Neodymium trinitrate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May to 05 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PIDC / NDNC-191202_BZ
- Expiration date of the lot/batch: 12 December 2020
- Purity test date: 12 Dec 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any):
- Preparation of a nanomaterial dispersion (incl. dilution):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: At the start of the test, samples were taken from excess test solutions and at the end of the test one replicate of the dilution water control and each test concentration was sampled. Plastic HDPE sample vessels were rinsed with 70% analytical grade nitric acid prior to sample addition. 50 mL of each sample and 0.5 mL of 70% analytical grade nitric acid were added to each vessel and inverted to mix. Samples were stored frozen prior to shipment to CEMAS for analysis. The samples taken at 72 hours, containing algae, were analysed post centrifugation.

- Sample storage conditions before analysis:

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock concentrate of Neodymium Trinitrate, with a nominal concentration of 50 mg/L was prepared by adding a nominal 0.025 g of test substance to 500 mL with dilution water in a volumetric flask. The stock concentrate was stirred for approximately 5 minutes, which resulted in a clear and colourless solution.
This stock concentrate was then used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of stock concentrate to 1000 mL of dilution water in a volumetric flask.
- Eluate: Water
- Differential loading:
- Controls: Yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
- Other relevant information:

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: laboratory
- Age of inoculum (at test initiation): A 4-day old culture of the alga in the exponential growth phase was used as inoculum for the test.
- Method of cultivation: cultures maintained under axenic conditions

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21-24°C

pH:

7.58 to 7.68

Nominal and measured concentrations:

Nominal; 0.032, 0.01, 0.32, 1.0 and 3.2 mg/L
Measured; 0.029, 0.088; 0.30; 0.94 and 3.1 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical glass flask
- Type: closed with a foam bung
- Material, size, headspace, fill volume: 100mL of test solution in a 250mL flask
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 0.521 x E04 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):1
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: AAP
- Detailed composition if non-standard medium was used:

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: Continuous
- Light intensity and quality: 6000 lux
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.67 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.85 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

The validity criteria specified in the OECD 201 guideline are;

(i) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 hour test period. In this test cell particle density increase (measured as a surrogate for biomass) was 83.3 times over the 72 hours for the control.

(ii) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 9.277% for the control.

(iii) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and was calculated as 1.629% for the control.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the ErC50, ErC20 and ErC10 (growth rate) of Neodymium trinitrate to algae, over a 72 h exposure period based on mean measured test concentrations were determined to be 2.67, 1.59 and 1.13 mg/L, respectively. The corresponding NOEC and LOEC values were 0.2 mg/L and 0.85 mg/L, respectively.
The EyC50, EyC20 and EyC10 (yield) of Neodymium trinitrate to algae, over a 72 h exposure period based on mean measured test concentrations were determined to be 1.60, 0.40 and 0.011 mg/L, respectively. The corresponding NOEC and LOEC values were 0.012 mg/L and 0.2 mg/L, respectively.

Executive summary:

At the request of PIDC, 4788 Runway Blvd, Ann Arbor, MI 48108, USA, the toxicity of Neodymium Trinitrate to the green alga Pseudokirchneriella subcapitata was determined. The experimental start and completion dates for the study were 26 May 2020 and 05 June 2020, respectively, and the study number was 1010.01003.

All original data generated at the Test Facility, together with other relevant records, are filed in the Scymaris archive. The final phase report, phase specific raw data, printed hardcopies of electronic raw data and other relevant documents generated at the Test Site are filed in the Scymaris archive. Electronic raw data generated at the Test Site are archived in accordance with CEMAS procedures.

The procedures employed were in accordance with the OECD 201 test guideline. The study is also considered acceptable when compared to the procedural requirements of the relevant EU and international guidelines.

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 4-day old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.

The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 21-24°C controlled at  2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.

Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. The position of each test replicate vessel in the incubator was randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration sampling occasion.

The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 μm respectively. Each replicate test vessel was inoculated with 0.35 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.521 × 104 cells/mL. This value was used for growth calculations.

The data for cell counts obtained at 24, 48 and 72 hours was entered into electronic data files and statistically analysed using CETIS (Ref 5). Appropriate procedures were applied to test for significant differences (p <0.05) between the control and test concentrations and determine EC50, EC20 and EC10 values and the associated 95% confidence intervals, where appropriate The individual statistical procedures applied to each set of data are detailed within the results. Lowest Observed Effect Concentration (LOEC) is defined as the lowest tested concentration at which the substance is observed to have a statistically significant reducing effect on growth (p <0.05) when compared with the controls, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. The No Observed Effect Concentration (NOEC) is defined as the test concentration immediately below the LOEC.

After 24, 48, and 72 hours, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.

At the end of the test microscopic observations were made on samples taken from a single replicate of the control and each test substance concentration.

This study was run with a culture medium control and nominal exposure concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L.

A stock concentrate of Neodymium Trinitrate, with a nominal concentration of 50 mg/L was prepared by adding a nominal 0.025 g of test substance to 500 mL with dilution water in a volumetric flask. The stock concentrate was stirred for approximately 5 minutes, which resulted in a clear and colourless solution.

This stock concentrate was then used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of stock concentrate to 1000 mL of dilution water in a volumetric flask.

All test solutions were assessed to be clear and colourless prior to use. The appropriate test solution (100 mL volume) was dispensed into each test and blank vessel.

To establish what concentrations were achieved, analysis for Neodymium in the test solutions was undertaken using the inductively coupled plasma-mass spectrometry (ICP-MS) validated method detailed in Appendix 4. The analysis was carried out at the test site (CEMAS).

The pH of the control and test solutions was measured at the start of the test with a calibrated pH meter, using the excess remaining after filling the test vessels. At the end of the test the pH of one replicate vessel (containing algae) from each test concentration was determined.

Temperature values were monitored using a Max/Min thermometer checked against a liquid in glass thermometer. Current, maximum and minimum temperatures were recorded daily in an additional test vessel (without algae). The light intensity was measured once during the study, in each of four representative positions, using a photometer reading in lux (cosine).

Description of key information

In conclusion, the EC50 (growth rate) of Neodymium trinitrate to algae, over a 72 h exposure period based on mean measured test concentrations was determined as 0.2 mg/L. The corresponding NOEC value was 0.85 mg/L.

The EC50 (yield) of Neodymium trinitrate to algae, over a 72 h exposure period based on mean measured test concentrations was determined as 0.012 mg/L. The corresponding NOEC value was 0.2 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.2 mg/L

EC10 or NOEC for freshwater algae:

0.85 mg/L