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Diss Factsheets

Administrative data

short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-12-14 to 2013-12-25
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted 1992-07-17
according to guideline
other: OECD Series on testing and assessment, Number 29. Guidance document on transformation/dissolution of metals and metal compounds in aqueous media. ENV/JM/MONO (2001)9, 23-July 2001.
GLP compliance:
yes (incl. QA statement)
signed 2011-02-07

Test material

Constituent 1
Chemical structure
Reference substance name:
Nickel iron chromite black spinel
EC Number:
EC Name:
Nickel iron chromite black spinel
Cas Number:
Molecular formula:
Nickel iron chromite black spinel
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):nickel iron chromite black spinel
- Molecular formula: Ni1-xCr2-y-zFeyMnx+zO4 (spinel structure type (Ni, Fe)(Fe, Cr)2O4)
- Physical state: solid, black powder, odourless
- Stability: December 31, 2023
- Storage condition of test material: store separate from food and drinks, dry in closed containers and protected places. Keep containers air-tight.
- Relate density: 5.16
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method/Sample storage conditions before analysis:
Duplicate samples of approximately 20 mL were taken. Solutions were examined for soapy bubbles or particles floating on the surface. The samples were filtered (0.22 μm polyether sulphone membrane syringe filter) at room temperature (20-25°C), transferred into disposable polyethylene vials (Scintillation vials), acidified with HNO3 (final concentration 1-3 % HNO3) and were stored until further analysis. The Cr content of the test item is 18.1 %, the Fe content is 19.0 %, the Mn content is 12.2 % and the Ni content is 20.9 %. The concentrations of dissolved chromium, iron, manganese and nickel of freshly prepared WAF solutions at test start were determined by ICP-OES.

Please note the following:
After 96 hours, the test media of all test concentrations (control, 1 and 100 mg/L) were accidently discarded before samples for further chemical analysis could be collected. The remaining solution present in test vessels and in vessels used for pH measurements was therefore collected and analysed. The volume of the remaining liquid was approximately 5 mL so that a duplicate sample could not be taken. Analytical data of solutions sampled after 96 hours were not included in the data evaluation.
The reliability of the study, including the analytical measurements at test start, is not compromised by this deviation. Also, the conclusions of the study remain the same since mortality of the fish was not observed in any of the treatments.

Test solutions

Details on test solutions:
- Method:
As the test item is a multi-component substance, toxicity was determined using water accommodated fractions (WAFs). The WAFs for the various test concentrations were prepared individually in accordance with the OECD guidance document on transformation/dissolution of metals and metal compounds in aqueous media No. 29 (2001)*.

Amounts of 2.502 mg and 250.009 mg of nickel iron chromite black spinel were transferred into glass flasks and filled up with 2.502 L and 2.5009 L of dilution water to obtain WAF loadings of 1 and 100 mg/L, respectively. Flasks were stirred at 100 rpm for 7 days at 21.5°C. For each loading, the insoluble part was then separated from the aqueous phase and sterilised by filtration through a 0.22 μm filter (Syringe Filters DIAFIL PS (Polyether sulfone, PES) DIA-Nielsen GmbH & Co. KG, Düren, Germany) under sterile conditions. The filtrated WAFs were directly used in the acute fish toxicity test. The test vessels were filled with 2.5 L of the filtered WAFs containing the dissolved test item. Dilution water was used as blank control.

- Controls: dilution water was used as negative control.

- OECD Series on testing and assessment, Number 29. Guidance document on transformation/dissolution of metals and metal compounds in aqueous media. ENV/JM/MONO (2001)9, 23-July 2001.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: zebrafish
- Species: Danio rerio (Teleostei, Cyprinidae)
- Source: test facility breed
- Length at study initiation (subsample of the fish used in the test was weighed (n = 7)): 2.1 to 2.6 cm; mean ± SD: 2.4 ± 0.2 cm
- Weight at study initiation: 0.20 to 0.26 g; mean ± SD: 0.24 g ± 0.02 g
- The fish were not fed 24 h prior to the test start and during the test.

- Acclimation period: at least 12 days prior to the test
- Acclimation conditions: fish were held under equivalent water quality and illumination conditions to those applied in the subsequent test.
- Type and amount of food: fish were fed throughout the holding period with live brine shrimp (Artemia spp.) nauplii and ground flake food TetraMinR (Tetra Werke, Melle, Germany).
- Feeding frequency: once daily; ad libitum
- Health during acclimation (any mortality observed): only healthy fish without diseases and abnormalities were used in the study. Mortality of the batch was less than 5 % in the week preceding the start of the study.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h

Test conditions

1.1 mmol/L
Test temperature:
22.8 to 23.2°C
between 8.1 and 8.6
Dissolved oxygen:
Oxygen saturation: 83 to 97%
not applicable
Nominal and measured concentrations:
Nominal concentrations: 1.0 mg/L and 100 mg/L
Details on test conditions:
- Test vessel: 3 L glass beakers filled with 2.5 L of test solution
- Aeration: test media in the vessels were slightly aerated via a glass capillary.
- No. of organisms per vessel: 7 fish
- The mean weight of fish resulted in a loading of 0.67 g/L test medium

- Source/preparation of dilution water: tap water was sourced from the Schmallenberg district water production plant, mostly fed by small springs and percolation, and met hardness and pH recommendations of the OECD-Guideline 203. The purification took place on-site at the laboratory and included filtration with activated charcoal, passage through a lime-stone column and aeration until oxygen saturation. To avoid copper contamination, plastic water pipes are used in the test facility. The water was aerated to the point of oxygen saturation.
Typical water chemistry data of the holding and dilution water are presented below.
- Conductivity: 238.6 µS/cm
- pH: 8.3
- dissolved oxygen: 104%
- nitrate: 1.3 mg/L
- nitrite: <0.005 mg/L*
- ammonium: <0.010 mg/L*
- phosphate: <0.100 mg/L*
- total hardness: 1.1 mmol/L
- alkalinity: 1.8 mmol/L
- calcium: 0.8 mmol/L
- magnesium: 0.3 mmol/L
- NPOC: 0.336 mg/L
- cadmium: < 0.286 µg/L*
- copper: < 1.83 µg/L*
- iron: < 2.20 µg/L*
- manganese: < 0.567 µg/L*
- nickel: < 0.882 µg/L*
- lead: < 2.61 µg/L*
- zinc: < 2.80 µg/L*
- total residual chlorine: < 0.02 mg/L*
*<: value below the respective limit of detection

- The pH of solutions in test vessel was not affected by the test substance and remained constant over the course of study.
- Photoperiod: 12 light / 12 dark

- mortality and clinical signs (sub-lethal and abnormal behavior) were recorded at 3, 24, 48, 72, and 96 hours of exposure of the fish to the test item.

- Range finding study
A non-GLP range-finding test was performed with nickel iron chromite black spinel prior to the definite test using water accommodated fractions (WAFs) with loadings of 1 mg/L, 10 mg/L and 100 mg/L. Toxic effects were not observed over the whole test period at any of the tested loadings.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities/Mortality/Clinical signs: mortality of the fish was not observed in any of the treatments. Neither clinical signs nor
mortality were observed throughout the study. The ecotoxicological evaluation was based on WAF loadings of the test item.

Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Since mortality could not be observed throughout the study, a statistical analysis to determine LC10 and LC50 values was not required. The NOEC is ≥ the highest WAF loading tested and reported as such. The evaluation of the effects was based on WAF loadings.

Applicant's summary and conclusion

Validity criteria fulfilled:
Neither clinical signs nor mortality could be observed at any loading throughout the study.
Based on the results of this study, the LC50 of nickel iron chromite black spinel is >100 mg test item/L (WAF loading). The NOEC based on the visual observation as amounts to ≥100 mg/L (WAF loading).