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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
all validity criteria of the test have been fulfilled
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
30 June 2016
Oxygen conditions:
aerobic
Inoculum or test system:
other: river water
Details on inoculum:
Origin: River water was sampled from “L’Adour”, situated downstream of a plant treating domestic wastewater (Aureilhan, France).
Pre-conditioning: The river water was aerated for about 7 days before use as inoculum at the test temperature to reduce endogenous respiration.
Duration of test (contact time):
ca. 28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test water: Mineral medium, Reconstituted water, as prescribed by the OECD Guideline No. 301
Composition of the mineral medium : KH2PO4 (8.50 g/L), K2HPO4 ( 21.75 g/L), NA2HPO4. 2H2O (33.40 g/L), NH4CL (0.50 g/L) CaCl2. 2H2O (36.40 g/L), MgSO4. 7H2O (22.50 g/L), FeCl3. 6H2O (0.25 g/L).
Test environment: Controlled environment cabinet (22°C ± 2°C); the test was performed in the dark.

TEST SYSTEM
Culture apparatus: All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity.
Inoculum: A concentration of inoculum of approximately 10^5 cells.L-1 was used.
Number of culture flasks/concentration including blank and control system:
- 10 bottles containing inoculum, mineral medium and silica gel (inoculum blank with silica gel)
- 10 bottles containing test item, inoculum, mineral medium and silica gel (test suspension)
- 10 bottles containing inoculum and mineral medium (inoculum blank without silica gel)
- 6 bottles containing reference substance, inoculum and mineral medium (procedure control)

SAMPLING
Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels were made at day 0, 7, 14, 21, 29, 35, 42, 49 and 56 for the “test suspension” and “inoculum blank with silica gel” series, at day 0, 7, 14, 21 and 29 for the “procedure control” and “inoculum blank without silica gel” series, and only at the start of the test and after 7 and 14 days of incubation for the “toxicity control” series.
Temperature: Measured continuously in the growth chamber
Reference substance:
benzoic acid, sodium salt
Remarks:
2 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 68
Sampling time:
28 d
Details on results:
Test item was biodegraded by 68% at day 28 in the Closed Bottle test. The pass level was reached after less than 7 days.
Test item is considered as readily biodegradable under the experimental conditions.
Results with reference substance:
The calculated ThOD of the test item were calculated from their empirical formulae to be ThOD = 3.29 mgO2.mg-1

Percentage biodegradation of the test item, the reference substance and 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

70.52

84.73

76.29

68.09

Replicate 2

0.00

61.25

86.85

68.09

67.93

Mean

0

66

86

72

68

Procedure Control

(2 mg reference substance.L-1)

Replicate 1

0.00

81.14

82.63

N.A.

N.A.

Replicate 2

0.00

81.44

82.04

N.A.

N.A.

Mean

0

81

82

N.A.

N.A.

* % biodegradationtox.Control=    BOD tox. cont. x 100

                                           ThODref. subst.

 

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.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
According to guideline OECD 301 D, the test item is considered as readily biodegradable (68% of biodegradation was observed at day 28).
Executive summary:

A ready biodegradability study was performed to assess the biotic degradation of the test item. Test method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 301 D, "Ready biodegradability – Closed Bottle Test" (1), referenced as Method C.4-E of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 (2) and with ISO 10634 (7).

Ten BOD bottles were filled with mineral medium inoculated with river water and test item added at a nominal concentration of 2 mg/L 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) were tested in order to check the procedure.

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 (28 days for each group except the procedure control until 14 days).

Test item alpha-pinene multiconstituent was biodegraded by 68% at day 28. The pass level of 60% was reached after less than 7 days (approximately 5 days) upon achieving 10% biodegradation. Therefore, alpha-pinene is considered as readily biodegradable based on the biodegradation percentage of 68% at day 28 and fulfilling the 10-day time window requirement (ca. less than 7 days).

The validity of the test was demonstrated by an endogenous respiration.

Description of key information

(-)-alpha-Pinene is readily biodegradable according to the criteria of OECD 301 D guideline.

The test item was biodegraded by 68% at day 28. The pass level of 60% was reached after less than 7 days (approximately 5 days) upon achieving 10% biodegradation.

An enhanced biodegradability study was performed to assess the biotic degradation of the test itemaccording to OECD Guideline 301 D and in compliance with GLP.

18 BOD bottles were filled with mineral medium inoculated with river water (5 mL/L) and test item added at a nominal concentration of 2 mg/L with the help of silica gel.

The test item was biodegraded by 5 % at day 29.

It is therefore considered that the test substance is not readily biodegradable according to the criteria of OECD 301 D guideline.

The result of this key study is proposed as end point value for the chemical safety assessment.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A ready biodegradability study was performed to assess the biotic degradation of alpha-pinene. Test method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 301 D, "Ready biodegradability – Closed Bottle Test" (1), referenced as Method C.4-E of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 (2) and with ISO 10634 (7).

Test item alpha-pinene multiconstituent was biodegraded by 68% at day 28. The pass level of 60% was reached after less than 7 days (approximately 5 days) upon achieving 10% biodegradation. Therefore, (-)-alpha-pinene is considered as readily biodegradable.

The result of this key study is proposed as end point value for the chemical safety assessment.