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Environmental fate & pathways

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:
August - September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
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:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
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/activated sludge (e.g. location, sampling depth, contamination history, procedure): 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: The sludge was kept under continuous aeration until further treatment.
- Storage length: Not recorded.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (45 minutes) and the liquid was decanted for use as inoculum at the amount of 1 0 mL/L of mineral medium.
- Pretreatment: None recorded.
- Concentration of sludge: The concentration of suspended solids was 3.6 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Initial cell/biomass concentration: Not recorded.
- Water filtered: yes/no Not recorded.
- Type and size of filter used, if any: Not recorded.
Duration of test (contact time):
28 d
Initial conc.:
12 mg/L
Based on:
other: TOC/L
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
Modified sturm test
Details on study design:
TEST CONDITIONS
- Composition of medium:
Stock solutions of mineral components:
A) 8.50 g KH2PO4.
21.75 g K2HPO4
67.20 g Na2HPO4.12H20
0.50 g NH4CI dissolved in Milli-Q water and made up to 1 Litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H20 dissolved in Milli-Q water and made up to 1 Litre.
C) 36.40 g CaCI2.2H20 dissolved in Miili-Q water and made up to 1 Litre.
D) 0.25 g FeCI3.6H20 dissolved in Milli-Q water and made up to 1 Litre.

- Additional substrate:
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), (Boom, the Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

- Solubilising agent (type and concentration if used):
Since EA-3098 was poorly soluble in water, weighed amounts of EA-3098 were added to the test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 32.9 mg; test substance bottle B: 32.3 mg and toxicity control bottle: 31.7 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and the test organisms, since the test substance tended to float on the water surface.

- Test temperature: The temperature recorded in a vessel with water in the same room varied between 21.6 and 22.3 deg C.
- pH: 7.5 - 7.7
- pH adjusted: yes/no: Not stated.
- CEC (meq/100 g): Not applicable.
- Aeration of dilution water: Not stated.
- Suspended solids concentration: The concentration of suspended solids was 3.6 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Continuous darkness: yes/no: Not stated.
- Other: Milli-RO/Milli-Q water:
Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Miili-Q) (Millipore Corp., Bedford, Mass., USA).

TEST SYSTEM
- Culturing apparatus: 2 Litre all-glass brown coloured bottles.

- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

- Method used to create aerobic conditions:
Synthetic air (C02< 1 ppm):
A mixture of oxygen (21 %) and nitrogen (79%) was passed through a bottle, containing 0.5 - 1 Litre 0.0125 M Ba(OH), solution to trap CO2 which might be present in smail 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).

- Method used to create anaerobic conditions: Not applicable.

- Measuring equipment: Not stated.

- Test performed in closed vessels due to significant volatility of test substance: No

- Test performed in open system: No

- Details of trap for CO2 and volatile organics if used:
Experimental CO2 production:
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCI (1 :20 dilution from 1 M HCI (Titrisol® ampul), Merck KGaA; Darmstadt, Germany).

- Other: Preparation of bottles:
Pre-incubation medium: Mineral components, MiIIi-RO water (ca. 80% total volume) and inoculum (1 % final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Preparation:
The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). 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.

SAMPLING
Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck KGaA, Darmstadt, Germany) was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 mL of concentrated HCI (37%, Merck KGaA, Darmstadt, Germany) was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Reference substance:
A solution of sodium acetate was prepared by dissolving 395.5 mg in Milli-RO water and making this up to a total volume of 100 mL. Volumes of 20 mL from this stock solution were added to 2 Litres of the test medium of the positive control bollie and the toxicity control bottIe, resulting in a final concentration of 40 mg/L sodium acetate (12 mg TOC/L).
- Inoculum blank: Prepared in the same manner.

STATISTICAL METHODS:
Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula.

Data evaluation:
ThC02, expressed as mg CO2/mg test substance, that can be generated by a test substance was calculated as follows:

ThCO 2 = No. of carbon atoms in test substance X Molecular weight CO2/Molecular weight test substance

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 material is determined by the difference (in mL of titrant) between the experimental and blank Ba(OH)2 traps.

The amount of 0.05 N HCI titrated is converted into mg of CO2 produced:

mg CO2 = 0.05 x ▲ mL HCI titrated/2 X 44 = 1.1 X ▲ mL HCl titrated

Relative degradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production based on the total carbon content of the amount of test material present in the test bottles. They were plotted versus time together with the relative degradation of the positive control.

A figure of more than 10% degradation was considered as significant.

Toxicity control: if less than 25% degradation (based on ThC02) occurred within 14 days, the test substance 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)z traps and fresh Ba(OH)2.
Reference substance:
other: Sodium Acetate
Preliminary study:
Not applicable.
Test performance:
1. The positive control substance was degraded by at least 60% (74%) within 14 days.
2. The difference of duplicate values for %-degradation of EA-3098 was always less than 20.
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (41.8 mg CO2 per 2 Litres of medium, corresponding to 20.9 mg/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
28 d
Remarks on result:
other: Modified sturm test
Details on results:
THEORETICAL CO2 PRODUCTION:
The Theoretical CO2 production (ThCO2) of EA-3098 was calculated to be 2.71 mg CO2/mg.
The amount of EA-3098 was 32.9 (bottle A) and 32.3 mg (bottle B) in 2 Litres test medium. Hence, the theoretical CO2 production following complete degradation was 89.2 mg per 2 Litres for bottle A and 87.5 mg per 2 Litres for bottle B.

The toxicity control contained 79.1 mg sodium acetate and 31.7 mg EA-3098 in 2 Litres of test medium. Hence, the theoretical CO2 production following complete degradation of EA-3098 plus sodium acetate was 170.5 per 2 Litres.

BIODEGRADATION
The results are present in Appendix 1 (see attached background material).

Points of degradation plot (test substance):
0 % degradation after 2 d
0 % degradation after 5 d
1 % degradation after 7 d
2 % degradation after 9 d
3 % degradation after 14 d
4 % degradation after 19 d
4 % degradation after 23 d
6 % degradation after 27 d
7 % degradation after 29 d

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of EA-3098.

In the toxicity control more than 25% degradation occurred within 14 days (38%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The positive control contained 79.1 mg sodium acetate (ThC02= 1.07 mg CO2/mg) in 2 Litres test medium, resulting in a theoretical CO2 production following complete degradation of 84.6 mg per 2 Litres.

The positive control obtained 81% degradation after 29 days.

Points of degradation plot (reference substance):
2 % degradation after 2 d
39 % degradation after 5 d
55 % degradation after 7 d
64 % degradation after 9 d
74 % degradation after 14 d
78 % degradation after 19 d
80 % degradation after 23 d
80 % degradation after 27 d
81 % degradation after 29 d

pH values of the different test media ranged from 7.5 - 7.7 throughout the study.

Validity criteria fulfilled:
yes
Remarks:
(refer to test performance section)
Interpretation of results:
other: not readily biodegradable
Conclusions:
In conclusion, EA-3098 was not readily biodegradable under the conditions of the modified Sturm test performed.
Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with EA-3098.

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, OECD guideline No. 301 B July 17, 1992 and ISO Standard 9439 (1999).

The Theoretical CO2 production (ThCO2) of EA-3098 was calculated to be 2.71 mg CO2/mg.

EA-3098 was a white powder and was treated as 100% pure. EA-3098 was tested in duplicate at 32 mg per 2 Litres, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.

The study consisted of six bottles:

- 2 blank controls (no test material),

- 2 test bottles (EA-3098, 16 mg/L),

- 1 positive control (sodium acetate, 40 mg/L) and

- 1 toxicity control (EA-3098, 16 mg/L; plus sodium acetate, 40 mg/L).

Since EA-3098 was poorly soluble in water, weighed amounts of EA-3098 were added to the 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 substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test.

Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and the test organisms, since the test substance tended to float on the water surface.

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of EA-3098 (7% after 29 days).

In the toxicity control, EA-3098 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, EA-3098 was not readily biodegradable under the conditions of the modified Sturm test performed.

Description of key information

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of EA-3098 (7% after 28 day).

EA-3098 was not readily biodegradable under the conditions of the modified Sturm test performed.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with EA-3098.

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, OECD guideline No. 301 B July 17, 1992 and ISO Standard 9439 (1999).

The Theoretical CO2production (ThCO2) of EA-3098 was calculated to be 2.71 mg CO2/mg.

EA-3098 was a white powder and was treated as 100% pure. EA-3098 was tested in duplicate at 32 mg per 2 Litres, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.

The study consisted of six bottles:

- 2 blank controls (no test material),

- 2 test bottles (EA-3098, 16 mg/L),

- 1 positive control (sodium acetate, 40 mg/L) and

- 1 toxicity control (EA-3098, 16 mg/L; plus sodium acetate, 40 mg/L).

Since EA-3098 was poorly soluble in water, weighed amounts of EA-3098 were added to the 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 substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test.

Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and the test organisms, since the test substance tended to float on the water surface.

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of EA-3098 (7% after 28 day).

In the toxicity control, EA-3098 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, EA-3098 was not readily biodegradable under the conditions of the modified Sturm test performed.