Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28th June 1993 to 2nd July 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 97.3%
Batch No.: 9236-23

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The test sample containing 100% v/v of the test substance extract and three samples of the frozen stock solutions (nominal test substance concentration 1000 mg/L) were analysed.
- Sampling method: Each test medium has been twice sampled; at the start and at the end of the study.
- Sample storage conditions before analysis: The samples were immediately frozen and transmitted frozen at the end of the experimental period to PHARMAKON EUROPE for analytical determination of the test substance concentration levels.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Owing to the test substance's low water solubility (0.0478 mg/L), and in order to prevent perturbation by solid test substance particles (water oxygenation, ...), the study was performed using an extract of test substance. It was prepared as follows :
• dilution water: similar to experimental water; its pH was adjusted to pH 7 with diluted NaOH
• a test substance stock suspension (1000 mg/L) was prepared in dilution water
• this stock solution was submitted to magnetic stirring for two hours
• it was then coarsly filtered through filter paper, and the filtrate was used for the preparation of the test media
• pH of the extract was adjusted to pH 7, with diluted NaOH.
To obtain respectively the theoretical concentrations of 25, 50, 75, 90 and 100% (Volume by Volume) of test substance extract in test medium, appropriate volumes of test substance extract were directly added into experimental water (4 litre test media).
- Controls: The control group was not exposed to the test substance but was otherwise treated in an identical manner.
- Chemical name of vehicle: Not applicable - the test solution was dissolved in dilution water.
- Concentration of vehicle in test medium: Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: Société d'Elevage Piscicole Contrôlé (La Grande Rivière - F - 69490 Saint-Forgeux)
- Age at study initiation (mean and range, SD): Not specified
- Length: Mean of 3.3 cm ranging from 3.0 to 3.5 cm (measurements made on ten fish taken at random at the end of the experimental period).
- Weight at study initiation (mean and range, SD): Not specified
- Method of breeding: Not specified
- Feeding during test: No food was provided during the study.

ACCLIMATION
- Acclimation period: At least 12 days
- Acclimation conditions: The fish were kept under identical conditions. Container: tank of approximately 200 L capacity; System: semi-static system; Water was continuously aerated and at least 1.5 times the volume was renewed every week ; pH of water remained between 6.28 and 7.00 during the last 12 days of the acclimatization period; Temperature of the water remained between 20.6°C and 21.0°C during the last 12 days of the acclimatization period; Lighting: artificial, 12 hours out of 24 (photoperiod = 07h30 - 19h30).
- Type and amount of food: Dehydrated complete flakes for aquaria fish "Tetra-Min" was available ad libitum.
- Feeding frequency: Daily. Feeding was stopped at least 30 hours before the start of the study.
- Health during acclimation (any mortality observed): A mortality rate lower than 5% (0.5%) was observed during a 7 day-period preceding the study, the batch of fish selected for this study was therefore accepted.

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Permanent hardness of water: between 70 and 125 mg of CaCO3 per litre.
Test temperature:
20.0°C to 22.2°C
pH:
pH of the test media remained between 6.77 to 7.34 during the study (the pH was re-adjusted in the test media approximately 24 hours after the start of the study).
Dissolved oxygen:
The dissolved oxygen concentration was maintained at greater than or equal to 95% of the air saturation value.
Salinity:
Not specified
Nominal and measured concentrations:
Nominal concentrations: 0, 25, 50, 75, 90 and 100 % v/v
Details on test conditions:
TEST SYSTEM
- Test vessel: Containers: tanks of 4 litre capacity.
- Type: All the containers were covered.
- Material, size, headspace, fill volume: Not specified
- Aeration: Aeration was maintained during the study by air bubbling in each medium.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 10 fish per treated group
- No. of vessels per concentration (replicates): 1 vessel
- No. of vessels per control (replicates): 1 vessel
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: Maximum biological loading = 1 g/L (actual value = 0.6 g/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water as well as acclimatization water was reverse osmosed natural water (from 60 m depth) and to which appropriate salts were added to get the required characteristics. No contaminant was present, (e.g. chlorine) at a concentration which could have prejudiced the study. Permanent hardness of water was between 70 and 125 mg of CaCO3 per litre.
- Conductivity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: Measurement of pH, dissolved oxygen and temperature were performed for each test medium and the control at the start of the study and then approximately 24, 48, 72 and 96 hours later, for each test medium.

OTHER TEST CONDITIONS
- Adjustment of pH: pH of the dilution water used for the preparation of the test media had been adjusted to pH 7 with diluted NaOH.
- Photoperiod: 12 hours daylight out of 24 (photoperiod = 07h30 - 19h30)
- Light intensity: artificial lighting

EFFECT PARAMETERS MEASURED
Any death was recorded two hours and then approximately 24, 48, 72 and 96 hours after the start of the study. The fish were considered as being dead if touching of the caudal pedoncule produced no response and if no respiratory movements were visible.
Any abnormal behaviour associated with swimming, respiration, pigmentation, loss of equilibrium, etc., was recorded two hours after the start of the study and then approximately every 24 hours.
Observation of the test media was performed at the start of the study and then approximately 24, 48, 72 and 96 hours later. Test medium abnormalities (films, deposits, blurs, ...) if any, were recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not specified
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: This study is a range-finding study and was performed using the concentrations of 25, 50, 75, 90 and 100% (v/v) of test substance extract in the test medium. No mortality was recorded during these 96-hour study period, until 100% of test substance extract. The range-finding study was then considered as a limit test, and no LC50 evaluation was performed.
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: LC50 is higher than the limit of solubility in water
Details on results:
No mortality was recorded during the 96 hour study period, until the concentration of 100% of the test substance extract was tested. Thus, no determination of LC50 was performed.

- Behavioural abnormalities: No abnormalities were recorded for any test extract concentration throughout the study.
- Observations on body length and weight: Not specified. The size of the fish were only measured at the end of the study.
- Other biological observations: None
- Mortality of control: The mortality did not exceed 10% in the control group by the end of the study (no death was observed in the control group by the end of the study period).
- Other adverse effects control: No abnormalities were recorded.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No abnormalities were observed in the test media at each observation time point.
- Effect concentrations exceeding solubility of substance in test medium: The water solubility of the test substance is 0.0478 mg/L. No effects were seen up to the highest dose tested; hence, the effect concentration must exceed the limit of solubility.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data available

Any other information on results incl. tables

The test samples were analysed by HPLC. The measured concentrations of test material were <0.04 mg/L i.e. below the limit of solubility in water.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No mortality was recorded in the experimental conditions during the 96-hour study period. The LC50 is higher than the limit of solubility.
Executive summary:

Freshwater fish (Brachydanio rerio) were exposed to a test substance extract of Millad 3988 for 96 h in accordance with OECD Guideline No. 203 and EEC Directive 84/449 Annex V Method C1. No mortality was recorded in the experimental conditions during the 96-hour study period, until 100% of test substance extract in test medium was used (containing less than 0.04 mg of test substance per litre of test medium as determined by analysis) which is the maximum possible concentration of exposure.The 96 h LC 50 for fish in freshwater medium is therefore higher than the limit of solubility (i.e. >0.04 mg/L).