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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge: Domestic waste water treatment plant, sewage plant Rossdorf, Germany
- Storage conditions: acivated sludge was fed and aerated according to the guideline
- Storage length: 2 d
- Pretreatment: activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged; this procedure was repeated twice
- Concentration of sludge: 1.5 g/L on dry matter base
Duration of test (contact time):
28 d
Initial conc.:
25 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to the guideline
- Test temperature: climatic chamber at 22 °C
- pH: 7.6 (measured at test start) and 7.7-7.9 (measured at test end)
- Suspended solids concentration: 5 mL of a stock suspension,1.5 g/L on dry matter
- Continuous darkness: yes
- Other: test item was weighed directly into test flasks

TEST SYSTEM
- Culturing apparatus: Manometric test flasks with a volume of 500 mL; Closed flasks were incubated in a climatic chamber under continous stirring
- Number of culture flasks/concentration: 3
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic, Germany
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2: potassium hydroxide solution (45%)

SAMPLING
- Sampling method: by measuring the pressure decrease in the reaction vessels

CONTROL AND BLANK SYSTEM
- Inoculum control: 2 flasks
- Abiotic sterile control: 1 flask (poisoned with HgCl2)
- Toxicity control: 1 flask
- Other: procedure control: 1 flask

Reference substance:
benzoic acid, sodium salt
Remarks:
25 mg/L
Parameter:
% degradation (O2 consumption)
Value:
9
Sampling time:
28 d
Remarks on result:
other: mean
Results with reference substance:
The reference item sodium benzoate was degraded to 96% after 14 days and to 101% after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum.

Toxicity control

In the toxicity control containing both, the test item and the reference item sodium benzoate, 41% biodegradation was noted within 14 days and 42% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed not to be inhibitory on the activated sludge microorganisms.

Results of BOD-Measurement:

The degradation rate of the test item based on BOD was found to be 4% (mean) after 14 days of incubation and 9% (mean) after 28 days at the end of the test.

Results of DOC-Measurement:

The DOC-content in the test item treatments was reduced by about 12% (mean) after 14 days and about 15% (mean) after 28 days.

Results of HPLC-Measurement:

The test item specific analysis resulted in a reduction of the test item of 7% and 19% after 14 and 28 days, respectively . The possible degradation productions 4-chloro­ phenol and 4-chlorocatechol were not found on day 14 sampling. On day 28, minor amounts of 4-chlorophenol (about 20 µg/L) were found in the culture broth.

Solubility of Chlorophene:

The solubility was tested by measuring the test item concentration within 1 hour and 24 hours after application. Within one hour after application, the test item was not dissolved completely and test item particles were visible in aqueous phase. The chemical analysis revealed in a test item concentration of about 50% of initial and the solubility increased after 24 h to about 76%. After 14 days of incubation, the test item concentration in the test vessels was about 93% of initial concentration. The test item is soluble in test water; the complete dissolution time is more than 24 h.

Adsorption of the test item to activated sludge:

The test item was degraded to about 9% of initial concentration (based on BOD) and the remaining test item in the solution was about 81% of initial concentration after 28 days. Therefore it is considered that the test item does not adsorb to activated sludge. The degradation rate of the test item did not reach the pass level for ready biodegradability of 60% based on BOD, but there is evidence that the test item is biodegradable under less stringent conditions.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The degradation rate of the test item did not reach the pass level for ready biodegradability of 60% based on BOD, but there is evidence that the test item is biodegradable under less stringent conditions.The test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was > 25% within 14 days. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Nov - 20 Dec 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
92/69/EC
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Remarks:
predomeinantly domestic
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (Yorkshire Water), which works with a waste-water catchment that is predominantly domestic.
- Preparation of inoculum for exposure: The activated sludge inoculum was not acclimatised or adapted to the test item before exposure to the test substance in this study.
- Pretreatment: On arrival at the laboratory, the sludge sample was aerated by means of a compressed air supply delivered through a diffuser block.
- Concentration of sludge: 90 mg/L (nominal solids concentration)
- Initial cell/biomass concentration: 30 mg/L (suspended solids concentration per vessel)
- Pretreatment for determination of suspended soilds concentration: suspended solids concentration was determined by filtering a 25 mL subsample through a pre-dried and pre-weighed glass microfibre filter (Whatman GF/C). The filter and retained solids were then dried by microwave oven and re-weighed. The contribution made by the sludge solids was determined by difference.
Duration of test (contact time):
28 d
Initial conc.:
0.042 other: g test item
Based on:
other: corresponds to 10 mg organic carbon /L
Initial conc.:
0.042 other: g test item
Based on:
other: corresponds to 10 mg organic carbon /L
Initial conc.:
0.042 other: g test item
Based on:
other: corresponds to 10 mg organic carbon /L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to OECD guideline 301 B
- Test temperature: 20.0 to 23.7 °C
- pH: 7.77 - 8.20 (days 0 and 28 for blank, test item and sodium benzoate)
- Aeration of dilution water: The air used in this study was nominally CO2-free, produced by a TOC Gencompressor (Model 45M35090, Schmidlin Labor & Service AG). As an added precaution, the flow passed through a column packed with ‘Carbosorb AS‘ (Merck), a self-indicating, artificial silicate CO2 absorber, before entering the test vessels. Adjustments were made as necessary to maintain a flow rate in the range of 50 to 100 mL per min.
- Suspended solids concentration: On the basis of the suspended solids determination described above, the medium concentrate was inoculated with activated sludge to provide a nominal solids concentration of 90 mg/L. One litre of medium was added to each test vessel and made up to 3 L by addition of reverse-osmosis water. The final suspended solids concentration in all vessels was thus nominally 30 mg/L.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Test vessels were incubated in darkness within a specified temperature range for 28 days and the medium continually sparged with a supply of CO2-free air. The exhaust air from each vessel was passed through as series of dedicated CO2 scrubbers containing a barium hydroxide (Ba(OH)2) solution.
- Number of culture flasks/concentration: 2 (blank, test item, reference item), 1 (toxicity control)
- Method used to create aerobic conditions: The air flow in this study was regulated in two stages. Initial control was provided by a gas regulator and the air flow to each vessel controlled by individual needle valves. Measurements were made, with a bubble flow meter and stop watch, at intervals not exceeding three days, of the flow rate exiting each test vessel through its train of scrubbers.
- Measuring equipment: A stock was made from the primary by diluting 20 mL of the latter to 1 L with reverse-osmosis water. The stock, whose TOC concentration was nominally 45 mg C/L, was subjected to confirmatory analysis, performed in triplicate on duplicate samples by means of a Rosemount Dohrmann DC-80 TOC analyser. For the purpose of this confirmatory analysis, the analyser was calibrated against a 400 mg C/L potassium hydrogen phthalate standard.

SAMPLING
- Sampling frequency: 0, 2, 4, 5, 6, 8, 10, 12, 14, 18, 23 and 28 days after application.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes
- Other: reference (sodium benzoate)

Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
68
Sampling time:
28 d
Remarks on result:
other: mean degradation
Remarks:
(69% replicate 1 and 66% replicate 2)
Results with reference substance:
Final degradation values were 85% and 81% in the two replicates. Maximum divergence between the replicates was 4% at Day 28.

Table 1: Biodegradability as a percentage of theoretical CO2 yield

Time (days)

BCP

Sodium benzoate

replicate 1

replicate 2

toxicity control

replicate 1

replicate 2

2

0

1

22

22

21

4

1

14

40

39

38

5

17

37

53

50

49

6

31

49

62

56

54

8

46

54

71

60

58

10

54

54

77

63

61

12

56

54

80

66

64

14

56

54

81

69

67

18

59

54

81

75

73

23

63

58

83

79

76

28

69

66

88

85

81

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Although the test item has failed to qualify for classification as readily biodegradable under the conditions employed in this study, the extent of mineralisation achieved in 28 days strongly suggests that the test item is unlikely to persist or accumulate in the aerobic environment.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: council Directive 87/302/EEC
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge from a domestic waste water treatment plant was supplied by the sewage plant of Rossdorf, Germany
- Storage conditions: sludge was aerated until use
- Preparation of inoculum for exposure: An aliquote of the final sludge suspension was weighed dried and the ratio of the wet sludge to its dry weight was determined.
- Pretreatment: activated sludge was washed by centrifugation and the supernatant liquid phase was decanted and the solid material was re-suspended in tap-water and centrifuged again. This procedure was repeated three times.
- Concentration of sludge: 4 g dry material per Liter
- Initial cell/biomass concentration: 0.2 g/L suspended soilds concentration
Duration of test (contact time):
28 d
Initial conc.:
87.4 mg/L
Based on:
test mat.
Remarks:
corresponding to 62.4 mg/L dissolved organic carbon
Initial conc.:
80.2 mg/L
Based on:
test mat.
Remarks:
Reference item
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: according to the guideline
- Test temperature: climatic chamber at 20 - 22 °C
- pH: 7.2 - 7.5 during the experiment in all treatments
- Dissolved oxygen concentration: 8.3 - 9.1 during the experiment in all treatments
- Aeration of dilution water: The test flasks were aerated with purified, moistened air and was adjusted to achieve a concentration of > 1 mg/L in the test media during the test period. On each sampling date water evaporation loss was compensated by adding deionised water and deposits on the test flasks were scraped off. The air flow was controlled and the loss of water in the gas washing flask (moistening of air) was compensated
- Suspended solids concentration: 4 g/L suspended soild (correspond to 100 mL of inoculum)
- Continuous darkness: yes (or diffuse illumination)

TEST SYSTEM
- Culturing apparatus: Cylindrical glass flasks with 3 litre volume were covered with a plastic lid and aerated using a glass tube
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The air was led through a column filled with glass wool and a bottle containing deionised water to clarify and moisture the air. For the abiotic controls, the air was filtrated by a sterile tube containing sterilised glass wool to prevent contamination of airborne microorganisms.
- Measuring equipment: DOC was measured with HPLC analysis
- Test performed in open system: yes

SAMPLING
- Sampling frequency: Samples were taken on day 0 at test start and after 3 hours test duration, and on days 2, 5, 7, 12, 14, 21, 27 and 28.
- Sampling method: 50 mL per treatment was filtered and were analysed for DOC measured

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes (2x)
- Toxicity control: yes
- Other: Reference control

Reference substance:
diethylene glycol
Parameter:
% degradation (DOC removal)
Value:
97
Sampling time:
28 d
Remarks on result:
other: 3% of the initial concentration based on DOC was found in the test medium
Results with reference substance:
The reference item diethylene glycol was sufficiently degraded to about 40% after 7 days, and to about 99% after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Results Biodegradation of Test Item:

Based on the results of DOC and HPLC measurements the biodegradation is considered to be higher than 97%, although at test start a rapid adsorption of Chlorophene to activated sludge was found.

Results Biodegradation of Toxicity Control:

Based on DOC removal, the initial DOC content in the toxicity control was about 1% of initial, showing a complete degradation of both, the test item and the reference item. The test item can be considered  not to be toxic to bacteria  in the tested concentration.

Abiotic Controls:

Whereas in replicate 2 the complete amount of Chlorophene was present after 28 days, in the first replicate of the abiotic control only 62% of initial concentration were left. This was due to unusual precipitation of Chlorophene. The reason for the precipitation could not be clarified and the second replicate was used for result evaluation only.

Tables

Table 1: Measurement of Dissolved Organic Carbon (mg DOC/L) in Test Flasks During the Test Period of 28 Days

Treatment Group

DOCmg/L

day O (0h)*

day 0 (3h)*

day 2

day S

day 7

day 12

day 14

day 21

day 27

day 28

Control 1

6.59

6.80

4.29

5.71

3.32

5.54

5.94

6.44

6.56

5.26

Control 2

4.76

5.36

2.51

6.06

3.56

4.87

6.24

5.80

5.87

5.00

Mean

5.68

6.08

3.40

5.89

3.44

5.21

6.09

6.12

6.22

5.13

Chlorophene 1

Corrected**

28.90

23.22

31.80

25.72

16.03

12.63

10.74

4.85

10.25

6.81

9.40

4.19

11.25

5.16

10.77

4.65

8.60

2.38

7.42

2.29

Chlorophene 2

Corrected**

36.13

30.45

33.09

27.01

12.15

8.75

9.54

3.65

9.26

5.82

9.82

4.61

10.43

4.34

8.64

2.52

8.34

2.12

6.96

1.83

Diethylene glycol

92.59

94.26

76.47

79.36

52.32

6.20

2.68

7.82

6.54

5.15

Corrected**

86.91

88.18

73.07

73.47

48.88

0.99

-3.41

1.70

0.32

0.02

Toxicity control

Corrected**

113.18

107.50

111.07

104.99

91.65

88.25

86.19

80.30

33.26

29.82

11.81

6.60

11.00

4.91

13.21

7.09

7.61

1.39

7.01

1.88

LOQ organic carbon: 2 mg/L;

* The start concentration of Chlorophene was 87.4 mg/L corresponding to 62.4 mg/L dissolved organic carbon.

**measured value minus mean value of control

Table 2: Percentage Biodegradation of Test Item and Diethylene Glycol During 28 Days of Incubation

Treatment Group

% of Initial DOC Concentration (1)

day 0

day 2

day 5

day7

day 12

day 14

day 21

day 27

day 28

Chlorophene  1

37.2

20.2

7.8

10.9

6.7

8.3

7.5

3.8

3.7

Chlorophene 2

48.8

14.0

5.8

9.3

7.4

7.0

4.0

3.4

2.9

mean

43.0

17.1

6.8

10.l

7.1

7.6

5.7

3.6

3.3

Diethylene glycol

108.4

91.1

91.6

60.9

1.2

-4.3

2.1

0.4

0.0

Toxicity control

75.4

61.9

56.3

20.9

4.6

3.4

5.0

1.0

1.3

LOQ organic carbon: 2 mg/L

(1) Initial DOC content:

Chlorophene 1: 62.4 mg/L dissolved organic carbon (calculation based on measured concentration (HPLC)); Chlorophene 2: 62.4 mg/L dissolved organic carbon (calculation based on measured concentration (HPLC)); Diethylene glycol 80.2 mg/L dissolved organic carbon (calculation based on nominal concentration); Toxicity control: 142.6 mg/L dissolved organic carbon (calculation based on nominal concentration (DEG) and measured concentration (Chlorophene))

Table 3: Measurement of Test Item by HPLC in Test Flasks During the Test Period of 28 Days

Treatment Group

Chlorophene mg/L

 

Day 0 (0h)

day 0 (3h)

day 28

% of initial on day 28

Abiotic Control 1

88.86

--

54.74**

61.6

Abiotic Control 2

88.14

--

92.79

105.3

Chlorophene 1*

57.40

47.95

2.88

3.3

Chlorophene 2*

54.80

45.98

2.81

3.2

Toxicity Control*

--

--

2.83

3.2

*Initial Chlorophene concentration in test solution without activated sludge: 87.4 mg/L LOQ organic carbon: 2 mg/L

---: not determined

**: precipitation of test item observed

 

Table 4: Measurement of Adsorption of Test Item to different Filters

Filter

Relative recovery* %

Cellulose acetate

70.5

Cellulose, regenerated

86.8

Cellulose ester (mixed)

96.4

Cellulose nitrate

97.5

*: mean of two measurements

 

Table 5: Measurement of Adsorption of Test Item to Activated Sludge

 

Chlorophene

 

mg/L

µg

%

Treatment Group

Liquid phase

Activated sludge*

Activated sludge

of Initial

Chlorophene 1** Chlorophene 2**

b.q.

b.q.

0.55

0.50

3.6

3.7

0.0041

0.0042

Toxicity control**

b.q.

0.35

2.7

0.0031

b.q. = measured value below limit of quantification LOQ test item: 0.1 mg/L

---: not determined

*: Acetonitrile extracts

**: Initial concentrations: Chlorophene 1: 87.4 mg/L; Chlorophene 2: 87.4 mg/L; Toxicity control 87.4 mg/L

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The test item Chlorophene was found to be biodegradable under the test conditions. On day 28, the initial concentration of Chloro­ phene based on DOC was reduced by 97%.

Description of key information

Not readily biodegradable (OECD 301 F and 301B, Reis 2007a and Bealing and Watson 2002)

Inherently biodegradable (OECD 302, Reis 2007b).

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

Three biodegradation screening studies according to guideline OECD 301 F, 302B and 301B (GLP) are available for the test substance (Reis 2007a, Reis 2007b and Bealing and Watson 2002).

According to Reis, K.H. (2007a) the test item Chlorophene (2-benzyl-4 -chlorophenol, CAS-No.: 120 -32 -1) was investigated for its ready biodegradability in a manometric respirometry test (OECD Guideline for Testing of Chemicals No. 301 F, adopted July 17, 1992) over a period of 28 days at 22 °C. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The degradation rate of Chlorophene did not reach the pass level for ready biodegradability of 60% based on BOD, but there is evidence that Chlorophene is biodegradable under less stringent conditions. Chlorophene is not readily biodegradable.

The second study was performed following to OECD-Guideline No. 302B (Reis, K.H. (2007b). The inherent biodegradability of Chlorophene (2-benzyl-4-chlorophenol) was determined at a concentration of 87.4 mg/L and was conducted for a test period of 28 days. The biodegradation process of the test substance was monitored by analytical determination of the test item content (HPLC) and DOC determination. Elimination of Chlorophene was ≥ 97% after 28 days. The reference substance revealed a degradation of about 99% after 28 days. Based on the results of DOC and HPLC measurements the biodegradation is considered to be higher than 97%, even at test start a rapid adsorption of Chlorophene to activated sludge was found. It can be concluded that Chlorophene is inherently biodegradable.

The third study was performed following OECD-Guideline No. 301B (revision 1992): CO2-Evolution Test. The test substance BCP (2-benzyl-4 -chlorophenol) was incubated with activated sludge for a period of 28 days at 22 ± 2 °C (Bealing, D.J. and Watson, S., 2002). The mean carbon dioxide evolution from the test item reached 68% of the theoretical maximum at the applied concentration over the course of the 28 day incubation. This exceeds the 60% level that conventionally represents complete mineralisation. Nevertheless, the results of this study show that BCP is ultimately biodegradable.

Additionally anaerobic biodegradation of chlorophene in anaerobically digesting sewage sludge was assessed by Reis (2007c).

A manometric test was conducted with a nominal application rate of 140 mg/L over a period of 60 days at 30 - 37 °C in darkness. Chlorophene was found to be not biodegradable under the anaerobic conditions of the test system. No net carbon-production (as methane and carbon dioxide) was found.

 

 

[Type of water: freshwater]