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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:
2017-02-22 to 2017-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
17th July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
- Origin: sewage plant for domestic sewage in Balatonfüred, Hungary (sampling date: 17 February 2017)
- Preparation of Activated Sludge Inoculum: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium and then aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.69, just before use the pH was: 7.28. A pH adjustment of activated sludge inoculum was not performed.
- Pre-conditioning of Activated Sludge Inoculum: Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium ) for 6 days (from February 17 to February 23, 2017) at test temperature (the actual temperature: 20.3 – 21.4 oC). During the aeration the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, 10-1, 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. Plates were incubated at 37 °C for 24 hours. The inoculum was not pre-adapted to the test chemical.
- Initial cell concentration: 105-106 cells/L

Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: According to guideline
- Additional substrate: No
- pH adjusted: No
- Aeration of dilution water: Yes
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 10 (+ 2 reserve) containing the test item and inoculum
- Measuring equipment: built-in thermometer, oxygen and pH meter with appropriate O2 and pH electrode,
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No

SAMPLING
- Sampling frequency: Oxygen measurements were performed in all duplicate bottles in all test groups on days 0, 7, 14, 21 and 28.
- Sampling method: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].


CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 (+2 reserve) bottles containing only inoculum
- Positive control: 10 (+2 reserve) bottles containing the sodium benzoate and inoculum
- Abiotic sterile control: Not performed
- Toxicity control: 10 (+2 reserve) bottles containing the test item, sodium benzoate and inoculum
Reference substance:
benzoic acid, sodium salt
Remarks:
Supplier: Reanal, Budapest Hungary, Manufacturer: ACRÕS
Preliminary study:
Preliminary Experiments
The pre-experiments on solubility of the test item, and the 14-day toxicity test for the determination of the test concentration for the main test were conducted non-GLP, and these pre-experiments are excluded from the Statement of Compliance in the final report. The raw data of these tests will be archived under the study code of the present study.

Preliminary Toxicity Test
The test item solubility, behavior, and toxicity were tested in a 14-day preliminary experiment. The test design was the same as described at the main experiment. In the preliminary experiment the test item was investigated at the concentration of 4 mg/L. No toxic effect of the test item was found at this investigated concentration.
Test performance:
The chosen test item concentration of 4.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. The chemical oxygen demand (COD) of 2.41 mg O2/ mg test item of Leuco Sulfur Blue 13 was determined at the start of the main experiment. Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of Leuco Sulfur Blue 13 reached a mean of 9.9 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 7th day of the experiment. From this day the slight changes were considered as being within the biological variability range of the applied test system.

The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the slight changes in nitrite and nitrate concentrations in the 21-day and 28-day samples were caused likely by a technical effect: turbidity and/or discoloration). Therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed.

The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 31.4 % biodegradation was noted within 14 days and 32.4 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).
Key result
Parameter:
% degradation (O2 consumption)
Value:
> 9.4 - <= 10.4
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Leuco Sulfur Blue 13 reached a mean of 9.9 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 7th day of the experiment. From this day on, the slight subsequent changes were considered as being within the biological variability range of the applied test system. The test item can be considered to be not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 31.4 % biodegradation was noted within 14 days and 32.4 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).
Key result
Parameter:
COD
Value:
2.41 g O2/g test mat.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Table 1: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Bottle

mg O2/L after n days of exposure

[mg/L]

No.

0

7

14

21

28

Test item

 

1a

8.67

7.07

6.95

6.67

6.49

4.0

1b

8.59

7.05

6.87

6.62

6.50

 

mean

8.63

7.06

6.91

6.65

6.50

Reference item

 

2a

8.69

4.07

3.87

3.70

3.58

3.0

2b

8.76

4.05

3.69

3.65

3.49

 

mean

8.73

4.06

3.78

3.68

3.54

Inoculum control

3a

8.73

8.03

7.86

7.71

7.67

3b

8.71

7.95

7.78

7.63

7.41

mean

8.72

7.99

7.82

7.67

7.54

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

8.54

3.46

3.24

2.92

2.72

4b

8.49

3.71

3.01

2.73

2.70

mean

8.52

3.59

3.13

2.83

2.71

 

Table 2: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Bottle

Percent of biodegradation after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

 

1a

9.0

8.5

9.8

10.4

4.0

1b

8.4

8.5

9.5

9.4

 

mean

8.7

8.5

9.7

9.9

Reference item

 

2a

77.8

78.4

78.8

78.6

3.0

2b

79.6

83.4

81.2

81.8

 

mean

78.7

80.9

80.0

80.2

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

30.5

30.8

32.0

32.5

4b

28.4

32.1

33.0

32.3

mean

29.4

31.4

32.5

32.4

Nitrate Concentrations

Analytical occasions

Measured nitrate concentration (mg/L) in the test bottles

1a

1b

3a

3b

4a

4b

0 day

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

7thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

14thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

21stday

<0.4

0.5

0.7

0.6

<0.4

<0.4

28thday

0.9

0.9

0.9

0.9

<0.4

<0.4

Remarks:            LOQ of nitrate determination: 0.4 mg NO3/L

1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers.

 

Table 3  Measured nitrite concentration (mg/L) in the test bottles

 

Nitrite Concentrations

 

Analytical occasions

Measured nitrite concentration (mg/L) in the test bottles

 

 

1a

1b

3a

3b

4a

4b

 

 

0 day

---

---

---

---

---

---

 

 

7thday

---

<0.03

---

---

---

---

 

 

14thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

21stday

0.24

0.56

0.67

0.73

0.11

0.19

 

 

28thday

0.76

0.67

0.81

0.76

0.21

0.24

 

Remarks:            LOQ of nitrite determination: 0.03 mg NO2/L

                              1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers,

                              --- : nitrite concentrations were not detectable.

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was considered to be not readily biodegradable (9.9 % biodegradation on day 28).
Executive summary:

The ready biodegradability of the test item was assessed in a GLP-compliant study according to OECD Guideline 301 D, EU Method C.4 -E and US EPA OPPTS 835.3110. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally inoculum (containing the filtered inoculum only) and toxicity (containing both the test item and reference item) controls were examined. The chosen test item concentration of 4.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. The biodegradability value of the test item was calculated based on its COD as concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. In result, the percentage biodegradation of the test item reached a mean of 9.9 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 7thday of the experiment. From this day the slight changes were considered as being within the biological variability range of the applied test system. The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 31.4 % biodegradation was noted within 14 days and 32.4 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). The validity criteria of the guidelines were fulfilled.

Description of key information

The substance was considered to be not readily biodegradable (9.9 % biodegradation on day 28).

Key value for chemical safety assessment

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

Additional information

The ready biodegradability of the test item was assessed in a GLP-compliant study according to OECD Guideline 301 D, EU Method C.4 -E and US EPA OPPTS 835.3110. The test item (4.0 mg/L) was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. Additionally, a positive control with the reference substance Sodium benzoate, an inoculum control and a toxicity control (containing both the test item and reference item) were examined. The biodegradability value of the test item was calculated based on its COD as concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. In result, the percentage biodegradation of the test item reached a mean of 9.9 % after 28 days based on its COD. The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3,thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 31.4 % biodegradation was noted within 14 days and 32.4 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). The validity criteria of the guidelines were fulfilled.