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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO standard 10634, 1995.
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Terephthalaldehyde
EC Number:
210-784-8
EC Name:
Terephthalaldehyde
Cas Number:
623-27-8
Molecular formula:
C8H6O2
IUPAC Name:
benzene-1,4-dicarbaldehyde
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch (Lot) Number: 180401
Expiry date: 01 April 2020 (retest date)
Physical Description: Light yellow powder
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment: Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment concentration of suspended solids (SS) was determined to be 3.1 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9.3 mg/L.

Reason for selection: The test has been accepted internationally for determining 'ready' biodegradability of test items under aerobic conditions.
Duration of test (contact time):
>= 28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 17 mg/L
Based on:
test mat.
Initial conc.:
ca. 12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Terephthaldehyde was a light yellow powder with a purity of 99.6%. Test item was tested in duplicate at a target concentration of 17 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula.
Since Terephthaldehyde 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 (test item bottle A: 33.97 mg; test item bottle B: 33.99 mg and toxicity control bottle: 33.91 mg). To this end, 10 mL of Milli- RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms.
Any residual volumes were discarded.

Test duration 28 days for inoculum blank and test item (last CO2 measurement on day 29).
14 days for procedure and toxicity control (last CO2 measurement on day 15).
During the test period, test media were aerated and stirred continuously.
Test vessels 2 litre brown coloured glass bottles.
Milli- RO water Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.

Stock solutions of
mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2

B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and
made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and
made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and
made up to 1 litre.
Mineral medium 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
Barium hydroxide 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from air.
Synthetic air (CO2 < 1 ppm) 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).
Illumination Test media were excluded from light.

Preparation of Bottles
Pre-incubation medium The day before start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Procedure control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
Preparation At the start of the test (day 0), test and reference item were added to bottles containing microbial organisms and mineral components.
Volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before.
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.

Measurements and Recordings
pH At the start of the test (day 0) and on the penultimate day (day 14 for procedure and toxicity control and day 28 for inoculum blanks and test item), before addition of concentrated HCl.
Temperature of medium Continuously in a vessel with Milli- RO water in the same room.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
9.1. Theoretical CO2 Production
ThCO2 of Terephthaldehyde was calculated to be 2.62 mg CO2/mg.
ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
If applicable ThCO2 per test bottle is given in the subscript of the tables
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
ca. 78
Sampling time:
28 d
Details on results:
All data are presented in Appendix 1. Results of CO2 production and biodegradation in blank bottles, background bottles and each test bottle are listed in Table 2 to 8. Table 9 compares biodegradation of Terephthaldehyde in bottles A and B.
Figure 1 shows curves for biodegradation of the bottles with Terephthaldehyde, procedure control and toxicity control.
Relative biodegradation values calculated from measurements performed during the test period revealed 75% and 80% biodegradation of Terephthaldehyde, for vessel A and B, respectively (based on ThCO2).
Furthermore, average biodegradation of Terephthaldehyde in vessel A and B reached ≥60% within a 10-day window.
In the toxicity control, more than 25% biodegradation occurred within 14 days (86%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

BOD5 / COD results

Results with reference substance:
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve. 1. Reference item was biodegraded by at least 60% (actual result: 77%) within 14 days.

Any other information on results incl. tables

Monitoring of Temperature and pH

Temperature recorded in a vessel with water in the same room varied between 22 and 23°C, and complied with the requirements as laid down in the study plan.The pH values of different test vessels are presented in Table1.

Table1          
pH Values of Different Test Media

Test medium:

At the start of the test:

On day 14:

On day 28:

Blank control (A)

7.6

-

7.6

Blank control (B)

7.6

-

7.6

Procedure control

7.6

7.81

-

Terephthaldehyde (A)

7.5

-

7.6

Terephthaldehyde (B)

7.5

-

7.6

Toxicity control

7.6

7.94

-

Note: Negative produced CO2-values are expressed as 0.00 mL HCl.

Table2          
HCl Titrated in Duplicate Blank Bottles

 

Day

HCl (0.05 N) titrated (mL)

 

 

Blank A

Blank B

Mean Value

 

 

2

47.95

47.01

47.48

 

 

5

46.13

44.88

45.51

 

 

8

45.16

44.12

44.64

 

 

12

43.15

42.96

43.06

 

 

15

43.11

43.03

43.07

 

 

19

43.04

42.99

43.02

 

 

23

43.31

43.27

43.29

 

 

291)

41.31

40.13

40.72

 

 

291)

45.30

46.30

45.80

 

 

291)

47.02

47.67

47.35

 

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

 

Table3          
HCl Titrated in Ba(OH)2Solution (Background Bottles)

Day

HCl (0.05 N) titrated (mL)

Bottle A

Bottle B

Mean value

2

50.40

50.30

50.35

5

50.54

50.58

50.56

8

50.70

50.19

50.45

12

49.40

49.46

49.43

15

49.17

49.29

49.23

19

49.26

48.49

48.88

23

48.67

48.21

48.44

29

49.11

49.36

49.24

29

51.66

50.75

51.21

29

51.20

50.72

50.96


  Table4          
CO2Production in the Blank

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

 

Ba(OH)21)

Blank (mean)

 

2

50.35

47.48

2.87

3.2

3.2

 

5

50.56

45.51

5.06

5.6

8.7

 

8

50.45

44.64

5.81

6.4

15.1

 

12

49.43

43.06

6.38

7.0

22.1

 

15

49.23

43.07

6.16

6.8

28.9

 

19

48.88

43.02

5.86

6.4

35.3

 

23

48.44

43.29

5.15

5.7

41.0

 

292)

49.24

40.72

8.52

9.4

50.4

 

292)

51.21

45.80

5.41

5.9

56.3

 

292)

50.96

47.35

3.62

4.0

60.3

 

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 activity was ended on day 28 by addition of HCl.

 

Table5          
CO2Production and Percentage Biodegradation of the Reference Item

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Procedure

control

2

47.48

35.90

11.58

12.7

12.7

15

5

45.51

26.86

18.65

20.5

33.2

39

8

44.64

33.93

10.71

11.8

45.0

52

12

43.06

35.02

8.04

8.8

53.9

63

152)

43.07

31.73

11.34

12.5

66.3

77

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 by addition of HCl.

 

 

Table6          
CO2Production and Percentage Biodegradation of the Test Item (Bottle A)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Bottle A

2

47.48

47.68

0.00

0.0

0.0

0

5

45.51

27.26

18.25

20.1

20.1

23

8

44.64

22.43

22.21

24.4

44.5

50

12

43.06

32.45

10.61

11.7

56.2

63

15

43.07

38.81

4.26

4.7

60.9

68

19

43.02

41.05

1.97

2.2

63.0

71

23

43.29

41.85

1.44

1.6

64.6

73

292)

40.72

39.13

1.59

1.7

66.3

75

292)

45.80

46.94

0.00

0.0

66.3

75

292)

47.35

49.89

0.00

0.0

66.3

75

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 89.0 mg CO2/2L.

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

 


 

Table7
CO2Production and Percentage Biodegradation of the Test Item (Bottle B)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Bottle B

2

47.48

47.09

0.39

0.4

0.4

0

5

45.51

24.77

20.74

22.8

23.2

26

8

44.64

23.06

21.58

23.7

47.0

53

12

43.06

32.52

10.54

11.6

58.6

66

15

43.07

39.06

4.01

4.4

63.0

71

19

43.02

41.27

1.75

1.9

64.9

73

23

43.29

42.65

0.64

0.7

65.6

74

292)

40.72

38.33

2.39

2.6

68.2

77

292)

45.80

43.35

2.45

2.7

70.9

80

292)

47.35

47.24

0.10

0.1

71.0

80

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 89.1 mg CO2/2L.

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

 

 

Table8
CO2Production and Percentage Biodegradation of the Toxicity Control

Day

HCl(0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Toxicity

control

2

47.48

36.46

11.02

12.1

12.1

7

5

45.51

18.36

27.15

29.9

42.0

24

8

44.64

4.21

40.43

44.5

86.5

49

12

43.06

12.65

30.41

33.4

119.9

69

152)

43.07

14.88

28.19

31.0

150.9

86

1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and reference item: 174.7 mg CO2/2L (ThCO2test item: 88.8 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 by addition of HCl.

 

Table9    
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

23

26

25

3

8

50

53

52

3

12

63

66

65

3

15

68

71

70

3

19

71

73

72

2

23

73

74

74

1

292)

75

77

76

2

292)

75

80

78

5

292)

75

80

78

5

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:
readily biodegradable
Conclusions:
In conclusion, Terephthaldehyde was readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objective of the studywas to evaluate test item Terephthaldehyde for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

Study procedures described in this report were in compliance with OECD guideline No. 301 B, 1992. In addition, procedures were designed to meet test methods of ISO standard 10634, 1995.

Terephthaldehyde was a light yellow powder with a purity of 99.6%. The test item was tested in duplicate at a target concentration of 17 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Theoretical CO2production (ThCO2) of Terephthaldehyde was calculated to be 2.62 mg CO2/mg.

The study consisted of six bottles:

·        2 inoculum blanks (no test item),

·        2 test bottles (Terephthaldehyde),

·        1 procedure control (sodium acetate) and

·        1 toxicity control (Terephthaldehyde plus sodium acetate).

Since Terephthaldehyde 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, 10 mL of Milli- RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test to ensure optimal contact between test item and test organisms. Test duration was28 days for inoculum blank and test item (last CO2measurement on day 29) and 14 days for procedure and toxicity control (last CO2measurement on day 15).

Relative biodegradation values calculated from measurements performed during the test period revealed 75% and 80% biodegradation of Terephthaldehyde, for vessel A and B, respectively (based on ThCO2). Furthermore, average biodegradation of Terephthaldehyde in vessel A and B reached60% within a 10-day window.

In the toxicity control, Terephthaldehyde was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion,Terephthaldehyde was designated as readily biodegradable.