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Environmental fate & pathways

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:
15 November 2017 to 12 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
Appearance: Clear light amber liquid
Purity/Composition: >90%
Test item storage: At room temperature
Additional information
Test Facility test item number: 209077/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Glyceryl Triacetyl Ricinoleate
CAS number: 101-34-8
Molecular structure: Not indicated
Molecular formula: C63H110O12
Molecular weight: 1059
Highly reactive to water: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
Stability in water: Stable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Source
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 4 g/L in the concentrated sludge. The magnetically stirred sludge was used as inoculum at the amount of 15 mL/L of mineral medium, leading to a concentration SS of 30 mg/L.

Reason for selection
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.
Duration of test (contact time):
ca. 28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 17 mg/L
Based on:
test mat.
Initial conc.:
ca. 12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test concentration and preparation of test solutions:
The test item was a clear light amber liquid with a purity of >90%. The test item was tested in duplicate at a target concentration of 17 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.
Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 33.59 mg; test item bottle B: 33.45 mg and toxicity control bottle: 34.03 mg). To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test suspensions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Any residual volumes were discarded.

Test System :
Source:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment:
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 4 g/L in the concentrated sludge. The magnetically stirred sludge was used as inoculum at the amount of 15 mL/L of mineral medium, leading to a concentration SS of 30 mg/L.

Reason for selection:
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

- Test Procedure and Conditions:
Test duration:
28 days for the inoculum blank and test item (last CO2 measurement on day 29).
14 days for the positive and toxicity control (last CO2 measurement on day 15).
During the test period, the test media were aerated and stirred continuously.

Test vessels:
2 litre brown coloured glass bottles.

Milli- RO water:
Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.

Stock solutions of mineral components:
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl, dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
Mineral medium:
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.

Barium hydroxide:
0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm):
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination:
The test media were excluded from light.

- Preparation of Bottles:
Pre-incubation medium:
The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle.
This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles:
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).

Preparation:
At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
All criteria for acceptability of the test were met, this study was considered to be valid.
% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 7
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 5
Sampling time:
28 d
Details on results:
Theoretical CO2 Production:
The ThCO2 of Dermol GTR was calculated to be 2.62 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation:
Figure 1 (attached) shows the curves for biodegradation of the two bottles with Dermol GTR, the positive control and the toxicity control.
The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of the test item (7% and 5%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (44%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve
.
Monitoring of Temperature and pH:
The temperature recorded in a vessel with water in the same room varied between 22 and 23°C. The pH values of the different test media are presented in Table 1.

Table 1 pH Values of Different Test Media:
Test medium: At the start of the test: On day 14: On day 28:
Blank control (A) 7.5 - 7.5
Blank control (B) 7.5 - 7.5
Positive control 7.6 7.7 -
Dermol GTR (A) 7.6 - 7.5
Dermol GTR (B) 7.6 - 7.5
Toxicity control 7.6 7.6 -

BOD5 / COD results

Results with reference substance:
The reference item (sodium acetate) showed a normal biodegradation curve.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In conclusion, EC 202-935-1 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objective of the study was to evaluate the test item EC 202-935-1 for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge; Carbon dioxide (CO2) evolution test (modified Sturm test).

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995.

EC 202-935-1 was a clear light amber liquid with a purity of >90%. The test item was tested in duplicate at a target concentration of 17 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of EC 202-935-1 was calculated to be 2.62 mg CO2/mg.

The study consisted of six bottles: 

          2 inoculum blanks (no test item), 

          2 test bottles (test item), 

          1 positive control (sodium acetate) and 

          1 toxicity control (test item plus sodium acetate).

Since EC 202-935-1 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test suspensions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of EC 202-935-1 (7% and 5%, based on ThCO2).

In the toxicity control, EC 202-935-1 was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.