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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:
02-Feb-2017 to 28-Sept-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance OECD 301B guideline without deviation. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
July 1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: confidential
- Purity test date: confidential

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not relevant
- Specific activity: not relevant
- Locations of the label: not relevant
- Expiration date of radiochemical substance: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated at 2-8 °C
- Stability under test conditions: Presumed stable
- Solubility and stability of the test substance in the solvent/vehicle: not relevant
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not relevant

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: none
- Final preparation of a solid: none

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: not relevant
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): Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht
- Laboratory culture: No
- Method of cultivation: Not relevant
- Storage conditions: Not relevant
- Storage length: Not relevant
- Preparation of inoculum for exposure: 12.9 mL activated sludge was filled up to 1500 mL with 1487.1 mL mineral medium. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers.
- Pretreatment: The dry solid content of the activated sludge was 3.5 g/L. It was determined by weight measurements after drying at 105 °C for 3.5 hours (mean of triplicate measurements). The activated sludge was washed twice with chlorine free tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
- Concentration of sludge: 30 mg/L dry solids
- Initial cell/biomass concentration: no data
- Water filtered: demineralised water used to make mineral medium
- Type and size of filter used, if any: no data
Duration of test (contact time):
28 d
Initial conc.:
>= 19.8 - <= 20.1 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
A: KH2PO4: 8.50 g
K2HPO4: 21.75 g
Na2HPO4 * 2 H2O: 33.40 g
NH4Cl: 0.50 g
are dissolved in demineralised water and made up to 1 litre.
B: CaCl2 * 2H2O: 36.4 g
is dissolved in demineralised water and made up to 1 litre.
C: MgSO4 * 7H2O: 22.5 g
is dissolved in demineralised water and made up to 1 litre.
D: FeCl3 * 6H2O: 0.25 g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.

For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.

- Additional substrate: inoculum as previously described
- Solubilising agent (type and concentration if used): none
- Test temperature: 21.3 – 22.4 °C
- pH: 7.4 ± 0.2
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: 30 - 100 mL / min visually determined as 1.6 – 5.5 bubbles/second (counted bubbles: 2.4 – 4.3 bubbles/second).
- Suspended solids concentration: 30 mg/L dry solids
- Continuous darkness: no; under diffuse light
- Other:

TEST SYSTEM
- Culturing apparatus: 1000 mL gas wash bottles with Teflon-sealing, Thoma, Freiburg
- Number of culture flasks/concentration: 3 replicates for test item, 3 replicates for reference item, 1 replicate for toxicity control (i.e. reference and test item)
- Method used to create aerobic conditions: aerobic conditions were kept by bubbling CO2-free air through the test vessels.
- Method used to create anaerobic conditions: not relevant
- Measuring equipment: IC measurement was performed with a total carbon analyser (TOC-L Shimadzu with an autosampler ASI-L) by purging the inorganic carbon with H3PO4 (25%) using a non-dispersive infrared (NDIR) detector.
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no -test system was closed in order to measure CO2 generated in the system during degradation of the test item.
- Details of trap for CO2 and volatile organics if used: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH.
The absorption medium was made as follows: 80.42 g NaOH was dissolved in 10000 mL deionised water in closed vessels (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined (IC = 2.464 mg/L).
- Other:

SAMPLING
- Sampling frequency: Day 0, 3, 7, 10, 14, 21 & 28
- Sampling method: 6 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air.
On the 28th day 2 mL of 4M hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.
- Sterility check if applicable: not applicable
- Sample storage before analysis: not applicable
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; containing inoculum only as previously described.
- Abiotic sterile control: no
- Toxicity control: Yes; 39.9 mg of the test item and 5.15 mL of the reference stock solution (10 g/L) were added to the toxicity control vessel corresponding to a concentration of 40.1 mg/L organic carbon.
- Other:

STATISTICAL METHODS:
The theoretical CO2 amount of the test item (ThCO2) is calculated as follows:

ThCO2 [mg] = weight of added test item [mg] * carbon-content [mg/mg] * 44/12

The amount of CO2 released from the reactors is calculated through IC-measurements in the CO2-absorber flasks as follows:

CO2 [mg/1500 mL] = IC [mg/L]* Volumeabsorber flask [L] *44/12

Hereby the volume of the absorber flask at the beginning of the test was 0.2 litre (200 mL) and the volume was reduced through each sampling by 6 mL. The factor 44/12 corresponds the molar weight-ratio of CO2 and C.

The amount of CO2 removed for IC-measurement is considered by adding up the CO2 content of each sampling to the current CO2 content of each absorber flask:

CO2 total (i, x) = CO2 absorber flask (i, x) + CO2 sampling (i, x-1)

with

CO2 total (i, x) = Total amount of CO2 [mg] absorbed in the ith absorber flask at the xth sampling including the amount removed by sampling

CO2 absorber flask (i, x) = CO2 [mg] absorbed in the ith absorber flask derived from IC-measurement at the xth sampling.

CO2 sampling (i, x-1) = Sum of CO2 [mg] removed from the ith absorber flask with the 1th to x-1thsampling (sampling volume 6 mL each CO2 removed = IC [mg/L] * 0.006 [L]*44/12]).

The percentage biodegradation is calculated from:

BiodegradationCO2 [%] = 100*(CO2 Test reactor [mg] - CO2 Blank reactor [mg])/ThCO2 [mg]

CO2 test reactor= Total CO2 evolution in the test reactor [mg]

CO2 Blank reactor = Total CO2 evolution in the blank reactor [mg] (mean of two vessels)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None
Test performance:
The test satisfied all validation criteria as indicated below:
- The IC content in the test vessel was less than 5% of the TOC introduced with the test item.
- The CO2 evolution in the inoculum blank at the end of the test was below 40 mg/L.
- The difference of extremes of replicate values of the test item at the end of the test was less than 20%.
- The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 3.
- The degradation extent in the toxicity control was above 25% in 14 days based on ThCO2.

No deviations from the guideline were seen.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
92.9
St. dev.:
2.89
Sampling time:
28 d
Details on results:
Although the substance is a mixture of similar chemicals, and the 10-d window criteria need not be applied, nonetheless, the criteria of the 10-d window was met. On day 7 the degradation extent is already at 67.9% the degradation of the test item is therefore higher than 60% within 10 days after 10 % degradation of the substance is acheived.
Results with reference substance:
The reference compound sodium benzoate reached the pass levels for ready biodegradability within 3 days.

Table 1 Degradation of reference item, test item and toxicity control at x days

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

3

73.13

31.77

49.00

7

84.47

67.93

75.00

10

88.53

77.17

81.30

14

89.93

85.50

88.70

21

92.53

88.90

91.90

28

94.13

93.13

93.30

29

93.83

92.90

95.20

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
An OECD 301B CO2 Evolution Test (1992) was conducted with the test item. This was to assess the substances ready biodegradability. The test met all guideline validity criteria.
The test item had no inhibitory effect on the inoculum according to the criterion of the guideline. The degradation of the test item was 92.9% after 28 days and the criteria of the 10-d-window was met.

The test item reached the criteria for ready biodegradability.
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The study was in concordance with OECD (1992) 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

The test item was added to activated sludge from a municipal wastewater treatment plant (sewage sludge contained 30 mg/L of dry solids). The test item was applied to the system at a concentration of ~20 mg TOC/L and left for 28 days in diffuse light at approximately 21 -22 °C.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item (sodium benzoate) together with a toxicity control (reference item and test item) were used for validation purposes.

Although the substance is a mixture of similar chemicals, and the 10-d window criteria need not be applied, nonetheless, the criteria of the 10-d window was met. On day 7 the degradation extent is already at 67.9 % the degradation of the test item is therefore higher than 60 % within 10 days of 10 % degredation being reached.

Therefore the test item reached the criteria for ready biodegradability according to the OECD criteria (≥ 60% ThCO2 and fulfilment of 10-d window).

The test item is ready biodegradable.

Description of key information

28 d biodegradation 92.9 % (readily biodegradable); OECD 301B; A. Brunswik-Titze (2017)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One key study for the assessment of ready biodegradability was available for the test item. The study was in concordance with OECD (1992) 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

The test item was added to activated sludge from a municipal wastewater treatment plant (sewage sludge contained 30 mg/L of dry solids). The test item was applied to the system at a concentration of ~20 mg TOCC/L and left for 28 days in diffuse light at approximately 21 -22 °C.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item (sodium benzoate) together with a toxicity control (reference item and test item) were used for validation purposes.

Although the substance is a mixture of similar chemicals, and the 10-d window criteria need not be applied, nonetheless, the criteria of the 10-d window was met. On day 7 the degradation extent is already at 67.9 % the degradation of the test item is therefore higher than 60 % within 10 days of 10 % degradation being reached.

Therefore the test item reached the criteria for ready biodegradability according to the OECD criteria (≥ 60% ThCO2 and fulfilment of 10-d window).

The test item is ready biodegradable.

[Type of water: freshwater]