Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-heptanoic, n-octanoic and n-decanoic acids

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 mg/L
- Sampling method: HPLC
- Sample storage conditions before analysis: Room temperature in the dark

Vehicle:

no

Details on test solutions:

Nominal amounts of test item (25, 50, 100, 200 and 400 mg) were each separately added to the surface of 20 L of test water to give the 1.25, 2.5, 5.0, 10 and 20 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. The aqueous phase or WAF was removed by mid-depth siphoning (the first 100 mL discarded) to give the 1.25, 2.5, 5.0, 10 and 20 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. Prior to dispensing the test item, the siphon tube was inserted into the test media in order to avoid disturbing test item on the surface.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test was carried out using newly fertilized eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were obtained from Osage Catfisheries, Osage Beach, Missouri, USA and maintained in-house since 24 June 2021 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 24 to 25 °C with a dissolved oxygen content of greater than or equal to 6.7 mg O2/L. The breeding stock fish were fed daily with ZM small granular food and frozen brine shrimp.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

120 to 152 mg/L as CaCO3 at the start of the test and from 122 to 154 mg/L as CaCO3 at termination of the test

Test temperature:

25 °C with a maximum deviation of ± 1.5 °C

pH:

7.9 to 8.4

Dissolved oxygen:

> 7.1 mg/L

Conductivity:

513 μS/cm at 20 °C

Nominal and measured concentrations:

Nominal: 1.25, 2.5, 5.0, 10 and 20 mg/L loading rate WAF

Measured: Analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.000108 to 0.00184 mg/L. Analysis of the old test preparations on Days 1, 8, 15, 22, 29 and 33 showed measured test concentrations to range from 0.000110 to 0.00407 mg/L.

Details on test conditions:

TEST SYSTEM
In the definitive test 1 L glass vessels were used from Day 0 to Day 15 and from Day 15 to the end of the test 5 L glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 8, 800 mL from Day 8 to Day 15 and 4000 mL from Day 15 to Day 33. Four replicate flasks were used for each control and test concentration. A semi-static test regime was employed in the test involving a renewal of the test preparations three times per week from the start of the test to prevent the build-up of nitrogenous waste products.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.
The test vessels were aerated via narrow bore glass tubes from Day 8 onwards. The eggs and larvae were not individually identified. The larvae were fed freshly hatched brine shrimp nauplii from Day 7 to Day 32.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heartbeat, white opaque coloration and lack of reaction to mechanical stimulus.
At the end of the test, the length and wet weight of each surviving fish was determined.

VEHICLE CONTROL PERFORMED: N/A

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L loading rates WAF
- Results used to determine the conditions for the definitive study: Yes

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

Number of Eggs Hatching
The numbers of dead eggs observed during the definitive test are given in tabular form below in the section entitled "Any other information on results incl. tables". The number of dead eggs observed was low throughout the test. One egg was recorded to be dead on Day 1; however, 20 eggs were later confirmed to be present on Day 4. Considering that 20 eggs had been confirmed to have been added on Day 0 and as the numbers recorded post Day 4 corresponded with the number of live fish measured at the end of the test, the dead egg found on Day 1 is considered to have been recorded in error and therefore, not to have impacted the test. The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 6 of the test. Statistical analysis of the hatching data was carried out for the control and all loading rates. There were no statistically significant differences (P≥0.05), between the control and 1.25, 2.5, 5.0, 10 and 20 mg/L loading rate WAF test groups and therefore the NOEL based on the number of eggs hatching was 20 mg/L loading rate WAF. Correspondingly the LOEL based on the number of eggs hatching was not determined.

Post-hatch Survival
The number of hatched (live) larvae observed during the definitive test are given in tabular form below in the section entitled "Any other information on results incl. tables". 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 control and all loading rates. There were no statistically significant differences (P≥0.05), between the control and 1.25, 2.5, 5.0, 10 and 20 mg/L loading rate WAF test groups and therefore the NOEL based on post-hatch survival was 20 mg/L loading rate WAF. Correspondingly the LOEL based on post-hatch survival was not determined.

Length and Weight Data
The body length and wet weight data for all fish alive at the end of the test are given in tabular form below in the section entitled "Any other information on results incl. tables". Statistical analysis of the fish body length data was carried out for the control and all loading rates. There were no statistically significant differences (P≥0.05), between the control and 1.25 mg/L loading rate WAF test group, however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on body length was 1.25 mg/L loading rate WAF. Correspondingly the LOEL based on body length was 2.5 mg/L loading rate WAF.

Weight Concentration
Statistical analysis of the fish wet weight data was carried out for the control and all loading rates. There were no statistically significant differences (P≥0.05), between the control, 1.25 and 2.5 mg/L loading rate WAF test groups however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on wet weight was 2.5 mg/L loading rate WAF. Correspondingly the LOEL based on wet weight was 5.0 mg/L loading rate WAF.

Observations
Abnormal observations were seen in the Control and 1.25, 2.5 and 10 mg/L loading rate test groups and included the following observations: pale fish and loss of equilibrium. The numbers of fish recorded with these observations were very low, therefore, these observations are considered to be due to defects occurring in a natural population, and not necessarily attributable to exposure to the test item. There were no sub-lethal effects observed in the 5.0 and 20 mg/L test groups throughout the test.

Endpoint		Concentration (mg/L loading rate WAF) [95% Confidence Limits]
Number Hatched	^LL10	10 (not possible to calculate 95% confidence limits)
	^LL20	>20 [Not determined]
	^LL50	>20 [Not determined]
	No Observed Effect Loading Rate	20
	Lowest Observed Effect Loading Rate	Not determined
Post-hatch Survival	^LL10	>20 [Not determined]
	^LL20	>20 [Not determined]
	^LL50	>20 [Not determined]
	No Observed Effect Loading Rate	20
	Lowest Observed Effect Loading Rate	Not determined
Body Length	^EL10	>20 [Not determined]
	^EL20	>20 [Not determined]
	^EL50	>20 [Not determined]
	No Observed Effect Loading Rate	1.25
	Lowest Observed Effect Loading Rate	2.5
Wet Weight	^EL10	3.1 [0.91 to 11]
	^EL20	12 [3.2 to 50]
	^EL50	>20 [Not determined]
	No Observed Effect Loading Rate	2.5
	Lowest Observed Effect Loading Rate	5.0

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the study, the substance does not meet the classification requirements for hazardous to aquatic life

Executive summary:

In an OECD 210 GLP study, Fathead Minnows were exposed to 0. 1.25, 2.5, 5, 10 and 20 mg/L WAF for 21 d. Endpoints measured were number of hatched eggs, post-hatch survival, body length and body weight. No effects were observed for number of hatched eggs or post-hatch survival (NOELR 20 mg/L). Effects were observed on body length and body weight (NOELR 1.25 and 2.5 mg/L, respectively). However, these effects occurred at a dose that does not require classfication; as such the substance is not classified as hazardous to aquatic life.

Description of key information

In this key study, fathead minnow (Pimephales promelas) were exposed to a Water Accommodated Fraction of the substance at nominal loading rates of 1.25, 2.5, 5, 10 or 20 mg/L under semi-static conditions for 21 days. The LL50 (21 d) was > 10 mg/L loading rate WAF. The No Observed Effect Loading Rate was 10 mg/L loading rate WAF. The substance is not harmful to fish after prolonged exposure.