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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th Jan to 5th Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene
EC Number:
830-582-9
Cas Number:
1951440-04-2
Molecular formula:
C32H50O2
IUPAC Name:
1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AS455433
- Expiration date of the lot/batch: 01 November 2020
- Purity: 98.5%
-CAS number: 1951440-04-2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from the light
- Solubility and stability of the test substance in the solvent/vehicle: Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2-litre test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer weighed amounts of test item to respective test bottles.


Study design

Oxygen conditions:
aerobic
Remarks:
aerated and continuously stirred
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): municipal sewage treatment plant, Waterschap Aa en Maas', 's-Hertogenbosch, the Netherlands.
- Storage length: used immediately
- Preparation of inoculum for exposure: before use, the sludge was coarsely sieved (1mm)
- Pretreatment: none
- Concentration of sludge: After preparation, 3.0g/l
- Initial cell/biomass concentration: not stated
- Water filtered: tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon
- Type and size of filter used, if any: 1mm
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
14.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium as per guidelines.
- Additional substrate: None
- Solubilising agent (type and concentration if used): not used
- Test temperature: 22 to 23°C measured
- pH: 7.4 to 7.6 measured
- pH adjusted: no
- CEC (meq/100 g): not provided
- Suspended solids concentration: 9mg/l (3ml of 3g/l added per litre)
- Continuous darkness: brown coloured glass bottles. Test media were excluded from light.

TEST SYSTEM
- Culturing apparatus: flasks, capable of holding 2L.
- Number of culture flasks/concentration: 2 flasks (test bottles),
- Method used to create aerobic conditions: continuous aeration and stirring
- Test performed in closed vessels due to significant volatility of test substance: not stated
- Details of trap for CO2 and volatile organics if used: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Other: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency: days 2, 5, 8, 12, 15, 19, 23, 29 (test vessels).
- Sampling method: CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany). Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator. On the penultimate day, pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) Bottles were aerated overnight to drive off CO2 present in the test suspension. Final titration was made on day 15 (procedure and toxicity control) and on day 29 (remaining vessels).
- Sterility check if applicable: not stated
- Sample storage before analysis: not stated

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 flasks, measurements taken over 28 day period.
- Abiotic sterile control: not included
- Toxicity control: 1 flask (toxicity control), measurements were made over a period of 14 days for the procedure and toxicity control.
- Other: 1 flask (procedure control), measurements were made over a period of 14 days for the procedure and toxicity control.

Reference substance
Reference substance:
other: Sodium acetate, CAS number 127-09-3
Remarks:
Purity 99.7%, batch AM1176568

Results and discussion

Preliminary study:
Not conducted
% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
28 d
Remarks on result:
other: bottle A
Key result
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
28 d
Remarks on result:
other: Bottle B
Details on results:
- In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:
The reference substance was degraded by 75% within 14 days, which fits the acceptibility criteria of the test.

Any other information on results incl. tables

Table 1. HCl titrated in duplicate blank bottles.

Day

HCl (0.05 N) titrated (mL)

Blank A

Blank B

Mean Value

2

42.04

43.44

42.74

5

40.05

41.80

40.93

8

38.96

40.31

39.64

12

40.60

42.19

41.40

15

40.67

43.56

42.12

19

40.21

43.63

41.92

23

291)

291)

291)

41.05

38.49

39.21

41.81

43.61

41.85

42.20

43.16

42.33

40.17

40.71

42.49

1): CO2measured on day 29 is actually part of CO2productionof day 28, since microbial activity was ended on day 28 by addition of HCl.

Table 2. HCl Titrated in Ba(OH)2Solution (Background Bottles)

Day

HCl (0.05 N) titrated (mL)

Bottle A

Bottle B

Mean value

2

45.68

45.35

45.52

5

46.26

46.29

46.28

8

46.30

46.67

46.49

12

49.57

49.28

49.43

15

49.32

49.03

49.18

19

49.31

49.41

49.36

23

48.73

48.80

48.77

29

48.86

48.45

48.66

29

46.40

45.33

45.87

29

45.41

45.00

45.21

Table 3 CO2 Production in the Blank

 

Day

HCl (0.05 N) titrated (mL)

ProducedCO2

(mL HCl)

ProducedCO2

(mg)

CumulativeCO2

(mg)

Ba(OH)21)

Blank (mean)

2

45.52

42.74

2.78

3.1

3.1

5

46.28

40.93

5.35

5.9

8.9

8

46.49

39.64

6.85

7.5

16.5

12

49.43

41.40

8.03

8.8

25.3

15

49.18

42.12

7.06

7.8

33.1

19

49.36

41.92

7.44

8.2

41.3

23

292)

292)

292)

48.77

48.66

45.87

45.21

42.33

40.17

40.71

42.49

6.44

8.49

5.16

2.72

7.1

9.3

5.7

3.0

48.3

57.7

63.3

66.3

1): "Strength" of untreated 0.0125 M Ba(OH)2solution

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activitywas ended on day 28 by addition of HCl.

Table 4. CO2Production and Percentage Biodegradation of the Reference Item

 

Day

HCl (0.05 N) titrated (mL)

ProducedCO2

(mL HCl)

ProducedCO2

(mg)

CumulativeCO2

(mg)

Biodegradation1)

(%)

Blank(mean)

Procedurecontrol

2

5

8

12

152)

42.74

40.93

39.64

41.40

42.12

33.28

21.08

25.05

32.84

35.63

9.46

19.85

14.59

8.55

6.49

10.4

21.8

16.0

9.4

7.1

10.4

32.2

48.3

57.7

64.8

12

38

56

67

75

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.9 mg CO2/2L.

2): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 byaddition of HCl.

 

Table 5. CO2Production and Percentage Biodegradation of the Test Item (Bottle A)

 

Day

HCl (0.05 N) titrated (mL)

ProducedCO2

(mL HCl)

ProducedCO2

(mg)

CumulativeCO2

(mg)

Biodegradation1)

(%)

Blank(mean)

Bottle A

2

5

8

12

15

19

23

292)

292)

292)

42.74

40.93

39.64

41.40

42.12

41.92

42.33

40.17

40.71

42.49

42.54

42.99

39.79

43.17

43.77

43.20

43.83

41.01

40.45

42.78

0.20

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.25

0.00

0.2

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.3

0.0

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.5

0.5

0

0

0

0

0

0

0

0

1

1

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 87.1 mg CO2/2L.

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 byaddition of HCl.

Table 6. CO2Production and Percentage Biodegradation of the Test Item (Bottle B)

 

Day

HCl (0.05 N) titrated (mL)

ProducedCO2

(mL HCl)

ProducedCO2

(mg)

CumulativeCO2

(mg)

Biodegradation1)

(%)

Blank(mean)

Bottle B

2

5

8

12

15

19

23

292)

292)

292)

42.74

40.93

39.64

41.40

42.12

41.92

42.33

40.17

40.71

42.49

43.87

41.60

41.63

42.16

42.35

41.81

42.03

38.96

39.88

42.30

0.00

0.00

0.00

0.00

0.00

0.11

0.30

1.21

0.82

0.19

0.0

0.0

0.0

0.0

0.0

0.1

0.3

1.3

0.9

0.2

0.0

0.0

0.0

0.0

0.0

0.1

0.5

1.8

2.7

2.9

0

0

0

0

0

0

1

2

3

3

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 87.5 mg CO2/2L.

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 byaddition of HCl.

Table. 7. CO2Production and Percentage Biodegradation of the Toxicity Control

 

Day

HCl (0.05 N) titrated (mL)

ProducedCO2

(mL HCl)

ProducedCO2

(mg)

CumulativeCO2

(mg)

Biodegradation1)

(%)

Blank(mean)

Toxicitycontrol

2

5

8

12

152)

42.74

40.93

39.64

41.40

42.12

31.15

20.78

25.29

32.91

34.00

11.59

20.15

14.35

8.49

8.12

12.7

22.2

15.8

9.3

8.9

12.7

34.9

50.7

60.0

68.9

7

20

29

35

40

1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and reference item: 173.2 mg CO2/2L (ThCO2test item: 87.2 mg CO2/2L + ThCO2sodium acetate: 85.9 mg CO2/2L).

2): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 byaddition of HCl.

Table. 9. Comparison of Biodegradation of the Test Item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

2

0

0

0

0

5

0

0

0

0

8

0

0

0

0

12

0

0

0

0

15

0

0

0

0

19

0

0

0

0

23

292)

292)

292)

0

0

1

1

1

2

3

3

1

1

2

2

1

2

2

2

1): Absolute difference in biodegradation between bottles A and B

2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of 4,4’-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene) (1% and 3%, based on ThCO2) and it was concluded that the test substance was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

A biodegradation in water screening test was carried out on 4,4’-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene) according to the OECD 301B guidelines and to GLP. Duplicate test vessels were run at a TOC concentration of 12mg/l (test substance concentration of 14.5mgl). In addition, 2 inoculum blanks, 1 procedural control and 1 toxicity control vessel were included. The biodegradation profile was measured over a 28 day period (14 days for the procedure and toxicity controls) by titration of the sample with barium carbonate. After 28 days no biologically relevant biodegradation was measured in the test vessels (1% and 3%, based on ThCO2). No toxicity to the microbes of the test substance was found. It was concluded that the test substance was not readily biodegradable under the conditions of the test.

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