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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 November 2017 to 18 January 2018
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)
Version / remarks:
17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: Viscous amber liquid (determined by Charles River Den Bosch)
Purity/Composition: >85%
Test item storage: At room temperature
Additional information
Test Facility test item number: 209080/A
Purity/Composition correction factor: No correction factor required
Organic carbon (w%): 71.4%
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Glyceryl Triacetyl Hydroxystearate
CAS number: 139-43-5
Molecular structure: Not indicated
Molecular formula: C63H116O12
Molecular weight: 1065.6
Highly reactive to water: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
Stability in water: Stable


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 conc.:
ca. 17 mg/L
Based on:
test mat.
Initial conc.:
ca. 12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Reference item
Identification number RS186
Container E1
Identification Sodium acetate
CAS Number 127-09-3
Molecular formula CH3COONa
Molecular weight 82.03
Appearance White powder (determined at Charles River Den Bosch)
Batch AM1027668
Purity 99.5%
Storage conditions At room temperature
Stable under storage conditions until 30 June 2018
Supplier Merck KGaA, Darmstadt, Germany
Article number 1.06268
Certified Yes

Reference Item Concentration and Preparation of Test Solutions

A solution of sodium acetate was prepared by dissolving 799.41 mg in Milli- RO water and making this up to a total volume of 200 mL. Volumes of 20 mL from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/L).

Test Concentration and Preparation of Test Solutions

Hetester HCA was a viscous amber liquid with a purity of >85%. 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 Hetester HCA 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: 33.75 mg; test item bottle B: 34.31 mg and toxicity control bottle: 34.04 mg). To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Any residual volumes were discarded.



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.

Acceptability of the Test

1. The reference item was biodegraded by at least 60% (80%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 14%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (79.7 mg CO2 per 2 litres of medium, corresponding to 39.9 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Analysis
ThCO2, expressed as mg CO2/mg test item, was calculated as follows:

ThCO2 = (No.of carbon atoms in test item × Molecular weight CO2) / (Molecular weight test item)

The first step in calculating the amount of CO2 produced is to correct for background (endogenous) CO2 production. Thus the amount of CO2 produced by a test item is determined by the difference (in mL of titrant) between the experimental and blank Ba(OH)2 traps.
The amount of 0.05 M HCl titrated is converted into mg of CO2 produced:

mg CO2 = (0.05 × ∆ mL HCl titrated) / 2 × 44 = 1.1 × ∆ mL HCl titrated

Relative biodegradation values were calculated from the cumulative CO2 production relative to the ThCO2. A figure of more than 10% biodegradation was considered biologically relevant.
The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control. Assessment of ready biodegradability was made based on the average biodegradation in test item bottle A and B. If applicable, the number of days was calculated from the attainment of 10% biodegradation until 60% biodegradation. If this period was ≤ 10 days (10-day window) the test item was designated as readily biodegradable.
Toxicity control: if less than 25% biodegradation (based on the combined ThCO2 of the test item and reference item) occurred within 14 days, the test item was assumed to be inhibitory.
The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in mL of titrant) between the blank Ba(OH)2 traps and untreated Ba(OH)2 (background).
Reference substance:
acetic acid, sodium salt
Test performance:
Since all criteria for acceptability of the test were met, this study was considered to be valid.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 2
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 16
Sampling time:
28 d
Details on results:
Theoretical CO2 Production
The ThCO2 of Hetester HCA was calculated to be 2.60 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation
The relative biodegradation values calculated from the measurements performed during the test period revealed 2% and 16% biodegradation of Hetester HCA (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, 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 -
Hetester HCA (A) 7.6 - 7.5
Hetester HCA (B) 7.6 - 7.5
Toxicity control 7.6 7.6 -
Results with reference substance:
In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, 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.

Acceptability of the test

1.    The reference item was biodegraded by at least 60% (80%) within 14 days.

2.    The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 14%).

3.    The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (79.7 mg CO2per 2 litres of medium, corresponding to 39.9 mg CO2/L).

4.    The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

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

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

The relative biodegradation values calculated from the measurements performed during the test period revealed 2% and 16% biodegradation of Hetester HCA (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, 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.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In conclusion, substance EC 205-363-0 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objective of the study was to evaluate EC 205 -363 -0 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.

The test item was a viscous amber liquid with a purity of >85%. 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 Hetester HCA was calculated to be 2.60 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 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. To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test solutions 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 2% and 16% biodegradation of Hetester HCA (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, the test item was found not to inhibit microbial activity.

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

In conclusion,Hetester HCA was designated as not readily biodegradable.

Description of key information

Key value for chemical safety assessment

Additional information

In conclusion, substance EC 205-363-0 was not readily biodegradable under the conditions of the modified Sturm test presently performed.