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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:
16 - 22 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (18 Nov 2014)
Analytical monitoring:
yes
Remarks:
HPLC-UV/VIS
Details on sampling:
- Concentrations: Control, 100 mg/L
- Sampling method: Two samples of the test and control medium each were taken directly after preparation (0 h) and at the end of exposure (96 h). One sample was used for analytics and the other one was used as backup.
- Sample storage conditions before analysis: Samples were stored at ≤ -20 °C until further analysis of the test item concentration. All samples were diluted 1:1 with diluent (50% Milli-Q-Water/50% Acetonitrile) before freezing. The stability of the test item during freezing has been demonstrated in a separate non-GLP study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock preparation with a test item concentration of 100 mg/L was directly weighed into reconstituted water and stirred for 1 h.
- Controls: Reconstituted water
- Evidence of undissolved material: The test medium was clear at the start of exposure. After 96 h the test medium was slightly turbid.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Tübinger strain
- Source: Max-Planck-Institut, Tübingen, Germany
- Length at study initiation: 2.0 ± 1.0 cm

ACCLIMATION
- Acclimation period: At least 12 d.
- Acclimation conditions: Before the start of exposure, the fish were kept in a glass aquarium in an air conditioned laboratory at a water temperature of 21 - 25 °C and a 12 h light and 12 h dark regime to ensure similar conditions as in the experiment. Aerated reconstituted water was used as holding medium.
- Feeding frequency during acclimation: Daily. The last feeding took place 24 h before the start of the treatment.
- Health during acclimation: After a period of 48 h of settling in, mortalities were recorded and the following criteria applied: (1) Mortalities of greater than 10% of population in seven days: rejection of entire batch. (2) Mortalities of 5 - 10% of population: acclimatization continued for seven additional days. (3) Mortalities of less than 5% of population: acceptance of batch.

FEEDING DURING TEST
- Food type: The zebrafish were not fed during the exposure test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
228 mg CaCO3/L (reconstituted water before test)
Test temperature:
21.4 - 23.1 °C
pH:
0 h: 7.76 (control), 6.42 (treatment)
96 h: 7.51 (control), 7.23 (treatment)
Dissolved oxygen:
8.68 mg/L (95.9%, reconstituted water before test)
0 h: 96.7% (control), 95.5% (treatment)
96 h: 85.1% (control), 82.6% (treatment)
Conductivity:
679 µS/cm (reconstituted water before test)
Nominal and measured concentrations:
Control, 100 mg/L (nominal)
< LOD, 101.252 mg/L (0 h, measured)
< LOD, 91.739 mg/L (96 h, measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquariums
- Type: The aquariums were covered in a way to allow air exchange.
- Material, size, headspace, fill volume: Material: all-glass; Fill volume: 10 L
- Aeration: The media was not aerated during the exposure.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: max. 1 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, according to the guideline.
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: The fish were acclimatized in similar conditions as in the experiment for at least 12 d before the start of the test.
- Intervals of water quality measurement: pH and oxygen concentrations of the test media were measured at the start of the exposure period and then daily. The water temperature was measured in a separate vessel containing fully demineralized water only kept under the same experimental conditions using MCPS.

OTHER TEST CONDITIONS
- Photoperiod: 12 h light/ 12 h dark
- Other: At the start of the experimental phase the zebrafish were transferred into the test vessels containing test media (test item group) or reconstituted water (control group) using a landing net. The zebrafish were randomly allocated to different groups.

EFFECT PARAMETERS MEASURED:
- Mortality: Daily
- Clinical symptoms/Signs of mortality (loss of equilibrium, changes in swimming behavior, respiratory function, pigmentation, etc. ): Daily
- Length and Body Weight: At the end of the experimental part all surving fish were killed with a mixture of ethyl 3-aminobenzoate methanesulfonate (150 mg/L) and sodium bicarbonate (300 mg/L) dissolved in reconstituted water. Afterwards, body weight and length was determined.

TEST CONCENTRATIONS
- Range finding study: Yes.
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, no effect was seen in a static non-GLP pre-test at a concentrations of 100 mg/L in open vessels. In a separate non-GLP study the stability of the test item in reconstituted water for fish was investigated. In this test it was shown that the test item concentration in reconstituted water for fish was stable over a period of 96 h in open vessels and that samples can be deep-frozen until analysis.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Observations on body length: 2.5 - 2.8 cm (control), 2.4 - 2.8 cm (100 mg/L)
- Observations on body weight: 0.5 - 0.7 g (control), 0.4 - 0.8 g (100 mg/L)
- Mortality of control: No mortality was seen.
- Any observations that might cause a difference between measured and nominal values: The test medium was a clear preparation at start of exposure. After 96 h the test medium was slightly turbid.
- Effect concentrations exceeding solubility of substance in test medium: No

ANALYTICAL RESULTS

The measured concentration of the test item was 101.3% at the start of exposure and 91.7% after 96 h (Table 1). The measured concentrations were ≥ 80% of the nominal concentration. Therefore, the LC50 is based on the nominal concentration.

 

Table 1. Analytical results

 

0 h

96 h

 

mg/L

% of nominal

mg/L

% of nominal

Control

< LOD

-

< LOD

-

100.0 mg/L

101.252

101.3

91.739

91.7

 

BIOLOGICAL RESULTS

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L test item revealed no aquatic toxicity in the test system.

Description of key information

LC50 (96 h) > 100 mg/L (nominal, OECD 203, D. rerio)

Key value for chemical safety assessment

Additional information

There is one study available assessing the short-term toxicity of N-Acetyl-DL-tryptophan (CAS 87-32-1) to fish according to the OECD guideline 203 and GLP.

In a static limit test, 10 zebrafish (Danio rerio) were exposed to a nominal concentration of 100 mg/L test item in an open system for 96 h. Test item concentrations were analytically verified at the beginning (0 h) and at the end of the test (96 h) by HPLC-UV/VIS. Mortality and signs of toxicity were recorded daily and compared with control values.

The measured concentration of the test item was 101.3% at the start and 91.7% at the end of exposure. The measured concentrations were ≥ 80% of the nominal concentration. Therefore, the LC50 is based on the nominal concentration. After 96 h, no acute toxicity was observed. The LC50 (96 h) was > 100 mg/L (nominal).