 28 days.
The main factors influencing the results include the guideline/method used, the dosing method and the variable composition of the complex, hydrophobic test items.
It is expected that the extent of biodegradation observed in the (standard) tests performed was reduced due to lack of bioavailability and abiotic losses. Therefore, where studies show high rates of degradation this can be considered to be indicative of the potential for high degradation in the environment, and such studies should be given higher weight in any overall assessment.
In general terms, degradation can be seen to relate to the carbon range present in test material, which is consistent with water solubility limiting the rate of uptake by microorganisms. In studies of any multi-constituent test substance, there will be uptake of the more bioavailable constituents first. If homologous series are present, it is possible that microorganisms will adapt to the general structural types present, but it is inevitable that rates will overall appear to be slower than for pure substances.
It should be noted that within the carbon number range relevant to the registration substance (C25-150), individual components with lower carbon numbers and the less branched components may be susceptible to biodegradation; this is also shown by the degradation behaviour of closely related substances covering the low molecular weight fraction of the registration substance up to C30.
In fact, the lowest viscosity Gas-to-liquids (GTL) product GTL base oil 3 (covering the carbon range from C18 to C30) meets the criteria for ready biodegradability: the substance achieved 65% biodegradability in 28 days in a biodegradability test conducted according to OECD 301B (Best, 2015).
In addition, the degradation behaviour of the related GTL product 'Hydrocarbons, C18-C24, n-alkanes, isoalkanes, <2% aromatics' (covering the carbon range C18 to C24) is consistent with the conclusion that related substances containing lower carbon number constituents are readily biodegradable (ignoring the inapplicable 10-day window criterion): the substance achieved 74% biodegradability in 28 days in a biodegradability test conducted according to OECD 301F (Best, 2014b).
Finally, the BIOHCWIN estimation program provides a method for predicting primary aerobic biodegradation half-lives in surface water (see supporting study). The results show that individually all constituents less than C20 are expected to be readily biodegradable.
Constituents above C20 may be potentially persistent or very persistent as defined in the REACH technical guidance, Chapter R.11, (ECHA, June 2017), but they will all degrade completely given sufficient time.
Conclusion:
Based on the test results for GTL base oil with different viscosities it is concluded that low molecular constituents of the registration substance [C18-30 linear and branched alkanes (paraffins)] may meet the criteria for ready degradability, however, overall the registration substance containing higher molecular weight constituents (C25-150, about 30-55 % >C50) is not expected to be readily biodegradable, but is likely to be inherently biodegradable.
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