Registration Dossier

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-11 to 2015-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Version / remarks:
adopted July 26, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Various concentrations of the test item 1-Propanaminium, N,N,N-1-Propanaminium, N,N,N-trimethyl-3-[(1-oxohexadecyl)amino]-, chloride and the control were analytically verified by LC-MS/MS from freshly prepared test media and corresponding 24 hours aged test media of two sampling intervals, respectively. The samples from the freshly prepared media were taken directly from the test concentrations and the control. The samples of the 24 hours aged solutions were pooled per test concentration and control from at least 10 alternating replicates each.- Sample storage conditions before analysis: room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A stock solution of 5.25 mg test item/L was prepared with test medium (river water) for each renewal day. This solution was stirred at a temperature of 40°C ± 2°C for 24 ± 1 hours. Thereafter, the saturated solution was stirred for a further 24 ± 1 hours and cooled down to the temperature of the test (26 ± 1°C). The test concentrations were prepared from the stock solution by dilution with natural river water.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Source: brood fish: Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany
- Age at study initiation: fertilised eggs were used within 2-3 h post fertilization
- Method of breeding: A breeding stock of unexposed, mature zebrafish with an age of 9 months was used for egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60 % of air saturation value
- pH-value: 6.5 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
- Diffuse light (0.1 - 10 µmol photons • m-2 • s-1on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum; Daphnia magna, juvenile and adult daphnids, ad libitum; dry food sera vipan SERA, ad libitum
- No disease treatments were administered.
- Feeding during test: no
Approximately 2 h post fertilization, eggs were checked for fertilization. Every embryo was checked under a stereo microscope (40-fold magnification) for its blastomer phase. Eggs with only a 2 cell blastomer were regarded as not fertilised. These eggs as well as coagulated eggs were discarded.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
194 - 201 mg CaCO3/L
Test temperature:
25.3 - 26.0°C
pH:
7.92 - 8.49
Dissolved oxygen:
91-100 % of air saturation value
Nominal and measured concentrations:
nominal: 0.480, 0.864, 1.56, 2.80, 5.04 mg a.i./L
measured (geom. mean): 0.0320, 0.0430, 0.0587, 0.0806, 4.55 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 24 well micro-titre plates pretreated with the appropriate test solutions for 72 hours. Before the start of exposure, the test vessels were emptied and refilled with freshly prepared test solutions
- Fill volume: 2 mL per well
- Aeration: no
- Renewal rate of test solution: daily renewal: test organisms were retained in the test vessels whilst a proportion of at least 75 % of the test medium was gently removed with a pipette; freshly prepared media were then added gently to the test vessels with a pipette to avoid stressing of the test organisms
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 24
- No. of vessels per internal control (replicates): 24

TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 3.98 to 4.98 (mean value 4.53) µmol photons • m-2 • s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Coagulated eggs: 24, 48, 72, 96 h
Absence of somite formation: 24, 48, 72, 96 h
Non-detachment of the tail: 24, 48, 72, 96 h
Lack of heartbeat: 48, 72, 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
Range finding study
- Test concentrations: 0.0480, 0.480, 4.80, 48.0 mg a.i./L (nominal)
- Results used to determine the conditions for the definitive study: 100, 95, 100, 0, 0% survival at 0.0480, 0.480, 4.80, 48.0 mg a.i./L, respectively
Reference substance (positive control):
yes
Remarks:
3,4-Dichlororaniline
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% c.l. 0.0431 – 0.0560 mg a.i./L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% c.l. 0.0431 – 0.0555 mg a.i./L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% c.l. 0.0431 – 0.0555 mg a.i./L
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% c.l. 0.0431 – 0.0555 mg a.i./L
Details on results:
The mean fertility rate was ≥ 96 % for all eggs collected for test initiation.
The mortality in the positive control was 55 % after 96 h. The hatching rate in the negative control was 98 % after 96 hours.
Results with reference substance (positive control):
- Results with reference substance valid?
- Mortality: 100% mortality at 6.50 mg/L
- LC50: 2.72 mg/L, 95% c.l. 2.65 – 2.79 mg/L
- Other: tested 2015-03-15 to 2015-03-20
Reported statistics and error estimates:
LC10,20,50
-values and corresponding confidence intervals after 24, 48, 72 and 96 hours were calculated by sigmoidal dose-response regression. The values for LC0 and LC100 were determined directly from the test results.
Sublethal observations / clinical signs:

Geometric mean measured test item loading level [mg/L]

Effect

24 h

48 h

72 h

96 h

Control*

Survival

47

47

47

47

Mortality endpoints

 

          Coagulated / Dead

1

1

1

1

          Lack of somites

-

-

-

-

          Non-detached tail

-

-

-

-

          Lack of heartbeat

n.a.

-

-

-

Other observations

 

          Hatching rate [%]

-

-

96

98

          Hatched

-

-

45

47

          Scoliosis

-

-

-

-

          Lack of circulation

n.a.

-

-

-

          Malformation of heart

 n.a.

-

-

-

          Oedema

-

-

-

-

          No pigmentation

n.a.

-

-

-

0.0320

Survival

20

20

20

18

Mortality endpoints

 

          Coagulated / Dead

-

-

-

-

          Lack of somites

-

-

-

-

          Non-detached tail

-

-

-

-

          Lack of heartbeat

n.a.

-

-

2

Other observations

 

          Hatching rate [%]

-

-

80

100

          Hatched

-

-

16

20

          Scoliosis

-

-

-

-

          Lack of circulation

n.a.

-

-

-

          Malformation of heart

n.a.

-

-

-

          Oedema

-

-

-

-

          No pigmentation

n.a.

-

-

-

0.0430

Survival

16

16

16

16

Mortality endpoints

 

          Coagulated / Dead

4

4

4

4

          Lack of somites

-

-

-

-

          Non-detached tail

-

-

-

-

          Lack of heartbeat

n.a.

-

-

-

Other observations

 

          Hatching rate [%]

-

-

65

80

          Hatched

-

-

13

16

          Scoliosis

-

-

1

1

          Lack of circulation

n.a.

-

-

1

          Malformation of heart

n.a.

-

-

-

          Oedema

-

-

1

1

          No pigmentation

n.a.

-

-

-

0.0587

Survival

0

0

0

0

Mortality endpoints

 

          Coagulated / Dead

20

20

20

20

          Lack of somites

-

-

-

-

          Non-detached tail

-

-

-

-

          Lack of heartbeat

n.a.

-

-

-

Other observations

 

          Hatching rate [%]

-

-

-

-

          Hatched

-

-

-

-

          Scoliosis

-

-

-

-

          Lack of circulation

n.a.

-

-

-

          Malformation of heart

 n.a.

-

-

-

          Oedema

-

-

-

-

          No pigmentation

n.a.

-

-

-

0.0806

Survival

0

0

0

0

Mortality endpoints

 

          Coagulated / Dead

20

20

20

20

          Lack of somites

-

-

-

-

          Non-detached tail

-

-

-

-

          Lack of heartbeat

n.a.

-

-

-

Other observations

 

          Hatching rate [%]

-

-

-

-

          Hatched

-

-

-

-

          Scoliosis

-

-

-

-

          Lack of circulation

n.a.

-

-

-

          Malformation of heart

 n.a.

-

-

-

          Oedema

-

-

-

-

          No pigmentation

n.a.

-

-

-

4.55

Survival

0

0

0

0

Mortality endpoints

 

          Coagulated / Dead

20

20

20

20

          Lack of somites

-

-

-

-

          Non-detached tail

-

-

-

-

          Lack of heartbeat

n.a.

-

-

-

Other observations

 

          Hatching rate [%]

-

-

-

-

          Hatched

-

-

-

-

          Scoliosis

-

-

-

-

          Lack of circulation

n.a.

-

-

-

          Malformation of heart

 n.a.

-

-

-

          Oedema

-

-

-

-

          No pigmentation

n.a.

-

-

-

* = All replicates, including the internal control replicates, were considered

 

Geometric mean measured test item loading level
[mg/L]

Effect

[n/20 or 48]

24 h

48 h

72 h

96 h

Survival at 96 h
[%]

Control*

Survival (overall)

47

47

47

47

98

Mortality (overall)

1

1

1

1

0.0320

Survival (overall)

20

20

20

18

90

Mortality (overall)

0

0

0

2

0.0430

Survival (overall)

16

16

16

16

80

Mortality (overall)

4

4

4

4

0.0587

Survival (overall)

0

0

0

0

0

Mortality (overall)

20

20

20

20

0.0806

Survival (overall)

0

0

0

0

0

Mortality (overall)

20

20

20

20

4.55

Survival (overall)

0

0

0

0

0

Mortality (overall)

20

20

20

20

* = All replicates, including the internal control replicates, were considered

Validity criteria fulfilled:
yes
Conclusions:
The 96 h LC50 of C16 Alkylamidopropyltrimethylammonium Chloride to embryonic stages of zebrafish (Danio rerio) was 0.0455 mg a.i./L (95% c.l. 0.0431 – 0.0560 mg a.i./L) based on geometric mean measured concentrations.
Executive summary:

In a 96-h acute toxicity study according to OECD Guideline 236, adopted July 26, 2013, embryonic stages of zebrafish (Danio rerio) were exposed to C16 Alkylamidopropyltrimethylammonium Chloride at nominal concentrations of 0 (control), 0.480, 0.864, 1.56, 2.80, 5.04 mg a.i./L, corresponding to measured (geom. mean) concentrations of 0, 0.0320, 0.0430, 0.0587, 0.0806, 4.55 mg/L under semi-static conditions.

The test item is known to sorb to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results from such tests depend from the test settings applied. Therefore, natural river water was used which contains particulate as well as dissolved organic carbon to which the test item can sorb partially to reduce the difficulties encountered in tests with synthetic water (e.g. preventing that the test item settles onto surfaces). The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries at the end of the exposure. Nevertheless the test item is present in the test system and therefore available for exposure (dissolved in water and sorbed).

Additionally, the test vessels were pre-treated with the test solutions one day before the start of the respective exposure interval to saturate the glass walls with sorbed test item in the definitive test. This procedure was carried out to achieve stable exposure concentrations in the test vessels and to reduce losses of the test item by adsorption onto the glass walls.

The 96-h LC50 0.0455 mg a.i./L (95% c.l. 0.0431 – 0.0560 mg a.i./L) based on geometric mean measured concentrations

This toxicity study is classified as acceptable and satisfies the guideline requirement for anacute toxicity study with embryonic stages of zebrafish (Danio rerio).

 

Results Synopsis

Test Organism Size/Age: Danio rerio embryos, 2 -3 h post fertilisation

Test Type: Static Renewal

96 h LC50: 0.0455 mg a.i./L (95% c.l. 0.0431 – 0.0560 mg a.i./L)

Endpoint(s) Effected: Mortality 

Description of key information

96 h LC50 = 45.5 µg a.i./L (95% c.l. 43.1 – 56.0 µg a.i./L) (geom. mean measured); embryonic stages of Danio rerio, semi-static, river-water; OECD Guideline 236 (2013); RL1, GLP

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
45.5 µg/L

Additional information

In a 96-h acute toxicity study according to OECD Guideline 236, adopted July 26, 2013, embryonic stages of zebrafish (Danio rerio) were exposed to C16 Alkylamidopropyltrimethylammonium Chloride at nominal concentrations of 0 (control), 0.480, 0.864, 1.56, 2.80, 5.04 mg a.i./L, corresponding to measured (geom. mean) concentrations of 0, 0.0320, 0.0430, 0.0587, 0.0806, 4.55 mg/L under semi-static conditions.

The test item is known to sorb to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results from such tests depend from the test settings applied. Therefore, natural river water was used which contains particulate as well as dissolved organic carbon to which the test item can sorb partially to reduce the difficulties encountered in tests with synthetic water (e.g. preventing that the test item settles onto surfaces). The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries at the end of the exposure. Nevertheless the test item is present in the test system and therefore available for exposure (dissolved in water and sorbed).

Additionally, the test vessels were pre-treated with the test solutions one day before the start of the respective exposure interval to saturate the glass walls with sorbed test item in the definitive test. This procedure was carried out to achieve stable exposure concentrations in the test vessels and to reduce losses of the test item by adsorption onto the glass walls.

The 96-h LC50 0.0455 mg a.i./L (95% c.l. 0.0431 – 0.0560 mg a.i./L) based on geometric mean measured concentrations