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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Sep 1992 to 18 Feb 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: BBA-IV section 5-1
Version / remarks:
December 1990
Deviations:
yes
Remarks:
Soil:water ratio was 1:2 and not 1:4, however was considered as a minor deviation from the guideline and does not compromise the scientific validity of this study.
Qualifier:
according to guideline
Guideline:
other: Dutch Registration Guideline, G2
Version / remarks:
January 1987
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: EPA 162-4
Version / remarks:
October 18, 1982
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment: freshwater
Details on source and properties of surface water:
Properties of the water are provided in Table 1 in 'Any other information in materials and methods incl. tables'.
SOURCE
- Pond: small natural Pond (AG, Switzerland)
- River: Rhine river (AG, Switzerland)
Details on source and properties of sediment:
Properties of the sediment are provided in Table 1 in 'Any other information in materials and methods incl. tables'.
SOURCE
- Pond: small natural Pond (AG, Switzerland)
- River: Rhine river (AG, Switzerland)
- Preparation: Before use, the sediments were sieved through a 2 mm sieve. The sediment height in the aquatic systems was about 2 to 2.5 cm.
Duration of test (contact time):
360 d
Initial conc.:
0.14 mg/L
Based on:
test mat.
Remarks:
in the water compartment
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of water/sediment: Wet sediment was fillled to a hight of about 2 - 2.5 cm (56.5 ± 1.1 and 139.0 ± 2.6 g based on sediment dry weight for Pond and Rhine, respectively). Afterwards exactly 500 mL of the corresponding water were added to reach a height of about 6 cm.
- Equilibration: After preparation, the aquatic systems were equilibrated in climatization chambers under aeration by gentle stirring from the top without disturbing the sediment. During equilibration pH and oxygen content of the water and redox potential of water and sediments were followed.
- Test temperature: 20 ± 2 °C (during the equilibration and incubation time)
- Aeration of dilution water: Yes
- Continuous darkness: Yes
- Details of trap for CO2 and volatile organics if used: The trapping system consisted of one bottle with ethyleneglycol (50 mL), one bottle with 0.25N H2SO4 (50 mL) and two bottles with 2N NaOH (50 mL each).
- Volume and application of test solution stock: Volumes of exactly 0.151 ± 0.0019 µL of the solution A and 0.151 ± 0.0014 µL of the treatment solution B were transferred into small glass vials for Pond and Rhine application, respectively. The total content of this vials was transferred by means of suitable syringes to the water phase of the aquatic systems. Thereafter, the vials were rinsed with acetone/water and the solution likewise applied to the water phase. The total amounts of test substance 1 and test substance 2 applied to each aquatic system, were 0.0657 ± 0.0078 mg and 0.0678 ± 0.0073 mg, respectively. This amount correspond to four times the recommended field rate of 0.1 kg/ha assuming uniform distribution of the insecticide in the top-30 cm of surface waters. This higher rate was used to achieve sufcient analytical accuracy.

TEST SYSTEM
- Culturing apparatus: 1 L glass flasks
- Number of culture flasks/concentration: 14 (Per aquatic system 12 samples and 2 reserve samples)

SAMPLING
- Sampling frequency: Samples were taken up to one year. On 0, 3, 7, 14, 21, 30, 59, 90, 119, 182, 268 and 360 days after treatment.
A detailed sampling schedule is provided in Table 2 in 'Any other information on materials and methods incl. tables'.

Test performance:
Stability of the test substance
HPLC-analysis of aliquots of the corresponding treatment solutions of the test substance 1 and 2 after the treatment showed that the test substances had still purities of 97.7 and 96.6%, respectively, i.e. they proved to be stable in the vehicle.
Compartment:
natural water / sediment: freshwater
% CO2:
1.5
% Other volatiles:
4.1
% Recovery:
96.2
Remarks on result:
other: 14C-Dichlorophenylring labelled; Pond; Day 360
Compartment:
natural water / sediment: freshwater
% CO2:
4
% Other volatiles:
8.5
% Recovery:
100
Remarks on result:
other: 14C-Dichlorophenylring labelled;Rhine; Day 360
Compartment:
natural water / sediment: freshwater
% CO2:
49.4
% Other volatiles:
0
% Recovery:
94.9
Remarks on result:
other: 14C-Difluorophenylring labelled; Pond; Day 360
Compartment:
natural water / sediment: freshwater
% CO2:
40
% Other volatiles:
0
% Recovery:
96.4
Remarks on result:
other: 14C-Difluorophenylring labelled; Rhine; Day 360
Parent/product:
parent
Compartment:
total system
% Degr.:
81.8
Parameter:
radiochem. meas.
Sampling time:
360 d
Remarks on result:
other: 14C-Dichlorophenylring labelled; Pond
Parent/product:
parent
Compartment:
total system
% Degr.:
76.3
Parameter:
radiochem. meas.
Sampling time:
360 d
Remarks on result:
other: 14C-Dichlorophenylring labelled; Rhine
Parent/product:
parent
Compartment:
total system
% Degr.:
85.4
Parameter:
radiochem. meas.
Sampling time:
360 d
Remarks on result:
other: 14C-Difluorophenylring labelled; Pond
Parent/product:
parent
Compartment:
total system
% Degr.:
69.3
Parameter:
radiochem. meas.
Sampling time:
360 d
Remarks on result:
other: 14C-Difluorophenylring labelled; Rhine
Key result
Compartment:
natural water / sediment: freshwater
DT50:
159.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: Dichlorophenylring label; Rhine River
Key result
Compartment:
natural water / sediment: freshwater
DT50:
187 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: Difluorophenylring label; Rhine River
Key result
Compartment:
natural water / sediment
DT50:
33.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: Dichlorophenylring label; Pond
Key result
Compartment:
natural water / sediment: freshwater
DT50:
67.2 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: Difluorophenylring label; Pond
Transformation products:
not specified
Remarks:
M4, M6 and M9
Details on transformation products:
An overview of the results is provided in Table 3 - Table 12 in 'Any other information on results incl. tables'
- Pathway of the test substance: The test substance degraded into M4 and assumable 2,6-Difluorobenzoic acid reaching its highest concentration with 47.5 and 19.8% of the radioactivity applied after 59 and 120 days of incubation in Pond and Rhine aquatic system, respectively. Under aerobic conditions, the Dichlorophenylring labelled moiety was further degraded to M6 showing its highest concentrations with 26.0 and 12.8% of the dose applied after 120 and 182 days of incubation in Pond and Rhine aquatic system, respectively. Finally, in Pond aquatic system M6 was further metabolised to metabolite M9, reaching highest concentrations with 6.7% of the radioactivity initially applied after 187 days. However, at the end of the incubation period, all extracted metabolites of the dichlorophenylring labelled moiety had decreased under all incubation conditions, demonstrating their transient character. M6, M4 and the metabolite M9 declined in Pond aquatic system to 6.3, 17.4 and 3.8% of the dose applied, respectively. In Rhine aquatic system M6 and M4 declined to 8.3 and 11.2% of the radioactivity applied, respectively. For the difluorophenylring labelled test compound practically no metabolites extractable from sediment and in water were found. The amounts detected ranged from 0.2 to 3.9 % of the radioactivity applied.

- Rates of Degradation of the test substance and Metabolites: The test substance was broken down in Pond aquatic system, with calculated half-lives 33.6 and 67.2 days, for the l4C-dichlorophenylring labelled and the 14C-difluorophenylring labelled parent molecule, respectively. The calculated DT-90 values in the same aquatic system were 111.6 and 223.2 days, respectively. In Rhine aquatic system the test substance was broken down more slowly with calculated half-lives of 159.7 and 187 days, respectively.
The half-lives of the degradation products were calculated based on the concentration of the molecule found at various time intervals after reaching the maximum concentration in the system. The degradation rate constants were determined by applying pseudo first order reaction kinetics. M6 disappeared with half-lives of 53.9 and 45.4 days from Pond and Rhine aquatic system, respectively. M4 disappeared with half-lives of 116.9 and 101.4 days from Pond and Rhine aquatic system, respectively. The metabolite M9 disappeared from Pond aquatic system with a calculated half-life of 247.1 days.
Evaporation of parent compound:
not specified
Volatile metabolites:
yes
Residues:
yes
Details on results:
An overview of the results is provided in Table 3 - Table 12 in 'Any other information on results incl. tables'
- Microbial Biomass of Sediment: In Pond sediment, a high microbial biomass was present with 240 mg C per 100g sediment dry weight. In Rhine sediment the microbial biomass was 125 mg C per 100 g sediment dry weight.
- Redox Potential: In Pond aquatic system the redox potential of the sediment was always negative, ranging from -41 to -137 mV during equilibration and from -122 to -67 mV during incubation, demonstrating reductive/anaerobic conditions in deeper sediment layers. In the corresponding water, the redox potential ranged from 105 to 156 mV, after 358 and 21 days of incubation, respectively, figuring oxidative/aerobic conditions in the surface layer of the sediment. In Rhine aquatic system the redox potential of the sediment continuously decreased during equilibration from 100 to -80 mV at day 8 after application. During incubation the redox potential of the sediment was always negative, ranging between -23 and -83 mV, demonstrating slightly reductive/anaerobic conditions in deeper sediment layers. In Rhine water, the redox potential ranged from 86 to 158 mV, after 358 and 3 days of incubation, respectively, figuring oxidative/aerobic conditions in the surface layer of the sediment.
- Recovery of Radioactivity: The recovery of radioactivity was, on average, 96.8 ± 2.55% and 99.1 ± 3.12% for Pond and Rhine aquatic system, respectively, treated with dichlorophenylring labelled test substance (test substance 1). For the aquatic systems treated with difluorophenylring labelled test substance (test substance 2) the corresponding figures were 97.8 ± 2.43% and 96.7 ± 3.13%.
- Distribution of Radioactivity: The test substance 1 and 2 disappeared very fast from the water of both sediment/water aquatic systems. The disappearance times for 50 and 90% of the applied activity from water were <1 and < 3 days for both aquatic systems. Thereafter, the radioactivity in water was always 6% of the radioactivity applied under all incubation conditions, demonstrating the very low availability of the test substance in water of aquatic system.
- Radioactivity in Extractables of Sediment and Water: For the test substance 1 incubation, total radioactivity in extractables of sediment and water decreased after 360 days to 46.7 and 49.8% of the radioactivity applied to Pond and Rhine aquatic system, respectively. The non-extractable fractions accounted at the same time for 43.9 and 40.3% of the dose applied. The radioactivity in extractables of sediment and water of test substance 2 incubation in Pond and Rhine aquatic system decreased after 360 days to 15.4 and 35.7% of the radioactivity applied, respectively. The non-extractable fractions of the same samples accounted for 30.2 and 20.7% of the dose applied.
The radioactivity liberated by harsh extraction procedures of sediment samples from test substance 1 incubation after 360 days ranged from 9.7 and 9.3% of the dose applied for Pond and Rhine sediment. respectively. For the corresponding the test substance 2 incubated sediments the extractable radioactivity ranged only from 1.6 and 1.3% of the radioactivity applied, respectively. However, HPLC- and TLC-chromatograms of these extracts demonstrate that only matrix radioactivity could be extracted by this procedure, i.e. no discrete peaks showed up.
- Volatile Radioactivity: For test substance 1 incubated in Pond and Rhine aquatic system the evolution of 14C-CO2 yielded within 360 days to 1.5 and 4.0% of the dose applied, respectively, indicating a slow mineralization of the dichlorophenylring moiety. Other volatile radioactivity gathered in ethylene glycol traps accounted to 4.1 and 8.5% of the radioactivity applied in Pond and Rhine aquatic system, respectively. These figures indicate, that small amounts of the dichlorophenylring moiety are metabolised to volatile compounds during degradation.
For the incubation of test substance 2 the evolution of 14C-CO2 accounted for 49.4 and 40.0% of the radioactivity initially applied to Pond and Rhine aquatic system, respectively, clearly demonstrating the high mineralization of the difluorophenylring moiety of the test substance.

Table 3. Distribution Pattern of Radioactivity for the Degradation of 14C-Dichlorophenylring labelled test substance in Pond aquatic system (Values are given in % of the radioactivity applied)

Incubation Time (Days)

Water

CO2

Volatiles

Sediment Extractables

Sediment Non-Extr.

Recovery

0

23.1

0.0

0.0

78.4

0.1

101.6

3

1.7

0.0

0.0

98.5

0.1

100.3

7

2.2

0.0

0.1

95.0

0.4

97.7

14

3.0

0.0

0.2

91.0

0.6

94.8

21

2.3

0.0

0.2

88.8

1.4

92.7

30

4.0

0.0

0.2

91.3

2.0

97.5

59

2.1

0.1

0.6

85.6

5.2

93.6

90

2.0

0.2

1.2

78.8

14.8

97.0

120

1.2

0.2

1.3

81.0

13.3

97.1

182

1.3

0.5

1.6

64.6

29.7

97.6

269

0.0

1.0

4.4

56.2

33.4

95.0

360

1.0

1.5

4.1

45.7

43.9

96.2

 

Table 4. Distribution Pattern of Radioactivity for the Degradation of 14C-Dichlorophenylring labelled test substance in Rhine aquatic system (Values are given in % of the radioactivity applied)

Incubation Time (Days)

Water

CO2

Volatiles

Sediment Extractables

Sediment Non-Extr.

Recovery

0

33.6

0.0

0.0

67.4

0.0

101.0

3

5.5

0.0

0.0

96.5

0.1

102.1

7

1.3

0.0

0.2

101.7

0.5

103.6

14

2.5

0.0

0.1

96.2

0.4

99.2

21

4.4

0.0

0.2

95.5

0.7

100.9

30

4.5

0.0

0.3

86.9

1.9

93.6

59

4.3

0.1

0.5

90.1

3.7

98.6

90

1.5

0.2

0.7

88.3

6.0

96.6

120

4.2

0.4

1.3

84.2

9.4

99.6

182

5.8

1.8

5.2

62.9

19.5

95.2

269

6.0

3.1

7.3

61.7

18.6

96.6

360

4.4

4.0

8.5

45.4

40.3

102.6

Table 5. Distribution Pattern of Radioactivity for the Degradation of 14C-Difluorophenylring labelled the test substance in Pond Aquatic (Values are given in % of the radioactivity applied)

Incubation Time (Days)

Water

CO2

Volatiles

Sediment Extractables

Sediment Non-Extr.

Recovery

0

35.8

0.0

0.0

60.3

0.1

96.1

3

3.9

0.8

0.0

94.9

1.0

100.6

7

3.6

1.5

0.0

88.8

5.3

99.2

14

3.0

3.0

0.0

82.6

7.7

96.2

21

4.5

6.7

0.0

73.9

15.4

100.5

30

4.7

8.0

0.0

64.8

19.9

97.5

59

4.2

18.1

0.0

46.2

26.2

94.7

90

2.4

35.7

0.0

27.3

37.2

102.5

120

2.1

30.2

0.0

31.8

32.6

96.8

182

0.9

41.2

0.0

18.0

38.0

98.1

269

0.8

43.1

0.0

18.0

34.9

96.7

360

0.8

49.4

0.0

14.6

30.2

94.9

 

Table 6. Distribution Pattern of Radioactivity for the Degradation of 14C-Difluorophenylring labelled the test substance in Rhine Aquatic (Values are given in % of the radioactivity applied)

Incubation Time (Days)

Water

CO2

Volatiles

Sediment Extractables

Sediment Non-Extr.

Recovery

0

31.4

0.0

0.0

68.6

0.0

100.0

3

3.3

0.2

0.0

97.5

0.5

101.5

7

2.4

0.7

0.0

93.9

1.4

98.4

14

1.9

1.2

0.0

91.5

1.6

96.1

21

2.6

3.3

0.0

84.5

3.4

93.8

30

2.9

3.6

0.0

87.1

4.0

97.5

59

2.6

7.2

0.0

77.8

7.2

94.9

90

1.3

11.8

0.0

73.1

12.0

98.2

120

2.7

15.2

0.0

58.4

16.8

93.2

182

2.0

34.4

0.0

48.0

15.1

99.5

269

2.6

34.7

0.0

32.8

20.6

90.7

360

1.1

40.0

0.0

34.6

20.7

96.4

Table 7. Distribution of labelled14C-Dichlorophenylring labelled test substance and Metabolites in Pond Aquatic System (Values are given in % of the radioactivity applied, NA = not analysed, UK= unknown)

Incubation time (days)

 

 

Radioactive Fraction/Code-Number

 

Parent substance

M4

M6

UK

M9

UK

UK

NA

 Total

0

101.3

0.0

0.0

0.0

0.0

0.0

0.0

0.3

101.5

3

97.7

0.0

2.4

0.0

0.0

0.0

0.0

0.1

100.2

7

88.1

0.0

8.8

0.0

0.0

0.0

0.0

0.2

97.2

14

79.2

0.0

14.6

0.0

0.0

0.0

0.0

0.2

94.0

21

64.8

1.9

24.4

0.0

0.0

0.0

0.0

0.2

91.1

30

49.0

3.1

43.0

0.0

0.0

0.0

0.0

0.2

95.2

59

23.6

14.6

47.5

0.0

0.0

0.0

0.0

2.1

87.7

90

11.7

20.8

44.4

0.0

1.8

0.0

0.0

2.0

80.8

120

23.0

26.0

27.7

0.0

4.3

0.0

0.0

1.2

82.2

182

19.9

24.6

13.4

0.0

6.7

0.0

0.0

1.3

65.9

269

17.3

21.4

11.7

0.0

5.8

0.0

0.0

0.0

56.2

360

18.2

17.4

6.3

0.0

3.8

0.0

0.0

1.0

46.7

 

Table 8. Distribution of labelled14C-Dichlorophenylring labelled test substance and Metabolites in Rhine Aquatic System (Values are given in % of the radioactivity applied, NA = not analysed, UK= unknown)

Incubation time (days)

 

 

Radioactive Fraction/Code-Number

 

Parent substance

M4

M6

UK

M9

UK

UK

NA

 Total

0

100.5

0.0

0.0

0.0

0.0

0.0

0.0

0.6

101.0

3

101.5

0.0

0.4

0.0

0.0

0.0

0.0

0.1

102.0

7

100.6

0.0

2.2

0.0

0.0

0.0

0.0

0.1

103.0

14

95.4

0.0

3.1

0.0

0.0

0.0

0.0

0.2

98.7

21

92.0

0.0

7.5

0.0

0.0

0.0

0.0

0.3

99.9

30

77.0

1.6

12.4

0.0

0.0

0.0

0.0

0.4

91.4

59

74.1

2.6

13.3

0.0

0.0

0.0

0.0

4.3

94.3

90

 

4.4

10.5

0.0

0.0

0.0

0.0

1.5

89.7

120

56.0

8.4

19.8

0.0

0.0

0.0

0.0

4.2

88.5

182

38.3

12.8

11.8

0.0

0.0

0.0

0.0

5.8

68.7

269

 

12.6

11.5

0.0

0.0

0.0

0.0

6.0

67.6

360

23.7

11.2

8.3

2.1

0.0

0.0

0.0

4.4

49.8

Table 9. Distribution of 14C-Difluorophenylring labelled test substance and Metabolites in Pond Aquatic System (Values are given in % of the radioactivity applied, NA = not analysed, UK= unknown)

Incubation time (days)

 

 

Radioactive Fraction/Code-Number

 

Parent substance

M4

M6

UK

M9

UK

UK

NA

 Total

0

93.5

0.0

0.0

0.4

0.0

0.0

0.0

2.2

96.1

3

96.4

0.0

0.0

0.4

0.0

0.2

0.0

1.9

98.8

7

88.8

0.0

0.0

0.0

0.0

0.0

0.0

3.6

92.4

14

82.6

0.0

0.0

0.0

0.0

0.0

0.0

3.0

85.6

21

73.9

0.0

0.0

0.0

0.0

0.0

0.0

4.5

78.3

30

64.8

0.0

0.0

0.0

0.0

0.0

0.0

4.7

69.6

59

46.2

0.0

0.0

0.0

0.0

0.0

0.0

4.2

50.4

90

27.3

0.0

0.0

0.0

0.0

0.0

0.0

2.4

29.7

120

31.8

0.0

0.0

0.0

0.0

0.0

0.0

2.1

33.9

182

18.0

0.0

0.0

0.0

0.0

0.0

0.0

0.9

18.9

269

18.0

0.0

0.0

0.0

0.0

0.0

0.0

0.8

18.8

360

14.6

0.0

0.0

0.0

0.0

0.0

0.0

0.8

15.4

 

Table 10. Distribution of 14C-Difluorophenylring labelled test substance and Metabolites in Rhine Aquatic System (Values are given in % of the radioactivity applied, NA = not analysed, UK= unknown)

Incubation time (days)

 

 

Radioactive Fraction/Code-Number

 

Parent substance

M4

M6

UK

M9

UK

UK

NA

 Total

0

97.4

0.0

0.0

0.5

0.0

0.0

0.0

2.1

100.0

3

98.5

0.0

0.0

0.8

0.0

0.0

0.0

1.5

100.8

7

93.9

0.0

0.0

0.0

0.0

0.0

0.0

2.4

96.3

14

91.5

0.0

0.0

0.0

0.0

0.0

0.0

1.9

93.3

21

84.5

0.0

0.0

0.0

0.0

0.0

0.0

2.6

87.1

30

87.1

0.0

0.0

0.0

0.0

0.0

0.0

2.9

89.9

59

77.8

0.0

0.0

0.0

0.0

0.0

0.0

2.6

80.4

90

73.1

0.0

0.0

0.0

0.0

0.0

0.0

1.3

74.4

120

58.4

0.0

0.0

0.0

0.0

0.0

0.0

2.7

61.1

182

48.0

0.0

0.0

0.0

0.0

0.0

0.0

2.0

50.0

269

32.8

0.0

0.0

0.0

0.0

0.0

0.0

2.6

35.4

360

30.7

0.0

0.0

0.0

0.0

0.0

3.9

1.1

35.7

Table 11. DT50 and DT 90 of test substance

Aquatic system

Dichlorophenylring label

Difluorophenylring label

 

DT50 (days)

DT90 (days)

DT50 (days)

DT90 (days)

Pond

33.6

111.6

67.2

223.2

Rhine

159.7

530.6

187

622

Table 12. DT50 of major metabolites

Aquatic system

M4

M6

 

DT50 (days)

DT50 (days)

Pond

116.9

53.9

Rhine

101.4

45.4

  

Validity criteria:
The total recovery at the end of the esperiment should be between 90% and 100% for radiolabelled substance
Observed value:
River system = 96.4 - 100%; Pond system = 94.9 - 96.2%
Validity criteria fulfilled:
yes
Conclusions:
In a biodegradation study with fresh water-sediment systems, performed in accordance with BBA IV 5-1 guideline, the test substance was broken down with half-lives of 33.6 and 159.7 days (14C-Dichlorophenylring labelled test substance) in the Pond and Rhine system, respectively. The corresponding values for the 14C-Difluorophenyl labelled test substance were 67.2 and 187 days. Metabolites (M4 and M6), CO2 and non-extractable are the major degradation products of the microbial breakdown observed for 14C-dichlorophenylring labelled test substance under aerobic conditions. In addition to these metabolites, metabolite M9 was observed. The formation of 14C-CO2 demonstrates the slow but significant mineralization of the dichlorophenlring labelled moiety with time. Degradation of the difluorophenylring labelled test substance mainly proceeded via the formation of 14C-CO2 and bound residues. Other metabolites were practically not observed.
Executive summary:

The degradation of 14C Dichlorophenylring-labelled test substance and 14C Difluorophenylring labelled test substance were investigated in two aerobically incubated fresh water-sediment systems, Rhine river and pond. The study was performed with following BBA IV 5-1 and Dutch registration guideline (section G2) and also taking the EPA 162-4 guideline into consideration. The study was in compliance with GLP criteria. The test substances were applied to the water surface at approximately 0.14 mg/L. This rate correspond to four times the total seasonal field rate of 100 g test substance/ha assuming uniform distribution in the top 30 cm of surface water. The test systems were incubated in the dark at 20 ± 2°C and sampled at 0, 3, 7, 14, 21, 30, 59, 90, 119, 182, 268 and 360 days. The systems were maintained in a flow-through atmosphere with gentle stirring from the top, ensuring that the sediments were not disturbed. The distribution of radioactivity between the surface water and the sediment layer was measured at intervals throughout the incubation. The amount of produced CO2, radioactivity and the degraded metabolites were analysed using HPLC, TLC and MS.

 The recovery of radioactivity was, on average, 96.8 ± 2.55% and 99.1 ± 3.12% for Pond and Rhine aquatic system, respectively, treated with dichlorophenylring labelled test substance. For the aquatic systems treated with difluorophenylring labelled test substance the corresponding figures were 97.8 ± 2.43% and 96.7 ± 3.13%. The test substance degraded into M4 and assumable 2,6-Diuflorobenzoic acid reaching its highest concentration with 47.5 and 19.8% of the radioactivity applied after 59 and 120 days of incubation in Pond and Rhine aquatic system, respectively. The Dichlorophenylring labelled moiety was further degraded to M6 showing its highest concentrations with 26.0 and 12.8% of the dose applied after 120 and 182 days of incubation in Pond and Rhine aquatic system, respectively. Finally, in Pond aquatic system M6 was further metabolised to metabolite M9, reaching highest concentrations with 6.7% of the radioactivity initially applied after 187 days. However, at the end of the incubation period, all extracted metabolites of the dichlorophenylring labelled moiety had decreased under all incubation conditions, demonstrating their transient character. M6, M4 and the metabolite M9 declined in Pond aquatic system to 6.3, 17.4 and 3.8% of the dose applied, respectively. In Rhine aquatic system M6 and M4 declined to 8.3 and 11.2% of the radioactivity applied, respectively. For the difluorophenylring labelled test compound practically no metabolites extractable from sediment and in water were found. The amounts detected ranged from 0.2 to 3.9 % of the radioactivity applied. Overall, M4, M6, CO2 and non-extractable are the major degradation products of the microbial breakdown observed for 14C-dichlorophenylring labelled test substance under aerobic conditions. In addition to these metabolites, metabolite M9 was observed. The formation of 14C-CO2 demonstrates the slow but significant mineralization of the dichlorophenlring labelled moiety with time. Degradation of the difluorophenylring labelled test substance mainly proceeded via the formation of 14C-CO2 and bound residues.

Based on these results, the half-lives of the test substance were calculated to be 33.6 and 159.7 days (14C-Dichlorophenylring labelled test substance) in the Pond and Rhine system, respectively. The corresponding values for the 14C-Difluorophenyl labelled test substance were 67.2 and 187 days. The major metabolite, M6, disappeared with half-lives of 53.9 and 45.4 days from Pond and Rhine aquatic system, respectively. While, M4 disappeared with half-lives of 116.9 and 101.4 days from Pond and Rhine aquatic system, respectively.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Data waiving:
study technically not feasible
Justification for data waiving:
the study does not need to be conducted because the substance is highly insoluble in water
other:
Transformation products:
not specified

Description of key information

All available data was assessed. The study representing the worst-case effects of the water-sediment system was included as key study and its effect values were used as key values in a weight of evidence approach.

Arithmetic mean DT50 value of the test substance in freshwater-sediment = 112 d, BBA-IV 5 -1 guideline followed study, Gonzalez-Valero 1994

Arithmetic mean DT50 values of the major metabolites, M4 and M6, in freshwater-sediment were 49.7 and 109 days, respectively, BBA-IV 5 -1 guideline followed study, Gonzalez-Valero 1994

The biodegradation of the substance in surface water : simulation test is waived due to the high insolubility of the substance. In addition, no further study on biodegradation in surface water is needed beyond the available simulation studies on soil and sediment.

Key value for chemical safety assessment

Whole System

Half-life in whole system:
112 d
at the temperature of:
20 °C
Type of system:
fresh water and sediment

Additional information

Two standard test guideline followed and GLP complied studies are available for this endpoint. One study (Gonzalez-Valero 1994; Reliability 1) was selected as key study, because it represents the worst-case effects (i.e. showed higher DT50 values) of the water-sediment systems. The effect values from this study were used in a weight of evidence approach for the CSA. The 14C-dichlorophenyl- and 14C-difluorophenyl-labelled test substances were studied in the key study. The test conditions and results were summarized in the Table A below. Two major metabolites, M4 and M6 were found in both pond and river systems. The arithmetic average DT50 values for M4 and M6 were identified to be 109 and 49.7 days (see Table B below).

The supporting study (Volz 2003; Reliability 2) is a biodegradation and bioaccumulation study with outdoor fresh water-sediment systems. This 190-day study includes communities of phyto- and zooplankton, macroinvertebrates and plants. The 14C-labelled test substance very rapidly disappeared from the aquatic systems. The DT50 of the test substance was calculated to be 15 minutes in the water compartment and 63 days in the sediment compartment. Three major metabolites, M4, M6 and M7 were found in the water compartment and showed DT50 < 2 days. 

Table. DT50 values for the substance and its major metabolites in freshwater-sediment system under aerobic standard test conditions (key study)

A) Parent substance

Test system

Test condition

Mineralisation / Non-extractable residues / Major metabolites (% applied)

Compartment

DT50 (d) – Kinetic model

Author/ year

Pond (loam), Aargau, CH

56.5 g sediment (DW) + 500 mL corresponding

water.

Incubation in the dark for 360 days at 20°C under aerobic conditions

14C- Difluorophenyl- label:

Miner.: max. 49.4 % (after 360 d)

Non-extr.: max. 38.0 % (after 182 d)

Maj. met.: none

Water

Total system

< 1 - SFO

67.2 - SFO

Gonzalez-Valero 1994

Pond (loam), Aargau, CH

56.5 g sediment (DW) + 500 mL corresponding

water.

Incubation in the dark for 360 days at 20°C under aerobic conditions

14C- Dichlorophenyl- label: Miner.: max. 1.5 % (after 360 d)

Non-extr.: max. 43.9 % (after 360 d)

Maj. met.: max. 26.0 % M6 (day 120); max. 47.5 % M4 (day 59)

Water

Total system

< 1 - SFO

33.6 - SFO

Gonzalez-Valero 1994

Rhine river (loamy sand), Aargau, CH

139 g sediment (DW) + 500 mL

corresponding water.

Incubation in the dark for 360 days at 20°C under aerobic conditions

14C- Difluorophenyl- label: Miner.: max. 40.0 % (after 360 d)

max. 20.7 % (after 360 d) Maj. met.: none

Water

Total system

< 1 - SFO

187 - SFO

Gonzalez-Valero 1994

Rhine river (loamy sand), Aargau, CH

 

139 g sediment (DW) + 500 mL

corresponding water.

Incubation in the dark for 360 days at 20°C under aerobic conditions 1

14C- Dichlorophenyl- label: Miner.: max. 4.0 % (after 360 d)

Non-extr.: max. 40.3 % (after 360 d)

Maj. met.: max. 12.8 % M6 (day 182); max.

19.8 % M4 (day 120)

Water

Total system

< 1 - SFO

159.7 - SFO

Gonzalez-Valero 1994

 

 

Arithmetic mean DT50 (days)

Water

< 1

 

 

 

Arithmetic mean DT50 (days)

Total system

112

 

 

b) Metabolites: M4 and M6

Test system

Test condition

Mineralisation / Non-extractable residues / Major metabolites (% applied)

Compartment

 

 

DT50 (d) – Kinetic model

Author/ year

Pond (loam), Aargau, CH

56.5 g sediment (DW) + 500 mL corresponding

water.

Incubation in the dark for 360 days at 20°C under aerobic conditions

14C- Dichlorophenyllabel:

M6: max. 26.0

% (120d) down to 17.4

(360d); M4:

max. 47.5 % (59d) down

to 6.3 (360d)

Total system

117 - SFO (M4)

53.9 - SFO (M6)

Gonzalez-Valero 1994

Rhine river (loamy sand), Aargau, CH

139 g sediment (DW) + 500 mL

corresponding water.

Incubation in the dark for 360 days at 20°C under aerobic conditions

14C- Dichlorophenyllabel:

M6: max. 12.8

% (182d) down to 11.2

(360d); M4:

max. 19.8 % (120d) down

to 8.3 (360d)

Total system

101 - SFO (M4)

45.4 - SFO (M6)

Gonzalez-Valero 1994

 

 

Arithmetic mean DT50 (days)

Total system (M4)

109

 

 

 

Arithmetic mean DT50 (days)

Total system (M6)

49.7