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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:
2017-12-13 / 2018-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant AZV Staufener Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 140,000 inhabitant equivalents. Sampling date of activated sludge was on 9 January 2018. The dry solid content of the activated sludge was 4.3 g/L. It was determined by weight measurements after drying at 105°C for 6 hours (mean of triplicate measurements). The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
Duration of test (contact time):
29 d
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Mineral medium
A: Potassium dihydrogenphosphate KH2PO4 8.50 g, Dipotassium hydrogenphosphate K2HPO4 21.75 g, Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g, Ammonium chloride NH4Cl 0.50 g are dissolved in demineralised water and made up to 1 litre. The pH of the solution should be 7.4.
B: Calcium chloride dihydrate CaCl2 * 2H2O 36.4 g is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate 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.
- CO2-absorption medium
56.01 g NaOH was dissolved in 7000 mL deionised water in closed vessels (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined (IC = 8.2 mg/L).
- Test item
Due to the insolubility, the test item was added directly into the test vessels. Carrier material was inert PE film. 130.7 – 132.1 mg of the test item were added into the test vessels corresponding to a concentration of 19.9 – 20.1 mg/L organic carbon.
- Reference substance
A stock solution of 10.0 g/L sodium benzoate in water was prepared. 5.15 mL of this stock solution were added into the reference vessels corresponding to a concentration of 20.0 mg/L organic carbon.
- Toxicity control
131.7 mg of the test item and 5.15 mL of the reference stock solution (10 g/L) were added into the toxicity control vessel corresponding to a concentration of 40.0 mg/L organic carbon.
- Test temperature: 20.0 - 23.7°C


TEST SYSTEM
- Experimental set up
The CO2-free air production system consists of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.1 M NaOH (sodium hydroxide). At the end of the system is one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A color change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with two input and 22 output channels and through PE-tubes Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septa were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by magnetic stirrers with 2 cm stir bars. 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. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes.

- Procedure
In total three reactors containing the test item with inoculum, three reactors containing only inoculum (blank), three reactors containing the reference compound with inoculum and one reactor containing the test item and reference compound with inoculum (toxicity control) were set up. 10.5 mL activated sludge was filled up to 1500 mL with 1489.5 mL mineral medium corresponding to 30 mg/L dry solids. Only the abiotic control vessel was filled with 1500 mL mineral medium without inoculum. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers. On the next day, the absorber wash bottles were filled with 0.2 M NaOH and the test substance was added into the three test vessels and into the toxicity control vessel. The reference compound was added into the reference vessels and into the toxicity control vessel. The aeration rate was kept at a rate of 30 - 100 mL / min (1.6 - 5.5 bubbles / second) and determined visually daily on working days. The determination by counting the gas bubbles over a defined period using a stop watch was made on days 11 and 29. The CO2-free air production system, the air-tightness of the whole experimental set-up, the aeration of the absorber flasks and the magnetic stirrers were controlled daily on working days. At the beginning of the study the IC concentration of the 0.2 M NaOH used for the CO2-absorption flasks was determined as 8.2 mg/L. The IC in the reactors at the beginning of the test was 0.80 mg/L. On the 4th, 7th, 11th, 14th, 21st and 28th day 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. IC measurement was performed with a total carbon analyser (TOC-L Shimadzu) by purging the inorganic carbon with H3PO4 (25%) using a non-dispersive infrared (NDIR) detector.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Reference substance:
benzoic acid, sodium salt
Test performance:
The degradation extent of the test item at the end of the test was 80.0% of ThCO2 (28 d after acidification, mean of three replicates).
Since the mean degradation on day 11 was >60% and the beginning of the 10-d window (degradation >10%) can be on day 1 at the earliest, the degradation of 60% was reached within 10 days.
Therefore, the test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).
Parameter:
% degradation (CO2 evolution)
Value:
80
Sampling time:
28 d
Results with reference substance:
The reference compound sodium benzoate reached the pass level for ready biodegradability within 4 days.

Test item

The degradation extent of the test item at the end of the test was 80.0% of ThCO2 (28 d after acidification, mean of three replicates). Since the mean degradation on day 11 was >60% and the beginning of the 10-d window (degradation >10%) can be on day 1 at the earliest, the degradation of 60% was reached within 10 days. Therefore, the test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).

Toxicity control

The degradation extent in the toxicity control was 76.9% within 14 days. According to the guideline, the test substance had no inhibitory effect on the inoculum.

Reference item

The reference compound sodium benzoate reached the pass level for ready biodegradability within 4 days.

Blank

The mean CO2-evolution of the blank flasks was 29.8 mg/L on day 28 after acidification.

Test Parameters

- Before adding the test item, the IC in the reactor was determined, but only insignificant amounts of IC (0.80 mg/L) were found.

- The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 7 ppm and was not considered in the data processing, because CO2 absorption from room air was its source.

- The temperature was 20.0 – 23.7°C throughout the whole study and therefore within the required range of 22 ± 2°C.

- The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 2.3 – 4.3 bubbles/second).

Criteria of Validity

- 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 4.

- The degradation extent in the toxicity control was above 25% in 14 days based on ThCO2.

The test is valid according to OECD Test Guideline 301 B (July 1992).

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The degradation of the test item at the end of the test was 80.0% (28 d after acidification, mean of two replicates). The test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10-d window). The degradation of the toxicity control after 14 days was 76.9%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.
Executive summary:

A CO2 evolution test was performed according to OECD Guideline 301B (July 1992) to investigate the ready biodegradability of the test item. The degradation of the test item at the end of the test was 80.0% of ThCO2 (28 d after acidification, mean of three replicates). The test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10 -d window). The degradation of the toxicity control after 14 days was 76.9%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline. The test is valid according to OECD Guideline 301B.

Description of key information

A CO2 evolution test was performed according to OECD Guideline 301B (July 1992) to investigate the ready biodegradability of the test item.The degradation of the test item at the end of the test was 80.0% of ThCO2 (28 d after acidification, mean of three replicates). The test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10-d window). The degradation of the toxicity control after 14 days was 76.9%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline. The test is valid according to OECD Guideline 301B.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

One reliable experimental study is available and is selected as key study (Hydrotox, 2018). It was performed according to OECD Guideline 301B and under GLP principles.