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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 29th, 2015 - February 27th, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test item: SE7B
CAS-No.: 1365345-64-7
Lot No. of test item: 0000140602
Purity: 98%
Organic Carbon: 77.0% (analysed by ASG Analytik-Service GmbH)
Water solubility: insoluble (information from the sponsor)

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:

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 January 27th, 2015. Dry solid of the activated sludge was determined as 4.5 g/L by weight measurements after drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice with tap water and one time with mineral medium by settling the sludge, decanting the supernatant and resuspending the sludge.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Method
The CO2-evolution test determines the ready biodegradability of test items according to the Modified Sturm Test. A solution or suspension of the test item in a mineral medium, corresponding to 10 - 20 mg TOC/L, is inoculated with activated sludge (30 mg d. s./L). 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 IC-measurement (IC = inorganic carbon). The amount of carbon dioxide produced from the test item less the amount derived from the blank inoculum is expressed as a percentage of ThCO2 (theoretical amount of CO2). The pass level for ready biodegradability is 60% of ThCO2 and must be reached within a 10-d window. The 10-d window begins when the degree of biodegradation reaches 10%. The test method is suitable for water soluble and non-soluble test items which are non-volatile and not toxic under the test conditions.

Solutions
-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.

CO2-absorption medium
72.14 g NaOH was dissolved in 9 L deionised water in closed recipients (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined.

Test item
Due to the insolubility of the test item in water, the test item was added directly into the test vessels. Carrier material was inert PE film. 38.9 – 39.1 mg of the test item was weighed on PE film and added into each vessel. This corresponds to a test concentration of 20 mg/L organic carbon. PE-film was used as an inert carrier material.

Reference substance
A stock solution of 10 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 mg/L organic carbon.

Toxicity control
39.0 mg of the test item and 5.15 ml of the reference stock solution were added to the toxicity control vessel corresponding to a concentration of 40 mg/L organic carbon.

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 was one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A colour 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.

In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.

Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septums were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by a magnetic stirrer with 2 cm stir bars. 38.9 – 39.1 mg of the test item were added into the three test vessels (corresponding to a TOC concentration of 20.0 mg/L). The reference compound (5.15 mL of a 10 g/L stock solution) was added to the reference vessels. 39.0 mg of the test item and 5.15 ml of the reference stock solution were added to the toxicity control vessel.

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
10 mL activated sludge was filled up to 1500 mL with 1490 mL mineral medium corresponding to 30 mg/L dry solids. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers. On the next day, after filling the absorber wash bottles with 0.2 M NaOH, the test item and reference compound were introduced and the test started. 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 at day 11 and 28. 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 2.9 ppm. The IC in the reactor at the beginning of the test showed insignificant amounts of IC (0.5 mg/L). On the 4th, 7th, 11th, 14th, 21st and 28th day 4 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.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
89.5
Sampling time:
29 d
Details on results:
Summary of the results
Test item:
The degradation extent of the test item at the end of the test was 89.5% (28 d after acidification, mean value of three replicates).
The mean degradation extent on day 11 was already higher than 60% (81.1%, mean value of three replicates). Therefore the criteria for ready biodegradation was met (10-d window).
The test item is ultimately biodegradable and reached the criterion for ready biodegradation.

Toxicity control:
The degradation extent in the toxicity control was 66.5% within 14 days, the test substance had no inhibitory effect on the inoculum.

Reference item:
The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days.

Blank:
The mean CO2-evolution of the blank flasks was 33.3 mg/L on day 28 after acidification.

Before adding the test item, the IC in the reactor was determined, but only insignificant amounts of IC (0.6 mg/L) were found. The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 10 ppm and was not considered in the data processing, because CO2 absorption from room air was its source.
The temperature was 20 – 23.°C throughout the whole study. The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 2.6 – 5.0 bubbles/second).
The degradation extents after acidification are lower than the one before acidification. This is caused by a higher CO2-evolution after the acidification in the blanks than in the test vessels.

Any other information on results incl. tables

Reactor

Day

0

4

7

11

14

21

28

29

7

Test flasks

0

56.6

79.6

92

94.1

104.7

102.6

99.6

8

0

51.6

72.1

76

77.2

82

80.2

78.2

9

0

37.8

63.1

75.3

79.5

91.4

92.3

90.7

4

Reference Flasks

0

72.2

81.1

84.6

88.4

93.7

93

91.9

5

0

73.4

84.8

91.5

91.2

98.6

99.1

97.3

6

0

84

95.7

99.1

99.7

106.9

104.1

103

19

Toxicity control:
Test item

0

39.8

51.3

62.8

66.5

85.4

96

96.4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The degradation extent of the test item at the end of the test was 89.5% (28 d after acidification, mean value of three replicates).
The mean degradation extent on day 11 was already higher than 60% (81.1%, mean value of three replicates). Therefore the criteria for ready biodegradation was met (10-d window).