2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 23: Aquatic Toxicity Testing of Difficult Substances and Mixtures

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The Water Soluble Fraction and the control were analytically verified from freshly prepared test media after 0 and 72 hours and from corresponding 24 hours aged test media after 24 and 96 hours.
The solutions of the test item are very sensitive to light. Therfor, samples the for determination of test item analysis were handled under light exclusion.

- Sample Storage: All original and prepared samples were stored at room temperature until the start of the analysis, if necessary.

Vehicle:

no

Details on test solutions:

- Water Accommodated: Fraction (WAF):
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a
complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.
- Preparation of the Water Soluble Fraction (WSF):
One Water Soluble Fraction (WSF) was prepared under red light with a nominal loading of 10 mg/L. The loading rate of nominal 10 mg/L was chosen with regard to the expected water solubility given as 0.75 mg/L (buffer solution pH 7) and 0.03 mg/L (buffer solution pH 9), thus clearly exceeding the reported solubility values. An appropriate amount of the test item was weighed on a glass slide and inserted in a brown glass bottle with an appropriate amount of dilution water (dechlorinated tap water, pH adjusted to 7 ± 0.2 with HCL 1 M). The test item dispersion was stirred with a magnetic stirrer for 24 hours at room temperature and under light exclusion. After the completion of mixing, undissolved particles were removed by membrane filtration (membrane filter 0.45 μm, RC, MACHEREY-NAGEL). During filtration, the filter was always covered with the test item dispersion. The resulting WSF was used for testing.
- Limit loading:
The effects of the WSF was tested in a limit test. The limit loading was based on the results of a non-GLP preliminary range finding test carried out in the dark and under semi-static conditions with a WSF prepared as described above.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test originated from a single brood stock at DR.NOACK-LABORATORIEN (supplier: Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany).
- Length at study initiation (length definition, mean, range and SD): Average total body length at the start of the exposure: 2.7 cm.
- Weight at study initiation (mean and range, SD): Average body weight at the start of the exposure: 0.24 g.
- Method of breeding:
- Feeding during test: No
- Food type: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany.
- Amount: Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.

ACCLIMATION
- Holding: Fish were held at the test facility at 20 - 25 °C and diffuse light {0.1 - 10 μmol photons · m-2 · s-1, natural photoperiod). The water
was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value.
Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within this period of time were used in the test. No disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

70 mg CaCO3/L

Test temperature:

23 ± 2 °C

pH:

6.0 - 8.5

Dissolved oxygen:

Not less than 60 % of air saturation value

Nominal and measured concentrations:

Nominal (loading rate): 10 mg/L (= 142 μg/L geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 3 L were used, filled with 2 L of dilution water (dimensions: 11.5/13/20 cm, depth of water: 13.5 cm), and covered with glass plates.
- Aeration: Gentle aeration was provided.
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Fish density in the tanks was 0.84 g fish per litre test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 hours from freshly prepared and aged test media.
In the control, total hardness of the dilution water was determined at the beginning of the exposure.
During exposure, the water temperature was recorded continuously (once per hour) by a data logger.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity: Due to the light sensitivity of the test item the test was carried out in the dark.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were made after 2, 24, 48, 72 and 96 hours of exposure.
Fish were considered dead if there was no visible movement (e.g. gill movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.).

TEST CONCENTRATIONS
- Test concentrations: The limit loading was based on the results of a non GLP preliminary range finding test carried out in the dark and under semi-static conditions with a WSF prepared as described above. No effects occurred after 96 h of exposure to the test substance.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality (fish)

Remarks on result:

other: No acute toxic effects occur within the range of solubility.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.142 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No acute toxic effects occur within the range of solubility.

Details on results:

The test solution was visually clear throughout the exposure period.

Validity criteria fulfilled:

yes

Conclusions:

The test material showed no toxicity to fish up to the limit of solubility (96 hour EC50 >10 mg/l). The test material is therefore considered to be non-toxic to fish.

Executive summary:

In this guideline (OECD 203) study conducted with GLP certification, the 96 hour EC50 of the test material (EC 404-360-3) was determined to be above the limit of solubility (>10 mg/l). The study was conducted as a semi-static limit test, on Danio rerio. The test solution was prepared as a Water Soluble Fraction/Water Accomodated Fraction, with undissolved test material filtered out. As no toxicity was observed up to the limit of toxicity (>10 mg/l), the test material is not considered as acutely hazardous to the environment under the criteria of the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Description of key information

 Study carried out according to guideline and with GLP compliance. 

Additional information

A GLP guideline study according to OECD 203 has been conducted (DR. U. NOACK-LABORATORIEN 2014). Due to the low solubility and the light sensitivity of the test item a semi-static test with daily renewal of the Water Soluble Fraction was carried out. The loading rate of nominal 10 mg/L was chosen with regard to the expected water solubility given as 0.75 mg/L (buffer solution pH 7) and 0.03 mg/L (buffer solution pH 9), thus clearly exceeding the reported solubility values. The measured initial concentrations were within this range. Additionally, due to the light sensitivity of the test substance solutions all preparation steps were carried out under red light. The stirring phase (24 hours) and the test were done in the dark. Samples for the determination of test item analysis were handled under light exclusion.

After 96 h of exposure no effects occurred within the range of solubility and the LC50 was determined to be > 10 mg/L (loading rate) respectively > 0.142 mg/L (geometric mean measured). Therefore, the test substance is with high probability not acutely harmful to fish