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Short-term toxicity to fish

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Reference
Endpoint:
fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 17 Aug 2018 (biological part) and 20 - 21 Aug 2018 (analytical part)
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:
26 Jul 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requuirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (13 Mar 2017)
Analytical monitoring:
yes
Remarks:
LC-MS
Details on sampling:
- Concentrations: Control, 0.94, 1.88, 3.75, 7.50, and 15.0 at test start, each water renewal (24, 48, and 72 h) and at test end
- Sampling method: One sample was taken from the freshly prepared stock solution (2 samples at test start) and duplicate samples from the freshly prepared test media of all treatment groups were taken at test start and at each medium renewal (Day 1, 2 and 3 of exposure). For the determination of the stability of test item concentrations, duplicate samples of aged test media of all treatment groups were taken on day 1, 2, and 3 and at the end of the last renewal period on day 4. Wells were pooled to obtain sufficient volumes. All samples were directly diluted by a factor of two with acetonitrile.
- Sample storage conditions before analysis: In a freezer (≤ - 20 °C) protected from light until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the pre-condition of the test vessels, a stock solution of 100 mg test item/L was prepared (64.7 µL test item in 200 mL) by intense stirring for 30 min. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations. For the main test, a 100 mg/L stock solution was prepared on Day 0, 1, 2, and 3 in the same way and the desired final concentrations were prepared by dilution of appropriate amounts of this stock solution. All test media were freshly prepared just before pre-conditioning, introduction of the embryos (= start of the test) and at renewal (on day 1, 2, and 3).
- Differential loading: No
- Controls: Test water without test item or reference item.
- Evidence of undissolved material: A slightly pronounced turbidity of the test medium, combined with a slightly pronounced flocculation was observed in the test item concentration of 15.0 mg/L (nominal).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: In-house breeding
- Brood fish: Breeding stock of known holding conditions
- Age at study initiation: Embryos; The cell stage of the embryos introduced at test start: 2 to 8
- Sex: Male and female
- Quality of eggs (fertilisation rate): 87% (n = 10, sub-sample)

FEEDING DURING TEST
None
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
196 - 231 mg CaCO3/L (fresh media)
196 - 231 mg CaCO3/L (aged media)
Test temperature:
25.5 - 25.8 °C
pH:
7.3 - 7.7 (fresh media)
7.5 - 7.8 (aged media)
Dissolved oxygen:
99 - 116 mg/L (fresh media)
69 - 100 mg/L (aged media)
Conductivity:
513 - 732 µS/cm (fresh media)
558 - 580 µS/cm (aged media)
Nominal and measured concentrations:
Control, 0.94, 1.88, 3.75, 7.50, and 15.0 mg/L (nominal)
Control, 0.67, 1.44, 2.95, 6.14, and 10.70 mg/L (geometric mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 24-well polystyrene well plates with > 2 mL test medium
- Renewal rate of test solution: Daily . The same well plates were used for test water renewals. Most of the old test media was removed, leaving sufficient volume for the fish embryo not to be exposed to air, and freshly prepared test media (approximately 2 mL) was introduced into each well. With this procedure minimal disturbance or stress was exerted on the embryo.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20 (20 eggs on 1 plate for each test concentration)
- No. of vessels per negative control (replicates): 24 (24 eggs in 1 plate)
- No. of vessels per positive control (replicates): 20 (20 eggs in 1 plate)
- No. of vessels as internal plate control: 4 (4 eggs per plate)
- Other: Three days prior to the test, all wells were pre-conditioned with test media. Each well was filled with at least 2 mL freshly prepared test water with the respective concentration of the test item, reference item or negative control.
- Fish embryo handling: Eggs were immersed in the test media containing the five test concentrations, the positive, negative and internal controls at cell stages 2 to 8. The eggs were inspected with a stereo microscope in order to determine fertilisation rate and cell stages. The introduction of the eggs in the test media indicated the initiation of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (ISO medium) in deionised water
- Alkalinity: 0.8 mmol/L
- Conductivity: ≤ 10 µScm^-1
- Ca/mg ratio: 4:1 (based on molarity)
- Na/K ratio: 10:1 (based on molarity)
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: Dissolved oxygen and pH were determined daily in the freshly prepared and aged test media of each treatment group. Water hardness and conductivity were measured at test start and termination. In the aged test media, after each water exchange and at test end, parameters were measured in pooled replicates. Temperature was recorded daily in a separate vessel with the test chamber. pH was measured in all test item concentrations and the control at the start and end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 14 h light, 10 h dark
- Light intensity: 660 - 740 lux
- Test environment: Controlled incubator

EFFECT PARAMETERS MEASURED:
- Apical observations: The embryos were inspected at test start and after approximately 24, 48, 72, and 96 h after test start for coagulated embryos, lack of somite formation and non-detachment of the tail. The lack of heartbeat was recorded after 48, 72, and 96 h. All these test parameters were determined with a steroscopic microscope.
- Hatcvhing: Recorded in all test chambers after 48, 72, and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study : Yes, non-GLP
Reference substance (positive control):
yes
Remarks:
3,4-Dichloroaniline
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.256 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence interval: 2.95 - 6.14 mg/L
Details on results:
- Behavioural abnormalities: Lethargy and coagulation were observed at the highest two concentrations at 24, 48, 72, and 96 h
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: No remarkable observations, clear test medium
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: 100% (4.0 mg/L Dichloroaniline)
Reported statistics and error estimates:
Data analyses were performed with ToxRate Professional (version 3.2.1, ToxRat Solutions GmbH). The LC50 and its confidence interval were calculated by a binomial distribution test. The NOEC was calculated by Step-dwon Cochran-Armitage test procedure and the LOEC was derived empirically.
Sublethal observations / clinical signs:

VALIDITY CRITERA

The study met all validity criteria of the guideline (Table 1) and is therefore considered valid.

Table 1: Validity criteria for OECD 236.

Criterion

Outcome

Validity criterion fulfilled

Fertilization rate

The overall fertilization rate of all eggs collected was 87% in the batch test.

Yes

Water temperature

The water temperature was maintained at 25.5. to 25.8 °C at the chamber at any time during the test.

Yes

Survival of embryos

The overall survival of embryos in the negative control (test water) was 100% until test termination (96 h).

Yes

Hatching Rate

Hatching rate in the negative control was 100% at test termination 896 h).

Yes

Dissolved oxygen

At test termination (96 h), the dissolved oxygen concentration in the negative control and the highest test concentration was 98% and 96% of saturation, respectively.

Yes

Positive control

The mortality in the positive control was 100%.

Yes

 

ANALYTICAL RESULTS

Measured test item concentrations were 85 – 92% of nominal in fresh media and 71 to 82% of nominal in aged media (Table 2). Since the concentrations of the test item were not within ± 20% of the initial concentrations during the test, results were based on the geometric mean measured concentration.

Table 2. Summary of analytical results.

Nominal concentration

fresh % of nominal1

aged % of nominal1

% of nominal1

Geometric mean measured

n

[mg test item/L]

[mg test item/L]

Control

n.a.

n.a.

n.a.

n.a.

n.a.

0.94

92

57

71

0.672

16

1.88

85

70

77

1.44

16

3.75

87

72

79

2.95

16

7.5

87

77

82

6.14

15

15.0

88

58

71

10.7

16

1           mean value of all measured samples per treatment group

n           Number of analyzed samples

n.a.       Not applicable

 

BIOLOGICAL RESULTS

After 96 h, all fish embryos survived up to the test item concentration of 2.95 mg/L. In the test item concentration of 6.14 and 10.70 mg/L, all embryos died during the exposure. Hatching success in the control and in all test item concentration up to 2.05 mg/L was 100%. The internal plate controls showed 100% hatching success and thus confirmed the validity of the testing procedure (Table 3).

 

Table 3. Observed mortality and hatching success of the zebrafish embryos (D. rerio) after 96 h

Geometric Mean Measured Concentrations

96-hour

96-hour (internal plate control)

mg test item/L

% mortality

% hatching

% mortality

% hatching

Control

0

100

-

-

0.67

0

100

0

100

1.44

0

100

0

100

2.95

0

100

0

100

6.14

100

0

0

100

10.70

100

0

0

100

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

LC50 (96 h) = 4.256 mg a.i./L (geom. mean measured, OECD 236, D. rerio)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
4.256 mg/L

Additional information

One GLP study is available, in which the short-term toxicity of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) to fish was assessed according to OECD guideline 236.

In a semi-static test with daily test medium renewal, Zebrafish embryos (≤ 16 cell stage) were exposed to the five nominal test item concentrations of 0.94, 1.88, 3.75, 7.50, and 15.0 mg a.i./L (corresponding to 3.04, 6.08, 12.14, 24.27, and 48.54 mg test item/L) for 96 h. A negative control, positive control and internal (procedural) control were run in parallel. Hatching rates were determined and acute lethality was assessed by daily assessment of the coagulation of embryos, lack of somite formation, the non-detachment of the tail-bud from the yolk sac, and the lack of heart beat. The actual test item concentrations were analytically verified by LC-MS every 24 h in fresh and aged media.

The average measured test item concentration at the start of the test and at the renewal of the test media was 88% of nominal in fresh media and 67% of nominal in aged media after 24, 48, 72 and 96 h. Since the concentrations of the test item were not within ± 20% of the initial concentrations during the test, results were based on the geometric mean measured concentrations of 0.67, 1.44, 2.95, 6.14, and 10.70 mg a.i./L (corresponding to 2.17, 4.66, 9.55, 19.87, and 34.63 mg test item/L).

After 96 h, all embryos survived up to the test item concentration of 2.95 mg a.i./L. All embryos died during exposure to the test concentrations of 6.14 and 10.7 mg a.i./L. Hatching success in the control and in all test item concentration up to 1.44 mg a.i./L was 100%. Hatching success in the test item concentratin of 2.95 mg a.i./L was 95%. The internal plate controls showed 100% hatching success and thus confirmed the validity of the testing procedure. The obtained LC50 (96 h) is 4.256 mg a.i./L based on the geometric mean measured concentrations.

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