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EC number: 700-351-6 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- Environmental data
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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- Specific investigations
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- Additional toxicological data
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Biodegradation in water: screening tests
Administrative data
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 November 2014 to 24 December 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
- Deviations:
- yes
- Remarks:
- room temperature variations with no impact on results or integrity of study (see below)
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
- Deviations:
- yes
- Remarks:
- room temperature variations with no impact on results or integrity of study (see below)
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).
- Deviations:
- yes
- Remarks:
- room temperature variations with no impact on results or integrity of study (see below)
- GLP compliance:
- yes (incl. QA statement)
Test material
- Test material form:
- solid
- Details on test material:
- - Appearance/physical state: Black solid
- Storage conditions: Room temperature, protected from light under nitrogen
Constituent 1
Study design
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic (adaptation not specified)
- Details on inoculum:
- TEST SYSTEM
- A mixed population of activated sewage sludge micro-organisms was obtained on 24 November 2014 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 twice by settlement and re-suspension in mineral me
dium 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 sludge was carried out by filtering a sample
(100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using
a Buchner funnel. The filter paper had been rinsed three times with 20 mL deionised reverse osmosis wa
ter prior to drying in an oven.
- Filtration was continued for a further 3 minutes after rinsing of the filter three successive times with 10
mL of deionised reverse osmosis water.
- The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to c
ool before weighing.
- The process was repeated until a constant weight was attained.
- The suspended solids concentration was equal to 3.2 g/L prior to use. - Duration of test (contact time):
- 28 d
Initial test substance concentration
- Initial conc.:
- 10 mg/L
- Based on:
- other: carbon content of test item
Parameter followed for biodegradation estimation
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- REFERENCE SUBSTANCE
- Identification: Sodium benzoate
- Description: white granular solid
- Batch: SLBG9515V
- Purity: > 99.5 %
- Expiry Date: 09 July 2015
- Storage conditions: room temperature over silica gel
MEDIUM
- The mineral medium used in this study was that recommended in the OECD guidelines (see Appendix 2,
attached).
PRELIMINARY SOLUBILITY WORK
- Information provided by the Sponsor indicated that test item was insoluble in water.
- Preliminary solubility/dispersibility work was therefore performed in order to determine the most suitable
method of preparation (see Appendix 3, attached).
TEST ITEM PREPARATION
- Following the recommendations of the International Standards Organisation (ISO, 1995) and published literature (Handley et al, 2002), the test item was dissolved in an auxiliary solvent prior to adsorption onto Whatmann GF/A (70 mm diameter) filter paper.
- High shear mixing was also applied to break up the filter paper containing the test item.
- Using this method the test item was evenly distributed throughout the test medium and the surface area
exposed to the test organisms was increased thereby increasing the potential for biodegradation.
- A nominal amount of test item (1000 mg) was dissolved in 10 mL of dimethylformamide to give a 1000 mg/10 mL
solvent stock solution. At the request of the sponsor, oxygen had been removed from the dimethylformamide by purging with nitrogen for approximately 30 seconds.
- An aliquot (384 μL) of the solvent stock solution was dispensed onto a filter paper and the solvent was
allowed to evaporate to dryness for approximately one hour.
- The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear m
ixing (approximately 7500 rpm for 5 minutes) prior to addition of inoculated mineral medium.
- The volume was then adjusted to 3 L to give a final concentration of 12.8 mg/L (equivalent to 10 mg car
bon/L as recommended in the test guideline).
- Since it is not a requirement of the test guideline, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This was an exception with regard to GLP and was reflected in the GLP compliance statement.
REFERENCE ITEM
- A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels.
- An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes.
- An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 L to give a final test concentration of 17 .1 mg/L (equivalent to 10 mg carbon/L).
- The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
- A Whatman GF/A (70 mm diameter) filter paper was added to each vessel in order to maintain consistency between the test and procedure control vessels.
- Dimethylformamide (384 μL) which had been purged with nitrogen for approximately 30 seconds was dispensed onto each filter paper and evaporated to dryness for approximately one hour.
- The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.
TOXICITY CONTROL
- A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
- An aliquot (384 μL) of the test item solvent stock solution was dispensed onto a Whatman GF/A (70 mm diameter) and the solvent allowed to evaporate for approximately one hour.
- The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel containing inoculated mineral medium.
- An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 L to give a final concentration of 12.8 mg test item/L plus 17 .1 mg sodium benzoate/L (equivalent to a total of 20 mg carbon/L).
PREPARATION OF THE TEST SYSTEM
- Test preparations were prepared and inoculated in 5 L test culture vessels each containing 3 L of solution.
(a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a Whatman GF/A (70 mm diameter) filter paper.
(b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a Whatman GF/A (70 mm diameter) filter paper to give a final concentration of 10 mg carbon/L.
(c) The test item on a Whatman GF/A (70 mm diameter) filter paper, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(d) The test item on a Whatman GF/A (70 mm diameter) filter paper 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).
- A filter paper with dimethylformamide evaporated to dryness 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 temperatures of between 18 to 24 °C, in darkness. This slight deviation to the study plan was considered to have had no adverse effect on the study given that all validation criteria were met.
- Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 28.1 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 HQ 160 Flexi handheld meter. If necessary the pH 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 L 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 C02-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb) granules.
- The C02 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The C02 absorbing solutions were prepared using purified water.
OBSERVATIONS
- The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.
MEASUREMENT of pH
- The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ160 Flexi handheld meter.
IC ANALYSIS
- 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 analysed for IC immediately.
- On Day 28, concentrated hydrochloric acid (1 mL ) 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 IC using a Tekmar-Dohrmann Apollo 9000 TOC analyser or a Shimadzu TOC-VCSH TOC analyser.
- Samples (300 or 50 μL) were injected into the IC channel of the TOC analyser.
- IC analysis occurs by means of the conversion of an aqueous sample to C02 by orthophosphoric acid using zero grade air as the carrier gas.
- Calibration was by reference solutions of sodium carbonate (Na2C03).
- Each analysis was carried out in triplicate.
IC/TC RATIO
- 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 analysed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 μL) were injected into the TC and IC channels of the TOC analyser. 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 (C8H5K04) and sodium carbonate (Na2C03) in deionized water. Each analysis was carried out in triplicate.
DATA EVALUATION
- The test item contains 78.30% carbon (data supplied by the Sponsor) and so for a concentration
of 10 mg C/L the total organic carbon present was 30 mg C.
- The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) = (No of C atoms * mol wt of C / mol wt of sodium benzoate) * 100.
- The calculation used was therefore (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.
PERCENTAGE BIODEGRADATION
- The percentage biodegradation or percentage of Theoretical Amount of Carbo Dioxide (ThCO2) produced was calculated by substituting the inorganic carbon values (see Table 1, attached) into the equation % ThCO2 = (mg IC in test flask - mg IC in control flask) / (mg TOC added as test chemical) x 100
- The mean values of replicates R1 and R2 were obtained for the inoculum, control, test and reference items before substitution into the equation.
- The % ThCO2 result equals % biodegradation where the conversion factor for carbon to carbon dioxide is 3.67.
TOTAL CO2 EVOLUTION
- The total CO2 evolution in the inoculum control vessels at the end of the test was calculated using the equation Total CO2 evolution (mg C/L) = (mg IC in control) x (100 / % C of CO2) x (1 / test volume) = (mg IC in control) x (100 / 27.29) x (1 / 3)
- The mean inorganic carbon values for replicates R1 and R2 on Day 28 were obtained before substitution into the equation.
VALIDATION CRITERIA
- The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60 % degradation (in a 10-day window) 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 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, is less than 20 %.
- The total CO2 evolution in the inoculum control vessels on Day 28 of the test should not normally exceed 40 mg/L medium (= 120 mg/3 L 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
- Reference substance:
- benzoic acid, sodium salt
Results and discussion
% Degradation
- Key result
- Parameter:
- % degradation (CO2 evolution)
- Value:
- 1
- Sampling time:
- 28 d
- Details on results:
- DEFINITIVE TEST
- Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1 (attached).
- Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 (attached)
- The biodegradation curves are presented in Figure 1 (attached).
- Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3 (attached).
- The pH values of the test preparations on Days 0 and 28 are given in Table 4 (attached).
- Observations made on the contents of the test vessels are given in Table 5 (attached).
VALIDATION CRITERIA
- The total C02 evolution in the inoculum control vessels on Day 28 was 30.51 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3, attached) 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 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.
BIODEGRADATION
- 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 procedure control replicates 1 and 2 and the toxicity control.
- 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 1 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301 B.
- The toxicity control attained 33% biodegradation after 14 days and 35% 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.
BOD5 / COD results
- Results with reference substance:
- - Sodium benzoate attained 78 % biodegradation after 14 days and 79 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Applicant's summary and conclusion
- Validity criteria fulfilled:
- yes
- Interpretation of results:
- not readily biodegradable
- Conclusions:
- The test item attained 1 % biodegradation 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:
GUIDELINE
A 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; C02 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)).
METHODS
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 18 to 24 °C for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.
RESULTS
The test item attained 1 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
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