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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:
02 August 2017 to 01 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", (1995)
GLP compliance:
yes
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Storage conditions: kept under aeration
- Settlement length: approximately 47 minutes
- Pretreatment: allowed to settle, the supernatant is used as inoculum
- Concentration of sludge: 10 mL/L medium
- suspended solids: 3.2 g/L
Duration of test (contact time):
29 d
Initial conc.:
18.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium according to the guideline
- Test temperature: 22.3 - 23.2°C.
- pH: 7.5-7.6 (positive control 7.6-7.8)
- pH adjusted: yes with 1M HCl (initial pH 7.8)
- Aeration of dilution water: yes
- Suspended solids concentration: 3.2 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown glass vessels
- Number of culture flasks/concentration: 2 for inoculum controls, 2 for test substance, 1 for reference substance, 1 for toxicity control
- Method used to create aerobic conditions: Synthetic air was sparged through the solutions
- Measuring equipment: 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 sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Test performed in closed vessels due to significant volatility of test substance: NA
- Details of trap for CO2: 3 BaOH traps in serie

SAMPLING: day 1, 4, 6, 8, 11, 15, 18, 22, 25 and 29 (+after elution with HCl)

STATISTICAL METHODS: none applied
Reference substance:
acetic acid, sodium salt
Remarks:
40 mg/L, 12 mg/L as TOC
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
29 d
Details on results:
1. The positive control item was biodegraded by at least 60% (70%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 0%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (59.1 mg CO2 per 2 litres of medium, corresponding to 29.6 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).

CO2production and percentage biodegradation

Day

HCl (0.05 N) titrated (mL)

Cumulative CO2

(mg) Bottle A

Cumulative CO2

(mg) Bottle B

Biodegradation1)

(%) Bottle A

Biodegradation1)

(%) Bottle B

Blank (mean)

Bottle A

Bottle B

1

46.91

47.53

47.48

0.0

0.0

0

0

4

43.65

45.13

44.96

0.0

0.0

0

0

6

44.28

45.75

45.76

0.0

0.0

0

0

8

44.87

46.48

46.26

0.0

0.0

0

0

11

46.59

47.43

47.13

0.0

0.0

0

0

15

45.16

46.78

46.31

0.0

0.0

0

0

18

45.31

46.11

46.35

0.0

0.0

0

0

22

43.88

44.80

45.60

0.0

0.0

0

0

25

44.82

44.95

45.75

0.0

0.0

0

0

292)

43.55

44.68

43.41

0.0

0.1

0

0

292)

46.26

46.74

47.45

0.0

0.1

0

0

292)

48.77

48.53

48.76

0.3

0.2

0

0

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 87.3 mg CO2/2L.

2): CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The substance is considered not readily biodegradable under the conditions of the test
Executive summary:

CH01610 was tested in duplicate at a target concentration of 18.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The study consisted of six bottles:

·      2 inoculum blanks (no test item),

·      2 test bottles (CH01610),

·      1 positive control (sodium acetate) and

·      1 toxicity control (CH01610 plus sodium acetate).

Weighed amounts of the substance were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. Test duration was 28 days for the inoculum blank and test item (last CO2measurement on day 29) and 14 days for the positive and toxicity control (last CO2measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed no biological relevant biodegradation of CH01610 (0% in both test vessels).

In the toxicity control, CH01610 was found not to inhibit microbial activity. All criteria for acceptability of the test were met.

In conclusion, CH01610 was designated as not readily biodegradable.

Description of key information

The substance was tested in duplicate at a target concentration of 18.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.The study consisted of six bottles:

·      2 inoculum blanks (no test item),

·      2 test bottles (CH01610),

·      1 positive control (sodium acetate) and

·      1 toxicity control (CH01610 plus sodium acetate).

Weighed amounts of the substance were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. Test duration was28 days for the inoculum blank and test item (last CO2measurement on day 29) and 14 days for the positive and toxicity control (last CO2measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed no biological relevant biodegradation of the substance (0% in both test vessels).

CH01610was tested in duplicate at a target concentration of 18.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.The study consisted of six bottles:

·      2 inoculum blanks (no test item),

·      2 test bottles (CH01610),

·      1 positive control (sodium acetate) and

·      1 toxicity control (CH01610 plus sodium acetate).

Weighed amounts of the substance were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. Test duration was28 days for the inoculum blank and test item (last CO2measurement on day 29) and 14 days for the positive and toxicity control (last CO2measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed no biological relevant biodegradation of CH01610 (0% in both test vessels).

In the toxicity control, CH01610 was found not to inhibit microbial activity.All criteria for acceptability of the test were met.

In conclusion,CH01610 was designated as not readily biodegradable.

The substance was designated as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information