2-imino-1-methylimidazolidin-4-one

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:

11 - 28 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken from the highest test item concentration and from the control after 0, 24, 48 and 72 hours. All samples were analysed at 0 and 72 hours to verify the actual test concentration using a validated analytical method.

Vehicle:

no

Details on test solutions:

The limittest (range-finding test in limittest design) was performed with the following test item concentrations (spaced by a factor of 10): 1.00, 10.0 and 100 mg/L. There were six replicates for the control (0 mg/L) and the highest test item concentration (100 mg/L) and three replicates for the other test item concentrations (1.00 and 10.0 mg/L).
A stock solution (S1) was prepared by directly weighing 50 mg in 500 mL AAP-medium. This stock solution was homogenized by shaking. Afterwards
the solution was clear and transparent. V1 and V2 were made by diluting the appropriate solutions to give the required test concentrations. All stock and test solutions were prepared with AAP-medium (prepared in accordance with Annex 3 of OECD 201) containing algae cells with a density of 0.5 × 104 algae per mL. One replicate per treatment with algae was run in parallel which was used for the analytical sampling.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindak
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, 37075 Göttingen, Germany
- Method of cultivation: The algae were grown semi-continuously in the laboratory in liquid cultures under permanent illumination. Old medium was periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures were ordered regularly from the culture collection. Culture conditions were as follows:
- Illumination: from the top by light tubes, 60 – 120 µE m-2 s-1 at cell culture level
- Temperature: 21 – 24 °C
- Culture flasks: 100 mL Erlenmeyer flasks
- Visually healthy cells were used for the test
- CO2 supply: by shaking on rotating shaker (approx. 105 rpm)
Cells from this semi-continuous liquid stock culture were used for the test.

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.

Hardness:

no data

Test temperature:

23.0 - 23.5 °C

pH:

7.68 - 8.11

Dissolved oxygen:

no data

Salinity:

no data (freshwater)

Nominal and measured concentrations:

Nominal test item concentration: 1.00, 10.0, 100 mg/L
Measured test item concentrations from the highest test item concentration (100 mg/L nominal) after 0 h: 95.7 mg/L
Measured test item concentrations from the highest test item concentration (100 mg/L nominal) after 72 h: 102 mg/L
Measured test item concentrations from the control (0 mg/L nominal) after 0 h: < LOD
Measured test item concentrations from the control (0 mg/L nominal) after 72 h: < LOD

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps
- Type: closed
- Initial cells density: 0.5 x 10^4 cells/mL
- No. of vessels per control and concentration: There were six replicates for the control and the highest test item concentration and three replicates for the other test item concentrations.

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium, according to Annex 3 of OECD 201)

OTHER TEST CONDITIONS
- Light intensity and quality: continuously illumination from the side, 65 - 116 µEm^-2s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The daily fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan icontrol, 1.10.4.0).
At defined dates (24, 48 and 72 hours after beginning of the test), the number of cells in each replicate was determined in duplicate. The determination was performed by fluorescence measurement. By the means of a calibration curve, where fluorescence signals (y axis) were plotted versus cell numbers (x axis), the cell numbers were derived from the fluorescence signals. To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata culture. Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
The limittest (range-finding test in limittest design) was performed with the following test item concentrations (spaced by a factor of 10): 1.00, 10.0 and 100 mg/L. There were six replicates for the control (0 mg/L) and the highest test item concentration (100 mg/L) and three replicates for the other test item concentrations (1.00 and 10.0 mg/L).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

The morphology of the algae cells was observed microscopically. The cells were considered normal for the control and all test item concentrations.

Results with reference substance (positive control):

Potassium dichromate was tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against
compounds with known effects under the test conditions. The EC50 values calculated in this reference test were considered to be within an
acceptable range therefore it can be considered that the algae cells are sensitive.

Reported statistics and error estimates:

No statistically significant inhibitory effects were observed after 72 hours at any of the tested concentrations up to 100 mg/L (nominal).

The average cell numbers for each concentration and time of sampling are shown inTable 1. No decrease of cell numbers compared to the control was observed at any test item concentration and the growth curves were alike. Table 2 shows the percentage inhibition of growth rate andTable 3 the percentage inhibition of yield. No inhibition of growth rate or yield was found at any sampling time for any of the employed test item concentrations.

Table 1:       Average cell number for each sampling time and concentration

Test item concentration	Test solution No.	Average cell numbers/mL^1)
[mg/L]		0 h	24 h	48 h	72 h
0	Control	0.57	1.60	8.19	42.59
1.00	V2	0.57	1.78	9.00	44.31
10.0	V1	0.57	1.85	8.83	47.73
100	S1	0.57	1.73	9.01	48.71

¹⁾Algae counts are divided by 10000.

Table 2:   Percentage inhibition of growth rate

Test item concentration		Test solution No.		% Inhibition of growth rate
[mg/L]				0 – 24h		0h – 48h		0h – 72h
0		Control		0		0		0
1.00		V2		-10.4		-3.4		-1.0
10.0		V1		-14.2		-2.7		-2.7
100		S1		-7.6		-3.4		-3.1

Table 3:       Percentage inhibition of yield

Test item concentration	Test solution No.	% Inhibition of yield
[mg/L]		24 – 0 h	48 – 0 h	72 – 0 h
0	Control	0	0	0
1.00	V2	-17.5	-10.6	-4.1
10.0	V1	-24.3	-8.4	-12.2
100	S1	-12.6	-10.8	-14.5

 Analytical Results

Since no inhibitory effects on growth rate or yield occurred at any of the employed test item concentrations up to 100 mg/L the test is considered as limit test. Hence, the Creatinine concentration was only analysed in the control and the nominal concentration of 100 mg/L at test start and end.

The test was performed under static conditions. The concentration of the test item was verified in the test medium by analysing the content of Creatinine in the samples at 0 and 72 hours.The results are presented in Table 4.

Table 4:       Concentrations of Creatinine in the test medium

Test item		Creatinine		Sampling time		Creatinine		% of nominal
nominal [mg/L]		nominal [mg a.i./L]			found [mg a.i./L]
0		0		0 h		< LOD		-
			72 h aged		< LOD		-
100		99.5		0 h		95.7		96
			72 h aged		102		103

Limit of quantification (LOQ) =10 mg/L test item corresponds to 10 mg/L Creatinine

The initial measured content of Creatinine was 96% in the highest test item concentration. At test end the measured concentration was 103% of nominal. The toxicological endpoints were evaluated using nominal concentrations.

Statistical Evaluation of the Results

In Table 5 the endpoint values are summarised.

 

Table 5:        Endpoints for test item after 72 h

Endpoint		Test item[mg/L] nominal
ErC50		> 100
EyC50		> 100
NOEC^1)		> 100

¹⁾for growth rate and yield (Dunnett’s t-test, one-sided smaller, p<0.05)

No statistically significant inhibitory effects were observed after 72 hours at any of the tested concentrations up to 100 mg/L (nominal).

 Results of pH, Temperature and Light Measurements

The results from the measurement of the growth conditions during the test are shown in the following tables (Table 6, Table 7).

Table 6:   pH measurements during the test

Test item concentration nominal [mg/L]	Test solution No.	Sampling time (t = 0 h)	Sampling time (t = 72 h)
0	Control	7.68	8.11
1.00	V2	7.77	8.14
10.0	V1	7.77	8.17
100	S1	7.64	8.21

Table 7: Temperature during the test

Time	Temperature [°C]
[h]	min	max
0	23.2
24	23.1	23.5
48	23.1	23.5
72	23.0	23.3

 

The mean light intensity at test start (hour 0) was 76.8 µEm^-2s^-1, which was within the permitted range of 65.2 – 88.3 µEm^-2s^-1

Validity criteria fulfilled:

yes

Conclusions:

No inhibitory effects were observed for the test substance Creatinine up to the highest concentration of 100 mg/L (nominal). The EC50-value for growth rate (ErC50) and yield (EyC50) was > 100 mg/L (nominal). The LOEC was determined to be > 100 mg/L (nominal) and the NOEC was determined to be 100 mg/L (nominal) for the parameters growth rate and yield.

Executive summary:

A study was performed in order to evaluate the toxic potential of Creatinine towards Pseudokirchneriella subcapitata according to OECD Guideline No. 201 ("Freshwater Alga and Cyanobacteria, Growth Inhibition Test"). Initial concentration of 0.5 × 10⁴ cells/mL in each test vessel, were exposed in a static test system for 72 hours. A static limit test (range-finding test in limit test design) with concentrations of 1.00, 10.0, 100 mg/L and control was performed. Six replicates were tested for the control and the highest test item concentration and three for the other test item concentrations. After 24, 48 and 72 hours, the cell growth was determined by fluorescence detection. The mean value of the cell concentration was plotted versus time to produce growth curves for each concentration. EC values (ErC₅₀, E_yC₅₀, NOEC, LOEC) were calculated. Analytical samples were taken from the highest test item concentration and from the control after 0, 24, 48 and 72 hours. All samples were analysed at 0 and 72 hours to verify the actual test concentration using a validated analytical method.

No inhibitory effects were observed for the test substance Creatinine up to the highest concentration of 100 mg/L (nominal). The EC50-value for growth rate (ErC50) and yield (EyC50) was > 100 mg/L (nominal). The LOEC was determined to be > 100 mg/L (nominal) and the NOEC was determined to be 100 mg/L (nominal) for the parameters growth rate and yield.

Description of key information

No inhibitory effects were observed for the test substance Creatinine up to the highest concentration of 100 mg/L (nominal).

The EC50-value for growth rate (ErC50) and yield (EyC50) was > 100 mg/L (nominal).

The LOEC was determined to be > 100 mg/L (nominal) and the NOEC was determined to be 100 mg/L (nominal) for the parameters growth rate and yield.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A study was performed in order to evaluate the toxic potential of Creatinine towards Pseudokirchneriella subcapitata according to OECD Guideline No. 201 ("Freshwater Alga and Cyanobacteria, Growth Inhibition Test").Initial concentration of 0.5 × 10⁴ cells/mL in each test vessel, were exposed in a static test system for 72 hours. A static limit test (range-finding test in limit test design) with concentrations of 1.00, 10.0, 100 mg/L and control was performed. Six replicates were tested for the controland the highest test item concentrationand three for the other test item concentrations. After 24, 48 and 72 hours, the cell growth was determined by fluorescence detection. The mean value of the cell concentration was plotted versus time to produce growth curves for each concentration. EC values (ErC₅₀, E_yC₅₀, NOEC, LOEC) were calculated.Analytical samples were taken from the highest test item concentration and from the control after 0, 24, 48 and 72 hours. All samples were analysed at 0 and 72 hours to verify the actual test concentration using a validated analytical method.

No inhibitory effects were observed for the test substance Creatinine up to the highest concentration of 100 mg/L (nominal). The EC50-value for growth rate (ErC50) and yield (EyC50) was > 100 mg/L (nominal). The LOEC was determined to be > 100 mg/L (nominal) and the NOEC was determined to be 100 mg/L (nominal) for the parameters growth rate and yield.