2-ethylhexyl 3,5,5-trimethylhexanoate

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

Study period:

December 2012 - April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully compliant OECD guideline study performed under GLP

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:

For determination of the concentrations of the test item, duplicate samples were taken from all test concentrations and from the solvent control at one treatment period of the first, second and last week of the test (Day 0, 7 and 16, respectively).
To determine the maintenance of the test item concentration in the test media, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).
The following stability samples were taken in duplicate:
a) Samples taken from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal period.
b) Samples incubated during the renewal periods under the test conditions, however without food and daphnids.
Immediately after the samples were taken, 3 mL of ethyl acetate plus dodecan was added to each 10 mL sample to stabilize the test item during the storage period. Then the samples were stored deep-frozen (at about –20 °C) till date of analysis. According to pre-experiments to the storage stability the test item is stable in test media samples under these storage conditions.

Vehicle:

yes

Details on test solutions:

Due to the low solubility of the test item in test water, the organic solvent N,N-Dimethylformamide p.a. (DMF) was used to dose the test item. The solvent was chosen based on its solubilizing properties and its relative non-toxicity to daphnids. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
The feasibility of this dosage approach was tested and confirmed in a pre-experiment. The application solution for the dosage of the highest test concentration was prepared by completely dissolving 20 mg (effective weights in the range of 20.09 to 20.24 mg) of the test item in 10 mL of DMF using stirring for 5 minutes at room temperature in a closed volumetric flask. Further dilutions of this application solution with DMF were performed to obtain the application solutions for the dosage of the lower test concentrations.
The application solutions were prepared three times during the test period (Day 0, 7 and 14) and were used during the following test medium preparations. Between each use, the application solutions were stored in the refrigerator (2 – 8 °C) in the dark. In a previously performed pre-experiment the test item was shown to be stable in DMF for at least 5 days.
For preparation of the test media, adequate volumes of the application solutions were added to the test water (335 µL per 3350 mL of test water) using intense stirring (15 minutes) to obtain the test concentrations as stated above. The concentration of DMF was the same in all test media and in the solvent control (100 µL DMF per liter of test water). For the control, test water without addition of test item or solvent was used.

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with females of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium identical to the medium used for the test. The temperature and light conditions were identical to those of the test.
During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).
Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.
The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old. The test method and test species, Daphnia magna, are recommended by the test guidelines.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

The water temperature was maintained at 20 - 21 °C.

pH:

During the test period, the pH of the test media and controls ranged from 7.5 to 8.0

Dissolved oxygen:

The dissolved oxygen concentrations were at least 7.9 mg/L (ranged from 7.9 to 9.2 mg/L)

Salinity:

Alkalinity: 0.9 mmol/L

Nominal and measured concentrations:

The nominal concentrations tested were 1.25, 2.5, 5.0, 10 and 20 µg/L. Additionally, a control and a solvent control were tested in parallel.
The concentrations of the test item were measured in the duplicate test media samples from the nominal concentration of 20 µg/L from all sampling dates. From the solvent control, one of the duplicate samples was analyzed per sampling date. The samples of the nominal test concentrations of 1.25 to 10 µg/L were not analyzed since the concentrations were below the NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Details on test conditions:

This test was performed in a temperature-controlled room (water temperature 20 – 21 °) with continuous monitoring of the room temperature. A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used.
In this semi-static test, the test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16 and 19 of the test period (every Monday, Wednesday, and Friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into new test vessels with freshly prepared test medium.
The test animals were fed daily with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use.
The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 8.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 8.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 8.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 8.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The test item had no toxic effects on survival, reproduction and the growth of Daphnia magna after the exposure period of 21 days up to the mean measured concentration of 8.6 µg/L. Thus, the 21-day NOEC of the test item was determined to be at least 8.6 µg/L. The 21-day LOEC was above the highest test concentration of 8.6 µg/L.
The 21-day EC 50 for the inhibition of the reproduction rate and the growth of the daphnids was higher than 8.6 µg/L.
In conclusion, the test item 2-ethylhexyl 3,5,5-trimethylhexanoate had no toxic effect on Daphnia magna up to its solubility limit in test water under the conditions of the test.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

See "any other information on results including tables"

Analytical Results: The measured test item concentrations in the freshly prepared test media (without food) of the nominal concentration of 20 µg/L were between 22 and 24 µg/L (corresponding to 108% and 120% of the nominal value) at the start of the test medium renewal periods. This measured test item concentration corresponded to the solubility of the test item which was determined in a pre-experiment to be 20 µg/L.

In the stability samples without food particles and daphnids in the closed system, the measured concentrations were between 31% and 66% of the nominal value at the end of the test medium renewal periods of 48 to 72 hours.

In the stability samples taken from the actual test including food particles, the measured concentrations at the end of the test medium renewal periods were lower than in the stability samples without food particles (< limit of quantification (LOQ = 1.42 µg/L) to 22% of the nominal concentration of 20 µg/L). This result indicates that a part of the test item adsorbed onto the food particles.

The biological results are based on the mean measured concentration of 8.6 µg/L corresponding to nominal 20 µg/L (calculated as the time-weighted mean over all measurements with food). The measured concentration at the end of the second sampling interval was <LOQ (LOQ: 1.42 µg/L). Therefore the ½ LOQ was used to calculate the time-weighted mean.

Biological Results: In the control, solvent control and at all test concentrations up to and including 8.6 µg/L (mean measured), the survival of the test animals at the end of the test was at least 90% or higher. The immobility of 10% determined at the nominal concentration of 10 µg/L is regarded as being within the natural range and is also allowed in the control of a test to be valid according to the OECD test guideline 211. Moreover, the lack of a concentration-response relationship suggests that the immobility of this daphnia was not test item related. Therefore, the immobility of 10% at the nominal concentration of 10 µg/L is considered as inadvertent immobility. Thus, the survival of Daphnia magna over 21 days was not affected by the test item up to and including the highest test concentration of 8.6 µg/L.

The first young offspring released from their parent animals were recorded in the control, solvent control and at the higher test concentrations up to and including 8.6 µg/L at observation on Day 8. Thus, the time of the first brood was not affected by the test item up to and including the highest test concentration of 8.6 µg/L.

The mean reproduction rate of the daphnids in the control was 143 ± 12 living offspring per surviving adult (mean ± standard deviation). In the solvent control the corresponding value was 142 ± 11. The mean reproduction rate of the daphnids in the solvent control was not significantly different from the control (results of a Student-t test, a = 0.05, two-sided). The results obtained from the different test concentrations were compared to the solvent control. No significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the highest test concentration of 8.6 µg/L (Dunnett t-test, one-sided smaller, a = 0.05) compared to the solvent control.

The mean body lengths of the daphnids in the control and solvent control were 4.17 ± 0.11 mm and 4.10 ± 0.12 mm (mean ± standard deviation), respectively. The mean body length of the daphnids in the solvent control was not significantly different from the control (results of a Student-t test, a = 0.05, two-sided). At any test concentration up to and including the highest mean measured concentration of 8.6 µg/L, the mean body length was not significantly smaller compared to the solvent control (Dunnett t-test, one-sided smaller, a = 0.05).

No visible abnormalities were observed at the test animals during the test.

General Results: During the test period, the pH of the test media and controls ranged from 7.5 to 8.0. The dissolved oxygen concentrations were at least 7.9 mg/L. The water temperature was between 20 and 21°C during the test period. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods.

Validity criteria fulfilled:

yes

Conclusions:

The test item had no toxic effect on Daphnia magna (mortality, growth and reporduction) up to its solubility limit in test water under the conditions of the test.

Executive summary:

The impact of the test item on the survival, growth (body length) and reproduction of daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2012) and the Commission Regulation (EC) No 440/2008, C.20: “Daphnia magna Reproduction Test”.

The nominal concentrations tested were 1.25, 2.5, 5.0, 10 and 20 µg/L. Additionally, a control and a solvent control were tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000). The highest test concentration corresponded to the solubility of the test item in test water which was determined in a stirring experiment to be 20 µg/L.

The biological results are based on the mean measured concentration of 8.6 µg/L corresponding to nominal 20 µg/L (calculated as the time-weighted mean over all measurements with food). The measured concentration at the end of the second sampling interval was <LOQ (LOQ: 1.42 µg/L). Therefore, the ½ LOQ was used to calculate the time-weighted mean (8.6 µg/L, equivalent to 43% of nominal concentration).

The test item had no toxic effects on survival, reproduction and body length of daphnia magna after the exposure period of 21 days up to the mean measured concentration of 8.6 µg/L. Thus, the 21-day NOEC of the test item was determined to be at least 8.6 µg/L. The 21-day LOEC was above the highest test concentration of 8.6 µg/L. The 21-day EC 50 for the inhibition of the reproduction rate of the daphnids was higher than 8.6 µg/L.

In conclusion, the test item had no toxic effect on daphnia magna up to its solubility limit in test water under the conditions of the test.

Description of key information

The test item had no toxic effects on survival, reproduction and body length of Daphnia magna after the exposure period of 21 days up to the mean measured concentration of 8.6 µg/L. Thus, the test item had no toxic effect on Daphnia magna up to its solubility limit in test water under the conditions of the test (NOEC >= 8.6 µg/L). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.6 µg/L

Additional information

The impact of the test item on the survival, growth (body length) and reproduction of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2012) and the Commission Regulation (EC) No 440/2008, C.20: “Daphnia magna Reproduction Test”.

The test item had no toxic effects on survival, reproduction and body length of Daphnia magna after the exposure period of 21 days up to the mean measured concentration of 8.6 µg/L. Thus, the 21-day NOEC of the test item was determined to be at least 8.6 µg/L. The 21-day LOEC was above the highest test concentration of 8.6 µg/L and the 21-day EC50 for the inhibition of the reproduction rate of the daphnids was higher than 8.6 µg/L.

In conclusion, the test item had no toxic effect on Daphnia magna up to its solubility limit in test water under the conditions of the test.