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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:
12 March 2018 to 26 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to guideline
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",
Version / remarks:
1995
GLP compliance:
yes
Specific details on test material used for the study:
The test mateiral was a yellowish powder (UVCB) and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. A sample of the test material was taken for determination of the Total Organic Carbon (TOC) content. TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose (C6H12O6, 99.5%, Sigma,
Steinheim, Germany) as total carbon (TC) standard and Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) as inorganic carbon (IC) standard. The Total Organic Carbon
(TOC) content of the test material was determined to be 44.76%. The test material was tested in duplicate at a target concentration of 27 mg/L, corresponding to 12 mg TOC/L. No correction was made for the purity/composition of the test material.
On the day of testing weighed amounts of the test material were added to the 2 litre test bottles containing medium with microbial organisms and mineral components (test material bottle A: 54.1 mg; test material bottle B: 54.0 mg and toxicity control bottle: 54.0 mg). To this end, small watch glasses were used to transfer the weighed amounts of test material to the respective test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test material and the test organisms. Furthermore, the test medium was swirled around daily, since the test material tended to float on the water surface.
Any residual volumes were discarded.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): 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. After treatment the concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
27 mg/L
Based on:
test mat.
Remarks:
(target)
Initial conc.:
12 mg/L
Based on:
TOC
Remarks:
(target)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water. The stock solutions (A-D) were composed of the following mineral components:
Stock solution 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)
Stock solution B: 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
Stock solution C: 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
Stock solution D: 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
- 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.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: Test suspension: containing test material and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles) Procedure control: containing reference item and inoculum (1 bottle). Toxicity control: containing test material, reference item and inoculum (1 bottle).
- Preparation of the bottles: At the start of the test (day 0), test material 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. 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.
- Measuring equipment:
Determination of 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 standardised HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test material. Titrations for the procedure and toxicity control were made over a period of at least 14 days. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were 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) was used as pH-indicator. On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37 %, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (procedure and toxicity control) and on day 29 (remaining vessels).
Theoretical CO2 production: The theoretical CO2 production was calculated from the results of the TOC-analysis.
- Measurements and Recordings: At the start of the test (day 0) and on the penultimate day (day 14 for the procedure and toxicity control and day 28 for the inoculum blanks and test material), the pH was measured before addition of concentrated HCl. The temperature of the medium was measured continuously in a vessel with Milli- RO water in the same room.

ANALYSIS
ThCO2, expressed as mg CO2/mg test material, was calculated from the results of carbon analysis.
ThCO2 = (Fraction organic carbon × 44) / 12

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 mateiral 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 × Δ mL HCl titrated) / 2] x 44 = 1.1 × Δ 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 procedure control. Assessment of ready biodegradability was made based on the average biodegradation in test material bottle A and B. If not clear from the experimental data 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 material was designated as readily biodegradable.
Toxicity control: if less than 25 % biodegradation (based on the combined ThCO2 of the test material and reference item) occurred within 14 days, the test material 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
Remarks:
40 mg sodium acetate per litre (12 mg TOC/L)
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
(ThOC)
Value:
0
Sampling time:
29 d
Details on results:
- Theoretical CO2 Production
The ThCO2 of the test material was calculated to be 1.64 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 no biologically relevant biodegradation of the test material (0 % and 1 %, based on ThCO2).
In the toxicity control, more than 25 % biodegradation occurred within 14 days (39 %, based on ThCO2). Therefore, the test material 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 throughout the study period.

Table 1: CO2 production and percentage biodegradation of the test material

Day

HCl (0.05N) titrated (mL)

Produced CO2

(mL HCl)

Produced CO2(mg)

Cumulative CO2(mg)

Biodegradation (%)†

Blank (mean)

Bottle A

3

47.71

48.79

0.00

0.0

0.0

0

6

45.71

47.72

0.00

0.0

0.0

0

9

44.92

45.88

0.00

0.0

0.0

0

12

46.14

47.18

0.00

0.0

0.0

0

15

46.14

47.40

0.00

0.0

0.0

0

19

43.55

46.17

0.00

0.0

0.0

0

23

42.89

45.73

0.00

0.0

0.0

0

26

43.26

46.13

0.00

0.0

0.0

0

29*

44.70

45.90

0.00

0.0

0.0

0

29*

46.24

48.45

0.00

0.0

0.0

0

29*

47.70

48.73

0.00

0.0

0.0

0

Day

HCl (0.05N) titrated (mL)

Produced CO2(mL HCl)

Produced CO2(mg)

Cumulative CO2(mg)

Biodegradation (%)†

Blank (mean)

Bottle B

3

47.71

48.15

0.00

0.0

0.0

0

6

45.71

45.66

0.05

0.1

0.1

0

9

44.92

45.96

0.00

0.0

0.1

0

12

46.14

46.63

0.00

0.0

0.1

0

15

46.14

46.54

0.00

0.0

0.1

0

19

43.55

44.66

0.00

0.0

0.1

0

23

42.89

44.22

0.00

0.0

0.1

0

26

43.26

44.32

0.00

0.0

0.1

0

29*

44.7

44.29

0.41

0.4

0.5

1

29*

46.24

47.3

0.00

0.0

0.5

1

29*

47.7

47.84

0.00

0.0

0.5

1

† Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 88.6 mg CO2/2L.

* 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 test material was not readily biodegradable under the conditions of the modified Sturm test performed.
Executive summary:

The ready biodegradability of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 301-B and ISO 10634, under GLP conditions.

The test mateiral was found to be insufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test material was determined to be 44.76 %. Based on the TOC content the ThCO2 of the test material was calculated to be 1.64 mg CO2/mg. The test material was tested in duplicate at a target concentration of 27 mg/L, corresponding to 12 mg TOC/L.

The study consisted of six bottles: 2 inoculum blanks (no test material), 2 test bottles (test material), 1 procedure control (sodium acetate) and 1 toxicity control (test material plus sodium acetate).

Since the test material 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 litre test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer the weighed amounts of test material to the respective test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test material and test organisms. Furthermore, the test medium was swirled around daily, since the test material tended to float on the water surface. Test duration was 28 days for the inoculum blank and test material (last CO2 measurement on day 29) and 14 days for the procedure 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 test material (0 % and 1 %, based on ThCO2).

In the toxicity control, 39% biodegradation was observed by day 14. Therefore, the test material was found not to inhibit microbial activity.

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

The procedure control reached 78 % biodegradation by day 14 and CO2 production in the blank was 31.2 mg/L at the end of the study (day 28).

In conclusion, the test material cannot be considered to be readily biodegradable.

Description of key information

The test material 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
Type of water:
freshwater

Additional information

The ready biodegradability of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 301-B and ISO 10634, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test mateiral was found to be insufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test material was determined to be 44.76 %. Based on the TOC content the ThCO2 of the test material was calculated to be 1.64 mg CO2/mg. The test material was tested in duplicate at a target concentration of 27 mg/L, corresponding to 12 mg TOC/L.

The study consisted of six bottles: 2 inoculum blanks (no test material), 2 test bottles (test material), 1 procedure control (sodium acetate) and 1 toxicity control (test material plus sodium acetate).

Since the test material 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 litre test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer the weighed amounts of test material to the respective test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test material and test organisms. Furthermore, the test medium was swirled around daily, since the test material tended to float on the water surface. Test duration was 28 days for the inoculum blank and test material (last CO2 measurement on day 29) and 14 days for the procedure 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 test material (0 % and 1 %, based on ThCO2).

In the toxicity control, 39 % biodegradation was observed by day 14. Therefore, the test material was found not to inhibit microbial activity.

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

The procedure control reached 78 % biodegradation by day 14 and CO2 production in the blank was 31.2 mg/L at the end of the study (day 28).

In conclusion, the test material cannot be considered to be readily biodegradable.