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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2012 to 29 August 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum: Aeration stage of sewage treatment plant which treats predominantly domestic sewage.
- Storage conditions: Continuously aerated at 21°C
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: Washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may be present. Test vessels containing 2400 mL mineral medium and 22.5 mL inoculum were aerated with CO2 free air overnight prior to the addition of the test and reference items. The volume in all vessels was finally adjusted to 3 litres.
- Concentration of sludge: 2.9g suspended solids/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: Total carbon content
Initial conc.:
13.4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: OECD guideline mineral medium containing KH2PO4 (0.0850g/L), K2HPO4 (0.2175 g/L), Na2HPO4.2H2O ().3340 g/L), NH4Cl (0.005 g/L), CaCl2 (0.0255 g/L), MgSO4.7H2O (0.0225 g/L) and FeCl3 (0.00025 g/L).
- Test temperature: 22 ± 2°C
- pH: 7.4 to 7.6
_ pH adjustment: If necessary, the pH was adjusted to 7.4 ± 0.2 using dilute hydrochloric acid or sodium hydroxide solution prior to aeration of the mineral medium
- Aeration of dilution water: Test vessels were sealed and CO2-free air was bubbled through the solutions at rates of 30 to 100 mL/minute/vessel and stirred continuously with a magnetic stirrer. CO2-free air was produced by passing compressed air through a glass column containing self-indicating lime soda.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5L test culture vessel
- Number of culture flasks/concentration: Two for test item, inoculate control and reference item. One for toxicity control (test item plus reference item).
- Method used to create aerobic conditions: Continuous aeration with C02-free air.
- Measuring equipment: Inorganic carbon (IC) and total carbon (TC) channels of TOC analyser.
- Details of trap for CO2: Two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH solutions with purified degassed water.

SAMPLING
- Sampling: 2 mL samples were taken from the first CO2 absorber vessels on days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and analysed for CO2 immediately using IC channel of TOC analyser. Second absorber vessels were sampled on days 0 and 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated mineral medium containing glass filter paper pre-washed with chloroform, which was allowed to evaporate before use.
- Procedure control: Reference item (sodium benzoate) in inoculate medium plus glass filter paper pre-washed with chloroform, which was allowed to evaporate, to give a final concentration of 10 mg carbon/L.
- Toxicity control: Test item plus reference item in inoculated mineral medium plus glass filter paper pre-washed with chloroform, which was allowed to evaporate, to give a final concentration of 20 mg carbon/L.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable
Test performance:
The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed a decrease in all replicate vessels with the exception of procedure control replicate R2, which was considered to be due to sampling/analytical variation. Inorganic carbon analysis of samples from the second absorber vessels on day 29 showed no significant carry-over of CO2.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
31
Sampling time:
28 d
Details on results:
The test item did not satisfy the 10-day window validation criterion, whereby 60% degradation must be obtained within 10 days of the degradation exceeding 10%. The test item therefore, cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 44% degradation after 14 days and 56% degradation after 28 days thereby confirming the test item was not toxic to the sewage treatment micro-organisms used in the test.
Results with reference substance:
The reference substance (sodium benzoate) attained 96% degradation after 14 days and 91% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling /analytical variation. The slight decrease in degradation between days 14 and 28 was also considered to be due to sampling/analytical variation (see table below).

Table 1. Percentage Biodegradation Values

Day

 

Percentage Biodegradation

Test item

Sodium Benzoate Procedure Control

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

19

61

17

6

27

83

27

8

23

80

30

10

30

108

43

14

24

96

44

21

22

101

55

28

28

89

59

29*

31

91

56

*  Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validity criteria fulfilled:
yes
Remarks:
The total CO2 evolution in the inoculum control was < 40 mg/L (actual 24.28 mg/L). The IC content of the test item in the mineral medium at test start was < 5% (actual 0%). The difference in CO2 production at test end between replicates was < 20%.
Interpretation of results:
other: Partly biodegradable but does not satisfy the readily biodegradable criteria.
Conclusions:
The substance as a 50% w.w. concentration in pharmaceutical white oil reached 31% degradation after 28 days and therefore cannot be considered to be readily biodegradable.
Executive summary:

The substance as a 50% w.w. concentration in pharmaceutical white oil reached 31% degradation after 28 days and therefore cannot be considered to be readily biodegradable. The biodegradability of the test item was determined in a GLP-compliant CO2 evolution test following OECD guideline 301B (Harlan 2012). The study is considered reliable and relevant for use as a supporting study for this endpoint. The substance is not considered to be inhibitory to sewage treatment micro-organisms.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
7 August 2012 to 5 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None, see test material information
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum: Aeration stage of sewage treatment plant which treats predominantly domestic sewage.
- Storage conditions: Continuously aerated at 21°C
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: Washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may be present. Test vessels containing 2400 mL mineral medium and 22.5 mL inoculum were aerated with CO2 free air overnight prior to the addition of the test and reference items. The volume in all vessels was finally adjusted to 3 litres.
- Concentration of sludge: 4.0 g suspended solids/L prior to use..
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: Total carbon content
Initial conc.:
15.4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:OECD guideline mineral medium containing KH2PO4 (0.0850g/L), K2HPO4 (0.2175 g/L), Na2HPO4.2H2O (0.3340 g/L), NH4Cl (0.005 g/L), CaCl2 (0.0255 g/L), MgSO4.7H2O (0.0225 g/L) and FeCl3 (0.00025 g/L).
- Test temperature: 21°C
- pH: 7.6 to 7.7
- pH adjustment: If necessary, the pH was adjusted to 7.4 ± 0.2 using dilute hydrochloric acid or sodium hydroxide solution prior to aeration of the mineral medium
- Aeration of dilution water: Test vessels were sealed and CO2-free air was bubbled through the solutions at rates of 30 to 100 mL/minute/vessel and stirred continuously with a magnetic stirrer. CO2-free air was produced by passing compressed air through a glass column containing self-indicating lime soda.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5L test culture vessel
- Number of culture flasks/concentration: Two for test item, inoculate control and reference item. One for toxicity control (test item plus reference item).
- Method used to create aerobic conditions: Continuous aeration with CO2-free air.
- Measuring equipment: inorganic carbon (IC) and total carbon (TC) channels of TOC analyser.
- Details of trap for CO2: Two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH solutions with purified degassed water.

SAMPLING
- Sampling: 2 mL samples were taken from the first CO2 absorber vessels on days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and analysed for CO2 immediately using IC channel of TOC analyser. Second absorber vessels were sampled on days 0 and 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated mineral medium.
- Procedure control: Reference item (sodium benzoate) in inoculate medium to give a final concentration of 10 mg carbon/L.
- Toxicity control: Test item plus reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable
Test performance:
The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed a decrease in all replicate vessels, which was considered to be due to sampling/analytical variation. Inorganic carbon analysis of samples from the second absorber vessels on day 29 showed no significant carry-over of CO2.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
79
Sampling time:
28 d
Details on results:
The test item satisfied the 10-day window validation criterion, whereby 60% degradation must be obtained within 10 days of the degradation exceeding 10%. The test item is therefore considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 64% degradation after 14 days and 87% degradation after 28 days thereby confirming the test item was not toxic to the sewage treatment micro-organisms used in the test.
Results with reference substance:
The reference substance (sodium benzoate) attained 82% degradation after 14 days and 92% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1. Percentage Biodegradation Values

Day

 

Percentage Biodegradation

Test item

Sodium Benzoate Procedure Control

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

21

56

36

6

28

68

46

8

40

70

53

10

52

80

51

14

64

82

64

21

61

83

69

28

75

90

79

29*

79

92

87

*  Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validity criteria fulfilled:
yes
Remarks:
The total CO2 evolution in the inoculum control was < 40 mg/L (actual 27.36 mg/L). The IC content of the test item in the mineral medium at test start was < 5% (actual 2%). The difference in CO2 production at test end between replicates was < 20%.
Interpretation of results:
readily biodegradable
Conclusions:
Aluminum, benzoate C16-18-fatty acids complexes reached 79% degradation after 28 days, meeting the 10 day window, and therefore is considered to be readily biodegradable.
Executive summary:

Aluminum, benzoate C16-18-fatty acids complexes reached 79% degradation after 28 days, meeting the 10 day window, and therefore is considered to be readily biodegradable. The ready biodegradability of the test item was determined in a GLP-compliant CO2 evolution test following OECD guideline 301B (Harlan 2012). The study is considered reliable and relevant for use for this endpoint. Aluminum, benzoate, C16 -18 fatty acids complexes is not considered to be inhibitory to sewage treatment micro-organisms.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Read across data
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.

The substances consist of aluminium salts of fatty acids, with the source substance having additionally a benzoate moiety linked to the aluminium cation as well. As such, both substances have common functional groups and as the fatty acid moieties are considered not to be hazardous to humans since they are an endogenous part of every living cell and are an essential dietary requirement, the toxicity of the substances will be driven by the presence of the aluminium species (and additionally the benzoate for the source substance). The substances have common starting materials and methods of manufacture, principally the reaction of aluminium with organic acids, with the target substance often being used as a pre-cursor reacted with benzoic acid in the manufacture of the source substance. The substances have common breakdown products and, in the environment, the substances will dissociate and biodegrade into inorganic aluminium species and fatty acids then carbon dioxide and water.

2. CHEMICAL(S)

Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)

See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.

3. ANALOGUE APPROACH JUSTIFICATION

The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.

The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the two substances are therefore expected to be exempt from registration. Fatty acids are an endogenous part of every living cell and are an essential dietary requirement. They are absorbed, digested and transported in animals and humans. When taken up by tissues, they can either be stored as triglycerides or can be oxidised via the ß-oxidation and tricarboxylic acid pathways. The ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.

The read across substance also contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.

Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data. On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicate that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment. Although the read-across substance met the criteria for ready biodegradability and the target substance did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.

There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases. The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener. These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

For the biodegradation of the substances, read across from the source to the target substance is considered justified as both substances have very low solubility in water, would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances, in the environment, would dissociate and degrade into inorganic aluminium species and firstly, fatty acids (plus benzoic acid for the source substance), then ultimately carbon dioxide and water. Neither substance is inhibitory to micro-organism.

4. DATA

T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance

- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T). Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
- In vitro gene mutation in mammalian cells: No data available for T, Negative (RA)
- Short-term repeated dose toxicity, oral route: No data available for T, NOAEL: > 225 mg/kg (RA)
- Reproductive toxicity: No data available for T, NOAEL (P): > 225 mg/kg (RA)
- Developmental toxicity: No data available for T, NOAEL (F1): > 225 mg/kg (RA)
Reason / purpose:
read-across source
Preliminary study:
Not applicable
Test performance:
The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed a decrease in all replicate vessels, which was considered to be due to sampling/analytical variation. Inorganic carbon analysis of samples from the second absorber vessels on day 29 showed no significant carry-over of CO2.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
79
Sampling time:
28 d
Details on results:
The test item satisfied the 10-day window validation criterion, whereby 60% degradation must be obtained within 10 days of the degradation exceeding 10%. The test item is therefore considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 64% degradation after 14 days and 87% degradation after 28 days thereby confirming the test item was not toxic to the sewage treatment micro-organisms used in the test.
Results with reference substance:
The reference substance (sodium benzoate) attained 82% degradation after 14 days and 92% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1. Percentage Biodegradation Values

Day

 

Percentage Biodegradation

Test item

Sodium Benzoate Procedure Control

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

21

56

36

6

28

68

46

8

40

70

53

10

52

80

51

14

64

82

64

21

61

83

69

28

75

90

79

29*

79

92

87

*  Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validity criteria fulfilled:
yes
Remarks:
The total CO2 evolution in the inoculum control was < 40 mg/L (actual 27.36 mg/L). The IC content of the test item in the mineral medium at test start was < 5% (actual 2%). The difference in CO2 production at test end between replicates was < 20%.
Interpretation of results:
readily biodegradable
Conclusions:
Aluminum, benzoate C16-18-fatty acids complexes reached 79% degradation after 28 days, meeting the 10 day window, and therefore is considered to be readily biodegradable.
Executive summary:

Biodegradation results have been read across from a structural analogue, aluminium salts of benzoate, C16-18 fatty acids complexes, to provide data on the substance in an isolated form. Based on the read across, the substance in its isolated form is expected to be readily biodegradable. Indeed, although the aluminium ion is inorganic, and therefore biodegradation is not applicable, read across from the acids indicates that the fatty acid components biodegrade rapidly.

Proprietary data are read across from aluminum, benzoate C16-18-fatty acids complexes (CAS No. 94166-87-7, EC No. 303-385). This substance is considered suitable for read-across as it similarly contains a fatty acid moiety coordinated to an aluminium atom, although it also contains a coordinated benzoate ion. Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and was shown to be readily biodegradable and non-inhibitory to microorganisms (Harlan 2013).

Aluminum, benzoate C16-18-fatty acids complexes reached 79% degradation after 28 days, meeting the 10 day window, and therefore is considered to be readily biodegradable. The ready biodegradability of the test item was determined in a GLP-compliant CO2 evolution test following OECD guideline 301B (Harlan 2012). The study is considered reliable and relevant for use for this endpoint. Aluminum, benzoate, C16 -18 fatty acids complexes is not considered to be inhibitory to sewage treatment micro-organisms.

Description of key information

As contradictory data are available, the data on the substance, in the form in which it is marketed and used, have been used for concluding on this endpoint. Therefore, the information to be taken into account for any hazard/risk/persistence assessment is that the substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Biodegradation results have been read across from a structural analogue, aluminium salts of benzoate, C16-18 fatty acids complexes, to provide data on the substance in an isolated form. Based on the read across, the substance in its isolated form is expected to be readily biodegradable. Indeed, although the aluminium ion is inorganic, and therefore biodegradation is not applicable, read across from the acids indicates that the fatty acid components biodegrade rapidly.

Proprietary data are read across from aluminum, benzoate C16-18-fatty acids complexes (CAS No. 94166-87-7, EC No. 303-385 -6). This substance is considered suitable for read-across as it similarly contains a fatty acid moiety coordinated to an aluminium atom, although it also contains a coordinated benzoate ion. Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and was shown to be readily biodegradable and non-inhibitory to microorganisms (Harlan 2013).

However, as the substance is only manufactured in situ in a generic inert carrier, typically base oil, the ready biodegradability test was also conducted on the substance in situ in medicinal white oil as a carrier. Based on the study conducted on the substance in situ in medicinal white oil, the substance in the form in which it is marketed and used, is not readily biodegradable.

Proprietary data are available (Harlan 2013) showing the substance at 50% w.w. pharmaceutical white oil reached 31% degradation after 28 days and therefore cannot be considered to be readily biodegradable. However, the study should be considered carefully due to the presence of white oil, which constituted 50% by weight of the substance tested. Pharmaceutical white oil is known not to be readily biodegradable (31% biodegradation after 28 days) (ECHA Dissemination Portal CAS 8042-47-5). It can therefore be concluded that the test results are significantly determined by the properties of the pharmaceutical white oil. However, it did demonstrate that the substance is not inhibitory to micro-organisms at the concentration tested.

Additional testing on the biodegradation of the substance in water and sediment are considered not scientifically necessary based on the substance having very low solubility in water. The water solubility of the substance as a 50% w.w. concentration in pharmaceutical white oil has been determined to be less than 0.00015 g/L of solution at 20.0 ± 0.5°C, based on test substance. The substance is not expected to leach out or partition from the grease base into water due to its poor water solubility and strong physical interactions with the grease matrix.