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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:
2017-05-29 to 2017-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
HPLC-DAD in the first definitive test and LC-MS/MS in the second definitive test
Details on sampling:
- Concentrations: In the first definitive test the limit concentration of nominal 1.20 mg/L (corresponding to a target exposure concentration of 0.600 mg/L), the solvent control and the negative control were analysed. In the second definitive test nominal concentrations of 22.5, 45.0, 90.0, 180.0, 360 mg/L, solvent control and negative control were analysed.
- Sample storage conditions before analysis: Sample storage was different for the respective analyttic method: HPLC-DAD: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis. LC-MS/MS: All samples were stored at room temperature until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions were prepared once in dimethyl formamide (DMF) three days prior to the start of the exposure for both definitive tests.
- Controls: see table 1
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide with a loading of 0.100 mL/L dilution water
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio
- Length at study initiation (length definition, mean, range and SD): not specified, Average body length at the test end: 2.7 cm
- Weight at study initiation (mean and range, SD): not specified, Average body weight at the test end: 0.17 g
- Maintenance of the brood fish: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (7 - 750 Lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value.

ACCLIMATION
- Acclimation period: At least 12 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany. The amount of food was 4 % of the fish body weight per feeding day.
- Feeding frequency during acclimation: Food was provided 3 times per week.
- Health during acclimation (any mortality observed): Zebrafish with < 5% mortality during acclimation were used in the test
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
10 - 250 mg CaCO3/L
Test temperature:
23 ± 2 “C, controlled at ± 1 “C
pH:
6.0 - 8.5
Dissolved oxygen:
Not less than 60 % of air saturation value.
Conductivity:
151 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 1200 µg/L corresponding to a target exposure concentration of 0.600 mg/L, in the first definitive test and 360, 180, 90.0, 45.0, 22.5 µg/L in the second definitive test
Measured concentrations: 782 µg/L (test vessel) and 1400 µg/L (mixing chamber supply) in the first definitive test and arithmetic mean measured test item concentrations of 240, 108, 53.4, 27.0 and 12.7 µg/L in the second definitive test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass-aquaria of 3.3 L volume, filled with 3 L of dilution water (dimensions: 12/13/21 cm, depth of water: 19 cm), covered with glass plates were used. The aquaria were uniquely identified.
- Type (delete if not applicable):closed
- Aeration: No aeration was provided in the test vessels. The dilution water supply tank was aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Membrane piston pumps provided the water flow-through.
- Renewal rate of test solution (frequency/flow rate): Water exchange in the test aquaria (approx. Vol. 3 L) was about 50 times per day (6.250 LIh).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): One replicate per test concentration, solvent control and control.
- Biomass loading rate: Fish density in the tanks was 0.0793 g fish per litre of test solution within 24 h (required: < 1 g x L-1 x d-1).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin
- Total organic carbon: 10-250 mg CaCO3/L
- Chlorine: The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
- Alkalinity: 0.60 mmol/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h cycle
- Light intensity: 7-750 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation, etc.). For measurements of size and body weight the control fish were used at the end of the exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 2
- Range finding study
- Test concentrations: The target exposure concentration (threshold concentration) was based on the toxicity data of an acute toxicity test with Daphnia magna (under flow through conditions) and an alga toxicity test.
- Results used to determine the conditions for the definitive study: A nominal test concentration of 1.20 mg/L (corresponding to a target exposure concentration of 0.600 mg/L) was prepared and tested as a threshold concentration in a limit test. Since the test item is known to hydrolyse quickly (Half life T1/2 0.149 d (aqueous solution pH 7, 20 °C), preliminary investigations on feasibility of dosing via the flow through system were carried out. The nominal test concentration of 1.20 mg/L was based on the results of preliminary investigations of dosing the test item via the flow-through system. The concentrations in the concentration-response test were based on a preliminary range finding test and a first definitive study under threshold approach.
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
433 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
240 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Other biological observations: One animal was found dead at a test concentration of 27 µg/L after 96 h. For the animals of the highest concentration group (240 µg/L) quiescence was recorded at all observation time points.
- Mortality of control: no mortality was detected in the control group

Reported statistics and error estimates:
The LC50-values and corresponding confidence intervals after 96 h were calculated by standard procedures, as revised in OECD Series on Testing and Assessment No. 54 (2006). For evaluation the results of both definitive tests were considered In the first definitive test the nominal threshold concentration of 1200 µg/L induced a mortality of 100 %. In the second definitive test the highest nominal test concentration of 360 µg/L caused no mortality. Therefore the LC50-value was calculated with straight line regression of both concentration levels. The concentrations enclosed to the LC50-value were chosen as lower and upper confidence limits. Results are given based on the arithmetical mean measured test item concentrations. All data were computer generated and rounded for presentation from the full derived data.

Table 2: Cumulative Mortality of the first definitive test:

Measured test item concentration [µg/L]

Cumulative mortality [%] after different exposure periods [hours]

3

24

48

72

96

782

100

-

-

-

-

Control

0

0

0

0

0

Table 3: Cumulative Mortality of the second definitive test:

Arithmetic mean measured test item concentrations [µg/L]

Observation

Number of fish effected after different exposure periods [hours]

3

24

48

72

96

240

Quiescence

7

7

7

7

7

108

normal

7

7

7

7

7

53.4

normal

7

7

7

7

7

27.0

exitus letalis (1),

0

0

0

0

1

normal (6)

7

7

7

7

6

12.7

normal

7

7

7

7

7

Solvent control

normal

7

7

7

7

7

Control

normal

7

7

7

7

7

Validity criteria fulfilled:
yes
Conclusions:
The test item was found to be toxic to zebrafish with a LC50 (arithmetic mean measured concentration) after 96 hours of 433 (240 to 782) µg test item/L.
All effect levels are given based on the arithmetic mean measured concentrations of the test item 2,4-Bismaleimidotoluene.

Description of key information

- OECD guideline study (203, adopted 1992), 7 Danio rerio for each treatment group, exposure 96h under flow-through conditions, LC50 = 433 µg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
0.433 mg/L

Additional information

The acute toxicity of the test item 2,4-Bismaleimidotoluene to fish (Danio rerio) was determined according to OECD TG 203 (adopted 1992). Fish were exposed to 1200 µg/L (measured concentration: 782 µg/L) in a first experiment following the threshold approach. Since 3h exposure to the test item at this concentration resulted in 100% mortality a second test was performed with the nominal concentrations of 22.5 - 45.0 - 90.0 - 180 - 360 µg/L (factor 2; arithmetic mean measured concentrations: 12.7 - 27.0 - 53.4 - 108 - 240 µg/L) for 96h. Both tests were performed using a flow-through test design. Seven test organisms were exposed to each test concentration and the control. In the second definitive test various concentrations of the test item 2,4-Bismaleimidotoluene were analytically verified via LC-MS/MS on study day 0 and daily thereafter until the end of exposure in all tested concentration levels and the control. The measured concentrations of 2,4-Bismaleimidotoluene in the test vessels were in the range of 47 to 77 % of the nominal values during exposure. The measured concentrations of the mixing chamber supplies were in the range of 80 to 92 % at study day 0 and in the range of 81 to 87 % at study day 3 of the nominal values. The LC50 value for the observation time points 3, 24, 48, 72 and 96h was in each case 433 µg/L (arithmetic measured mean). The LC0 was determined to be 240 µg/L.