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Biodegradation in water and sediment: simulation tests

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Reference
Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 June 2018 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Exception(s) to GLP compliance: Aquatic sediments were obtained and measurements made by LRA Labsoil; and physical and chemical characteristics of the aquatic sediments were determined by NRM.
Specific details on test material used for the study:
Radiolabeled Test Item
Radiochemical purity: The radiochemical purity was measured to be 97.1% prior to application

Non-Radiolabeled Test Item
Purity: UVCB substance
Description: Light yellow to tan solid
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
See Tables 1 and 2 (below)
Details on source and properties of sediment:
See Tables 1 and 2 (below)
Duration of test (contact time):
100 d
Initial conc.:
52.6 µg/L
Based on:
other: Application rate calculated from radioactivity and volume applied, and specific activity of the test material
Remarks:
The radiochemical purity of [14C] test material for the main experiment was 97.1%
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
A solution of radiolabeled (14C) test item was prepared in tetrahydrofuran (THF) at a nominal concentration of 50 µg/mL, such that the proportion of solvent in the test system would not exceed 1% of the volume of water in the vessel. The 14-C test item was applied to the water layer of samples of two aquatic sediment types at a nominal rate of 50 µg/L. The volume ratio of water to wet sediment in each sample was approximately 3:1. Aquatic sediment systems were acclimatized under aerobic conditions prior to test item application until reasonable stability had been established with respect to the pH, oxygen concentration and redox potential in the water and the pH and redox potential in the sediment.

For each aquatic sediment, 16 vessels were set up for treatment with radiolabeled test item at a nominal rate of 50 µg/L. In addition, six vessels of each aquatic sediment were set up for microbiological activity measurements and two vessels of each sediment type were set up for the determination of the microbiological activity at the start and end of the incubation period and for measurement of the pH and redox potential in both phases and oxygen content in the water phase. These vessels were not treated with the test item.

Samples were arranged in flow-through systems designed to trap volatile radiolabeled compounds including 14-CO2. Air was drawn through each system at a flow rate of approximately 50 mL/minute. Flow rates were checked and adjusted throughout the incubation period. During the acclimatization period (19 days) all traps except the humidifying water bottle were empty.

The samples assigned for measurement of microbiological activity measurements were connected, in series, into a separate flow-through system for each aquatic sediment. Humidified air was passed through these vessels but no traps were included.

The samples assigned for system parameter measurements were connected in series (one for each aquatic sediment) as for treated vessels. Throughout the incubation period, these samples were used for measurement of pH and redox potential in both phases and oxygen content in the water phase. Measurements were made twice weekly throughout the acclimatization period and then, following test item application, at every sampling interval.

The systems were incubated in darkness at 12 +/- 2 deg C for up to 100 days prior to analysis. Deviations from the acceptable temperature range occurred between Day 24 and Day 33 due to failure of the cooling unit of the room, with an overall duration of approximately 147 hours. The temperature reached a maximum of 2.5°C above tolerance and a minimum of 0.9°C below tolerance. It is considered that these temperature deviations did not affect the integrity or the outcome of the study

Two samples of each aquatic sediment were taken for analysis immediately after application, and after 1, 7, 14, 30, 59 and 100 days of incubation. Trapping solutions were taken for analysis when the associated sample was taken for analysis. Additionally, all remaining traps were taken for analysis and replaced with fresh media as necessary at 14, 30, 44, 59, 73, 87 and 100 days after application. Sediment and water phases were analyzed separately.


Compartment:
natural water / sediment: freshwater
Remarks on result:
other: 90.9 to 100.5% applied radioactivity
Remarks:
Exceptions: four Calwich Abbey Lake samples (Day 7, 89.3%; Day 59, 86.0%; Day 100, 89.2% and 85.3%).
Compartment:
natural water: freshwater
DT50:
2.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Compartment:
natural sediment: freshwater
DT50:
811 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Key result
Compartment:
natural water / sediment: freshwater
DT50:
657 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Calwich Abbey Lake
Compartment:
natural water: freshwater
DT50:
10.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Emperor Lake
Compartment:
natural sediment: freshwater
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
not determinable
Key result
Compartment:
natural water / sediment: freshwater
DT50:
636 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other:
Remarks:
Emperor Lake
Transformation products:
yes
No.:
#1
Details on transformation products:
Mostly parent compound was detected in the water organic extracts and sediment extracts throughout the study, with only minor amounts of unidentified polar material (up to 5.6% applied radioactivity at time zero).
Residues:
yes
Details on results:
Recovery and Distribution of Radioactivity

Total recoveries of radioactivity (‘mass balances’, i.e. the sum of radioactivity in the water layer, extractable and non-extractable sediment radioactivity and volatile radioactivity) were in the range 90.9 to 100.5% applied radioactivity, except for Calwich Abbey Lake samples CA05 (Day 7, 89.3% AR), CA11 (Day 59, 86.0% AR), CA13 (Day 100, 89.2% AR) and CA14 (Day 100, 85.3% AR).

In Calwich Abbey Lake aquatic sediment, total radioactivity in the water layer declined from a mean of 85.7% applied radioactivity at time zero to 0.9% after 100 days. In the sediment, total radioactivity increased to a mean maximum of 87.5% applied radioactivity after 30 days and decreased slightly, accounting for 80.9% applied radioactivity at 100 days. Non-extractable radioactivity in the sediment (bound residues) increased from a mean value of 0.6% applied radioactivity at time zero to 4.8% applied radioactivity after 100 days. Volatile radioactivity, all presumed to be associated with 14CO2, accounted for a mean maximum of 5.6% applied radioactivity after 100 days.

Dissipation of radioactivity followed a similar pattern in the Emperor Lake aquatic sediment system. Total radioactivity in the water layer declined from a mean of 88.5% applied radioactivity at time zero to 3.1% after 100 days. In the sediment, total radioactivity increased to a mean maximum of 87.7% applied radioactivity at 100 days. Non-extractable radioactivity in the sediment (bound residues) increased from a mean value of 2.4% applied radioactivity at time zero to 8.7% applied radioactivity after 100 days. Volatile radioactivity, all presumed to be associated with 14CO2, accounted for a mean of 3.3% applied radioactivity after 100 days.

Chromatographic Analysis

The identity of [14C]-test material could not be established by co-chromatographic correspondence with a reference item, since the non-radiolabeled test material was not pure (UVCB substance). However, TLC analysis was used to determine whether degradation of the parent compound was occurring, by comparison of the plates/ chromatograms from each sampling interval with the time zero results. The amount of test item in the respective phases of Calwich Abbey Lake and Emperor Lake systems is summarized in Tables 3 and 4 (below).

Mostly parent test item was detected from the TLC analysis of the water organic extracts and sediment extracts, with only minor amounts of unidentified polar material separating (up to 5.6% applied radioactivity in Calwich Abbey Lake water and up to 3.6% applied radioactivity in Emperor Lake water, both at time zero). The retention factor of test item in the TLC analyses shifted considerably throughout testing (approximately 0.4 ± 0.2). To investigate any possible degradation, two concentrated extract pool samples from each sediment type with different retention factors were analyzed together, spotting the TLC plate so each pair of bands partially overlapped. The TLC plate was developed and imaged, confirming that the main band in all plates could be attributed to the parent compound.


 Table 3: Mean Distribution and Recovery of Radioactivity in Aquatic Sediment [Calwich Abbey Lake]

Fraction

Sampling time (days)

0

1

7

14

30

59

100

Water

85.7

72.5

22.3

9.0

6.0

1.4

0.9

Partition of water:

 

 

 

 

 

 

 

Organic phase (DCM)

85.3

69.3

17.8

4.1

2.2

0.2

0.1

Aqueous phase

1.2

1.7

2.4

2.5

2.8

0.6

0.5

Sediment:

 

 

 

 

 

 

 

Extract 1 (DCM)

6.0

8.8

34.2

36.0

32.1

21.2

20.7

Extract 1a (Aqueous)

nd

0.2

0.5

1.2

0.9

0.7

0.8

Extract 2 (DCM)

2.0

3.2

12.0

14.7

15.5

7.0

6.9

Extract 3 (DCM)

0.5

1.5

5.2

6.6

5.9

na

na

Extract 4 (THF)

na

3.4

10.6

14.1

18.2

42.5

39.7

Extract 5 (THF)

na

na

na

na

5.8

7.4

8.0

Vessel rinse (THF)

nd

0.7

1.3

3.1

5.6

0.8

na

Total extractable

8.4

17.7

63.8

75.6

83.9

79.5

76.1

Non-extractable

0.6

1.1

4.7

6.2

3.7

4.1

4.8

Total in sediment

9.0

18.7

68.5

81.8

87.5

83.5

80.9

Volatiles:

 

 

 

 

 

 

 

Organics

na

na

0.0

nd

nd

nd

nd

CO2

na

na

0.5

0.9

1.6

3.6

5.6

Total volatiles

na

na

0.5

0.9

1.6

3.6

5.6

Total recovered

94.7

91.2

91.3

91.6

95.1

88.5

87.3

Results expressed as % applied radioactivity. 

nd       Not detected

na        Not applicable

Partitioning of water with DCM not considered for the calculation of total recovery.

Extractions 1 to 5 and vessel rinse performed at ambient temperature. Solvent used indicated in brackets.

Table 4: Mean Distribution and Recovery of Radioactivity in Aquatic Sediment [Emperor Lake]

Fraction

Sampling time (days)

0

1

7

14

30

59

100

Water

88.5

85.1

67.4

40.9

12.0

8.0

3.1

Partition of water:

 

 

 

 

 

 

 

Organic phase (DCM)

86.7

79.9

63.2

35.6

9.1

3.1

0.8

Aqueous phase

1.2

2.9

3.5

3.6

2.3

2.6

1.6

Sediment:

 

 

 

 

 

 

 

Extract 1 (DCM)

5.6

4.0

7.4

19.6

14.3

15.3

7.1

Extract 1a (Aqueous)

0.1

0.3

0.5

0.9

0.8

0.7

0.4

Extract 2 (DCM)

2.9

1.5

3.2

5.4

5.5

5.2

3.0

Extract 3 (DCM)

0.5

0.4

0.9

1.4

0.9

na

na

Extract 4 (THF)

na

na

11.3

16.8

35.9

45.3

57.3

Extract 5 (THF)

na

na

na

na

9.2

8.5

11.5

Vessel rinse (THF)

0.1

0.4

1.4

2.9

7.9

0.1

na

Total extractable

9.1

6.4

24.5

46.9

74.4

74.8

79.1

Non-extractable

2.4

5.3

5.1

8.4

6.0

6.8

8.7

Total in sediment

11.4

11.6

29.6

55.2

80.4

81.6

87.7

Volatiles:

 

 

 

 

 

 

 

Organics

na

na

nd

nd

nd

nd

nd

CO2

na

na

0.4

0.5

1.6

4.2

3.3

Total volatiles

na

na

0.4

0.5

1.6

4.2

3.3

Total recovered

99.9

96.7

97.3

96.6

93.9

93.7

94.1

Results expressed as % applied radioactivity.
nd       Not detected

na        Not applicable

Partitioning of water with DCM not considered for the calculation of total recovery.

Extractions 1 to 5 and vessel rinse performed at ambient temperature. Solvent used indicated in brackets.

Validity criteria fulfilled:
yes
Conclusions:
The test material dissipated rapidly from the water of both aquatic sediment systems, with DT50 values of 2.6 days (Calwich Abbey Lake) and 10.4 days (Emperor Lake). The DT50 value in the sediment phase of Calwich Abbey Lake was 811 days, based on two sampling intervals. The DT50 value for the decline in the sediment phase of Emperor Lake could not be calculated, as the test material (as % applied parent) continued to increase until the last sampling interval. Decline in the overall aquatic sediment system was slow and corresponded to DT50 values of 657 days (Calwich Abbey Lake) and 636 days (Emperor Lake).
Executive summary:

The fate of test material was studied in two natural aquatic sediment systems under laboratory conditions based on the aerobic test design in OECD Guideline for the Testing of Chemicals No. 308 (April 2002). The sediment from Emperor Lake was a sandy clay loam with an acidic pH and low organic carbon content, while that from Calwich Abbey Lake was an approximately neutral silt loam with a higher organic carbon content. Samples of each aquatic sediment system were allowed to acclimatize before being treated with [14C]-test material at a rate of 52.6 µg/L, based on the amount of water in the test vessel including that present within the sediment. The samples were incubated under aerobic conditions at approximately 12 deg C in darkness for periods of up to 100 days.

Total recoveries of radioactivity (mass balances) were in the range 90.9 to 100.5% applied radioactivity, except for four Calwich Abbey Lake samples (Day 7, 89.3%; Day 59, 86.0%; Day 100, 89.2% and 85.3%).

In Calwich Abbey Lake aquatic sediment, total radioactivity in the water layer declined from a mean of 85.7% applied radioactivity at time zero to 0.9% after 100 days. In the sediment, total radioactivity increased to a mean maximum of 87.5% applied radioactivity after 30 days andthen decreasedslightly, accounting for 80.9% applied radioactivity at 100 days. 

Non-extractable radioactivity in the sediment (bound residues) increased to a mean value of 4.8% applied radioactivity after 100 days.

Dissipation of radioactivity followed a similar pattern in the Emperor Lake aquatic sediment system. Total radioactivity in the water layer declined from a mean of 88.5% applied radioactivity at time zero to 3.1% after 100 days. In the sediment, total radioactivity increased to a mean maximum of 87.7% applied radioactivity at 100 days. Non-extractable radioactivity in the sediment (bound residues) increased to a mean value of 8.7% applied radioactivity after 100 days.

For both aquatic sediments, volatile radioactivity increased to a mean maximum of 5.6% applied radioactivity after 100 days, all presumed to be associated with 14-CO2.

DT50 values for the decline of test item in the water, the sediment and from the total aquatic sediment system are shown below.

Compartment

Calwich Abbey Lake

Emperor Lake

Water

2.6

10.4

Sediment

811

Not applicable

Total system

657

636

Kinetic model: SFO = single first order.

Mostly parent compound was detected in the water organic extracts and sediment extracts throughout the study, with only minor amounts of unidentified polar material (up to 5.6% applied radioactivity at time zero).

Description of key information

In an OECD 308 study, conducted according to GLP, the test material dissipated rapidly from the water of both aquatic sediment systems, with DT50 values of 2.6 days (Calwich Abbey Lake) and 10.4 days (Emperor Lake).  The DT50 value in the sediment phase of Calwich Abbey Lake was 811 days, based on two sampling intervals.  The DT50 value for the decline in the sediment phase of Emperor Lake could not be calculated, as the test material (as % applied parent) continued to increase until the last sampling interval.  Decline in the overall aquatic sediment system was slow and corresponded to DT50 values of 657 days (Calwich Abbey Lake) and 636 days (Emperor Lake).

Key value for chemical safety assessment

Half-life in freshwater:
2.6 d
at the temperature of:
12 °C
Half-life in freshwater sediment:
811 d
at the temperature of:
12 °C

Additional information

Due to the low solubility of the test material and rapid partitioning from the aqueous phase to the sediment, aerobic mineralisation in surface water – simulation biodegradation testing according to OECD Testing Guideline No. 309 is considered technically not feasible.