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Ecotoxicological information

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:
Experimental start date: 21 June 2004. Experimental completion date: 3 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and 100 mg/l loading rate WAF
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from the test medium and the control at the start of the test, after 48 hours test duration, and at the end of the test after 96 hours.
The concentration of the test item was analytically determined in all test medium samples.
From the control samples, only one of the duplicate samples was analysed from the corresponding sampling times.

- Sample storage conditions before analysis:
The samples were analysed immediately after sampling without prior storage.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In order to assess the toxicity of the test substance to fish, a water accommodated fraction (WAF) with a loading rate of 100 mg/L was tested.
Additionally, a control (test water without test item) was tested in parallel. Thus, a limit test was performed in accordance with the test guidelines to demonstrate that WAFs of the test item have no toxic effects on the fish up to the loading rate of 100 mg/L. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The WAF was prepared as follows: A dispersion with a loading rate of 100 mg/L was prepared by dispersing 500.5 mg of the test item in 5000 mL of test water using intense stirring on a magnetic stirrer. The dispersion was stirred for 3 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. Ultrasonic treatment was not applied to avoid accelerating hydrolysis.
The short stirring period of 3 hours was chosen because the components of the test item are not stable in water (results of a pre-experiment to determine the solubility and stability of the test item in test water, without GLP).
Then, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 pm) after the 3 hour stirring period. The test medium was prepared just before introduction of the fish (= start of the test).
The actual concentration of the test item in the test medium was analytically determined.
The limit test was based on the results of a range-finding test and the results of a pre-experiment to determine the solubility of the test item (without GLP). However, loading rates above 100 mg/L were not tested according to the guidelines.

- Controls: a control (test water without test item) was tested in parallel.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain: Not reported
- Source: Zoohaus Schaub, CH-4410 Liestal, Switzerland.
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): 3.2 =/- 0.1 cm
- Weight at study initiation (mean and range, SD): 0.28 =/- 0.05 g
- Method of breeding: Not reported
- Maintenance of the brood fish: The fish were held in the laboratories of RCC for more than 2 weeks without any medication. The fish were fed with a commercial fish diet.

ACCLIMATION
- Acclimation period: One week
- Acclimation conditions (same as test or not): Yes
- Type and amount of food during acclimation: During holding and acclimatisation until one day before the start of the test, the fish were fed with a commercial fish diet (TETRA MIN Hauptfutter, supplied by TETRA-Werke, D-49304
Melle, Germany).
- Feeding frequency during acclimation: Not reported
- Health during acclimation (any mortality observed): During the last two weeks prior to the test, the mortality rate in the test fish batch was lower than 5% and all fish were healthy.

FEEDING DURING TEST - None
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
228 mg/l as CaCO3
Test temperature:
21 °C
pH:
8.1 to 8.3
Dissolved oxygen:
8.0 to 8.8 mg/L or higher therefore higher than 60% oxygen saturation.
Nominal and measured concentrations:
Nominal: 0 (control) and 100 mg/l loading rate WAF.
Measured:
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquarium
- Type (delete if not applicable): open / closed: not reported
- Material, size, headspace, fill volume: Glass, filled with 5 litres test medium
- Aeration: Yes - slight aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- 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): n/a
- Biomass loading rate: 0.4 g fish wet weight per litre test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Local tap water (non chlorinated well water of drinking water quality), reduced for total hardness by ion exchange.
- Total organic carbon:
- Particulate matter:
Lindane <0.05
Heptachlor <0.05
Malathion <0.05
DDT, total <0.05
Dieldrin <0.05
Cadmium <0.5
Arsenic <3
Lead <3
Mercury <1
Selenium <3
Copper <4
PCBs (28, 52, 101, 138, 153, 180) <0.05
Nitrosamines, total (DMN, DEN, NAP, NMORPH) <0.002
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement: The water temperature, pH-values, and oxygen concentrations were measured at the start of the test and once every day during the test in the single test concentration and the control. At the same dates, the appearance of the test medium was recorded. The total hardness of the water used in the test was measured at the start of the test in the control.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 hours light and 8 hours darkness (with a 30 minute transition period)
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 3, 24, 48, 72, and 96 hours test duration for mortality and visible abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: n/a
- Justification for using less concentrations than requested by guideline: Multi-constituent substance with low solubility so 100 mg/l loading rate WAF was prepared according to the OECD difficult substances guidance..
- Range finding study
The limit test was based on the results of a range-finding test and a pre-experiment to determine the solubility of the test item, but the results are not reported in the study report.
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: Not reported
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Remarks on result:
other: no toxic effects
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Remarks on result:
other: no toxic effects
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: none reported
- Other biological observations: none
- Mortality of control: None
- Other adverse effects control: none reported
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: Not relevant
Results with reference substance (positive control):
n/a
Reported statistics and error estimates:
No effects - no statistics reported

Table: Mortality and observed visible abnormalities

 Loading rate (mg/l)  Measured concentration (µg/l)  Number of affected fish*/number of dead fish        
     3 h  24 h  48 h  72 h  96 h
 Control  -  0/0  0/0  0/0  0/0  0/0
 100  <0.1  0/0  0/0  0/0  0/0  0/0

*number of dead fish plus number of fish with visible abnormalities

Table: Concentrations of the test item in test medium

 Nominal concentration

100 mg/l WAF loading rate

 Sampling date (day)  Age of sample (hours)

 Measured concentration

mg/l

 Measured concentration

% of nominal

   0  0  <LOD* n/a
   2  48  <LOD*  n/a
   4  96  <LOD*  n/a

*LOD (Limit of Detection) = 0.0001 mg/l

Validity criteria fulfilled:
yes
Conclusions:
A 96 hour LL50 value of >100 mg/l (nominal, WAF loading rate) (highest concentration tested) has been determined for the effects of the submission substance (CAS 352230-22-9) on mortality of Danio rerio, in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Each constituent of the test substance has a slow hydrolysis half-life at pH 7 (predicted to be 200 to 630 hours). However, the hydrolysis rate greatly increases with higher pH; 2 to 5.3 hours at pH 9. As this test was conducted at pH 8.1 to 8.3, hydrolysis will have occurred in the timescale of the test. In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed to a mixture of the parent substance and the hydrolysis products of each constituent of the tested substance.

Description of key information

96-hour LL50 >100 mg/L (nominal, WAF loading rate) (highest concentration tested); mortality of Danio rerio.

Key value for chemical safety assessment

Additional information

A 96-hour LL50 value of >100 mg/L (nominal, WAF loading rate) (highest concentration tested) has been determined for the effects of the submission substance (CAS 352230-22-9) on mortality of Danio rerio, in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test).

The study reports an analytically determined test substance concentration of <0.1 µg/L (<LOD, mean measured). This value reflects the low water solubility of the test substance, but losses may also have occurred as a result of filtering of the test solution prior to addition of test organisms, and the potential for adsorption of the test substance onto the filter matrix.

Each constituent of the test substance has a slow hydrolysis half-life at pH 7 (predicted to be >200 to >630 hours). However, the hydrolysis rate greatly increases with higher pH; 2 to 5.3 hours at pH 9. As this test was conducted at pH 8.1 to 8.3, hydrolysis will have occurred in the timescale of the test. In view of the test media preparation method and static exposure regime it is likely that the test organisms were exposed to a mixture of the parent substance and the hydrolysis products of each constituent of the tested substance.