2-ethyl-3-methylpyrazine

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:

from 2017-03-10 to 2017-04-28

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 to 100 mg/L
- Sampling method: Duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae).
- Sample storage conditions before analysis: frozen (at -20 ± 5 °C)

Vehicle:

no

Details on test solutions:

The test medium was prepared by dissolving 102 μL of test item completely in 1000 mL of test water. This volume is equivalent to the concentration of 100 mg/L, considering the relative density of the test item of 0.9830. The test item was mixed into test water by intensive stirring for 30 minutes at room temperature in the dark.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: No. 61.81 SAG
- Source: cultivated in the testing laboratory
- Method of cultivation: under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 3 days before test initiation
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

22 °C

pH:

at the start of the test: 7.4 - 7.6
during test: 7.7 - 8.0

Dissolved oxygen:

no data

Salinity:

NA

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 0.32, 1.0, 3.2, 10.0, 32.0 and 100 mg/L
Measured concentrations (% of nominal concentration): 97 - 106 % at the start of the test and 104 % at the end of the test

Details on test conditions:

TEST SYSTEM
- Test vessel: 75-mL Erlenmeyer flasks
- Type: closed with glass stoppers
- Material, size, headspace, fill volume: with headspace of about 40 mL; 30 mL test solution
- Initial cells density: algal cell density of 5000 cells/mL
- Control end cells density: algal cell density of 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM
- Standard medium used: AAP Medium modified according to ISO 14442 (modification is as a precaution to be sure that enough carbon is available to obtain appropriate growth of the algae)
- Detailed composition if non-standard medium was used: The concentration of NaHCO3 was increased to 250 mg/L and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to keep the pH of the test media as constant as possible.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: LED light with intensity of approx. 66 μE s-1 m-2 (range: 65 to 68 μE s-1 m-2)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1, 10 and 100 mg/L

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:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CL: 0.25 - 13 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The test item had a significant inhibitory effect on the yield Y and the growth rate μ of the algae after the test period of 72 hours at the concentration of 32 mg/L and at the highest test concentration of 100 mg/L.
The 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):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50 for growth rate was 0.9 mg/L (October 2016) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.9 - 1.5 mg/L).

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and the 72-hour EC50 value for the inhibition of yield could not be determined because of the absence of a significant inhibitory effect of the test item on the algae at the tested concentrations. The 72-hour EC10 and EC20 values for the inhibition of yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. 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 Williams t-test.

Validity

- In the control, the biomass increased by a factor of 89 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled.

- The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 15 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled.

- The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 4.1 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7.0 %. Thus, the validity criterion was fulfilled.

Table 1. Average Growth Rates (µ)

Nominal Test Item Concentration [mg/L]	Average Growth Rateμ(day-1) and Inhibition ofμ(Ir)
	0 – 24 h		0 – 48 h		0 – 72 h
	μ	Ir [%]	μ	Ir [%]	μ	Ir [%]
Control 0.32 1.0 3.2 10 32 100	1.314 1.232 1.022* 1.155* 0.980* 0.890* 0.944*	0.0 6.2 22.2 12.1 25.4 32.3 28.1	1.514 1.476 1.352* 1.400* 1.406* 1.288* 1.313*	0.0 2.5 10.7 7.6 7.1 14.9 13.3	1.490 1.503 1.443 1.476 1.476 1.417* 1.374*	0.0 -0.9 3.2 0.7 0.9 4.9 7.8

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

Table 2. Yield (Y)

Nominal Test Item Concentration [mg/L]	Yield Y (x 104) and Inhibition of Y (Iy)
	0 – 24 h		0 – 48 h		0 – 72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control 0.32 1.0 3.2 10 32 100	3.45 3.08 2.25* 2.76* 2.13* 1.83* 1.99*	0.0 10.9 34.7 20.1 38.2 46.9 42.3	25.03 23.00 17.69* 19.57* 19.90* 15.42* 16.35*	0.0 8.1 29.3 21.8 20.5 38.4 34.7	110.70 113.74 94.79 106.38 105.42 87.52* 77.02*	0.0 -2.7 14.4 3.9 4.8 20.9 30.4

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

Table 3. Results for Test Samples – Analytical measurements

Sampling Day/Sample Age   [d/h]	Nominal Concentration of Test Item Cnom [mg/L]	Measured Concentration of Test Item x [mg/L]	Sample Preparation Factor   F	Determined Concentration of Test Item c [mg/L]	% of Nominal Concentration   [%]
0/0 (fresh)	Control 10 32 100	n.d. 5.28 15.5 49.9	2 2 2 2	< LOQ 12.6 31.0 99.7	n.a. 106 97 100
3/72 (aged)	Control 10 32 100	n.d. 5.19 16.6 52.0	2 2 2 2	< LOQ 10.4 33.2 104.0	n.a. 104 104 104

n.d. = no test item detected

n.a. = not applicable

LOQ: 4 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata are determined as follows:
72h-ErC10 (growth rate) >100 mg/L
72h-ErC50 (growth rate) >100 mg/L

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel. The measured concentrations of the test item in the test media of the test concentrations 10 to 100 mg/L were between 97 and 106 % of the nominal values at the start of the test and 104 % at the end of the test. Thus, the correct dosing of the test item was confirmed and it was stable in the test media over the test period of 72 hours.

The biological results were related to the nominal concentrations of the test item. The determined 72-hour ErC50 and ErC10 for growth rate was >100 mg/L, respectively.

Description of key information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata are determined as followed:

72h-ErC10 (growth rate) >100 mg/L (nominal)

72h-ErC50 (growth rate) >100 mg/L (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel. The measured concentrations of the test item in the test media of the test concentrations 10 to 100 mg/L were between 97 and 106 % of the nominal values at the start of the test and 104 % at the end of the test. Thus, the correct dosing of the test item was confirmed and it was stable in the test media over the test period of 72 hours.

The biological results were related to the nominal concentrations of the test item. The determined 72-hour ErC50 and ErC10 for growth rate was >100 mg/L, respectively.