Reaction mass of Willemite, white and zinc iron chromite brown spinel

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:

2013-08-06 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2011-07-28

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 2008

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

dichromium(3+) diiron(3+) lambda2-iron(2+) trizinc(2+) octaoxidandiide silicate

EC Number:

936-897-9

Cas Number:

1373399-58-6

Molecular formula:

(Zn, Fe)(Fe, Cr)2O4-Zn2SiO4

IUPAC Name:

dichromium(3+) diiron(3+) lambda2-iron(2+) trizinc(2+) octaoxidandiide silicate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reaction mass of willemite, white and zinc iron chromite brown spinel
- Substance type: inorganic pigment
- Physical state: solid, light brown powder, odourless
- Storage condition of test material: store separate from food and drinks, dry in closed containers and protected places. Keep containers air-tight.
- Relative density: 4.58

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentrations of aluminium, chromium, iron and zinc in the WAFs were measured at the start and the end of the test. Dissolved aluminium, chromium, iron and zinc concentrations were analysed in all test solutions sampled at the beginning of the test prior to addition of the algae and in pooled samples from each treatment level at the end of the exposure period. The samples were filtered (0.2 μm polyether-sulphone membrane syringe filter) at room temperature (20 - 25°C). The solutions were 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 aluminium, chromium, iron and zinc were determined by ICP-OES or ICP-MS, depending on concentrations and potential interferences. 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)*.
Appropriate amounts of the test item was transferred into sterile glass flasks and 1 L growthmedium was added, respectively, to obtain nominal loadings. 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 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 was 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: 8.01 at test start and between 8.14 and 8.17 at test end
Test vessels:
- start of test: 8.02 to 8.07
- end of test: 8.15 to 9.04

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.01, 0.10, 1.00 and 10.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel/material, size, headspace, fill volume: 250 mL conical glass flasks covered with air-permeable siliconesponge caps; Test vessels were acid-washed (10 % HNO3) and rinsed six times with purified water. The test vessels and caps were sterilised by autoclaving prior to use.
- Initial cells density: algal pre-culture (583 μL at a cell density of 1.72 x 10^6 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. All test preparations were performed under sterile conditions.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
Every flask was filled with 100 mL of the respective test medium, i.e. the filtered WAF solution.
All test preparations were performed under sterile conditions.

GROWTH MEDIUM
The sterilised synthetic OECD medium according to OECD 201 (2011) was used as growth medium. As stated in the guideline OECD 201, 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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: light intensity (day light) ranged from 63.80 to 72.20 μmol s-1m-2. The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm. The light intensity was measured daily using the illuminance meter LI-250 with a cosine (2π) receptor in μmol s-1m-2 (at level of the test media surface).
- 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 (with observation intervals if applicable) :
- 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 hours in samples taken directly from the test vessels.
The appearance of the algae was also checked by microscope (Axiostar plus) at the end of the test to verify any visual effect of the test item.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
A non-GLP range-finding-test was performed with WAFs of the test item at loadings of 1, 10, and 100 mg/L without a chemical analysis. Growth rate inhibition was estimated with 21.5, 92.6 and 91.7 % compared to control at 1, 10 and 100 mg/L loadings, respectively.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.394 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

water accommodated fraction after 7 days

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.58 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

water accommodated fraction after 7 days

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

water accommodated fraction at 0.1 mg/L after 7 days

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.071 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

water accommodated fraction after 7 days

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.415 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

water accommodated fraction after 7 days

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

water accommodated fraction at 0.01 mg/L after 7 days

Basis for effect:

other: yield

Details on results:

- there were loading-dependent effects on the growth of the green algal species
- microscopy revealed normal appearances of the algae cells despite increasing cell debris with increasing growth inhibition, which is a normal appearance not related to the test item.
Please also refer to table 1 in the field "Any other information on results incl. tables" and to "Attached background material" below.

Results with reference substance (positive control):

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in non-GLP tests. The nominal ErL50 value of 3.39 mg/L (95% confidence limits 3.27 - 3.50 mg/L, August 2013) 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 the 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) were determined since there was a loading-dependent inhibition > 50 %.
- calculation of the percent inhibition of growth rate [r] and yield [y] was performed according to the OECD 201 guideline.

The test results were statistically analysed to determine EL50, EL20 and EL10, and the respective 95 % confidence intervals, using Probit-analysis (Finney, 1984)* assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis. The NOEL and LOEL values for growth rate and yield were determined with the Williams Multiple Sequential t-test (Williams, 1971 & 1972)* using the computer program ToxRat Solutions (ToxRat® Professional 2.10)*.

*References:
- Finney. D.J.: Statistical Method in Biological Assay. 2nd ed., London. 1984.
- 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

Validity of the test:

The test fulfils all validity criteria of the OECD guideline 201 (2011) as:

- the cell number in the control cultures increased by a factor of 195 within the 72-hour test period (validity criterion: > 16).

- evaluation of the sectional growth rates of the controls: the mean of the replicate coefficients of variations (CV %) in the section-by-section growth rate of controls was 18.9 % during the test period (validity criterion ≤ 35 %).

- the coefficient of variation of average specific growth rate at test end in replicate control cultures was 2.3 % (validity criterion ≤ 7 %).

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

WAF loading [mg/L]	% Inhibition of growth rate	% Inhibition of yield
Control	0	0
0.010	1.73	7.03
0.100	2.77	14.1
1.00	25.5	74.2
10.0	82.4	99.2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test evaluation was based on WAF loadings. A dose-related growth inhibition was observed during the 72 hour test.
The ErL50 value for inhibition of growth rate and the EyL50 for algal yield were 2.58 and 0.415 mg test item/L (WAF loading). The corresponding ErL10 and EyL10 values were 0.394 and 0.071 mg/L (WAF loading).
The NOEL values were 0.100 and 0.010 mg test item/L (WAF loading) for growth rate and yield, respectively.