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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Administrative data

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
The mineralization of chloroform in river sediments
Author:
van Beelen P, van Vlaardingen PLA
Year:
1993
Bibliographic source:
Netherlands Journal of Aquatic Ecology 27(1), 51-58

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroform
EC Number:
200-663-8
EC Name:
Chloroform
Cas Number:
67-66-3
Molecular formula:
CHCl3
IUPAC Name:
chloroform
Test material form:
liquid
Radiolabelling:
yes

Study design

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.

Results and discussion

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.
Half-life of parent compound / 50% disappearance time (DT50)
Key result
Compartment:
sediment
DT50:
> 0.9 - < 37 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Transformation products:
yes
Identity of transformation products
No.:
#1
Reference
Reference substance name:
Unnamed
IUPAC name:
dioxomethane
Inventory number:
InventoryMultipleMappingImpl [inventoryEntryValue=EC 204-696-9]
CAS number:
124-38-9
Identity:
Carbon dioxide
Molecular formula:
CO2
Molecular weight:
44.01
SMILES notation:
O=C=O
InChl:
InChI=1/CO2/c2-1-3
Evaporation of parent compound:
not measured
Volatile metabolites:
yes
Residues:
yes
Details on results:
See Table 3

Any other information on results incl. tables

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

Applicant's summary and conclusion

Validity criteria
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.