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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2005 through 25 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
NECTARYL :
- Batch No. : 9000606028
- Purity : 97.2%
- Expiry Date : 18 May 2006
Analytical monitoring:
yes
Details on sampling:
The experimental set-up employed semi-static renewal (renewal frequency - at least three times per week).

Water samples, both fresh and aged media, were taken at intervals throughout the exposure :
- Fresh Media Samples : Day 0, 7, 14, 20, 28
- Aged Media Samples : Day 9, 16, 20, 22, 24, 26, 28, 30
Vehicle:
yes
Remarks:
Ethanol (final volume in test media = 0.02 mL/L)
Details on test solutions:
Preparation of test solutions started with stock solutions in Ethanol. To maintain ethanol as low as possible, the concentrations in the stocks were a factor of 50,000-times higher than those in the final test solutions (0.02 mL/L). The test solutions were kept in closed bottles and stirred for at least 10 minutes to optimise mixing and dissolving of the test substance in the medium.

Nominal Exposure Concentrations Tested :
1.0 mg/L
0.46 mg/L
0.22 mg/L
0.10 mg/L
0.046 mg/L
Solvent Control
Control
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
In-house culture.

Medium : In re-circulated, UV sterilised tap water. The tap-water is filtered by bio-filtration
Water Conditions : NO3/NO2 < 1 ppm, NH3/NH4 < 1 ppm, O2 > 5 ppm
Temperature : 27 °C
Age of Brood Stock : 2 - 9 months
Ratio of Male-to-Female : 1 : 1 or 1 : 2
Spawning Tank : Equipped with a false mesh bottom to enable colection of the fertilised eggs
Feeding Brood Stock : Crumble feed - Own formulation (50% protein, 6% fat, 9% ash, 4% fibre)
Time of Fertilisation : Males and females admitted in to spawning tank and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.
Hardness of Holding Water : Approx. 180 mg CaCO3/L
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Remarks on exposure duration:
Media renewal - 3 times per week.
Hardness:
180 mg CaCO3/L
Test temperature:
Temperature range : 23.4 to 25.2 °C
pH:
pH range : 7.2 to 8.1
Dissolved oxygen:
Dissolved Oxygen range : 5.1 to 9.2 mg O2/L
Nominal and measured concentrations:
The Nominal test concentrations were : 1.0, 0.46, 0.22, 0.10 and 0.046 mg/L

Analyses of the fresh media had mean recovery values of > 90% and a coeff. of variation of < 20%.
During the initial-phase of the study, the first five days of exposure of the fertilised eggs, there was likely to be little variation in the expsoure concentrations since daily media renewal was performed. However, once the larvae were transferred to the bigger, 1 Litre, tanks, the losses became more significant.
Details on test conditions:
Test Type : Semi-static (frequency of renewal - at least three times per week)
Test Duration : 30 days
Introduction of Eggs : 2 - 4 hours following fertilisation
Test Vessels : Embryonic-Phase - Sterile petri-dishes (diameter 10 cm) until hatching of all eggs (3 - 5 days)
Larval-Phase - All glass vessels with a minimum volume of 1 L
Medium : Frshly prepared ISO-Medium (not more than 1 week old)
Experimental Design : Started with 60 fresh and healthy fertilised eggs, between 2 - 4 hours old, per concentration, randomly distributed to 3 individual replicates (petri dishes containing 100 mL of test medium). The Control agroups consisted of 180 eggs distributed to nine petri-dishes, of which three replicates for the blank Control and six replicates for the Solvent Control.
Larval and Post-Larval Exposure : On Day 5 the surviving hatched larvae were transferred to all-glass vessels with a volume of 1 litre. The glass vessels were closed to avoid volatile losses of the test item.
Light / Dark Period : 16 hours Light / 8 hours Dark
Feeding : Embryo-stage - No feeding
Newly-hatched larvae - Brachionus suspension
Juvenile stage - Brine shrimp Naupli 24 hours old
Food was supplied ad libitum and one hour after each feeding any remaining residues of food were removed.
Euthanasia : At the end of the test the surviving larvae were rapidly killed by exposing them to approx. 1.2% ethylene glycol monophenylether in water.
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Overall NOEC based on results for time to hatch, post-hatch survival and body growth
Duration:
30 d
Dose descriptor:
LC50
Effect conc.:
0.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Details on results:
- Nectaryl induced significant effects on the development of Zebrafish embryos at a nominal concentration of 1.0 mg/L, corresponding with an actual concentration of 0.84 mg/L;
- Nectaryl tended to affect time of hatching at nominal concentrations of 0.46 and 1.0 mg/L, corresponding with actual exposure concentrations of 0.32 and 0.84 mg/L, but it did not affect hatching success at any of the concentrations tested;
- Nectaryl affected larval and post-larval survival significantly at nominal concentrations of 0.46 and 1.0 mg/L, corresponding with actual exposure concentrations of 0.32 and 0.84 mg/L. Based on actual exposure concentrations the 30-day LC50 was 0.34 mg/L with a 95% confidence interval of 0.25 to 0.49 mg/L;
- Nectaryl did not significantly affect growth of the larvae at sub-lethal levels, i.e. levels up to a nominal concentration of 0.22 mg/L corresponding to an actual exposure concentration of 0.14 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
The overall NOEC determined for Zebrafish in this early-life stage test was 0.14 mg/L based on actual exposure concentrations (0.22 mg/L nominal exposure concentration).
Executive summary:

The overall NOEC determined for Zebrafish in this early-life stage test was 0.14 mg/L based on actual exposure concentrations (0.22 mg/L nominal exposure concentration).

- Nectaryl induced significant effects on the development of Zebrafish embryos at a nominal concentration of 1.0 mg/L, corresponding with an actual concentration of 0.84 mg/L;

- Nectaryl tended to affect time of hatching at nominal concentrations of 0.46 and 1.0 mg/L, corresponding with actual exposure concentrations of 0.32 and 0.84 mg/L, but it did not affect hatching success at any of the concentrations tested;

- Nectaryl affected larval and post-larval survival significantly at nominal concentrations of 0.46 and 1.0 mg/L, corresponding with actual exposure concentrations of 0.32 and 0.84 mg/L.  Based on actual exposure concentrations the 30-day LC50 was 0.34 mg/L with a 95% confidence interval of 0.25 to 0.49 mg/L;

- Nectaryl did not significantly affect growth of the larvae at sub-lethal levels, i.e. levels up to a nominal concentration of 0.22 mg/L corresponding to an actual exposure concentration of 0.14 mg/L.

Description of key information

The overall NOEC determined for Zebrafish in this early-life stage test was 0.14 mg/L based on actual exposure concentrations (0.22 mg/L nominal exposure concentration).

- Nectaryl induced significant effects on the development of Zebrafish embryos at a nominal concentration of 1.0 mg/L, corresponding with an actual concentration of 0.84 mg/L;

- Nectaryl tended to affect time of hatching at nominal concentrations of 0.46 and 1.0 mg/L, corresponding with actual exposure concentrations of 0.32 and 0.84 mg/L, but it did not affect hatching success at any of the concentrations tested;

- Nectaryl affected larval and post-larval survival significantly at nominal concentrations of 0.46 and 1.0 mg/L, corresponding with actual exposure concentrations of 0.32 and 0.84 mg/L.  Based on actual exposure concentrations the 30-day LC50 was 0.34 mg/L with a 95% confidence interval of 0.25 to 0.49 mg/L;

- Nectaryl did not significantly affect growth of the larvae at sub-lethal levels, i.e. levels up to a nominal concentration of 0.22 mg/L corresponding to an actual exposure concentration of 0.14 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.14 mg/L

Additional information