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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:
22 Oct. - 01 Dec. 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
use of radiolabelled test substance, low initial concentration (range of water solubility); reduction of the ratio headspace to water volume (50 : 200 mL = 1 : 4)
Qualifier:
according to guideline
Guideline:
other: ISO 14593:2005 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)"
Principles of method if other than guideline:
Ultimate biodegradation/mineralisation by measuring the formation of 14CO2 from radiolabelled precursor
GLP compliance:
yes (incl. QA statement)
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):
activated sludge from the activation basin of a predominantly domestic biological sewage treatment plant
(community STP, Stadtentsorgung Neustadt, Im Altenschemel, Lachen-Speyerdorf)
- Method of cultivation: freshly prepared
- Storage conditions: no storage
- Preparation of inoculum for exposure: The sludge was washed with tap water twice, then filtrated through a cloth,
resuspended in test medium and aerated. An aliquot was added to the test medium.
- Pretreatment: no pretreatment
- Concentration of sludge: 3.36 g dw suspended solid/L
Duration of test (contact time):
56 d
Initial conc.:
ca. 0.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: standard medium according to test guideline
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 22 ± 2 °C
- pH: no data
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: ca. 10 ± 1 mg/L
- Continuous darkness: no, no direct light, bottles used were made of brown glass

TEST SYSTEM
- Culturing apparatus: size of test flasks was 250 mL (fill volume 200 mL). The flasks were closed with a silicon septum and a lid. Flasks were set up in a rotation shaker in an air-conditioned room.
- Number of culture flasks/concentration: test vessels, negative and positive controls: 2; medium blank and abiotic control: 1
- Method used to create aerobic conditions: prior to test aeration of dilution water, during test air supply of the head space
- Measuring equipment: carbon analyzer TOC multi N/C 2100S; liquid scintillation counter Thriathler, Hidex Oy, Turku, Finland
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: 1 M NaOH (2 mL) as absorbing agent for CO2 in head space located in an external micro-scrubber attached to test vessels via a needle passing the cover septum of the flask; activated charcoal in small absorber tubes to collect volatile organics in head space

SAMPLING
- Sampling frequency: day 0, 4, 8, 14, 21, 28, 40, and 56
- Sampling method: see above "Details on analytical methods"
- Sample storage before analysis: only charcoal absorber tubes were stored.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: 1, poisoned with 5 mg HgCl2/L
- Toxicity control: no (not relevant due to low test concentration)
- Other: 1 medium blank without test substance and inoculum

STATISTICAL METHODS: no statistics
Reference substance:
aniline
Remarks:
initial concentration: 20 mg organic carbon/L
Parameter:
% degradation (CO2 evolution)
Value:
<= 0.1
Sampling time:
56 d
Remarks on result:
other: based on CO2 formation, no biodegradation was observed
Parameter:
% degradation (radiochem. meas.)
Remarks:
distribution of 14C marker
Value:
ca. 15
Sampling time:
56 d
Results with reference substance:
Reference substance (aniline, 20 mg C/mL) performed as expected. Biodegradation was ca. 99% after 14 days.
day 4 8 14 21 28 40 56
% degradation 76.2 73.1 98.5 100.5 95.4 101.7 98.0
(mean of 2 replicates)

Reference radioactivity for initial test substance concentration (40 µg test substance)

Determination of total radioactivity for initial test substance concentration as described under "Any other information on materials and methods incl, tables" resulted in an activity of 330770 cpm.

 

CO2 formation

In CO2 headspace samples, virtually no radioactivity was detected (< 0.05%) during the total 56 day test period (see table below). There was no ultimate biodegradation of diisopropylnaphthalene.

 

Activated charcoal from absorber

The amount of radioactivity in the charcoal of the absorber from sampling of headspace was ≤ 0.02%. No organic radioactive substances were found in the headspace withdrawn from the test vessels.

 

Residual radioactivity in test vessels

Non polar substances (xylene extractable fraction under acidic condition)

In xylene extracts of the test medium from test vessels, ca. 65 to 85% of radioactivity was detected. Information about the nature of chemical substances present is not available.

Polar substances (fraction remaining in aqueous phase)

Radioactivity in the residual aqueous phase accounted for 10 to 21% of total radioactivity (single values, mean 15 %), indicating to the formation of polar substances from diisopropylnaphthalene, probably by biotic and abiotic transformation of the side chain.

 

Abiotic control

Distribution of radioactivity in abiotic controls was similar to that in test vessels. Depending on the incubation period, xylene extracts of test medium accounted for approx.. 70 to 85% of radioactivity. In the residual aqueous phase ca. 10 to 20% of total radioactivity (single values, not shown) was found, on the average apoprox. 15% from day 28 through 56.

 

 

Distribution of radioactivity (in %) (from Report Table 8.3 -i and 8.2 -j)

 

Day

Headspace

Test medium

 

CO2

Xylene extract

Aqueous phase (after extraction)

 

Test 1

Test 2

Abiotic control

Test 1

Test 2

Abiotic control

Test 1

Test 2

Abiotic control

4

0.005

0.00

0.002

85.4

84.7

106.3

no data 

 no data

 no data

8

0.005

0.003

0.002

89.7

78.3

86.0

no data

 no data

 no data

14

0.009

0.006

0.005

73.1

60.8

85.0

 no data

no data 

 no data

21

0.009

0.011

0.008

65.8

80.3

83.8

 no data

no data 

no data 

28

0.004

0.016

-0.003

71.7

67.8

73.7

15.5

15.7

16.8

40

0.014

0.102

0.006

72.4

70.8

76.1

12.8

17.0

19.7

56#

0.032

0.019

0.007

66.6

70.4

72.4

15.6

12.2

11.7

  # Day 56: Average value of three replicates.

 

Ultimate degradation (in %) of test substance as determined by CO2 measurements (radioactive CO2)

 

Day

Test 1

Test 2

Abiotic control

4

0.01

0.00

0.00

8

0.00

0.00

0.01

14

0.01

0.01

0.01

21

0.01

0.01

0.01

28

0.00

0.02

0.00

40

0.01

0.10

0.01

56

0.03

0.02

0.01

 

 

Information on primary biodegradation was investigated in a separate experiment and is reported in LAUS GmbH 2010b.

Validity criteria fulfilled:
yes
Interpretation of results:
other: under test conditions no ultimate biodegradation observed
Executive summary:

In a sealed vessels ready biodegradability test according to OECD test guideline 310, 14C-labelled diisopropylnaphthalene (200 µg/L) was incubated for 56 days with activated sludge (10 mg/L SS) from a predominantly domestic sewage treatment plant. Formation of CO2 was determined on day 4, 8, 14, 21, 28, 40, and 56 by counting of CO2 radioactivity isolated from test vessels.

No radioactive CO2 (ultimate biodegradation) could be detected during the test period. Radioactivity was found prevalently in the organic extract of the test medium (extraction with xylene under acidic condition). Radioactivity in the organic extract decreased over time from ca. 85% at day 4 to ca. 60 to 75% at the end of test period. On the average, the residual aqueous phase accounted for approx. 15% (only examined from day 28).

Approx. 15% radioactivity was not detected anymore in the fractions examined. There may be some loss of test material due to adsorption to test vessel walls. An abiotic control showed the same pattern. Radioactivity was found in the organic extract and the residual aqueous phase of the test medium. Distribution of radioactivity was similar to experiments with inoculum.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Oct. - 19.11.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
low initial concentration (range of water solubility); reduction of the ratio headspace to water volume (50 : 200 mL = 1 : 4); GC analysis
Principles of method if other than guideline:
Primary biodegradation/elimination of single components of diisopropylnaphthalene by analysing the GC profile over time.
GLP compliance:
yes (incl. QA statement)
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): activated sludge from the activation bassin of a predominantly domestic biological sewage treatment plant (community STP, Stadtentsorgung Neustadt, Im Altenschemel, Lachen-Speyerdorf)
- Method of cultivation: freshly prepared
- Storage conditions: no storage
- Preparation of inoculum for exposure: The sludge was washed with tap water twice, then filtrated through a cloth, resuspended in test medium and aerated. An aliquot was added to the test medium
- Pretreatment: no pretreatment
- Concentration of sludge: 3.36 g dw suspended solid/L
Duration of test (contact time):
56 d
Initial conc.:
ca. 0.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
; GC-analysis of diisopropylnaphthalene isomers
Details on study design:
TEST CONDITIONS
- Composition of medium: standard medium according to test guideline
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 22 ± 2 °C
- pH: no data
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: ca. 10 ± 1 mg/L
- Continuous darkness: no, no direct light

TEST SYSTEM
- Culturing apparatus: size of test flasks was 250 mL (fill volume 200 mL). The flasks were made of DURAN 3.3 and closed with red lids and silicon septa. Flasks were set up in a rotation shaker in an air-conditioned room.
- Number of culture flasks/concentration: test vessels, negative and positive controls: 2; medium blank and abiotic control: 1
- Method used to create aerobic conditions: prior to test aeration of dilution water, during test air supply of the head space
- Measuring equipment: carbon analyzer TOC multi N/C 2100S; HP Model 6890N GC
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: positive controls: CO2 was measured as carbonate after adjustment of test vessels pH with 1 mL 7M NaOH in closed vessel. Test vessels: no carbon dioxide measurementsTest substance was extracted in 10 mL iso-hexane in closed vessel.

SAMPLING
- Sampling frequency: day 0, 4, 8, 14, 21, 28, 40, and 56
- Sampling method: for CO2 measurement, 1 mL sample was taken directly from test medium 1 hour after addition of NaOH solution.
for analysis of test substance,
for test substance measurements, iso-hexane extracts of test medium (see above "Details on analytical method") were concentrated and aliqots were injected into GC.
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM (to be checked)
- Inoculum blank: 2
- Abiotic sterile control: 1, poisoned with 50 mg HgCl2/L
- Toxicity control: no (not relevant due to low test concentration)
- Other: 1 medium blank without test substance and inoculum

STATISTICAL METHODS: no statistics
Reference substance:
aniline
Key result
Parameter:
% degradation (test mat. analysis)
Value:
21 - 30
Sampling time:
56 d
Remarks on result:
other: primary biodegradation; range of results from day 21 to 56
Results with reference substance:
Reference substance (aniline, 20 mg C/mL) performed as expected. Biodegradation was ca. 99% after 14 days.
day 4 8 14 21 28 40 56
% degradation 76.2 73.1 98.5 100.5 95.4 101.7 98.0
(mean of 2 replicates)

Within the test period of 56 days, primary degradation of ca. 50% was observed. Abiotic degradation contributed ca. 20 - 30%.

Average biodegradation was approx. 25%. The time course of primary degradation is presented in the following table.

After a test period of ca. 21 days, there was no more substantial increase in total biodegradation.

 

Mean percentage of degradation as sum of all peaks (total of diisopropylnaphthalene) [Report, Table 9.2 -d]

Day

Primary degradation
total

Primary degradation
abiotic

Primary degradation
biodegradation

0

0

0

0

4

5

6

-1

8

19

7

12

14

24

16

8

21

32

11

21

28

43

20

23

40

47

17

30

56

53

32

21

 

 

Individual isomers of the diisopropylnaphthalene technical product mixture showed different susceptibility to degradation as is illustrated in the following table.

 

 

Percentage of degradation for individual diisopropylnaphthalene (DIPN) isomers as mean of replicate determinations calculated from peak areas (isomers are characterised by substituent position numbers) [from Report, Table 9.2 -b and 9.2 -c]

Day

Peak 1
(1,3-DIPN)

Peak 2
(1,7- DIPN)

Peak 3
(2,6- DIPN)

Peak 4
(2,7- DIPN)

Peak 5
(1,6- DIPN)

Peak 6
(1,4- DIPN)

Peak 7
(1,5- DIPN)

 

total

biotic

total

biotic

total

biotic

total

biotic

total

biotic

total

biotic

total

biotic

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4

-3

-10

0

-8

18

12

11

5

7

2

-3

-7

7

0

8

7

2

11

3

40

33

28

20

24

17

7

2

15

7

14

3

-10

9

-8

59

40

37

18

41

24

5

-9

15

1

21

8

-1

13

3

82

70

45

33

38

27

7

-1

30

9

28

8

-10

14

-5

100

76

59

35

78

59

7

-6

33

14

40

12

-3

19

3

100

81

68

49

87

69

13

-1

34

17

56

17

-12

28

-6

100

>81

78

36

92

56

21

-9

52

15

  Negative percentages indicate that the abiotic value was determined to be higher than the biotic portion.

 

Total primary degradation was found for all isomers (between 17 and 100%). But biodegradation was only observed for 4 of 7 isomers (between ca. 15% - 1,5 -DIPN to >81% - 2,6 -DIPN). For 1,3 -DIPN, 1,4-DIPN, and 1,7 -DIPN, no primary biodegradation could be demonstrated in the present experiment.

 

Capability of biodegradation appears to be dependent on isomeric structure. In general, biodegradation appeares to proceed the better if the two isopropyl-substituents are attached to different rings of naphthalene and the further apart the two substituents are from each other (2,6- > 1,6- > 2,7- > 1,5-).

 

Obviously there was no relevant increase in primary biodegradation after day 28 of the experiment. Variable results do not show a clear trend and are considered to be caused by variances of individual test vessels and by measurement inaccuracy.

 

 

Averaged peak areas from single runs (corrected by ISTD) (DIPN isomers are characterised by substituent position numbers)

 

Day

Sample

Area
Peak 1 (1,3-)

Area
Peak 2 (1,7-)

Area
Peak 3 (2,6-)

Area
Peak 4 (2,7-)

Area
Peak 5 (1,6-)

Area
Peak 6 (1,4-)

Area
Peak 7 (1,5-)

0

Replic. 1

44.42

54.15

48.65

40.45

43.28

47.97

27.44

Replic. 2

47.62

57.97

52.73

43.74

46.36

50.93

27.28

Abiotic

56.27

69.27

64.04

52.82

55.03

60.07

29.42

4

Replic. 1

45.88

54.76

40.14

36.11

39.97

48.98

20.77

Replic. 2

48.56

57.53

42.54

38.57

43.59

53.15

29.87

Abiotic

52.40

64.14

59.67

49.60

52.18

58.05

27.15

8

Replic. 1

43.29

50.12

29.94

30.15

33.76

46.16

20.34

Replic. 2

42.22

49.45

30.62

30.71

34.05

46.19

26.06

Abiotic

53.48

63.84

59.16

48.93

51.23

57.36

27.02

14

Replic. 1

44.70

50.82

20.17

26.60

25.50

46.81

21.10

Replic. 2

44.84

51.47

21.34

26.41

27.37

47.25

25.31

Abiotic

49.07

57.84

51.63

42.63

45.47

51.80

25.31

21

Replic. 1

42.34

48.22

6.86

22.15

26.00

45.39

18.01

Replic. 2

42.70

48.84

11.12

24.28

29.69

46.93

20.40

Abiotic

51.27

61.91

56.37

46.35

49.16

55.66

23.39

28

Replic. 1

41.66

47.07

< LOD

17.27

8.82

44.29

17.09

Replic. 2

42.80

48.86

< LOD

17.57

10.63

47.41

19.34

Abiotic

46.30

55.78

48.83

40.57

44.23

51.92

23.70

40

Replic. 1

36.23

39.75

< LOD

7.48

4.82

38.54

14.78

Replic. 2

45.35

51.53

< LOD

19.53

7.28

48.17

21.55

Abiotic

48.27

58.15

51.77

42.62

45.46

51.84

24.56

56

Replic. 1

37.08

41.15

< LOD

12.75

4.75

40.09

15.55

Replic. 2

37.31

42.49

< LOD

12.92

4.65

41.34

16.81

Replic. 3

38.67

35.91

< LOD

8.04

3.58

34.65

14.25

Replic. 4

36.34

45.57

< LOD

-

-

44.32

2.75

Replic. 5

38.71

41.23

< LOD

10.80

4.14

39.11

15.09

Replic. 6

39.95

38.02

< LOD

14.11

4.92

37.20

15.71

Abiotic 1

43.76

51.16

< LOD

35.87

39.18

46.26

19.66

Abiotic 2

40.10

42.56

< LOD

29.46

32.81

39.42

18.54

Abiotic 3

38.48

47.00

< LOD

30.99

35.72

44.37

17.93

 

 

In diisopropylnaphthalene test vessels, CO2 development (ultimate biodegradation) was not measured. Ultimate biodegradation was tested in a separate test using 14C radiolabelled test substance (LAUS GmbH 2010a).

 

Procedure controls performed as expected.

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable; only partial primary biodegradation
Conclusions:
In a sealed vessels ready biodegradability test according to OECD test guideline 310, diisopropylnaphthalene (200 µg/L) was incubated for 56 days with activated sludge (10 mg/L SS) from a predominantly domestic sewage treatment plant. Primary degradation was followed by analysing the decrease of diisopropylnaphthalene levels in test vessels. Diisopropylnaphthalene was analysed by a GC-method measuring seven individual diisopropylnaphthalene isomers (individual GC peaks). Degradation was measured on day 4, 8, 14, 21, 28, 40, and 56.
Total primary degradation of diisopropylnaphthalene was ca. 50% within the test period. 20 to 30% was caused by abiotic degradation. Primary biodegradation was found to be about 20 to 30% for all isomers. There appeared no relevant increase in overall biodegradation from day 21 to day 56 (varying levels of biodegradation between 20 to 30%)
Distinct diisopropylnaphthalene isomers showed variable biodegradation. For three isomers, no primary biodegradation could be demonstrated. For four isomers, 15 to 100% primary biodegradation was noted at the end of the test period. Abiotic degradation was similar for all isomers within certain variations. Between day 14 and 40, abiotic biodegradation seemed to level off at around 15 to 20%. On day 56, rates of around 30 to 35% abiotic primary degradation was observed.
Executive summary:

In a sealed vessels ready biodegradability test according to OECD test guideline 310, diisopropylnaphthalene (200 µg/L) was incubated for 56 days with activated sludge (10 mg/L SS) from a predominantly domestic sewage treatment plant. Primary degradation was followed by analysing the decrease of diisopropylnaphthalene levels in test vessels.

Diisopropylnaphthalene was analysed by a GC-method measuring seven individual diisopropylnaphthalene isomers (individual GC peaks). Degradation was measured on day 4, 8, 14, 21, 28, 40, and 56.

Total primary degradation of diisopropylnaphthalene was ca. 50% within the test period. 20 to 30% was caused by abiotic degradation. Primary biodegradation was found to be about 20 to 30% for all isomers. There appeared no relevant increase in overall biodegradation from day 21 to day 56 (varying levels of biodegradation between 20 to 30%).

The diisopropylnaphthalene isomers showed variable biodegradation. For three isomers, no primary biodegradation could be demonstrated. For four isomers, 15 to > 81% primary biodegradation was noted at the end of the test period. Abiotic degradation was similar for all isomers within certain variations. Between day 14 and 40, abiotic biodegradation seemed to level off at around 15 to 20%. On day 56, rates of around 30 to 35% abiotic primary degradation were observed.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Nov 2004 - 25 Jan 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
including non-adapted and adapted inoculum
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
other: Activated sludge (non-adapted or actively adapted) mixed with extract from soil
Details on inoculum:
Source and preparation of inoculum/activated sludge:
- Mixture of sewage microorganisms: derived from a municipal sewage treatment plant (sewage treatment plant at Taunusstein-Bleidenstadt, Germany) processing predominantly domestic sewages, and soil microorganisms (derived from soil on the Institute Fresenius area at Taunusstein, Germany).
- Laboratory culture of soil microorganisms: The soil inoculum was freshly prepared on the day of use. A mixture of 100 g soil was suspended in 1000 ml drinking-water and shaken on a shaking machine for approx. 30 minutes. After settling, the suspension was filtered through a folded paper filter. The first 200 ml were discarded. The filtrate was used as the second source of inoculum.

First test series (without special adaptation of microorganisms):
- Preparation of inoculum for exposure: Equal volumes of soil inoculum and non-adapted inoculum of sewage were mixed for the test. 5000 ml of the test solutions were inoculated with 2 ml of the mixed inoculum.

Second test series (with special adaptation of microorganisms):
Pretreatment of the sewage sludge for adaptation:
Every 2-3 days, approximately 200 mg of the test item were added to the 5 L of inoculum (in total 1023.3 mg) (for details see: Report, page 7, chapter 8.2.1.2). Equal volumes of soil inoculum and adapted inoculum of sewage were mixed for the test. 5000 ml of the test solutions were inoculated with 2 ml of the mixed inoculum.

Aeration by stirring on a magnetic stirrer.
Duration of test (contact time):
ca. 28 d
Initial conc.:
>= 3.3 - <= 3.5 mg/L
Based on:
other: ThOD (first test series)
Initial conc.:
>= 3.7 - <= 3.9 mg/L
Based on:
other: ThOD (second test series)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Preparation of test medium: Aliquots of the test substance were dosed from a stock solution in dichloromethane (51 - 55 mg/50 mL solvent),
0.3 mL pipetted into each test flask. After evaporation of the solvent, 290 mL of test medium were added.
- Reference substance: Sodium benzoate (final test concentration: 3.3 - 3.5 mg ThOD/L, first test series; 3.7 - 3.9 mg ThOD/L, second test series)
- Solvent: Dichloromethane (evaporated for 1 h before test)
- Test temperature: 20°C - 22°C (first test series), 20°C - 21°C (second test series)
- pH: adjusted: no data
- Continuous darkness: yes
- Oxygen content: 9.24 mg O2/L (prior to start of first test series), 9.12 mg O2/L (prior to start of second test series)

TEST SYSTEM
- Number of culture flasks: 15
- Number of blank control flasks flasks: 15
- Number of reference controls: 15
- Test performed in closed vessels due to significant volatility of test substance: Yes

SAMPLING
- Sampling: At start (2 flasks), day 7, day 14, day 21 (3 flasks), day 28 (4 flasks);
2-4 flasks each series (test solutions with the test item, the reference item and the toxicity control) were analysed for oxygen content at each
sampling time with an oxygen electrode (Oximeter WTW model OXI 530 with Tri Oxmatic EO 200 electrode.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test Item
- Abiotic sterile control: No
- Toxicity control: Series of test solutions containing both test item and reference substance (equal to concentrations in test solutions) was
examined in parallel.

CALCULATION
- Degradation and biochemical oxygen demand (BOD): % degradation= (BOD (mg/L) / AS (mg/L)xThOD) x 100
- Theoretical oxygen demand (ThOD):
Sodium benzoate (MW: 144.1 g7mol): ThOD=16x (2x7C + 0.5 x 5 H –2O +0.5Na)/ 144.1=1.665 O2/mg
DIPN (MW: 212 g/mol; purity: 98 %): ThOD=16 x (2 x 16C + 0.5 x20H) x 0.98/ 212 = 3.1064 mg O2/mg
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
ca. 3
Sampling time:
28 d
Remarks on result:
other: with non-adapted microflora
Parameter:
% degradation (O2 consumption)
Value:
ca. 17
Sampling time:
28 d
Remarks on result:
other: with adapted microflora
Details on results:
- First test series (non-adapted): 3 % degradation of DIPN within 28 days (table 1, attached documen))
- Second test series (adapted): 17 % degradation within 28 days (table 5, attached document)
- The threshold of 'Ready biodegradability' of 60 % degradation and the 14-days-window (required by OECD Guideline 301 D for 'Ready
biodegradability') was not met.
- No toxicity was observed in both test series.
- The biodegradation profiles showed a significant tendency for better degradation in the presence of sodium benzoate.
Parameter:
BOD5
Value:
>= 0.068 - <= 0.178 other: mg O2/L test solution after 28 d incubation (with non-adapted microflora)
Parameter:
BOD5
Value:
>= 0 - <= 0.595 other: mg O2/L test solution after 28 d incubation (with adapted microflora)
Results with reference substance:
- First test series (non adapted): 69 % degradation within 28 days (see table 2, attached document)
- Second test series (adapted): Aprox. 80 % degradation within 28 days (see table 6, attached document)
- The threshold of 'Ready biodegradability of 60 % degradation was reached within 7 days in both test series.
Validity criteria fulfilled:
yes
Interpretation of results:
other: not clearly evaluable
Conclusions:
The test item DIPN is not readily biodegradable. One reason for this result may be: poor water solubility, poor bioavailability.
(Note: the test solutions of the closed bottle test are not moved by stirring or shaking.)
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15-July-2005 - 09-Nov-2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 D Draft (Inherent Biodegradability - Concawe Test)
Principles of method if other than guideline:
- The "inherent biodegradability" of DIPN was to be tested according to OECD-Test Guideline 302 D [Draft Version, October 2001 (Concawe Test)]
after a ready biodegradation test (OECD 301 D, Closed-Bottle-Test) failed to show that DIPN was readily biodegradable.
(Inst. Fresenius IF-04/00281301, 25 Jan. 2005)
- Appropriate test system to investigate the inherent aerobic biodegradability of a test compound and useful for insoluble and/or volatile materials
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge: Mixture of sewage microorganisms derived from a municipal sewage treatment plant (sewage treatment
plant at Taunusstein-Bleidenstadt, Germany) dealing with predominant domestic sewages, and soil microorganisms (derived from soil on the
Institute Fresenius area at Taunusstein, Germany) being both adapted to the test item
- Laboratory culture of soil microorganisms: A mixture of 100 g soil was suspended in 1000 ml drinking-water and shaken on a shaking machine
for approx. 30 minutes. After settling, the suspension was filtered through a coarse cotton filter. The first 200 ml were discarded.
The filtrate was used as the second source of inoculum.
- Preparation of inoculum for exposure: 4500 ml of sewage sludge and 500 ml of filtrate of soil microorganism were mixed in a 5-l Erlenmeyer flask.
Thereafter, necessary mineral salts were added from stock solutions. Additionally, 250 mg yeast extract were added to the solution as vitamin and
macronutrient. This mixture was incubated on a magnetic stirrer and aerated by means of compressed air.
- Pretreatment: Every 2-3 days, approximately 200 mg of the test item were added to the 5 L of inoculum until 10 days (in total 1006.3 mg).
The Incubation of the sludge was continued for up to day 14 in order to degrade the remaining test item.
Thereafter, this mixture was washed twice with the mineral nutrient medium in order to eliminate remaining organic components and inorganic
matter (CO2 and carbonates).
Thereafter, homogenisation by means of a waring blender and filtration through a coarse cotton filter being previously rinsed with deionized water
in order to eliminate organic impurities from the cotton material. The prepared inoculum (filtrate) was acidified to a value of pH 6.5 using diluted
phosphoric acid and then aerated with CO2-free compressed air for the duration of approx. 1h in order to eliminate CO2.
Thereafter, the pH was adjusted to a value 7.45.
- Initial biomass concentration: 500 ml of the prepared inoculum was added to a final volume of 10 L of test solution resulting in 8 mg dry solids
each litre.
Duration of test (contact time):
>= 56 d
Initial conc.:
20 mg/L
Based on:
other: TOC/L
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Preparation of test medium: 7 µl of the test item were injected in one step into closed serum flasks with the test medium.
- Preparation of reference substance: 1 ml from stock solution (399.7 mg dissolved in 50 ml dichloromethane) was transferred to serum flasks.
Thereafter, the solvent was evaporated. 300 ml of inoculated medium was added and closed with the Teflon septum.
- Additional substrate: see above
- Solvent: Dichloromethane (only used for reference item)
- Test temperature: 20°C
- pH: adjusted to 7.45
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Shaking machine: Buhler, VKS
- Number of culture flasks: 20
- Number of blank control flasks flasks 20
- Number of reference controls: 40
- Measuring equipment: Carbon analyser for TIC determination: LiquiTOC, Foss-Heraeus
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Details of trap for CO2 and volatile organics if used: CO2 being formed due to biodegradation processes of the test item (reference item) is
accumulated in the headspace above the test solution and within the test solution itself.

SAMPLING
- Sampling frequency: About weekly, 2-3 bottles each series (test solutions with the test item, the reference item and the toxicity control)
- Sampling method: Using a syringe, 2 ml of a freshly prepared 10 M NaOH was transferred through the septa into the liquids, and shaking was
continued for about 1h. Within that time, CO2 in the headspace was transferred into the liquid.
The prepared solutions are analysed for TIC (Total Inorganic Carbon).

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test Item
- Abiotic sterile control: No
- Toxicity control: 7 µl of the test item were injected into the prepared closed serum flasks with the test medium. Thus, these toxicity controls
contained both the test and the reference item at the same concentration as within the individual test solutions with the test and the reference item alone, respectively.
Reference substance:
other: Rape oil, refined (see report p. 6 for details)
Details on results:
Under the chosen conditions, a degradation of about 26 % was obtained after 7 d, but thereafter was increasing only slowly to 37 % (mean of the last 4 measurements) within 56 d using an adapted microflora (for details see tables 1 and 2 below and report Fig. 1)
No toxicity towards the microflora at the concentration of the test item was noted (see report table 4 and fig. 1).
Results with reference substance:
The reference item rape oil was degraded by 83 % within 56 d (mean of the last 4 measurements) using the microflora adapted to the test item
(see report table 3 and fig. 1)

Table 1:       Results TIC Determinations (Report p 12)

Date

Time [d]

TIC-Values[mgTIC/L]

Blank

Mean

Blank

Reference

Mean

Reference

Test Item

Mean

Test Item

Tox-Control

Mean

Tox-Control

05.08.05

7

0.26

0.36

14.09

14.57

4.88

5.56

18.55

18.94

0.45

15.04

6.24

19.33

12.08.05

14

2.45

1.94

18.74

18.03

7.66

7.46

25.31

23.31

1.42

17.32

7.26

21.30

19.08.05

21

1.35

1.31

20.05

19.51

7.37

7.47

23.30

23.09

1.27

18.96

7.57

22.88

26.08.05

28

1.22

1.90

16.98

17.83

7.39

7.72

22.15

22.47

2.57

18.67

8.04

22.79

02.09.05

35

2.31

2.19

20.58

18.95

8.23

10.38

24.32

23.51

2.07

17.32

12.52

22.69

09.09.05

42

1.70

1.41

(33.57)')

17.62

7.21

7.69

25.21

25.01

1.12

17.62

8.16

24.81

16.09.05

49

1.63

1.40

19.02

18.58

10.21

9.21

36.84

35.11

1.16

18.13

8.21

33.38

23.09.05

56

1.79

1.87

18.27

18.27

9.28

9.10

22.58

28.21

2.11

(29.63)*)

8.60

39.29

1.71

(38.80)*)

9.42

22.77

*)This value is not taken for the final calculation because it is beyond the maximum of about 20mg/Ltheoretically possible.

Table 2:         % Degradation of the Test Item (Report p 12)

Date

Time [d]

Test Item

Test [mg C/L]

Test Blank [mg C/L]

% Degradation

Mean % Degradation

05.08.05

7

4.88

4.53

22.40

25.77

6.24

5.89

29.13

12.08.05

14

7.66

5.73

28.34

27.35

7.26

5.33

26.36

19.08.05

21

7.37

6.06

30.02

30.51

7.57

6.26

31.00

26.08.05

28

7.39

5.50

27.20

28.81

8.04

6.15

30.42

02.09.05

35

8.23

6.04

29.92

40.53

12.52

10.33

51.14

09.09.05

42

7.21

5.80

28.70

31.07

8.16

6.75

33.43

16.09.05

49

10.21

8.81

43.62

38.68

8.21

6.82

33.74

23.09.05

56

9.28

7.41

36.68

35.79

8.60

6.73

33.30

9.42

7.55

37.38

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
In the presence of adapted inoculum, teh degradation rate increased to 37 %/56 d, while in the presence of non-adapted only biodegradation rate of 18%/56 d had been attained (latter not shown). In either case, no significant continuation of biodegradation was observed within the observation time of 56 d.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given; comparable to guideline study; test was performed by a laboratory with an high reputation for delivery of solid data.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Principles of method if other than guideline:
Similar or according to later OECD test guideline 302 C (MITI II Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sludge was sampled and prepared according to Japanese standard procedure (samples taken from 10 different sites: 3 city sewage plants, 1 industrial sewage plant, 3 rivers and 3 bays; sampling 4 times every year)
- Laboratory culture: sludge was kept in culture for 3 months
- Method of cultivation: Sludge was cultured at 25 ± 2°C. Every day about 30 min after cessation of aeration, 1/3 of the supernatant was removed and replaced by an equal amount of dechlorinated water. Aeration was continued and synthetic sewage (5% w/v of glucose, peptone and monopotassium phosphate, pH adjusted to 7 ± 1 with sodium hydroxide) was added.
- Storage conditions: at 25 ± 2°C
- Storage length: 3 months
- Preparation of inoculum for exposure: measurement of concentration of suspended solid according to JIS K 0102 method 14.1
Duration of test (contact time):
14 - 28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 3 mL of solution A, B, and C as described in JIS K 0102 method 21 were made up to 1000 mL with purified water and the pH was adjusted to 7.0
- Test temperature: 25 ± 2
- pH: 7
- pH adjusted: if required
- Aeration of dilution water: no data
- Suspended solids concentration: 100 ppm
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measuring apparatus; 300 mL glas vessels
- Number of culture flasks/concentration: 3 for test substance, 2 for test substance blanks, 1 for abiotic control, 1 for positive control
- Method used to create aerobic conditions: production of O2 as O2 was consumed.
- Measuring equipment: Coulometer Ohkura Electric Co., Ltd.
- Test performed in closed system: yes
- Details of trap for CO2 and volatile organics if used: soda lime No. 1 (extra pure reagent, Wako Pure Chemical Industries, Ltd.)
- Other: test solution was stirred by a magnetic stirrer

SAMPLING
- Sampling frequency: continuous registration of O2 consumption; test substance determination at the beginning and at the end of the test

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic control: yes
- Toxicity control: no
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
7.6
Sampling time:
28 d
Results with reference substance:
Biodegradation as required (60% within 5 days)

The high test-substance concentration far above solubility may explain the inhibition of degradation (compare to entries Yoshida 1978).

Validity criteria fulfilled:
yes
Remarks:
; criteria as required by MITI II method
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

In a sealed vessel CO2 headspace test using 14C ring labelled diisopropylnaphthalene, no ultimate biodegradation could be demonstrated. Analysing DIPN content in test medium, primary biodegradation (ca. 20 - 25%) was observed. In two previous screening studies (OECD 301D and 302D), partial biodegradation was noted, in particular when adapted microflora was applied. The mono-isopropylated naphthalene, MIPN, was rapidly biodegraded, the 60% window slightly failing with 12 rather than 10 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The biodegradation of diisopropylnaphthalene (DIPN) has been examined in several studies (see table below). The most recent studies are from 2011 (LAUS GmbH 2011a and b). In one test, biodegradation was studied using DIPN 14C labelled at one position of the naphthalene ring system (position 1, 4, 5, or 8, respectively). In the second test, primary degradation was studied following the decrease in DIPN concentration in the test medium. Previous testing according to OECD 301D (closed-bottle test) (Fresenius 2005a) and a follow-up test according to OECD 302 D (Fresenius 2005b) failed to show ultimate biodegradation after extension of the incubation period to 56 d: While the ready-test did not exceed 18% O2 -consumption, the inherent test after adaptation of the inoculum attained O2 consumption of 37%, apparently levelling off after 56 d.

Results of a MITI II test (OECD 302 C) for inherent biodegradability, performed probably in 1977, are reported by the Chemicals Evaluation and Research Institute Japan (CERI). Despite very limited reporting, the reliability is assigned to be 2 (CERI 1977/CITI 1992), as the institute reporting this data has a reputation to perform tests according to high standards and to provide data of high reliability.

In the study of Yoshida and Kojima 1978, DIPN radiolabelled with 14C at position 1 of the naphthalene nucleus was incubated under continuous aeration of test vessels. Developing CO2 (including any volatile radioactivity) was transported by the air stream into or through a trap containing ethanolamine dissolved in methanol.

Summary table of biodegradation tests of DIPN  

Report

Method

Guideline
followed

TS concentration

[mg/L]

Sludge
concentration

 [mg/L]

Bio-
degrad.

[%]

Time

 

[d]

Result

Reliability

Fresenius 2005a

O2consumption

OECD 301D
(- and + adaptation)

1.1

No data

 3 (-)

17 (+)

28

not ready

1

Fresenius 2005b

CO2evolution

OECD 302D (with adaptation)

20

8 (SS, dry)

26

37

7

56

partial biodegradation: not fully evaluable

1

LAUS GmbH 2011a

14C CO2evolution

ISO 14593:2005
OECD 310

0.2

10 (SS)

0.05

56

not biodegradable

1 KS

LAUS GmbH 2011b

Decrease of DIPN concentrat.

ISO 14593:2005
OECD 310

0.2

10 (SS)

primary
ca. 25

56

not biodegradable
only partial primary biodegradation

1 KS

CERI 1977/ CITI 1992

MITI II

OECD 302 C

30

100

0

14/ 28

not biodegradable

2 SS

Yoshida and Kojima 1978

14C CO2evolution

Similar to
OECD 301 B

0.4

1

84

28

readily biodegradable

3

Yoshida and Kojima 1978

14C CO2evolution

Similar to
OECD 301 B

10 and 1.6

100 and 30

20 - 70

28

inherently biodegradable

3

 

LAUS GmbH 2011

Ultimate biodegradation

In the study by LAUS, no ultimate biodegradation of DIPN was observed. Radioactivity of CO2 samples was of the order of background samples. Biodegradation was determined to be < 0.05%. In addition, no radioactivity was observed in samples of absorption material from traps for organic volatiles in the air stream. In acidic organic extracts (xylene) of the test medium, about 85% of total radioactivity was determined at day 4 decreasing to ca. 70% at day 56 (abiotic control100 % at day 4, 73% at day 56). Following day 28, there was no substantial change/increase in the content of radioactivity in the xylene extracts. In the remaining aqueous phase, the percentage of total radioactivity from day 28 to 56 was between 10 and 21 % for single samples, the mean about 15 % (abiotic control 11 to 20%). There was no substantial change over time in the fraction of radioactivity in the aqueous phase.

Mean percentage of radioactivity in distinct test fractions:

Day

14CO2 (%)

Absorbant for
volatile organics (%)

Xylene extract (%)

Residual aqueous phase (%)

Balance

4

0.03

0.002

85.1

-

-

21

0.01

0.008

73.1

-

-

40

0.058

0.006

71.5

14.9

86.5

56

0.026

0.007

68.5

13.9

82.4

Overall conclusion:

1. Deficit in the radioactive mass balance: approx. 15% (not found initially)

2. Volatiles (CO2 or organic compounds): approx. 0%.

3. Xylene extractable material (day 28, 40, 56): approx. 70% (apolar under acidic condition)

4. Xylene non-extractable material (day 28, 40, 56): approx. 15% (polar under acidic condition)

It is concluded that of the 100 % of initial radioactivity approx. 15 % were transformed to polar substances.

This appeared to occur in the abiotic control to similar extent.

Primary biodegradation

Primary degradation (total and abiotic) was determined by GC-analysis of DIPN content in test vessels and abiotic controls. Biodegradation was the difference between total and abiotic degradation. Degradation of total DIPN was calculated by summation of the seven DIPN isomers analysed. The seven positional isomers measured in GC-analysis were (in order following peak elution): 1,3-DIPN, 1,7-DIPN, 2,6-DIPN, 2,7-DIPN, 1,6-DIPN 1,4-DIPN, and 1,5-DIPN (for quantitative distribution of isomers see study record).

Total primary degradation was ca. 50% with ca. 20 to 30% biodegradation. Biodegradation of total DIPN seemed to level off after 21 days, while abiotic degradation appeared to increase from day 40 to day 56 after a plateau between day 14 and day 40.

Abiotic primary degradation was similar for all DIPN isomers. Final abiotic degradation was between 28 and 39%, average 32%.

Mean primary degradation of total DIPN

Day

Primary degradation total (%)

Primary degradation abiotic (%)

Primary degradation biodegradation (%)

4

5

6

-1

21

32

11

21

40

47

17

30

56

53

32

21

Regarding primary biodegradation, there were substantial differences between isomers. 1,3- DIPN, 1,4-DIPN and 1,5-DIPN did not show any primary biodegradation. 2,6- DIPN was biodegraded by > 81%, 1,6-DIPN by 56%, 2,7-DIPN by 36% and 1,5-DIPN by 15%. Except 2,6-DIPN there was no substantial increase in primary biodegradation following day 28 (see table in study record).

The ability of biodegradation appears to be dependent on isomeric structure. In general, biodegradation appears to proceed the better if the two isopropyl-substituents are attached to different rings of naphthalene and the further apart the two substituents are from each other (2,6- > 1,6- > 2,7- > 1,5-).

Mean percentage of primary degradation for DIPN isomers at test end (56 days) (%)

1,3-DIPN

1,4-DIPN

1,5-DIPN

1,6 DIPN

1,7 DIPN

2,6 DIPN

2,7-DIPN

total

biotic

total

biotic

total

biotic

total

biotic

total

biotic

total

biotic

total

biotic

17

-12

21

-9

52

15

92

56

28

-6

100

>81

78

36

Negative percentages indicate that the abiotic degradation was faster than the biotic portion.

IF 2005a/b

These studies demonstrated that there was partial but very limited biodegradation under the stringent test conditions applied. Degradation could be enhanced by using adapted inoculum. In the second test (OECD 302D), there were signs of inhibition following rapid initial biodegradation (IF 2005b).

CERI 1977/ CITI 1992

The results of the MITI II test are in accordance with the results of the LAUS study. In this test for inherent biodegradability, no ultimate biodegradation was observed. Primary degradation was only 7.6% (determined by GC analysis after extraction of test substance with n-hexane from test medium). Test substance concentrations (30 mg/L) were far above water solubility of the test substance. This fact may contribute that no biodegradation was demonstrated.

Yoshida and Kojima 1978

Dissolved substrate (0.4 mg/L)

In this ready biodegradation test similar to OECD TG 301 B, test substance concentration was in the range of water solubility. 84% biodegradation (measured as radioactivity in CO2 trap) was determined within 4 weeks. Pass levels for ready biodegradability were met. Percentage of radioactivity in medium and sludge (separated by centrifugation) was 5.3 and 1.5% respectively. In total, recovery of radioactivity was 90.8%.

This result is contradictory to the findings of LAUS. Differences cannot easily be explained. As DIPN can be evaporated from aqueous solution, there may have been some transport of DIPN to the CO2 trap followed by absorption in the methanol phase. Radioactivity counted was then the sum of CO2 generated and unchanged test substance. If DIPN was present in the trap, the extent of biodegradation should have been reduced respectively. Furthermore, with increasing test concentration, the unchanged volatile fraction should have increased, i.e. more radioactivity should have accumulated in the trap. But the contrary came true: the higher the concentration in the test vessel, the lower the radioactiovity in the trap (see below). Presently there is no possibility to assess if biodegradation was overestimated.

Non dissolved substrate (1.6 and 10 mg/L)

In biodegradation tests, different incubation conditions (substrate, sludge concentrations) were used. Test substance concentrations were 1.6 or 10 mg/L. Sludge concentration were 30 or 100 mg/L

Under all conditions, biodegradation was demonstrated decreasing from ca. 70 to ca. 20% with increasing test substance concentrations. Higher sludge concentrations on the other hand enhanced biodegradation again. Higher test substance concentrations resulted in increase radioactivity in supernatant medium and in sludge. Total recovery was approximately constant (between 84 to 94%) indicating that loss of radioactivity was minor and not result of change in test conditions.

Identification of metabolites

Using large volume biodegradation tests, metabolites could be separated, and three were isolated. Based on m.p., NMR, and MS data, the structure of metabolites was proposed as 3-isopropyl salicylic acid, 4-isopropyl salicylic acid, and 2-(6-isopropylnaphth-2-yl)propionic acid indicating ring cleavage of 1 and 2 substituted isopropylnaphthalenes and oxidation of a isopropyl substituent to the corresponding carboxylic acid. This is considered valuable information on the biological fate of DIPN.

Conclusions

The studies conducted at Institute Fresenius show some evidence that DIPN isomer mixture undergoes partial biodegradation, but only or significantly after induction of relevant metabolic pathways (adaptation). There is evidence to suggest that induced microbial degradation does not precede well for all isomers, a fact that was clearly demonstrated in the follow-up study under the conditions of the OECD TG 310 (Laus 2011a/b).

There is no obvious reason why in the Yoshida study biodegradation could be demonstrated but not in the LAUS study. One factor could be the constitution of the inoculum that may have contained cultures better or faster adapted to DIPN in the Yoshida study than in the LAUS study.

The LAUS study was attended closely by the registrant, and the report was scrutinised. The test method is more suited for testing of substances which are able to volatilise. On the other hand, performance of the Yoshida study cannot be assessed in depth. On the basis of the data available it is not possible to evaluate why biodegradation was observed, and if there were methodological deficiencies or not. Thus, reliability of the Yoshida biodegradation experiments is rated 3, and the LAUS studies are selected as key information. Nevertheless, the information on metabolites of Yoshida are considered valuable.