Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

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:

20 April - 24 May 2018

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:

adopted 23 March 2006, Annex 5 corrected: 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt Baden-Württemberg, Karlsruhe, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: highest test item loading rate and control
- Sampling method: Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions. For each sampling also a retain sample was taken. 500 μL samples were stabilised with 500 μL acetonitrile.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions of the three highest concentrations (100, 10 and 1 mg/L) were prepared as Water-Accommodated Fractions (WAFs) due to poor solubility of the test substance. These stock solutions were stirred in the dark at room temperature for 24 h (based on OECD Series on Testing and Assessment No. 23).The two lowest concentrations were prepared by appropriate dilution of the 1 mg/L test solution). Algae were added to each solution individually.
- Eluate: no
- Differential loading: yes, for the three highest test concentrations
- Controls: yes, blank control
- Evidence of undissolved material: After stirring of the three stock solutions, the solutions were clear and transparent but not the entire test item went into solution. White deposits were observed on the surface and on the bottom of the solutions. Subsequently the undissolved test item was allowed to sediment and/or float for a period of 30 min until the phases had separated. After the settling the necessary volume for the test was withdrawn via a Teflon tube from the medium level of the stock solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: SAG 61.81
- Source: purchased from a commercial supplier, MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum: 3-4 days
- 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: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 μEm^-2s^-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: none observed

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.3 – 23.1 °C

pH:

7.62 to 8.63

Nominal and measured concentrations:

nominal loading rates: 100, 10, 1, 0.1, 0.01 mg/L and control
measured: 100 mg/L: 3% of nominal at 0 h (fresh); < LOQ at 72 h (aged); Control: < LOQ at 0 h (fresh) and 72 h (aged)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution.
- Initial cells density: 0.5 × 10E+04 cells/mL
- Control end cells density: 43.7 × 10E+04 cells/mL (mean, 72 h)
- 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
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at 0, 24, 48 and 72 h.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: adjusted to 7.5 ± 0.1 with NaOH
- Photoperiod: continuous light
- Light intensity and quality: 88.1 μEm^-2s^-1 (mean); laterally placed fluorescent tubes

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Fluorescence measurement, performed with fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0). At tets start (t = 0h), the number of cells in each control replicate was determind in duplicates. At defined intervals (24, 48 and 72 h), the number of cells in each replicate was determined in duplicates.
- The morphological appearance of the cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Based on the results of the range-finding test, the final test was designed as a limit test. Further information is not included in the report.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate; tested twice a year in a separate study

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

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: yes
- Observation of abnormalities: At test end, the cells were considered normal for the control and up to and including a nominal test item loading rate of 100 mg/L.
Any observation (e.g. precipitation) that might cause a difference between measured and nominal values: After 24 h, 48 h and 72 h white particles at the surface of the solutions at 100 mg/L and 10.0 mg/L loading rate could be observed.
- Other: No LOELR was observed.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 0.971 mg/L, ErC10: 0.582 mg/L

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR were determined by using a multiple comparison method (Jonckheere Terpstra test, left sided, for growth rate and yield). The calculation of the EL10, 20, 50 was not indicated since the inhibition was below 10 % at the highest test item loading rate for both, yield and growth rate and hence the database was inappropriate for probit analysis.

Table 1: Percentage inhibition of growth rate

Loading rate [mg/L]	% Inhibition of growth rate
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.0100	9.0	2.9	-0.7
0.100	-5.0	-2.7	-3.5
1.00	-25.7	-14.3	-13.8
10.0	-22.4	-11.8	-10.8
100	-26.1	-12.7	-12.8

Table 2: Percentage inhibition of yield

Loading rate [mg/L]	% Inhibition of yield
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.0100	16.3	10.5	-1.5
0.100	-7.5	-7.0	-15.2
1.00	-53.1	-56.6	-81.2
10.0	-44.4	-44.4	-58.9
100	-53.8	-48.5	-74.2

Table 3: Analytical results

Test item nominal [mg/L]	Sampling	Test item found
		[mg/L]	% of nominal
Control	0 h fresh	< LOQ	-
	72 h aged	< LOQ	-
100	0 h fresh	3.15	3
	72 h aged	< LOQ	-

Table 4: Validity criteria

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.	82.45	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%	23%	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%.	5.9%	yes

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

ErL50 > 100 mg/L (nominal, OECD 201)

ErL10 ≥ 100 mg/L (nominal, OECD 201)

Additional information

The substance showed no toxicity to aquatic algae up to the highest tested nominal loading rate (100 mg/L; OECD 201, Pseudokirchneriella subcapitata).