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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-06-30 to 2013-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 2011-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
, 2008
Deviations:
no
Qualifier:
according to guideline
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.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2011-02-07

Test material

Constituent 1
Chemical structure
Reference substance name:
Nickel iron chromite black spinel
EC Number:
275-738-1
EC Name:
Nickel iron chromite black spinel
Cas Number:
71631-15-7
Molecular formula:
(Ni,Fe)(Fe,Cr)2O4
IUPAC Name:
Nickel iron chromite black spinel
Test material form:
solid: particulate/powder
Remarks:
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 (structure type (Ni, Fe)(Cr, Fe)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:
yes
Details on sampling:
- Sampling method/Sample storage conditions before analysis:
Dissolved chromium, iron, manganese and nickel concentrations were measured in all test solutions sampled at the beginning of the test prior to addition of the algae. After 72 hours, samples were taken from each replicate of each treatment and pooled. The samples were filtered (0.2 μm polyether-sulphone membrane syringe filter) at room temperature (20 - 25°C). A volume of 10 mL of the solutions was then transferred into disposable polyethylene vials (Scintillation vials), acidified with HNO3 (final concentration 1 - 3 % HNO3), and stored at 4°C until further analysis.

The concentrations of dissolved chromium, iron, manganese and nickel were determined by ICP-OES. The results were assured by recovery experiments and the analysis of certified reference materials.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Since the test item is a multi-component substance and of low water solubility, toxicity was determined using water accommodated fractions (WAFs). The WAFs for the different test loadings were prepared individually in accordance with the OECD guidance document on transformation/dissolution of metals and metal compounds in aqueous media No. 29 (2001)*.

For the preparation of the test solutions, amounts of 1 and 100 mg of nickel iron chromite black spinel were added to 1 L of the growth medium. If required, additional test media were added to obtain the exact nominal loadings. All flasks were agitated on laboratory shakers at 100 rpm at 21.5 ± 1.5°C for 7 days.
To separate the insoluble part from the aqueous phase and to sterilise the WAF by filtration, the test preparations were filtered through a 0.22 μm filter (e.g. 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 algal growth test.

- Controls: growth medium only was used as control.

*Reference:
- 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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Pseudokirchneriella subcapitata, Chlorophycea, Chlorophyta
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller
Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.

ACCLIMATION
- Culturing media and conditions: before the onset of a test, a pre-culture is established in the OECD 201 growth medium under test conditions to obtain exponentially-growing algae for the test.
- Acclimation period: culture duration of the pre-cultures was 3 days.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
no data
Test temperature:
20.5 to 21.0 °C
pH:
Controls: test start: 8.01; test end: 8.15 to 9.21
WAF loadings: test start: 8.05 to 8.06; test end: 8.20 to 9.14
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 1.00 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps (vessels and caps sterilised by autoclaving prior to use).
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: algal pre-culture (583 μL at a cell density of 1.72 x 106 cells/mL) was added to the test vessels to achieve the initial cell concentration of 10,000 cells/mL. At the beginning of the test, the initial cell concentration was calculated based on the cell number of the pre-culture.
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates):
Every flask was filled with 100 mL of the respective test medium, i.e. the filtered WAF solution.

GROWTH MEDIUM
The sterilised synthetic OECD medium according to OECD 201 (2011) was used as growth medium. The molar ratio of medium-EDTA to medium-iron slightly exceeds unity in this medium. This prevents iron precipitation and at the same time, chelation of heavy metal ions is minimised (molar ratio EDTA / Fe(III+) = 1.135). Considering the molar ratio and the stability constants of EDTA with Fe(III+), the chelation of other metals remains negligible in comparison with nominal test concentrations. All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. Please also refer to table 1 in the field "Any other information on materials and methods incl. tables" below.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality: culture vessels were incubated with a light intensity (day light: OSRAM Standard “cool white”) adjusted to approximately 60 - 120 μmol m-2 s-1 (4440 - 8880 lux).
The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm (Incubation Shaker Multitron®, INFORS, Switzerland). The light intensity was measured daily using an illuminance meter LI-250 (LI-COR, Lincoln, USA) with a cosine (2π) receptor in μmol m-2 s-1 at level of the test media surface. Light intensity ranged from 63.8 to 72.2 μmol m-2 s-1.
- During the exposure period, the incubation temperature was measured daily with a calibrated thermometer in an additionally prepared control vessel kept under the same conditions. The pH values were measured in the additionally prepared replicate at the beginning of the test and directly in the test vessels at the end of the test.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany)
The correctness of the electronic counts was checked by microscope counts following internal standard operation procedures.
The cell density of the pre-culture was determined at test initiation and the volume required to achieve the initial cell concentration of 10,000 cells/mL was calculated.
During the test, the cell concentrations were determined after 24, 48 and 72 h in samples taken directly from the test vessels.
The appearance of the algae was also checked by microscope at the end of the test to verify any visual effect of the test item.

- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
A non-GLP range-finding-test with WAFs of nickel iron chromite black spinel was performed at loadings of 1.00, 10.0 and 100 mg/L without a chemical analysis.
Toxic effects were not observed over the whole test period at any of the tested loadings.
The WAF loadings of the definite test were based on the results of the range-finding test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water accommodated fraction at 100 mg/L after 7 days
Basis for effect:
other: yield
Details on results:
- there were not any loading-dependent effects on the growth of the alga up to and including the WAF loading of 100 mg nickel iron chromite black spinel/L
- corresponding microscopy revealed normal appearances of the algae cells despite cell debris in vessels with slight growth inhibition, which is a normal appearance not related to the test item.
- the ErL50 value for inhibition of growth rate and the EyL50 value for inhibition of yield were estimated to be > 100 mg/L (WAF loading).
- the NOEL for both growth rate and yield was ≥ 100 mg test item/L
Please also refer to "Any other information on results incl. tables" and "Attached background material" below.
Results with reference substance (positive control):
The nominal ErL50 value of 3.39 mg/L (95% confidence limits 3.27 - 3.50 mg/L, August 2013) for the reference substance 3,5-dichlorophenol is in good agreement with the results of an international ring test with ErL50 of 3.38 ± 1.30 mg/L (International Organisation for Standardisation, 1993)*.

*Reference:
- International Organisation for Standardisation (1993). ISO 8692 Water quality - Algal growth inhibition test.
Reported statistics and error estimates:
- The evaluation of the loading-effect-relationships and the calculations of effect loadings were based on WAF loadings.
- The mean value of the cell counts for each WAF loading was used for plotting growth curves.
- The EL values for growth rate and yield after 72 hours (growth rate) could not be determined since there was not a loading-dependent inhibition > 10 %.
- Calculation of the percent inhibition of growth rate [r] and yield [y] was performed according to the OECD 201 guideline.

The NOEL values for growth rate and yield were determined with the Williams Multiple Sequential t-test (Williams, 1972 61972)* using the computer program ToxRat Solutions*.

*References:
- Williams, D.A. (1971): A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103-117.
- Williams, D.A. (1972): The comparison of several dose levels with a zero dose control. Biometrics 28, 519-531.
- ToxRat® Professional 2.10. ToxRat® Solutions GmbH, Naheweg 15, 52477 Alsdorf, Germany.

Any other information on results incl. tables

Table: Percent inhibition of growth rate and yield by the test item compared to controls after 72 h.

WAF loading [mg nickel iron chromite black spinel/L]

% Inhibition of growth rate

% Inhibition of yield

Control

0.0

0.0

1.00

4.16

19.9

100

-0.13*

-0.15*

*negative values indicate increase in the observed parameter

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test evaluation was based on WAF loadings. A dose-related growth inhibition was not observed during the 72-hour test.
The ErL50 value for inhibition of growth rate and the EyL50 value for inhibition of yield were estimated to be > 100 mg/L (WAF loading). The NOEL is ≥ 100 mg nickel iron chromite black spinel/L (WAF loading) for both parameters.

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