6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Nov 2017 - 27 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany (10 Dec 2015)

Test material

Test material information

Constituent 1

Reference substance name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

EC Number:

254-074-6

EC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Cas Number:

38668-46-1

Molecular formula:

C9H13N7

IUPAC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

- Concentrations: Chemical analysis was performed on the control and highest test item concentration (100 mg/L) at the beginnin (0 h) and end (72 h) of the test.
- Sampling method: From all test item concentrations and the control, 10 mL were sampled from fresh solutions at 0 h and from aged test solutions after 72 h. For each sampling a retain sample was taken.
- Sample storage conditions before analysis: All samples were deep frozen (≤ -18 °C) until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Clear and transparent stock solutions (500 mL) with nominal concentrations of 9.53, 17.1, 30.9, 55.6, and 100 mg/L were prepared by homogenisation of test item in test medium (AAP-medium) by shaking and subsequent ultrasonication for 5 min.
- Differential loading: Yes
- Controls: Untreated test medium (AAP-medium)
- Evidence of undissolved material: The stock solutions were clear and transparent.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green microalga
- Strain: SAG 61.81
- Source: Commercial supplier MBM Sciencebridge GmbH, Göttingen, Germany. Stock cultures are ordered regularly.
- Age of inoculum (at test initiation): 3 - 4 d in exponential growth phase, reared under test conditions
- Method of cultivation: Semi-continuous liquid cultures (sterile) in 100 mL Erlenmeyer flasks on a rotary shaker (approx. 105 rpm), with continuous illumination (4440 - 8880 lux/60 -120 µEm^-2s^-1) at 21 -24 °C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0 - 22.8 °C

pH:

7.28 - 8.02 (control)
7.33 - 8.36 (treatments)

Nominal and measured concentrations:

Control, 9.53, 17.1, 30.9, 55.6, and 100 mg/L (nominal)
Control, -- , -- , -- , -- , and 105 mg/L (0 h)/111 mg/L (72 h) (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps, filled with 50 mL test solution
- Initial cell density: 0.52E+04 cells/mL
- Control end cell density: 42.64E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (AAP-medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: The pH was recorded at test start and test end. Temperature was measured continuously and recorded daily. Light intensity of the continuous illumination was measured at test start.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: 88.8 µEm^-2s^-1 at cell culture level
- Other: Test vessels were placed in an incubator on a pivoted bogie, which induces shaking by regular sudden stops (continuous agitation).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorescence microplate reader (Infinite 200Pro with Tecan i-control software 1.11.1.0) at an emission wavelength of 670 nm
- Chlorophyll measurement: After 0, 24, 48, and 72 h
- Morphology (by microscope): At test end

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Yes
- Test concentrations: Control, 0.01, 0.1, 1.00, 10.0, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, after 72 h no concentration response relationship was observed for inhibition of growth rate and yield. The inhibition of growth rate peaked at 11.8% at a nominal concentration of 100.0 mg/L and the inhibition of yield peaked at 39.0% at a nominal test item concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (tested regularly)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (72 h): 1.23 mg/L potassium dichromate (95% confidence limits: 1.11 - 1.36 mg/L)
- Other: The reference study dated from 05 - 13 Jul 2017.

Reported statistics and error estimates:

Where possible NOEC and LOEC were determined by a multiple comparison method and the EC10, 20, and 50 values were determined by Probit analysis.

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria of the guideline (Table 1).

 

Table 1: 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.	Cell numbers in the controls increased by a factor of 82 (i.e. a growth rate of 1.47/d) within 72 h	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%	The section-by-section coefficient of variation of the control cultures was 21%.	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.	The coefficient of variation of the average growth rate in replicate control cultures was 1.9%.	Yes

 

ANALYTICAL RESULTS

The measured initial test item concentration was 105% of nominal.

In the aged sample taken at test end, the measured test item concentration was 111% of nominal.

Therefore, the effect values were based on the nominal test item concentrations.

 

BIOLOGICAL RESULTS

Two main definitive tests were carried out to confirm the test outcome.

After 72 h, no inhibition of growth rate or yield was observed for nominal test item concentrations of 9.53 - 100 mg/L in both studies. No statistically significant inhibitory effects on growth rate and yield were observed. Thus, the overall LOEC was not determinable and the overall NOEC was set at 100 mg/L (nominal). The results and derived effect values are summarized in Tables 2 and 3, respectively.

Table 2. Percentage inhibition of growth rate.

Concentration [mg/L]	% inhibition of growth rate
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
9.53	-15.6	-9.7	-5.6
17.1	0.2	-5.8	-4.1
30.9	-2.2	-5.9	-3.0
55.6	-2.5	-7.0	-4.1
100	7.0	-2.8	-1.6

 

Table 3. Effect values for inhibition of growth rate and yield after 72 h.

	Nominal test item concentration [mg/L]
Growth rate inhibition
ErC10	> 100
ErC20	> 100
ErC50	> 100
Inhibition of yield
EyC10	> 100
EyC20	> 100
EyC50	> 100
NOEC^1)	≥ 100
LOEC^1)	--

- No LOEC was observed

¹⁾For growth rate and yield (Dunnett’s t-test)

Table 3. Effect values for inhibition of growth rate and yield after 72 h.

	Nominal test item concentration [mg/L]
Growth rate inhibition
ErC10	> 100
ErC20	> 100
ErC50	> 100
Inhibition of yield
EyC10	> 100
EyC20	> 100
EyC50	> 100
NOEC^1)	≥ 100
LOEC^1)	--

- No LOEC was observed

¹⁾ For growth rate and yield (Dunnett’s t-test)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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