Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

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

Study period:

17 May - 04 Nov 2021

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)

Version / remarks:

2013

Deviations:

yes

Remarks:

Due to a technical problem, the temperature at one day was too high, resulting in a deviation in temperature between successive days. This deviation is considered to have no impact on integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations (nominal): 0 (control, solvent control), 1.0 mg/L
- Sampling method: Duplicate water samples were taken from freshly prepared test medium and aged media (appr. 1 d).
Days 0, 7, 14, 21, 28 and 32 and from the old media on Days 1, 8, 15, 22, 29 and 33 (from a single replicate of each treatment group, changing systematically amongst replicates throughout the test).
- Sample storage conditions before analysis: Samples were analyzed immediately on the day of sampling.

Vehicle:

yes

Remarks:

Dimethylformamide

Details on test solutions:

PRELIMINARY STUDY
Preliminary media preparation: Preliminary information indicated the water solubility of the test item to be less than 0.050 mg/L. A preliminary media preparation trial indicated that a solvent spike method of preparation at a nominal concentration of 1.0 mg/L, followed by the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap Filter (first approximate 500 mL discarded in order to pre-condition the filter) was most appropriate for this test item.
Preliminary stability: A 1.0 mg/L was prepared as described for the definitive test with and without filtration. Chemical analysis on days 0 and 3 indicated, that the test item is unstable under test conditions for 3 d. Therefore, a semi-static test design with daily renewal was considered appropriate.

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (100 mg) was dissolved in dimethylformamide and the
volume adjusted to 10 mL to give a 10 mg/mL solvent stock solution. This stock solution was dispersed in test water with the aid of magnetic stirring for 15 min to give a 1.0 mg/L test solution. To remove any undissolved test material, this test solution was filtrated (days 0 - 15: 0.2 µm Gelman Acrocap filter, preconditioned with appr. 500 mL test solution; days 16 - 33: 0.2 mm Sartorius Sartopore filter, preconditioned with appr. 1000 mL test solution)
- Controls: water control and solvent control
- Chemical name of vehicle (organic solvent): dimethylformamide
- Concentration of vehicle in test medium: 100 µl/L for test concentration and solvent control
- Evidence of undissolved material: none reported after filtration

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Adults for egg production from commercial supplier
- Breeding conditions: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the 7 days preceding the start of the test, the water temperature was controlled at approximately 25 °C with a dissolved oxygen content of greater than or equal to 7.3 mg O2/L. The breeding stock fish were fed daily with frozen brine shrimp. Fish were hold in dechlorinated tap water with activated charcoal and biological filtration systems.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized.
- Subsequent handling of eggs: The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage


POST-HATCH FEEDING
- Start date: 29 Jul 2021
- Type of feed: brine shrimp nauplii, less than 24 h old
- Amount given: ad libitum

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

33 d

Hardness:

134 - 170 mg/L as CaCO3

Test temperature:

23 - 25°C

pH:

7.6 - 8.5

Dissolved oxygen:

≥ 82% air saturation (6.8 mg/L]

Nominal and measured concentrations:

Nominal: 0 (control, solvent control), 1.0 mg/L
Geometric mean measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessel
- Material: glass
- Size: day 0 - 13: 1 L, day 14 - 33: 5 L
- Fill volume: day 0 - 6: 400 mL, day 7 - 13: 800 mL, day 14 - 33 : 4000 mL
- Aeration: constant aeration after day 8
- Renewal rate of test solution: daily renewal of test solution, expect for day 3 (to avoid causing premature hatching of the eggs)
- No. of fertilized eggs/embryos per vessel: 20
- 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: The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination unit) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. The tap water is checked at least yearly for pesticides, chlorine and metals.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark, with 20 min transition period
- Light intensity: 715 - 877 lux

EFFECT PARAMETERS MEASURED:
Hatching of eggs, post-hatch survival (daily observation)
body length, individual wet weight ( at the end of the definitive test)

VEHICLE CONTROL PERFORMED: yes


POST-HATCH DETAILS
- Begin of post-hatch period: day 5
- No. of hatched eggs (alevins)/treatment released to the test chamber: ≥15 eggs per replicate hatched
- Post-hatch feeding: The larvae were fed less than 24 hour old brine shrimp nauplii from day 7 to day 32.

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.081 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.081 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.081 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.081 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

- Mortality/survival at embryo and juvenile stages: Hatching rate: Control: 80%, Solvent control: 86%, 0.081 mg/L: 84%; mean survival post-hatch: Control: 100%, Solvent control: 100%, 0.081 mg/L: 99%
- Days to hatch: 6 d
- Observations on body length and weight of young: No difference between controls and test item concentration
- Type of and number with morphological abnormalities: none
- Type of and number with behavioural abnormalities: none
- Type and number of developmental effects: none
- Incidents in the course of the test which might have influenced the results: none

Reported statistics and error estimates:

Analysis of Number of Eggs Hatching
Analysis of the number of eggs successfully hatching from the pooled controls and
0.081 mg/L test group were compared using the Chi squared 2x2 Table test. The total
number of eggs hatched from the pooled controls and 0.081 mg/L test group were used for
this analysis.

Analysis of Post-Hatch Survival
Analysis of the post-hatch survival from the pooled controls and 0.081 mg/L test group were
compared using the Chi squared 2x2 Table test. The total post-hatch survival of the pooled
controls and 0.081 mg/L test group were used for this analysis.

Analysis of Fish Length Data
Analysis of the fish length data obtained at termination of the test from the pooled controls
and 0.081 mg/L test group were compared using a Student t-test incorporating Levene’s Test
on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution. Mean and
standard deviation values from the fish length data of the pooled control and 0.081 mg/L test
group were used for this analysis.

Analysis of Fish Wet Weight Data
Analysis of the fish weight data obtained at termination of the test from the pooled controls
and 0.081 mg/L test group were compared using a Student t-test incorporating Levene’s Test
on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution. Mean and
standard deviation values from the fish wet weight data of the pooled controls and
0.081 mg/L test group were used for this analysis.

This study showed that there were no adverse effects on the post hatch survival and growth of P. promelas at the limit of solubility of the test item after exposure to 33 d.

Table: Summary of observations in fish

(n=20)

Geometric mean measured concentration (mg/L)	Replicate	Number of dead eggs	Hatching rate (%)	Number of dead fish	Survival rate (%)	Average length at test end (mm)	Average weight at test end (mg)
Control	1	5	75	0	100	22.07	87.1
	2	5	75	0	100	22.39	89.3
	3	4	80	0	100	21.88	82.8
	4	2	90	0	100	22.24	85.5
Solvent control	1	1	95	0	100	21.72	83.1
	2	3	85	0	100	21.86	84.9
	3	2	90	0	100	21.84	81.4
	4	5	75	0	100	21.91	83.2
0.081	1	2	90	1	94	21.70	81.5
	2	5	75	0	100	21.84	82.1
	3	1	95	0	100	21.35	77.7
	4	5	75	0	100	21.49	81.3

 

Analysis of the fresh test preparations on Days 0, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.00027 to 0.53 mg/L. Analysis of the corresponding aged test preparations on Days 1, 8, 15, 22, 29 and 33 showed measured test concentrations to range from 0.00011 to 0.43 mg/L.
Given the variability in the measured test concentrations obtained from the freshly prepared test media over the 33-Day test period it was considered justifiable to calculate the results based on the geometric mean measured test concentration which was determined to be 0.081 mg/L.

Table: Summary of analytical results

Time point (d)	Description of solution	Sample concentration nominal (mg/L)	Measured concentration (mg/L)	Nominal recovery (%)	Range of Procedural recovery
0	-	Control	<LOD	-	101 – 106%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.000329	<1
1	Old medium	Control	<LOD	-	102 – 112%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.000184	<1
7	Fresh medium	Control	<LOD	-	111 – 120%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.000270	<1
8	Old medium	Control	<LOD	-	115%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.000111	<1
14	Fresh medium	Control	<LOD	-	102 – 110%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.529	52.9
15	Old medium	Control	<LOD	-	105 – 110%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.305	30.5
21	Fresh medium	Control	<LOD	-	109 – 115%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.00109	<1
22	Old medium	Control	<LOD	-	109 – 115%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.0111	1
28	Fresh medium	Control	<LOD	-	108 – 131%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.00743	<1
29	Old medium	Control	<LOD	-	89 - 94%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.00151	<1
32	Fresh medium	Control	<LOD	-	89 - 94%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.00120	<1
33	Old medium	Control	<LOD	-	89 - 94%, <LOD in negative control
		Solvent control	<LOD	-
		0.081	0.000896	<1

 

Table 3: Validity criteria of the guideline OECD 210 (2013)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.	>82%	yes
The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2).	Water temperature: Between test chambers ±0.7°C Between successive days ±1.8°C	yes*
The analytical measure of the test concentrations is compulsory.	Analytical values showed different test item concentration at different time points, therefore, geometric mean measured values were used for evaluation.	yes
Overall survival of fertilised eggs and post-hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to the limits defined in Annex 2.	Hatching success 80% and post-hatch survival 100% in control vessels.	yes

* = On Day 12 of the definitive test the circulation pump present in the water bath was noted to have stopped working. As a consequence the temperatures measured in each of the replicate vessels had dropped to 23 °C. Such a decline resulted in a daily temperature fluctuation in excess of the permitted 1.5 °C. This deviation is considered to have had no adverse effect on the outcome or integrity of the test given that no mortalities or abnormalities in appearance or behaviour were noted at this time.

Validity criteria fulfilled:

yes

Remarks:

for details see Table in "Any other information on results incl. tables"

Description of key information

NOEC 0.081 mg/L (water solubility limit) for hatching rate, post-hatch survival, length and weight after 33 d in Pimephales promelas

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

weight

Effect concentration:

0.081 mg/L

Fresh water fish

Dose descriptor:

NOEC

Remarks:

length

Effect concentration:

0.081 mg/L

Fresh water fish

Dose descriptor:

NOEC

Remarks:

post-hatch survival

Effect concentration:

0.081 mg/L

Fresh water fish

Dose descriptor:

NOEC

Remarks:

Hatching success

Effect concentration:

0.081 mg/L

Additional information

One study evaluating the long-term toxicity of propane-1,2,3 triyl 3,5,5-trimethylhexanoate (CAS No. 56554-53-1) to fish species is available. This test was conducted according to OECD 210, under GLP conditions. Starting with freshly fertilized eggs, Pimephales promelas were exposed to the test substance for 33 days within a semi-static (daily renewal, except day 3) test, at a nominal concentration of 1.0 mg/L (limit test). Dechlorinated and partially softened (140 mg CaCO3/L) tap water was used as test medium.

A preliminary media preparation test was performed due to the poorly soluble nature of the substance (water solubility < 0.05 mg/L). The results from this pre-test showed that a solvent spike method was the most appropriate method for media preparation. The test solution was then prepared using a solvent spike method (with dimethylformamide, DMF). Therefore, an initial concentration of 1 mg/L propane-1,2,3 triyl 3,5,5-trimethylhexanoate (CAS No. 56554-53-1) was stirred for approximately 15 minutes prior to removal of undissolved test substance by filtration (0.2 µm pore size). Furthermore, a control with untreated test medium and a solvent control with DMF, but without test substance, were performed. The test medium concentration of propane-1,2,3 triyl 3,5,5-trimethylhexanoate (CAS No. 56554-53-1) was analytically determined by means of LC-MS/MS. Samples were taken every 7 d (5 d for the last interval) from fresh and aged solution in the treatment, control and solvent control groups. Concentration of test item in the solutions showed values >LOD/LOQ for control and solvent control at all time points. Analysis of the fresh test preparations on days 0, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.00027 to 0.53 mg/L. Analysis of the corresponding aged test preparations on days 1, 8, 15, 22, 29 and 33 showed measured test concentrations to range from 0.00011 to 0.43 mg/L. Therefore, effect values are based on the geometric mean measured concentration of 0.081 mg/L.

After 33 d of exposure of eggs and sac-fry stages to propane-1,2,3 triyl 3,5,5-trimethylhexanoate (CAS No. 56554-53-1), no effects on hatching success, post-hatch survival, length or weight were observed at the tested concentration. Therefore, the 33 d NOEC for all endpoints was determined to be ≥ 0.081 mg/L (geometric mean measured concentration). Hence, no toxicity was observed up to the limit of water solubility.