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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:
2017-01-09 to 2017-08-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 301 D. All validity criteria were fulfilled
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
10 January 2017
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
other: river water
Details on inoculum:
- Origin: River water was sampled from “La Mourachonne”, situated downstream of a plant treating domestic wastewater (Pegomas, France).
- Pre-conditioning: The river water was aerated for about 6 days before use as inoculum at the test temperature to reduce endogenous respiration.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Test water: Mineral medium, Reconstituted water, as prescribed by the OECD Guideline No. 301D
- Composition of medium: The mineral medium was prepared by adding one mL of each of the following stock solutions (a to d) to each litre of ultrapure water: a) 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.40 mg Na2HPO4•2H2O, 0.50 mg NH4Cl. The pH of this solution was approximately 7.4 ± 0.1; b) 36.40 mg CaCl2•2H2O, c) 22.50 mg MgSO4•7H2O and d) 0.25 mg FeCl3•6H2O.). This reconstituted water was aerated for at least 20 min and thereafter set aside at the test temperature for about 20 h before use.
- Test temperature: Temperatures recorded continuously in the growth chamber and situated between 20.8 and 21.3°C throughout the test (average value: 21.1°C), and complied with the requirements as laid down in the study plan (22°C ± 2°C, constant within 1°C).
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of the start of the biodegradability test (=J0) and type of series.
- Inoculum: A concentration of inoculum of approximately 6.10^4 cells.L-1 was used.
- Number of culture flasks/concentration: 10 bottles containing test item, inoculum, mineral medium and silica gel (test suspension)

SAMPLING
- Sampling frequency: Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels taken from each group (except
the procedure control) were made at day 0, 7, 14, 21 and 28, and only at the start of the test and after 7 and 14 days of incubation for the procedure and toxicity control.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 bottles containing inoculum, mineral medium and silica gel (inoculum blank + with silica gel); 10 bottles containing inoculum and mineral medium (inoculum blank without silica gel)
- Procedure control: 6 bottles containing reference substance, inoculum and mineral medium
- Toxicity control: 6 bottles containing test item, reference substance, inoculum and mineral medium
Reference substance:
benzoic acid, sodium salt
Remarks:
2 mg/L
Preliminary study:
None
Key result
Parameter:
% degradation (O2 consumption)
Value:
5
Sampling time:
28 d
Details on results:
Substances are considered to be readily biodegradable in the Closed Bottle test if oxygen consumption is equal to or greater than 60% of the ThOD value in the ten-day window within the 28-day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).
Test item NOPOL was biodegraded by 5% at day 28 in the Closed Bottle test. It should be noted that the test item had no inhibitory effect on the activity of the microbial inoculum, as demonstrated by the test results of the toxicity control, in agreement with OECD 301 (oxygen consumption in the toxicity control must be greater than 25% of the ThOD value within 14 days).

Calculated ThOD of the test item: 2.89 mg O2/mg
Results with reference substance:
Calculated ThOD of the reference substance: 1.67 mg O2/mg. The biodegradation percentage of the reference substance, sodium benzoate, was 75 % at day 14.

Table 5.2.1/1: Percentage biodegradation of the test item, the reference substance and of the toxicity control throughout the test

 

Time (days)

0

7

14

21

28

Test Suspension

(2 mg test item.L-1)

Replicate 1

0.00

0.61

-0.26

4.15

5.62

Replicate 2

0.00

-0.95

-1.64

5.19

4.93

Mean

0

0

-1

5

5

Procedure Control

(2 mg reference substance.L-1)

Replicate 1

0.00

77.69

74.70

N.A.

N.A.

Replicate 2

0.00

74.70

74.40

N.A.

N.A.

Mean

0

76

75

N.A.

N.A.

Toxicity Control *

(2 mg reference substance.L-1

+ 2 mg test item.L-1)

Mean

0

75

71

N.A.

N.A.

N.A.: not applicable

* % biodegradationtox. Control=  (BOD tox. cont / ThODref. subst.) * 100

with BOD tox. cont = (mTox(o) - mTox(n)) - (ma(o) - ma(n)) / mg test ref susbst.L-1in vessel

and where:

 n = number of days after the start of the test

mTox(0) = mean toxicity control value at day 0

 mTox (n) = mean toxicity control value at day n

 ma(0) = mean test suspension value at day 0

 ma(n) = mean test suspension value at day n.

Table 5.2.1/2: Dissolved oxygen concentrations (mg.L-1) throughout the test

 

Time (days)

0

7

14

21

28

 

Inoculum Blank

+ silica gel

9.28

8.56

8.38

8.51

8.04

9.15

8.64

8.54

8.64

7.90

Mean

9.22

8.60

8.46

8.58

7.97

Test Suspension

(2 mg test item.L-1)

9.26

8.61

8.52

8.38

7.69

9.19

8.63

8.53

8.25

7.66

Mean

9.23

8.62

8.53

8.32

7.68

Inoculum Blank

(without silica gel)

9.33

9.05

8.59

8.51

7.95

9.44

8.91

8.77

8.39

8.15

Mean

9.39

8.98

8.68

8.45

8.05

Procedure Control(2 mg reference substance.L-1)

9.16

6.16

5.96

N.A.

N.A.

9.06

6.16

5.87

N.A.

N.A.

Mean

9.11

6.16

5.92

N.A.

N.A.

Toxicity Control

(2 mg reference substance.L-1

+ 2 mg test item.L-1)

9.14

6.08

5.95

N.A.

N.A.

9.17

6.03

6.26

N.A.

N.A.

Mean

9.16

6.06

6.11

N.A.

N.A.

Validity criteria of the test:   

Inoculum blank: Oxygen depletion in the inoculum blank did not exceed 1.5 mg dissolved oxygen/L after 28 days.

Residual [O2]: Oxygen concentrations were > 0.5 mg/L in all bottles during the test period.

Replicate values: The differences of the replicate (test suspension) values at day 28 were less than 20%.

Reference substance: The biodegradation percentage of the reference substance, sodium benzoate, was 75% at day 14.

Thus all validity criteria of the test have been fulfilled in the present study.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Test item NOPOL was biodegraded by 5% at day 28. Therefore, NOPOL cannot be considered as readily biodegradable under the experimental conditions.
Executive summary:

A ready biodegradability study was performed to assess the biotic degradation of test item NOPOL according to OECD Guideline 301 D and in compliance with GLP.

Ten BOD bottles were filled with mineral medium inoculated with river water and test item was added at a nominal concentration of 2 mg.L-1 with the help of silica gel. In the meantime, two series of ten blanks (with and without silica gel) were filled with inoculated mineral medium. Furthermore, six bottles containing reference substance sodium benzoate (2 mg.L-1) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum.The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test.

The validity of the test was demonstrated by an endogenous respiration < 1.5 mgO2.L-1and by a biodegradation of the reference substance of 75% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg/L in all bottles during the test period. Furthermore, the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum.

In this study, test item NOPOL was biodegraded by 5% at day 28. Therefore, NOPOL cannot be considered as readily biodegradable under the experimental conditions.

Description of key information

In this study, test item NOPOL was biodegraded by 5% at day 28. Therefore, NOPOL cannot be considered as readily biodegradable under the experimental conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

For that endpoint, an experimental ready biodegradability study was available in order to assess the biotic degradation of the test item according to OECD Guideline 301 D and in compliance with GLP. The validity of the test was demonstrated by an endogenous respiration < 1.5 mg O2.L-1 and by a biodegradation of the reference substance of 75% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg.L-1 in all bottles during the test period. Furthermore, the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum.

The validity criteria of the OECD Guideline 301 D test were respected. The study satisfies guidelines requirements for ready biodegradability studies and was therefore considered as acceptable for that endpoint.