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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline, GLP
Qualifier:
according to
Guideline:
other: OECD Guideline 236: Fish Embryo Acute Toxicity (FET) Test, 2013
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the definitive test, the concentration of MDIPA-Esterquat C16-18 and C18 unsatd. was measured in the control and all test substance treatments in samples collected from parent solutions at initiation, 24-, 48-, and 72 hours, and in samples composited from spent replicate solutions at 24-, 48-, 72-, and 96 hours.
At each sampling point, 5-mL samples were collected into appropriately labeled culture tubes. The samples were diluted to 10 mL with isopropanol formate buffer mix and further diluted, if necessary, with 50:50 isopropanol formate buffer mix:water to provide final concentrations within the analytical standard concentration range (i.e., 0.200 to 25.0 ng a.i./mL). Two quality control (QC) fortification spikes at concentrations that bracket the expected high and low test substance treatment concentrations were prepared and analyzed in a similar manner.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A 0.025 mg MDIPA-Esterquat C16-18 and C18 unsatd./mL primary standard was prepared daily from 30 September to 04 October 2013 by diluting approximately 0.0250 grams of MDIPA-Esterquat C16-18 and C18 unsatd. to a volume of 1,000 mL with dilution water. The dilution water used to prepare the primary standard was heated to approximately 55 °C. The molten test substance (e.g. preheated to approximately
55-60 °C) was then be added to the heated dilution water and allowed to stir for approximately five to ten minutes, then allowed to cool to test temperature. The primary standard was used as the high test concentration and appropriate aliquots of the primary standard were diluted to a volume of 0.25 L with dilution water to prepare test solutions at concentrations of 1.6, 3.1, 6.3, and 13 mg/L. The control consisted of dilution water only. All test plates were labeled with the study number, treatment, and replicate, and placed in a temperature-controlled chamber.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
Eggs of Danio rerio
- Common name: Zebrafish
- Source: in-house culture
breeding stock of unexposed mature zebrafish with an age of approximately 11 months was used for egg production. Fish were inspected and free of macroscopically discernable symptoms of infection and disease. Spawning fish were maintained in aquaria with a loading capacity of at least one liter of water per fish and had a photoperiod of 16 hours of light and eight hours of darkness.

24 hours prior to test initiation, control and treatment solutions were placed in the respective wells to acclimate the plates. On day 0 (day of initiation), old solutions were removed and new solutions were added to the plates. Adult zebrafish were placed in breeding aquaria on 30 September 2013, and eggs were collected 12 to 17 minutes post fertilization, i.e. after onset of light the next morning. After egg collection, a representative sample of approximately 200 embryos was examined for viability. Of the examined embryos, 11 appeared to not be fertilized resulting in an estimated 95% viability. After assessment of embryo viability, the approximately 200 embryos were returned to the remaining collected embryos for use in the study. At 30 minutes post fertilization, while embryos were at the 2-cell and 4-cell-stage, approximately 80 zebrafish eggs were impartially transferred with a pipette into each petri dish containing control or test substance solution. Under a microscope with at least 30-fold magnification, fertilized eggs were visually identified in the control and test solutions and within less than three hours post fertilization, were transferred to a 24 well plate, one egg per well, until there was a total of 24 eggs in the control and 20 eggs in the test solution. Additionally, on each plate containing treatment solution, an additional 4 wells were assigned as internal plate controls.

- Age at study initiation (mean and range, SD): age of eggs ≤ 1 day
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
142-144 mg CaCO3/L
Test temperature:
25.2-26.5°C
pH:
8.3-8.4
Dissolved oxygen:
7.7-7.8mg/L (99-100% saturation)
Nominal and measured concentrations:
Nominal Concentrations: 0 (control), 1.6, 3.1, 6.3, 13, and 25 mg/L
Geometric Mean Measured Concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: 24-well polystyrene microtiter plate covered with a plastic lid containing approximately 2 mL of test solution or dilution water.

- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20 (24h), 20 (48h), 20 (72h), 20 (96h)
- No. of vessels per control (replicates): 24x4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: biologically aged laboratory freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO). The well water and RO water were blended together to yield a total hardness of 130 to 160 mg CaCO3/L. Prior to use, the dilution water was passed through a sediment filter.

- Metals: analysed, not critical
- Pesticides: analysed, not critical
- Conductivity: 296-362 (test solutions bevore, during and after testing)
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hr light:8-hr dark
- Light intensity: 263 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
coagulation 24, 48, 72, and 96 (± 1) hours after exposure initiation
lack of somites 24, 48, 72, and 96 (± 1) hours after exposure initiation
non-detached tail 24, 48, 72, and 96 (± 1) hours after exposure initiation
lack of heartbeat 48, 72, and 96 (± 1) hours after exposure initiation
post hatch mortality 72, and 96 (± 1) hours after exposure initiation


TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2
- Range finding study: no
- Results used to determine the conditions for the definitive study: Definitive test substance treatment concentrations were selected based on previously generated toxicological data.
Reference substance (positive control):
yes
Remarks:
3,4-DCA
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
11.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 9.91-13.8
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 10.5-13.8
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
10.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 8.68-12.5
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
9.81 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 8.12-11.8
Details on results:
- Other biological observations:
There were no coagulated embryos in the control throughout the test. Coagulated embryos were observed in the 1.90, 8.05, and 16.0 mg/L test treatments throughout the test. After 96 hours, the number of coagulated embryos in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test substance treatments was 0, 1, 0, 3, and 13 respectively, out of an initial 20 embryos.
None of the embryos in the control or test substance treatments lacked somites throughout the test, with the exception of two embryos in the 8.05 mg/L test substance treatment at 24 hours, and all embryos in the control and test substance treatments exhibited detached tails after 96 hours.
Out of an initial 24 embryos, one embryo had no visible heartbeat in the control at 48 hours. Two embryos in the 8.05 mg/L treatment and eight embryos in the 16.0 mg/L treatment lacked heartbeats after 48 hours; three embryos in the 16.0 mg/L lacked heartbeats after 72 hours; and one embryo lacked a heartbeat in the 8.05 mg/L treatment after 96 hours.
The hatching rate after 96 hours was 96% in the control and 100, 90, 95, 55, and 25% in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test treatments, respectivel. Hatching rates of the internal controls for the treatment plates were 17% and 100% at 72 and 96 hours respectively.



- Mortality of control: 24h:0, 48h: 1 (4%), 72h: 0, 96h 0
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: The control and all test substance solutions were clear and colorless throughout the test with no visible particulates, surface films, precipitates, or undissolved test substance.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: 96h-LC100=4.0mg/L
- Other:
After 96 hours of exposure to the reference toxicant, 3,4-DCA, mortality was 0% in the control and 100% in the 4.0 mg/L nominal test substance treatment (Table 7XH). Out of an initial 20 embryos, four embryos were coagulated after 24 hours in the 4.0 mg/L treatment solution and 10 embryos were coagulated after 96 hours. None of the embryos lacked somites or detached tails in the control or 3,4-DCA treatment during the test, with the exception of 3 embryos with non-detached tails at 24 hours. In the 3,4-DCA treatment four embryos lacked heartbeats at 48 hours, 11 embryos lacked heartbeats at 72 hours, and 10 embryos lacked heartbeats at 96 hours. The hatching rate in the 3,4-DCA treatment was 0% at 72 and 96 hours. Embryos and eleutheroembryos in the 3,4-DCA treatment exhibited abnormal yolk sacks and deformed heads (Table 8). There was no abnormal development in the internal controls, which showed a hatch rate of 100% at 96 hours.
Reported statistics and error estimates:
All statistical analyses were performed with SAS software (version 9.3). Estimates of LC50 values and their 95% confidence limits were calculated using the Spearman-Karber method. When the p-value for Goodness of Fit was > 0.05 and there was no other evidence of questionable convergence, the probit method was selected for reporting. When this criterion was not achieved, the untrimmed Spearman-Karber method was selected for reporting.

After 96 hours of exposure to the test substance, mortality was 0, 0, 5, 0, 20, and 75% in the 0 (control), 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test treatments, respectively. The 24-hour LC50 was estimated to be 9.81 mg/L with 95% confidence limits of 8.12 and 11.8 mg/L. The 48-hour LC50 was estimated to be 10.4 mg/L with 95% confidence limits of 8.68 and 12.5 mg/L. The 72-hour LC50 was estimated to be 13.0 mg/L with 95% confidence limits of 10.5 and 16.2 mg/L. The 96-hour LC50 was estimated to be 11.7 mg/L with 95% confidence limits of 9.91 and 13.8 mg/L.

There were no coagulated embryos in the control throughout the test. Coagulated embryos were observed in the 1.90, 8.05, and 16.0 mg/L test treatments throughout the test. After 96 hours, the number of coagulated embryos in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test substance treatments was 0, 1, 0, 3, and 13 respectively, out of an initial 20 embryos.

None of the embryos in the control or test substance treatments lacked somites throughout the test, with the exception of two embryos in the 8.05 mg/L test substance treatment at 24 hours, and all embryos in the control and test substance treatments exhibited detached tails after 96 hours.

Out of an initial 24 embryos, one embryo had no visible heartbeat in the control at 48 hours. Two embryos in the 8.05 mg/L treatment and eight embryos in the 16.0 mg/L treatment lacked heartbeats after 48 hours; three embryos in the 16.0 mg/L lacked heartbeats after 72 hours; and one embryo lacked a heartbeat in the 8.05 mg/L treatment after 96 hours.

The hatching rate after 96 hours was 96% in the control and 100, 90, 95, 55, and 25% in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test treatments, respectively. Hatching rates of the internal controls for the treatment plates were 17% and 100% at 72 and 96 hours respectively.

Mortality Endpoints of Zebrafish, Danio rerio, Exposed to HH-2012-459 for 96 Hours Under Static-Renewal Test Conditions

 

Treatment

Level

Geometric Mean Measured Concentration

(mg/L)

Initial Number

Time

Point

Coagulateda

Lack of Somites

Non-Detached Tail

Lack of Heartbeat

Mortality Post Hatch

Overall Survival

% Survival

Overall Mortality

% Mortality

Control

0.0

24

24h

0

0

0

--

--

24

100%

0

0%

L1

0.986

20

24h

0

0

0

--

--

20

100%

0

0%

L2

1.90

20

24h

1

0

0

--

--

19

95%

1

5%

L3

4.15

20

24h

0

0

0

--

--

20

100%

0

0%

L4

8.05

20

24h

2

2

4*

--

--

14

70%

6

30%

L5

16.0

20

24h

10

0

8

--

--

2

10%

18

90%

Ref Tox

4.0 (nominal)

20

24h

4

0

3

--

--

13

65%

7

35%

Internal Controls

0.0

24

24h

0

0

0

--

--

24

100%

0

0%

 

 

 

 

 

 

 

Control

0.0

24

48h

0

0

0

1

--

23

96%

1

4%

L1

0.986

20

48h

0

0

0

0

--

20

100%

0

0%

L2

1.90

20

48h

1

0

0

0

--

19

95%

1

5%

L3

4.15

20

48h

0

0

0

0

--

20

100%

0

0%

L4

8.05

20

48h

3

0

0

2

--

15

75%

5

25%

L5

16.0

20

48h

10

0

1

8**

--

2

10%

18

90%

Ref Tox

4.0 (nominal)

20

48h

5

0

0

4

--

11

55%

9

45%

Internal Controls

0.0

24

48h

0

0

0

1

--

23

96%

1

4%

Control

0.0

24

72h

0

0

0

0

0

24

100%

0

0%

L1

0.986

20

72h

0

0

0

0

0

20

100%

0

0%

L2

1.90

20

72h

1

0

0

0

0

19

95%

1

5%

L3

4.15

20

72h

0

0

0

0

0

20

100%

0

0%

L4

8.05

20

72h

3

0

0

0

0

17

85%

3

15%

L5

16.0

20

72h

10

0

1

3*

0

7

35%

13

65%

Ref Tox

4.0 (nominal)

20

72h

5

0

0

11

0

4

20%

16

80%

Internal Controls

0.0

24

72h

0

0

0

0

0

24

100%

0

0%

 

 

 

 

 

 

 

Control

0.0

24

96h

0

0

0

0

0

24

100%

0

0%

L1

0.986

20

96h

0

0

0

0

0

20

100%

0

0%

L2

1.90

20

96h

1

0

0

0

0

19

95%

1

5%

L3

4.15

20

96h

0

0

0

0

0

20

100%

0

0%

L4

8.05

20

96h

3

0

0

1

0

16

80%

4

20%

L5

16.0

20

96h

13

0

0

0

2

5

25%

15

75%

Ref Tox

4.0 (nominal)

20

96h

10

0

0

10

0

0

0%

20

100%

Internal Controls

0.0

24

96h

0

0

0

0

0

24

100%

0

0%

 

 

 

 

 

 

 

 

 

 

 

 

 

a If egg is observed as coagulated, observations for somites, tail-detachment and heartbeat are not made

* two embryos not counted in other mortality endpoints included in final overall mortality

** seven embryos not counted in other mortality endpoints included in final overall mortality

 

Validity criteria fulfilled:
yes
Remarks:
96 h mortality < 10 percent in control, > 30% in reference toxicant 96h hatching rate control > 80% overall fertilization rate of all eggs collected > 70%. water temp. 26 ± 1 °C after 96h dissolved oxygen control + highest test concentration > 80%
Conclusions:
The 96-hour LC50 was estimated to be 11.7 mg/L with 95% confidence limits of 9.91 and 13.8 mg/L.
Executive summary:

The 96–hr-acute toxicity of MDIPA-Esterquat C16-18 and C18 unsatd. to eggs of Danio rerio was studied under semi-static conditions according to guideline OECD guideline 236 (2013). Eggs were exposed to control and test chemical at analytically determined geometric mean concentrations of 0, 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L under semi-static conditions with daily renewal. Analytics and all observations were made daily.

The 96-hour LC50 was estimated to be 11.7 mg/L with 95% confidence limits of 9.91 and 13.8 mg/L.

After 96 hours, the number of coagulated embryos in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test substance treatments was 0, 1, 0, 3, and 13 respectively, out of an initial 20 embryos.

None of the embryos in the control or test substance treatments lacked somites throughout the test, with the exception of two embryos in the 8.05 mg/L test substance treatment at 24 hours, and all embryos in the control and test substance treatments exhibited detached tails after 96 hours.

Out of an initial 24 embryos, one embryo had no visible heartbeat in the control at 48 hours. Two embryos in the 8.05 mg/L treatment and eight embryos in the 16.0 mg/L treatment lacked heartbeats after 48 hours; three embryos in the 16.0 mg/L lacked heartbeats after 72 hours; and one embryo lacked a heartbeat in the 8.05 mg/L treatment after 96 hours

The hatching rate after 96 hours was 96% in the control and 100, 90, 95, 55, and 25% in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test treatments, respectively

Description of key information

96 h LC50 > 11.7 mg a.i./L (geom. mean) (Zebra fish, Danio rerio), OECD guideline 236, semi-static, GLP, RL1

Key value for chemical safety assessment

LC50 for freshwater fish:
11.7 mg/L

Additional information

The 96–hr-acute toxicity of MDIPA-Esterquat C16-18 and C18 unsatd. to eggs of Danio rerio was studied under semi-static conditions according to guideline OECD guideline 236 (2013). Eggs were exposed to control and test chemical at analytically determined geometric mean concentrations of 0, 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L under static conditions with daily renewal. Analytics and all observations were made daily.

The 96-hour LC50 was estimated to be 11.7 mg/L with 95% confidence limits of 9.91 and 13.8 mg/L.

After 96 hours, the number of coagulated embryos in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test substance treatments was 0, 1, 0, 3, and 13 respectively, out of an initial 20 embryos.

None of the embryos in the control or test substance treatments lacked somites throughout the test, with the exception of two embryos in the 8.05 mg/L test substance treatment at 24 hours, and all embryos in the control and test substance treatments exhibited detached tails after 96 hours.

Out of an initial 24 embryos, one embryo had no visible heartbeat in the control at 48 hours. Two embryos in the 8.05 mg/L treatment and eight embryos in the 16.0 mg/L treatment lacked heartbeats after 48 hours; three embryos in the 16.0 mg/L lacked heartbeats after 72 hours; and one embryo lacked a heartbeat in the 8.05 mg/L treatment after 96 hours

The hatching rate after 96 hours was 96% in the control and 100, 90, 95, 55, and 25% in the 0.986, 1.90, 4.15, 8.05, and 16.0 mg/L test treatments, respectively.

 

Similar results were obtained with the closely related read-across substance MDEA-Esterquat C16-18 and C18 unsatd.:

In a 96 h acute toxicity study according to OECD TG 203, the Zebra fish (Danio rerio), was exposed to MDEA-Esterquat C16-18 and C18 unsatd. at nominal concentrations of 0, 1.0, 1.6, 2.5, 4.0, 6.3 and 10 mg/L under static conditions. The nominal 96 h LC50 value based on mortality was 5.2 mg/L (95% C.I.: 4.4 to 6.3 mg/L). Mortality occurred in the first 48 h. After this time, seemingly moribund organisms recovered. Within the first 48 h the test solutions at concentrations of 2.5, 4.0, 6.3, and 10 mg/L showed a Tyndall effect. The intensity increased with increasing concentrations. Precipitates were observed on the bottom of the test aquarium in the exposure concentrations of 2.5 to 10 mg/L 48 h after preparation of the test solution. These observations suggest that mortality is most likely a physical and not a toxic effect due to the undissolved particles in the water phase. 5 additional fish were added to the 10.0 mg/L solution after 48 h (the time the precipitates were observed). The animals survived until test termination which supports the assumption of physical effects.