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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
equivalent or similar to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
according to guideline
other: DIN 38 412, L15 (1982)
Principles of method if other than guideline:
Statistical methods: Probit analysis
GLP compliance:
Analytical monitoring:
Details on test solutions:
- Method: the test item was added to the test water without any pretreatment
- Controls: positive control with Chloroacetamide as reference compound, negative control (test water only)
- Test concentration separation factor: 2.2
- Evidence of undissolved material: no
Test organisms (species):
Leuciscus idus
Details on test organisms:
- species: Golden Orfe (Leuciscus idus L., golden variety)
- supplier: Fischzucht Paul Eggers, D-2354 Hohenwestedt, GER
- age: no data
- length: 7.7 cm (range: 6.9 - 8.9)
- weight: 4.3 g (range: 2.8 - 6.6)
- loading: 4.3 (g fish / L test water)
- pretreatment: twice with 0.05 mg/L malachite green chloride, once with 10 mg/L tetracycline hydrochloride
- diet: growing feed ad libitium (F/B 50, SSNIFF Spezialdiaeten GmbH, D-4770 Soest, FRG)
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
2.5 mmol/L
Test temperature:
20 °C
7.6 - 8.0
Dissolved oxygen:
6.0 - 8.6 mg/L
not applicable
Nominal and measured concentrations:
Nominal: 0 (control), 46.4, 100, 215, 464, 1000 mg/L; measured concentrations not available
Details on test conditions:
- static
- the product was added to the test water without any pretreatment, subsequently the fish were placed into the aquaria
- nominal concentrations: 46.4, 100, 215, 464 and 1000 mg/L
- exposure vessel type: all-glass aquaria (30 cm x 22 cm x 24 cm), slight aeration
- photoperiod: 16 hours light and 8 hours darkness
- one vessel per concentration/control, 10 fish per replicate
- Water chemistry in test

- reconstituted freshwater according to DIN 38 412; part 11, October 1982
- preparation from fully demineralised tap water
- conductivity: max. 10 µmho/cm
- The water is carried out by adding
294.0 mg/L CaCl2 x 2 H20
123.3 mg/L MgS04 x 7 H20
63.0 mg/L NaHCO3
5.5 mg/L KCl
- continuous aeration with oil free air
- total hardness: 2.5 mmol/L
- acid capacity: 0.8 mmol/L
- ratio Ca/Mg ions: 4:1
- ratio Na/K ions: 10:1
- pH: about 8.0

- Apathy
- Exophthalmos
- Hyperreflexia
- Gasping
- Discoloration
- Accelerated respiration
- Abdominal distension
- Escape relfex
- Convulsions
- Narcotic-like state
- Restelessness
- Headstand
Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
>= 46 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
< 215 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Observations on body length and weight: no
- Other biological observations: tumbling
- Mortality of control: no
- Other adverse effects control: no
- Any observations that might cause a difference between measured and nominal values: no (test item concentrations not analyzed)
- Effect concentrations exceeding solubility of substance in test medium: no

The study was assessed to be valid with restrictions in accordance with the validity criteria of the current OECD TG 203 (18 June 2019). The test was carried out in 1988 according to a national guideline. To this time analytical measurement of the test item concentration was not compulsory and nominal test item concentrations were thus not analytically verified. However, the substance is considered relatively stable at test duration of 96 hours (4 days) and the applied test conditions as it does not hydrolyse rapidly in water. From the water surface, the substance will slowly evaporate into the atmosphere. The Henry´s law constant (H) at 25°C was estimated to be 21.5 Pa*m3/mol by HENRYWIN v3.20 (see IUCLID section 5.4.2). In the OECD Guidance Document No. 23 on aqueous phase aquatic toxicity testing of difficult test chemicals (second edition, 8. February 2019) it is stated that “losses due to volatilisation may become significant for test chemicals with Henry's law constants of 1-10 Pa*m3 mol under vigorous mixing conditions where the opportunity for water air exchange is high”. It is pointed out that “if H is greater than 100 Pa*m3/mol , more than 50% of the test chemical could be lost from the water phase within 3-4 hours”. In this study the test item was added to the test media without any pretreatment and the media were not vigorously mixed. The Henry’s Law constant of the substance is far below 100 Pa*m3/mol and the evaporation of the test item during the study is considered relatively low. Moreover, the biodegradation of the substance was assessed in a valid GLP guideline study to be 5 % after 7 days (see IUCLID section 5.2.1). Further, based on its calculated Koc the substance has only very low potential for adsorption. Therefore, it is expected that the actual test substance concentration was only slightly below the nominal concentrations. In addition, no mortality was observed in the negative control and the oxygen concentration was confirmed to be >= 60 % of saturation concentration throughout the test. Additionally, the suitability of the test system was verfied by a positive control. In sum, the study is regarded valid and acceptable for assessment.
Results with reference substance (positive control):
LC50 (48 hours) = 26 mg/L
Reported statistics and error estimates:
The lethal concentrations (LCx) were derived for 1, 24, 48 72 and 96 hours of treatment using Probit analysis acc. Finney (1971).
Sublethal observations / clinical signs:
- biological observations: at the concentration of 1000 mg/L after 4 hours tumbling of the fish were observed

- cumulative mortality:

(mg/L)1 h24 h48 h72 h96 h

- lowest test substance concentration causing 100% mortality: 215 mg/L
- mortality of controls: none
- abnormal responses: none
- reference substances: positive control of animals conducted with chloroacetamide;
LC 50 after 48 hours: about 26 mg/L (corresponds to normal sensitivity)
Validity criteria fulfilled:
In a 96-hour acute toxicity study with Leuciscus idus the LC50 was determined at >100 mg/L, based on nominal test concentrations.

Description of key information

With high probability acutely not harmful to fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
Effect concentration:
> 100 mg/L

Additional information

In an acute fish test according to German national standards (DIN 38412), the LC50 (96h) for Leuciscus idus was determined to be > 100 mg/L 1.2 -epoxybutane [BASF AG, 1988].

Animals were exposed for 96 hours in a static regime to nominal test item concentrations of 0 (control), 46.4, 100, 215, 464 and 1000 mg/L (separation factor: 2.2). In addition, a positive control with Chloroacetamide as reference compound was performed. One vessel per concentration/control with 10 fish per replicate was prepared. All-glass aquaria (30 cm x 22 cm x 24 cm) were used as test vessels. The photoperiod was 16 hours light and 8 hours darkness and media were continuously aerated with oil free air.  The study was evaluated to be valid [BASF, 1988].