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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
Study period:
From Feb. 14, 2013 to Sept.18, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals Draft Proposal for a New Guideline: Fish Embryo Acute Aquatic Toxicity (FET) Test with modifications
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to GLP principles of US EPA and OECD
Specific details on test material used for the study:
The test substance was Colipa A050 (CAS #54381-16-7; Batch Number 20110103), also known as G11-A11117 (Blauentwickler); 4-(Di(2-hydroxyethyl)amino)-Anilin-sulfat (1:1), hydrat; 2,2`-[4-aminophyenyl))imino]bis(ethanol) sulphate; Ethanol, 2,2`-[(4-aminophenyl))imino]bis-,sulphate (1:1) (salt); 2-[[(4-aminophenyl)-(2-hydroxyethyl)]amino]ethanol sulphate; INCI name: N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate.
The Certificate of Analysis reported the active ingredient in the form of free base for Colipa A050 as 62.5% by weight. Consequently the data in this record are expressed as active ingredient in conformity with the study report.
Since the active ingredient is not the registered substance a new Certificate of Analysis was performed of the tested substance, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulphate monohydrate, with a purity level of 99.24%. So the final results of the study were re-calculated taking into account the revised purity level and the LC50 is expressed as the tested substance

Analytical monitoring:
yes
Details on sampling:
- Concentrations (nominal): 0 , 12.5, 25.0, 50.0, 100, and 200 mg a.i./L
- Sampling method: The concentration of Colipa A050 was measured in the control and all test substance treatments in samples collected from parent solutions at initiation and 72-hours, and in samples composited from spent replicate solutions at 24- and 96-hours. At each sampling point, 9-mL samples were collected into appropriately labeled culture tubes. The samples were diluted to 10 mL with 2% erythorbic acid and further diluted, if necessary, with 0.2% erythorbic acid to provide final concentrations within the analytical standard concentration range (i.e., 0.0150 to 0.598 mg a.i./L). 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.
- Sample storage conditions before analysis: The samples were placed in vials appropriate for use in HPLC-UV and were analyzed by HPLC-UV.
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION
- Method: Test solutions were prepared by directly adding approximately 0.020, 0.040, 0.080, 0.1600, and 0.320 g (0.0125, 0.0250, 0.050, 0.100, and 0.200 g as active ingredient) to a volume of 1.0 L of dilution water.
- Controls: The control consisted of dilution water only
- Evidence of undissolved material: The test substance was completely dissolved at all concentrations tested.
- Evidence of any colored tinge in solution. The control solutions were clear and colorless throughout the test with no visible particulates, surface films, precipitates, or undissolved test substance. The 12.5 mg a.i./L test solutions were clear with a pink or light pink tint throughout the test. The freshly prepared 25.0 mg a.i./L test solutions were clear with a light pink tint and the corresponding spent solutions were clear with a light pink, pink, dark pink, or tan tint. The fresh 50.0 mg a.i./L test solutions were clear with a pink tint and the corresponding spent solutions were clear with a tan, purple, dark purple, and dark pink tint at 24, 48, 72, and 96 hours, respectively. The fresh 100 mg a.i./L test solutions were clear with a dark pink tint and the corresponding spent solutions were clear with a brown tint at 24, 48, and 72 hours, and a dark purple tint at 96 hours. The fresh 200 mg a.i./L test solutions were clear with a dark pink tint and the corresponding spent solutions were clear with a dark brown tint at 24, 48, and 72 hours, and a very dark purple tint at 96 hours.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Not reported
- Source: In-house culture
- Culturing of fish: A 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 1 L of water per fish and had a photoperiod of 16 hours of light and 8 hours of darkness.
- Egg collection: Fish were transferred to spawning aquaria the night before egg collection. An egg trap was placed in spawning aquaria before addition of the fish. The egg trap consisted of a 10 mm
square plastic grid placed at the bottom of the spawning chamber with a 2 mm nylon mesh completely covering the plastic grid. The fish and the egg trap were removed for egg collection the following morning. Eggs were collected 12 minutes post fertilization, i.e. after onset of light the next morning.

- Age of eggs at test initiation: Within approximately 2 hours post fertilization, fertilized eggs were transferred to test plate.
- Feeding during test: No
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
148 to 158 mg CaCO3/L
Test temperature:
Range 25.2-26.3 °C
pH:
5.5 to 8.1
Dissolved oxygen:
Fresh test solutions: 6.0 to 7.9 mg/L (77 to 101% saturation)
Spent test solutions: 1.9 to 7.8 mg/L (24 to 100% saturation)
Nominal and measured concentrations:
Nominal concentrations: 0, 12.5, 25.0, 50.0, 100, and 200 mg a.i./L
Geometric mean measured concentrations: Details on geometric mean measured concentrations are provided in Table 1 under ‘Any other information on materials and methods incl. tables section’
Details on test conditions:
TEST SYSTEM
- Test vessel: Micro-titre plates
- Type: Close
-Material: 24 well polystyrene micro-titre plates with 2 mL/well freshly prepared test solutions or dilution water.
- Aeration: No
- Renewal rate of test solution: 24 h renewals
- No. of eggs/well: 1 (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)
- No. of plates per concentration: 1
- No. of plates per control: 1
- Biomass loading rate: 1 egg/2 mL test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was 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, adjusted to a pH of approximately 7.0, and aerated.
The detail composition of dilution water is mentioned in 'Appendix C' of the study report
- Conductivity (during the test): 427 to 550 μS
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature, dissolved oxygen concentration, and pH were measured in parent solutions of freshly prepared and on pooled spent treatment solution on a daily basis. In addition, a continuous record of the temperature from a surrogate chamber containing dilution water and placed in the testing area was also maintained. Total hardness and conductivity were also measured daily on fresh parent and pooled spent solutions.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hr fluorescent lighting:8-hr dark
- Light intensity: 257 lux

EFFECT PARAMETERS MEASURED: Organisms were observed at 24, 48, 72 and 96 h for the following four apical endpoints: Coagulation of fertilized eggs, lack of somite formation, nondetachment of the tail-bud from the yolk sac and lack of heart-beat

-RANGE FINDING STUDY: Yes
- Nominal test concentrations: 0, 0.10, 1.0, 10, and 100 mg a.i./L
- Results used to determine the conditions for the definitive study: Yes, at test termination, approximately at 96 hours, mortality was 20, 10, 20, 100, and 0% in the control, 0.10, 1.0, 10, and 100 mg a.i./L. After 96 hours, the number of coagulated embryos in the 0.10, 1.0, 10, and 100 mg a.i./L test substance treatments was 2, 1, 2, 10, and 0, respectively, out of an initial 10 embryos
-Second Exposure Trial: After the range-finder completed, a definitive test was attempted in which the results conflicted with the range-finder. In order to determine whether this was due to solution preparation differences between the two tests, a second exposure trial was run.
The 24-hour static-renewal second exposure trial was performed at nominal concentrations of 0, 10, and 100 mg a.i./L. At test termination, approximately at 24 hours, mortality was 6, 65, and 5% in the control, 10, and 100 mg a.i./L. After 24 hours, the number of coagulated embryos in the 10 and 100 mg a.i./L test substance treatments was 13 and 1, respectively, out of an initial 20 embryos. The data indicated differences in solution preparation did not affect the results of the test.
Reference substance (positive control):
yes
Remarks:
3,4-dichloroaniline at 0 and 4.0 mg a.i./L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 235 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- The LC50 was initially calculated as > 148 mg a.i./L and recalculated as > 235 mg/l based on the tested substance and taking into account the updated certificate of analysis (see the explanations provided in "Specific details on test material used for the study").
- Mortality in test group: Mortality observed was 5, 40, 5, 5, and 5% in the 12.5, 25.0, 50.0, 100, and 200 mg a.i./L nominal test substance treatments, respectively.
-Mortality in control: After 96 h exposure to test substance, 8% mortality was observed in negative control group and 4% mortality was observed in internal controls
- Hatching rate: The hatching rate after 96 hours was 92% in the control and 95, 60, 95, 95, and 95% in the 12.5, 25, 50, 100, and 200 mg a.i./L test treatments, respectively. Hatching rates of the internal controls for the treatment plates were 96% after 96 hours.
-Other biological observations: Out of an initial 24 embryos, one embryo was coagulated and no heartbeat was observed in a second embryo in the control after 72 hours and throughout the test. After 96 hours, the number of coagulated embryos in the 12.5, 25.0, 50.0, 100, and 200 mg a.i./L test substance treatments was 0, 8, 1, 1, and 0, respectively, out of an initial 20 embryos. None of the embryos in the control or test substance treatments lacked somites throughout the test. All embryos in the control and test substance treatments exhibited detached tails except for one embryo in the 200 mg a.i./L test treatment. All embryos developed heartbeats with the exception of one embryo in the control and one embryo in the 12.5 mg a.i./L test treatment after 72 hours of exposure.

Details on mortality and other biological effects are provided in Table 1' of under “Any other information on results incl. tables”
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Mortality: 70% mortality was observed after 96 h exposure of test substance
- LC50: Not reported
- Other biological observations: Out of an initial 20 embryos, five embryos were coagulated after 24 hours in the 4.0 mg a.i./L treatment solution and eight embryos were coagulated after 96 hours. None of the embryos lacked somites or detached tails. Nine embryos lacked heartbeats at 48 hours, five embryos lacked heartbeats at 72 hours, and six embryos lacked heartbeats at 96 hours. The hatching rate in the 3,4-DCA treatment was 30 % at 72 and 96 hours. Embryos and eleutheroembryos in the 3,4-DCA treatment exhibited abnormal yolk sacks, deformed heads, and kinked tails.

Details on mortality and other biological effects of reference substance are provided in Table 1' of under “Any other information on results incl. tables”
Reported statistics and error estimates:
No statistical analyses were performed based on the lack of treatment related mortality.

Table 1: Mortality endpoints of Zebrafish,Danio rerio, exposed to Colipa A050 for 96 hours under static-renewal test conditions (Study # 68340)

Mortality

Concentration
of Substance
(mg a.s./L)

Initial
Number

Timepoint

Coagulated
b

Lack of
Somites

Nondetached
tail

Lack of
Heartbeat

Overall
Survival

%
Survival

Overall
Mortality

% Mortality

Control

0

24

24h

1

0

0

-

23

96%

1

4%

L1

12.5

20

24h

0

0

0

-

20

100%

0

0%

L2

25

20

24h

7

0

0

-

13

65%

7

35%

L3

50

20

24h

1

0

0

-

19

95%

1

5%

L4

100

20

24h

1

0

0

-

19

95%

1

5%

L5

200

20

24h

0

0

1

-

19

95%

1

5%

Ref Tox c

4

20

24h

5

0

0

-

15

75%

5

25%

Internal Controls

0

24

24h

1

0

0

-

23

96%

1

4%

Control

0

24

48h

1

0

0

0

23

96%

1

4%

L1

12.5

20

48h

0

0

0

0

20

100%

0

0%

L2

25

20

48h

8

0

0

0

12

60%

8

40%

L3

50

20

48h

1

0

0

0

19

95%

1

5%

L4

100

20

48h

1

0

0

0

19

95%

1

5%

L5

200

20

48h

0

0

1

1

19

95%

1

5%

Ref Tox c

4

20

48h

5

0

0

9

6

30%

14

70%

Internal Controls

0

24

48h

1

0

0

0

23

96%

1

4%

Control

0

24

72h

1

0

0

1

22

92%

2

8%

L1

12.5

20

72h

0

0

0

1

19

95%

1

5%

L2

25

20

72h

8

0

0

0

12

60%

8

40%

L3

50

20

72h

1

0

0

0

19

95%

1

5%

L4

100

20

72h

1

0

0

0

19

95%

1

5%

L5

200

20

72h

0

0

1

1

19

95%

1

5%

Ref Tox c

4

20

72h

7

0

0

5

8

40%

12

60%

Internal Controls

0

24

72h

1

0

0

0

23

96%

1

4%

Control

0

24

96h

1

0

0

1

22

92%

2

8%

L1

12.5

20

96h

0

0

0

1

19

95%

1

5%

L2

25

20

96h

8

0

0

0

12

60%

8

40%

L3

50

20

96h

1

0

0

0

19

95%

1

5%

L4

100

20

96h

1

0

0

0

19

95%

1

5%

L5

200

20

96h

1

0

0

0

19

95%

1

5%

Ref Tox c

4

20

96h

8

0

0

6

6

30%

14

70%

Internal Controls

0

24

96h

1

0

0

0

23

96%

1

4%

a Individual embryos may display multiple endpoints

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

c One of the effects of 3,-4 DCA on embryos is either to delay heartbeat or darken embryos making the heart beats more difficult to verify. Mortality in the Ref Tox decreases between 48 and 72 hours because the heart beat was not clearly visible at 48 hours, but became visible at 72 hours, perhaps because the heart beat was delayed in those embryos. By 96 hours, heart beats were more easily identified and observations reflect the most accurate results based on heart beat visibility.

Validity criteria fulfilled:
yes
Conclusions:
The 96 h LC50 of N,N-BIS(2-HYDROXYETHYL)- p-PHENYLENEDIAMINE SULPHATE to Zebrafish (Danio rerio) embryos was > 235 mg /L, based on geometric mean measured concentration of the tested substance.
Executive summary:

The 96 h LC50 of N,N-BIS(2 -HYDROXYETHYL)- p-PHENYLENEDIAMINE SULPHATE to Zebrafish (Danio rerio) embryo was determined under semi-static conditions following OECD draft guideline for the Fish Embryo Toxicity (FET) Test with modifications.

The nominal test concentrations were 0, 12.5, 25.0, 50.0, 100, and 200 mg active ingredient/L (the active ingredient being the tested substance without sulphate and monohydrate). The geometric mean measured concentrations were <MQL (Minimum Quantifiable Limit), 8.50, 17.8, 35.2, 70.6, and 148 mg a.i./L. The geometric mean measured concentrations were in the range of 65 to 74% of the nominal concentrations. Because the measured concentrations were not within twenty percent of the nominal concentrations, the biological response results are reported based upon the geometric mean measured concentrations.

Test vessels (24-well microtiter plates) were used (1 egg per well). There were 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.

After 96 hours of exposure to the test substance, mortality was 8, 5, 40, 5, 5, and 5% in the 0 (control), 12.5, 25.0, 50.0, 100, and 200 mg a.i./L nominal treatments, respectively.

After 96 hours, the number of coagulated embryos in the 12.5, 25.0, 50.0, 100, and 200 mg a.i./L treatments was 0, 8, 1, 1, and 0, respectively, out of an initial 20 embryos. None of the embryos in the control or treatments lacked somites throughout the test. All embryos in the control and treatments exhibited detached tails except for one embryo in the 200 mg a.i./L treatment. All embryos developed heartbeats with the exception of one embryo in the control and one embryo in the 12.5 mg a.i./L treatment after 72 hours of exposure.

The hatching rate after 96 hours was 92% in the control and 95, 60, 95, 95, and 95% in the 12.5, 25, 50, 100, and 200 mg a.i./L treatments, respectively. Hatching rates of the internal controls for the treatment plates were 96% after 96 hours.

Reference toxicant, 3,4-DCA, at 4.0 mg a.i./L resulted in mortality of 70% after 96 h of exposure.

The 96 h LC50 of N,N-BIS(2-HYDROXYETHYL)- p-PHENYLENEDIAMINE SULPHATE to Zebrafish (Danio rerio) embryos was > 235 mg /L (> 148 mg/L a.i.), based on geometric mean measured concentration of the tested substance.

This acute toxicity test is classified as acceptable and satisfies the guideline requirement of OECD draft guideline for the Fish Embryo Toxicity (FET) Test with modifications.

Description of key information

The 96 h LC50 of N,N-BIS(2-HYDROXYETHYL)- p-PHENYLENEDIAMINE SULPHATE to Zebrafish (Danio rerio) embryos was > 235 mg /L, based on geometric mean measured concentration of the tested substance.

Key value for chemical safety assessment

LC50 for freshwater fish:
235 mg/L

Additional information

The 96 h LC50 of N,N-BIS(2 -HYDROXYETHYL)- p-PHENYLENEDIAMINE SULPHATE to Zebrafish (Danio rerio) embryo was determined under semi-static conditions following OECD draft guideline for the Fish Embryo Toxicity (FET) Test with modifications.

The nominal test concentrations were 0, 12.5, 25.0, 50.0, 100, and 200 mg active ingredient/L (the active ingredient being the tested substance without sulphate and monohydrate). The geometric mean measured concentrations were <MQL (Minimum Quantifiable Limit), 8.50, 17.8, 35.2, 70.6, and 148 mg a.i./L. The geometric mean measured concentrations were in the range of 65 to 74% of the nominal concentrations. Because the measured concentrations were not within twenty percent of the nominal concentrations, the biological response results are reported based upon the geometric mean measured concentrations.

Test vessels (24-well microtiter plates) were used (1 egg per well). There were 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.

After 96 hours of exposure to the test substance, mortality was 8, 5, 40, 5, 5, and 5% in the 0 (control), 12.5, 25.0, 50.0, 100, and 200 mg a.i./L nominal treatments, respectively.

After 96 hours, the number of coagulated embryos in the 12.5, 25.0, 50.0, 100, and 200 mg a.i./L treatments was 0, 8, 1, 1, and 0, respectively, out of an initial 20 embryos. None of the embryos in the control or treatments lacked somites throughout the test. All embryos in the control and treatments exhibited detached tails except for one embryo in the 200 mg a.i./L treatment. All embryos developed heartbeats with the exception of one embryo in the control and one embryo in the 12.5 mg a.i./L treatment after 72 hours of exposure.

The hatching rate after 96 hours was 92% in the control and 95, 60, 95, 95, and 95% in the 12.5, 25, 50, 100, and 200 mg a.i./L treatments, respectively. Hatching rates of the internal controls for the treatment plates were 96% after 96 hours.

Reference toxicant, 3,4-DCA, at 4.0 mg a.i./L resulted in mortality of 70% after 96 h of exposure.

The 96 h LC50 of N,N-BIS(2-HYDROXYETHYL)- p-PHENYLENEDIAMINE SULPHATE to Zebrafish (Danio rerio) embryos was > 235 mg /L (> 148 mg/L a.i.), based on geometric mean measured concentration of the tested substance.