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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2013 to April 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
The protocol stated that the vessels used in the study would be shaken at 22 +/- 2°C. Minimum temperatures went slightly outside this range on days 1,2, and 3 of the first test. This deviation is not considered to have affected the integrity of the test.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
No IUPAC name is currently defined for Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (“Distilled Residue Grade”).
EC Number:
941-212-1
Molecular formula:
Cardanol (saturated side chain): Formula: C21 H36 O Cardanol (monoene): Formula: C21 H34 O Cardanol (diene): Formula: C21 H32 O Cardanol (triene): Formula: C21 H30 O
IUPAC Name:
No IUPAC name is currently defined for Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (“Distilled Residue Grade”).
Details on test material:
- Name of test material (as cited in study report): Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (Distilled Residue Grade)

- Physical state: Dark brown liquid

- Expiration date of the lot/batch: December 2014

- Stability under test conditions: Stable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Three or five days before the start of the tests, samples of activated sludge were obtained from the aeration tank of Worlingworth sewage treatment works (Suffolk, UK), which treats predominantly domestic waste. On the days of collection, the samples were passed through a sieve with a mesh of ca. 1 mm2. A sub-sample (ca. 1 L) was then removed and centrifuged at ca. 3000 rpm for ca. 1 minute, before the supernatant was removed. The sample was then made up to volume with mineral salts medium (MSM, as detailed in Appendix 2) and centrifuged again. This procedure was repeated twice and the sample was then aerated until required. Aliquots (10 mL) of a homogenised sample of the washed activated sludge were filtered through dried (approximately 105C) and pre-weighed Whatman GF/C filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume was used to inoculate control and test media to give a final suspended solids concentration (MLSS) of 4 mg/L for the test substance without enhancement measures and 0.5 mg/L with enhancement measures. The sample of activated sludge was not adapted to the test substance before the start of the tests.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
This study was conducted in two phases. The first phase measured the biodegradability of the test substance after it was applied on to the walls of the test vessel using a solvent (acetone) that was subsequently removed by evaporation. Although the test substance was not found to be readily biodegradable, there was evidence of biodegradation. Therefore, further formulation trials were undertaken to identify a suitable method to enhance the potential for biodegradation of CNSL Distillation residue. The method selected resulted in the substance being applied to glass fibre (GF/C) paper and the surfactant (Tween 85) was added to the vessels followed by the dilution medium.
For each phase, appropriate volumes of inoculated mineral salts medium (MSM) were prepared in either five or ten-litre clear glass vessels and aerated for three or five days with CO2-free air. On the day of test initiations, control cultures containing inoculated MSM, reference cultures containing inoculated MSM and the reference substance (sodium benzoate) and the test cultures containing inoculated MSM and CNSL Distillation residue were established. The concentration of reference substance and test substance employed was nominal 10 mgC/L for the phase without enhancement and 5 mgC/L for the test in which enhancement measures were employed to help increase the sensitivity of the assay.
Aliquots (100 mL) of each type of culture medium were transferred to groups of Wheaton vials (160 mL), which were sealed with Teflon-coated septa and aluminium crimp seals. The final suspended solids concentration (MLSS; mixed liquor suspended solids) was also reduced for the test with enhancement measures from 5 to 0.5 mg/L.
Test, control and reference mixtures were incubated for 28 days on a reciprocating shaker, at
a temperature of 19.7 to 22.7C for the initial test where CNSL Distillation residue was tested without enhancement measures, and 21.2C to 23.3C for the final test where enhancement measures where employed. On each occasion of analysis, the appropriate number of vials were injected with 1 mL of NaOH (nominally 7M) and shaken for at least one hour. The total inorganic carbon content of each sample was then determined using a carbon analyser.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
26.4
Sampling time:
5 d
Remarks on result:
other: With enhancement measures.
Parameter:
% degradation (CO2 evolution)
Value:
27.6
Sampling time:
7 d
Remarks on result:
other: With enhancement measures.
Parameter:
% degradation (CO2 evolution)
Value:
40.8
Sampling time:
14 d
Remarks on result:
other: With enhancement measures.
Parameter:
% degradation (CO2 evolution)
Value:
68.3
Sampling time:
28 d
Remarks on result:
other: With enhancement measures.
Details on results:
Test substance (no enhancement measures)
Sodium benzoate alone had been biodegraded by 74.6% by Day 7, and by 78.4% at Day 21 and 77.5% at the end of the test (Day 28). The production of CO2 in the control cultures, expressed as a percentage of the nominal organic carbon load as test substance in the test system (mean 12.6% on Day 28) was acceptable for this assay system (recommended maximum, 15%). These results confirm that the inoculum was viable and that the test was valid.
Biodegradation of CNSL Distillation residue was calculated as 26.5% on Day 7, and increased to 35.3% by Day 18, with a maximum of 42.0% on Day 28. Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. CNSL Distillation residue therefore cannot be considered to be readily biodegradable under the conditions of this test, when added directly.
The pH values of the test/control and reference MSM at the start of the test were 7.68 and 7.82, respectively. The temperature of a 100 mL volume of water held under test conditions ranged from 19.7C to 22.7C during the test period (See protocol deviations).

Test substance (with enhancement measures)
The biodegradation of sodium benzoate alone was 62.1% of the theoretical maximum after 7 days and 103% by the end of the test on Day 28. Although slightly in excess of that theoretically available (100%), this was not considered significant as this is within the level of experimental error for this type of study. The mean production of CO2 in the control cultures, expressed as a percentage of the nominal organic carbon load as test substance in the test system was 9.1% on Day 28. These results confirm that the inoculum was viable and that the test was valid.
Biodegradation of CNSL Distillation residue, using GF/C paper and Tween 85 surfactant, was calculated as 26.4% on Day 5; this had increased to 40.8% on Day 14, with a maximum of 68.3% by Day 28. Since CO2 production was less than 60% of the theoretical value within ten days of the level achieving 10%, CNSL Distillation residue cannot be considered to be readily biodegradable under the conditions of this phase of the study where surface area enhancement measures were employed.
The pH values of the test/control and reference MSM at the start of the test were 7.75 and 7.78, respectively. The temperature of a 100 mL volume of water held under test conditions ranged from 21.2C to 23.3C during the test period.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: Potentially ultimately biodegradable
Conclusions:
Although not ready biodegradable, the profile of the biodegradation for the test substance when enhanced with Tween surfactant and a GF/C paper support was similar to that for the test substance without enhancement measures. However, the extent of the degradation was increased by about 26% (to 68.3%), and exceeded the 60% threshold after 28 days.
Using the surface area enhancement measures as described, CNSL Distillation residue is considered to be potentially ultimately biodegradable. However, it should be noted that as the test substance is a mixture, different rates of degradation may occur under realistic conditions.
Executive summary:

The mean production of CO2 in the control cultures (for the phase with no enhancement measures), expressed as a percentage of the nominal organic carbon load as test substance in the test system was 12.6% on Day 28. With enhancement measures this was determined to be 9.1%. These results confirm that the inocula were viable and that all the tests were valid. Substances are considered to be readily biodegradable in this type of test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. CNSL Distillation residue cannot be considered to be readily biodegradable, under the conditions of this test, with or without enhancement measures.