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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:
03 April 2020 - 08 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted in accordance with the relevant OECD test guideline and in accordance with GLP. All validity criteria were met.
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 Standard 10634:2018
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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): 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.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: N/A
- Storage length: N/A
- Preparation of inoculum for exposure: Freshly obtained sludge was kept under continuous aeration until further treatment. Before the start of the test (Day -1) mineral components, Milli-RO water (ca. 80 % of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Pretreatment: The concentration of suspended solids (SS) in the activated sludge as sampled could not accurately be determined. Before use, sludge was coarsely sieved (1 mm2 mesh). After treatment the concentration of SS was determined to be 11.2 g/L in the concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 6 mL per vessel. Due to the high SS concentration of 11.2 g/L, inoculated medium was diluted four times after overnight pre-incubation, by making up 500 mL of inoculated medium to a final volume of 2 L, leading to a final SS concentration of 8.4 mg/L.
- Concentration of sludge: 8.4 mg/L
- Initial cell/biomass concentration: Not reported
- Water filtered: no, Milli-RO water used (Tap-water purified by reverse osmosis and subsequently passed over activated carbon.)
- Type and size of filter used, if any: N/A
Duration of test (contact time):
29 d
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 L mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B), (C), (D), and Milli-RO water:
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.

- Additional substrate: N/A
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 22 - 23 ºC (measured continuously in a vessel with Milli-RO water in the same room)
- pH: 7.5 - 7.9 (measured at Day 0 and on the penultimate day [Day 14 for procedural and toxicity control and Day 29 for inoculum blanks and test item], before addition of concentrated HCl)
- pH adjusted: yes
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Suspended solids concentration: 8.4 mg/L
- Continuous darkness: yes
- Other: A solution of sodium acetate (reference substance) was prepared by dissolving 810.1 mg in Milli-RO water and making this up to a total volume of 200 mL. Volumes of 20 mL of this stock solution were added to the test medium (2 L) of the procedural control and toxicity control bottle, resulting in a final concentration of 40.5 mg sodium acetate per litre (corresponding to 12 mg TOC/L).

TEST SYSTEM
- Culturing apparatus: 2 L amber glass bottles
- Number of culture flasks/concentration:
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Procedural control: containing procedural control item and inoculum (1 bottle).
Toxicity control: containing test item, procedural control item and inoculum (1 bottle).
On the day of testing weighed amounts of test item were added to 2 L test bottles containing medium with microbial organisms and mineral components (test item bottle A: 37.1 mg; test item bottle B: 37.6 mg; toxicity control bottle: 38.1 mg). At the start of the test (Day 0), test and procedural control item were added to bottles containing microbial organisms and mineral components. Volumes were made up to 2 L 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 aeration line of each test bottle.
- Method used to create aerobic conditions: Synthetic air was passed 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: N/A
- Measuring equipment: 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).
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: A mixture of oxygen (ca. 20 %) and nitrogen (ca. 80 %) was passed through a bottle, containing 0.5 - 1 L 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts.
- Other:

SAMPLING
- Sampling frequency: 2, 5, 8, 12, 14 19, 23, 29 & 30
- Sampling method:
The amount of CO2 produced was determined by titration of Ba(OH)2 with HCl.
CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until Day 29, for inoculum blank and test item. Titrations for procedural and toxicity control were made over a period of at least 14 days. Phenolphthalein (1 % solution in ethanol, Merck) was used as pH-indicator.
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. On the penultimate day, pH of respective test suspensions was measured and 1 mL of concentrated HCl (37 %, Merck) was added to the inoculum blank and test suspension. Bottles were aerated overnight to drive off CO2. Final titration was made on Day 15 (procedural and toxicity control) and on Day 30 (remaining vessels).
- Sterility check if applicable: N/A
- Sample storage before analysis: N/A
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, containing only inoculum as previously described
- Abiotic sterile control: No
- Toxicity control: Yes; containing test item, procedural control item and inoculum.
- Other:

STATISTICAL METHODS:

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

Total CO2 evolution in inoculum blank was determined by cumulative difference (in mL of titrant) between blank Ba(OH)2 traps and non-exposed Ba(OH)2 (stored in closed bottles).
The total CO2 evolution of the test item, procedural control and toxicity control was determined by the cumulative difference (in mL of titrant) between experimental Ba(OH)2 traps and blank Ba(OH)2 traps. The difference in 0.05 M HCl titrated was converted into mg of CO2 produced:

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

Biodegradation values were calculated as a percentage of the cumulative CO2 production relative to the ThCO2. Relative biodegradation values were plotted versus time together with relative biodegradation of the procedural control. Assessment of ready biodegradability was made based on average biodegradation in test item bottle A and B.
Reference substance:
acetic acid, sodium salt
Preliminary study:
N/A
Test performance:
1. Procedural control item was biodegraded by at least 60 % (actual result: 86 %) within 14 days.
2. Difference between duplicate values for %-degradation of the test item was always less than 20 % (actual result: ≤ 8 %).
3. Total CO2 release in the blank at the end of the test did not exceed 40 mg/L (58.5 mg CO2 per 2 litres of medium, corresponding to 29.3 mg CO2/L).
4. Inorganic Carbon content (IC) of the test item (suspension) in 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 validity criteria were satisfied the study was considered to be valid.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
10
Sampling time:
29 d
Remarks on result:
other: Bottle B
Key result
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
29 d
Remarks on result:
other: Bottle A
Details on results:
ThCO2 of the test item was calculated to be 2.33 mg CO2/mg.
Relative biodegradation values calculated from measurements performed during the test period showed limited biodegradation of the test item (7 % and 10 %, for bottle A and B respectively, based on ThCO2).
In the toxicity control, more than 25 % biodegradation occurred within 14 days (47 %, based on ThCO2). Therefore, the test item was considered not to inhibit microbial activity. The test item was concluded not to be readily biodegradable, as the % biodegradation values (7 & 10 %) fell below the 28 d cut off value of 60 % biodegradation as stipulated by the OECD guideline.
Results with reference substance:
ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg. Functioning of the test system was confirmed, whereby the reference substance demonstrated a normal biodegradation curve.

Table 1. Biodegradation values for the test item, reference substance and toxicity control and CO2 production values for the blank control

Time Days

HCl (0.05 N) titrated (mL)

Biodegradation (%)

 

Blank (mean) 

Test item

Bottle A

Test item

Bottle B

Sodium acetate

Toxicity control

2

47.34

0

2

12

12

5

46.30

1

6

41

29

8

44.05

2

10

61

40

12

42.53

3

10

73

44

15

42.91

5

10

86

47

19

42.57

6

10

-

-

23

43.13

6

10

-

-

29

41.86

6

10

-

-

30

43.54

7

10

-

-

30

40.10

7

10

-

-

30

47.42

7

10

-

-

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was not readily biodegradable under the conditions of this test, whereby 7 and 10 % biodegradation (based on CO2 evolution) were observed.
Executive summary:

A test was conducted in accordance with OECD 301 B (1992), in order to determined the biodegradation potential of the test item.

The test item was added to an an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge, obtained from a municipal sewage treatment plant.

Before use, sludge was coarsely sieved (1 mm2 mesh). After treatment the concentration of suspended sludge (SS) was determined to be 11.2 g/L in the concentrated sludge as used for the test. Due to the high SS concentration of 11.2 g/L, inoculated medium was diluted four times after overnight pre-incubation, by making up 500 mL of inoculated medium to a final volume of 2 L, leading to a final SS concentration of 8.4 mg/L. The test concentration was 19 mg/L, corresponding to 12 mg TOC/L.

The test was run in the dark for 29 days for the inoculum blank and test item (last CO2 measurement on Day 30); and 14 days for the procedural and toxicity control (last CO2 measurement on Day 15). 2 bottles each were prepared for for the test suspension and the inoculum blank; and 1 bottle each was prepared for the procedural control (sodium acetate) and the toxicity control.

To assess the biodegradation of the test item, determination of CO2 was performed by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HC. 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). Theoretical CO2 production was calculated from the results of the TOC-analysis.

After 29 days, for replicates Bottle A and Bottle B, 7 and 10 % biodegradation values were observed respectively. These values fall under the 60 % biodegradation cut off value stipulated in the guideline, in order to designate a substance as biodegradable (the 10-d window does not apply to UVCB substances).

All validity criteria were fulfilled. Therefore the test item was concluded not to be readily biodegradable under the conditions of the test.

Description of key information

OECD 301B, Timmer (2020), not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

A test was conducted in accordance with OECD 301 B (1992), in order to determined the biodegradation potential of the test item.






The test item was added to an an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge, obtained from a municipal sewage treatment plant.








The suspended sludge concentration was 8.4 mg/L. The test concentration was 19 mg/L, corresponding to 12 mg TOC/L. The test was run in the dark for 29 days for the inoculum blank and test item (last CO2 measurement on Day 30); and 14 days for the procedural and toxicity control (last CO2 measurement on Day 15). 2 bottles each were prepared for for the test suspension and the inoculum blank; and 1 bottle each was prepared for the procedural control (sodium acetate) and the toxicity control.








To assess the biodegradation of the test item, determination of CO2 was performed by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HC. TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A. Theoretical CO2 production was calculated from the results of the TOC-analysis.


After 29 days, for replicates Bottle A and Bottle B, 7 and 10 % biodegradation values were observed respectively. These values fall under the 60 % biodegradation cut off value stipulated in the guideline, in order to designate a substance as biodegradable (the 10-d window does not apply to UVCB substances).


All validity criteria were fulfilled. Therefore the test item was concluded not to be readily biodegradable under the conditions of the test.





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