Carnosine

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:

28 Apr - 21 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L
- Sampling method: Samples without algae were taken at test start and stability samples with algae were taken, after 24 and 48 hours and at the end of the test (72 hours).
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item (50.16 mg) was solved in 500 mL test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.
- Controls: yes, test medium control
- Evidence of undissolved material: After the dissolving procedure the single test concentration of 100 mg/L was a clear solution.
- Eluate: No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source: supplied by the Collection of Algal Cultures
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Cultivated at test facility under standardized conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

maintained at 24 °C

pH:

7.4 - 8.0 (Control and 100 mg/L)

Nominal and measured concentrations:

Control and 100 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with a glass dish)
- Material, size, headspace, fill volume: 75 mL Erlenmeyer flasks, 30 mL test volume, 45 mL headspace
- Initial cells density: 0.64 x 10E+04 cells/mL
- Control end cells density: 140.1 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Others: Test vessels were placed in a temperature controlled orbital shaker and were positioned randomly and repositioned daily.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium according to the OECD 201 guideline
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- pH: 7.5
- Photoperiod: continuously
- Light intensity and quality: LED light; approx 66 μE s-1 m-2 (range: 65 to 67 μE s-1 m-2)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The algal biomass was determined by fluorescence measurement. The measurements were performed at an excitation of 440 nm and emission of 680 nm. At the end of the test, a sample was taken from the control and from the single test concentration of 100 mg/L to determine a potential influence of the test item on the algal cells.
- Other: The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable limit test
Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Since no effects were observed up to and including 100 mg/L a limit test was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

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

Key result

Duration:

72 h

Dose descriptor:

EC10

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

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: Microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this test concentration.

Results with reference substance (positive control):

The 72 h EC50 for growth rate in the reference test was 1.0 mg/L (May 2017) and showed that the sensitivity of the test system was within the range recommended by the guideline (72 h EC50 for the growth rate 0.9-1.5 mg/L).

Reported statistics and error estimates:

The 72 h EC10, EC20 and EC50 values of the test item could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Two-sample t-test. Statistical analysis was performed using ToxRat Professional®.

 

Table 1: Biological Results Cell numbers

Nominal Test Item Concentration [mg/L]	Repl. No.	Biomass of Algae*
		24 Hours	48 Hours	72 Hours
Control	1	4.0	25.8	143.4
	2	3.6	26.0	142.7
	3	3.4	24.9	134.6
	4	3.2	26.0	144.6
	5	3.4	25.3	137.0
	6	3.0	25.1	138.4
	Mean	3.4	25.5	140.1
	SD	0.4	0.5	4.0
100	1	4.4	29.2	146.6
	2	3.5	26.7	140.0
	3	4.3	30.4	150.1
	4	3.3	28.7	146.9
	5	3.0	26.4	143.3
	6	3.1	25.4	138.3
	Mean	3.6	27.8	144.2
	SD	0.6	1.9	4.5

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 10E+04). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.64 x 10E+04 relative fluorescence units.

Table 2: Biological results inhibition of growth rate

Nominal Test Item Concentration	Average Growth Rate μ (day-1) and Inhibition of μ (Ir)
	0-24 h		0-48 h		0-72 h
[mg/L]	μ	Ir [%]	μ	Ir [%]	μ	Ir [%]
Control	1.677	0.0	1.846	0.0	1.798	0.0
100	1.715	-2.3	1.888	-2.3	1.808	-0.5

No statistically significant effect at the single test concentration compared to control (according to Two-Sample t-test, one-sided smaller, α= 0.05).

Table 3: Validity criteria for OECD 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	220	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	11%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.5%	Yes

Table 4: Analytical Results for Test Samples

Sampling Day/Sample Age	Nominal Concentration of Test Item cnom	Measured Concentration of Test Item x	Sample Preparation Factor F	Determined Concentration of Test Item c	% of Nominal Concentration
[d/h]	[mg/L]	[mg/L]		[mg/L]	[%]
0/0	Control	n.d.	1	<LOQ	n.a.
(fresh)	100	95.6	1	95.6	96
1/24	Control	n.d.	1	<LOQ	n.a.
(aged)	100	95.5	1	95.5	95
2/48	Control	n.d.	1	<LOQ	n.a.
(aged)	100	95.9	1	95.9	96
3/72	Control	n.d.	1	<LOQ	n.a.
(aged)	100	96.9	1	96.9	97

n.d. = no test item detected

n.a. = not applicable

LOQ: 24.2 mg/L

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

In conclusion, the test item had no toxic effects on Pseudokirchneriella subcapitata during the 72-hour test period up to and including the concentration of 100 mg/L.

Description of key information

ErC50 (72 h) >100 mg/L (Pseudokirchneriella subcapitata, OECD 201, nominal)

ErC10 (72 h) >100 mg/L (Pseudokirchneriella subcapitata, OECD 201, nominal)

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata. Since no effects were observed in the range-finding test, a limit-test was performed testing control and 100 mg/L. The measured concentrations at test start and test end were within a range of 80 - 120% of nominal. No statistically significant differences on growth rate and yield were determined compared to the control. Thus, the EC50 and EC10 after 72 h were derived to be >100 mg/L.