N-amidinosarcosine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2004 - 02 December 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

not specified

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 * H2O
- Physical state: colourless - light yellow powder
- Analytical purity: > 99.9 %
- Purity test date: 2004-11-18
- Lot/batch No.: 309931
- Expiration date of the lot/batch: 2006/03
- Stability under test conditions: stable
- Storage condition of test material: ambient temperature
- Water solubility: 14 g/L

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

Reconstituted water (see "Details on test conditions").

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna STRAUSS
- Source: "Landesamt für Wasserwirtschaft München", Munich, Germany
- Age at study initiation: < 24h
- Method of breeding: every 3-4 days cultures of daphnia magna were transferred into new medium and young animals were separated from the older ones by sieving; daphnids were fed daily with an aliquot of a culture of single cell green algae.
- Feeding during test: no


ACCLIMATION
- Acclimation period: none -> directly before starting the test the daphnia cultures were sieved, the young animals were trasnsferred into new reconstituted water and then transferred with a micropipette into the test vessels.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

n.a.

Hardness:

not reported

Test temperature:

Room temperature (approx. 20 °C)

pH:

7.5 - 8.2

Dissolved oxygen:

8.4 - 8.8 mg/L

Salinity:

n.a.

Nominal and measured concentrations:

Nominal concentrations:
- Preliminary test: 0 (control in reconstituted water), 1, 2, 10, 100, 333, and 500 mg/L
- Main test: 0 (control in reconstituted water), 250, 333, 500, and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers, volume 100 mL, filled with 20 mL test medium
- Type (delete if not applicable): open
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


RECONSTITUTED WATER:
- For exposure reconstituted water according to ISO 6341 was used.
- For hatchung reconstituted water according to ISO 6341 was used, which was supplied with the following trace element solutions:

Solution A (cation solution, 0.1 mL/L hatching water):
MnCl2 * 4 H2O 3.605 g/L
LiCl 3.06 g/L
RbCl 0.71 g/L
SrCl2 * 6 H2O 1.52 g/L
CuCl2 * 2 H2O 0.168 g/L
ZnCl2 0.13 g/L
CoCl2 * 4 H2O 0.10 g/L

Solution B (anion solution, 0.5 mL/L hatching water):
NaNO3 548 mg/L
H3BO3 5719 mg/L
NaBr 32 mg/L
Na2MoO4 * 2 H2O 126 mg/L
KJ 6.5 mg/L
NaSeO3 4.38 mg/L
BH4VO3 1.15 mg/L

Solution C (silicon solution, 0.2 mL/L hatching water):
Na2SiO3 21.475 g/L

Solution D (iron-EDTA-solution, 5 mL/L hatching water):
Na2EDTA *2 H2O 250 mg/L
FeSO4 * 7 H2O 99.6 mg/L

Solution E (phospahte solution, 0.5 mL/L hatching water):
KH2PO4 286 mg/L
K2HPO4 368 mg/L

Solution F (vitamin solution, 0.1 mL/L hatching water):
Thiamine HCl 750 mg/L
Cyanocobalamine 10 mg/L
Biotin 7.5 mg/L

- Distilled water was used for the preparation of the solutions. The vitamin solution F must be always prepared freshly or frozen until use.
- The water was aerated and the pH and the dissolved oxygen concentration was checked before use.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water, see above


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: natural day/night alternation
- Light intensity: natural

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilization (at 48 h)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Creatine Monohydrate

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 880 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Effect concentration of creatine calculated from effect concentration of creatine monohydrate.

Basis for effect:

mobility

Remarks on result:

other: Effect concentration of creatine calculated from effect concentration of creatine monohydrate.

Details on results:

IMMOBILIZATION:
48 hours after the beginning of exposure to the test substance the daphnids were observed for their swimming bahviour. Animals not able to swim within approx. 15s after gentle agitation of the test vessel are avaluated as immobilised. Additionally immobilised animals trapped at the surface were counted to evaluate an influence of the test substance to the water surface tension.

WATER QUALITY:
The dissolved oxygen concentration and pH of the test media were determined at the beginning in one test vessel of each concentration and the endin the pooled test media of each concentration.

QUALITY CRITERIA:
- Immobilization of control group daphnids was 0 % at the end of the test (guideline: maximum 10 %).
- There were no daphnids trapped at the water surface.
- The dissolved oxygen concentration was more than 3 mg/L at each determination.
- The pH deviation was not more than one unit in the test solution during the test.

Results with reference substance (positive control):

The EC50 value was within the limit of 0.6 - 1.7 mg/L indicated in ISO 6341.

Reported statistics and error estimates:

n.a.

Table 3: Preliminary test, results

Group	Dilution factor G	Test substance concentration [mg/L]	Immobilized daphnids / total number of daphnids	Total [%]
Control	-	Reconstituted water	0 / 5	0
Test substance	2	500	0 / 5	0
Test substance	3	333	0 / 5	0
Test substance	10	100	0 / 5	0
Test substance	100	10	0 / 5	0
Test substance	500	2	0 / 5	0
Test substance	1000	1	0 / 5	0

 

Validity criteria fulfilled:

yes

Conclusions:

The 48h EC50 of creatine monohydrate for Daphnia magna is > 1000 mg/L.
The corresponding EC50 (48h) of creatine for Daphnia magna is > 880 mg/L.

Executive summary:

The acute toxicity of creatine monohydrate to Daphnia magna was determined in a 48 hours static test following the pattern of 92/69/EEC C.2 (“Acute Toxicity for Daphnia magna”).  

Animals of Daphnia magna, not more than 24 hours old, were exposed to different concentrations of the test substance in reconstituted water according to ISO 6341. A negative control group was exposed to reconstituted water only.  

In the test 20 daphnids, divided into 4 replicates (5 daphnids each) were used for each concentration and for the control group.  

Immobilization 48 hours after the start of the test, pH in the control and test media at the start and at the end of the test, as well as dissolved oxygen concentration in the control and test media at the start and at the end of the test were recorded.  

Immobilization of the control and test group daphnids was 0% at the end of the test. The study shows that the EC50 (48h) for Daphnia magna is > 1000 mg/L under the conditions of the test.  

Justification for read-across:

For the aquatic REACH endpoint 9.1.1 ”Short-term toxicity testing on invertebrates”, the results obtained with creatine monohydrate can be used for the registration of creatine. There is no chemical difference between “creatine monohydrate” and “creatine” when dissolved in water. The crystal water contained in creatine monohydrate does not account for the toxicity profile of the compound. However, it has to be considered that the results obtained with "creatine monohydrate" have to be converted to the respective "creatine" concentration as "creatine" has a lower molecular weight (131.1332 g/mol) compared to "creatine monohydrate" (149.1484 g/mol). 1000 mg creatine monohydrate per liter correspond to approx. 880 mg creatine per liter. Therefore, the corresponding EC50 (48h) of creatine for Daphnia magna is > 880 mg/L.

Description of key information

The acute toxicity of Creatine monohydrate was determined according to 92/69/EEC C.2.

No effects were observed at the maximum concentration tested (1000 mg/L nominal).
This result was used for the anhydrous form Creatine in a read-across approach.

Additional information

The acute toxicity of creatine monohydrate to Daphnia magna was determined in a 48 hours static test following the pattern of 92/69/EEC C.2 (“Acute Toxicity for Daphnia magna”).  

Animals of Daphnia magna, not more than 24 hours old, were exposed to different concentrations of the test substance in reconstituted water according to ISO 6341. A negative control group was exposed to reconstituted water only.  

In the test 20 daphnids, divided into 4 replicates (5 daphnids each) were used for each concentration and for the control group.  

Immobilization 48 hours after the start of the test, pH in the control and test media at the start and at the end of the test, as well as dissolved oxygen concentration in the control and test media at the start and at the end of the test were recorded.  

Immobilization of the control and test group daphnids was 0% at the end of the test. The study shows that the EC50 (48h) for Daphnia magna is > 1000 mg/L under the conditions of the test.

Therefore, the corresponding EC50 (48h) of creatine for Daphnia magna is > 880 mg/L.