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Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June - 17 June, 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R,2R)-1-amino-2-difluoromethyl)-N- (1-methylcyclopropylsulfonyl)cyclo propanecarboxyamide hydrochloride
EC Number:
Cas Number:
Molecular formula:
(1R,2R)-1-amino-2-difluoromethyl)-N- (1-methylcyclopropylsulfonyl)cyclo propanecarboxyamide hydrochloride
Test material form:
solid: particulate/powder
Details on test material:
White solid

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A fresh sample of activated sludge was collected from a sewage treatment plant as recommended by the guidelines. A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 5 day before the test was started. It is a well functioning treatment plant receiving predominantly domestic sewage.

Concentration of microbial inoculum: not greater than 30 mg/L (as suspended solids (SS))

Preparation of Inoculum:
The inoculum was pretreated as follows: coarse particles were removed by filtration through a fine sieve and the sample was settled. After the supernatant was
discarded, the sludge was washed twice with mineral medium and the concentrated sludge was suspended in the mineral medium. The suspended sludge was kept in an aerobic environment at 21. 7- 23.1 °C. 4 replicates of 10 mL suspended sludge were weighed and dried at 105°C for 2 hours and 7 minutes and reweighed to calculate initial sludge concentration. The measured concentration of suspended sludge was 5.875 g/L as suspended solids (SS) and was prepared with mineral medium to yield a concentration of 4.0 g/L as SS. On the day of the test 5 replicates of 10 mL suspended sludge were weighed and dried at 105°C for 1 hour and 35 minutes and reweighed to calculate initial sludge concentration. The measured concentration of suspended sludge was 3306 mg/L as suspended solids (SS). According to the prepared concentration of suspended sludge, the added amount of wet sludge was calculated.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
44.51 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test Conditions:
Concentration of test item: 44.51 mg/L (equivalent to 70.33 mg/Las ThODNo3)
Concentration of microbial inoculum: not greater than 30 mg/L (as suspended solids (SS))
Test temperature: 22°C + 1 °C (actual temperature range 21.4 - 22.9 °C)
Test period: 28 days
The test was conducted in diffuse light.

Test Methods:
Preparation of Mineral Medium:
The stock solutions were prepared using analytical grade reagents, except that Na2HP04·2H20 was HPLC grade. For 1 liter of mineral medium, 10 mL stock solution A was added to approximately 800 mL distilled water and the mixture stirred before adding, sequentially, 1 mL each of stock solutions B, C and D. After mixing the mixture was made up to 1 liter with distilled water. Double 10 L mineral medium were prepared on the day the study started. The pH were checked to be 7.83 and 7.78, and adjusted to 7.45 and 7.46 with HCI.

Test Item Test Vessels:
This method for test item addition was as followed: 0.04416 g and 0.04486 g of test item was weighed and added directly to test vessel to give a final concentration of test item of 44.51 mg/L (average, 2 replicates), equivalent to 70.33 mg/L ThODN in the test suspension. Test item test vessels were prepared in duplicate as test suspension (identified as TS-1 and TS-2).

Reference Item Test Vessel:
A weighed quantity (0.03117 g) of reference item was added directly into test vessel. The concentration of the reference item was 31.17 mg/L sodium benzoate ( equivalent to 52.05 mg/L as ThOD). Reference item test vessel was prepared in one replicate (procedure control identified as PC).

Inoculum Blank Controls Test Vessels:
Test vessels containing only 1 L inoculated mineral medium were prepared in duplicate and served as inoculum controls identified as IC-1 and IC-2.

Abiotic Sterile Control Test Vessel:
0.04382 g oftest item was weighed into test flask and 1000 mL of sterilized mineral medium (sterilized at 121 °C for 20 min by autoclave) was added. The concentration of the test item was 43.82 mg/L, equivalent to 69.24 mg/L ThODN03. No inoculums was added to test flask which served as an abiotic sterile control identified as ASC. One replicate sufficed.

Toxicity Control Test Vessel:
Toxicity control test vessel contained both test item and reference item. Both a weighed quantity (0.04430 g) of test item and a weighed quantity (0.03202 g) of
reference item were weighed and added into test vessel. The concentration of test item was 44.30 mg/L (equivalent to 69.99 mg/L as ThODN03) and the concentration of reference substance was 32.02 mg/L (equivalent to 53.47 mg/L as ThOD). The concentration expressed as total ThOD (based on ThODN03) was 123.5 mg/L. One replicate (toxicity control identified as TC) sufficed.

Measuring of Oxygen Uptake:
Oxygen uptake values in all test flasks were recorded at 24 hour interval for the 28 day period by the automated respirometer. At the end of incubation, collect the data stored in the controller and save it on the PC's fixed disc and then convert the raw data files into Excel file. Incubation temperature was measured continuously with minimum/maximum thermometer and the minimum and maximum values were recorded at least on every working day during the test, the measured range was 21.4 -22.9 °C. In addition, the magnetic stirrer was checked on every working day to determine proper operation.

Physico-Chemical Measurements
The pH of the various test vessels was measured at the end of the incubation period (on Day 28)
Reference substance
Reference substance:
other: Sodium benzoate

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
According to the results of nitrate and nitrite determination, the complete nitrification occurred during the biodegradation course, the ThODNo was used to calculate the percentage biodegradation. Based on the percentage biodegradation expressed as an average, the percentage biodegradation of the test item after 12 days averaged 10.4% (2 replicate), which was the initial day on which 10% biodegradation was exceeded, and when the 10-day window began. The percentage biodegradation in activated sludge was 17.3% (2 replicate) within a 10 day window (after 22 days), and 19.3% after 28 days. The percentage degradation of the test item did not reach the pass level of 60% at the end of the test.

BOD5 / COD results

Key result
Results with reference substance:
Biodegradation of the reference substance (Sodium benzoate) attained 62.0% after Day 5, 86.4% after Day 14 and 96.5% after Day 28. In the Toxicity Control (TC) test mixture, 42.4% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test
item could be assumed to be not inhibitory under the conditions of this test. In the Abiotic Sterile Control (ASC) Test Vessel, 19.6% degradation occurred after 28 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Based on these results, the test item, Difluorosulfonamide HCl was not readily biodegradable under the described test conditions.