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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:
02 November 2016 - 08 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998

Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Date of production: 26.03.2015
Expiration date: 26.03.2020
Oxygen conditions:
anaerobic
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, on 27 October 2016
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 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.61, just before use the pH was: 7.31. 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, mineral medium ) for 7 days (from October 27 to November 03, 2016) at the test temperature (the actual temperature: 20.3 – 21.9 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, usually 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of ~109/L; therefore on the day of the test this inoculum was diluted 10000 x with mineral medium to reach the necessary 105-106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning 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.:
3 mg/L
Based on:
other: based on the results of the preliminary solubility and toxicity tests
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test solutions used in the test were prepared by mechanical dispersion freshly, at the beginning of the experiment, in the testing laboratory as follows:
At first the suitable amount (60 mg) of C. I. Leuco Sulfur Green 2 was suspended (using ultrasonic bath for about 10 min.) in the respective volume (1000 mL) of aqueous test medium (mineral medium) and a stock solution with a concentration of 60 mg/L was prepared. The test item stock solution was continuously stirred until use to ensure a good dispersion and homogeneity (extra care was taken to avoid of air bubbles in the stirred solution). This stock solution was adequately diluted in the test item containing test groups. During the incubation period the test solutions were not stirred.
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 18.20 g
Water ad 500 mL
C) Solution: MgSO4 x 7 H2O 11.25 g
Water ad 500 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).

Preparation of Mineral Medium (Ratio of Ingredients):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 8.76 mg/L. The pH of the mineral medium was 7.23.

Environmental Conditions:The test was carried out in a controlled environment room at a temperature of 22 ± 2°C according to the guideline. The actual temperature range was 20.5 - 21.9 °C. The test bottles were incubated in incubator at 22 ± 2 °C, in the dark. During the incubation (28 days) of the test units the temperature range was 20.0-20.3 °C. The oxygen concentration of test water (mineral medium) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.76 mg/L.
The pH was checked prior study start and found to be 7.23; further pH adjustment was considered as not necessary.

Test Units: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
The number of test bottles was the following:
10 (+2 reserve) bottles containing the test item and inoculum (source: bottle 1a and 1b)
10 (+2 reserve) bottles containing the sodium benzoate and inoculum (source: bottle 2a and 2b)
10 (+2 reserve) bottlescontaining only inoculum (inoculum control) (source: bottle 3a and 3b)
10 (+2 reserve) bottlescontaining the test item, sodium benzoate and inoculum (toxicity control) (source: bottle 4a and 4b)
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
3 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 45.2 % biodegradation was noted within 14 days .
Test performance:
The chosen test item concentration of 3.0 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 1.753 mg O2/ mg test item 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 reached a mean of 23.6 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values increased slowly until the 21st day of the experiment and a higher increase was obtained between the 21st and 28th days.
Key result
Parameter:
% degradation (O2 consumption)
Value:
>= 11.6 - <= 23.6
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 1.38 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 1.75 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 (~13 %) between duplicate values for degradation was calculated in the test item group on the 21st 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 79.0 % on the 14th day. All validity criteria were met as required by the test guideline OECD 301.

Biodegradation of the Test Item: 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 28-day samples; furthermore in the inoculum control, in the toxicity control and in one parallel of the test item samples in the 21-day samples. In the 21-day inoculum control samples in average 0.25 mg/L, in the 21-day test item samples in average 0.17 mg/L, in the 21-day toxicity control samples in average 0.09 mg/L nitrite was detected. The nitrate concentration of the samples was less than 0.4 mg/L on day 0, on 7th, 14th and on 21st day; but in the 28-day inoculum control samples in average 0.6 mg/L, in the 28-day test item samples in average 0.6 mg/L nitrate was detected. The measured quantities of nitrate in the 28-day toxicity control samples were below the LOQ. According to the referred OECD 301 guideline (Annex V) 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 reached a mean of 23.6 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values increased slowly until the 21st day of the experiment and a higher increase was obtained between the 21st and 28th days. 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 45.2 % biodegradation was noted within 14 days and 53.0 % 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:
1.753 other: mg O2/ mg test item
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % 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 45.2 % biodegradation was noted within 14 days and 53.0 % biodegradation after 28 days of incubation.

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

8.73

7.22

6.83

6.53

6.18

3.0

1b

8.72

7.21

6.80

6.62

6.04

 

mean

8.73

7.22

6.82

6.58

6.11

Reference item

 

2a

8.79

4.44

3.68

3.21

3.14

3.0

2b

8.78

4.12

3.51

2.99

2.81

 

mean

8.79

4.28

3.60

3.10

2.98

Inoculum control

3a

8.71

7.95

7.61

7.41

7.42

3b

8.76

7.72

7.38

7.36

7.30

mean

8.74

7.84

7.50

7.39

7.36

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

8.76

3.55

2.99

2.30

2.12

4b

8.73

3.31

2.75

2.37

1.75

mean

8.75

3.43

2.87

2.34

1.94

 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

3.0

1a

0.61

0.66

0.85

1.18

1b

0.61

0.68

0.75

1.31

Reference item

3.0

2a

3.45

3.87

4.23

4.28

2b

3.76

4.03

4.44

4.60

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

4.31

4.53

5.11

5.27

4b

4.52

4.74

5.01

5.61

 

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

3.0

1a

0.20

0.22

0.28

0.39

1b

0.20

0.23

0.25

0.44

Reference item

3.0

2a

1.15

1.29

1.41

1.43

2b

1.25

1.34

1.48

1.53

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

0.72

0.76

0.85

0.88

4b

0.75

0.79

0.84

0.93

 

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

11.6

12.5

16.2

22.3

3.0

1b

11.6

12.9

14.3

24.8

 

mean

11.6

12.7

15.2

23.6

Reference item

 

2a

69.0

77.4

84.6

85.5

3.0

2b

75.2

80.6

88.8

91.9

 

mean

72.1

79.0

86.7

88.7

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

42.0

44.2

49.8

51.3

4b

44.1

46.2

48.8

54.6

mean

43.0

45.2

49.3

53.0

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

<0.4

<0.4

<0.4

<0.4

<0.4

28thday

0.6

0.6

0.6

0.6

<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.

 

 

 

Nitrite Concentrations

 

Analytical occasions

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

 

 

1a

1b

3a

3b

4a

4b

 

 

0 day

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

7thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

14thday

0.04

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

21stday

0.18

0.16

0.25

0.25

0.10

0.08

 

 

28thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

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

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

 

 

 

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

2.0

-*   no data

 

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

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The test item was inherently biodegradable under the conditions of the performed 28 Day Closed Bottle Test (OECD Guideline 301 D).
Executive summary:

The test item was investigated for its ready biodegradability in a closed bottle test over 28 days based on the OECD Guideline for Testing of Chemicals No  301 D (1992). The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The chosen test item concentration of 3.0 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 1.753 mg O2/ mg test item was determined at the start of the main experiment. The percentage biodegradation of the test item reached a mean of 23.6 % after 28 days based on its COD. The concurrently conducted analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore 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 79.0 % after 14 days, and to a mean of 88.7 % 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 45.2 % biodegradation was noted within 14 days and 53.0 % 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).

Description of key information

The test item was not ready biodegradable under the conditions of the performed 28 Day CO2 Evolution (Modified Sturm) Test according to the Method C.4-E of Commission Regulation (EC) No 440/2008, and the OECD Guideline for Testing of Chemicals, No. 301 D (1992) and the US EPA Draft Ecological Effects Test Guidelines OPPTS 835.3110).

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

The test item was investigated for its ready biodegradability in a closed bottle test over 28 days based on the OECD Guideline for Testing of Chemicals No  301 D (1992). The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The chosen test item concentration of 3.0 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 1.753 mg O2/ mg test item was determined at the start of the main experiment. The percentage biodegradation of the test item reached a mean of 23.6 % after 28 days based on its COD. The concurrently conducted analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore 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 79.0 % after 14 days, and to a mean of 88.7 % 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 45.2 % biodegradation was noted within 14 days and 53.0 % 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).