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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, relevant guideline,sufficient substance identification and justification for guideline modifications.
Reason / purpose for cross-reference:
reference to other study
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
WAF (Water accomodated Fraction) Method used. No chemical analysis conducted as stability was previously demonstrated in other GLP studies
Principles of method if other than guideline:
The half life of the test chemical was such that degradation products were tested as these were considered most relevant for determining the
aquatic hazard. A semi static regime was used to allow a more stable exposure. A WAF (Water Accommodated Fraction) method was used as the
test substance itself is intentionally mixed with iso dodecane for stability / safety reasons and the active component degrades to multiple breakdown
products all of which could potentially contribute to toxicity. Separation and testing of individual components is therefore not possible.
Quantification of all breakdown products was not possible . For this reason the dissapearance / degradation of the active ingredient was confirmed
analytically in the algae and daphnia studies (not in this test) and hence the resulting degradation product mixture was tested.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Details on test solutions:
Due to the test substance being a multi component substance and rapidly hydrolyzing to multiple degradation products. The test solution was prepared as a water accommodated fraction. A traditional stock solution was not made. The desired amount of test medium (DSW) (5L) was tapped from the laboratory DSW system and the appropriate amount of the test chemical required to achieve the desired loading in the total volume of test medium (approximately 500 mg) was accurately weighed out. Test chemical was then added to the test medium in a sealable glass vessel and the vessel was then closed. The test solution was gently stirred to avoid generation of emulsions for 24 hours. A control without test substance was treated in the same manner. Due to the nature of the instability of the test chemical and its short half life, 24 hours was sufficient to allow breakdown of the parent compound and formation of the breakdown products. The resulting mixture of breakdown products is treated as the test substance. After being allowed to stand for at least one hour the pH of the test solutions will be checked and adjusted if required with HCL or NaOH, and then transferred to the test
vessels, to which the test organisms were subsequently added.

For the solution refreshment a new WAF solution was made 24 hours prior to the solution replacement and identical procedures were followed as described above.

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The test was performed with juvenile Danio rerio (zebra fish), were accepted for use in testing according to laboratory standard operating procedures (Ref 6) and as recommended in the corresponding guideline (Ref1). The original parent batch was obtai¬ned from the “Dieren vriend” in Arnhem the Netherlands . The fish in stock were fed one to three times per day with commercially available dry, deep frozen food, or fresh Artemia salina nauplii / juvenile Daphnia magna neonates. Feeding was stopped 24 hours before the test was started, and the animals were not fed during the test. A
representative number of test fish (4 at random) were weighed prior to the test and measured after the test to assess compliance with guideline

The average length of a sample of 14 fish randomly taken from the test animals at the end of the test was < 3.0 (2.82) cm. The mean biomass loading was 0.79 g/L. The general criteria set for the test animals in the study plan and guideline was therefore met.

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:
Length was recorded post exposure and any abnormalities noted if observed
12.3 º dH which is equivalent to a maximum of 219 mg/L as calcium carbonate
Test temperature:
24.55 to 25.0°Cduring the study
8.0 to 8.3 during the study
Dissolved oxygen:
7.4 to 8.4 mg/Lduring the study
Nominal and measured concentrations:
Due to degradation products being tested measured concentrations of the active ingredient are not relevant for expressing the study endpoints. Nominal or loading concentrations of the tested mixture were therefore used : A limit test was conducted 100 mg/L and a control were therefore tested.
Details on test conditions:
The test was performed as a semi static limit test which means that the test media was replaced after 48 hours to ensure test solution stability and
only a single test concentration was tested. The minimum requirement of 7 fish per test concentration and control were used for ethical reasons.

Under otherwise identical test conditions, the fish were exposed to the chosen concentration of the test substance as described below and mortality
and sub-lethal effects were recorded at least at 30 minutes, 1, 2 and 4 hours after exposure and then and then at approximately 24, 48, 72 and 96

The fish were considered dead when a lack of opercular movement was observed and touching of the caudal peduncle produced no reaction. Dead fish were removed from the test vessels directly after being observed, which was checked several times up to 4 hours on the first day for ethical reasons and at least twice each day on subsequent days. In addition to death, sub-lethal effects such as erratic swimming, loss of reflex, increased excitability, lethargy, changes in physiology, discoloration, pigmentation, excessive mucous production, hyperventilation, opaque eyes, curved spine or
hemorrhaging were recorded if observed. Fish that were convulsing or showing other severe forms of distress not considered transient in nature and likely to become more severe before the exposure is terminated, were sacrificed for humane reasons if observed. These fish were treated as having
died in the test.

The fish were randomly placed in the test vessels which were positioned in the test room in a random manner. During the test the vessels were
covered with glass plates. The test solutions present in the test vessels were aerated gently with water-saturated air purified by an active coal filter a
nd a cotton filter during testing.

- Test vessel: 3 liter glass aquarium
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 3 L
- Aeration:yes
- No. of organisms per vessel:7
- Biomass loading rate:0.79 g/L

- Source/preparation of dilution water: Synthetic Dutch Standard Water See other information

- Adjustment of pH: yes pH in 100 mg/L replicate was adjusted to that of the control (7.0-8.2)
- Photoperiod: 16 light 8 Dark
- Light intensity: Ambient


- Range finding study NOELR = 100mg/L
- Test concentrations: 100 mg/L limit test
- Results used to determine the conditions for the definitive study:

Previous non GLP research and a GLP hydrolysis study had identified the breakdown products and indicated the short halflife. The test design was
therefore chosen using this information and reccomendations in the "OECD 2000 GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF

Breakdown products include : Isobutyric acid, Isopropanol, propene and acetone
Reference substance (positive control):
Not permitted by Animal Welfare Group
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.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No effects observed in limit test EL50 not reached
Details on results:
100 mg/L of degraded test chemical caused no effects to D.Rerio. The test substance would not require classification for its toxicity to fish.
Results with reference substance (positive control):
Reported statistics and error estimates:
Not always required with limit test control and test concentration are identical and hence no statistical calculations were conducted.
Validity criteria fulfilled:
The study can be considered a reliable representation of the effects of the test substance degradation products in an open system. Without
significant restrictions.
Executive summary:

A GLP study to appropriate guidelines with sufficient substance identification and justification for guideline modifications. No analysis was conducted as the degradation of the active component hand been previously demonstrated under GLP in other studies (Algae, Daphnia and Hydrolysis). No quantification of the degradation products was carried out. Due tothe lack of toxic effects observed and the ready biodegradability of the degradation products this is not considered a critical flaw to the study.

Description of key information

Due to the extremely short half-life of the test material determined in the hydrolysis study, the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. All test concentrations were prepared separately as Water Accommodated Fractions to allow the determination of the toxicity of the resulting degradation mixture as a whole to be determined. It is known that the test chemical degrades to multiple degradation products and is itself present in an isododecane solvent. The effects cannot therefore be attributed to a single component with 100% certainty however the main degradation product isobutryic acid is the most likely cause of any toxicity that may be observed.  A loading of 100 mg/L of test material left to degrade for 24 hours resulted in no visible toxicity to the test organisms. A 96h-LL50 could therefore not be determined and may be expressed as LL50 >100 mg/L.

ECHA data on Isobutyric acid gives a 96h-LC50 of 146.6 mg/L for Leuciscus idus (ECHA dossier CAS 79 -31 -2).

Key value for chemical safety assessment

Additional information

Due to the extremely short half-life of the test material determined in the hydrolysis study the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. No toxicity to Danio Rerio was observed. The primary degradation product of the test substance isobutyric acid and this is itself of low toxicity and is ready biodegradable. The test material should not be considered of concern for this endpoint.