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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 22 March 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Limit test design without replication and without reference item.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

Adopted: 17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt Baden-Württemberg, Karlsruhe, Germany

Analytical monitoring:

yes

Details on sampling:

- Sampling method and concentrations: Analytical samples were taken in 24 h intervals from fresh and aged test solutions from the control and the 100 mg/L loading rate. For each sampling also a retain sample was taken. 500 µL samples were taken and stabilized 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 necessary amounts of test item for preparing the stock solutions were weighed on a weighing scoop and transferred to separate Schott bottles for each test item loading rate. These bottles were filled with 2 L of test medium and these solutions were stirred at room temperature in the dark for 24 hours. Afterwards the undissolved test item was allowed to sediment and/or float for a period of two hours. The solutions were withdrawn from the middle of the vessel using a Teflon tube and transferred to the test vessels (2 L-beakers). Transfer of undissolved test item was avoided. For each test loading rate, a separate solution was prepared to achieve the corresponding nominal loading rate.
- Eluate: no
- Differential loading: yes
- Controls: blank control
- Evidence of undissolved material: The test item solutions showed a slight flocculation on the surface. At 100 mg/L, the test item solution was turbid.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio Hamilton
- Source: Originally from Fraunhofer IME (Schmallenberg, Germany), bred from wild strain zebra fish; Now reared in the laboratory of the testing facility.
- Method of breeding: The fish were cultured at a constant temperature of 21 to 25 °C within a range of 2 °C in a climate controlled room with 16 hours of illumination and 8 hours of darkness. The animals were fed with commercial fish flake food (TetraMin) and protein-enriched pellet food (Caviar) at least once per day.

ACCLIMATION
- Acclimation conditions not stated
- Health during acclimation: Only fish in good health and free from any apparent malformation were used.

FEEDING DURING TEST
- no feeding during test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Ca:Mg ratio 4:1
Na:K ratio 10:1
13°dH corresponding to 231 mg CaCO3/L

Test temperature:

23.9 – 24.8 °C

pH:

7.32 – 7.69

Dissolved oxygen:

85 ± 7 % oxygen saturation

Conductivity:

679 µS/cm

Nominal and measured concentrations:

nominal loading rates: blank control, 1, 10 and 100 mg/L
measured, geometric mean (only blank control and nominal 100 mg/L): < LOQ, 3.16 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2L glass beakers filled with 1.5 L of test solution
- Aeration: none
- No. of organisms per vessel: 7 for control and 100 mg/L; 3 at test loading rates 1.00 and 10.0 mg/L
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: max. loading of 0.138 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, pH-value and % oxygen saturation of the test solutions, measured after 0, 24, 48, 72 and 96 hours from fresh and aged test solutions, are reported. Hardness of the test water was measured at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hours light / 8 hours darkness

EFFECT PARAMETERS MEASURED: Fish were observed at 0, 4, 24, 48, 72 and 96 hours after test start. Fish were considered dead if there was no visible movement (e.g. gill movement), and if touching of the caudal peduncle produced no reaction. Records were made on visible abnormalities as: loss of equilibrium, swimming behaviour, respiratory function, pigmentation and all other observed events. Dead fish were removed if observed and mortality, length and weight was recorded. At termination of the test, all remaining fish were euthanized and all fish were weighed and measured.

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):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 3.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: could not be statistically determined, but assumed to be greater than the highest test concentration

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: could not be statistically determined, but assumed to be greater than the highest test concentration

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: No sublethal effects were observed in the control and test item loading rate 100 mg/L test item (nominal) after 96 h.
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item solutions showed a slight flocculation on the surface. At 100 mg/L, the test item solution was turbid.
- Due to a lack of mortality, no LL50 values could be statistically determined.
- All fish were weighed and measured at test end. The average weight of the test organisms was 0.0256 ± 0.0094 g; the average length was 15 ± 2 mm.

Reported statistics and error estimates:

The LL50-values are the estimated test item loading rates causing 50 % mortality in the test organism population.
Due to a lack of mortality, no LL50 values could be statistically determined.
The NOELR was established based on the highest test item loading rate at which fish are alive within the allowed control mortality.
All toxicological endpoints were evaluated using nominal loading rates and geometric mean measured concentrations.

Sublethal observations / clinical signs:

Table 1: Statistical Results:   LL₅₀/LC₅₀ and NOELR/NOEC-values for fish exposed to the test item based on nominal loading rates and mean measured concentrations

N,N',N''-Tris(methoxymethyl)-N,N',N''-tris[(octadecyloxy)methyl]-1,3,5-triazine-2,4,6-triamine nominal and geometric mean measured
Time [h]	NOELR mortality nominal [mg/L]	NOEC mortality Mean measured [mg/L]	LL50 nominal [mg/L]	LC50 mean measured [mg/L]	95 % confidence limit of LL50 [mg/L]
4	100	3.16	>100^1)	>3.16^1)	-
24	100	3.16	>100^1)	>3.16^1)	-
48	100	3.16	>100^1)	>3.16^1)	-
72	100	3.16	>100^1)	>3.16^1)	-
96	100	3.16	>100^1)	>3.16^1)	-

¹⁾        could not be statistically determined, but assumed to be greater than the highest test concentration

-      not calculable due to inappropriate data or mathematical reasons

Table 2: Determined concentrations of the test item during the test

Test item nominal [mg/L]	Sampling	N,N',N''-Tris(methoxymethyl)-N,N',N'' -tris[(octadecyloxy)methyl]-1,3,5-triazine-2,4,6-triamine		Mean fresh [%]	Test item mean measured [mg/L]
		[mg/L]	% of nominal
0	0 h fresh	n.d.	< LOQ	-	-
	96 h fresh	n.d.	< LOQ
100	0 h fresh	2.56	2.56	3.16	3.16
	96 h fresh	3.89	3.89

- = not calculable, n.d. = not detectable

Table 3: Validity Criteria of the Study

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0%	yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	≥ 74 %	yes

 

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

LL50 (96 h) > 100 mg/L (nominal, OECD 203)

LC50 (96 h) > 3.16 mg/L (geometric mean measured, OECD 203)

NOEC (96 h) = 100 mg/L (nominal, OECD 203)

NOELR (96 h) = 3.16 mg/L (geometric mean measured, OECD 203)

Additional information

In a GLP limit study conducted according to the OECD guideline 203 and using the zebrafishDanio rerioas the test organism, the LL50/LC50 (96 h) of the test item was determined to be > 100 mg/L (nominal) and > 3.16 mg/L (geometric mean measured), respectively. The corresponding NOELR/NOEC (mortality) (96 h) was 100 mg/L (nominal) and 3.16 mg/L (geometric mean measured), respectively.

No sublethal effects were observed in the control and test item loading rate 100 mg/L test item (nominal) after 96 h.