Reaction product of: 2,4-diamino-6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine and cyanuric acid

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 Feb - 14 Mar 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 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 Jul 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L at 0, 24 and 72 h
- Sampling method: Analytical samples (10 mL) were taken at 0 h (initial value) from fresh test solutions and after 24 and 72 h from aged test solutions. Two samples sets were taken and for each sampling a retain sample was also taken.
- Sample storage conditions before analysis: Deep frozen (≤ -18 °C)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The necessary amount of test item for preparing the 100 mg/L stock solution (nominal) was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenized by shaking and treated with 25 min of ultrasonication. Algae were added to the stock solution at a target cell density of 0.5 E+04 cells per mL. Lower test solutions were prepared by dilution of appropriate solutions with AAP-medium containing algae cells with a target density of 0.5 E+04 cells/mL.
- Differential loading: No
- Controls: Same medium and treatment but without test item
- Evidence of undissolved material: The stock solution was clear and transparent.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Single cell green alga
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Göttingen, Germany (commercial supplier). Stock cultures are re-ordered regularly.
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Culture conditions: Continuous illumination (4440 - 8880 lux or 60 - 120 µEm^-2^-1 at cell culture level); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply: Shaking on a rotating shaker (approx. 105 rpm)
- Age of inoculum (at test initiation): 3 - 4 d old pre-culture in the state of exponential growth

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

22.2 - 23.2 °C

pH:

0 h: 7.21 - 7.29
72 h: 7.93 - 8.29

Nominal and measured concentrations:

Control, 100 mg/L (nominal)
< LOD, 92 mg/L (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: 0.57 E+04 cells/mL
- Control end cell density: 33.66 E+04 cells/mL
- No. of vessels per concentration (replicates): 3 replicates each for 0.01, 0.10, 1.0, and 10.0 mg/L treatments, and 6 for the 100 mg/L treatment
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- pH: 7.5 ± 0.1 (1 h after preparation)
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: The pH was measured at 0 and 72 h, temperature was measured continuously and recorded at 0, 24, 48, and 72 h.

OTHER TEST CONDITIONS
- Photoperiod: Continuously
- Light intensity: 88.1 µEm^-2s^-1 (mean)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorimeter (fluorescence microplate reader infinite 200Pro, emission 670 nm) after 0, 24, 48, and 72 h
- Other: Microscopic evaluation of morphological appearance at test end (72 h).

TEST CONCENTRATIONS
- Range finding study: A static limit test was performed as range-finding test in limit test design (6 replicates for the control and the highest test item concentration and 3 replicates for the other test item concentrations)
- Test concentrations: Control, 0.01, 0.10, 1.0,10.0, and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes, cell numbers in the controls increased by a factor of 59.05 between 0 and 72 h (> required threshold of 16), corresponding to a growth rate of 1.36^-1 d
- Observation of abnormalities: The cells appeared normal up to and including the highest test item concentraton of 100 mg/L.
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the test fulfilled the validity criteria.
- ErC50 (72 h): 0.971 mg/L
- Other: Last test dating from 15 - 25 Jan 2018

Reported statistics and error estimates:

Statistical analyses were performed with SAS (2002 - 2010). The following tests were applied: normality test (Shapiro-Wilk statistic), homogeneity of the variance of the data (Levene test). The NOEC and LOEC were determined by the multiple comparison method (Bonferroni-Holms corrected Welch test, left-sided). The effect concentrations (EC10, 20, and 50) were not indicated because growth inhibition was < 10% at all test item concentration levels for both yield and growth rate.

Any other information on results incl. tables

VALIDITY CRITERIA

The study met the validity criteria laid down by 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, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 59.05, which exceeds the threshold of 16.It corresponds to a growth rate of 1.36103 d^-1.	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 mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 16% and did not exceed 35%.	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 average growth rate in replicate control cultures was 2.2% and did not exceed 7% for the whole test period.	Yes

 

ANALYTICAL RESULTS

The measured initial concentration was 92% of nominal. In the aged sample taken at test end after 72 h the measured concentration was 93% of nominal (Table 2). Therefore, the toxicological endpoints were based on the nominal concentrations of the test item.

 

Table 2. Measured test item concentrations.

nominal concentration [mg/L]	sampling	test item
		[mg/L]	% of nominal
Control	0 h (fresh media)	< LOD	-
	24 h (aged media)	< LOD	-
	24 h (aged media)	< LOD	-
100 mg/L	0 h (fresh media)	92	92
	24 h (aged media)	92	92
	24 h (aged media)	93	93

- = not calculated; LOD = 0.30 mg/L test item; LOQ = 1.00 mg/L test item

 

BIOLOGICAL RESULTS

After 72 h, no concentration response relation was observed for the inhibition of growth rate and yield (Table 3). The inhibition of growth rate peaked at 1.00% at a nominal test item concentration of 10.0 mg/L (Table 4) and the inhibition of yield peaked at 3.4% at a nominal concentration of 100 mg/L.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) at test end. Thus, the overall LOEC was not determinable ant the overall NOEC was set at ≥ 100 mg/L (nominal). The effect concentrations for growth and yield inhibition are summarized in Table 5.

 

Table 3. Average cell numbers for each sampling time and concentration.

Concentration [mg/L]	Average cell numbers [1 E+04 cells/mL]
	0 h	24 h	48 h	72 h
Control	0.57	1.90	7.28	33.66
0.0100	0.57	2.01	9.12	43.32
0.100	0.57	2.37	9.30	45.61
1.00	0.57	1.79	7.63	34.30
10.0	0.57	2.39	8.89	32.56
100	0.57	2.26	8.77	36.40

 

  Table 4. 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
0.0100	-3.5	-8.5	-6.0
0.100	-17.3	-9.4	-7.3
1.00	6.4	-1.3	0.1
10.0	-18.3	-7.6	1.0
100	-13.3	-6.9	-1.7

 

Table 5. Toxicological endpoints for the test item.

	nominal test item concentration [mg/L]
Growth inhibition
ErC10 (72 h)	> 100
ErC20 (72 h)	> 100
Yield inhibition
EyC20 (72 h)	> 100
EyC50 (72 h)	> 100
NOEC*	≥ 100
LOEC*	-

* Following Bonferroni-Holms corrected Welch test (left-sided, p < 0.05) for growth rate and yield.

- No LOEC was observed

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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