Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April 2018 - 28 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 301F. GLP study.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was aerated for 6 days, at the test temperature of about 22 °C, until application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids. Before application the sludge was washed in a mineral medium.
- Concentration of sludge: 5.00 g suspended solids/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Remarks:
(88.2 mg/l of organic carbon)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 10ml of solution (A) in 800 ml water, plus 1 ml solutions (B), (C), (D) and dilution water up to 1 L. The following stock solutions were used, prepared with analytical grade reagents: Solution (A) contains: 8.50 g monopotassium dihydrogen orthophosphate (KH2PO4), 21.75 g dipotassium monohydrogen orthophosphate (K2HPO4), 33.40 g disodium monohydrogen orthophosphate dihydrate (Na2HPO4·2H2O), 0.50 g ammonium chloride (NH4Cl), in 1 L water. Solution (B) contains: 27.50 g calcium chloride, anhidrous (CaCl2) in 1L water. Solution (C) contains: 22.50 g magnesium sulphate heptahydrate (MgSO4·7H2O) in 1L water. Solution (D) contains: 0.25 g iron(III) chloride hexahydrate (FeCl3·6H2O) in 1L water.
- The water used is double-distilled, containing 4.4±0.5 mg/L of organic carbon (< 10% of the organic carbon content introduced by the test item), checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents.
- Test temperature: 22 ± 2ºC
- pH: 7.4 ± 0.2
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Volume of test solution in flask, V: 0.164 L

TEST SYSTEM
- Number of culture flasks/concentration: 3 flasks were used containing test item (100 mg/l) and inoculum (30mg/L SS).
- Measuring equipment: O2 uptake was measured by a closed WTW OxiTop OC 110 repirometer.
- Details of trap for CO2 and volatile organics if used: potassium hydroxide solution.

SAMPLING
- Sampling frequency: O2 uptake data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recorded and stored in the measuring heads of the sample bottles.
- Sampling method: Readings from apparatus (closed WTW OxiTop OC 110 repirometer).

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 flasks containing only inoculum 30mg/L SS.
- Procedure control: 3 flasks containing reference item (sodium acetate 100 mg/l) and inoculum 30mg/L SS.
- Toxicity control: 3 flasks containing test item, reference item at the same concentrations as in the individual solutions and inoculum 30mg/L SS.

STATISTICAL METHODS: The calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.
Reference substance:
acetic acid, sodium salt
Remarks:
CAS No: 127-09-3, purity ≥ 99.7%, source: CHEMPUR, Piekary Śląskie, Polska.
Key result
Parameter:
% degradation (O2 consumption)
Value:
63.5
Sampling time:
28 d
Remarks on result:
other: The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 41.7%.
Details on results:
-The biodegradability attained 63.5% within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 41.7%. Lag time was 4 days and degradation time was 18.5 days.
-The reference item reached 89% of biodegradation and the level for ready biodegradability by 4 days
-In the toxicity test the biodegradation was equal to 34.7% in 14 days
-The oxygen uptake of the inoculum blank was equal to 33.1 mg/L in 28 days
-The pH values of flasks containing test item were inside the range 7.51-7.58.
Results with reference substance:
The reference item reached 89% of biodegradation and the level for ready biodegradability by 4 days (>60% reached before day 14)

Table 2. Sample oxygen uptake: biodegradability.

 

time, days

1

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/L

a1

2.3

19.1

57.2

85.8

112.9

141.0

155.6

169.4

180.8

200.3

210.8

221.9

241.9

a2

4.8

18.7

62.0

95.0

124.6

157.9

173.6

188.1

201.2

219.7

228.8

238.0

249.8

a3

4.8

14.5

55.0

80.6

109.2

138.1

154.1

169.5

184.4

204.3

213.5

222.4

234.7

am. avg

4.0

17.4

58.1

87.1

115.6

145.7

161.1

175.7

188.8

208.1

217.7

227.4

242.1

Blank test O2uptake mg/L

b1

4.1

9.7

12.1

14.5

17.0

20.0

21.8

24.0

24.2

25.4

27.7

28.7

29.2

b2

4.3

10.1

12.5

16.0

18.3

20.9

24.1

25.5

26.7

27.7

29.9

31.2

33.1

b3

6.1

10.5

12.6

15.9

19.3

22.8

26.1

28.1

28.6

30.9

33.0

34.9

37.1

bm. avg

4.8

10.1

12.4

15.5

18.2

21.2

24.0

25.9

26.5

28.0

30.2

31.6

33.1

Reference item O2uptake. mg/L

w1

11.6

48.8

60.4

69.1

78.1

84.8

89.4

93.9

94.5

96.7

100.5

102.9

104.6

w2

16.7

53.3

66.4

76.6

82.0

87.6

91.5

92.8

95.2

96.8

98.1

99.2

100.6

w3

14.0

48.6

60.8

69.5

76.4

84.7

88.3

90.0

92.8

97.1

99.1

101.3

104.1

wm. avg

14.1

50.2

62.5

71.7

78.8

85.7

89.7

92.2

94.1

96.9

99.2

101.1

103.1

Toxicity control O2uptake. mg/L

tox1

11.7

47.6

63.1

85.2

106.6

136.7

156.1

173.9

192.8

222.8

239.8

254.1

273.8

tox2

13.2

49.4

65.5

89.3

114.1

146.3

169.7

195.2

217.5

244.0

256.9

269.9

289.4

tox3

15.0

53.6

80.1

106.2

129.5

156.0

171.8

183.4

197.5

217.6

228.6

239.2

252.8

toxm.avg

13.3

50.2

69.6

93.6

116.7

146.4

165.9

184.1

202.6

228.1

241.8

254.4

272.0

Corrected

test item O2uptake, mg/L

(a1-bm)

-2.5

8.9

44.8

70.3

94.7

119.8

131.6

143.5

154.3

172.3

180.6

190.3

208.7

(a2-bm)

0.0

8.5

49.6

79.5

106.4

136.7

149.6

162.3

174.7

191.7

198.6

206.4

216.7

(a3-bm)

0.0

4.3

42.6

65.1

91.0

116.9

130.1

143.7

157.9

176.3

183.3

190.8

201.6

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/L

R1(w1)

8.7

49.6

61.5

68.7

76.8

81.6

83.8

87.3

87.2

88.0

90.1

91.5

91.6

R2(w2)

15.2

55.3

69.2

78.3

81.8

85.2

86.6

85.8

88.0

88.3

87.0

86.6

86.5

R3(w3)

11.7

49.3

62.0

69.2

74.6

81.4

82.5

82.2

85.0

88.6

88.4

89.4

91.0

Rwavg

11.9

52.5

65.4

73.5

79.3

83.4

85.2

86.6

87.6

88.1

88.6

89.1

89.0

Test item% degradation

ThOD = 3.29

mgO2/mg

C = 100 mg/L

R1(a1)

0.0

2.7

13.6

21.4

28.8

36.4

40.0

43.6

46.9

52.4

54.9

57.8

63.4

R2(a2)

0.0

2.6

15.1

24.2

32.3

41.6

45.5

49.3

53.1

58.3

60.4

62.7

65.9

R3(a3)

0.0

1.3

13.0

19.8

27.7

35.5

39.5

43.7

48.0

53.6

55.7

58.0

61.3

Raavg

0.0

2.2

13.9

21.8

29.6

37.8

41.7

45.5

49.3

54.7

57.0

59.5

63.5

Toxicity test

% degradation

 

R1(tox1)

1.7

9.2

12.5

17.1

21.7

28.4

32.5

36.4

40.9

47.9

51.5

54.7

59.1

R2(tox2)

2.1

9.7

13.0

18.1

23.6

30.7

35.8

41.6

46.9

53.1

55.7

58.6

63.0

R3(tox3)

2.5

10.7

16.6

22.3

27.4

33.1

36.3

38.7

42.0

46.6

48.8

51.0

54.0

Rtoxavg

2.1

9.8

14.0

19.2

24.2

30.7

34.9

38.9

43.3

49.2

52.0

54.7

58.7

 

Table 3. The pH values of test flasks (no adjustment of pH was conducted).

flask #

7

8

9

1

2

3

4

5

6

10

11

12

Test item

Inoculum blank

Reference item

Toxicity test

initial

7.56

7.58

7.55

7.46

7.49

7.54

7.55

7.53

7.54

7.56

7.54

7.56

final

7.57

7.51

7.58

7.51

7.58

7.58

8.66

8.87

8.80

8.17

7.55

7.90

 

Validity criteria fulfilled:
yes
Remarks:
See "overall remarks".
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Under the ready aerobic biodegradability test, the test item attained 63.5% biodegradation within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 41.7%.
Executive summary:

A ready biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test item, according to OECD 301F / EC C.4 – D manometric respirometry methods, in accordance with GLP principles. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 2ºC) for 28 days. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 34.9%. Thus, the test item is not inhibitory. All validity criteria were fulfilled. The  biodegradability of the test item attained 63.5% within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 41.7%.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test: Test material was evaluated for degradation by cultures derived from coniferous forest soil, diluted and used directly without any prior enrichment.
- Short description of test conditions: see below
- Parameters analysed / observed: see below
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural soil
Details on inoculum:
- Source of inoculum: The primary inocula for this study were prepared from extracts of soil samples collected from a coniferous forest (soil A).
- Preparation of inoculum for exposure: Soil extracts were prepared by passing soil/water mixtures through a 500-µm sieve, followed by 2 h settling. The resulting supernatants were used as the inoculum.
Initial conc.:
>= 0.5 - <= 3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
other: biomass concentration
Details on study design:
First biodegradation experiment:
Glass flask (2 l) equipped with two glass/Teflon valves and a septum-sealed port was used.
The reactor was flushed with pure oxygen and then 1.4 l oxygen saturated minimal medium was added. Test substance was tested at concentrations in the range 0.5-3 mg/l. After the addition of the undiluted monoterpene, the reactor was then crimp-sealed with Teflon-lined septa. After 24 h equilibration, soil A extract was added to the reactor at 1% (v/v) through the bottom glass/Teflon valve. A sodium-azide-amended control was also set up.
Incubation took place in the dark at 23ºC with continuous mixing using magnetic stirrers (at approx. 300 rpm). At regular intervals, duplicate gas and liquid samples were removed and analyzed for test substance and CO2.

Second biodegradation experiment:
A second experiment was performed using replicate, 18x150-mm (26 ml volume) serum tubes (Bellco Glass, Vineland, N.J.). The tubes were flushed with pure oxygen and crimp sealed with Teflon-lined septa.
Inoculum drawn from the reactor used in the previous experiment was directly injected into replicate serum tubes from a microsyringe and then quickly crimp-sealed. The initial terpene concentration in the replicate tubes was uniform.
Azide amended controls were also prepared and incubated following the procedures used for the live cultures.
Headspace gas analysis of serum tubes showed an oxygen content of up to 90%, which was suficient for the complete mineralization of the terpene at the levels tested. The serum tubes were continuously rotated (at 1 rpm) and incubated in the dark at 23ºC. At diferent intervals analyses were performed by sacrificing duplicate serum tubes.
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.044
Sampling time:
182 h
Remarks on result:
other: Experiment 1 (CSR1)
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.4
Sampling time:
15 h
Remarks on result:
other: Experiment 2 (serum tubes)
Key result
Parameter:
other: Normalized degradation rate (h-1)
Value:
0.076
Sampling time:
15 h
Remarks on result:
other: Experiment 2 (serum tubes)
Details on results:
The detection of CO2, the increase in biomass concentration and lack of any substantial change in the concentration of terpene in the azide-amended control reactor demonstrated that biodegradation of d-limonene took place and that its disappearance was not the result for hydrolysis or any other physicochemical process (e.g., volatilization of the hydrocarbon monoterpenes).

Experiment/ compound

Reactor type

Inoculum

Lag period (h)

Maximum degradation rate (mg l-1h-1)

Normalized degradation rate (h-1)

Experiment 1

d-limonene

CSR1

Unacclimated soil A extract

182

0.044

NM

Experiment 2

d-limonene

Serum tubes

Acclimated (from CSR1)

15

0.40

0.076

The normalized degradation rate is the maximum degradation rate normalized to biomass concentration expressed as volatile suspended solids

CSR 1: continuously-stirred reactor 1

NM: not measured because of lack of accurate biomass data

Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
d-limonene was readily degraded under aerobic conditions at 23ºC by mixed cultures derived from forest soils.
Executive summary:

In a ready biodegradation study, d-limonene was tested under aerobic conditions at 23ºC and concentrations of 0.5-3 mg/L. Forest-soil extract cultures were used as inocula for the experiments conducted first without (experiment 1), then with prior microbial acclimation to the test material (experiment 2). The degradation of the test material was assessed by the determination of the biomass, concentration of the test material and headspace CO2. The test treatments and control (sodium azide, 2.5 g/L) were measured in duplicates. The lack of any substantial change in d-limonene concentration in the azide-amended control reactor demonstrated that disappearance of the test item in the test reactor was not the result of hydrolysis or any other physicochemical process. The normalised degradation rate in experiment 2 was 0.076 h-1. The maximum degradation rate in experiments 1 and 2 were 0.044 and 0.40 mg/L/h, respectively. The lag period in experiments 1 and 2 were 182 and 15 h, respectively. Under the test conditions, d-limonene was readily degraded by cultures derived from forest soils.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test: Test material was evaluated for degradation by cultures derived from coniferous forest soil, diluted and used directly without any prior enrichment.
- Short description of test conditions: see below
- Parameters analysed / observed: see below
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural soil
Details on inoculum:
- Source of inoculum: The primary inocula for this study were prepared from extracts of soil samples collected from a coniferous forest (soil A).
- Preparation of inoculum for exposure: Soil extracts were prepared by passing soil/water mixtures through a 500-µm sieve, followed by 2 h settling. The resulting supernatants were used as the inoculum.
Initial conc.:
>= 0.5 - <= 3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
other: biomass concentration
Details on study design:
First biodegradation experiment:
Glass flask (2 l) equipped with two glass/Teflon valves and a septum-sealed port was used.
The reactor was flushed with pure oxygen and then 1.4 l oxygen saturated minimal medium was added. Test substance was tested at concentrations in the range 0.5-3 mg/l. After the addition of the undiluted monoterpene, the reactor was then crimp-sealed with Teflon-lined septa. After 24 h equilibration, soil A extract was added to the reactor at 1% (v/v) through the bottom glass/Teflon valve. A sodium-azide-amended control was also set up.
Incubation took place in the dark at 23ºC with continuous mixing using magnetic stirrers (at approx. 300 rpm). At regular intervals, duplicate gas and liquid samples were removed and analyzed for test substance and CO2.

Second biodegradation experiment:
A second experiment was performed using replicate, 18x150-mm (26 ml volume) serum tubes (Bellco Glass, Vineland, N.J.). The tubes were flushed with pure oxygen and crimp sealed with Teflon-lined septa.
Inoculum drawn from the reactor used in the previous experiment was directly injected into replicate serum tubes from a microsyringe and then quickly crimp-sealed. The initial terpene concentration in the replicate tubes was uniform.
Azide amended controls were also prepared and incubated following the procedures used for the live cultures.
Headspace gas analysis of serum tubes showed an oxygen content of up to 90%, which was suficient for the complete mineralization of the terpene at the levels tested. The serum tubes were continuously rotated (at 1 rpm) and incubated in the dark at 23ºC. At diferent intervals analyses were performed by sacrificing duplicate serum tubes.
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.039
Sampling time:
168 h
Remarks on result:
other: Experiment 1 (CSR1)
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.27
Sampling time:
13 h
Remarks on result:
other: Experiment 2 (serum tubes)
Key result
Parameter:
other: Normalized degradation rate (h-1)
Value:
0.056
Sampling time:
13 h
Remarks on result:
other: Experiment 2 (serum tubes)
Details on results:
The detection of CO2, the increase in biomass concentration and lack of any substantial change in the concentration of terpene in the azide-amended control reactor demonstrated that biodegradation of gamma terpinene took place and that its disappearance was not the result for hydrolysis or any other physicochemical process (e.g., volatilization of the hydrocarbon monoterpenes).

Experiment/ compound

Reactor type

Inoculum

Lag period (h)

Maximum degradation rate (mg l-1h-1)

Normalized degradation rate (h-1)

Experiment 1

γ-terpinene

CSR1

Unacclimated soil A extract

168

0.039

NM

Experiment 2

γ-terpinene

Serum tubes

Acclimated (from CSR1)

13

0.27

0.056

The normalized degradation rate is the maximum degradation rate normalized to biomass concentration expressed as volatile suspended solids

CSR 1: continuously-stirred reactor 1

NM: not measured because of lack of accurate biomass data

Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
Gamma terpinene was readily degraded under aerobic conditions at 23ºC by mixed cultures derived from forest soils.
Executive summary:

In a ready biodegradation study, gamma terpinene was tested under aerobic conditions at 23ºC and concentrations of 0.5-3 mg/L. Forest-soil extract cultures were used as inocula for the experiments conducted first without (experiment 1), then with prior microbial acclimation to the test material (experiment 2). The degradation of the test material was assessed by the determination of the biomass, concentration of the test material and headspace CO2. The test treatments and control (sodium azide, 2.5 g/L) were measured in duplicates. The lack of any substantial change in gamma terpinene concentration in the azide-amended control reactor demonstrated that disappearance of the test item in the test reactor was not the result of hydrolysis or any other physicochemical process. The normalised degradation rate in experiment 2 was 0.056 h-1. The maximum degradation rate in experiments 1 and 2 were 0.039 and 0.27 mg/L/h, respectively. The lag period in experiments 1 and 2 were 168 and 13 h, respectively. Under the test conditions, gamma terpinene was readily degraded by cultures derived from forest soils.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test: Test material was evaluated for degradation by cultures derived from coniferous forest soil, diluted and used directly without any prior enrichment.
- Short description of test conditions: see below
- Parameters analysed / observed: see below
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural soil
Details on inoculum:
- Source of inoculum: The primary inocula for this study were prepared from extracts of soil samples collected from a coniferous forest (soil A).
- Preparation of inoculum for exposure: Soil extracts were prepared by passing soil/water mixtures through a 500-µm sieve, followed by 2 h settling. The resulting supernatants were used as the inoculum.
Initial conc.:
>= 0.5 - <= 3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
other: biomass concentration
Details on study design:
First biodegradation experiment:
Glass flask (2 l) equipped with two glass/Teflon valves and a septum-sealed port was used.
The reactor was flushed with pure oxygen and then 1.4 l oxygen saturated minimal medium was added. Test substance was tested at concentrations in the range 0.5-3 mg/l. After the addition of the undiluted monoterpene, the reactor was then crimp-sealed with Teflon-lined septa. After 24 h equilibration, soil A extract was added to the reactor at 1% (v/v) through the bottom glass/Teflon valve. A sodium-azide-amended control was also set up.
Incubation took place in the dark at 23ºC with continuous mixing using magnetic stirrers (at approx. 300 rpm). At regular intervals, duplicate gas and liquid samples were removed and analyzed for test substance and CO2.

Second biodegradation experiment:
A second experiment was performed using replicate, 18x150-mm (26 ml volume) serum tubes (Bellco Glass, Vineland, N.J.). The tubes were flushed with pure oxygen and crimp sealed with Teflon-lined septa.
Inoculum drawn from the reactor used in the previous experiment was directly injected into replicate serum tubes from a microsyringe and then quickly crimp-sealed. The initial terpene concentration in the replicate tubes was uniform.
Azide amended controls were also prepared and incubated following the procedures used for the live cultures.
Headspace gas analysis of serum tubes showed an oxygen content of up to 90%, which was suficient for the complete mineralization of the terpene at the levels tested. The serum tubes were continuously rotated (at 1 rpm) and incubated in the dark at 23ºC. At diferent intervals analyses were performed by sacrificing duplicate serum tubes.
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.053
Sampling time:
174 h
Remarks on result:
other: Experiment 1 (CSR1)
Key result
Parameter:
other: Maximum degradation rate (mg/L/h)
Value:
0.47
Sampling time:
28 h
Remarks on result:
other: Experiment 2 (serum tubes)
Key result
Parameter:
other: Normalized degradation rate (h-1)
Value:
0.089
Sampling time:
28 h
Remarks on result:
other: Experiment 2 (serum tubes)
Details on results:
The detection of CO2, the increase in biomass concentration and lack of any substantial change in the concentration of terpene in the azide-amended control reactor demonstrated that biodegradation of terpinolene took place and that its disappearance was not the result for hydrolysis or any other physicochemical process (e.g., volatilization of the hydrocarbon monoterpenes).

Experiment/ compound

Reactor type

Inoculum

Lag period (h)

Maximum degradation rate (mg l-1h-1)

Normalized degradation rate (h-1)

Experiment 1

terpinolene

CSR1

Unacclimated soil A extract

174

0.053

NM

Experiment 2

terpinolene

Serum tubes

Acclimated (from CSR1)

28

0.47

0.089

The normalized degradation rate is the maximum degradation rate normalized to biomass concentration expressed as volatile suspended solids

CSR 1: continuously-stirred reactor 1

NM: not measured because of lack of accurate biomass data

Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
Terpinolene was readily degraded under aerobic conditions at 23ºC by mixed cultures derived from forest soils.
Executive summary:

In a ready biodegradation study, terpinolene was tested under aerobic conditions at 23ºC and concentrations of 0.5-3 mg/L. Forest-soil extract cultures were used as inocula for the experiments conducted first without (experiment 1), then with prior microbial acclimation to the test material (experiment 2). The degradation of the test material was assessed by the determination of the biomass, concentration of the test material and headspace CO2. The test treatments and control (sodium azide, 2.5 g/L) were measured in duplicates. The lack of any substantial change in terpinolene concentration in the azide-amended control reactor demonstrated that disappearance of the test item in the test reactor was not the result of hydrolysis or any other physicochemical process. The normalised degradation rate in experiment 2 was 0.089 h-1. The maximum degradation rate in experiments 1 and 2 were 0.053 and 0.47 mg/L/h, respectively. The lag period in experiments 1 and 2 were 174 and 28 h, respectively. Under the test conditions, terpinolene was readily degraded by cultures derived from forest soils.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 July 1980 - 06 August 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
yes
Remarks:
(test duration lower than 28 d)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
14 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
not specified
Key result
Parameter:
% degradation (O2 consumption)
Value:
73
Sampling time:
14 d
Remarks on result:
other: Indirect analysis (BOD): 41, 81 and 98%; Direct analysis (GC): 100, 100 and 100%
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, dipentene was readily biodegradable.
Executive summary:

In a ready biodegradation study, dipentene was tested at concentrations of 100 mg/L and the inoculum was activated sludge (30 mg/L). The degradation of the test material was assessed by the determination of the oxygen consumption. At 100 mg/L test concentration, 73% degradation (biochemical oxygen demand) was reached in 14 days. Under the test conditions, dipentene was determined to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 February 1988 - 02 March 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(Test method according to OECD guideline 301 C)
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
94
Sampling time:
28 d
Remarks on result:
other: Indirect analysis (BOD): 102, 92 and 89%; Direct analysis (GC): 100, 100 and 100%
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, gamma terpinene was readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 C, gamma terpinene was tested at concentrations of 100 mg/L and the inoculum was activated sludge (30 mg/L). The degradation of the test material was assessed by the determination of the oxygen consumption. At 100 mg/L test concentration, 94% degradation (biochemical oxygen demand) was reached in 28 days. Under the test conditions, gamma terpinene was determined to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 301 C. No data on GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
not specified
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(%ThOD)
Value:
>= 1 - <= 4
Sampling time:
28 d

Camphene reached 1-4 % of its theoretical BOD after four weeks incubation, so it is not ready biodegradable.

Validity criteria fulfilled:
not specified
Remarks:
(No data on control nor reference substance)
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Camphene reached 1-4 % of its theoretical BOD after four weeks incubation, so it is not ready biodegradable.
Executive summary:

Camphene, present at 100 mg/L, reached 1 -4 % of its theoretical BOD after four weeks incubation with activated sludge inoculum (30 mg/L), using a test according to the Japanese MITI-I test (OECD Guideline 301 C). Under test conditions no biodegradation was observed so biodegradation of this substance is not a fast environmental fate process in water.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25 March 1997 - 22 April 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(Test method according to OECD guideline 301 C)
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
72
Sampling time:
28 d
Remarks on result:
other: Indirect analysis (BOD): 67, 70 and 79%; Direct analysis (HPLC): 100, 100 and 100%
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, terpinolene was readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 C, terpinolene was tested at concentrations of 100 mg/L and the inoculum was activated sludge (30 mg/L). The degradation of the test material was assessed by the determination of the oxygen consumption. At 100 mg/L test concentration, 72% degradation (biochemical oxygen demand) was reached in 28 days. Under the test conditions, terpinolene was determined to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD guideline 301 C. No data on GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
not specified
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(%ThOD)
Value:
>= 90 - <= 95
Sampling time:
28 d
Remarks on result:
other: Indirect analysis (BOD): 91, 90 and 95%; Direct analysis (GC): 100, 100 and 100%
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, alpha pinene was readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 C, alpha pinene was tested at concentrations of 100 mg/L and the inoculum was activated sludge (30 mg/L). The degradation of the test material was assessed by the determination of the oxygen consumption. At 100 mg/L test concentration, 90 -95% degradation (biochemical oxygen demand) was reached in 28 days. Under the test conditions, alpha pinene was readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 June 2010 - 08 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to OECD guideline 301 D under GLP with only minor deviations (ammonium chloride was omitted from the medium without resulting in nitrogen limitation; activated sludge was used as inoculum)
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation); activated sludge instead of an effluent/extract/mixture was used as inoculum
GLP compliance:
yes (incl. certificate)
Remarks:
2010-07-21
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 (Nieuwgraaf, Duiven, The Netherlands)
- Preconditioning: 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week and then diluted in the BOD bottles
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
- Composition of medium: Nutrient medium contained per liter of deionised water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCI2, 0.25 mg FeCI3.6H2O
- Source/preparation of dilution water: Deionised water containing no more than 0.01 mg/L Cu was prepared in a water purification system.
- Test temperature: 22-24 °C
- pH (at start of test): 7.0; pH (at end of test): 6.8 (control, and control with silica gel) and 6.9 (test)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Concentration of sludge: The sludge was diluted in the BOD bottles at 2 mg DW/L according to van Ginkel and Stroo (1992)
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel dosed with test substance, and 6 bottles containing sodium acetate and inoculum.
- The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. Administration of L-limonene was accomplished by dosing 0.03 g of L-limonene on 10 g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with alumiminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with L-limonene was added to the test bottles.

MEASURING EQUIPMENTS:
- Dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands)
- pH was measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Germany).
- Temperature was measured and recorded with a sensor connected to a data logger.

SAMPLING
- Sampling frequency: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Days 0, 7, 14, 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
- Other: Containing inoculum and silica gel
Reference substance:
acetic acid, sodium salt
Remarks:
Acros organics, Geel, Belgium. CAS reg. No. 127-09-3; Purity >99%; Batch/lot number A0206783001
Preliminary study:
No data
Test performance:
The pass level of 60% was reached after 7 to 8 days upon achieving 10% biodegradation. L-Limonene therefore fulfilled the 14-day time window (10-day for other OECD 301 tests) criterion for ready biodegradable compounds. The 14-day window is considered acceptable for the Closed Bottle test because the number of bottles to evaluate the 10-day window causes the test to become too unwieldy (OECD, 1992). L-Limonene is therefore classified as readily biodegradable.
Although the substance was degraded within a time window of 14 days it should be emphasized that time window should not be used as pass/fail criterion for
substances which consists of mixture of compounds (stereoisomers of limonene). The degradation curve is therefore the sum of two growth curves. The time window criterion was developed on the assumption that a single compound is degraded according to a “standard” growth curve in ready biodegradability tests.
Key result
Parameter:
% degradation (O2 consumption)
Value:
85
Sampling time:
28 d
Details on results:
Initial test material concentration: 2 mg/L
- Theoretical oxygen demand (ThOD) = 3.3 mg/mg
- % biodegradation on Day 8: > 60%
- % biodegradation on Day 28 = 85%
Results with reference substance:
- Theoretical oxygen demand (ThOD) = 0.8 mg/mg
- % biodegradation on Day 7 > 60%
- % biodegradation on Day 14 = 83%

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

 

Time (days) 

 Oxygen concentration (mg/L) 

 Ocs 

 Ot 

 Oc 

 Oa 

 0 

 8.6 

 8.6 

 8.6 

 8.6 

 

 8.6 

 8.6 

 8.6 

 8.6 

 Mean (M) 

 8.6 

 8.6 

 8.6 

 8.6 

 7 

 7.8 

 4.2 

 8.0 

 3.9 

 

 8.0 

 4.0 

 7.9 

 3.9 

 Mean (M) 

 7.9 

 4.1 

 8.0 

 3.9 

 14 

 7.8 

 2.6 

 7.7 

 3.4 

 

 7.6 

 2.8 

 7.8 

 3.2 

 Mean (M) 

 7.7 

 2.7 

 7.8 

 3.3 

 21 

 7.7 

 2.2 

 7.8 

 

 

 7.6 

 2.5 

 7.6 

 

 Mean (M) 

 7.7 

 2.4 

 7.7 

 

 28 

 7.6 

 2.0 

 7.5 

 

 

 7.6 

 2.0 

 7.6 

 

 Mean (M) 

 7.6 

 2.0 

 7.6 

 

Ocs: Mineral nutrient solution without test material but with inoculum and silica gel

Ot: Mineral nutrient solution with test material (2.0 mg/L), silica gel, and inoculum

Oc: Mineral nutrient solution with only inoculum

Oa: Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum

 

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

 

Time (days) 

 Oxygen consumption (mg/L) 

 Biodegradation (%) 

Test substance

 Acetate 

 Test substance

 Acetate 

 0 

 0.0 

 0.0 

 0 

0

 7 

 3.8 

 4.1 

 58 

76

 14 

 5.0 

 4.5 

 76 

83

 21 

 5.3 

 

 80 

 

 28 

 5.6 

 

 85 

 

Validity criteria fulfilled:
yes
Remarks:
The validity of the test is demonstrated by: an endogenous respiration at Day 28 of 1.0 mg/L; differences of the replicate values at Day 28 < 20%; degradation in reference material of 83% at Day 14 and O2 concentration during the test> 0.5 mg/L.
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, l-limonene was readily biodegradable.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

l-limonene did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. l-limonene was biodegraded 85% at day 28 in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 83% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The pass level of 60% was reached after 7 to 8 days upon achieving 10% biodegradation. L-Limonene therefore fulfilled the 14-day time window (10-day for other OECD 301 tests) criterion for ready biodegradable compounds. Hence, this substance should be classified as readily biodegradable.

Description of key information

Key study: Test method according to OECD 301F / EU C.4 (Manometric respirometry methods), GLP study. The test item attained 63.5% biodegradation but not reached within the 10 day window. However, the substance could be considered as readily biodegradable assuming that a sequential biodegradation of the individual structures of the UVCB is presumably taking place according to the content of the test substance and previous information of the individual components (supporting studies mentioned below).

Supporting studies: Individual biodregradabilities of the main components are available from experimental tests. Terpinolene, d-limonene, l-limonene, dipentene, gamma terpinene and alpha pinene were found as readily biodegradable. Only camphene was determined to be not ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:
freshwater

Additional information

Key study. A ready biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test item, according to OECD 301F / EC C.4 – D manometric respirometry methods, in accordance with GLP principles. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 2ºC) for 28 days. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 34.9%. Thus, the test item is not inhibitory. All validity criteria were fulfilled. The  biodegradability of the test item attained 63.5% within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 41.7%. However, according to the "Revised introduction to the OECD guidelines for testing of chemicals, SECTION 3, PART 1", 23 March 2006, and considering that the test substance is a mixture of structurally similar chemicals with different degree of isomers, the 10-day window should not be applied to interpret the results since a sequential biodegradation of the individual structures is presumably taking place according to the content of the test substance and previous information of the individual components (supporting studies mentioned below). Thus, the test substance could be classed as readily biodegradable.

Supporting studies: Individual biodegradabilities of the main components are available from experimental tests:

Terpinolene: Test method equivalent to OECD 301C (MITI, 1997). The test substance is ready biodegradable.

Terpinolene: Peer reviewed publication (Misra G, 1996). The test substance was readily degraded under aerobic conditions by mixed cultures derived from forest soils.

Camphene: Test method according to OECD 301C (MITI, 1992). The test substance is not ready biodegradable.

L-Limonene: Test method according to OECD 301F (Van Ginkel CG, 2010). The test substance is ready biodegradable.

D-Limonene: Peer reviewed publication (Misra G, 1996). The test substance was readily degraded under aerobic conditions by mixed cultures derived from forest soils.

Dipentene: Test method equivalent to OECD 301C (MITI, 1980). The test substance is ready biodegradable.

Alpha pinene: Test method according to OECD 301C (MITI, 2005). The test substance is ready biodegradable.

Gamma Terpinene: Test method equivalent to OECD 301C (MITI, 1988). The test substance is ready biodegradable.

Gamma Terpinene: Peer reviewed publication (Misra G, 1996). The test substance was readily degraded under aerobic conditions by mixed cultures derived from forest soils.