1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996-draft

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline 72-4 (a) (1982)

Deviations:

no

Principles of method if other than guideline:

The afore mentioned guidelines were harmonized for various test parameters (i.e. temperature, light, etc.) to achieve optimal environmental conditions for the test organism. Scientific discretion was implemented where guideline parameters do not fully converge.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared as needed (approximately weekly). The appropriate amount of compound was weighed into a volumetric flask (corrected for percent a.i.). The flask was brought to volume with dimethylformamide and sonicated 25 minutes. A stir bar was added to the flask and the stock was placed on the diluter system.
- Controls: yes
- Chemical name of vehicle: Dimethylformamide
-

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow
- Source: In-house fathead minnow breeding facility, original source of organisms was Aquatic Biosystems, Ft. Collins, Colorado.
- Organism Age at Initiation: < 24 hours old eggs in the pre-blastula and blastula and neurula stages (determined via a light microscope)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs):
Adults per Aquaria: 2 males and 4 females
Number of Spawning Substrates per Aquaria: 3

- Method of collection of fertilised eggs: Eggs were removed from substrates contained in breeding tanks on the morning of the experimental start with nonviable eggs being discarded. Eggs appropriate for testing were pooled for impartial placement of eggs into randomly positioned egg cups
- Subsequent handling of eggs: 35 eggs at initiation, thinned to 20 alevin after hatching phase.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Hardness:

50 to 60 mg/L as CaCO3.

Test temperature:

23.3 to 26.2 °C

pH:

7.3 to 8.1

Dissolved oxygen:

5.0 to 7.8 mg/L (61 to 94% saturation)

Salinity:

-

Nominal and measured concentrations:

Nominal Test Concentrations: Control, Solvent Control, 63, 125, 250, 500 and 1000 μg a.i./L
Measured Test Concentrations: Control (<5.95), Solvent Control (<5.95), 59.11, 142.1, 286.0, 465.3 and 1013 μg a.i./L

Details on test conditions:

TEST SYSTEM
- Emybro cups: One oscillating egg cup per replicate placed in each replicate through the egg hatching phase
- Test vessel: All glass
- Size, headspace, fill volume: 8.4-L with a testing Volume of 7-L (21.6 x 12.7 x 25.5 cm)
- Type of flow-through: diluter system used for intermittent delivery of test solutions to test chambers. Splitter cups were used to equally divide (± 0%) test solutions between replicates at each level.
- Renewal rate of test solution (flow rate): Volume Turnover/Vessel/24 hours: Approximately 7 to 10
- No. of fertilized eggs/embryos per vessel: 35
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark (w/ 30 minute dawn/dusk transition period of lower light intensity)
- Light Intensity: 595 to 724 lux
- Light Source: Cool white fluorescents

EFFECT PARAMETERS MEASURED:
- Egg Observations: Daily for mortality (nonviable eggs removed) and hatching
- Thinning Phase: When at least 90% of all viable control eggs had hatched, observations were made and the alevin were impartially thinned to 20 per replicate by releasing them from the egg cup to the test vessel in which the egg cup had been suspended. One additional alevin was retained at thinning (21 instead of 20) in the Control B, and 500 μg a.i./L replicate A vessels. This error did not have a negative impact on the study results.
- Thinning Day: Day 5
- Post Hatch Phase: Begins after thinning
- Post Hatch Phase Observations: Daily for abnormal behavior, abnormal physical changes and mortality (dead organisms were removed).
- Length Measurements: Standard Length (nearest 0.5 mm) via a millimeter scale (all surviving fish on Day 35) after sacrificing fish via warm water emersion
- Wet Weight Measurements: Taken at termination for control fish to establish biomass loading estimate (nearest 0.01 g)
- Dry Weight Measurements: All surviving fish at Day 35 (nearest 0.0001 g)

VEHICLE CONTROL PERFORMED: yes

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: fry survivorship

CONCLUSIONS:

The 35-day exposure to AE 1170437 technical resulted in a NOEC of 465.3 μg a.i./L and a LOEC of 1013 μg a.i./L based on fry survivorship.

Percent Hatch (Time to hatch):

Day of Evaluation:	Days 4 to 5
Percent Hatch Determination:	Percent hatch on Day 4 or 5 = (alevin/total # eggs on Day 0) x 100
Day 4 Percent Hatch :	Control (61%), Solvent Control (62%), 59.11 (57%), 142.1 (61%), 286.0 (64%), 465.3 (64%) and 1013 (59%) μg a.i./L
Day 5 Percent Hatch :	Control (92%), Solvent Control (99%), 59.11 (94%), 142.1 (94%), 286.0 (93%), 465.3 (92%) and 1013 (96%) μg a.i./L
Control and Solvent Control Comparison Result:	Pooled
Passed Criteria for Normality and Homogeneity of Variance:	Yes
Statistical Analysis:	Parametric
Treatment Groups Significantly Different (P≤0.05) Controls:	None

Alevin and Fry Survival:

Day of Evaluation:	Day 5 (Alevin) based on the number of eggs at experimental start & Day 35 (Fry) based on the number of fry after thinning
Alevin Survival
Day 5 Alevin Percent Survival:	Control (92%), Solvent Control (96%), 59.11 (94%), 142.1 (94%), 286.0 (90%), 465.3 (90%), and 1013 (93%) μg a.i./L
Percent Inhibition From Pooled Control:	59.11 (-0.4%), 142.1 (-0.4%), 286.0 (4.2%), 465.3 (4.2%), and 1013 (1.1%) μg a.i./L

Day 5 survival: the percent survival after 5 days was compared and ranged from 90 to 96%.

Statistical analysis of this data indicated that there were no significant differences in any

treatment group as compared to the controls.

Fry Survival
Day 35 Fry Percent Survival:	Control (94%), Solvent Control (99%), 59.11 (94%), 142.1 (96%), 286.0 (93%), 465.3 (99%), and 1013 (3%) μg a.i./L
Percent Inhibition From Pooled Control:	59.11 (2.6%), 142.1 (0.0%), 286.0 (3.9%), 465.3 (-2.6%), and 1013 (97.4%) μg a.i./L

Day 35 survival: the percent survival after 35 days post hatch, or the percentage of live fish

present at the start of the growth period still alive at 35 days post hatch. Survival during this

phase of the study ranged from 3 to 99%. Statistical analysis indicated that the day 35 survival

in the highest treatment group was statistically different from the combined control and solvent

control survival.

Growth:

After 35 days of growth, the fish were sacrificed and measured for standard length and dry

weight. The mean lengths ranged from 21.6 to 23.5 mm. A comparison of the control groups

indicated that they should be pooled for comparison to the treatment levels. The dry weights

showed no percent inhibition at any test level as well as no statistically significant difference

between the lowest treatment group (59.11 μg a.i./L) and the pooled control group.

Dry weight:

Mean dry weights for fish at day 35 ranged from 46.2 to 66.2 mg. Analysis of the control groups

indicated a statistical difference from one another and that treatment level comparisons should be

made to the solvent control group. The dry weights showed no percent inhibition at any test

level as well as no statistically significant difference between the lowest treatment group (59.11

μg a.i./L) and the solvent control.

Description of key information

The substance shows long term effects in the early life stage test  to fry survival (NOEC of 0.46 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.46 mg/L

Additional information

Information about long term toxicity is not required under Regulation (EC) No 1907/2006 within this tonnage band but to complete the data set additional information are provided

A 35-day early life stage test according to OECD 210 was conducted with the test substance under flow-through conditions in the Fathead Minnow (Pimephales promelas). Based on analytically verified concentrations the NOEC was 0.46 mg /L test item due to fry survival.