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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 November 2004 - 09 March 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- Name of test material (as cited in study report): Creatine monohydrate
- Molecular formula (if other than submission substance): C4H9N3O2 x H20
- Substance type: organic substance
- Physical state: colourless powder
- Analytical purity: > 99.9
- Purity test date: 2004-11-18
- Lot/batch No.: 309931
- Expiration date of the lot/batch: 2006/03
- Solubility in water: 14 g/L (20 °C)
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: stable under storage conditions
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: from effluent of sewage treatment works A-2500 Baden, waste-water catchment predominatly domestic
- Laboratory culture: no
- Method of cultivation: n.a.
- Storage conditions: n.a.
- Storage length: n.a.
- Preparation of inoculum for exposure: upon arrival in the laboratory ths sample was aerated by means of filtered compressed air for about 45 minutes before being used in the test
- Pretreatment: none
- Concentration of sludge: no
- Initial cell/biomass concentration: < 1 x 10^5 (except for abiotic sterile control)
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium:
a) KH2PO4: 8.50 g, K2HPO4: 21.75 g, Na2HPO4 x 2 H2O: 33.40 g, NH4Cl: 0.50 g
pH 7.4, dissolved in deionised water and filled up to 1000 mL.
b) MgSO4 x 7 H2O: 22.50 g
dissolved in deionised water and filled up to 1000 mL.
c) CaCl2 x 2 H2O: 36.40 g
dissolved in deionised water and filled up to 1000 mL.
d) FeCl3 x 6 H2O: 0.25 g
dissolved in deionised water and filled up to 1000 mL.
e) Trace element solution:
MnSO4 x H2O: 30.70 mg
H3BO3: 58.00 mg
ZnSO4 x 7 H2O: 42.90 mg
(NH4)6Mo7O24: 34.90 mg
Fe-Chelat (FeCl3, EDTA): 100.00 mg
dissolved in deionised water and filled up to 1000 mL.
f) Vitamine solution: 15.30 mg extract of yeast was dissolved in 100 mL deionised water (freshly prepared)

Chemicals for solutions a) to e) were analytical grade supplied by Merck, Darmstadt, Germany. For solution f) pure grade (SERVA, Heidelberg, Germany) was used
10 mL of solution a) were added to 800 mL deionised water and 1 mL each of the solutions b) to f) were added and then made up to a total volume of 1000 mL deionised water (-> 0 mg DOC/L).

- Additional substrate: none
- Test temperature: 21.5 +/- 0.4 °C
- pH: 7.2 - 7.4
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: < 1 x 10^5 cells (except for abiotic steril control); number of cells determined with Coulter Counter (Model ZF)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2L conical flask reaction vessels with 800 mL mineral medium + sufficient stock solutions of test and / or reference substance
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: test vessels were covered with aluminium foil in such a way that the exchange of air was garantueed; vessels were inserted in a shaking machine
- Method used to create anaerobic conditions: n.a.
- Measuring equipment: TOC-Analysator multi N/C 2000, Analytik Jena
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: n.a.


SAMPLING
- Sampling frequency: Days 0 (beginning of study), 2, 4, 7, 10, 14, 17, 21, 24, and 28
- Sampling method: 30 mL samples were taken from supernatant from each reaction vessel and centrifuged (4000 g, 15 min, room temperature)
- Sterility check if applicable: n.a.
- Sample storage before analysis: no data


CONTROL AND BLANK SYSTEM
- Negative control: medium plus inoculum
- Positive control: reference substance sodium benzoate, 20 mg DOC/L (C7H5NaO2, MW: 144.11, CAS: 532-32-1, AppliChem GmbH, 64291 Darmstadt, Germany)
- Abiotic sterile control: test substance (20 mg DOC/L) + 5 mL 1% HgCl2 solution per liter test medium, no inoculum
- Toxicity control: reference substance (20 mg DOC/L) + test substance (20 mg DOC/L) with inoculum
- Adsorption control: test substance (20 mg DOC/L) + 5 mL 1% HgCl2 solution per liter test medium, inoculum

STATISTICAL METHODS:
- Calculation of DOC removal (degradation):
Dt = (1- (ct - cblt)/(c0 - bl0)) x 100
Dt = degradation in % at time t
c0 = DOC concentration of the inoculated culture medium containing the test or the reference substance at the beginning (Day 0)
ct = DOC concentration of the inoculated culture medium containing the test or the reference substance at time t
cbl0 = DOC concentration of the negative control at the beginning
cblt = DOC concentration of the negative control at time t
Reference substance:
benzoic acid, sodium salt
Test performance:
No unforseen events occurred.
Key result
Parameter:
% degradation (DOC removal)
Value:
>= 96.18 - <= 96.95
Sampling time:
28 d
Details on results:
TEST SUBSTANCE:
In this Modified OECD Screening Test the biodegradation of “creatine monohydrate” was 96.95 & and 96.18 % with a mean of 96.56 % after 28 days. The “10-day window” as defined by the Guidelines started at day about Day 3. 70 % degradation was exceeded at approx. Day 10.

POSITIVE CONTROL:
The plateau of biodegradation was reached on Day 2and the degradation of the positive control substance sodium benzoate exceeded the pass level of 70 % within 14 days.

TOXICTY CONTROL:
Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of an inhibition of the microbial activity by the test substance.

ABIOTIC CONTROL:
On Day 0, the DOC concentration was between 20.95 and 21. 25 mg/L and on Day 28 between 20.54 and 20.61 mg/L, therefore, no significant degradation to inorganic carbon occurred in the abiotic sterile control.

ADSORPTION CONTROL:
On Day 0, the DOC concentration was between 20.93 and 20.95 mg/L and on Day 28 between 20.58 and 20.01 mg/L, therefore, no adsorption of the test substance to the inoculum occurred.
Results with reference substance:
See section "details on results".

Table 1: Individual DOC determinations

Vessel number

Sample

DOC (mg/L) on Day

mean

0

2

4

7

10

14

17

21

24

28

NK 1 negative control

NK 1/1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

NK 1/2

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

mean

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

NK 2 negative control

NK 2/1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

NK 2/2

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

mean

0.00

0.08

0.08

0.00

0.00

0.00

0.00

0.00

0.00

0.00

mean of NK 1 and NK 2

0.00

0.04

0.04

0.00

0.00

0.00

0.00

0.00

0.00

0.00

A 3 test substance

A 3/1

20.09

19.91

8.25

6.35

5.89

6.24

5.77

0.79

1.08

0.70

A 3/2

20.21

19.89

7.67

6.33

5.99

6.09

5.35

0.69

0.44

0.53

mean

20.15

19.90

7.96

6.34

5.94

6.17

5.56

0.74

0.76

0.62

A 4 test substance

A 4/1

20.59

20.16

7.00

6.54

5.72

6.28

5.54

0.87

0.96

0.82

A 4/2

20.61

19.72

7.28

6.92

5.90

5.78

5.82

1.45

0.58

0.76

mean

20.60

19.94

7.14

6.73

5.81

6.03

5.68

1.16

0.77

0.79

PK 5 positive control

PK 5/1

2012

3.08

1.60

1.43

0.62

0.56

0.41

1.33

1.55

0.50

PK 5/2

19.99

3.34

1.62

1.44

0.75

0.45

0.29

1.24

1.49

0.78

mean

10.06

3.21

1.61

1.44

0.68

0.51

0.35

1.29

1.52

0.64

TK 6 positive control

TK 6/1

39.98

20.68

7.23

7.42

6.43

6.95

5.43

1.88

1.71

2.09

TK 6/2

39.47

21.17

7.59

7.38

6.38

6.38

5.56

1.82

1.69

1.52

mean

39.73

20.93

7.41

7.40

6.41

6.67

5.50

1.85

1.70

1.81

Table 2: Blank-corrected DOCs (mg/L)

Day

Test substance

Positive control

Toxicity control

A 3

A 4

PK 5

TK 6

0

20.15

20.60

20.06

39.73

2

19.90

19.94

3.21

20.93

4

7.92

7.10

1.57

7.37

7

6.30

6.69

1.39

7.36

10

5.94

5.81

0.68

6.41

14

6.17

6.03

0.51

6.67

17

5.56

5.68

0.35

5.50

21

0.74

1.16

1.29

1.85

24

0.76

0.77

1.52

1.70

28

0.62

0.79

0.64

1.81

Validity criteria fulfilled:
yes
Remarks:
Validity criteria fulfilled.
Interpretation of results:
readily biodegradable
Conclusions:
The test substance can be classified as readily biodegradable.
Executive summary:

The ready biodegradability of „creatine monohydrate“ was determined by the measurement of DOC (dissolved organic carbon) at frequent intervals over a 28d period. The method used was that described in Part C.4-B of the EC Commission Directive 92/69/EEC and in the OECD Guideline 301 E.

Results:

-       The reference substance sodium benzoate was degraded by 97.5 % within 14 days.

-       Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of an inhibition of the microbial activity by the test substance.

-       The test substance “creatine monohydrate” with a nominal concentration of 20 mg DOC/L was degraded by 71.2 % after 10 days of incubation at 21. ± 0.4 °C. The “10-d window” as defined by the guidelines started at approx. Day 3. 70 % degradation was exceeded at approx. Day 10.

Classification: According to the Guideline a substance giving a result greater than 70 % loss of DOC in a 10-d window within 28 days is regarded as “readily biodegradable”.

Therefore, “creatine monohydrate” can be classified as readily biodegradable.

Description of key information

Creatine (CAS 57-00-1) is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of „creatine monohydrate“ was determined according to OECD Guideline 301 E at a nominal concentration of 20 mg DOC/L. Creatine monohydrate degraded by 71.2 % after 10 days of incubation at 21. ± 0.4 °C fulfilling the “10-d window” criterion. Therefore, creatine monohydrate can be classified as"readily biodegradable". This result is used for the classification of creatine (CAS 57 -00 -1) in a read-across approach.