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Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study generated according to valid and internationally accepted testing guideline and was performed under GLP.
according to guideline
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
first at preparation, then weekly and at the end of the test, for all test concentrations in duplicate.
The samples were filter sterilised over a 0.22 µm filter.
Details on test solutions:
Procedure: 1.28, 3.2 and 20 g of test substance were weighed and dissolved in 5, 5 and 2 L respectively of DSW.
The stock solutions were agitated for 1 to 5 hours to dissolve the test substance.
Test solutions were prepared by further diluting the stock solutions.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Supplier: Dierenspeciaalzaak Engelen Arnhem, The Netherlands.
-The broodstock is maintained in Akzo Nobel Environmental Chemistry laboratory.
-Eggs, Akzo Nobel Environmental Chemistry laboratory
- Wild caught: No
- Post-hatch transfer time: about 45 minutes
- Age/loading: between zygote and blastodisc cleavage stage /
- maximum 1 g of biomass per liter of test solution per 24 hours and not exceeding 2 g per liter of test solution any time.
-Finally 20 larvae per replicate.
- Pretreatment: no
-Feeding during test: 4 days after hatching with Paramecia species, 3 times per day to provide in total 2490 Paramecia per fish per day. From day 28 on the fish were fed with brine shrimp nauplii.
Test type:
Water media type:
Limit test:
Total exposure duration:
36 d
Total hardness: 8.43-13.1 mg/L
Test temperature:
Test temperature: 24.25-26.7 ºC, not more than +/- 1.5 ºC between successive days, except for two occasions during the test when maximum
difference over a 24h time period was 1.85 and 2.05 ºC. This was not considered to have an impact on the validity of the test.
pH: 7.8-8.1
Adjustment of pH: No
Dissolved oxygen:
7.9-8.4 mg O2-/L
Nominal and measured concentrations:
- Nominal/measured test concentrations: 0, 12.8, 32, 80, 200 and 500 mg/L
- Nominal is equal to measured concentrations.
Details on test conditions:
Dutch Standard Water:
- Hardness: approx. 97.5 mg/L as CaCO3
- pH: 6.0-8.5
- Oxygen content: >= 60% of the air saturation value
- Holding water: Dutch Standard Water
- Renewal of test solution: continuously; at least 5 volumes of test solution per day with a peristaltic pump.
- Exposure vessel type: 1.5 L glass aquaria
- Number of replicates, individuals per replicate: 2, test was started with 40 eggs per replicate, at day 1 it was reduced to 30 eggs and at day 6 the
number was reduced to 20 individuals per replciate.
- Conductivity: control 507-630 µS/cm; 12.8 mg/L 517-639 µS/cm; 32 mg/L 537-658 µS/cm; 80 mg/L 574-713 µS/cm; 200 mg/L
685-814 µS/cm; 500 mg/L 976-1113 µS/cm.
- Intensity of irradiation: No information
- Photoperiod: 14 h of ambient light per day
Reference substance (positive control):
36 d
Dose descriptor:
Effect conc.:
>= 500 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: all endpoints
Details on results:
Chemical analyses
All test solutions were found to be stable over the test period. Concentrations were observed to be between 100 and 111% of the nominals.

- Hatching: hatching started on day 3 except for one fish at 12.8 mg/L that hatched on day 2. Hatching was complete by day 4. All fish hatched at all concentrations.
- Mortality: No concentration related mortality was observed at any concentration at any of the stages during the test. In the test concentrations less fish died compared to the control.
- Morphology and behaviour: No abnormalities were observed.

- Number/percentage of animals surviving: 36 out of 40 survived. The post-hatch success (until the end of the test) was greater than 70% in the control.
- Length: 0.62-1.39 cm
Reported statistics and error estimates:
Chi-square test and Shapiro Wilks test for normality. Hartley's and Bartlett's tests for homogeneity of variance.
Bonferroni t-test and Dunnett's test was performed on weight and length data.
Validity criteria fulfilled:
Test conducted under GLP with analysis, analysis certificate and carried out to relevant guideline. This study can be considered reliable without
The NOEC is greater than or equal to 500 mg/l for all endpoints.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the early life stages of Danio rerio, in a 36-day flow-through test complying with the OECD Guideline No. 210,17 July 1992. The test criterion of toxicity used was the effects on hatching, larvae mortality, morphological abnormalities and growth of Danio rerio exposed to the test substance over the test period.

The nominal concentrations used in the study were as follows: 0, 12.8, 32.0, 80.0, 200 and 500 mg/l.

Analytical determinations of the test solutions were made on six occasions during the test. The concentrations were found to remain stable to within 20% of the nominals. The nominal concentrations were used to calculate the effect concentrations.

The validity criteria were respected:

• the dissolved oxygen concentration was between 60 and 100% of the air saturation value throughout the test,

• water temperature remained between 23 and 27°C over the test period and did not differ more than ±1.5°C between successive days except on 2 occasions during the test when maximum difference over a 24 h period was 1.85 and 2.05°C respectively. This was not thought to have any impact on the validity of the study.

• The post-hatch success (until the end of the test) was greater than 70% in the control.

The No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data using the appropriate statistical test.

However, as all embryos hatched at the highest concentration tested of 500 mg/l as well as in the control and post-hatch mortality was less than that of the control, a statistical test was not used for this parameter and the NOEC was considered to be at or greater than the highest concentration tested. No teratogenic malformations were noted for any larvae at any concentration.

Based on results from weight and length, the Lowest Observed Effect Concentration (LOEC) cannot be calculated and the No Observed Effect Concentration (NOEC) was determined as greater than or equal to the highest concentration tested, 500 mg/l.

Description of key information

The NOEC for fish is equal or greater than 500 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
500 mg/L

Additional information

One chronic fish study was found which is considered valid without restrictions, an early life stage test on Danio rerio. The test was performed according to the OECD 210 guideline without deviations and under GLP. Chemical analyses showed that the test substance concentrations were stable during the test and equal to nominal concentrations. The highest concentration tested was 500 mg/l which did not show any effect.

The NOEC for fish is equal or greater than 500 mg/l.