2-ethylhexyl benzoate

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:

16 June 2020 to 8 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Based on ECHA decision TPE-D-2114514926-44-01/F

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2021

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

fresh samples: from bulk preparations on day 0, 6*, 13* and 20*
old samples from pooled replicates on day 1, 7, 14 and 21

* kept frozen until analysis

Vehicle:

no

Details on test solutions:

A nominal amount of test item (220 mg) was dispersed in 11 L of test water using a magnetic
stirrer at a rate sufficient to form a dimple at the water surface for 48 hours. After 48 hours
the stirring was stopped and any undissolved test item was removed by filtration through a
0.2 µm Sartorius Sartopore filter (first approximate 2 L discarded in order to pre-condition
the filter) to give a 100% v/v saturated solution.

Preliminary media preparation trials indicated that a dissolved test item concentration of
between 0.21 and 0.33 mg/L was obtained from a 48-Hour stir saturated solution method of
preparation without any sample pre-treatment indicating this to be the limit of water
solubility of this item under test conditions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in house culture
- Justification for species other than prescribed by test guideline: default
- Stage and instar at study initiation: first instar < 24 hours old
- Method of breeding: parthenogenesis
- Age of parental stock: 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.
- Feeding during test: yes
- Food type: mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension.
- Amount: 5 to 20 µL suspension (0.1 to 0.2 mg carbon/daphnid/day)
- Frequency: daily on day 0, 1, 2 and 3

ACCLIMATION: NA

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
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) 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.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4 medium)

Limit test:

yes

Total exposure duration:

21 d

Hardness:

240-260 mg/L as CaCO3 measured in controls and at saturated concentration on day 0, 6, 13 and 20 in fresh solutions and on day 2, 7, 14 and 21 in old solutions

Test temperature:

19-21 °C measured daily before and after renewal

pH:

7.2-8.1 measured daily in fresh and old solutions

Dissolved oxygen:

8.4-9.3 mg/L measured daily in fresh and old solutions

Nominal and measured concentrations:

nominal 0 and 100% saturated (220 mg in 11 L)
measured

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessel containing 100 mL test solution
- Type: closed with plastic lid
- Aeration: dilution water was aerated prior to use
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium according to the guideline
- Hardness: 250 mg/L as CaCO3
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk
transition
- Light intensity: 767 to 986 Lux

EFFECT PARAMETERS MEASURED:
Daily: survival (parental and F1), number of offspring and number of unhatched eggs
At end of the test: length of surviving parental daphnids

TEST CONCENTRATIONS
- Spacing factor for test concentrations: NA
Range finding study: during 10 days with daily renewal with 1 daphia per replicate and 5 replicates per exposure group and controls
- Test concentrations: 0, 0.1, 1, 10 and 100% saturated concentrations (dilutions from saturated stock prepared according to a similar procedure as used for the main test)
- Results used to determine the conditions for the definitive study:
see attached table: the immobilization in one replicate at 10% was considered to be incidental, as no effects on day of first brood and total young in the other replicates were observed at this and the 100% concentrations.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental animals: 0/10 in controls 1/10 at 0.014 mg/L (on day 15)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

length on day 21

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Details on results:

see attached summary of results

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for reproduction in Daphnia manga is 0.014 mg/L

Executive summary:

In a test according to OECD 211  Daphnia magna were exposed (10 replicates of a single daphnid per group) to a solution of the substance at a nominal concentration of 100% v/v saturated solution (actual 24-h TWA 0.014 mg/L) for a period of 21 days. The test solutions were renewed daily throughout the test.

No effects on mortality/immobility, daphnia length and reproduction were seen at the saturated concentration. Therefore it can be concluded that the NOEC is 0.014 mg/L. 

Description of key information

In a test according to OECD 211  Daphnia magna were exposed (10 replicates of a single daphnid per group) to a solution of the substance at a nominal concentration of 100% v/v saturated solution (actual 24-h TWA 0.014 mg/L) for a period of 21 days. The test solutions were renewed daily throughout the test.

No effects on mortality/immobility, daphnia length and reproduction were seen at the saturated concentration. Therefore it can be concluded that the NOEC is 0.014 mg/L. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.014 mg/L

Additional information

A testing proposal for a study on long-term toxicity to aquatic invertebrates (Daphnia magna reproduction study, OECD 211) was included in a previous version of the dossier. This test was approved by ECHA (TPE-D-2114346840-50-01/F) and was initiated by Envigo Laboratories. The test was dicontinued due to the fact that desired concentrations could not be reached and maintained. The use of dispersants led to toxicity that could not be explained by toxicity of the substance, but was most likely related to the specific dispersant (Envigo 2018).

After ECHA decision TPE-D-2114514926-44-01/F, Covance performed a test with a saturated concentration, that is included as the key study in the present dossier.