Oligomerisation products of alpha-pinene and beta-pinene

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-20 to 2016-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

no

Remarks:

The study has been conducted in DRT laboratory, which is not a GLP laboratory. However, the results of this study allow to conclude on the ready biodegradability properties of the substance.

Oxygen conditions:

aerobic

Inoculum or test system:

other: Effluent water from industrial on-site waste water treatment plant

Details on inoculum:

- Origin: Effluent water from industrial on-site waste water treatment plant was sampled from “DRT”, located out of the plant treating industrial wastewater (Vielle Saint Girons, France).
- Pre-conditioning: The water was aerated for about 7 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. This mineral medium was prepared by dividing the prescribed reagent quantities by two.
a) 4.25 mg KH2PO4, 10.875 mg K2HPO4, 16.70 mg Na2HPO4•2H2O, 0.25 mg NH4Cl.
b) 13.75 mg CaCl2•2H2O,
c) 11.25 mg MgSO4•7H2O and
d) 0.125 mg FeCl3•6H2O.
This reconstituted water was aerated for at least 2 hours and the mineral medium container was closed by parafilm until use.
- Test temperature: Temperatures situated around 22°C throughout the test, 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 identified with replicate number, date of the start of the biodegradability test (=J0) and type of series.
- Inoculum: A nominal concentration of approximately 1 mL inoculum.L-1 of mineral medium 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 were made at day 0, 7, 14, 21 and 28 for the “test suspension” and “inoculum blank” series, only at the start of the test and after 14 and 28 days of incubation for the ''procedure control'' seriesand 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 bottles containing inoculum and mineral medium
- Test suspension: 10 bottles containing test item, inoculum and mineral medium
- 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:

other: Sodium acetate

Remarks:

2 mg/L

Preliminary study:

None

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

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 TERPENIC OLIGOMERS was biodegraded by 4% 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: 3.05 mg O2/mg

Results with reference substance:

Calculated ThOD of the reference substance: 0.78 mg O2/mg. The biodegradation percentage of the reference substance, sodium acetate, was 60 % at day 28.

Table 2: 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  8.89  -7.04  6.11  3.89  Replicate 2  0.00  0.74  -4.82  5.00  4.26  Mean  0.00  4.81  -5.93  5.56  4.08        Procedure Control (2 mg reference substance.L^-1)  Replicate 1  0.00    57.05    63.46  Replicate 2  0.00    69.87    57.05  Mean  0.00    63.46    60.26  Toxicity Control* (2 mg reference substance.L^-1 + 2 mg test item.L^-1)  Mean  0.00  62.0  79.0  NA  NA

N.A.: not applicable

* % biodegradation_{tox. 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 1: Dissolved oxygen concentrations (mg.L^-1) throughout the test

Time (days)  0  7  14  21  28 Inoculum Blank + silica gel        Mean  9.00  8.38  7.81  7.89  7.54  9.00  8.37  7.71  7.85  7.73  9.00  8.38  7.76 7.87   7.64 Test Suspension (2 mg test item.L^-1)     Mean     8.93  7.83  8.07  7.47  7.36  8.91  8.25  7.93  7.51  7.32  8.92  8.04  8.00  7.49  7.34 Procedure Control  (2 mg reference substance.L^-1)    Mean     8.86    6.73    6.51  8.86    6.53    6.61  8.86    6.63    6.56 Toxicity Control (2 mg reference substance.L-1 + 2 mg test item.L-1)        Mean  9.00  7.06  6.78  N.A.  N.A.  9.00  7.16  6.90  N.A.  N.A.  9.00  7.11  6.84  N.A.  N.A.

N.A.: not applicable

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 acetate, was 63% 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 TERPENIC OLIGOMERS was biodegraded by 4% at day 28 in the Closed Bottle test.
Therefore, TERPENIC OLIGOMERS cannot be considered as readily biodegradable under the experimental conditions.

Executive summary:

A study was performed to assess the biotic degradation of test item TERPENIC OLIGOMERS by performing a ready biodegradability test. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 301 D, ''Ready biodegradability - Closed Bottle Test44 (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 (4).

Ten BOD bottles were filled with mineral medium inoculated with water release of water treatment plant and test item added at a nominal concentration of 2 mg.L^-1 with silica gel. In the meantime, one serie of ten blanks (with silica gel) were filled with inoculated mineral medium. Furthermore, six bottles containing reference substance sodium acetate (2 mg.L¹) were tested in order to check the procedure. A toxicity control (a serie of six bottles), 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^-1 after 28 days and by a biodegradation of the reference substance of 63% of its ThOD after 14 days of incubation. Moreover, the difference 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.

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).

In this study, test item TERPENIC OLIGOMERS was biodegraded by 4% at day 28.

Therefore, TERPENIC OLIGOMERS cannot be considered as readily biodegradable under the experimental conditions

Description of key information

A study was performed to assess the biotic degradation of test item TERPENIC OLIGOMERS by performing a ready biodegradability test. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 301 D, ''Ready biodegradability - Closed Bottle Test44 (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 (4).

Test item TERPENIC OLIGOMERS was biodegraded by 4% at day 28 in the Closed Bottle test.

Therefore, TERPENIC OLIGOMERS 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