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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Remarks:
Algae growth inhibition test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July - 11 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 23rd March, 2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
published in the Official Journal of the European Union L 54 of 01 March 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
Test item: Neodymium fluoride oxide, magnesium doped
Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Five replicate samples were taken from the test solution and one control sample on both analytical occasions. Samples taken at the start of the study were stored at 21-24°C for two days before the analysis. Before the ICP-OES analysis 2% nitric acid was added to the samples.

Test solutions

Vehicle:
no
Details on test solutions:
For preparation of test solution a supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.1 g test item in 1000 mL test medium (OECD medium). This supersaturated solution was shaken for at least two days and thereafter the non-dissolved test material was removed by filtration through a 0.22 µm membrane filter to obtain the saturated test solution. The test solution was freshly prepared in the testing laboratory just before introduction of algae (start of the experiment).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata
(formerly known as Pseudokirchneriella subcapitata)
Strain number: 61.81 SAG
(identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The preculture was incubated for three days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment. Hardness is not specified.
Test temperature:
Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.3 – 22.5°C measured in the flask and between 22.1 and 23.2°C measured within the climate chamber.
pH:
The pH was measured in both control and test item treated group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium not increased by more than 1.5 units during the test. The range of the pH was 6.94 – 8.07 during the experiment.
Nominal and measured concentrations:
A saturated test item solution (limit concentration) and a concurrent control were included in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment.

The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than 20 % of the analytical quantification limit (LOQ)).
In the treated group mean of the measured concentrations was 0.041 mg/L at the start and 0.038 mg/L at the end of the test. The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 0.04 mg/L. This concentration was considered as the saturation concentration in the test medium (equivalent to 100 mg/L nominal concentration).
Details on test conditions:
Test Water (Algal Mineral Salts Test Medium)

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
Separate stock solutions were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX water purification system). The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

Table 2: Composition of the OECD medium
Stock solution Substance Final concentration in the prepared growth medium
Stock solution 1
(macro nutrients) NH4Cl 15.0 mg/L
MgCl2 5.6 mg/L*
CaCl2 × 2 H2O 18.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
KH2PO4 1.6 mg/L

Stock solution 2
(iron) FeCl3 × 6 H2O 64.0 microg/L
Na2EDTA × 2H2O 100.0 microg/L

Stock solution 3
(trace elements) H3BO3 185.0 microg/L
MnCl2 × 4 H2O 415.0 microg/L
ZnCl2 3.0 g/L
CoCl2 × 6 H2O 1.5 microg/L
CuCl2 × 2 H2O 0.01 microg/L
Na2MoO4 × 2 H2O 7.0 microg/L

Stock solution 4
(bicarbonate) NaHCO3 50.0 mg/L
* This corresponds to the concentration of 12.0 mg/L of MgCl2 × 6 H2O (cf. OECD no. 201 guideline)
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Slight growth stimulation (negative inhibition) was observed in the treated group (-0.3 % inhibition for growth rates and -1.1 % for yield) after 72 hours of exposure compared to the control which was considered to be within the normal biological range based on the statistical evaluation.
Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using 2 Sample t-Test (alpha = 0.05, TOXSTAT software). The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield at the treatment group (saturated test item concentration) was not statistically significantly different from the untreated control.
Results with reference substance (positive control):
For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.
The date of the last study (Study Number: 392-201-1333) with the reference item Potassium dichromate was: 01 – 04 March 2016.
The 72h ErC50: 0.70 mg/L, (95 % confidence limits: 0.64 – 0.78 mg/L)
The 72h EyC50: 0.39 mg/L, (95 % confidence limits: 0.36 – 0.43 mg/L)

Name and data of Reference Item

Name: Potassium dichromate (K2Cr2O7)
Batch No.: MKBN3524V
Description: Orange crystalline powder
Expiry Date: 04 March, 2018
Storage: Room temperature
Supplier: SIGMA-ALDRICH
Reported statistics and error estimates:
Average specific growth rate and yield were calculated for each test flask. Then the mean growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment. The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3)) in order to demonstrate exponential growth for the entire study period.
The inhibition of algae growth was determined from the average specific growth rate and yield using the following equations:

Average specific growth rate (µ):
The average specific growth rate for a specific period is calculated as follows:


where: µi-j = average specific growth rate from time i to j
Xj = biomass at time j
Xi = biomass at time i

Percent inhibition of growth rate (% Ir):


where: µC = mean value for average specific growth rate in the control group
µT = average specific growth rate for the treatment replicate

Yield (Y):
Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield value will be calculated.

Percent inhibition in yield (% Iy):


where: Yc = mean value for yield in the control group
YT = value for yield for the treatment replicate

Mean values and standard deviations were calculated for each treatment at the start, and at the end of the test using Excel for Windows software. Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (alpha = 0.05, TOXSTAT software). The ErC50 and EyC50 values were determined from the raw data.

Any other information on results incl. tables

Summary of the Biological Results

Endpoints
(0-72 h)

Growth rate (r)

Yield (y)

EC10

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

EC20

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

EC50

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

NOEC

0.04 mg/L
(equivalent to 100 mg/L nominal)

0.04 mg/L
(equivalent to 100 mg/L nominal)

LOEC

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

> 0.04 mg/L
(equivalent to > 100 mg/L nominal)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item Neodymium fluoride oxide, magnesium doped had no toxic effect at aquatic saturation (i.e. limit test concentration) on the growth of green algae; the EC10, EC20, EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to a nominal concentration of 100 mg/L (measured geometric mean concentration: 0.04 mg/L)
Executive summary:

The effect ofNeodymium fluoride oxide, magnesium dopedtest item was assessed on algal growth using the unicellular green algaRaphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata), over an exposure period of 72 hours.

A limit test was performed in which, algal cells were exposed to aqueous test media containing the test item for 72 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the growth of algae up to at least the saturation concentration.

The quantification of the test item Neodymium fluoride oxide, magnesium dopedwas performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of the experiment and analysed by ICP-OES method based on the measured neodymium content. The determined exposure concentration of the test item was 0.04 mg/L which was calculated as the geometric mean of the measured start and end concentrations.

Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates per test concentration and control. The alga cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals.

Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using 2 Sample t-Test (a= 0.05, TOXSTAT software).The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield in the treatment group (saturated test item concentration) was not statistically significantly different from the untreated concurrent control.

All validity criteria were met and therefore the study can be considered as valid.

In this 72-h algal growth inhibition test wit\h Raphidocelis subcapitata, the obtained results showed that the test itemNeodymium fluoride oxide, magnesium dopedhadno toxic effectat aquatic saturation (i.e. limit test concentration)on the growth of green algae; theEC10,EC20,EC50results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to a nominal concentration of 100 mg/L.