N-amidinosarcosine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-20 - 2012-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The temperature in the reference study was higher than stated in the guideline. Because the controls showed normal exponential growth, this can be stated as uncritical.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The temperature in the main study was higher than stated in the guideline. Because the controls showed normal exponential growth, this can be stated as uncritical.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The cell concentrations in the adjusted preculture were about 9 * 104 cells/mL in the main study and 4.5 * 104 cells/mL in the reference study. Because the controls showed normal exponential growth, this can be stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The stability was determined by measuring a standard solution containing 100 mg/L in algal test medium at the time levels 0 h and 72 h at room temperature.

- Concentrations: t = 0 h: 37.896 mg/l (mean); t = 72 h: 38.355 mg/l (mean

Vehicle:

yes

Details on test solutions:

A test solution containing 142 mg/L Creatine Monohydrate (corresponding to 125 mg/L Creatine) in deionised water was prepared (concentration in the test * 1.25, as the solution was diluted by nutrient medium and algae).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification:
Unicellular freshwater green alga
Genus, Species Desmodesmus subspicatus
Strain CHODAT
Family Scenedesmaceae
Order Sphaeropleales

Origin and Culture:
The culture of Desmodesmus subspicatus was obtained in Oct. 2011 by MBM Science-bridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 8 °C. From an aliquot of this stock culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observations were recorded.

Test temperature:

24 - 25 °C

pH:

The pH values ranged from 8.3 to 9.1.

Nominal and measured concentrations:

t = 0 h:
nominal: 100 mg/l
measured: 109 mg/l

t = 72h:
nominal: 100 mg/l
measured: 115 mg/l

Details on test conditions:

Treatments tested: 100 mg/L (anhydrous substance)
Number of replicates: six replicates for the control
six replicates for the treatment
Vessels: glass flasks 50 mL
Duration: 72 hours
Temperature: 24 – 25 °C
Lighting: 4800 Lux
Control: deionised water with nutrient medium and alga
Treatments: test solution with nutrient medium and alga

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Results with reference substance (positive control):

NOEC 72h: 0.16 mg/l
LOEC 72h: 0.25 mg/l
EC50 (growth): 1.40 mg/l
EC50 (biomass): 0.63 mg/l
EC50 (yield): 0.38 mg/l

Reported statistics and error estimates:

For the treatment with the test item concentration 100 mg/L, it was tested whether the differences between treatment and control were significant. The values for the area under the curve, Yield and the growth rate were used for the assessment of observed effect levels.

Nominal Conc. 100 mg/L
Endpoint µ AUC Yield
calculated t-value t-test 0.64 not applicable not applicable
tabulated t-value t-test 2.23 not applicable not applicable
calculated t-value WEIR-test not applicable 0.23 0.42
tabulated t-value WEIR-test not applicable 2.00 2.00
Significance given? no no no

Determination of the NOEC:
For the endpoints µ (growth rate), AUC (area under curve) and Yield, the differences between the treatment 100 mg/L and the control can be considered as not significant (level of significance: 97.5 %) as the calculated t-values were smaller than the limit of significance. Therefore, the concentration 100 mg/L is stated as NOEC for these endpoints.

Determination of the LOEC:
As for the endpoints µ, AUC and Yield, the differences between the treatment 100 mg/L and the control can be considered as not significant (level of significance: 97.5 %), no LOEC can be stated for these endpoints.

Cell Numbers

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Cell Number/mL Main Study

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	9051	66374	299437	1211326
0	SD	0	10484	16170	117538
100	Mean	9051	67883	303963	1155511
100	SD	0	12059	60134	266104

Microscopical Observation

In the following table the appearance of the alga at the end of the test were stated:

Microscopical Observation Main Study

Nom. Conc. in mg/L	Observation
0	Normal growth
100	Normal growth

Area under the Curve, Growth Rate, Yield

From the cell numbers, the Growth Rate µ, the Area under the Curve AUC, and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Area under the Curve AUC, Yield Main Study

Nom. Concentration in mg/L		Growth Rate [day^-1]	AUC [Cell Concentration/mL*day]	Yield [Cell Concentration/mL]
0	Mean	1.63	948847	1202275
	SD	0.03	61710	117538
100	Mean	1.61	926973	1146460
	SD	0.08	194546	266104

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ, the Area under the Curve AUC, and the Yield.

Inhibition Values Main Study

Nom. Concentration in mg/L	% Inhibition
	Growth Rate µ	Area under the Curve AUC	Yield
0	0!	0!	0!
100	1.31	2.31	4.64

Validity criteria fulfilled:

yes

Conclusions:

The following effects of the test item on the test system were observed under the conditions reported (concentration 100 mg/L, 72 h exposition, 24 - 25 °C): A NOEC of 100 mg/L and no LOEC were observed for all three endpoints. The endpoints biomass integral, growth rate and yield showed inhibitions below 50% in this study, therefore, the respective ECs lie above 100 mg/L.

Executive summary:

The study (according to OECD Guideline 201) was performed in order to evaluate the toxic potential of Creatine Monohydrate towards Desmodesmus subspicatus Chodat, an unicellular freshwater green alga, which belongs to the order of chlorococcales. This freshwater green alga was chosen as a typical part of phytoplankton.

One valid main study was performed.

The study was performed using one concentration containing 113.6 mg/L Creatine Monohydrate (corresponding to 100 mg/L Creatine). The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatusfor a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate µ, the area under the growth curve (AUC[1]) and the Yield[2]were determined.

At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The recovery after 72 hours was 105 % of the start concentration. The measured concentration was 109 % of the nominal concentration at the beginning of the test and 115 % of the nominal concentration at the end of the test.

Therefore, the determination of the results was based on the nominal concentrations.

The EC50s of potassium dichromate were tested in a current reference test. The values lay within the normal range of the laboratory.

The following results for Creatine were determined:

Endpoint	NOEC	LOEC	EC50
Growth Rate	≥100 mg/L	> 100 mg/L	> 100 mg/L
AUC	≥100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥100 mg/L	> 100 mg/L	> 100 mg/L

Description of key information

In the 72-h test that was conducted according to OECD Guideline 201 no toxic effects (based on growth rate)

of creatine monohydrate to the unicellular freshwater green alga Desmodesmus subspicatus could be observed at the tested maximum concentration of 113.6 mg/L. This concentration corresponds to 100 mg/L creatine.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The study was performed according to OECD Guideline 201 in order to

evaluate the toxic potential of Creatine Monohydrate towards Desmodesmus subspicatus Chodat, an unicellular freshwater green alga, which belongs to the order of chlorococcales.

The study was performed using one concentration containing 113.6 mg/L Creatine Monohydrate (corresponding to 100 mg/L Creatine). The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours, and the growth rate µ, the area under the growth curve (AUC[1]) and the Yield[2] were determined.

The following results for the test item Creatine were determined:

Endpoint	NOEC	LOEC	EC50
Growth Rate	≥100 mg/L	> 100 mg/L	> 100 mg/L
AUC	≥100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥100 mg/L	> 100 mg/L	> 100 mg/L

Justification for read-across:

For the aquatic REACH endpoint 9.1.2 ”Toxicity to aquatic algae and plants", 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). 113.6 mg creatine monohydrate per liter correspond to 100 mg creatine per liter. Therefore, the corresponding ErC50 (72h) of creatine forDesmodesmus subspicatusChodatis > 100 mg/L.