Isooctadecanoic acid, reaction products with tetraethylenepentamine

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:

2021-2022

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:

yes

Remarks:

Deviations from the study plan occurred (see attache repport) but these deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 1000352370
Purity: 95%
Physical State/Appearance: Amber viscous liquid

Analytical monitoring:

yes

Details on sampling:

VERIFICATION OF TEST CONCENTRATIONS
Water samples were taken from the control, solvent control and test group on one occasion prior to the addition of the eggs and on Days 0, 5, 7, 14, 21, 28 and 32 of the test for quantitative analysis.
On each sampling occasion, two samples were taken from one replicate vessel per control, solvent control and test group, changing systematically amongst replicates throughout the test. All samples were analyzed on the day of sampling.

Duplicate sets of samples on each occasion were taken and stored frozen for further analysisif necessary.

Vehicle:

no

Details on test solutions:

TEST 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. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

EXPERIMENTAL PREPARATION
On Days -2 (pre-exposure) 0, 7, 14, 21 and 28, a nominal amount of test item (10 mg) was dissolved in DMF and the volumes adjusted to 100 mL with the aid of ultrasonication for approximately 5 minutes to give the 0.10 mg/mL solvent stock solution.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 μL/L of DMF.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test was initiated with newly fertilized eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were bred at Osage Catfisheries on 13 May 2021 and maintained in dechlorinated tap water with an activated carbon and biological filtration system.
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.
Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 24 June 2021 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Hardness:

The water hardness values were observed to range from 142 to 146 mg/L as CaCO3 at the start of the test and from 156 to 192 mg/L as CaCO3 at termination of the test.

Test temperature:

The temperature measurements recorded in a control replicate by a Testo temperature logger were shown to have been maintained at between 25.2 and 26.2 °C. The temperature recorded in each test vessel ranged from 25.8 to 26.4 °C.

pH:

the pH ranged from 8.0 to 8.4

Dissolved oxygen:

The dissolved oxygen concentration was maintained at least at 5.7 mg/L (equivalent to 70% ASV).

Nominal and measured concentrations:

Chemical analysis of the test preparations on Days 0, 5, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0026 to 0.0056 mg/L and so the results are based on average measured test concentration only. The duplicate sample for the test concentration was analyzed for Day 21 as the original sample produced a measured test concentration of less than the Limit of Quantification (LOQ) of 0.0024 mg/L. This gave a measured test concentration of 0.0026 mg/L. Although the duplicate analysis was very close to the LOQ, this value was used in the calculation of results as it was considered to be more reliable than a value of below the LOQ.

Analysis of the test preparations on Days 0, 5, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0026 to 0.0056 mg/L and so the results are based on the mean measured test concentration only, determined to be 0.0041 mg/L.

Details on test conditions:

DEFINITIVE TEST
EXPERIMENTAL PREPARATION
On Days -2 (pre-exposure) 0, 7, 14, 21 and 28, a nominal amount of test item (10 mg) was dissolved in DMF and the volumes adjusted to 100 mL with the aid of ultrasonication for approximately 5 minutes to give the 0.10 mg/mL solvent stock solution.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 μL/L of DMF.

EXPOSURE CONDITIONS
For the purpose of the definitive test, approximately 4.5 L glass vessels were used each containing approximately 3.8 L of test preparation. Four replicate vessels were used for each control and test concentration.
Dynamic continuous flow test conditions were employed in the test. Dilution water was dosed at a flow rate of 120 mL per minute using Tacmina solenoid-driven metering pumps and the solvent stock solutions at a flow rate of 0.36 mL per hour using a KDS Legato Syringe Pump. The dilution water and solvent stock solutions were combined in separate mixing vessels at each concentration and delivered to the test vessels using a four way splitter at 30 mL per minute to each replicate, to give the required test concentration of 0.0050 mg/L.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 μL/L of DMF.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at approximately 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 32 days.
The dilution water in the header tank was aerated.
The larvae were fed freshly hatched brine shrimp nauplii from Day 6 to Day 31.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Key result

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

32 d

Dose descriptor:

LC50

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch Survival

Key result

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch Survival

Key result

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

5 d

Dose descriptor:

EC10

Effect conc.:

> 0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

NUMBER OF EGGS HATCHING
The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed.
The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 5 of the test. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.
Statistical analysis of the hatching data was carried out for the pooled control and 0.0041 mg/L test group. There were no statistically significant differences (P≥0.05), between the pooled control and 0.0041 mg/L test group and therefore the NOEC based on the number of eggs hatching was 0.0041 mg/L.

POST-HATCH SURVIVAL
The number of dead larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed. There were no significant mortalities observed in any of the test concentrations.
Statistical analysis of the post-hatch survival data was carried out for the pooled control and 0.0041 mg/L test group. There were no statistically significant differences (P≥0.05), between the pooled control and 0.0041 mg/L test group and therefore the NOEC based on post-hatch survival was 0.0041 mg/L.

LENGTH AND WEIGHT DATA
There were no statistically significant differences (P≥0.05), between the pooled control and 0.0041 mg/L test group and therefore the NOEC based on body length was 0.0041 mg/L.
There were no statistically significant differences (P≥0.05), between the pooled control and 0.0041 mg/L test group and therefore the NOEC based on wet weight was 0.0041 mg/L.

OBSERVATIONS
Abnormal observations were seen in the control, solvent control and test group and included the following observations; kinked spine, under-developed and lying on the bottom of the vessel.

WATER QUALITY CRITERIA
The temperature measurements recorded in a control replicate by a Testo temperature logger were shown to have been maintained at between 25.2 and 26.2 °C. The temperature recorded in each test vessel ranged from 25.8 to 26.4 °C. The dissolved oxygen concentration was maintained at least at 5.7 mg/L (equivalent to 70% ASV) and the pH ranged from 8.0 to 8.4. Throughout the test the light intensity was observed to be in the range 647 to 768 Lux.
The water hardness values were observed to range from 142 to 146 mg/L as CaCO3 at the start of the test and from 156 to 192 mg/L as CaCO3 at termination of the test.

OBSERVATIONS ON TEST ITEM SOLUBILITY
After dosing all control, solvent control and test concentrations were observed to be clear, colorless solutions by visual inspection.

RANGE-FINDING TEST

The results showed there was no difference between the controls and test concentrations in terms of hatching, survival and growth.
No sub-lethal effects were observed at the test concentrations of 0.010 and 0.10 mg/L. Abnormal swimming was observed in the control and at the test concentration of 0.0010 mg/L and kinked spine was observed in the solvent control.
Based on this information and further information of solubility provided by the sponsor, a single test concentration of 0.0050 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 0.0050 mg/L, no adverse reactions to exposure were observed.
Chemical analysis of the fresh test preparations on Day 0 showed measured test concentrations to range from less than the limit of detection (LOD) of the analytical method to 0.085 mg/L. Measured test concentration of the corresponding old test preparations on Day 2 showed measured test concentrations to range from less than the LOD to 0.066 mg/L indicating that the test item was not stable under test conditions.
Samples were also taken on Day 2 of the freshly prepared media which showed measured concentrations of between less than the LOD and 0.096 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Analysis of the test preparations on Days 0, 5, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0026 to 0.0056 mg/L and so the results are based on the mean measured test concentration only, determined to be 0.0041 mg/L.

There were no significant effects resulting from the exposure of fathead minnow eggs and larvae throughout the test at a concentration of 0.0041 mg/L.

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of the early-life stages of fathead minnow (Pimephales promelas) larvae to a test concentration of 0.0041 mg/L. The No Observed Effect Concentration (NOEC) was 0.0041 mg/L. This study showed that there were no toxic effects at the limit of solubility.

Executive summary:

INTRODUCTION
A study was performed to assess the effects of the test item on the early-life stages of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.

METHODSPreliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.Preliminary media preparation trials conducted for the Algal Growth Inhibition Test (Study Number GN21WB) indicated that the maximum solubility of the test item was 0.0050 mg/L, achievable in the test media via solvent addition.
Based on the results of a preliminary range-finding test and initial test, newly fertilized fathead minnow eggs (four replicates of 20 eggs per group) were exposed to a solution of the test item at a nominal concentration of 0.0050 mg/L for a period of 32 days (28 days post-hatch) at a temperature of approximately 26 ºC under dynamic test conditions. The test solutions were continuously renewed throughout the test. An initial solvent stock solution was prepared by dissolving 10 mg of test item in a final volume of 100 mL of dimethylformamide (DMF) to give a 0.10 mg/mL solvent stock solution. The bulk stock solutions were prepared weekly and stored refrigerated when not in use. The stock solution was sonicated for 5 minutes prior to being inverted several times to ensure adequate mixing and homogeneity. The dilution water and solvent stock solutions were combined in separate mixing vessels and delivered to the test vessels at 30 mL per minute to each replicate to give the required test solution of 0.0050 mg/L.
The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

RESULTS

Analysis of the test preparations on Days 0, 5, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0026 to 0.0056 mg/L and so the results are based on the mean measured test concentration only, determined to be 0.0041 mg/L.

There were no significant effects resulting from the exposure of fathead minnow eggs and larvae throughout the test at a concentration of 0.0041 mg/L.

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of the early-life stages of fathead minnow (Pimephales promelas) larvae to a test concentration of 0.0041 mg/L. The No Observed Effect Concentration (NOEC) was 0.0041 mg/L. This study showed that there were no toxic effects at the limit of solubility.

Description of key information

A fish early life test was conducted at test concentrations up to the limit of solubility for the test material. Analysis of the test preparations on Days 0, 5, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0026 to 0.0056 mg/L and so the results are based on the mean measured test concentration only, determined to be 0.0041 mg/L.

There were no significant effects resulting from the exposure of fathead minnow eggs and larvae throughout the test at the highest concentration of 0.0041 mg/L.

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of the early-life stages of fathead minnow (Pimephales promelas) larvae to the highest test concentration of 0.0041 mg/L. The No Observed Effect Concentration (NOEC) was 0.0041 mg/L. This study showed that there were no toxic effects at the limit of solubility.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

No effects observed at saturation

Effect concentration:

> 0.004 mg/L