Neodymium oxide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2020 to 08 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test preparations were verified by chemical analysis on Days 0, 6, 13 and 20 (fresh media from bulk test solutions) and on Days 1, 7, 14 and 21 (aged/ old media from pooled replicates). All samples were filtered through 0.45 µm cellulose acetate filter (first initial 10 mL discarded in order to pre-condition the filter). All samples were stored frozen prior to analysis. Duplicate sets of samples were also taken on each occasion and stored frozen for future analysis (if required).

Water samples were taken from the control and each surviving test group for quantitative analysis of dissolved neodymium. Samples of the fresh test preparations were taken from the bulk preparation on Days 0, 6, 13 and 20 and of the corresponding aged test preparations on Days 1, 7, 14 and 21 (pooled replicate). All samples were filtered through 0.45 µm cellulose acetate filter (first initial 10 mL discarded in order to pre-condition the filter). Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Range-Finding Test
A nominal amount of test material (18 mg) was dissolved in test water and the volume adjusted to 1 L to give a 10 mg/L test material stock solution from which a series of dilutions was made to give further test solutions of 0.010, 0.10 and 1.0 mg/L test material. The 10 mg/L stock solution and its dilutions were prepared at the beginning of the range-finding test and before each renewal period.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test material (9.0 mg) was dissolved in test water and the volume adjusted to 5 L to give a 1.0 mg/L stock solution. A series of dilutions was made from this stock solution to give the required test solutions of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L. The 1.0 mg/L stock solution and its dilutions were prepared at the beginning of the test and before each renewal period. The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Age at study initiation (mean and range, SD): The young daphnids produced overnight were less than 24 hours old.
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test. The young were removed from the cultures and used for testing; they were not first brood progeny.
- Source: The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Feeding during test : Each daphnid received approximately 5 to 20 µL of an algal suspension (Raphidocelis subcapitata) daily and approximately 20 µL of GEMMA Micro 300 fish food suspension on Days 0 to 3 only. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Adult Daphnia were maintained in 150 mL glass vessels containing ISO medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata)) and GEMMA Micro 300 fish food suspension. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

242 - 274 mg/L as CaCo3

Test temperature:

19 °C - 21 °C

pH:

5.6 - 6.5

Dissolved oxygen:

> 8.7 mg/L

Nominal and measured concentrations:

Nominal concentrations of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L.

Details on test conditions:

STABILITY AND FILTRATION TRIAL
At the request of the Sponsor a stability trial was conducted to indicate the media renewal regime to be used during testing. A nominal amount of test material (90.8 mg) was dispersed in 5 L of pH modified ISO media at pH 6 to a give a nominal concentration of 10 mg/L as test material. The stock solution was inverted several times to ensure homogeneity and then left to stand under test conditions for 72 hours. After 0, 24, 48 and 72 hours, the pH of the media was checked and adjusted to approximately 5.9 – 6.1 and a sample was taken for chemical analysis at 0, 24 and 72 hours. Duplicate samples were taken and stored frozen for further analysis if required.
From these results it was evident that there was no significant decline in the measured concentration of the test material at a nominal 10 mg/L neodymium trinitrate over the 72-hour period.
At the Sponsor's request, an additional trial was conducted to determine whether the test material formed a true solution.
A nominal amount of test item (36.3 mg) was dispersed in 2 L of pH modified ISO media at pH 6 to give a nominal concentration of 10 mg/L test material. The stock solution was inverted several times to ensure homogeneity and a sample was taken for chemical analysis after the following pre-treatment: Untreated; and filtration through a 0.2 µm Gelman Acrocap filter (initial 500 mL discarded). Untreated samples were expected to reflect total neodymium concentration, while filtered samples were expected to reflect dissolved neodymium concentration. Duplicate samples were taken and stored frozen for further analysis if required.
These results show no significant difference in the measured concentrations between the untreated and filtered samples; meaning that all measured neodymium was dissolved. Based on these results the test material appears to create a true solution when maintained at pH 6.0. The alignment between measured and nominal concentrations confirms that the test material was completely soluble in test media to a nominal concentration of 10 mg/L as test material.
However, the fact that all measured neodymium was dissolved was only demonstrated immediately after solution preparation; indeed comparison between measured concentrations in untreated and filtered samples was only made after 0 hour. As a safeguard against what could happen over long durations, the Sponsor requested the samples for chemical analysis were to be filtered through an acetate filter provided by the Analytical Chemistry Department prior to acidification for analysis

INITIAL EXPERIMENT
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test material at nominal concentrations of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L test material for a period of 14 days after which the test was terminated as two parental Daphnia in the control treatment did not produce any neonates over the 14 days, and therefore should fail the validity criteria that each surviving adult Daphnia should produce a mean of 60 living neonates by the end of the test in Day 21. The test was therefore repeated and the results from the initial experiment were not used for reporting purposes.

TEST SYSTEM
- Type: Closed with a plastic lid to reduce evaporation.
- Material, size, headspace, fill volume: 100 mL test preparation in 150 mL glass vessels.
- Aeration: The diluent water only was aerated prior to use.
- Renewal rate of test solution: The test preparations were renewed daily throughout the test. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and visually counted before being discarded.
- No. of organisms per vessel: One
- No. of vessels per concentration: Ten replicates of a single daphnid.
- No. of vessels per control: A control group was maintained under identical conditions but not exposed to the test material.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 294 mg/L CaCl2·2H2), 123 mg/L MgSO4·7H2O, 65 mg/L NaHCO3, 5.8 KCl. The reconstituted water had a pH of 6.0 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study. The reconstituted water (ISO medium) used for the stability and filtration trial, range-finding and definitive tests was the same as that used to maintain the stock animals. This medium is suitable when metal toxicity is investigated as it does not contain chelators such as EDTA.
- Intervals of water quality measurement: Dissolved oxygen concentrations, pH and temperature were recorded at the start and end of the test and before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a calibrated Hach HQ30d Flexi handheld meter whilst the temperature was measured using a calibrated digital thermometer. Measurements were made on one replicate for each test concentration. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the media was adjusted to 6.0 in order to aid solubilisation and stability of the test material.
- Photoperiod:16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: 713 - 995 lux.

EFFECT PARAMETERS MEASURED
Test Organism Observations: On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental daphnia as compared with the controls.
Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (i.e. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal (excluding the anal spine) was determined.

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal concentrations of 0.010, 0.10, 1.0 and 10 mg/L test material.
- In the range-finding test, for each concentration a single daphnid was placed in 100 mL of the test preparation in 120 to 200 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group five replicate test vessels were prepared. The water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °Cwith a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 10 days.
The control group was maintained under identical conditions but not exposed to the test material.
The test preparations were renewed on Days 1, 4, 6 and 8. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 20 µL of an algal suspension (Raphidocelis subcapitata) daily and approximately 20 µL of GEMMA Micro 300 fish food suspension on Days 0 to 3 only. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/ daphnid/ day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
A sample of each test concentration was taken from the freshly prepared bulk preparations for chemical analysis on Day 0 and from the corresponding old media (pooled replicates) on Day 1 in order to determine the stability of the test material under test conditions. All samples were filtered through 0.45 µm cellulose acetate filter (first initial 10 mL discarded in order to pre-condition the filter) and the Day 0 samples were stored frozen prior to analysis with the Day 1 samples. Duplicate samples were taken and stored frozen for further analysis if required
- Results used to determine the conditions for the definitive study: Yes.
No immobilisation was observed at the test concentrations of 0.010 and 0.10 mg/L, however, 80 and 100 % immobilisation were observed at 1.0 and 10 mg/L, respectively. Sub-lethal effects of exposure were observed at test concentrations of 0.010 and 1.0 mg/L. These responses were pale in colouration, reduced mobility, covered in algae and smaller in size.
The range-finding test was conducted with a semi-static design with renewals on Days 1, 4, 6 and 8. Chemical analysis was performed for the first 24-hour renewal period and showed measured concentrations of the fresh test preparations on Day 0 to range from less than the Limit of Quantification (LOQ) to 11 mg/L as test material (5.6 % to 106 % of nominal), equivalent to less than the LOQ to 4.6 mg/L as neodymium. Chemical analysis of the old/aged test preparations on Day 1 showed measured concentrations to range from less than the LOQ to 8.8 mg/L as test material (5.6 % to 88 % of nominal), equivalent to less than the LOQ to 3.8 mg/L as neodymium. The decline in the measured test concentrations in the aged test preparation over 1 day confirmed that the definitive test needed to be run with a daily renewal of test preparations.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

immobilisation

Remarks on result:

not determinable

Duration:

21 d

Dose descriptor:

LOEC

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

not determinable

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.078 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

Body length

Remarks on result:

other: 95 % confidence limits: 0.041 - 0.15 mg/L neodymium.

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Body length

Remarks on result:

other: 95 % confidence limits: 0.091 - 0.36 mg/L test material

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

Body length

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

Body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Body length

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

Body length

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Body length

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.079 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits: 0.037 - 0.17 mg/L.

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits: 0.081 - 0.41.

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Lethal effects on parental (P1) generation: One mortality was observed in the control, 0.045, 0.070 and 0.31 mg/L mean measured test groups on Days 21, 2, 10 and 3 respectively and two mortalities were observed in the 0.028 mg/L group which were observed on Days 2 and 6. No mortalities occurred in the 0.13 mg/L test group. Statistical analysis of the mortality data using the Fisher’s Exact Binomial test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts showed that the observed mortalities in the all test groups were not significantly different (P≥0.05) when compared to the control group.
- Observations on body length and weight: Sub-lethal effects on parental (P1) generation: Sub-lethal effects were observed in the control, 0.070, 0.13 and 0.31 mg/L groups. Two replicates of the control test group were observed to be pale in colouration, one was observed to be on Days 19 to 20 and the other replicate on Day 21. In the 0.070 mg/L test material concentration one replicate was observed to have reduced mobility on Day 3 and on Days 19 to 21 another replicate was observed to be pale in colouration. In the 0.13 mg/L test material concentration one replicate was observed to be pale in colouration on Days 19 to 21, and in the 0.31 mg/L test material concentration two replicates were observed to be pale in colouration on Days 19 to 21.
The daphnids at the remaining test concentrations were observed to be the same size and colour as the control daphnids. Statistical analysis of the results showed that there were no significant differences (P≥0.05) between the control and the 0.028, 0.045 and 0.070 mg/L test groups; however significant difference (P<0.05) was determined between the control and the 0.13 and 0.31 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test material. In these groups, the length was reduced from 9.3 % and 14.4 %, respectively, as compared to the control group.
- Effects on reproduction: After 21 days there were no statistically significant differences (P≥0.05) between the control and the 0.028, 0.045, 0.070 and 0.13 mg/L test group in terms of the number of live young produced per adult. The 0.31 mg/L test group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult. In this group, the number of live young produced per adult was indeed reduced by 14.7 % as compared to the control group.
- Effects on filial generation (F1): Information on the effects of the test material on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by 0.028, 0.045, 0.070 and 0.13 mg/L test groups were in the same general condition as the young produced by the controls over the duration of the test. The young produced by the 0.31 mg/L test group in three replicates, however, were observed to be smaller in size when compared to the control young on Days 17 and 18. Young were first produced in the control test group on Day 8 of the test. Young were also first produced on Day 8 in the 0.045, 0.070, 0.13, 0.31 mg/L test groups and on Day 9 in the 0.028 mg/L test groups
- Any observations that might cause a difference between measured and nominal values: At the start and throughout the test all control and test solutions were observed to be clear colourless solutions

- Verification of Test Concentrations: Analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.047 to 0.63 mg/L as test material (39 % to 112 % of nominal), equivalent to 0.021 to 0.27 mg/L neodymium. A decline in measured test concentration of the aged test preparations on Days 1, 7, 14 and 21 was observed to between less than the LOQ, which was determined to be 0.020 mg/L to 0.19 mg/L as test material (5.6 % to 40.4 % of nominal), equivalent to less than the LOQ, which was determined to be 0.010 mg/L to 0.084 mg/L as neodymium.

Reported statistics and error estimates:

- ECx Calculations
The 21-Day ECx (immobilisation, P1 generation) values were calculated by Probit Analysis using Linear Maximum Likelihood Regression.
The 21-Day ECx (reproduction, P1 generation) values and associated confidence limits were calculated by 3-Parameter Normal Cumulative Distribution Function.
The total number of living offspring produced at the end of the test per parent at the start of the test, excluding any parental accidental and/or inadvertent mortalities were used for this calculation.
The 21-Day ECx (length, P1 generation) values and associated confidence limits were calculated by 2-Parameter Normal Cumulative Distribution Function.
All statistical analysis was conducted using ToxRatPro software package (TOXRAT).

-Lowest Observed Effect Concentration and No Observed Effect Concentration Determinations
For the determination of the LOEC and the NOEC for survival (immobilisation), the number of surviving Daphnia over the duration of the test for the control and each test group were compared using Fisher’s Exact Binomial Test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts.
For the estimation of the LOEC and NOEC for reproduction, the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using Williams Multiple Sequential t-test incorporating Cochran’s test on Variance Homogeneity and Shapiro-Wilk's test on Normal Distribution.
For the estimation of the LOEC and NOEC for body length, results from the control and each of the test groups Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using Dunnett’s Multiple t-test incorporating Levene’s test on Variance Homogeneity and Shapiro-Wilk's on Normal Distribution. All statistical analyses were performed using the ToxRat Professional software package (TOXRAT).

Geometric Mean Measured Test Concentrations

Nominal Test Concentration (mg/LTest Material)	Geometric Mean Measured Test Concentration (mg/L as Neodymium)	Geometric Mean Measured Test Concentration (mg/L asTest Material)
0.056	0.013	0.028
0.10	0.020	0.045
0.18	0.032	0.070
0.32	0.058	0.13
0.56	0.13	0.31

 

Validation Criteria

	Required	Actual
Control mortality	≤20 %	10 %
Mean number of live young per surviving adult (control group)	≥60 after 21 days	178
Coefficient of variation for control group*	≤25 %	8.9%
No ephippia produced	0	0
Dissolved oxygen	> 3 mg O2/L	≥ 8.7 mg O2/L
pH (control group)	6 to 9 Variation £1.5	5.9 to 7.7 1.8

*Based on total number of living offspring per parent animal alive at the end of the test.

The variation of pH values in the control increased by more than 1.5 units on two occasions during the test, however given that there were no adverse effects or mortalities seen during these occasions and all other validity criteria were met, this is not thought to have affected the outcome or validity of the test.

 

Range-finding Test Data

Nominal Concentration (mg/L Test Material)	Percentage Immobilisation After 10 Days Exposure	Sub-Lethal Effects	Total Number of Live Young in First Brood
Control	0	Normal	91
0.010	0	Pale	90
0.10	0	Normal	90
1.0	80	Pale, covered in algae, small and reduced mobility	15
10	100	Normal	0

 

 

Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Geometric Mean Measured Test Concentrations (mg/L as Test Material)	Parental (P1) Generation Mortalities (Initial Population = 10)			Total Number of Live Young	Number of Live Young from Parents Surviving to the End of the Test		Number of Live Young at the End of the Test Excluding Parents that were Accidental or Inadvertent Mortalities
	Total	Accidental	Inadvertent		Total	Average per Parent	Total	Average per Parent
Control	1	0	1	1764	1602	178	1602	178
0.028	2	0	2	1524	1524	191	1524	191
0.045	1	0	1	1620	1620	180	1620	180
0.070	1	0	1	1632	1614	179	1614	179
0.13	0	0	0	1687	1687	169	1687	169
0.31	1	0	1	1367	1367	152	1367	152*

*Statistically different compared to the control, calculated by Probit analysis using Linear Maximum Likelihood regression and Fisher’s Exact Binomial Test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts.

 

Summary of Daily Observations

Day	Geometric Mean Measured Concentration (mg/L Test Material)
	Control		0.028		0.045		0.070		0.13		0.31
	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young
1	10	0	10	0	10	0	10	0	10	0	10	0
2	10	0	9	0	9	0	10	0	10	0	10	0
3	10	0	9	0	9	0	10	0	10	0	9	0
4	10	0	9	0	9	0	10	0	10	0	9	0
5	10	0	9	0	9	0	10	0	10	0	9	0
6	10	0	8	0	9	0	10	0	10	0	9	0
7	10	0	8	0	9	0	10	0	10	0	9	0
8	10	8	8	0	9	9	10	34	10	14	9	22
9	10	116	8	143	9	107	10	59	10	100	9	84
10	10	12	8	0	9	11	9	24	10	15	9	0
11	10	18	8	0	9	21	9	92	10	39	9	57
12	10	243	8	273	9	276	9	172	10	255	9	163
13	10	63	8	0	9	40	9	115	10	61	9	100
14	10	0	8	0	9	0	9	44	10	45	9	50
15	10	337	8	377	9	332	9	207	10	293	9	182
16	10	45	8	0	9	0	9	142	10	68	9	108
17	10	0	8	0	9	47	9	50	10	0	9	45
18	10	332	8	345	9	333	9	227	10	328	9	174
19	10	44	8	0	9	55	9	139	10	91	9	131
20	10	47	8	0	9	0	9	63	10	155	9	88
21	9	337	8	386	9	389	9	246	10	223	9	163
TOTAL	9	1602	8	1524	9	1620	9	1614	10	1687	9	1367

 

Total Cumulative Production of Live Young Excluding Offspring of Parents Accidentally or Inadvertently Dead

Geometric Mean Measured Test Concentration (mg/L as Test Material)	Day
	1		2		3		4		5		6		7		8		9		10
Control	0		0		0		0		0		0		0		8		124		136
0.028	0		0		0		0		0		0		0		0		143		143
0.045	0		0		0		0		0		0		0		9		116		127
0.070	0		0		0		0		0		0		0		34		93		117
0.13	0		0		0		0		0		0		0		14		114		129
0.31	0		0		0		0		0		0		0		22		106		106
Geometric Mean Measured Test Concentration (mg/L as Test Material)	Day
	11	12		13		14		15		16		17		18		19		20		21
Control	154	397		460		460		797		842		842		1174		1218		1265		1602
0.028	143	416		416		416		793		793		793		1138		1138		1138		1524
0.045	148	424		464		464		796		796		843		1176		1231		1231		1620
0.070	209	381		496		540		747		889		939		1166		1305		1368		1614
0.13	168	423		484		529		822		890		890		1218		1309		1464		1687
0.31	163	326		426		476		658		766		811		985		1116		1204		1367

 

Body Length of Surviving Adults at Day 21

Geometric Mean Measured Test Concentration (mg/L as Test Material)	Individual Daphnia Lengths (mm)
	1	2	3	4	5	6	7	8	9	10	Mean	SD
Control	-	4.0	4.2	3.9	4.3	4.1	3.9	4.3	4.0	4.1	4.1	0.15
0.028	4.3	4.0	-	3.9	4.2	4.3	-	4.1	4.0	4.1	4.1	0.15
0.045	3.9	3.8	4.0	4.3	3.9	4.0	3.9	3.8	-	4.1	4.0	0.16
0.070	4.3	4.0	4.1	3.9	3.8	4.0	3.9	-	4.0	4.2	4.0	0.16
0.13	3.4	3.8	3.5	3.8	3.6	3.7	3.5	4.0	4.1	3.7	3.7	0.22
0.31	3.4	-	3.4	3.9	3.5	3.6	3.2	3.4	3.9	3.2	3.5*	0.26

Body length measured excluding the anal spine.

 

- = No measurement, Daphnia died during the test

*= Statistically difference compared to the control, calculated by the 2-Parameter Normal Cumulative Distribution Function and Dunnett’s Multiple t-test incorporating Levene’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal Distribution.

 

Analytical Results for Range-Finding Samples

Time Point (Day)	Nominal Concentration of the Test Material in Range-Finding Samples (cnom) (mg/L Test Material)	Determined Concentration of Nd in Range-Finding Samples (c) (mg/L Nd)	Calculated Concentration of the Test Material in Range-Finding Sample (c) (mg/L Test Material)	Percentage Nominal Concentration (%) As Test Material
0† (fresh)	Control	< LOQ	< LOQ	-
	0.010	< LOQ	< LOQ	-
	0.1	0.0390	0.0892	89
	1.0	0.224	0.513	51
	10	4.62	10.6	106
P. Rec 1	5.0	4.90	-	98
P. Rec 2	5.0	5.12	-	102
1†	Control	< LOQ	< LOQ	-
	0.010	< LOQ / < LOQ*	< LOQ / < LOQ*	-/-*
	0.1	0.021 / 0.0153*	0.0480 / 0.0349*	48/35*
	1.0	0.0338 / 0.0271	0.0773 / 0.0620	8/6*
	10	3.84	8.79	88
P. Rec 1	5.0**	4.75	-	95
P. Rec 2	5.0**	5.39	-	108

† Day 0 and Day 1 range-finding samples analysed on the same day.

*Duplicate samples, stored frozen prior to analysis.

**Prepared and analysed alongside duplicate samples.

LOQ = Limit of Quantification (0.02 mg/L as anhydrous substance).

1. Rec = procedural recovery.

- = Not applicable.

Note that the concentration quoted for the procedural recovery samples are the actual fortified concentrations expressed in mg Nd/L.  The corresponding percentages of nominal concentration values relate to the percentage of fortified concentration recovered expressed in percentage as Nd.

The procedural recoveries prepared alongside the range finding samples were prepared using neodymium analytical standard.  For the validation and definitive test, the test material was used.

 

Precision and Accuracy/ Recovery Samples

Sample No.	Fortified Concentration of Test Material in the Spiked Sample (cfort) (mg/L Test Material)	Determined Concentration of Neodymium in the Spiked Sample (c) (mg/L Nd)	Calculated Concentration of Test Material in the Spiked Sample (c) (mg/L Test Material)	Mean Analytical Recovery Rate (R) (% Test Material)	Precision (Relative Standard Deviation of Recovery) (% Test Material)
1	0.0199	0.00869	0.0199	101	0.97
2	0.0199	0.00891	0.0204
3	0.0199	0.00874	0.0200
4	0.0199	0.00884	0.0202
5	0.0199	0.00878	0.0201
1	0.996	0.475	1.09	106	2.6
2	0.996	0.443	1.01
3	0.996	0.464	1.06
4	0.996	0.465	1.06
5	0.996	0.470	1.08
Acceptance target				70 - 110	< 20

 

Results for Test Samples

Time Point (Day)	Nominal Concentration of the Test Material in Range-Finding Samples (cnom) (mg/L Test Material)	Determined Concentration of Nd in Range-Finding Samples (c) (mg/L Nd)	Calculated Concentration of the Test Material in Range-Finding Sample (c) (mg/L Test Material)	Percentage Nominal Concentration (%) As Test Material
0 (fresh)	Control	< LOQ	< LOQ	-
	0.056	0.0217	0.0497	89
	0.10	0.0364	0.0832	83
	0.18	0.0640	0.146	81
	0.32	0.126	0.288	90
	0.56	0.231	0.530	95
P. Rec 1	0.203	0.0822	0.188	93
P. Rec 2	0.203	0.0822	0.188	93
1 (old)	Control	< LOQ	< LOQ	-
	0.056	< LOQ	< LOQ	-
	0.10	0.0102	0.0233	23
	0.18	0.0158	0.0361	20
	0.32	0.0350	0.0801	25
	0.56	0.0809	0.185	33
6 (fresh)	Control	< LOQ	< LOQ	-
	0.056	0.0217	0.0497	89
	0.10	0.0361	0.0827	83
	0.18	0.0653	0.150	83
	0.32	0.122	0.279	87
	0.56	0.196	0.450	80
P. Rec 1	0.203	0.0791	0.181	88
P. Rec 2	0.203	0.0864	0.198	96
7† (old)	Control	< LOQ	< LOQ	-
	0.056	0.00549	< LOQ	-
	0.10	0.00639	< LOQ	-
	0.18	0.00649	< LOQ	-
	0.32	0.0108	0.0248	8
	0.56	0.0517	0.118	21
13 (fresh)	Control	ND	ND	-
	0.056	0.0246	0.0563	101
	0.10	0.0433	0.0991	99
	0.18	0.0853	0.195	108
	0.32	0.157	0.359	112
	0.56	0.274	0.628	112
P. Rec 1	0.208	0.0902	0.207	99
P. Rec 2	0.208	0.0919	0.210	101
14† (old)	Control	ND	ND	-
	0.056	0.00987	0.0226	40
	0.10	0.0122	0.0279	28
	0.18	0.0140	0.0320	18
	0.32	0.0243	0.0556	17
	0.56	0.0842	0.193	34
20 (fresh)	Control	ND	ND	-
	0.056	0.0205	0.0470	84
	0.10	0.0355	0.0813	81
	0.18	0.0724	0.166	92
	0.32	0.134	0.307	96
	0.56	0.243	0.557	99
P. Rec 1	0.201	0.0875	0.200	100
P. Rec 2	0.201	0.0867	0.198	99
21†† (old)	Control	ND	ND	-
	0.056	0.00962	0.0220	39
	0.10	0.0157	0.0360	36
	0.18	0.0207	0.0475	26
	0.32	0.0321	0.0734	23
	0.56	0.0844	0.193	35

† Day 7 test sample analysed alongside Day 6 test samples.  Day 14 test sample analysed alongside Day 13 test sample.

†† Day 21 test sample analysed alongside Day 20 test sample.

LOQ = Limit of Quantification (0.02 mg/L as anhydrous substance).

1. Rec = Procedural recovery.

- = Not applicable.

ND = Not detected.

Note: The concentrations quoted for the procedural recovery samples are the actual fortified concentrations.  The corresponding percentage of nominal concentration values related to the Percentage of Fortified Concentration recovered.  For some samples (7 days old) in 0.056, 0.1 and 0.18 mg/L treatments) it was possible to determine neodymium concentrations whilst below the LOQ; these concentrations were provided for information only and the corresponding calculated test material concentrations were set less than the LOQ to avoid uncertain extrapolation.

 

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, exposure of Daphnia magna to the test material gave the following results based on the geometric mean measured test concentrations:
The EC10 and EC50 based on immobilisation were both > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).
The NOEC for immobilisation was ≥0.13 mg/L (as Nd) and≥0.31 mg/L (as test material) based on immobilisation.
The LOEC for immobilisation could not be determined for immobilisation.
The EC10 based on reproduction was > 0.13 mg/L (as Nd) with 95 % confidence limits of 0.037 – 0.17 mg/L Nd, and > 0.31 mg/L (as test material) with 95 % confidence limits of 0.081 – 0.41 mg/L test.
The EC50 for reproduction was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).
The NOEC for reproduction was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).
The LOEC for reproduction was 0.13 mg/L (as Nd) and 0.31 mg/L (as test material).
For body length, the EC10 was > 0.078 mg/L (as Nd) with 95 % confidence limits of 0.041 – 0.15 mg/L Nd, and > 0.18 mg/L (as test material) with 95 % confidence limits of 0.091 – 0.36 mg/L test material.
The EC50 for body length was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).
The NOEC for body length was 0.032 mg/L (as Nd) and 0.07 mg/L (as test material).
The LOEC for body length was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).

Executive summary:

A study was performed to assess the chronic toxicity of the test item to Daphnia magna according to OECD Test Guideline 211 and EU Method C.20, and in compliance with GLP.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test material at nominal concentrations of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L for a period of 21 days. Test solutions were renewed daily. In order to maximize the dissolved concentrations of the test material, daphnids were exposed in test media adjusted to pH 6.0. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension. On Days 0 to 3 the Daphnia were also fed GEMMA Micro 300 fish food suspension.

Analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.047 to 0.63 mg/L as test material (39 to 112 % of nominal), equivalent to 0.021 to 0.27 mg/L as neodymium. A decline in measured test concentration of the aged test preparations on Days 1, 7, 14 and 21 was observed to between less than the Limit of Quantification (LOQ), which was determined to be 0.020 mg/L to 0.19 mg/L as test material (5.6 to 40 % of nominal), equivalent to less than the LOQ, which was determined to be 0.010 mg/L to 0.084 mg/L as neodymium. Given the decline in test concentrations in the aged test preparations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.028, 0.045, 0.070, 0.13 and 0.31 mg/L as test material (equivalent to 0.013, 0.020, 0.032, 0.058 and 0.13 mg/L as neodymium.

Under the conditions of the study, exposure of Daphnia magna to the test material gave the following results based on the geometric mean measured test concentrations:

The EC10 and EC50 based on immobilisation were both > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for immobilisation was ≥0.13 mg/L (as Nd) and ≥0.31 mg/L (as test material) based on immobilisation.

The LOEC for immobilisation could not be determined for immobilisation.

The EC10 based on reproduction was > 0.13 mg/L (as Nd) with 95 % confidence limits of 0.037 – 0.17 mg/L Nd, and > 0.31 mg/L (as test material) with 95 % confidence limits of 0.081 – 0.41 mg/L test.

The EC50 for reproduction was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for reproduction was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).

The LOEC for reproduction was 0.13 mg/L (as Nd) and 0.31 mg/L (as test material).

For body length, the EC10 was > 0.078 mg/L (as Nd) with 95 % confidence limits of 0.041 – 0.15 mg/L Nd, and > 0.18 mg/L (as test material) with 95 % confidence limits of 0.091 – 0.36 mg/L test material.

The EC50 for body length was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for body length was 0.032 mg/L (as Nd) and 0.07 mg/L (as test material).

The LOEC for body length was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).

Description of key information

Under the conditions of the study, exposure of Daphnia magna to the test material gave the following results based on the geometric mean measured test concentrations:

The EC10 and EC50 based on immobilisation were both > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for immobilisation was ≥0.13 mg/L (as Nd) and ≥0.31 mg/L (as test material) based on immobilisation.

The LOEC for immobilisation could not be determined for immobilisation.

The EC10 based on reproduction was > 0.13 mg/L (as Nd) with 95 % confidence limits of 0.037 – 0.17 mg/L Nd, and > 0.31 mg/L (as test material) with 95 % confidence limits of 0.081 – 0.41 mg/L test.

The EC50 for reproduction was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for reproduction was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).

The LOEC for reproduction was 0.13 mg/L (as Nd) and 0.31 mg/L (as test material).

For body length, the EC10 was > 0.078 mg/L (as Nd) with 95 % confidence limits of 0.041 – 0.15 mg/L Nd, and > 0.18 mg/L (as test material) with 95 % confidence limits of 0.091 – 0.36 mg/L test material.

The EC50 for body length was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for body length was 0.032 mg/L (as Nd) and 0.07 mg/L (as test material).

The LOEC for body length was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

Reproduction

Effect concentration:

0.18 mg/L

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

body length

Effect concentration:

0.07 mg/L

Additional information

A study was performed to assess the chronic toxicity of the test item to Daphnia magna according to OECD Test Guideline 211 and EU Method C.20, and in compliance with GLP.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test material at nominal concentrations of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L for a period of 21 days. Test solutions were renewed daily. In order to maximize the dissolved concentrations of the test material, daphnids were exposed in test media adjusted to pH 6.0. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension. On Days 0 to 3 the Daphnia were also fed GEMMA Micro 300 fish food suspension.

Analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.047 to 0.63 mg/L as test material (39 to 112 % of nominal), equivalent to 0.021 to 0.27 mg/L as neodymium. A decline in measured test concentration of the aged test preparations on Days 1, 7, 14 and 21 was observed to between less than the Limit of Quantification (LOQ), which was determined to be 0.020 mg/L to 0.19 mg/L as test material (5.6 to 40 % of nominal), equivalent to less than the LOQ, which was determined to be 0.010 mg/L to 0.084 mg/L as neodymium. Given the decline in test concentrations in the aged test preparations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.028, 0.045, 0.070, 0.13 and 0.31 mg/L as test material (equivalent to 0.013, 0.020, 0.032, 0.058 and 0.13 mg/L as neodymium.

Under the conditions of the study, exposure of Daphnia magna to the test material gave the following results based on the geometric mean measured test concentrations:

The EC10 and EC50 based on immobilisation were both > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for immobilisation was ≥0.13 mg/L (as Nd) and ≥0.31 mg/L (as test material) based on immobilisation.

The LOEC for immobilisation could not be determined for immobilisation.

The EC10 based on reproduction was > 0.13 mg/L (as Nd) with 95 % confidence limits of 0.037 – 0.17 mg/L Nd, and > 0.31 mg/L (as test material) with 95 % confidence limits of 0.081 – 0.41 mg/L test.

The EC50 for reproduction was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for reproduction was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).

The LOEC for reproduction was 0.13 mg/L (as Nd) and 0.31 mg/L (as test material).

For body length, the EC10 was > 0.078 mg/L (as Nd) with 95 % confidence limits of 0.041 – 0.15 mg/L Nd, and > 0.18 mg/L (as test material) with 95 % confidence limits of 0.091 – 0.36 mg/L test material.

The EC50 for body length was > 0.13 mg/L (as Nd) and > 0.31 mg/L (as test material).

The NOEC for body length was 0.032 mg/L (as Nd) and 0.07 mg/L (as test material).

The LOEC for body length was 0.058 mg/L (as Nd) and 0.13 mg/L (as test material).