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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

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 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction 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:

A nominal amount of test item (40 mg) was added to the surface of 4 L of test water to give the 10 mg/L loading rate. 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. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 100 mL discarded) to give the 10 mg/L loading rate WAF. On one occasion on Day 14, plastic particles were observed to be present in the test media. As a precaution, a glass wool plug was inserted into the opposite end of the siphon tubing prior to removal as for the other renewal days. Microscopic observation of the WAF was performed after filtering and showed no micro-dispersions. Test solution observations also confirmed the test solution to be a clear colorless solution, therefore it was considered to have not impacted the test. As test solutions without a glass plug filter were observed to be a clear colorless solution with no micro-dispersions, the addition of the plug is considered to have not impacted the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M4 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata)) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The water hardness was observed to be in the range 250 to 270 mg/L as CaCO3 in the control and the 10 mg/L loading rate WAF test group throughout the test.

Test temperature:

Temperature was maintained at approximately 20 °C to 21 °C throughout the test

pH:

7.2 - 7.8

Dissolved oxygen:

8.4 - 9.4 mg O2/L

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal
0 and 10 mg/L

Measured
Chemical analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured test concentrations to range from 0.00085 to 0.020 mg/L. Chemical analysis of the corresponding aged test preparations on Days 3, 7, 14 and 21 showed measured test concentrations to range from 0.00038 to 0.019 mg/L.

Details on test conditions:

Test System:
For each concentration a single daphnid was placed in approximately 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed three times per week on Days 3, 5, 7, 10, 12, 14, 17 and 19. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
Each daphnid received approximately 5 to 20 μL of an algal suspension (Raphidocelis subcapitata) daily supplemented by the addition of approximately 20 μL of GEMMA Micro 300 fish food suspension on Days 0, 1, 2 and 3. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Water Quality Criteria:
Dissolved oxygen concentrations, pH and temperature were recorded at the start and end of the test and before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a digital thermometer. Measurements were made on one replicate for each test concentration. The temperature was also measured every hour in one surrogate vessele using a Testo temperature logger. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.

Test Concentrations Selections:
A range finding study showed no immobilization in the control and test groups. Sub-lethal effects of exposure were observed in the control and 100 mg/L Loading Rate WAF test group. Based on this information and at the request of the Sponsor, at single nominal loading rate of 10 mg/L was selected for the definitive test.

Effect parameters measured in the definitive study:
Immobilization
Reproduction
Body Length

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

not determinable

Details on results:

Lethal Effects on the Parental Generation (P1)
Mortality was observed in the control on Day 7 and at the test loading rate of 10 mg/L on Days 12 and 17. However, statistical analysis of the mortality data using the Fisher’s Exact Binomial test showed that the observed mortalities in the 10 mg/L loading rate WAF group were not significantly different (P≥0.05) when compared to the control group.

Sub-lethal Effects on the Parental Generation (P1)
The daphnids in the 10 mg/L test loading rate were observed to be the same size and color as the control daphnids. There was a statistically significant difference (P≥0.05) between the control and the 10 mg/L Loading Rate WAF test group in terms of length of the daphnids after 21 days exposure to the test item. However, inspection of the data indicated that the observed effect was less than 10% therefore it was considered that a repeat test would not be able to determine an EL10.

Effects on Reproduction
After 21 days there were no statistically significant differences (P≥0.05) between the control and the 10 mg/L loading rate WAF test group in terms of the number of live young produced per adult alive at the end of the test excluding parental accidental and/or inadvertent mortality.

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by the 10 mg/L loading rate WAF group were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control test group on Day 8 of the test. There were no unhatched eggs or dead young in the control and treatment groups

Reported statistics and error estimates:

The EL50 (immobilization), EL50 (reproduction) and EL50 (length) values after 21 days were estimated by inspection of the data. For the estimation of the Lowest Observed Effect Loading Rate (LOEL) and the No Observed Effect Loading Rate (NOEL) for immobilization, the control and 10 mg/L test groups were compared using Fisher’s Exact Binomial test. For the estimation of the LOEL and the NOEL, the numbers of live young over the duration of the test produced per parent Daphnia at the start of the test, excluding from the analysis parental inadvertent mortality for the control and 10 mg/L test group were compared using a Student t-test for Homogenous Variances incorporating Shapiro-Wilks test on Normal Distribution and Levene’s test on Variance Homogeneity. Results from the control and 10 mg/L test group Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using Student t-test for Homogenous Variances incorporating Shapiro-Wilks test on Normal Distribution and Levene’s test on Variance Homogeneity. All statistical analyses were performed using the ToxRat Professional computer software package.

Endpoint

Concentration (mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

Immobilization

^EL10

≤10

Not determined

^EL20

>10

Not determined

^EL50

>10

Not determined

NOEL

10

Not applicable

Reproduction

^EL10

>10

Not determined

^EL20

>10

Not determined

^EL50

>10

Not determined

NOEL

10

Not applicable

Body Length

^EL10

>10

Not determined

^EL20

>10

Not determined

^EL50

>10

Not determined

NOEL

10

Not applicable

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 211 GLP study, Daphnia were exposed to 0 mg/L and a single concentration of the substance at 10 mg/L WAF for 21 d. Endpoints measured were body length, immobilisation and reproduction. There were no adverse effect obsevered in any of these endpoints meaning the NOELR was 10 mg/L. The substance does not, therefore, require classification as hazardous to aquatic life

Description of key information

One reliable study is available. In this study (Sacker, 2022) performed under GLP according to OECD TG 211, EC Method C.20 and draft OPPTS 850.1300, Daphnia magna were exposed to the substance under semi-static conditions for 21 days. Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF). Based on the results of the acute toxicity test daphnids (10 replicates of a single daphnid per group) to WAFs of the test item at a nominal loading rate of 10 mg/L. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily.

The 21-Day EL50 (immobilization) value, based on nominal test concentrations, for the parental Daphnia generation (Pi) was > 10 mg/L loading rate WAF. The 21-Day EL50 (reproduction) value based on nominal test concentrations was > 10 mg/L.

The "No Observed Effect Loading Rate" were considered to be >10 mg/L based on the absence of effects.