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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:
22 February - 14 June, 2017
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
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 17 February 2017 (six days before the main test). The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 °C, for about 6 days.

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 °C). 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 viable cell number of the cultures was determined by manual colony counting. The approximately cell count of aerated inoculum was ~108/L; therefore on the day of the test this inoculum was diluted 1000x with mineral medium to reach the necessary 105-106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning (see above) improves the precision of the method.
The inoculum was not pre-adapted to the test chemical.
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
other: based on the preliminary toxicity test results
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:

Stock Solutions for Mineral Medium

In purified, deionized water analytical grade salts were added to prepare the following stock solutions:
A) Solution: KH2PO4 8.50 g
K2HPO4 21.75 g
Na2HPO4 x 12H2O 67.16 g
NH4Cl 0.50 g
Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 36.40 g
Water ad 1000 mL
C) Solution: MgSO4 x 7 H2O 22.50 g
Water ad 1000 mL
D) Solution: FeCl3 x 6 H2O 0.125 g
Water ad 500 mL
(The “D” stock solution was prepared on the day of the mineral medium preparation and was not further stored).

The mineral medium was prepared in the following ratio: 1 mL of the stock solutions A - D) were combined per 1000 mL total volume, filled with water (purified deionized) . The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found 8.97 mg/L. The pH of the mineral medium was 7.32.

- Additional substrate: No
- Test temperature: 20.7 - 21.3 °C.
- pH: The pH of the mineral medium was 7.32.
- pH adjusted: No (not necessary)
- Aeration of dilution water: Yes
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 10 replicates (+ 2 reserve)
- Method used to create aerobic conditions: Aeration of mineral medium prior start of the test
- Test performed in closed vessels due to significant volatility of test substance: Yes


SAMPLING
- Sampling frequency:
Oxygen: At the start of the test
pH: Prior study start
Temperature: Measured continuously using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator)
- Sampling method: Oxygen and pH meter with appropriate O2 and pH electrode

TEST UNITS
- Type and Size: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Identification: Each test bottle was uniquely identified with study number, test group, days of measurement and replicate number.

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 TESTS
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.

Reference substance:
benzoic acid, sodium salt
Preliminary study:
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. In the toxicity control containing both, the test item and the reference item, a mean of 43.8 % biodegradation was noted within 14 days.
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 1.29 mg O2/ mg test item of Brown 3 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 Brown 3 reached a mean of 17.8 % 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 43.8 % biodegradation was noted within 14 days and 43.4 % 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:
>= 17.3 - <= 17.9
Sampling time:
28 d
Details on results:
Validity of the Study

The study was considered as valid since oxygen depletion in the inoculum control was 1.18 mg O2/L on average, and did not exceed the validity criteria of 1.5 mg O2/L after 28 days. The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. The lowest value was 2.98 mg O2/L, which was measured on the 28th day in the toxicity control. The difference of duplicate values for the degradation at the plateau or at the end of the test was not greater than 20 %. At the biodegradation plateaus (test item, procedure control, and toxicity control groups) or at the end of the test the highest difference (~18 %) between duplicate values for degradation was calculated in the test item group on the 14th day of the test. The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. The percentage degradation of the reference item was 80.9 % on the 14th day. All validity criteria were met as required by the test guideline OECD 301.

Correction for Oxygen Uptake for Interference with Nitrification
Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5%), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels. However, for test substances containing N, serious errors can arise if the observed oxygen uptake is not corrected for the amount of oxygen used in oxidizing ammonium to nitrite and nitrate. For that reason at this N-containing test item, the oxidized nitrogen (nitrate and nitrite) concentrations were determined following each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2/L and 0.4 mg NO3/L, respectively.

The measured quantities of nitrite in the inoculum control, test item and toxicity control samples were below the LOQ in the 0-day, 7-day and 14-day samples. In the 21-day inoculum control samples in average 0.70 mg/L, in the 28-day inoculum samples in average 0.79 mg/L nitrite was detected. Similarly to the inoculum control samples, the nitrite concentrations in the 21-day and 28-day test item and toxicity control samples showed a slight increasing tendency.
The nitrate concentration of the samples was less than 0.4 mg/L on day 0, on 7th and on 14th day; but in the 21-day inoculum samples in average 0.7, in the 28-day inoculum control samples in average 0.9 mg/L, in the 21 and 28-day test item samples in average 0.7 mg/L, in the 21-day toxicity control samples in average 0.5 mg/L in the 28-day toxicity control samples in average 0.7 mg/L nitrate was detected.

According to the referred OECD 301 guideline the oxygen consumed in nitrate formation approximates 4.57 x increase of nitrate-N concentration, and the oxygen consumed in nitrite formation is 3.43 x increase of nitrite-N concentration.
In this study the change of the measured dissolved oxygen concentrations in the inoculum control, test item and procedure control bottles did not correspond to the consumed oxygen of ammonium oxidation processes. Nitrite and nitrate was measured even in the inoculum control samples.

The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Therefore any correction of the measured dissolved oxygen concentrations was considered as not necessary or not possible. The measured relatively higher nitrite-nitrate concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Biodegradation of the Test Item
Under the test conditions the percentage biodegradation of Brown 3 reached a mean of 17.8 % 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.

Biodegradation of the Toxicity Control
In the toxicity control containing both, the test item and the reference item, a mean of 43.8 % biodegradation was noted within 14 days and 43.4 % 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:
1.29 other: mg O2/ mg test item
Results with reference substance:
Biodegradation of the Reference Item: 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.

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

8.77

7.13

7.03

6.79

6.71

4.0

1b

8.79

7.18

6.88

6.84

6.65

 

mean

8.78

7.16

6.96

6.82

6.68

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

8.75

3.76

3.37

3.15

3.11

4b

8.69

3.34

3.20

3.05

2.98

mean

8.72

3.55

3.29

3.10

3.05

Oxygen Depletion 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.

7

14

21

28

Test item

4.0

1a

0.91

0.84

0.93

0.88

1b

0.88

1.01

0.90

0.96

Reference item

3.0

2a

3.89

3.92

3.94

3.93

2b

3.98

4.17

4.06

4.09

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

4.26

4.48

4.55

4.46

4b

4.62

4.59

4.59

4.53

 

oxygen depletion : (mt0- mtx) - (mbo- mbx), where:

 

mt0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 2)

mtx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 2)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 2)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 2)

BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Bottle

BOD after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

4.0

1a

0.23

0.21

0.23

0.22

1b

0.22

0.25

0.23

0.24

Reference item

3.0

2a

1.30

1.31

1.31

1.31

2b

1.33

1.39

1.35

1.36

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

0.61

0.64

0.65

0.64

4b

0.66

0.66

0.66

0.65

BOD = = mg O2/mg T.i and/or R.i.

where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

 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

17.6

16.2

18.0

17.0

4.0

1b

17.0

19.5

17.4

18.6

 

mean

17.3

17.9

17.7

17.8

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

41.1

43.2

43.9

43.1

4b

44.6

44.3

44.3

43.7

mean

42.9

43.8

44.1

43.4

Biodegradation % =

where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control


CODof the test item = 1.29 mg O2/mg test item

ThODNH3of reference item = 1.67 mg O2/mg reference item

 

The biodegradation in the toxicity control was calculated according to the following formula:

 

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

0.7

0.7

0.7

0.6

0.5

0.4

28thday

0.7

0.7

0.9

0.9

0.6

0.7

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

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

 

 

 

Analytical occasions

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

 

 

1a

1b

3a

3b

4a

4b

 

 

0 day

---

---

---

---

---

---

 

 

7thday

---

---

---

---

---

---

 

 

14thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

21stday

0.72

0.73

0.67

0.73

0.41

0.39

 

 

28thday

0.73

0.75

0.81

0.76

0.59

0.59

 

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.

 

 

Quality Control Samples

Analytical occasions

Nitrite

Nitrate

Nominal concentration

Nominal concentration

0.05 mg/L

0.5 mg/L

2 mg/L

0.5 mg/L

2 mg/L

(Measured concentrations mg/L)

7thday

0.05

0.50

2.00

0.5

2.0

21stday

0.05

0.50

-*

0.5

2.0

28thday

0.05

0.50

-*

0.5

2.1

-*   The quality control solution of 2 µg/mL concentration was prepared and measured only on March 02, 2017 (7thday of the test).

 

Measurement of the QC samples were performed for providing information about the method applicability.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In a valid and GLP-compliant ready biodegradability test according to OECD Guideline 301 D, EU Method C.4-E, and US EPA Guideline 712-C-98-076: OPPTS 835.3110, the test item was considered to be not readily biodegradable (17.8 % 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 Guideline 712-C-98-076: 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 chemical oxygen demand (COD) of 1.29 mg O2/ mg test item of the test item was determined at the start of the main experiment. 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 43.8 % biodegradation was noted within 14 days and 43.4 % 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). Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 17.8 % 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. In conclusion, the test item is regarded to be not readily biodegradable by OECD criteria. All validity criteria of the test guidelines were fulfilled.

Description of key information

The test item was considered to be not readily biodegradable (17.8 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.

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 Guideline 712-C-98-076: 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. in addition, a procedure control with the reference substance Sodium benzoate (3 mg/L), an inoculum control (containing the filtered inoculum only) and a toxicity control (containing both the test item and reference item) 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 chemical oxygen demand (COD) of 1.29 mg O2/ mg test item of the test item was determined at the start of the main experiment. As no nitrification occurred, the biodegradability value of the test item was calculated 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 43.8 % biodegradation was noted within 14 days and 43.4 % biodegradation after 28 days of incubation. Therefore, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). Under the test conditions ready biodegradation of this test item was not demonstrated. The percentage biodegradation of the test item reached a mean of 17.8 % after 28 days based on its COD. In conclusion, the test item is regarded to be not readily biodegradable by OECD criteria. All validity criteria of the test guidelines were fulfilled.

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