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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2016-02-18 to 2016-03-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot number: 151104
Purity: 99.1%
Appearance: Clear and colourless liquid
Storage conditions: Stored in an a dark storage place at room temperature
Oxygen conditions:
aerobic
Inoculum or test system:
other: Secondary effluent of municipal sewage treatment plant
Details on inoculum:
Secondary effluent was sampled from Kurume central sewage treatment centre (Kurume, Fukuoka, Japan).
Collected secondary effluent was filtered through a No. 2 filter paper and filtrate was used as inoculum. The inoculum was kept under aerobic condition until used.
Duration of test (contact time):
28 d
Initial conc.:
7.89 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Test preparations:
Mineral medium (5 L) was prepared by adding purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical) to each 1 mL of aliquot of solutions A, B, C and D (as prescribed in the OECD 301D test guideline) in order to prepare 1 L of solution. After the preparation, it was aerated for 35 minutes and stood still for 23 hours at test temperature. The prepared inoculum was added to the mineral medium at the rate of 0.05 mL of the inoculum per litre of mineral medium. Sodium benzoate was used to confirm the inoculum was sufficiently active.


Test solutions preparation:
a) Test solution (water + test item) (n=2)
Each test bottle was filled with purified water and 1.0 µL of test sample (0.789 mg as the test item) was added via microsyringe, so that test item concentration was 7.89 mg/L.

b) Test solution (inoculum + test item) (n=12)
Each test bottle was filled with the inoculated mineral medium and 1.0 µL of test sample (0.789 mg as the test item) was added via microsyringe, so that test item concentration was 7.89 mg/L.

c) Test solution (inoculum + sodium benzoate) (n=10)
Each test bottle was filled with 3.00 mg/L sodium benzoate solution prepared with the inoculated mineral medium.

d) Test solution (control blank) (n=11)
Each test bottle was filled with the inoculated mineral medium.

Test solutions were incubated for 28 days under dark conditions (incubator: SANYO Electronic, type MIR-552) at 20 ± 1°C and continuously stirred in sealed test bottle (stirrer: TGK, type F-626N), since the test item was insoluble in the test solutions. Test solutions were observed once a day during incubation.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
N/A
Test performance:
The average recovery rates in each test solution were >90% and the difference between replicate values in each were within 5%. In addition, no peak exceeding the limit of quantitation appeared around the peak of the test item on the chromatogram of the test solution (control blank). Therefore, the validity of the pre-treatment was confirmed.

All validity criteria set by the guideline were met, thus the tests were considered valid.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
other: mean value
Parameter:
% degradation (test mat. analysis)
Value:
0
Sampling time:
28 d
Remarks on result:
other: mean value
Details on results:
The test item did not biodegrade under the test conditions of the study, showing mean percentage biodegradation values of 0%.
Results with reference substance:
The reference substance, sodium benzoate, achieved a mean percentage biodegradation (by BOD) value of 70% after 14 days and a mean value of 89% after 28 days.

Percentage biodegradation:

 

Test solution (inoculum + test item)

The value in [ ] is a mean value

7 days

14 days

21 days

28 days

Percentage biodegradation by BOD

0

0

0

0

0

0

0

0

[0]

[0]

[0]

[0]

Percentage biodegradation of test item (by GC)

-

-

-

0

-

-

-

1

-

-

-

[0]

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No biodegradation was observed throughout the duration of the study, therefore the test item is concluded to be not readily biodegradable.
Executive summary:

The biodegradability of test item was investigated according to the OECD 301D guideline (Ready Biodegradability: Closed Bottle Test).

In the test solutions there was no discernible decline in the test item and no peak except for the test item peak was detected on the GC chromatograms. Furthermore, the mean percentage biodegradation by BOD was 0% after 28 days. These results indicated that the test item did not undergo biodegradation under the test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2018-08-30 to 2018-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Remarks:
SEPA GLP
Specific details on test material used for the study:
Batch No.: 707001
Purity: 98.5%
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 7 day before the test was started. It is a well functioning treatment plant receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: 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 mineral medium.
- Concentration: The measured concentration of suspended sludge was 17.45 g/L as suspended solids (SS) and was prepared with mineral medium to yield a concentration of 4 g/L as SS. Keep the suspended sludge aerobic at 20.9°C ~ 23.2 °C until the day of the test. On the day of the test 5 replicates of 10 mL suspended sludge was weighed and dried at 105°C for 1.5 hours and reweighed to calculate initial sludge concentration. The measured concentration of suspended sludge was 3724 mg/L as suspended solids (SS).
- Concentration of microbial inoculum: 30 mg/L (as suspended solids (SS))
Duration of test (contact time):
28 d
Initial conc.:
18.61 mg/L
Based on:
test mat.
Initial conc.:
52.47 mg/L
Based on:
ThCO2
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
The stock solutions were prepared as follows using analytical grade reagents, except that Na2HPO4•2H2O was HPLC grade.
(A) Dissolve 4.2505 g potassium dihydrogen orthophosphate (KH2PO4), 10.8756 g dipotassium hydrogen orthophosphate (K2HPO4), 16.7019 g disodium hydrogen orthophosphate dihydrate (Na2HPO4•2H2O), and 0.2503 g ammonium chloride (NH4Cl) in distilled water and make up to 500 mL. The pH of the solution was 7.55.
(B) Dissolve 3.6403 g Calcium chloride dihydrate (CaCl2•2H2O) in distilled water and make up to 100 mL.
(C) Dissolve 2.2504 g magnesium sulfate heptahydrate (MgSO4•7H2O) in distilled water and make up to 100 mL.
(D) Dissolve 0.0251 g iron (III) chloride hexahydrate (FeCl3•6H2O) in distilled water and make up to 100 mL.
There were no precipitates formed in above stock solutions, and the solutions were used for preparation of mineral medium.
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 was checked to be 7.56.
- Test temperature: 21.2°C ~ 22.8 °C
- pH: 7.10-7.34
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no, conducted in diffuse light.

TEST SYSTEM
- Number of culture flasks/concentration: duplicate for test item vessel (TS-1, TS-2)
- Measuring of oxygen uptake:
Respirometer system preparation: tighten the reference pressure vessel, fill the electrolyte bottle with 45 mL of 25% (w/v) CuSO4.5H2O solution and 2 ~ 3 g CuO and fit the electrolyte bottle into the probe housing and tighten.
After the test flasks were prepared, the following operation was performed as soon as possible: Insert the magnetic stirring rod into the flasks, fill CO2 trap cuvette with 4 mL of 10 mol/L NaOH solution and push the cuvette on to the sensor head. Attach the test flasks to the sensor head. Immerse the sample flasks in the constant temperature water bath (22°C ± 1°C) and start the stirring.
Oxygen uptake values in all test flasks were recorded at 24 hour interval for the 28 day period by the automated respirometer.
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. In addition, the magnetic stirrer was checked on every working day to determine proper operation.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 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: 0.01860 g of test item was weighed into test flask and 950 mL of mineral medium with 100 mL of 3,5-dichlorophenol solution in mineral medium at the concentration of 500 mg/L was added. The concentration of 3,5-dichlorophenol in final volume was 50 mg/L into the flask. The concentration of test item was 18.60 mg/L, equivalent to 52.45 mg/L ThOD NH3. No inoculums were added to test flask which served as an abiotic sterile control identified as ASC. One replicate sufficed.
- Toxicity control: oxicity control test vessel contained both test item and reference item. Both a weighed quantity (0.01796 g) of test item and a weighed quantity (0.03222 g) of reference item were weighed and added into test vessel. The concentration of test item was 17.96 mg/L (equivalent to 50.65 mg/L as ThODNH3) and the concentration of reference substance was 32.22 mg/L (equivalent to 53.81 mg/L as ThODNH3). The concentration expressed as total ThOD (based on ThOD NH3) was 104.46 mg/L. One replicate (toxicity control identified as TC) sufficed.
- Other: Procedure control: A weighed quantity (0.03122 g) of reference item was added directly into test vessel. The concentration of the reference item was 31.22 mg/L sodium benzoate (equivalent to 53.14 mg/L as ThODNH3). Reference item test vessel was prepared in one replicate (procedure control identified as PC).
Reference substance:
benzoic acid, sodium salt
Test performance:
The following validity criteria were met by this test:
1) The oxygen uptake of the inoculum blank was 28.37 mg O2/L in 28 days, which did not exceed 60 mg O2/L.
2) The pH values were in the range of 7.10 ~ 7.34, which were in the range of 6 ~ 8.5.
3) The difference of extremes of replicate values of the removal of the test item at the end of the test was 0.5%, it was less than 20%, which is specified criterion in test guideline.
4) The percentage degradation of the reference item reached 71.1% after day 3, and was 91.4% after day 14 which exceeded the required pass level (the percentage degradation of reference item after day 14 ≥ 60%).
5) In Toxicity Control (TC) test mixture, containing both test item and reference item, 28.0% ThOD degradation occurred within 2 days and 45.3% ThOD within 14 days based on total ThOD. Since these values exceeded the 25% ThOD limit for toxicity control, it was concluded that the test item is not inhibitory to sludge micro-organisms.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0.3
Sampling time:
28 d
Details on results:
Based on the percentage biodegradation expressed as average, the percentage biodegradation of test item after 28 days averaged 0.3% (2 replicate). The percentage degradation of the test item did not reach the pass level of 60% at the end of the test.

In the Toxicity Control (TC) test mixture, 45.3% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test item can be assumed to be not inhibitory under the conditions of this test.
In the Abiotic Sterile Control (ASC) Test Vessel, degradation was calculated to be 11.6% after 28 days.
Results with reference substance:
Biodegradation of the reference substance (Sodium benzoate) attained 71.1% after Day 3, 91.4% after Day 14 and 91.7% after Day 28.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item is not readily biodegradable under the described test conditions.
Executive summary:

The ready biodegradability test of test item was conducted with unacclimatised sewage micro-organism by measuring oxygen consumption over the 28-day test period according to OECD 301F.

Based on the percentage biodegradation expressed as average, the percentage biodegradation of test item after 28 days averaged 0.3% (2 replicate). The percentage degradation of the test item did not reach the pass level of 60% at the end of the test.

Biodegradation of the reference substance (Sodium benzoate) attained 71.1% after Day 3, 91.4% after Day 14 and 91.7% after Day 28.

In the Toxicity Control (TC) test mixture, 45.3% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test item can be assumed to be not inhibitory under the conditions of this test. 

In the Abiotic Sterile Control (ASC) Test Vessel, degradation was calculated to be 11.6% after 28 days.

Based on these results, the test item is not readily biodegradable under the described test conditions.

Description of key information

Two Ready Biodegradability tests are available for the submission substance.

 

Ready Biodegradability: Closed Bottle Test was conducted according to the OECD 301D.

In the test solutions there was no discernible decline in the test item and no peak except for the test item peak was detected on the GC chromatograms. Furthermore, the mean percentage biodegradation by BOD was 0% after 28 days. These results indicated that the test item did not undergo biodegradation under the test conditions.

 

The ready biodegradability: Manometric Respirometry Test of test item was conducted with unacclimatised sewage micro-organism by measuring oxygen consumption over the 28-day test period according to OECD 301F.

Based on the percentage biodegradation expressed as average, the percentage biodegradation of test item after 28 days averaged 0.3% (2 replicate). Therefore, the test item is not readily biodegradable under the described test conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information