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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:
From 2017-02-02 to 2017-06-27
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2016-08-29
Specific details on test material used for the study:
- Elementary analysis: C = 50.4% / H = 5.58% / N = <0.5% / O = 44.7% (analysed by ASG Analytik-Service Gesellschaft mbH)
- Idealised molecular formula: C41.97H55.36O27.94 (calculation by Hydrotox on the basis of the elementary analysis)
- ThOD: 1.33 mg O2/mg substance (calculated by Hydrotox on the basis of the idealised molecular formula)
- Analytical purity: >=99%
- Purity test date: 24 August 2016
- Lot/batch No.: BA5H22021
- Expiration date of the lot/batch: 24 August 2017
- Appearance: Colourless limpid liquid
- Storage: In darkness at room temperature


Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Remarks:
from municipal wastewater treatment plant Breisgauer Bucht
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant Breisgauer 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 600.000 inhabitant equivalents. Sampling date of activated sludge was on 03 April 2017. The dry solid content of the activated sludge was 5.09 g/L. It was determined by weight measurements after drying at 105°C for 4 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):
28 d
Initial conc.:
75.1 mg/L
Based on:
test mat.
Initial conc.:
99.9 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- 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.
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.
Before use, the mineral medium is aerated for about one hour and the pH is adjusted to 7.4 ± 0.2.
- Temperature: 21.9-22.6°C

TEST SYSTEM
- Experimental set up:
The system OxiTop®- Control from WTW, Weilheim and the Sensomat-System from Aqualytic GmbH & Co., Langen were used as test systems. They consists of narrow-necked glass bottles with rubber sleeve inserts for NaOH pellets, in which the carbon dioxide evolved is absorbed. The liquid volume was fixed as 164 mL each. Magnetic stir bars were introduced into the bottles which were positioned on a stirrer platform. The bottles were sealed tightly with the measuring heads with a measuring range of 500 – 1350 hPa. Every 112 minutes the current pressure was measured and stored by each measuring head. At the end of the experiment pressure data were read out via an infrared interface to the controller unit and afterwards data were transferred via a RS232 interface using the Achat OC software of WTW to an Excel file where further data processing was carried out. Additionally several measured values were randomly read out and recorded by hand and were afterwards compared with the printed excel table for quality control.

- Procedure:
In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound (reference) and three reactors containing reference compound and test item (toxicity control) were set up. 164 mL of the pre-aerated dilution water with a pH of 7.5 were filled into the blank flasks. The test, reference, toxicity control and abiotic control vessels were filled with 164 mL of the corresponding stock solutions. After tempering the flasks to the incubation temperature for about 1.25 hours, 967 µL of the inoculum were added into each flask, one sodium hydroxide pellet was added to each rubber quiver inserted in the flasks and the flasks were sealed tightly with the measuring heads and the test was started. After 28 days the data were read out and the pH in the flasks was measured on the next day.

- Test item:
A stock solution of 75.1 mg/L was prepared with mineral medium. This corresponds to a concentration of 99.9 mg ThOD/L. 164 mL of the stock solution were added into the test vessels. The pH was 7.3 and was not adjusted.

- Reference substance:
A stock solution of 128.2 mg/L sodium acetate in mineral medium was prepared. 164 mL of this stock solution were added into the reference vessels corresponding to a concentration of 100.0 mg ThOD/L. The pH was 7.5 and was not adjusted.

- Toxicity control:
A stock solution with 75.1 mg/L test item and 128.1 mg/L sodium acetate in mineral medium was prepared. 164 mL of this stock solution were added into the toxicity control vessel. This corresponds to a concentration of 199.8 mg ThOD/L. The pH was 7.3 and was not adjusted.










Reference substance:
acetic acid, sodium salt
Test performance:
Validity of the test
• The oxygen uptake of the inoculum blank was in the range of 20 - 30 mg O2/L and did not exceed 60 mg O2/L in 28 days.
• The pH value in the test and blank bottles was 7.5.
• The difference of extremes of replicate values was less than 20%.
• The biodegradation of the reference item reached the pass level of 60% ThOD by day 8.
• The degradation extent in the toxicity control was above 25% in 14 days based on ThOD.
The test is valid according to OECD Test Guideline 301 F (July 1992).
Key result
Parameter:
% degradation (O2 consumption)
Value:
87.4
Sampling time:
28 d
Remarks on result:
other: 10-day window criterion fulfilled
Details on results:
Test item
The biodegradation of the test item was 87.4% ThOD within 28 days (mean of three replicates). On day three, the degradation of the test item was for the first time higher than 10%, therefore the end of the 10-d window is on day 13. A degradation of 60% was reached on day 11. Therefore, the test item reaches the criteria for ready biodegradability (60% of ThOD within a 10-d window). The pH in the test vessels at the end of the test was 7.5.

Toxicity control
The degradation in the toxicity control reached 43.6% within 4 days (see table 2) and was thus above the criterion for inhibition effects to the inoculum (<25% on day 14). The test item had no toxic effect to the inoculum according to the validity criteria of OECD 301. The pH in the toxicity control vessel at the end of the test was 8.8 – 8.9.

Blank
The oxygen consumption of the blanks was 24.4 mg/L in 28 days (mean of three replicates). In one of the blank flasks a slight decrease of the oxygen consumption was measured between day 24 and day 28 (see table 1). This decrease is within the normal fluctuation range. The pressure values measured by the measuring heads do not remain completely stable but can vary slightly from one measuring point to another, possibly due to slight variations in temperature
The pH in the blank vessels at the end of the test was 7.5.

Test parameters
The temperature was 21.9 – 22.6°C throughout the whole study.



Results with reference substance:
The reference compound sodium acetate reached the pass level for ready biodegradability (60% ThOD within a 10-day window) within 8 days. The pH in the reference vessels at the end of the test was 9.0.

Biodegradation in % of ThOD

 

Test item*

Toxicity control**

Reference item***

Test item

x

x

x

x

x

x

 

Reference item

 

x

x

x

x

x

x

ThOD [mg/l]

99.9

99.9

99.9

199.8

199.8

199.8

100.0

100.0

100.0

Day

Degradation [%]

0

0

0

0

0

0

0

0

0

0

4

17.8

17.8

15.0

44.0

44.0

42.7

57.1

57.1

57.1

8

42.3

42.3

42.3

63.1

63.1

63.1

73.1

70.3

73.1

12

72.3

66.6

66.6

81.2

81.2

78.2

80.7

77.9

80.7

16

76.1

76.1

73.3

85.8

85.8

84.3

81.5

81.5

81.5

20

85.7

79.7

79.7

90.4

89.4

89.4

85.6

79.6

85.6

24

89.8

81.8

81.8

91.5

91.5

89.9

84.7

81.7

84.7

28

90.7

85.7

85.7

91.9

93.4

91.9

85.6

82.6

85.6

*164 mL of a stock solution with 75.1 mg/L test item

**164 mL of a stock solution with 75.1 mg/L test item and 128.1 mg/L reference item

***164 mL of a stock solution with 128.2 mg/L reference item

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The biodegradation of the test item within 28 days was 87.4% ThOD (mean of three replicates) and reached the criteria for ready biodegradability (60% ThOD within a 10-d window). The degradation of the toxicity control after 14 days was >25%; the test item had no inhibitory effect on the inoculum according to the criterion of the guideline.
Executive summary:

The aerobic biodegradability of the REACH substance was investigated in a GLP-compliant ready biodegradability study performed in accordance with OECD Guideline No. 301 F. The biodegradation of the test item within 28 days was 87.4% ThOD and reached the criteria for ready biodegradability (60% ThOD within a 10-d window). The degradation of the toxicity control after 14 days was >25%; the test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Description of key information

The aerobic biodegradability of the REACH substance was investigated in a GLP-compliant ready biodegradability study performed in accordance with OECD Guideline No. 301 F. The biodegradation of the test item within 28 days was 87.4% ThOD and reached the criteria for ready biodegradability (60% ThOD within a 10-d window). The degradation of the toxicity control after 14 days was >25%; the test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The aerobic biodegradability of the REACH substance was investigated in a GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.