Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 March 2010 to 12 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
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)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): "3-HYDROXY-2-(HYDROXYMETHYL)-2-METHYLPROPIONALDEHYDE"
- Molecular formula (if other than submission substance):C5H10O3
- CAS No.: 185146-18-2.
- Lot No.: CH 138067/001.
- Appearance: uncolored and high viscous liquid.
- Solubility: In water: Soluble
- Purity test date:
~ 82 % Dimethylpropanale
~ 10 % Hydroxymethanesulfonic acid, Na-salt (CAS No: 870-72-4)
~ 6 % water *)
~ 2 % Potassiumformiate (CAS No: 590-29-4).
- pH: 7.25.
- Conditions of storage: Room temperature.
- Stability at conditions of storage: Min. 1year.
- Expiry date: 30 November 2010.

*) The results presented in the report refer to the dry weight of the test substance not including the 6 % water content.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal concentrations: 0.0, 28.2, 59.2, 117.5, 235.0, 470.0, and 940.0 mg/L
- Sampling method: Samples of the test media for determination of the actual test substance concentration were taken immediately before introduction of the fish and after approximately 4, 24, 48, 72 and 96 hours. When the test media were changed after 24, 48 and 72 hours of exposure, the old as well as the new media were analysed.
test in all groups.
- Sample storage conditions before analysis: The samples were diluted appropriately and analysed on the day of sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared at the start of the test and 24, 48 and 72 hours thereafter by dilution of nominal 4 000 mg test substance (6 % water content) per 1000 mL dilution water by stirring for 30 to 40 minutes. (see Table 1)
The test media were prepared by diluting the stock solution with dilution water. In order to maintain the actual test substance concentration as high as possible new test media were prepared for test media change at 24, 48 and 72 hours after the start of the test. The control media was also changed after 24, 48 and 72 hours. The medium of the highest concentration was not changed at 48 and 72 hours as all fish were dead after 48 hours. (see Table 2)
- Controls: dilution water (local tap water + deionised water, 1:1)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio).
- Strain: not applicable.
- Source: Tomy's Zoo GmbH / Zoo & Co, Josef-Strebl-Gasse 6, 2345 Brunn am Gebirge.
- Length at study initiation (length definition, mean, range and SD): 2.89cm, SD: 0.20 (mean of control group at the end of study).
- Weight at study initiation (mean and range, SD): 0.37g, SD: 0.06 (mean of control group at the end of study).
- Feeding during test: none.

ACCLIMATION
- Acclimation period: Approx. 6 weeks.
- Acclimation conditions (same as test or not): Dilution water for holding and exposure.
Holding: Continuous aeration by water recirculation using an EHEIM/JBL filter pump. Full glass vessel (60 cm x 30 cm x 30 cm), each filled with
ca. 54 L and covered with a transparent plastic lid. Water was purified by recirculation (power filter pump EHEIM / JBL) through filter wool.
Exposure: No aeration. Full glass vessels (29 cm x 20 cm x 24 cm), filled with 10 L of control and test medium and covered with transparent plastic lids.
Maximum loading of 1.0 g fish/litre.
- Type and amount of food: TetraMin; Tetra Werke, Dr.rer.nat. Ulrich Baensch GmbH, D-49304 Melle, feeding ad libitum.
- Feeding frequency: daily (during exposure: no feeding)
- Health during acclimation (any mortality observed): Once daily for behaviour and mortality. Following a 48 hour settling-in period, mortalities are recorded and the following criteria applied:
• Mortalities of greater than 10 percent of population in seven days: rejection of batch.
• Mortalities of between 5 and 10 percent of population: acclimatisation continued for seven additional days.
• Mortalities of less than 5 percent of population: acceptance of batch.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none.

Test conditions

Hardness:
230 mg CaCO3/L
Test temperature:
20 - 21°C
pH:
7.9 - 8.1 (control), 8.0-8.3 (test)
Dissolved oxygen:
At least 83 % of the saturation value at the corresponding temperature throughout the test period.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal: 0.0, 28.2, 59.2, 117.5, 235.0, 470.0 and 940.0 mg/L.
Geometric mean of the actual concentrations: - , 24.02, 51.56, 102.72, 207.67, 432.25 and 795.74 mg/L.

Details on test conditions:
TEST SYSTEM
- Test vessel: full glass vessels , filled with 10 L of control and test medium and covered with transparent plastic lids.
- Material, size, headspace, fill volume: 29 cm x 20 cm x 24 cm
- Aeration: no aeration.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable.
- Renewal rate of test solution (frequency/flow rate): 1x/d
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable.
- Biomass loading rate: Maximum loading of 1.0 g fish/litre


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was local tap water which was mixed 1:1 with deionised water to achieve the appropriate water hardness as required by the guideline. Dilution water was also used for the negative control group.
- Culture medium different from test medium: no.
- Intervals of water quality measurement: pH, temperature and dissolved oxygen was measured at 0h and at 4h, 24 h, 48 h and 72 in each case before
and after the media change, and at 96 h. Total hardness was measured before the start of the study.


OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: Artificial light from 4 a.m. to 8 p.m.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
All animals were observed for alterations and behavioural changes at introduction of the fish and approximately 2, 4, 24, 48, 72 and 96 hours afterwards

TEST CONCENTRATIONS
- Spacing factor for test concentrations: <2.2
- Range finding study
- Test concentrations: A preliminary fish test with four nominal concentrations of 10 000, 1 000, 100 and 10 mg/L (dry weight), using 3 litres of test medium and three fish per concentration was performed. The test was conducted under static conditions for 96h.
- Results used to determine the conditions for the definitive study: According to the results of the preliminary experiment mean actual test substanc concentrations of 24.0, 51.6, 102.7, 207.7, 432.3 and 795.7 mg/L were used for the main study. Furthermore it was decided to use a semi static test design and 10 litre medium per concentration tested, to maintain the actual test substance concentrations as high as possible.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 800 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
590 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Slope: 11.0
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
590 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Slope: 11.0
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
530 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limit lower : 440 mg/L, upper: 650 mg/L, Slope: 6.2
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
ca. 800 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
ca. 430 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: All dead fish in group F showed highly decreased mobility and loss of equilibrium from 24 hours after the start of the test until their death. In group E decreased mobility was observed from 24 to 96 hours which was accompanied by loss of equilibrium between 48 and 96 hours. The fish in Group D displayed a slight decreased mobility between 24 and 96 hours. All other fish were inconspicuous in appearance and behaviour throughout the study.
- Observations on body length and weight: The fish had a length between 2.5 and 3.1 cm and a mean weight of 0.37 g.
- Other biological observations: none.
- Mortality of control: none.
- Other adverse effects control: none.
- Abnormal responses: none.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
Reported statistics and error estimates:
Not applicable.

Any other information on results incl. tables

Quality criteria

The following quality criteria were fulfilled:

·      Constant conditions were maintained (temperature, pH).

·      No mortality occurred in the negative control group.

·      The dissolved oxygen concentration was higher than 60% of the air saturation value throughout the test.

·      The for the LD50determination relevant test substance concentrations E and F have been satisfactorily maintained to within 80% of the initial concentration throughout the duration of the test. Nevertheless, due to the properties of the test substance, the measured concentration of the groups A to D decreased slightly to below of 80% of the initial starting concentration (maximum deviation 26 %). Therefore the actual concentrations are based on the geometric mean concentrations.

Mortality:


Group

Mean actual test substance concentration

Total number of dead fish / total number of fish exposed
at hours after start of exposure

 

(mg/L)

0

24

48

72

96

Control group (K)

0

0 / 7

0 / 7

0 / 7

0 / 7

0 / 7

A

24.0

0 / 7

0 / 7

0 / 7

0 / 7

0 / 7

B

51.6

0 / 7

0 / 7

0 / 7

0 / 7

0 / 7

C

102.7

0 / 7

0 / 7

0 / 7

0 / 7

0 / 7

D

207.7

0 / 7

0 / 7

0 / 7

0 / 7

0 / 7

E

432.3

0 / 7

0 / 7

0 / 7

0 / 7

1 / 7

F

795.7

0 / 7

0 / 7

7 / 7

7 / 7

7 / 7

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
LC50 96 h: 530 mg/L
LC100 96h: ca. 800 mg/L
LC0 96 h: ca. 430 mg/L
NOEC: 100 mg/L
Executive summary:

The acute toxicity of"3-HYDROXY-2-(HYDROXYMETHYL)-2-METHYLPROPIONALDEHYDE"toZebrafish (Danio rerio)was determined for regulatory purposes.

Regulation (EC) 440/2008: C.1. Acute Toxicity for Fish and OECD-Guideline 203, "Fish, acute toxicity test" (17thJuly 1992) were applied.

SevenZebrafish (Danio rerio), were exposed in each of the 6 test substance groups and in the control group.

The exposure of the Zebrafish was for 96 h in a semi-static test with renewal of test and control media after 24, 48 and 72 hours.

Mean actual test substance concentrations of 24.0, 51.6, 102.7, 207.7, 432.3 and 795.7 mg per litre dilution water were obtained by diluting the stock solutions prepared at the start of the test as well as at 24, 48 and 72 hours thereafter. The stock solutions were prepared by diluting the appropriate amount of test substance (6 % water content) in dilution water by stirring for 30 to 40 minutes.

The dilution water was local tap water which was mixed 1:1 with deionised water to achieve the appropriate water hardness as required by the guideline. Dilution water was also used for the negative control group.

LC50 96 h: 530 mg/L

LC100 96h: ca. 800 mg/L

LC0 96 h: ca. 430 mg/L

NOEC: 100 mg/L