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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 24 to August 29, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD guideline 310 with minor deviations: incomplete certificate of analysis of the test substance; CO2 in the headspace was not measured
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
incomplete certificate of analysis of the test substance; CO2 in the headspace was not measured
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of secondary activated sludge: Activated sludge plant treating predominantly domestic waste water (WWTP Nieuwgraaf, Duiven, The Netherlands)
- Preconditioning: 400 mg Dry Weight (DW)/L of activated sludge was aerated overnight and then diluted to a concentration of 4 mg DW/L in the test vessels
- Concentration of sludge: 400 mg DW/L
Duration of test (contact time):
28 d
Initial conc.:
25.7 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: Nutrient medium contained per liter of deionised water: 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4.2H2O, 5 mg NH4CI, 22.5 mg MgSO4.7H2O, 27.5 mg CaCI2, 0.25 mg FeCI3.6H2O
- Source/preparation of dilution water: Deionised water containing < 1 mg/L of organic carbon
- Test temperature: 19-21 °C
- pH: 6.8-7.4
- Suspended solids concentration: 4 mg DW/L
- Continuous darkness: Yes
- Agitation: Incubated bottles were agitated throughout the test on a rotary shaker at 180 rpm

TEST SYSTEM
- Culturing apparatus: Serum flaks (120 mL) with butyl septa and crimp-on aluminum seals; volume of the liquid phase: 80 mL
- Number of culture flasks/concentration: 36 vessels containing only inoculum, 36 vessels containing test substance and inoculum; 9 vessels containing reference substance and inoculum

MEASURING EQUIPMENTS:
- Inorganic carbon (IC) was measured in the alkaline medium (7 M NaOH) using the TOC apparatus (Shimadzu s'Hertogenbosch, The Netherlands). Sodium carbonate and sodium bicarbonate used as control in the IC analyses.
- Chemical oxygen demand (COD) of the stock solution with the test substance was determined by oxidation with an acid-dichromate mixture in which Cr6+ was reduced to Cr3+. The subsequent increase in Cr3+ was determined spectrophotometrically at 620 nm. Potassium hydrogen phthalate used as control in the COD determination.
- pH was measured in a sample taken before the addition of NaOH using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Germany).
- Temperature was measured and recorded with a thermo couple connected to a data logger.

SAMPLING
- Sampling frequency: Triplicate vessels of all series were withdrawn for analyses of the carbon dioxide formed at Day 3, 7, 10, 14, 17, 21 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; containing inoculum only
- Procedure control: Yes; containing reference substance (sodium acetate) with inoculated medium
- Toxicity control: No; test material was considered to be non-toxic to micro-organisms as inhibition of the endogenous respiration of the inoculum was not detected during the biodegradation test.

Reference substance:
acetic acid, sodium salt
Remarks:
68.4 mg/L; Source: Acros organics, Belgium
Preliminary study:
No data
Test performance:
The pass level of 60% was reached after approximately 10 days upon achieving 10% biodegradation. Terpineol therefore fulfilled the 10-day time window criterion for ready biodegradable compounds.
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
80
Sampling time:
28 d
Details on results:
- Initial test material concentration: 25.7 mg/L, representing organic carbon concentration of 20 mg/L
- Chemical oxygen demand (COD) of terpineol in the stock solution = 2.9 g/L
- Theoretical oxygen demand (ThOD) = 2.9 g/L
- Theoretical carbon content = 0.78 mg/mg
- % biodegradation on Day 5: > 10%
- % biodegradation on Day 12: > 60%
- % biodegradation on Day 28 = 80%
Results with reference substance:
- Calculated organic carbon = 0.29 mg/mg
- % biodegradation (Day 7) > 60%
- % biodegradation (Day 14) = 80%

Table 1: Inorganic carbon concentration (mg/L) in the aqueous phase (alkaline) of the test vessels

 

Time (days)

Inorganic carbon concentration (mg/L)

CLc

CLt

CLa

0

3.3

3.4

3.7

 

3.5

2.8

3.7

 

3.6

2.7

4

Mean (M)

3.5

3

3.8

3

4.5

4.5

 

 

3.6

4

 

 

4.2

4.1

 

Mean (M)

4.1

4.2

 

7

3.7

13.8

19.1

 

3.9

11.8

19.1

 

3.9

11.6

19.2

Mean (M)

3.8

12.4

19.1

10

4.9

17.6

 

 

4

14.5

 

 

4.3

14.8

 

Mean (M)

4.4

15.6

 

14

4.5

19.4

20.4

 

5

18

21.4

 

5.4

19.6

20.9

Mean (M)

5

19

20.9

17

5.6

20.6

 

 

4.7

19.9

 

 

4.7

19.7

 

Mean (M)

5

20.1

 

21

5.4

21.1

 

 

6.4

21.7

 

 

5.8

21.7

 

Mean (M)

5.9

21.5

 

28

5.7

22

 

 

5.3

21.2

 

 

6.4

21.8

 

Mean (M)

5.8

21.7

 

CLc:Mineral nutrient solution without test material but with inoculum;

CLt: Mineral nutrient solution with test material (20 mg/L organic carbon), and with inoculum;

CLa: Mineral nutrient solution with sodium acetate (20 mg/L organic carbon), and with inoculum

 

Table 2: Carbon dioxide formation (mg/L) and the percentages biodegradation of Terpineol (TIC/ThIC) and sodium acetate (TIC/ThIC) in the carbon dioxide headspace test

 

Time (days)

mg/L

Biodegradation (%)

Test

Acetate

Test

Acetate

0

 

 

0

2

3

0.1

 

1

 

7

8.6

15.3

43

77

10

11.2

 

56

 

14

14.0

15.9

70

80

17

15.1

 

76

 

21

15.6

 

78

 

28

15.9

 

80

 

Validity criteria fulfilled:
yes
Remarks:
% degradation in reference material was > 60% by Day 7; mean TIC in the blank control at the end of the test was > 3 mg C/L
Interpretation of results:
readily biodegradable
Conclusions:
Terpineol multiconstituent was readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 310 and GLP, terpineol multiconstituent was tested at concentrations of 25.7 mg/L and the inoculum was activated sewage sludge, domestic. The degradation of the test material was assessed by the determination of the inorganic carbon concentration. The test treatments, inoculum blank and reference (sodium acetate) were measured in triplicates.

 

At 25.7 mg/L test concentration, greater than 10% biodegradation of the test substance was reached on Day 5 and greater than 60% biodegradation was reached on Day 12. Hence, the test material met the 10-day window requirement for ready biodegradability. On Day 28, the biodegradation was 80%.

 

The reference material, sodium acetate, reached greater than 60% biodegradation on Day 7. alpha-Terpineol was considered to be non-toxic to micro-organisms as inhibition of the endogenous respiration of the inoculum was not detected during the test. Hence, it met the validity criteria for reference material and toxicity control. The mean amount of inorganic carbon concentration in the blank control at the end of the test was greater than 3 mg C/L.

 

Therefore, terpineol multiconstituent was considered as readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Terpinolene is an impurity of multiconstituent substance TERPINEOL MULTICONSTITUENT. Data on terpinolene are used as worst case data, in comparison with the main constituents of TERPINEOL MULTICONSTITUENT.
Reason / purpose:
read-across source
Preliminary study:
no data
Test performance:
no data
Key result
Parameter:
% degradation (O2 consumption)
Value:
81
Sampling time:
28 d
Details on results:
Initial test material concentration: 2 mg/L
Theoretical oxygen demand (ThOD) = 3.2 mg/mg

% biodegradation on Day 2: > 10 %
% biodegradation by Day 9: > 60 %
% biodegradation on Day 28 = 81 %
Results with reference substance:
- Theoretical oxygen demand (ThOD) = 0.8 mg/mg
- % biodegradation on Day 7: > 60 %
- % biodegradation on Day 14 = 78 %

Table 5.2.1/1: Dissolved oxygen concentrations (mg/L) in the closed bottles

 

Time (days)

Oxygen concentration (mg/L)

Ocs

Ot

Oc

Oa

0

9.1

9.1

9.1

9.1

 

9.1

9.1

9.1

9.1

Mean (M)

9.1

9.1

9.1

9.1

7

8.4

4.9

8.6

5.0

 

8.6

4.7

8.6

5.0

Mean (M)

8.5

4.8

8.6

5.0

14

8.2

3.0

8.3

4.0

 

8.2

3.5

8.3

4.2

Mean (M)

8.2

3.3

8.3

4.1

21

8.0

3.0

7.9

 

 

8.0

2.8

8.0

 

Mean (M)

8.0

2.9

8.0

 

28

8.0

3.0

7.9

 

 

7.9

2.6

8.0

 

Mean (M)

8.0

2.8

8.0

 

Ocs: River water with nutrients and silica gel but without test material

Ot: River water with nutrients, test material (2.0 mg/L), and silica gel

Oc: River water with nutrients

Oa: River water with nutrients and sodium acetate (6.7 mg/L)

 

Table 5.2.1/2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, terpinolene monoconstituent (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test

 

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Sodium acetate

Test substance

Sodium acetate

0

0.0

0.0

0

0

7

3.7

3.6

58

67

14

4.9

4.2

77

78

21

5.1

 

80

 

28

5.2

 

81

 

Validity criteria fulfilled:
yes
Remarks:
endogenous respiration at Day 28 was 1.1 mg/L; differences of the replicate values at Day 28 were < 20%; degradation in reference material was 78 % at Day 14; O2 concentration during the test was > 0.5 mg/L
Interpretation of results:
readily biodegradable
Conclusions:
Terpinolene was readily biodegradable in a test carried out according to OECD Guideline No 301 D.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline No 301 D and GLP, the test item was tested at a concentration of 2 mg/L and the inoculum was a river water, near to a domestic wastewater treatment plant (3 km upstream).

The degradation of the test material was assessed by the determination of the oxygen consumption. The test treatments, inoculum blank, and reference (sodium acetate) were measured in duplicates.

At 2 mg/L test concentration, greater than 10% of degradation was reached by Day 2 and greater than 60% of biodegradation was reached by Day 9.

The pass level for ready biodegradability was reached in the required 14-d window within the 28-d period of the test: 81% degradation on Day 28.

The reference material (sodium benzoate) reached greater than 60 % biodegradation on Day 7. The biodegradation curves obtained with the reference substance alone and with test material+reference substance show no toxic effect on the micro-organisms. Hence, it met the validity criteria for reference material and toxicity control. The endogenous respiration at Day 28 was 1.1 mg/L and oxygen concentration during the test was greater than 0.5 mg/L.

Therefore, terpinolene was readily biodegradable.

Description of key information

Terpineol multiconstituent is readily biodegradable according to the criteria of the OECD 310 guideline.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The registered substance is a multiconstituent substance with a well-defined composition for which the relative percentage of each constituent and impurity is known. The full composition of terpinolene multiconstituent is provided under Section 1.2.

One reliable screening study for biodegradability is available for a terpineol quality similar in composition to terpineol multiconstituent. A test was carried out according to OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test). The inoculum was activated sewage sludge, domestic. The degradation of the test material was assessed by the determination of the inorganic carboncon centration. At 25.7 mg/L as test concentration, greater than 10% biodegradation of the test substance was reached on Day 5 and greater than 60% biodegradation was reached on Day 12. On Days 28, the biodegradation was 80%. The test substance was found ready biodegradable according to the criteria of the OECD 310 guideline.

Terpineol multiconstituent also contains a hydrocarbon monoterpen fraction. For the purposes of chemical safety assessment, impurity terpinolene was considered as a representative hydrocarbon monoterpen.

Therefore, a reliable screening study for biodegradability, which is available on terpinolene, is provided as supporting study. The experimental study was conducted according to OECD 301 D Guideline and GLP without deviation. Terpinolene was tested at a concentration of 2 mg/L. The inoculum was a river water, near to a domestic wastewater treatment plant (3 km upstream). Degradation was assessed by measurement of oxygen consumption. The pass level for ready biodegradability was reached in the required time window within the 28-d period of the test (81% biodegradation on Day 28).

It is therefore considered that terpineol multiconstituent substance is ready biodegradable according to the criteria of the OECD test guideline.