N-amidinosarcosine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 * 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:

yes

Details on sampling:

For determination of the actual test substance concentration samples were taken immediately before the introduction of the fish and after approximately 4, 24, 48, and 96 hours after begin of exposure.

Vehicle:

no

Details on test solutions:

TEST MEDIUM PREPARATION:
The test medium with nominal 100 mg/L was prepared by dissolving 500.4 mg test substance in 5 L dilution water.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Brachydanio rerio)
- Source: Fa. Amazonas Aquarium, A-2840 Grimmenstein, Austria
- Age at study initiation (mean and range, SD):
- Length at study initiation: 2.63 +/- 0.11 cm (mean of control group at the end of the study)
The total lengths of the animals of the control group were determined for documentation of the appropriate size. As the allocation of the fish to their groups was performed using a randomisation procedure the control group was regarded as representative for the entity of all fish used.
- Weight at study initiation: 0.55 +/- 0.12 g (mean of control group at the end of the study)
- Feeding during test: no (last feeding about 24 h prior to start of exposure)


ACCLIMATION
- Acclimation period: approx. 25 weeks
- Acclimation conditions (same as test or not): similar to test conditions. However, fish were kept in bigger glass vessels (47 x 29 x 31 cm³; 40 L) and water was purified by recirculation through filter wool
- Type and amount of food: TetraMin (Tetra Werke, Dr. rer. nat. Ulrich Baensch GmbH, Melle, Germany)
- Feeding frequency: daily
- Health during acclimation:
* Mortalities > 10 % of population in 7 days: rejection of batch.
* Mortalities 5 - 7 % of population: acclimatisation continued for 7 additional days.
* Mortalities < 5 % of population: acceptance of batch.


OTHER:
- Allocation to test groups: random

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

n.a.

Hardness:

250 mg CaCO3/L (checked in control vessel at the start of the test)

Test temperature:

20 °C (throughout the test)
Determined in control and test medium before introduction of the fish and then approx. 24, 48, 72, and 96 h after the start of the test.

pH:

Control medium: 7.5 -7.6
Test medium: 7.9 - 8.0
Determined in control and test medium before introduction of the fish and then approx. 24, 48, 72, and 96 h after the start of the test.

Dissolved oxygen:

> 86 % of the air saturation value throughou the test period;
Determined in control and test medium before introduction of the fish and then approx. 24, 48, 72, and 96 h after the start of the test.

Salinity:

n.a.

Nominal and measured concentrations:

Nominal concentrations: 0 (control) and 100 mg/L
Measured geometric mean concentration: 96.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels (29 x 20 x 24 cm³) filled with 5 L of control and test medium
- Type (delete if not applicable): closed (test vesels covered with transparent plastic lid)
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 1.0 g fish/L maximum

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: local tap water was mixed 1:1 with deionised water to achieve the appropriate water hardness as required by the guideline. Residue analysis of the tap water showed that it was free of pesticides.
- Pesticides: none

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark = 16/8
- Light intensity: no data

EFFECT PARAMETERS MEASURED
- All animals were observed for alterations and behavioural changes at introduction of the fish and after approx. 2, 4, 24, 48, 72, and 96 h.
(Visual observations were made at the same time as the animal observation. Any change (like change in colour or precipitation) was recorded. )


TEST CONCENTRATIONS
- Test concentrations: 0, 100 mg/L

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Creatine Monohydrate

Basis for effect:

behaviour

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 96.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Creatine Monohydrate

Basis for effect:

behaviour

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Creatine Monohydrate

Basis for effect:

behaviour

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 96.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Creatine Monohydrate

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 84.6 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Effect concentration calculated from effect concentration of creatine monohydrate.

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 84.6 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Effect concentration calculated from effect concentration of creatine monohydrate.

Basis for effect:

behaviour

Details on results:

MORTALITY:
All animals survived until the scheduled termination of the test (96 h after start).

ANIMAL OBSERVATION:
All animals of the test substance group and the negative control group were inconspicuous in behaviour and appearance until the scheduled termination of the test (96 h after start).

ACTUAL TEST SUBSTANCE CONCENTRATION
After 48 h the actual test substance concentration was 90.71 mg/L (88.2 % of the actual starting concentration) and it was 92.44 mg/L after 72 h (89.9 % of the actual starting concentration). At 96 h the actual test substance cocentration was 95.1 mg/L (92.5 % of the actual starting concentration). The actual test substance concentrations show that the test substance was stable during the whole experiment. For the 96-h exposure period the geometric mean concentration between start and end of the exposure was 96.1 mg/L. The effective concentrations have been determined and expressed as the geometric mean of the measured concentrations. This is in accordance with the criteria of the respective guidelines.

VISUAL OBSERVATION OF THE TEST MEDIA
The test media were clear during the whole exposure time.

Results with reference substance (positive control):

n.a.

Reported statistics and error estimates:

n.a.

Sublethal observations / clinical signs:

Table 1: Mortality

Group	Nominal test substance concentration [mg/L]	Total number of dead fish at hours after start of exposure
		0	24	48	72	96
Control	0	0	0	0	0	0
Test substance	100	0	0	0	0	0

Table 2: Water quality of the control and the test medium

Nominal test substance concentration (mg/L)	Parameter	Hours after start of exposure
		0	24	48	72	96
Control (0)	Temperature	20	20	20	20	20
	Oxygen	98	88	87	88	87
	pH	7.5	7.6	7.6	7.6	7.6
Test substance (100)	Temperature	20	20	20	20	20
	Oxygen	99	89	89	87	86
	pH	7.9	7.9	8.0	7.9	7.9

Validity criteria fulfilled:

yes

Conclusions:

The 96h LC50 of creatine monohydrate for Brachydanio rerio is > 96.1 mg/L. (based on mean actual concentration).
The corresponding LC50 (96-h) of creatine for Brachydanio rerio is > 84.6 mg/L.

Executive summary:

The acute toxicity of Creatine Monohydrate to Zebrafish (Brachydanio rerio) was determined in a 96 hours static test following the pattern of 92/69/EEC C.1 (“Acute Toxicity for Fish”).

A limit test with a nominal concentration of 100 mg test substance/L dilution water was performed. The dilution water was local tap water which was mixed 1:1 with deionised water to achieve the appropriate water hardness as required by the guideline. The total hardness was 250 mg CaCO3/L. This water was used for fish-keeping and for the dilution water for the control and the test media.

7 fish each were used per test substance group and control group (dilution water only).

No mortality and no adverse effect were noted in the test substance group animals and in the control group. pH and temperature were throughout the test period. The dissolved oxygen concentration was always higher than 80 % of the air saturation value at the given temperature. The actual test substance concentration was always > 80 % of the actual starting concentration and the nominal concentration, respectively. The effective concentrations have been determined and expressed as the geometric mean of the measured concentrations.

The LC50 (96-h) of “creatine monohydrate” for Brachydanio rerio was > 96.1 mg/L (based on mean actual test substance concentration). LC0 (96-h) as well as the NOEC, based on mean actual test substance concentration, were ≥ 96.1 mg/l.

The corresponding LC50 (96 -h) of creatine for Brachydanio rerio based on mean actual test substance concentration is > 84.6 mg/L.

Description of key information

In a test on the acute toxicity of creatine monohydrate to Danio rerio

no toxic effects could be observed at the highest concentration tested (100 mg/L nominal creatine monohydrate).

Additional information

The acute toxicity of Creatine Monohydrate to Zebrafish (Danio rerio) was determined in a 96 hours static test following the pattern of 92/69/EEC C.1 (“Acute Toxicity for Fish”).

A limit test with a nominal concentration of 100 mg test substance/L dilution water was performed. The dilution water was local tap water which was mixed 1:1 with deionised water to achieve the appropriate water hardness as required by the guideline. The total hardness was 250 mg CaCO3/L. This water was used for fish-keeping and for the dilution water for the control and the test media.

7 fish each were used per test substance group and control group (dilution water only).

No mortality and no adverse effect were noted in the test substance group animals and in the control group. pH and temperature were throughout the test period. The dissolved oxygen concentration was always higher than 80 % of the air saturation value at the given temperature. The actual test substance concentration was always > 80 % of the actual starting concentration and the nominal concentration, respectively. The effective concentrations have been determined and expressed as the geometric mean of the measured concentrations.

The LC50 (96-h) of “creatine monohydrate” for Brachydanio rerio was > 96.1 mg/L (based on mean actual test substance concentration). LC0 (96-h) as well as the NOEC, based on mean actual test substance concentration, were ≥ 96.1 mg/l.

The corresponding LC50 (96 -h) of creatine for Brachydanio rerio based on mean actual test substance concentration is > 84.6 mg/L.