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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:
supporting study
Study period:
17 Mar - 10 Apr 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
quality criteria of guideline were not completely reached.
Qualifier:
according to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Version / remarks:
2002
Deviations:
yes
Remarks:
A minor short-term deviation (maximum deviation +0.3°C for 1 hour and 9 minutes) from the range occurred on day 23 of the incubation period. This was not considered to have had any impact on the results or the integrity of the study.
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of sediment:
Calwich Abbey Lake
- Details on collection: location: Staffordshire, UK (52° 59’ 6.4”N, 1° 48’ 38.3”W), sampling date: 19 Jun 2017, contamination history: no pesticide use in past 5 years, sampling depth: 5 cm
- Storage conditions: stored at approximately 4 °C
- Temperature (°C) at time of collection: 25 °C
- pH at time of collection: 8.27
- Oxygen concentration (mg/l): > 200%
- Total organic carbon (mg/L): water: 1.7 (post handling) - 8.94 (time 0), sediment: 3.7 (post handling) - 5.1 (time 0)
- Water filtered: yes, water was passed through a 0.2 mm sieve, sediment was passed through a 2 mm sieve

River Rhein
- Details on collection: location: Mumpf, Switzerland (47° 32’ 45.7”N, 7° 55’ 53.3”E), sampling date: 29 May 2017, contamination history: not known, sampling depth: not given
- Storage conditions: stored at approximately 4 °C
- Temperature (°C) at time of collection: 18.6 °C
- pH at time of collection: 8.13
- Oxygen concentration (mg/l): 9.5 mg/L
- Total organic carbon (mg/L): water: 2.3 (post handling) - 13.9 (day 30), sediment: 1.3 (post handling) - 1.83 (day 30)
- Water filtered: no, sediment was passed through a 2 mm sieve
Duration of test (contact time):
30 d
Initial conc.:
0.25 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
test mat. analysis
Details on study design:
TEST CONDITIONS
- Sediment volume of test solution/treatment: 84 g or 145 g, dry weight equivalent, of Calwich Abbey Lake or River Rhein sediment, respectively / 206 g of Calwich Abbey Lake sediment and 259 g of River Rhein sediment
- Water volume of test solution/treatment: 513 g from Calwich Abbey Lake water and 490 g from River Rhein water
- Test temperature: 20 °C
- pH: 7.1 - 8.8
- pH adjusted: no
- CEC (meq/100 g): 14.6 meq/100g (Calwich Abbey), 8.4 meq/100g (Rhein)
- Aeration of dilution water: yes, the test vessels were aerated through a dip tube immersed just below the surface of the water
- Suspended solids concentration: not measured (Calwich Abbey), 10 mg/L (Rhein)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: cylindrical 1-L glass bottles (diameter: approx. 9 cm) in an individual flow-through system
- Number of culture flasks/concentration: 24
- Method used to create aerobic conditions: teet system was aerated through a dip tube immersed just below the surface of the water (50 mL/min). Flow rates were checked and adjusted throughout the incubation period.
- Method used to control oxygen conditions: additional two vessels of each sediment type were set up for oxygen measurements
- Measuring equipment: not reported

- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: gas flow-through system
- Details of trap for CO2 and volatile organics if used: Test system included two vessels containing 2 N methanolic sodium hydroxide solution with phenolphthalein indicator (to trap 14CO2/14CS2)

SAMPLING
- Sampling frequency: after 0, 3, 8, 16, 24 hours and 2, 4, 7, 14 and 30 days of incubation
- Sampling method used per analysis type: Duplicate samples of each aquatic sediment were taken for analysis. One vessel, established for the determination of microbial activity, was taken for total viable plate count analysis on the day of application. Two vessels, one solvent treated and one untreated, were taken for analysis after 30 days of incubation.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: 5 untreated vessels of each aquatic sediment

STATISTICAL METHODS: Computer systems used for the generation, measurement and assessment of data included Laura (Radio HPLC data capture and evaluation), Debra (Metabolism LIMS system) and validated Excel spreadsheets. kinetic assessment was performed according to procedures and assessments
recommended in the FOCUS degradation kinetics guidance (2006, 2014) using the model KinGUI 2.1 (version 2.2014.224.1704). Single first-order (SFO), and bi-phasic models (FOMC, DFOP, HS) were evaluated. The kinetics were calculated from maximum % AR onwards and the application interval at this point was set to zero. Subsequent application intervals were adjusted accordingly.
Compartment:
natural water / sediment: freshwater
% Total extractable:
>= 15.9 - <= 18.2
% Non extractable:
>= 22.9 - <= 26.4
% CO2:
>= 0.1 - <= 0.3
% Other volatiles:
0.1
Remarks on result:
other: Calwich Abbey Lake / applied radioactivity
Remarks:
Day 30
Compartment:
natural water / sediment
% Total extractable:
>= 14.4 - <= 16.1
% Non extractable:
>= 15.2 - <= 25.1
% CO2:
>= 0.5 - <= 5.7
% Other volatiles:
0.1
Remarks on result:
other: River Rhein sediment / applied radioactivity
Remarks:
Day 30
Parent/product:
parent
Compartment:
total system
% Degr.:
100
Sampling time:
30 d
Remarks on result:
other: Calwich Abbey Lake and River Rhein aquatic sediment (Total System)
Compartment:
natural water / sediment
DT50:
0.24 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: Calwich Abbey Lake
Compartment:
natural water / sediment: freshwater
DT50:
0.57 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: River Rhein
Compartment:
natural water / sediment
DT50:
0.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: re-calculated to 12 °C using Arrhenius equation
Remarks:
Calwich Abbey Lake
Compartment:
natural water / sediment: freshwater
DT50:
1.2 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: re-calculated to 12 °C using Arrhenius equation
Remarks:
River Rhein
Transformation products:
yes
Remarks:
Only 2 transformation products were quantified in the report
No.:
#1
No.:
#2
Details on transformation products:
- Formation and decline of each transformation product during test: In the water phase of Calwich Abbey sediment, up to eighteen degradates were detected. Two were tentatively identified by co-chromatography as tetramethylthiuram monosulfide (up to 7.2% AR) and thiram (up to 38.0% AR). Two polar components were detected at levels of greater than 10%, component 1 (up to 18.4% AR) and component 3 (up to 23.5% AR). Mass spectrometry was used to confirm if these components could be identified from the reference standards provided but the attempts were not successful and the components were not identified. One component exceeded 5% AR on two consecutive occasions: component 10 accounted for a maximum of 6.2% AR. Up to 13 minor unidentified components were also detected and accounted for individual maxima of 3.9% AR. In the sediment extracts of Calwich Abbey sediment up to six degradates were detected. Two were tentatively identified by co-chromatography as tetramethylthiuram monosulfide (up to 4.2% AR) and thiram (up to 3.5% AR). Up to 4 minor unidentified components were also detected and accounted for individual maxima of 7.2% AR.
In the water phase of River Rhein sediment, up to eighteen degradates were detected. Two were tentatively identified by co-chromatography as tetramethylthiuram monosulfide (up to 9.1% AR) and thiram (up to 36.9% AR). Two polar components were detected at levels of greater than 10%, component 1 (up to 29.8% AR) and component 3 (up to 21.1% AR).
Identification using authentic references was attempted but was unsuccessful. One other degradate, component 9, accounted for a maximum of 10.4% in one replicate on one occasion and then declined. One component exceeded 5% AR on two consecutive occasions: component 2 accounted for a maximum of 6.7% AR. These were not identified. Up to 12 minor unidentified components were also detected and accounted for individual maxima of 3.5% AR. In the sediment extracts of River Rhein sediment up to ten degradates were detected. Two were tentatively identified by co-chromatography as tetramethylthiuram monosulfide (up to 2.1% AR) and thiram (up to 3.1% AR). The polar degradate, component 1, was detected at levels of up to 11.0% AR but was not identified. Up to seven minor unidentified components were also detected and accounted for individual maxima of 7.2% AR.

- Pathways for transformation: The target substance was degraded via cleavage and oxidation to a number of degradates, including thiram and tetramethylthiuram monosulfide, and finally incorporation into bound residues and mineralisation to carbon dioxide.

- Maximum occurrence of each transformation product: Thiram: 38.3% AR (8.0% AR (day 0.13, total system, Calwich Abbey Lake) and 40.0% AR (day 0.33, total system, River Rhein), tetramethylthiuram monosulfide: 8.0% AR (day 7, total system, Calwich Abbey Lake), 9.1% AR (day 7, total system, River Rhein)
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
not measured
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: Thiram: 34.1 (day 0), 40.0 (day 0.33), 20.1 (day 0.67), 12.5 (day 2) in River Rhine and 28.8 (day 0), 33.3 (day 0.13), 38.3 (day 0.33), 29.3 (day 0.67), 20.1 (day 1), 29.5 (day 2), 6.1 (day 4) in Calwich Abbey Lake, tetramethylthiuram monosulfide: 2.4 (day 0.67), 3.7 (day 2 and 4), 6.3 (day4), 9.1 (day 7) in River Rhine; 0.7 (day 1), 6.3 (day 4), 8.0 (day 7), 5.1 (day 14) in Calwich Abbey Lake
- Range of maximum concentrations in % of the applied amount at end of study period: thiram: 2.2 Calwich Abbey Lake and River Rhein, tetramethylthiuram monosulfide: 3.4 in River Rhein and 6.7 in Calwich Abbey Lake

MINOR TRANSFORMATION PRODUCTS
not specified

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.4 - 0.7 (Calwich Abbey Lake) and 0.6 - 1.1 (River Rhein)
- % of applied amount at end of study period: 15.9 - 18.2 (Calwich Abbey Lake) and 14.4 - 16.1 (River Rhein)

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.4 - 0.7 (Calwich Abbey Lake) and 1.9 -2.2 (River Rhein)
- % of applied amount at end of study period: 22.9 - 26.4 (Calwich Abbey Lake) and 15.2 - 17.0 (River Rhein)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 0.1 - 0.3 (Calwich Abbey Lake) and 0.5 - 5.7 (River Rhein)
VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: max. 0.1

Both aquatic sediments were microbiologically active throughout the incubation period and there were no appreciable differences in levels of the various organisms at Day 30 between untreated samples and samples treated with solvent.

Table 1: Distribution and recovery of radioactivity in Calwich Abbey Lake aquatic sediment

 

Time after application

0 hours

24 hours

7 days

14 days

30 days

CA01

CA02

CA10

CA22

CA15

CA16

CA17

CA18

CA19

CA20

Water

99.3

106.2

55.8

68.3

46.4

55.3

36.9

50.2

29.2

26.7

Sediment

 

 

 

 

 

 

 

 

 

 

Extract 1

0.6

0.3

7.7

6.3

9.5

9.2

9.8

8.2

12.2

10.6

Extract 2

-

-

2.5

2.2

3.3

3.2

3.2

2.6

3.8

3.2

Extract 3

-

-

1.8

1.5

2.0

1.9

1.6

1.2

1.5

1.4

Extract 4

0.1

0.1

1.0

0.8

0.8

0.7

1.0

0.7

0.7

0.7

Total extractable

0.7

0.4

13.0

10.8

15.6

15.0

15.6

12.7

18.2

15.9

Non-extractable

0.7

0.4

23.6

18.4

27.6

21.0

30.4

10.7

22.9

26.4

Total in sediment

1.4

0.8

36.6

29.2

43.2

36.0

46.0

23.4

41.1

42.3

Organics

-

-

nd

nd

nd

nd

nd

nd

nd

0.1

CO2

-

-

0.4

0.4

1.1

1.0

0.4

0.1

0.1

0.3

Total recovery

100.7

107.0

92.8

97.9

90.7

92.3

83.3

73.7

70.4

69.4

Results are expressed as % applied radioactivity

- :     not applicable

nd:      not detected

Extracts 1 - 3: acetonitrile:buffer (2:1, v/v), buffer: 0.01M NaH2PO4 + 0.01M Na2HPO4 at pH 9, ambient temperature Extract 4: acetonitrile, ambient temperature

Table 2: Distribution and recovery of radioactivity in River Rhein aquatic sediment

 

Time after application

0

24 hours

7 days

14 days

30 days

RH01

RH02

RH09

RH10

RH15

RH16

RH17

RH18

RH19

RH20

Water

103.5

105.1

50.9

72.3

67.0

68.4

39.1

39.3

29.7

24.6

Sediment

 

 

 

 

 

 

 

 

 

 

Extract 1

0.9

0.5

8.9

6.0

7.3

6.5

11.3

11.0

11.2

9.9

Extract 2

-

-

3.0

1.9

1.9

1.9

2.7

3.0

3.1

2.8

Extract 3

-

-

1.7

1.0

0.9

0.8

1.0

1.3

1.3

1.2

Extract 4

0.2

0.1

0.9

0.5

0.3

0.3

0.4

0.6

0.5

0.5

Total extractable

1.1

0.6

14.5

9.4

10.4

9.5

15.4

15.9

16.1

14.4

Non-extractable

2.2

1.9

24.9

12.2

8.1

7.7

11.9

17.0

15.2

25.1

Total in sediment

3.3

2.5

39.4

21.6

18.5

17.2

27.3

32.9

31.3

39.5

Organics

-

-

nd

nd

nd

0.1

nd

nd

0.1

nd

CO2

-

-

1.1

0.8

4.1

1.8

3.7

7.3

0.5

5.7

Total recovery

106.8

107.6

91.4

94.7

89.6

87.5

70.1

79.5

61.6

69.8

Results are expressed as % applied radioactivity

- : not applicable

nd: not detected

Extracts 1 - 3: acetonitrile:buffer (2:1, v/v), buffer: 0.01M NaH2PO4 + 0.01M Na2HPO4 at pH 9, ambient temperature Extract 4: acetonitrile, ambient temperature

Table 3: Proportions of radioactive components in Calwich Abbey Lake aquatic sediment (Total system)

Component

Retention time (minutes)

Time after application (days)

0

0.13

0.33

0.67

1

2

4

7

14

30

Target substancea

8.0

31.1

21.0

17.9

17.0

10.2

13.3

2.4

-

-

-

Component 1

3.5

13.9

14.8

14.4

18.9

18.7

17.2

19.7

25.6

18.4

9.9

Component 2

4.8

-

-

2.5

-

-

-

2.8

-

-

-

Component 3

5.0

20.6

23.5

17.9

15.7

13.7

18.7

9.5

13.3

8.5

3.3

Component 4

5.6

-

-

-

-

-

-

-

-

2.6

3.2

Component 5

7.0

-

-

-

-

2.2

2.3

2.5

1.0

0.8

0.8

Component 6

8.5

-

 

-

-

-

-

2.5

2.6

-

-

Component 7

9.4

-

-

-

-

-

-

-

-

3.0

2.6

Component 8

12.6

-

-

-

-

-

-

-

-

0.4

0.3

Component 10

15.7

-

-

-

-

-

-

1.1

4.2

5.5

6.2

Component 11

17.4

-

-

-

-

-

-

-

0.9

1.0

3.8

Component 12

18.5

-

-

0.5

1.6

4.3

0.7

3.2

4.2

3.2

2.0

Component 13

20.6

-

-

-

-

0.7

-

-

-

0.8

0.3

Component 14

23.6

0.4

-

-

-

-

0.5

-

-

0.1

-

Tetramethylthiuram monosulfide

24.2

-

-

-

-

0.7

2.2

6.3

8.0

5.1

6.7

Thiram

25.4

28.8

33.3

38.3

29.3

20.1

29.5

6.1

2.5

3.1

2.2

Component 16

27.3

1.7

1.5

3.9

1.3

0.5

0.6

-

-

0.3

0.2

Component 17

28.2

0.2

-

0.6

-

-

-

-

-

-

-

Others

 

6.3

4.5

2.4

5.3

3.2

3.2

4.3

4.1

5.3

3.8

Results are expressed as mean % applied radioactivity.

a: data are from ionpair HPLC analysis, all others are from reverse phase HPLC analysis

-: not detected

Others: radioactivity distributed throughout regions of the chromatogram other than those specified and which did not contain any discrete radioactive components

Table 4: Proportions of radioactive components in River Rhein aquatic sediment (Total system)

 

Component

Retention time (minutes)

Time after application (days)

0

0.13

0.33

0.67

1

2

4

7

14

30

Zirama

8.0

27.8

21.2

18.0

9.1

5.4

5.4

3.5

-

-

-

Polar

2.8

-

-

-

-

-

-

-

-

0.6

0.6

Component 1

3.5

15.6

19.4

21.9

21.1

25.7

27.4

33.2

27.7

18.5

11.0

Component 2

4.8

-

-

1.4

-

-

3.2

-

0.6

2.7

6.7

Component 3

5.0

19.4

21.1

17.5

15.2

14.4

16.6

16.2

15.5

11.6

2.8

Component 4

5.6

-

-

-

-

-

1.5

1.0

-

3.2

3.5

Component 5

7.0

-

-

-

0.6

2.2

2.8

2.2

0.6

0.7

0.8

Component 7

9.4

-

-

-

-

-

-

-

-

1.2

1.2

Component 7a

10.0

-

-

-

-

-

-

-

0.7

0.4

0.3

Component 9

14.5

-

-

-

-

-

0.9

3.6

9.7

3.3

3.9

Component 10

15.7

-

-

-

-

-

-

-

-

0.3

-

Component 11

17.4

-

-

-

-

-

-

1.7

6.4

0.9

2.5

Component 12

18.5

-

0.1

1.0

2.4

7.2

1.7

-

1.1

-

-

Component 13

20.6

-

-

-

1.1

0.6

0.2

-

-

0.2

0.2

Component 14

23.6

-

-

-

-

-

-

-

-

-

0.1

Tetramethylthiuram monosulfide

24.2

-

-

-

2.4

3.7

3.7

6.3

9.1

2.2

3.4

Thiram

25.4

34.1

35.2

40.0

20.1

8.8

12.5

3.2

1.1

2.4

2.2

Component 16

27.3

1.1

1.6

2.2

0.5

0.3

-

-

-

-

-

Component 18

31.5

-

0.5

-

-

-

-

-

-

-

-

Others

 

6.4

7.0

2.8

6.8

5.6

4.5

4.4

5.2

7.0

3.7

Results are expressed as mean % applied radioactivity.

a: data are from ion pair HPLC analysis, all others are from reverse phase HPLC analysis

-: not detected

Others: radioactivity distributed throughout regions of the chromatogram other than those specified and which did not contain any discrete radioactive components

Table 5: Summary of components in aquatic sediment systems (Calwich Abbey)

 

Ziram

Water

Sediment

Total System

Max. in Total System (mean)

Rule

Component 1

Yes

Yes

Yes

25.6, 7d

>10%, 2× >5%

Component 2

-

-

-

-

 

Component 3

Yes

-

Yes

23.5, 3h

>10%

Component 4

-

-

-

-

 

Component 5

-

-

-

-

 

Component 6

-

-

-

-

 

Component 7

-

-

-

-

 

Component 8

-

-

-

-

 

Component 10

Yes

-

Yes

6.2, 30d

>5% at end

Component 11

-

-

-

-

 

Component 12

-

-

-

-

 

Component 13

-

-

-

-

 

Component 14

-

-

-

-

 

Tetramethylthiuram monosulfide

-

-

Yes

8.0, 7d

2× >5%

Thiram

Yes

-

Yes

38.3, 8h

>10%

Component 16

-

-

-

-

 

Component 17

-

-

-

-

 

Table 6: Summary of components in aquatic sediment systems (River Rhein)

Ziram

Water

Sediment

Total System

Max. in Total System (mean)

Rule

Polar

-

-

-

-

 

Component 1

Yes

Yes

Yes

33.2, 4 days

>10%

Component 2

Yes

-

Yes

6.7, 30 days

>5% at end

Component 3

Yes

-

Yes

21.1, 3 hours

>10%

Component 4

-

-

-

-

 

Component 5

-

-

-

-

 

Component 7

-

-

-

-

 

Component 7a

-

-

-

-

 

Component 9

-

-

-

-

 

Component 10

-

-

-

-

 

Component 11

-

-

-

-

 

Component 12

-

-

-

-

 

Component 13

-

-

-

-

 

Component 14

-

-

-

-

 

Tetramethylthiuram monosulfide

Yes

-

Yes

9.1, 7 days

2× >5%

Thiram

Yes

-

Yes

40.0, 8 hours

>10%

Component 16

-

-

-

-

 

Component 18

-

-

-

-

 

Table 7: Quality criteria for OECD 308.

Criterion from the guideline

Outcome

quality criterion fulfilled

Recoveries should

range from 90% to 110% for labelled chemicals and from 70% to 110% for non-labelled chemicals.

Total recoveries of radioactivity (mass balances) were between 61.6% and 119.0% applied

radioactivity.

No


Validity criteria fulfilled:
no
Remarks:
For more details refer to field "any other information on results incl. tables".
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Feb 2014 - 24 Feb 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
a solvent was used for the study
Qualifier:
according to guideline
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Remarks:
pond known not to be subjected to discharges of effluent
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): location: pond in Biederthal, France (47.4702°N / 7.4341°E), sampling date: 04 Sep 2014,
- Temperature (°C) at time of collection: 20 °C
- pH at time of collection: 8.03
- Redox potential (mv): 278
- Oxygen concentration (mg/l): 9.21
- Dissolved organic carbon (%): 7.24
- Total organic carcon (mg/L): 7.24
- Ammonium (mg/L): 0.28
- Nitrate (mg/L): 0.80
- Redox potential (Eh): 137
- Water filtered: no
Duration of test (contact time):
30 d
Initial conc.:
100 µg/L
Based on:
test mat.
Initial conc.:
10 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
test mat. analysis
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 100 mL
- Solubilising agent: acetone (concentration not reported)
- Test temperature: 21.1 ± 0.1 °C
- pH: 8.23 - 8.37
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 300 mL all-glass Erlenmeyer flasks
- Number of culture flasks/concentration: 2 except for high dose sterile (1 flask)
- Method used to create aerobic conditions: Flask were aerated with moistened air pushed through the system and agitation facilitated oxygen transfer from headspace to liquid, in a way that aerobic conditions were maintained.

- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: Flasks were connected to a volatile trapping system. The exhaust air was pushed through three absorption traps, one containing ethylene glycol (50 mL)
and the others 2 N NaOH/methanol (1:1; v:v) (50 mL), in this sequence per flask, to trap organic volatiles and to trap 14CO2 and 14CS2, respectively

SAMPLING
- Sampling frequency: Water samples were taken immediately after treatment (day 0) and after 0.25, 1, 2, 5, 15 and 30 days of incubation. Trapping solutions of samples were taken on the corresponding sampling day. In addition, the 2 N NaOH/methanol trapping solutions were exchanged once for samples incubated for 15 days, and twice for samples incubated for 30 days. Degradation of [14C(U)]benzoic acid was monitored in order to test the microbial activity of the test water (control and solvent control). The same experimental set-up was used, except for the volatile trapping system. For samples treated with the reference test item, the exhaust air was only pushed through two absorption traps. The first contained ethylene glycol (50 mL) and the second 2 N NaOH (50 mL) to trap organic volatiles and 14CO2, respectively. Additionally, two solvent blanks were used to measure physico-chemical water parameters.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: not performed
- Abiotic sterile control: 1 high dose sterile control (Sterile samples, flasks containing surface water were autoclaved for 15 minutes at 121 °C and a pressure of about 106 kPa (Compact 40 Benchtop, Priorclave, UK))
- Toxicity control: not performed

STATISTICAL METHODS: The degradation rate of the test item in surface water was calculated using CAKE software according to FOCUS Kinetics Guidance (2006). Input data sets for modelling were derived from individual data for each time-point. Data only up to the interval where parent was no longer detected are included in the kinetic evaluations. All subsequent intervals were not considered.
Reference substance:
other: [14C(U)]Benzoic acid
Compartment:
natural water: freshwater
% CO2:
< 31.5
% Other volatiles:
2.9
% Recovery:
96.1
Remarks on result:
other: % applied radioactivity / sampling time: 30 d
Remarks:
high dose level
Compartment:
natural water: freshwater
% CO2:
< 9.5
% Other volatiles:
< 0.1
% Recovery:
104.7
Remarks on result:
other: % applied radioactivity / sampling time: 30 d
Remarks:
high dose level, sterile / Recovery: 104.7% AR
Compartment:
natural water: freshwater
% CO2:
56.9
% Other volatiles:
1
% Recovery:
100.5
Remarks on result:
other: % applied radioactivity / sampling time: 30 d
Remarks:
low dose level, recovery: 100.5% AR
Parent/product:
parent
Compartment:
water
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
30 d
Compartment:
natural water: freshwater
DT50:
2.47 d
Type:
(pseudo-)first order (= half-life)
Temp.:
21.1 °C
Remarks on result:
other: high dose
Compartment:
natural water: freshwater
DT50:
1.53 d
Type:
(pseudo-)first order (= half-life)
Temp.:
21.1 °C
Remarks on result:
other: high dose, sterile
Compartment:
natural water: freshwater
DT50:
5.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: high dose
Remarks:
re-calculated to 12 °C using Arrhenius equation
Compartment:
natural water: freshwater
DT50:
3.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: high dose, sterile
Remarks:
re-calculated to 12 °C using Arrhenius equation
Transformation products:
not specified
Remarks:
For details please refer to field "Details on transformation products".
Details on transformation products:
- Formation and decline of each transformation product during test: At the high dose level, the test item degraded into the five major radioactive fractions M1, M9, M13, M14 and M22 with maximum occurrences of 16.5% (day 30), 11.7%, (day 15) 14.8%, (day 15), 14.5% (day 0) and 38.2% AR (day 30), respectively. Furthermore, three minor radioactive fractions were observed, reaching maximum concentrations of 3.6% AR (M2 on sampling day 1), 6.0% AR (M15, only detected on sampling day 0.25) and 6.7% AR (M17 on sampling day 5). In sterile water treated at the high dose level, the four major radioactive fractions detected were M2, M14, M15 and M22, and which reached maximum occurrences of 28.1% (day 30), 23.0% (day 1), 17.3% (day 0.25) and 73.4% AR on day 15, respectively. In addition, four minor radioactive fractions were observed, reaching maximum concentrations of 9.6% AR (M1 on sampling day 30), 4.0% AR (M9 on sampling day 2), 5.0% AR (M17, only detected on sampling day 5) and 3.9% AR (M20, only detected on sampling day 30).
Evaporation of parent compound:
no
Volatile metabolites:
yes
Remarks:
CO2, CS2
Residues:
no
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Formation of volatile radioactivity trapped in the sodium hydroxide/methanol solutions increased over time, reaching a maximum of 31.5% AR (high dose), 9.5% AR (high dose, sterile) and 56.9% AR (low dose) on day 30. The vast majority of radioactivity trapped in the sodium hydroxide/methanol solutions could be precipitated with barium hydroxide and therefore attributed to 14CO2. Radioactivity remaining in the supernatants after precipitation never exceeded 1.1% AR, and was therefore not further characterised.

STERILE TREATMENTS
- Transformation of the parent compound: In sterile water treated at the high dose level, the four major radioactive fractions detected were M2, M14, M15 and M22, and which reached maximum occurrences of 28.1% (day 30), 23.0% (day 1), 17.3% (day 0.25) and 73.4% AR on day 15, respectively. In addition, four minor radioactive fractions were observed, reaching maximum concentrations of 9.6% AR (M1 on sampling day 30), 4.0% AR (M9 on sampling day 2), 5.0% AR (M17, only detected on sampling day 5) and 3.9% AR (M20, only detected on sampling day 30).
- Volatilization: 9.5% AR
Results with reference substance:
A fast mineralisation was observed in control as well as solvent control samples, reaching 49.0% and 79.4% of applied radioactivity after 14 days of incubation. Furthermore,
a fast degradation of the reference test item was confirmed by HPLC analysis. After 7 days of incubation, [14C(U)]benzoic acid was no longer detectable in the surface water of control, as well as solvent control samples. The results confirmed a sufficient microbial activity of the surface water.

Table 1: Mineralisation of the reference test item [14C(U)]benzoic acid in surface water (control). Values in percent of applied radioactivity.

Biederthal

 

[% AR]

 

Replicate

INCUBATION TIME IN DAYS

7

14

 

Aqueous phase

A

B

23.0

28.4

42.6

21.1

Mean

25.7

31.9

 

14CO2

A

B

57.5

47.7

37.7

60.3

Mean

52.6

49.0

 

Other volatiles in EG

A

B

0.2

0.1

<0.1

<0.1

Mean

0.2

<0.1

 

T O T A L

A

B

80.7

76.3

80.3

81.5

Mean

78.5

80.9

MEAN ± SD

 

79.7 ± 2.3

Table 2: Mineralisation of the reference test item [14C(U)]benzoic acid in surface water (solvent control). Values in percent of applied radioactivity.

Biederthal

 

[% AR]

 

Replicate

INCUBATION TIME IN DAYS

7

14

 

Aqueous phase

A

B

28.7

28.9

14.4

18.6

Mean

28.8

16.5

 

14CO2

A

B

71.2

71.3

85.9

72.9

Mean

71.3

79.4

 

Other volatiles in EG

A

B

0.1

0.1

0.1

<0.1

Mean

0.1

0.1

 

T O T A L

A

B

100.0

100.3

100.3

91.6

Mean

100.1

96.0

MEAN ± SD

 

98.0 ± 4.3

The mass balance of the control samples treated with the reference test item were <90% of applied radioactivity. A possible explanation is loss of volatile radioactivity (for example present in the sample head space or dissolved in the aqueous phase) during sample processing. But even if the missing radioactivity would be attributed as parent reference test item, the microbial activity could still be assessed as sufficient, since about 80% of applied reference test item were either degraded or mineralised.

Table 3: Balance of radioactivity in surface water following application of radiolabelled test item (high dose).Values in percent of applied radioactivity.

Biederthal

 

 

[% AR]

 

Replicate

INCUBATION TIME IN DAYS

0

0.25

1

2

5

15

30

 

Aqueous phase

A

B

97.4

97.4

98.7

99.3

97.9

93.7

100.7

100.4

76.6

81.7

63.1

70.8

57.1

66.4

Mean

97.4

99.0

95.8

100.5

79.2

66.9

61.8

Volatiles in NaOH/methanol

A

B

n.p.

n.p.

<0.1

<0.1

0.1

0.1

<0.1

0.4

0.4

13.9

22.8

12.1

32.9

30.1

Mean

n.p.

<0.1

0.1

0.2

7.2

17.4

31.5

 

Volatiles in EG

A

B

n.p.

n.p.

<0.1

<0.1

0.1

0.2

0.1

0.1

<0.1

0.2

0.8

0.2

3.4

2.3

Mean

n.p.

<0.1

0.1

0.1

0.1

0.5

2.9

 

T O T A L

A

B

97.4

97.4

98.7

99.3

98.1

93.9

100.8

100.8

77.0*

95.8

86.7*

83.0*

93.5

98.8

Mean

97.4

99.0

96.0

100.8

86.4

84.8

96.1

MEAN ± SD

 

94.4 ± 7.2

Table 4: Balance of radioactivity in surface water following application of radiolabelled test item (high dose, sterile).Values in percent of applied radioactivity.

Biederthal

 

 

[% AR]

INCUBATION TIME IN DAYS

0

0.25

1

2

5

15

30

Aqueous phase

97.6

100.6

95.5

104.0

102.4

98.5

95.2

Volatiles in NaOH/methanol

n.p.

0.2

0.1

2.4

4.7

5.8

9.5

Volatiles in EG

n.p.

<0.1

0.1

<0.1

<0.1

<0.1

<0.1

T O T A L

97.6

100.8

95.6

106.4

107.1

104.3

104.7

MEAN ± SD

102.4 ± 4.4

Table 5: Balance of radioactivity in surface water following application of radiolabelled test item (low dose).Values in percent of applied radioactivity.

Biederthal

 

 

[% AR]

 

Replicate

INCUBATION TIME IN DAYS

0

0.25

1

2

5

15

30

 

Aqueous phase

A

B

101.3

98.4

99.3

98.0

92.6

102.8

110.0

91.5

103.4

80.8

57.0

53.0

45.2

40.1

Mean

99.8

98.6

97.7

100.8

92.1

55.0

42.6

Volatiles in NaOH/methanol

A

B

n.p.

n.p.

1.1

<LOD

<LOD

2.8

<LOD

<LOD

<LOD

26.5

42.5

46.2

56.7

57.0

Mean

n.p.

0.6

1.4

<LOD

13.2

44.4

56.9

 

Volatiles in EG

A

B

n.p.

n.p.

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

1.5

<LOD 1.5

0.6

1.4

Mean

n.p.

<LOD

<LOD

<LOD

0.7

0.7

1.0

 

T O T A L

A

B

101.3

98.4

100.5

98.0

92.6

105.6

110.0

91.5

103.4

108.8

99.5

100.6

102.6

98.4

Mean

99.8

99.2

99.1

100.8

106.1

100.1

100.5

MEAN ± SD

 

100.8 ± 5.2

Table 6: Validity criteria for OECD 309.

Criterion from the guideline

Outcome

Validity criterion fulfilled

Reference substance should be degraded within the expected time interval ((for aniline and

sodium benzoate, usually less than two weeks).*

After 7 days of incubation, [14C(U)]benzoic acid was no longer detectable in the surface water of control, as well as solvent control samples.

yes

The total recovery (mass balance) at the end of the experiment should be between 90% and 110%

for radiolabelled substances, whereas the initial recovery at the beginning of the experiment should be

between 70% and 110% for non-labelled substances.**

The total mean recoveries were 94.4 ± 7.2% AR (high dose), 102.4 ± 4.4% AR (high dose,

sterile) and 100.8 ± 5.2% AR (low dose), respectively.

yes

Validity criteria fulfilled:
yes
Remarks:
For details refer to field "Any other information on results incl. tables"
Conclusions:
The test item degraded in natural surface water with a half-live of 2.5 days, and with a half-live of 1.5 days in sterilised water. The fast degradation of the test item was observed for biotic as well as sterilised surface water and indicated that abiotic degradation was the main degradation process. At the high dose level, the test item degraded into five major (M1, M9, M13, M14 and M22) and three minor (M2, M15 and M17) radioactive fractions. The highest mineralization to CO2 after 30 days was < 31.5% AR (high dose).
Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 October 1999 - 11 June 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
very long acclimation time of 10 weeks
Qualifier:
according to guideline
Guideline:
other: European Community Commission Directive 95/36/EC, para. 7.2.1.3.2. This method corresponds to that later included in the directive 68/548: C.24 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Version / remarks:
1995
Deviations:
no
Principles of method if other than guideline:
Guideline based on SETAC (Europe), Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995
GLP compliance:
yes (incl. QA statement)
Remarks:
The Swiss GLP Monitoring Authorities
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
- Details on collection: water and sediment were collected from two different locations (Rhein river, Rheinsulz AG/Switzerlandand and a pond, Ormalingen BL, Switzerland). The pond is located far from areas of human activity and it was not exposed to any effluent discharges. This was not however the case of the river. The water was collected at a depth of 10-30 cm.
- Storage conditions: 4°C
- Storage length: 1 week before start of acclimation period
- Water was passed through a 0.2 mm sieve and its parameters measured:

River
Temperature: 17.5-17.7°C
pH: 8.18-8.20
Redox potential: 200 mV
Oxygen content: 10-10.6 mg O2/L
NO2 N: 0.03 mg/L
NO3 N: 1.45 mg/L
NH4 N: 0.05 mg/L
Total Organic Carbon (TOC): 2.5 mg C/L
Hardness: 13

Pond
Temperature: 10.7-11.6°C
pH: 7.02-7.29
Redox potential: 130-200 mV
Oxygen content: 7.1-10.9 mg O2/L
NO2 N: < detection limit
NO3 N: 1.70 mg/L
NH4 N: 0.09 mg/L
Total Organic Carbon (TOC): 3.6 mg C/L
Hardness: 24
Details on source and properties of sediment:
- Details on collection: water and sediment were collected from two different locations (Rhein river, Rheinsulz AG/Switzerlandand and a pond, Ormalingen BL, Switzerland). The pond is located far from areas of human activity and it was not exposed to any effluent discharges. This was not however the case of the river. The sediment was sampled from the top 5-10 cm.
- Storage conditions: 4°C
- Storage length: 1 week before start of acclimation period
- Sediment was passed through a 2.0 mm sieve and it was allowed to settle before its parameters were determined:

River
pH at time of collection: 7.83
Organic carbon (g carbon/g dry sediment): 0.97
CEC (meq/100 g): 75.9 mVal/kg dry sediment
Redox potential (mv) initial/final: -36 mV
Microbial mass: 0.14 g microbial C/kg dry sediment
Dry weight (g wet sediment/g dry sediment): 1.23

Pond
pH at time of collection: 7.61
Organic carbon (g carbon/g dry sediment): 6.13
CEC (meq/100 g): 215.12 mVal/kg dry sediment
Redox potential (mv) initial/final: -79 mV
Microbial mass: 0.32 g microbial C/kg dry sediment
Dry weight (g wet sediment/g dry sediment): 2.32

- other: The sediment microbial biomass was determined using a modification of the respiration method reported by Anderson and Domsch (1978). The sieved sediments were mixed with seasand to facilitate aeration. Thereafter, subsamples were amended with increasing amounts of glucose and submitted for respiratory measurements, which were used to determine the microbial biomass of the soil. Aliquots of the mixture sediment/sand were packed into all-glass columns and measured semi-continuously by means of an IR-gas-analyser (UNOR 6N, Maihak/Germany) for a minimum of 4 hours. The total volume of CO2 evolved during approximately one hour, was calculated. For the calculations, the values which corresponded to a maximum initial and constant CO2-production, were used. The value obtained was extrapolated to 100 g dry sediment.
Duration of test (contact time):
101 d
Initial conc.:
0.29 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: Sediment was added to a depth of about 2.5-3.5 cm, corresponding to a wet weight of 200 g for river and 250 g for pond sediment. The respective water of 600 ml: river and 550 ml: pond, was added to the sediment to reach a depth of about 6 cm.
- Test temperature: 20+/-1°C
- Aeration of water: yes
- Continuous darkness: yes
- Other: The test systems were acclimated for 10 weeks before treatment at 20°C and in the dark. Thereafter, the radiolabelled test item was applied to the water (dropwise evently on the surface) at the target concentration of 0.29 mg a.i/L, corresponding to an application rate of 1.6 kg a.i/ha, assuming an uniform substance distribution within a 30 cm water layer. After treatment, the samples were connected to the flow-through system (adjusted to 60-80 mL/minute). During the entire incubation period, aeration was provided but the sediment remained undisturbed.

TEST SYSTEM
- Culturing apparatus: 1 L glass metabolism flasks (inner diameter: about 10.6 cm, area: about 88.2 cm^2). The flasks were connected to a trap containing only water during the equilibration.
- Number of culture flasks/concentration: 14 (8 sampling intervals (no duplicates) and 6 reserves)

- Test performed in closed vessels due to significant volatility of test substance:
- Details of trap for CO2 and volatile organics if used: After treatment with the test item the flasks were connected to a series of four traps: two traps containing 50 mL 2N KOH or NaOH/Methanol followed by one trap containing 50 mL ethylene glycol, and a fourth trap containing 50 mL 0.1N sulphuric acid.

SAMPLING
- Sampling frequency: Samples were taken at days 0, 1, 5, 7, 14, 31, 60 and 101. HPLC was used as primary method for quantification of the parent molecule in samples taken on day 0, 1 and 5.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Other: Untreated reserve samples to determine the microbial biomass at the beginning and at the end of the study and for the determination of the physical-chemical characteristics during equilibration and incubation.
Compartment:
natural water / sediment: freshwater
% Total extractable:
2.1
% Non extractable:
19.2
% CO2:
82.3
% Other volatiles:
1.9
% Recovery:
104.7
Remarks on result:
other: River, at study end (Day 101)
Compartment:
natural water / sediment: freshwater
% Total extractable:
4.5
% Non extractable:
37.8
% CO2:
47.3
% Other volatiles:
1.3
% Recovery:
90
Remarks on result:
other: Pond, at test end (Day 101)
Parent/product:
parent
Compartment:
total system
Remarks:
water/sediment system
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
7 d
Remarks on result:
other: River
Parent/product:
parent
Compartment:
total system
Remarks:
water/sediment system
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
1 d
Remarks on result:
other: Pond
Compartment:
other: water and whole system (River)
DT50:
0.3 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT90 = 1d
Compartment:
other: water and whole system (Pond)
DT50:
0.2 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT90 = 0.7d
Compartment:
other: water and whole system (River)
DT50:
0.6 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: re-calculated to 12 °C using Arrhenius equation
Compartment:
other: water and whole system (Pond)
DT50:
0.4 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: re-calculated to 12 °C using Arrhenius equation
Transformation products:
yes
Remarks:
For more details please refer to field "Details on transformation products" and attached document.
No.:
#1
Details on transformation products:
- Formation and decline of each transformation product during test: In the river system, the parent compound was degraded to more than 13 components. The major component was Thiram (M1) from day 0 until day 14. In the pond system the parent compound degraded to more than 12 components. Thiram was again the major transformation product. I reached a maximum of 47.5% after 6 h and desappeared after 14 d. After 101 days, Ziram degraded mainly to CO2. For more details please refer to attached document.
Evaporation of parent compound:
no
Volatile metabolites:
yes
Remarks:
CO2 and CS2
Residues:
yes
Details on results:
TEST CONDITIONS
- Aerobicity in water, temperature and other experimental conditions maintained throughout the study: River: Yes (pH: 8.16 ± 0.08, O2: 7.4 ± 1.0 mg/L, redox potential: 222 ± 14 mV), Pond: Yes (pH: 7.90 ± 0.06, O2: 5.9 ± 0.6 mg/L, redox potential: 220 ± 10 mV)

MAJOR AND MINOR TRANSFORMATION PRODUCTS
- Please refer to attached document.

The microbial biomass determined for the river sediment was 0.14 g microbial C/kg dry sediment at start of the incubation time and remained constant until the end of incubation.

The microbial biomass determined for the pond sediment was 0.32 g microbial C/kg dry sediment at start of the incubation time and remained constant at 0.29 g microbial C/kg until the end of incubation.

 

Total mean recoveries obtained during the whole incubation period were 100% +/- 3.4% and 95.3% +/-3.7% of the applied radioactivity in the pond and the river respectively.

 

Ziram was rapidly degraded to thiram and further to the metabolites M6, M8 and M9. Ziram is extensively converted to bound residue (19-38 % AR after 101 d, at 20°C) and mineralized to carbon dioxide (47-82% AR after 101 d, at 20°C). Thiram is the major metabolite. Metabolites M6 (molecular weight : 176), M8 and M9 are further major metabolites (total level of 10-13% AR in the water+sediment extracts at days 1 to 14). Detailed investigations were made by LC/MS in order to characterize these unknown fractions. It cannot be excluded that M6, M8 M9 consist each of several fractions. The possible structure of M6 would be (CH3)2 -N-CO-S-CO-N-(CH3)2 .

Table 1: Quality criteria for OECD 308.

Criterion from the guideline

Outcome

quality criterion fulfilled

Recoveries should

range from 90% to 110% for labelled chemicals and from 70% to 110% for non-labelled chemicals.

90 - 104.7%

yes

 

Validity criteria fulfilled:
yes
Remarks:
Please refer to field "any other information on results incl. tables"

Description of key information

- DT50 = 1.53 - 2.47 days based on test material analysis (3.7 - 5.8 days, recalculated to 12 °C, OECD 309, surface water)

- 31.5% (high dose system) - 56.9% (low dose system) mineralisation after 30 d

- DT50 = 0.3 and 0.2 days based on test material analysis (0.6 - 0.4 days, recalculated to 12 °C, Commission Directive 95/36/EC)

- 48.6% (pond system) - 84.2% (river system) mineralisation after 101 d


Key value for chemical safety assessment

Additional information

Three simulation tests are available assessing the biodegradablitiy of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) in water and sediment.

In the key study (2015), the aerobic degradation of the test item in surface water was assessed, according to OECD 309 and GLP. Natural pond water was treated in duplicate using a high nominal test item concentration of 100 µg/L and a low test item concentration of 10 µg/L for 30 days. Additionally, sterile samples treated at the high test item concentration were incubated under sterile conditions in order to gain information about abiotic degradability of the test item. The amount of radioactivity was determined by liquid scintillation counting (LSC). Aliquots of the water phase were submitted to high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC, selected samples only). The total mean recoveries were 94.4 ± 7.2% AR (high dose), 102.4 ± 4.4% AR (high dose, sterile) and 100.8 ± 5.2% AR (low dose), respectively. Mineralization (formation of CO2 and CS2) of the substance increased over time, reaching a maximum of 31.5% AR (high dose) and 56.9% AR (low dose) on day 30. The lower mineralization rate in the high dose application system could be attributed to an inhibitory effect on microorganisms as observed in further studies with microorganisms. The amount of radioactivity in the aqueous phases of sterile samples remained stable, accounting for 95.2% AR after 30 days of incubation. Mineralisation, reached a maximum of 9.5% AR. Based on test material analysis the derived DT50 values for high dose and the sterile high dose were 2.47 and 1.53 days (5.8 and 3.7 days at 12°C), respectively. In natural surface water treated at the high dose level, Ziram degraded into five major (M1, M9, M13, M14 and M22) and three minor (M2, M15 and M17) radioactive fractions, which could not be further identified.

The second key study (2001), was performed according to European Community Commission Directive 95/36/EC, Annex I, 7.2.1, Route and Rate of Degradation in Aquatic Systems (1995) and GLP. 14C-labeled Ziram was incubated in two different water/sediment systems (from a pond and a river) for 101 days at a concentration of 0.29 mg/L. Radioactivity in water and extractable radioactivity in sediment was analysed by HPLC and TLC. Total recoveries obtained during incubation were 100%±3.4% and 95.3±3.7% of the applied radioactivity for river and pond system, respectively. Based on test material analysis Ziram rapidly degraded in both systems, with DT50 values of 0.3 days and 0.2 days (0.6 and 0.4 d at 12°C) for river and pond, respectively. Mineralisation played an important role in the degradation of Ziram, where 14CO2 and 14CS2 at the end of the study (101 d) accounted for 84.2% and 48.6% of the total applied radioactivity for river and pond system, respectively. In addition to 14CO2, Thiram (CAS No. 137-26-8) was a major intermediate degradation product. This study in water/sediment system was chosen as key due to the longer study period and the higher recoveries of applied radioactivity obtained during incubation time (>90%).

In a supporting study (2018) the aerobic transformation of the substance in natural aquatic sediment systems was studied according to the OECD Guideline 308 and GLP. Two different sediment types (sandy loam with a low organic carbon content from River Rhein and sandy loam with a low organic carbon content from Calwich Abbey Lake) were used and incubated for 30 days in the dark at approx. 20°C. Total recoveries of radioactivity (mass balances) were between 61.6% and 70.4% applied radioactivity. In Calwich Abbey Lake aquatic sediment, the total radioactivity in the water layer declined from 99.3 - 106.2% applied radioactivity at time zero to 26.7 - 29.2% after 30 days. The proportion of radioactivity remaining unextracted in the sediment increased to a maximum of 28.0 - 32.7% of applied radioactivity after 4 days. Volatile radioactivity was minimal (maximum of 1.0 - 1.1% applied radioactivity after 7 days) and was almost all associated with 14CO2. Dissipation of radioactivity followed a similar pattern in the River Rhein aquatic sediment. The total radioactivity in the water layer declined from 103.5 - 105.1% applied radioactivity at time zero to 24.6 - 29.7% after 30 days. The proportion of radioactivity remaining unextracted in the sediment increased to 15.2 - 25.1% of applied radioactivity after 30 days. Volatile radioactivity accounted for a maximum of 3.7 - 7.3% applied radioactivity after 14 days and was almost all associated with 14CO2. Based on test material analysis the DT50 values were determined to be 0.24 hours for Calwich Abbey Lake sediment and 0.57 hours for River Rhein sediment. The degradation of Ziram in the total system proceeded largely via Thiram (up to 40.0% AR), and Tetramethylthiuram monosulfide (9.1% AR). This study in water/sediment system was chosen as supporting information, due to the short incubation time (30 d) and the low recoveries of the applied radioactivity at the end of the study (61.6 – 70.4%).