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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:
2017-11-10 to 2018-03-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
July 1992
Deviations:
yes
Remarks:
Between day 7 and 8 the temperature was 19.8 - 20.6 °C and therefore partially below the range required (22 ± 2 °C); no impact on the study as the underrun of temperature was only temporary
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
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): municipal wastewater treatment plant AZV Staufener Bucht. Treatment plant clarifies predominantly domestic wastewater and has a capacity of 140,000 inhabitant equivalents. The sludge was sampled on 2017-11-20, and the suspended solid content of the activated sludge was 5.1 g/L. This was determined by weight measurements after drying at 105 °C for 3.25 h (mean of triplicate measurements).
- Preparation of inoculum for exposure: washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge
- Concentration of sludge: 30 mg suspended solids per litre
Duration of test (contact time):
28 d
Initial conc.:
>= 19.9 - <= 20.1 mg/L
Based on:
TOC
Remarks:
The test item had unknown solubility so 40.0 to 40.5 mg of the test item were added into the test vessels
Parameter followed for biodegradation estimation:
inorg. C analysis
Remarks:
Determine carbon dioxide produced and absorbed to sodium hydroxide
Details on study design:
TEST CONDITIONS
- Composition of mineral medium:
a) 8.50 g KH2PO4, 21.75 g K2HPO4, 33.40 g Na2HPO4 * 2 H2O, 0.5 g NH4Cl were dissolved in demineralised water and made up to 1 litre. The pH was 7.4.
b) 36.4 g CaCl2 * 2H2O was dissolved in demineralised water and filled up to 1 litre
c) 22.5 g MgSO4 * 7H2O was dissolved in demineralised water and filled up to 1 litre
d) 0.25 g FeCl3 * 6H2O was dissolved in demineralised water, stabilised with one drop of concentrated HCl and filled up to 1 litre
For preparation of the mineral medium 10 mL of a) was mixed with 900 mL demineralised water, 1 mL each of solutions b), c), and d) were added and the volume was adjusted to 1 litre.
- Composition of CO2-absorption medium: 72.08 g NaOH was dissolved in 9 L deionised water in closed vessels (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined (IC = 6.5 mg/L).
- Test temperature: 19.8 - 22.2 °C
- pH: not specified
- Aeration of dilution water: in the tolerated range of 1.6 - 5.5 bubbles/second (counted bubbes: 2.1 - 4.9 bubbles/second)
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no; in diffuse light

TEST SYSTEM
- Culturing apparatus: Compressor NO10.AN 18, NF Neuberger, Freiburg with 1000 mL gas wash bottles with Teflon-sealing, Thoma, Freiburg and magnetic stirrer, 'MONO direct' with stir bars 2 cm, H+P Labortechnik AG, Oberschlessheim.
- Number of culture flasks/concentration: 3 replicates for test substances, 1 for toxicity control, 1 for abiotic control, 3 positive control replicates, 3 blank replicates. 8.8 mL activated sludge was filled up to 1500 mL with 1491 mL mineral medium corresponding to 30 mg suspended solids/L (the abiotic control was filled with 1500 mL minteral medium without inoculum). The day following incubation, the absorber wash bottles were filled with 0.2 M NaOH and the test substance was added into to the three test vessels, into the toxicity control vessel and the abiotic control vessel.
- Preparation of Test flask: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.
- Incubation: The closed test flasks were sealed and aerated with CO2-free air overight, kept mixed with magnetic stirrers.
- Measuring equipment: Total carbon analyser TOC-L, Shimadzu Deutschland, Duisburg, total carbon analyser TOC-5050A with autosampler ASI 5000A, Shimdzu Deutschland, Duisburg
- Details of trap for CO2 and volatile organics if used: CO2-free air production system concists 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. At end of system there is one gas wash bottle filled with demineralised water, followed by an empty one to catch drops of condensation. A colour change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air was 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
- Sampling frequency: On the 3rd, 7th, 10th, 14th, 21st, and 28th day as well as beginning and end of study
- Sampling method: Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes. On the 3rd, 7th, 10th, 14th, 21st, and 28th day, 6 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the inorganic carbon (IC) was 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 HCl was added into each reactor to release the CO2 dissolved in water. on day 29 the IC is determined in both CO2-absorber flasks in line.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 replicates
- Abiotic sterile control: 1 replicate
- Toxicity control: 1 replicate
- Other: Reference compound control (positive control): 3 replicates
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
-17.5
Sampling time:
28 d
Remarks on result:
not determinable
Remarks:
The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10 day window)
Details on results:
The test vessels are aerated by the passage of carbon dioxide-free air and are incubated under aerobic conditions in diffuse light for 28 days. Degradation is followed by determining the carbon dioxide produced and absorbed to sodium hydroxide via inorganic carbon measurement. The amount of carbon dioxide produced from the test item minus the amount derived from the blank inoculum is expressed as a percentage of ThCO2. The pass level for ready biodegradability is 60% of ThCO2 and must be reached within a 10 day window (which begins when the degree of biodegradation reaches 10%).
No degradation of the test item could be observed during the test duration (28 days after acidification). The test item did not reach the criteria for ready biodegradability according to the OECD criteria. The biodegradation extent of the test item at the end of the test showed a negative value (-17.5%, mean of three replicates). The negative degradation value at the end of the test may indicate a slight inhibitory effect on the inoculum.
Results with reference substance:
The reference compound sodium benzoate reached the pass level for ready biodegradability within 3 days.

Biodegradation of the Toxicity Control

In the toxicity control containing both the test item and the reference item sodium benzoate, 40.5% ThCO2 biodegradation was noted within 14 days. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Abiotic Control

The degradation of the abiotic control at the end of the test was 21.6%

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No degradation of 4,4'-Dimethoxytrityl chloride was observed during the test duration of 28 days after acidification. Therefore, 4,4'-Dimethoxytrityl chloride is not biodegradable in the test conditions based on OECD 301 B, CO2 Evolution Test according to the Modified Sturm Test.
Executive summary:

The biodegradation of 4,4'-Dimethoxytrityl chloride was studied in a suspension in mineral medium inoculated with activated sludge (30 mg suspended solids/L). The experiment was conducted in accorded with the OECD test guideline 301 B "CO2 Evolution Test / Modified Sturm Test" and in compliance with the OECD-GLP standards. The test system used a CO2-free air production system consisting of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime followed by one bottle filled with 0.1 M NaOH to monitor CO2 absorption capacity. Gas wash bottles of 2000 mL volume with lateral connecting pieces for butyl rubber septa were used as reactors, with liquid volume fixed at 1500 mL per bottle. CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles filled with NaOH and sampled with PE syringes. The amount of CO2 produced from the test item minus the amount derived from the blank inoculum is expressed as a percentage of theoretical amount of CO2.

4,4'-Dimethoxytrityl chloride never reached the pass level for ready biodegradability (60% ThCO2 in a 10 -day window, which begins when the degree of biodegradation reaches 10%). No degradation of the test item could be observed during the test duration (28 days after acidification). The biodegradation extent of the test item at the end of the test showed a negative value (-17.5%, mean of three replicates).

Description of key information

No degradation of 4,4'-Dimethoxytrityl chloride could be observed during the test duration (28 days after acidification).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The biodegradation of 4,4'-Dimethoxytrityl chloride was studied in a suspension in mineral medium inoculated with activated sludge (30 mg suspended solids/L). The experiment was conducted in accorded with the OECD test guideline 301 B "CO2 Evolution Test / Modified Sturm Test" and in compliance with the OECD-GLP standards.

4,4'-Dimethoxytrityl chloride never reached the pass level for ready biodegradability (60% ThCO2 in a 10 -day window, which begins when the degree of biodegradation reaches 10%). No degradation of 4,4'-Dimethoxytrityl chloride could be observed during the test duration (28 days after acidification). The biodegradation extent of the test item at the end of the test showed a negative value (-17.5%, mean of three replicates). The negative degradation value at the end of the test may indicate a slight inhibitory effect on the inoculum.