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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test did not follow any guideline, but test procedures and conditions are well-described and comparable to those of the OECD guideline 308 and interpretation is based on 14C-label found as chloroform or carbon dioxide.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Inoculum or test system:
natural sediment
Details on source and properties of sediment:
Sediment samples were taken at "Krinkel de Winkel", a little harbour in the River Merwede near the Dutch town of Gorinchem. The samples were taken from a pontoon situated at 10 m from the quay-wall at 0.5 till 1 m below the water-sediment interface. During sampling large quantities of gas escaped from the sediment which indicates that the sediment was methanogenic. Soil analysis showed 5 % organic carbon, 0.42 % nitrogen, 0.21 % phosphorous, 3.5 % iron, 11 % CaCO3 and a cation exchange capacity of 23 meq. per 100 g. The pH of the sediment measured in water was 7.5 and measured in KCl was 7.3.
Details on inoculum:
Within four hours the samples were transferred to the airlock of an anaerobic Braun type glove box and were gassed with nitrogen and evacuated three times. Samples were stored overnight at a temperature of 10 °C in the box under nitrogen atmosphere which contained less than 1 ppm oxygen. The sediment sample was pushed out of the sampling tube and 100 g sediment was homogenised in 100 mL distilled water in a Moulinex blender for 1 minute. Aliquots of 20 mL suspension were put into 100 mL-bottles which were crimpcap sealed with butylrubber stoppers. Samples were stored for 23 days at 4 °C.
Duration of test (contact time):
64 d
Initial conc.:
4 µg/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
14C labelled chloroform solutions were stored in bottles sealed with butylrubber stoppers at 20 °C. The bottles contained 10000 to 16000 cpm per liter of 14C labelled chloroform. Not radioactive compound was added to the stock solution in order to reach the test concentration of 4 microgram/L in the incubation bottles. With an injection needle 0.5 mL of the stock solution was transferred to the sample bottles.
Key result
Compartment:
sediment
DT50:
>= 10 - <= 14 d
Type:
(pseudo-)first order (= half-life)
Temp.:
10 °C
Key result
Compartment:
sediment
DT50:
ca. 2.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Transformation products:
yes
No.:
#1
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
yes
Validity criteria fulfilled:
yes
Conclusions:
The test using natural anaerobic sediments showed that chloroform was mainly mineralised to CO2 under methanogenic conditions with half-lives of a few days.
Executive summary:

The mineralisation of chloroform was tested with methanogenic natural sediment samples under anaerobic conditions using principles comparable to those of OECD guideline No. 308. Soil analysis showed 5 % organic carbon, 0.42 % nitrogen, 0.21 % phosphorous, 3.5 % iron, 11 % CaCO3 and a cation exchange capacity of 23 meq. per 100 g. The pH of the sediment measured in water was 7.5 and measured in KCl was 7.3. The test used 14C-labelled chloroform and the initial concentration was 4 ug/L. Inoculum was incubated at 10 °C or at 20 °C for 64 days. Most of the radioactivity was recovered as CO2. The degradation of chloroform was described by first order kinetics. The half-lives determined at 10 °C and 20 °C were 10 -14 days and 2.6 days, respectively.

In conclusion, the test demonstrated that chloroform is mineralised mainly to CO2 under methanogenic conditions using natural sediments.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
The test did not follow any guideline, but test procedures and conditions are well-described and comparable to those of the OECD guideline 308 and interpretation is based on 14C-label found as chloroform or carbon dioxide.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Inoculum or test system:
natural sediment
Details on source and properties of sediment:
Anaerobic mud samples were taken from locations in the Netherlands (Table 1). Samples were obtained from a depth of 10-40 cm below the sediment-water interface as described by van Beelen and van Keulen (1990). The sandy sediment samples were obtained by scraping the upper layer directly under the sediment-water interface. The wet mud or sand samples were stored in an icebox under a nitrogen atmosphere, transferred to the laboratory within three hours and placed in the airlock of an anaerobic glove box (Braun type MB 30 G). For sediment properties, see Table 2.
Details on inoculum:
Fresh mud was mixed with an equal amount of anaerobic double distilled water (bidest) and 20 mL of homogenised suspension was pipetted into incubation bottles in the anaerobic glove box. The sand samples were also mixed with an equal weight of anaerobic bidest. The upper suspension was decanted into a sterile vessel and the settled sand was homogenised with a spoon. Then 10 g of wet sand and 10 mL of suspension were subsequently put into an incubation bottle.
Duration of test (contact time):
40 d
Details on study design:
A stock solution of 14CHCl3 was prepared by adding gaseous labelled chloroform to a crimpcap sealed incubation bottle containing sterile anaerobic bidest. The stock solution was diluted and 0.5 mL aliquots were injected through the rubber stoppers into the incubation bottles containing mud or sandy sediment inoculums. Total bacteria numbers were counted by using epifluorescence microscopy. Either the FITC-DC (fluorescine isothiocyanate) or the AODC (acridine orange) method was used, both with small modifications. A 5 mL portion of a sediment/water suspension was sonified for 1 minute in order to remove bacteria from sediment particles. Dilutions made from this suspension were incubated for several weeks on agar plates with 100 x diluted nutrient broth at 20 °C. All manipulations were performed in the anaerobic glove box.
Test performance:
A good separation and recovery of pure 14CHCl3 or 14CO2 was achieved. The recovery of freshly added chloroform was about 90 % but due to sorption onto the stoppers the recovery dropped slowly to about 45 % after 30 to 40 days. Production of 14CO2 was not observed in autoclaved bottles with sediment suspension that were incubated during the mineralisation experiments.
Key result
Parameter:
CO2 evolution
Remarks on result:
not determinable because of methodological limitations
Key result
Compartment:
sediment
DT50:
>= 2 - <= 37 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Transformation products:
yes
No.:
#1
Evaporation of parent compound:
not measured
Volatile metabolites:
yes
Residues:
yes
Details on results:
See Table 3

Table 2: Sediment properties. *: Density of wet sediment derived from the % dry weight using a density of 2.55 g dry sediment per mL. The blank values in the table were not determined. The Gorinchem mud contained 0.3-2.5 mg nitrate N per kg and 390-460 mg ammonia N per kg while K-Sand-B contained 6 mg nitrate N per kg. Total N is expressed in g N per kg dry weight. M-50 is the median of the particle diameter. The mud samples produced methane while the sand samples did not.

Location and code

Sampling date

M-50 (um)

% clay

pH H2O

pH KCl

% org. carbon

% CaCO3

Total N

Density (g/mL)

% dry weight

Wageningen

W-Mud

89-01-11

--

--

7.5

7.6

--

--

--

1.79

73.3

Ketelmeer

K-Mud

89-03-17

--

10.8

7.4

7.1

3.2

11.7

2.4

1.54

64.7

K-Sand-A

89-03-17

220

0.5

8.3

8.2

0.2

4.7

0.2

2*

77.6

K-Sand-B

89-11-20

255

0.8

8.4

8.1

0.2

2.7

0.2

1.96

80.0

Biesbosch

B-Sand

89-04-18

220

1.9

8.0

7.8

0.3

2.8

0.2

2*

76.1

B-Mud

89-04-18

--

28.8

7.2

7.0

8.7

3.1

5.4

1.32

36.9

Oostvaardersplassen

O-Mud-A

89-06-26

63

29.2

8.0

7.6

2.9

6.6

2.1

1.2*

28.0

O-Mud-B

89-06-26

--

33.3

8.0

7.4

5.2

12.4

4.3

1.3*

36.3

Gorinchem

G-Mud-A

87-03-17

--

--

7.4

7.1

5.2

11.2

4.0

--

--

G-Mud-B

87-03-23

--

--

7.5

7.3

5.0

11.4

4.2

--

--

G-Mud-C

89-11-13

63

23.0

7.9

7.4

4.6

9.3

3.6

1.45

55.8

G-Mud-D

90-03-14

110

25.8

7.8

7.6

4.6

11.3

3.0

1.32

46.0

G-Mud-E

90-05-16

125

27.3

8.1

7.5

2.5

10.8

3.6

1.35

41.8

G-Mud-F

90-08-21

125

27.5

7.6

7.4

3.1

9.8

2.3

1.45

44.0

Table 3: The mineralisation rate of chloroform in methanogenic and sandy sediments. * Half-life of chloroform based on the production of carbon dioxide. AODC = Acridine Orange Direct Count. Blank values were not determined. The mineralisation data were fitted by first order kinetics.

log AODC

log CFUs air

Nitrogen

Carbon dioxide production* half-life

Max. % CO2

Chloroform removal half-life

Sediment

No.

(per g)

SD

(per g)

(per g)

(d)

SD

(d)

SD

W-Mud

1A

--

--

6.7

5.3

0.9

0.3

45

2

0

2A

--

--

--

--

2

0.6

54

2.5

0.2

W-Mud 10 °C

1B

--

--

--

--

4

1

36

5

1

2B

--

--

--

--

2

0.2

47

3

0.1

K-Mud

3

8.7

0.06

6.3

4.4

2

0.1

45

3

1

4

--

--

--

--

2

0.8

43

3

0.4

K-Sand-A

5

9.1

0.11

7.0

5.6

>100

--

--

--

--

6

--

--

--

--

>100

--

--

--

--

K-Sand B N2

7A

9.3

0.03

--

--

>500

--

--

--

--

K-Sand B H2

7B

--

--

--

--

>500

--

--

--

--

B-Sand

8

9.6

0.05

8.2

6.1

>130

--

--

--

--

9

--

--

--

--

37

21

36

19

0

B-Mud

10

9.6

0.06

6.8

5.2

11

7

35

17

8

11

--

--

--

--

37

10

20

28

16

O-Mud

12

10.3

0.03

6.8

--

6

0.3

44

6

3

13

--

--

--

--

11

0.1

38

9

2

G-Mud-C

14

9.9

0.03

--

--

4.5

2

48

10

2

G-Mud-D

15

10.1

0.03

--

--

5

0.2

53

4

0.1

G-Mud-E

16

--

--

--

--

5

2

63

5

0

G-Mud-F

17

10.1

0.03

--

--

9

0.6

62

6

0.5

Validity criteria fulfilled:
yes
Conclusions:
The test using natural anaerobic mud sediments and sandy sediments showed that chloroform was mainly mineralised to CO2 under methanogenic conditions with half-lives of a few days but that no mineralisation took place in the sandy sediments.
Executive summary:

The mineralisation of chloroform was tested with methanogenic natural sediment and sandy sediment samples under anaerobic conditions using principles comparable to those of OECD guideline No. 308. The initial concentrations of 14C labelled chloroform were 2.7 to 3.4 microgram/L and the radioactivity was 133 to 167 Bq per bottle with 20 mL sediment suspension. Preliminary tests showed a good separation and recovery of pure 14CHCl3 and 14CO2. The recovery of freshly added chloroform was about 90 %. It slowly dropped to 45 %, which was due to increasing sorption onto the stoppers. Chloroform was mineralised under anaerobic conditions with half-lives of 2 to 37 days at 20 °C in 12 methanogenic sediments producing mainly CO2. Relatively unpolluted samples showed similar mineralisation rates compared to heavily polluted samples from the Rhine and Meuse. Chloroform was not mineralised and persisted in the sandy sediments. This was not caused by the absence of chloroform mineralising bacteria but by the inactivity of these bacteria under the conditions present in the sandy sediments.

Description of key information

Biodegradation of chloroform is observed in anaerobic sediment. The corresponding half-life of 14 days will be considered here. The TGD proposes that 90 % of the sediment is anaerobic and suggests when only data is available for the anaerobic part correcting the half-life value in order to take into consideration the aerobic fraction of the sediment compartment. Therefore, taking into account the aerobic part of the sediment, only 45 % of the chloroform is biodegraded in 14 days and the actual half-life in the entire sediment is approximately 15 days. This value of 15 days will be used in the assessment for the sediment.

Key value for chemical safety assessment

Half-life in freshwater sediment:
14 d
at the temperature of:
283 K

Additional information

The anaerobic primary degradation of chloroform was studied by Gosset (1985) in batch studies with an inoculum based on municipal digested sludge at 35 °C. At a concentration of 5.1 mg/L, chloroform disappeared within 9 days. The main metabolite was dichloromethane (31%), which remained near constant for 21 days and then disappeared slowly over the remaining 60 days. The quantity of CH4 produced was negligible. Even at 1.7 mg/L, the gas production by the inoculum was inhibited by more than 60%, and by more than 80% at 17 mg/L. Bouwer et al. (1981) carried out a study on the degradation of chloroform with methanogenic bacteria over 112 days. At an initial concentration of 16 μg/L, 81 % of chloroform was degraded within two weeks. Degradation also occurred with initial concentrations of 34 μg/L (> 70% after 28 days) and 157 μg/L (43 % after 84 days). Degradation at the high concentration of 157 μg/L was less conclusive, but there appears to have been a gradual reduction in chloroform concentration. Removal percentages vary in an important way, as they are based on variable CHCl3 measurements in controls. Bouwer and McCarty (1983) found that in seeded cultures under methanogenic conditions, chloroform was almost completely oxidised to CO2. At initial concentrations of 15 and 40 μg/L a lag period of 40 and 20 days was observed respectively. 14C-measurements confirmed the removal by bio-oxidation. Rhee and Speece (1992) carried out a study with methanogenic bacteria under optimised conditions in a continuous fed anaerobic reactor. The feed contained a primary substrate (either formate, acetate or propionate) so as to maintain a concentration of 2000 mg/L of substrate in the reactor. The concentration of CHCl3 in the influent feed solution were 304, 1230 and 1960 mg/L in formate, acetate and proprionate enrichment cultures, respectively. The feed concentrations were chosen to produce a 50 % reduction in gas production. A degradation of 90, 89 and 93 % after 30 days of continuous operation was observed. The concentrations were monitored in the liquid and gas effluent. The removal by volatilisation was 6.2 - 10 % whereas the removal with the liquid effluent was < 0.08 %, corresponding to concentrations of <0.24, <0.98, <1.57 mg/L. Fathepure and Vogel (1991) determined a total decomposition of 83 % after two days in a sequential decomposition process in an anaerobic and aerobic column. A pre-adaptation of 4-6 weeks took place; the aerobic column was working for one year.

Mineralisation of chloroform mainly to CO2 was observed in a test carried out under anaerobic conditions and using muddy, methanogenic sediments (van Beelen and van Vlaardingen 1993). Half-lives between 2 and 37 days were observed at 20 °C. Mineralisation was also found at lower temperatures (10 °C). No mineralisation occurred in sandy sediment samples, which contained similar amounts of bacteria as the muddy sediments. The presence of organic matter in the muddy sediments seemed to be one important factor driving the mineralisation of chloroform. Another study testing the mineralisation of chloroform with methanogenic sediment samples at 10 °C or 20 °C (van Beelen and van Keulen 1990) came to the same conclusion, that chloroform is mineralised under anaerobic conditions resulting in the production of mainly CO2. The half-lives found in these tests were 10 -14 days at 10 °C and 2.6 days at 20 °C.

In conclusion, mineralisation of chloroform is likely to take place under environmental conditions in the anaerobic part of sediments with half-lives in the range of approximately 15 days. The most important degradation product is CO2. However, because no mineralisation occurred in the sandy sediment samples, it can be expected that chloroform may pass the sandy sediments of infiltrating rivers, thus polluting the groundwater.