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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
other information
Study period:
13 April 2018 to 14 September 2018
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: the main study was not performed
Remarks:
In a pre-test it was tried to generate concentrations that would be stable over 24 hours to be used in the daphnia reproduction study (daily renewal of the medium was foreseen). A large number of pre-tests with different dispersants was performed. In these attempts either non- stable concentrations were found or toxicity was observed that could be related to the dispersant used.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
no main test was performed
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
The test samples were analyzed on the day of receipt. The test samples (100 mL) were acidified with 1 mL formic acid and then extracted directly with 10 mL of hexane in the same bottle. An aliquot of the hexane layer was taken for analysis.

Preparation of Test Samples
Nominal
Concentration Sample Volume Final Volume Sample Preparation Final Nominal
Factor Concentration
[mg/L] [mL] [mL] F [mg/L]
0.02 100 10 0.1 0.2
0.2 100 10 0.1 2
2 100 10 0.1 20
20 100 10 0.1 200

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
exp 1: A nominal amount (20 mg) of 2-ethylhexyl benzoate was thoroughly combined with 100 µl Tween 20 using a glass rod, and then slowly added to 1 liter Elendt M7. The remaining concentrations were prepared by serial dilutions of the stock solution. A surfactant control was also prepared at 100 µL/L of Tween 20.
exp 2: A nominal amount (20 mg) of 2-ethylhexyl benzoate was thoroughly combined with 10 µL Tween 20 using glass rod and slowly combined with 1 liter Elendt M7. The final concentration of Tween 20 in each of the test solutions was 10 µL/L. A surfactant control of 10 µl/L was also prepared.
exp 3: Stock solutions at concentrations of 500, 100 and 20 mg/L were prepared as follows. Nominal amounts (50, 25 and 10 mg) of 2-ethylhexyl benzoate were thoroughly combined with 100, 250 and 500 µL Tween 20 using a glass rod and slowly combined with Elendt M7 to final volumes of 100, 250 and 500 mL. 10 mL of each of these stock solutions was then diluted to 1 litre in Elendt M7 to give the test concentrations of 0.20, 1.0 and 5.0 mg/L. The test concentration of 0.020 mg/L was prepared by adding 450 µL of Tween 20 to 50 mL of the 20 mg/L stock solution. 1.0 mL of this preparation was then diluted to 1 litre in Elendt M7. The final concentration of Tween 20 in each of the test solutions was 10 µL/L. A surfactant control was prepared, however, in error this was prepared at a concentration of 100 µL/L instead of 10 µL/L.
exp 4: Surfactants without substance: Tergitol NP10, Triton X 100, SPAN 80 and Castor Oil. All these surfactants were tested at 50 µL/L.
exp 5: 2-ethylhexyl benzoate at 1.0 mg/L in Tergitol NP10, Triton X 100 or SPAN 80 preparations. A nominal amount (20 mg) of 2-ethylhexyl benzoate was thoroughly combined with 200 µL of each surfactant using a glass rod and slowly combined with 1 liter Elendt M7. Each test concentration was then prepared by diluting 50 mL of the appropriate stock solution to 1 liter Elendt M7. Surfactant controls were also prepared at 10 µL/L for each surfactant.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnia magna
- Source: in-house first instar culture
- Age: less than 24 hours old
- Feeding during test: yes
- Food: 5 to 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
10 d
Remarks on exposure duration:
exp 1 3 and 4: 10 days; exp 2: 2 days; exp 5: 3 days
Test temperature:
18 to 22 °C with a maximum deviation of ±1 °C
Nominal and measured concentrations:
nominal concentrations
exp 1: control, vehicle control, 0.02, 0.2, 2.0 and 20 mg/L (100 uL/L Tween 20)
exp 2: control, vehicle control, 2.0 and 20 mg/L (10 uL/L Tween 20)
exp 3: control, vehicle control (100 uL/L), 0.02, 0.2, 2.0 and 20 mg/L (10 uL/L Tween 20)
exp 4: control, Tergitol NP10, Triton X 100, SPAN 80, Castor Oil, all at 50 ug/L
exp 5: Control, Tween 20 Control, Tween 20 (2.0 uL/L) + 2-ethylhexyl benzoate (1.0 mg/L), Tergitol NP10 Control, Tergitol NP10(50 uL/L) + 2-ethylhexyl benzoate (1.0 mg/L), Triton X 100 Control and Triton X 100 (50 uL/L)+ 2-ethylhexy benzoate (1.0 mg/L)

Exp 1: measured concentrations:
day 1: 57-104% of nominal; day 3 at 0.02 and 0.2 mg/L within background noise; day 3 at 2.0 and 20 mg/L 5 and 23% of nominal

Exp 3: measured concentrations:
day 1: 0-58% of nominal; day 3 within background noise

Exp 5: measured concentrations (see table)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass vessel containing 100 mL of test preparation in Elendt 7 medium
- Aeration: no
- Renewal rate of test solution: exp 1, 3 and 4 renewal on day 0, 3, 6 and 8; exp 2 no renewal; exp 5 daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 5

TEST MEDIUM / WATER PARAMETERS: Elendt 7 medium

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

EFFECT PARAMETERS MEASURED : immobility, Average Time to First Brood, Average Neonates per Adult in First Brood, Total Neonates Produced at Day 10, Total Number of Broods

TEST CONCENTRATIONS; only range finding studies were performed see analytical results
Remarks on result:
not measured/tested
Remarks:
no main test was perfromed
Details on results:
results see tables below

exp 1: No immobilization was observed in the Control, 0.020 and 0.20 mg/L; however, 80% immobilization was observed in the Tween 20 control, 40% immobilization at 2.0 mg/L and 100% immobilization at 20 mg/L; daphnia were small, pale, coated in algae and showed reduced mobility
exp 2: No immobilization was observed in the Control and Tween 20 Control; however, 100% immobilization was observed at 2.0 and 20 mg/L by Day 3. As there was 100% mortality in the two test concentrations the test was terminated; daphnia were coated in algae and showed reduced mobility.
exp 3: 20% immobilization was observed in the Control, 40% immobilization was observed at 0.020 and 0.20 mg/L, 60% immobilization was observed at 1.0 mg/L and 100% immobilization in the Tween 20 control and at 5.0 mg/L. The one immobilized daphnia observed in the control group was considered not to be significant but reflects the natural mortality rates within a population; daphnia were small, pale and coated with algae
exp 4: No immobilization was observed in the Control, however, 100% immobilization was observed in Tergitol NP10, Triton X 100 and SPAN 80. Only 20% immobilization was observed in the test group Castor Oil, however, observations of the test solution showed oily globules were visible on the surface of the test media.;in the SPAN 80 and Castor Oil test groups; daphnia were pale and coated with algae
exp 5: No immobilization was observed in the Control, Tween 20 Control, Tergitol NP10 Control and Triton X 100 Control, however, 60% immobilization was observed in the test group containing 2-ethylhexyl benzoate and Tween 20 and 100% was observed in the test groups containing 2-ethylhexyl benzoate and Tergitol NP10 and Triton X 100. the Tween 20, Tergitol NP10 and Triton X 100 test groups; daphnia showed reduced mobility and trapping on the surface of the test media (before becoming immobilized)
Reported statistics and error estimates:
NA

First Experiment Test Data (100 µL Tween 20/L)

Nominal Concentration of 2-ethylhexyl benzoate (CAS5444-75-7)

(mg/L)

Percentage Immobilization After
10 Days Exposure

Average Time to First Brood (days)

Average Neonates per Adult in First Brood

Total Neonates Produced at Day 10

Total Number of Broods

Control

0

8

12.0

77

6

Tween 20 Control

80

-

-

-

-

0.020

0

8

13.8

127

8

0.20

0

8

16.0

80

5

2.0

40

-

-

-

-

20

100

-

-

-

-

 

- = No brood was produced within the 10 day test

Second Experiment Test Data(10 µL Tween 20/L)

Nominal Concentration of 2-ethylhexyl benzoate (CAS5444-75-7)

(mg./L)

Percentage Immobilization After
3 Days Exposure

Average Time to First Brood (days)

Average Neonates per Adult in First Brood

Total Neonates Produced at Day 10

Total Number of Broods

Control

0

-

-

-

-

Tween 20 Control

0

-

-

-

-

2.0

100

-

-

-

-

20

100

-

-

-

-

 

Test terminated after three days due to 100% immobilization in both test concentrations

- = No brood was produced within the test

 

Third Experiment Test Data(10 µL Tween 20/L)

Nominal Concentration of 2-ethylhexyl benzoate (CAS5444-75-7)

(mg/L)

Percentage Immobilization After
10 Days Exposure

Average Time to First Brood (days)

Average Neonates per Adult in First Brood

Total Neonates Produced at Day 10

Total Number of Broods

Control

20

-

-

0

0

Tween 20 Control*

100

-

-

-

-

0.020

40

10

9.3

28

3

0.20

40

-

-

0

0

1.0

60

10

3.5

7

1

5.0

100

-

-

-

-

 

- = No brood was produced within the 10 day test

 

Fourth Experiment Test Data(50µL surfactant/L)

Nominal Concentration

(mg/L)

Percentage Immobilization After
10 Days Exposure

Average Time to First Brood (days)

Average Neonates per Adult in First Brood

Total Neonates Produced at Day 10

Total number of broods

Control

0

8

3.6

18

3

Tergitol NP10

100

-

-

-

-

Triton X 100

100

-

-

-

-

SPAN 80

100

-

-

-

-

Castor Oil

20

8

4.25

17

3

 

-: No brood was produced within the 10 day test

 

Fifth Experiment Test Data(50µL surfactant/L)

Nominal Concentration

(mg/L)

Percentage Immobilization After
3 Days Exposure

Control

0

Tween 20 Control*

0

Tween 20*+ 2-ethylhexyl benzoate (1.0 mg/L)

60

Tergitol NP10 Control

0

Tergitol NP10 + 2-ethylhexyl benzoate (1.0 mg/L)

100

Triton X 100 Control

0

Triton X 100 + 2-ethylhexy benzoate(1.0 mg/L)

100

 


*In error the Tween 20 Control was prepared at a concentration of 100µL/L

*Tween 20 concentration was 2.0µL/L

Validity criteria fulfilled:
not applicable
Conclusions:
As concentrations were not adequately attained and maintained and immobilization and/or reduced reproduction was seen not only in treatment groups but also in surfactant controls, it was decided not to perform the main study.
Executive summary:

A series of range-finding tests was performed to determine suitable test concentrations for a definitive test accordig to OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test".

In the these range-finding tests, Daphnia magna were exposed (5 replicates of a single daphnid per group) to a dispersion of the test item for a period of up to 10 days. The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and GM300 fish food suspension.

Exposure of Daphnia magna to the substance determined that parental survival was affected at concentrations of 1.0 mg/L and above. At a concentration of 0.020 and 0.20 mg/L with 100 µL Tween 20 as dispersant reproduction was unaffected over a 10 day exposure period, however, at a concentration of 1.0 mg/L reproduction was reduced and at 2.0 mg/L no neonates were produced. The controls with 100 µL Tween 20 showed 80-100% immobilization on Day 10.

The concentrations measured on Day 1 were between 22 and 58% of nominal when using 10 µL Tween 20 and between 65 and 104 % of nominal when using 100 µL Tween 20, but within background noise on Day 3 for all concentrations using 10 uL/L Tween 20.

As concentrations were not adequately attained and maintained and immobilization and/or reduced reproduction was seen not only in treatment groups but also in surfactant controls, it was decided not to perform the main study. During the third test,one immobilized daphnia was observed in the control group; however, this level of immobilization was considered not to be significant but reflects the natural mortality rates within a population.

Description of key information

A series of range-finding tests was performed to determine suitable test concentrations for a definitive test according to OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test".

In the these range-finding tests, Daphnia magna were exposed (5 replicates of a single daphnid per group) to a dispersion of the test item for a period of up to 10 days.The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and GM300 fish food suspension.

Exposure of Daphnia magna to the substance determined that parental survival was affected at concentrations of 1.0 mg/L and above. At a concentration of 0.020 and 0.20 mg/L with100 µL Tween 20 as dispersant reproduction was unaffected over a 10 day exposure period, however, at a concentration of 1.0 mg/L reproduction was reduced and at 2.0 mg/L no neonates were produced. The controls with 100 µL Tween 20 showed 80-100% immobilization on Day 10.

The concentrations measured on Day 1 were between 22 and 58% of nominal when using 10 µL Tween 20 and between 65 and 104 % of nominal when using 100 µL Tween 20, but within background noise on Day 3 for all concentrations using 10 µL/L Tween 20.

As concentrations were not adequately reached and maintained and immobilization and/or reduced reproduction was seen not only in treatment groups but also in surfactant controls, it was decided not to perform the main study.

Key value for chemical safety assessment

Additional information

A testing proposal for a study on long-term toxicity to aquatic invertebrates (daphnia reproduction study, OECD 211) was included in the previous version of the dossier. This test was approved by ECHA (TPE-D-2114346840-50-01/F) and was started by Envigo Laboratories.

In the short-term toxicity tests with the substance, THF was used as a solubilising agent to prevent formation of non-suspended precipitates. This was not very successful regarding maintenance of the test concentrations. In the daphnia study the concentrations, though initially within the expected ranges, were not properly maintained over 24 hour periods (semi-static design). In the acute fish study performed according to a flow-through design, a film was observed on the surface of the water, but the measured concentrations remained stable. With algae, no substance was measured at the end of the exposure period (72 h), although starting concentrations were adequate. No definitive conclusion can be drawn on the reasons for the dissipation of the substance, but biodegradation and adsorption are probable. The substance is readily biodegradable, but given that degradation over 1 day was 30% and over 3 days was 50%, the dissipation of the substance cannot wholly be attributed to biodegradation. The logPow of the substance is 6.2. which may lead to some sorption. The substance is hydrolytically stable.

Taking into account the above mentioned difficulties, an attempt was done to reach concentrations that would be stable over 24 hours to be used in the daphnia reproduction study (daily renewal of the medium was foreseen). A large number of pre-tests with different dispersants was performed (see Envigo 2018). In these attempts either inadequate, non-stable concentrations were found or toxicity was observed that could be related to the dispersant used. Therefore it was concluded that the daphnia reproduction study was technically unfeasible and was not performed.

According to the Guidance on information requirements and chemical safety assessment, Chapter R.7b, readily biodegradable substances can be expected to undergo rapid and ultimate degradation in most environments, including biological Sewage Treatment Plants (STPs) (ECHA, 2017). The substance has a logKoc value of 4.07, is poorly water soluble (0.4 mg/L), and thus will be extensively removed in the primary settling tank and fat trap. Only limited amounts will be in contact with activated sludge organisms and will be biodegraded (due to its ready biodegradability). The discharged concentrations of the substance into the surface water are likely to be low. Once present in the aquatic compartment, further biodegradation will occur and the substance will be bioavailable to aquatic organisms to a limited extent. This is confirmed by the results of the current risk assessment (CSR 2018). Therefore a waiver for the long-term daphnia study is included.