Neodymium trinitrate

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:

From 20 February 2020 to 21 june 2021

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:

2012

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control and test group with nominal concentrations of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L.
- Sampling method: The concentration and stability of the test item 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). ie, once a week before and after test medium renewal.
- Sample storage conditions before analysis: 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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (9.0 mg) was dissolved in test water and the volume adjusted to 5 liters to give a 1.0 mg/L neodymium trinitrate 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 neodymium trinitrate. The 1.0 mg/L neodymium trinitrate 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.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Test concentration separation factor: 1.8
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea (Daphnia magna)
- Age at study initiation (mean and range, SD): less than 24 hours old (not a first brood progeny)
- Method of breeding: Parental daphnids used for the test were obtained as follows. 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. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Source: 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The water hardness was observed to be in the range 242 to 274 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.

Test temperature:

19 to 21 °C throughout the test

pH:

5.8 to 6.5 in the fresh solutions; 6.3 to 7.7 in the old solutions

Dissolved oxygen:

≥ 8.7 mg/L

Nominal and measured concentrations:

-Nominal concentations: control, 0.056, 0.10, 0.18, 0.32 and 0.56 mg Nd(NO3)3/L
-Mesured concentration on Day 0 (start of experiment): < LOQ, 0.0217, 0.0364, 0.0640, 0.0126, 0.0231 mg Nd /L (i.e. < LOQ, 0.0497, 0.0832, 0.146, 0.288, 0.530 mg Nd(NO3)3/L)
-Mesured concentration on Day 1 (end of 1st renewal period): -Mesured concentration on Day 6 (fresh water): < LOQ, 0.0217, 0.0361, 0.0653, 0.122, 0.196 mg Nd /L (i.e. -Mesured concentration on Day 7 (old water): -Mesured concentration on Day 13 (fresh water): ND, 0.0246, 0.0433, 0.0853, 0.157, 0.274 mg Nd /L (i.e ND, 0.0563, 0.0991, 0.195, 0.359, 0.628, mg Nd(NO3)3/L)
-Mesured concentration on Day 14 (old water): ND, 0.00987, 0.0122, 0.0140, 0.0243, 0.0842 mg Nd /L (i.e ND, 0.0226, 0.0279, 0.0320, 0.0556, 0.193 mg Nd(NO3)3/L)
-Mesured concentration on Day 20 (fresh water): ND, 0.0205, 0.0355, 0.0724, 0.134, 0.243, mg Nd /L (i.e ND, 0.0470, 0.0813, 0.166, 0.307, 0.557 mg Nd(NO3)3/L)
-Mesured concentration on Day 21 (old water): ND, 0.00962, 0.0157, 0.0207, 0.0321, 0.0844 mg Nd /L (i.e ND, 0.0220, 0.0360, 0.0475, 0.0734, 0.193 mg Nd(NO3)3/L)
-Geometric Mean Measured Test Concentrations: control, 0.028, 0.045, 0.070, 0.13 and 0.31 mg Nd(NO3)3/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers filled with 100 mL of test medium
- Type (delete if not applicable): covered with a plastic lid
- Aeration: The test water was aerated prior to the start of the study. No aeration during the study.
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates):10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted ISO Medium (CaCl2.2H2O: 294 mg/L, MgSO4.7H2O: 123 mg/L, NaHCO3: 65 mg/L, KCl: 5.8 mg/L). The reconstituted water had a pH of 6.0 and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The pH of the medium was adjusted to 6.0 in order to aide solubilization and stability of the test item. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Photoperiod: 6 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 713 to 995 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-On a daily basis:
-numbers of live and dead of the "Parental" (P1) generation,
-numbers of live and dead "Filial" (F1) Daphnia,
-numbers of discarded unhatched eggs,
-general condition and size of the parental daphnia as compared with the controls.
-At the end of the test: length of each surviving parent animal.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 0.010, 0.10, 1.0 and 10 mg Nd(NO3)3/L (nominal)
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentrations of 0.010 and 0.10 mg Nd(NO3)3/L, however, 80 and 100% immobilization 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 coloration, reduced mobility, covered in algae and smaller in size. Test concentrations for the definitive study were selected on this basis.

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

95% CI:

0.081 - 0.41

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

neodymium trinitrate

Basis for effect:

reproduction

Remarks on result:

other: i.e.: 0.072 mg/L (CI95: 0.037 – 0.17) as neodymium element

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

neodymium trinitrate

Basis for effect:

reproduction

Remarks on result:

other: i.e.: 0.058 mg/L as neodymium element

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

neodymium trinitrate

Basis for effect:

immobilisation

Remarks on result:

other: i.e.: >0.13 mg/L as neodymium element

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

neodymium trinitrate

Basis for effect:

immobilisation

Remarks on result:

other: i.e.: ≥0.13 mg/L as neodymium element

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

95% CI:

0.091 - 0.36

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

neodymium trinitrate

Basis for effect:

other: Body Length

Remarks on result:

other: i.e.: 0.078 mg/L (CI95: 0.041 – 0.15mg/L) as neodymium element

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

neodymium trinitrate

Basis for effect:

other: Body Length

Remarks on result:

other: i.e.: 0.032 mg/L as neodymium element

Details on results:

- Behavioural abnormalities: no data available
- Observations on body length and weight: see results
- Other biological observations: no other data available
- Mortality of control: 10%
- Other adverse effects control: no
- Immobilisation of control: see mortality
- Abnormal responses: no data available
- Any observations that might cause a difference between measured and nominal values:
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 Nd(NO3)3 (39% to 112% of nominal). 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 Nd(NO3)3 (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 Nd.
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. The geometric mean measured concentrations were calculated for the following media renewal periods: Days 0 to 1, 6 to 7, 13 to 14 and 20 to 21 and the arithmetic mean of the four values determined. Where a measured concentration of less than the LOQ of the analytical method was determined, a value of half the LOQ was substituted into the equation.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

No data available

Reported statistics and error estimates:

The 21-Day ECx (immobilization, 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.

For the determination of the LOEC and the NOEC for survival (immobilization), 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 conducted using ToxRatPro software package (V3.3.0).

Lethal Effects on the Parental Generation (P₁)

One mortality was observed in the control, 0.045, 0.070 and 0.31 mg/L mean measured neodymium trinitrate test groups on Days 21, 2, 10 and 3 respectively and two mortalities were observed in the 0.028 mg/L group 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.

The following EC10, NOEC and LOEC values (immobilization) based on the geometric mean measured test concentrations were calculated by the Probit Analysis using Linear Maximum Likelihood Regression and Fisher’s Exact Binomial Test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts at 21 days:

Endpoint	Concentration (mg/L as neodymium)	Concentration (mg/L as neodymium trinitrate)
EC10	>0.13	>0.31
No Observed Effect Concentration	≥0.13	≥0.31
Lowest Observed Effect Concentration	Not determined	Not determined

 

Sub-lethal Effects on the Parental Generation (P₁)

Sub-lethal effects were observed in the control, 0.070, 0.13 and 0.31 mg/L neodymium trinitrate test groups.  Two replicates of the control test group were observed to be pale in coloration, one was observed to be on Days 19 to 20 and the other replicate on Day 21.

In the 0.070 mg/L neodymium trinitrate test 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 coloration.  In the 0.13 mg/L neodymium trinitrate test concentration one replicate was observed to be pale in coloration on Days 19 to 21, and in the 0.31 mg/L neodymium trinitrate test concentration two replicates were observed to be pale in coloration on Days 19 to 21.  

The daphnids at the remaining test concentrations were observed to be the same size and color as the control daphnids.

After 21 days the length of each surviving adult (excluding the anal spine) was determined:

Geometric Mean Measured Test Concentration (mg/L as Nd(NO3)3)	Individual Daphnia Body 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

-           = 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

 

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 neodymium trinitrate 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 item.  In these groups, the length was reduced from 9.3% and 14.4%, respectively, as compared to the control group.

The following EC10, NOEC and LOEC values (body length) based on geometric mean measured test concentrations were calculated by 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 methods at 21 days:

Endpoint	Concentration (mg/L as neodymium)	Concentration (mg/L as neodymium trinitrate)
EC10 (95% confidence limits)	0.078 (0.041 - 0.15)	0.18 (0.091 - 0.36)
No Observed Effect Concentration	0.032	0.070
Lowest Observed Effect Concentration	0.058	0.13

 

Effects on Reproduction

Young were first produced in the control 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 neodymium trinitrate test groups and on Day 9 in the 0.028 mg/L neodymium trinitrate test groups.

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 neodymium trinitrate test group in terms of the number of live young produced per adult.  The 0.31 mg/L neodymium trinitrate test group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult (see table below). In this group, the number of live young produced per adult was indeed reduced by 14.7% as compared to the control group.

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

Geometric Mean Measured Test Concentrations (mg/L as Nd(NO3)3)	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

The following EC₁₀, NOEC and LOEC values (reproduction) based on geometric mean measured test concentrations were calculated by 3-Parameter Normal Cumulative

Distribution and Williams Multiple Sequential t-test incorporating Cochran’s test on Variance Homogeneity and Shapiro-Wilk's test on Normal Distribution methods at 21 days:

Endpoint	Concentration (mg/L as neodymium)	Concentration (mg/L as neodymium trinitrate)
EC10 (95% confidence limits)	0.079 (0.037 - 0.17)	0.18 (0.081 - 0.41)
No Observed Effect Concentration	0.058	0.13
Lowest Observed Effect Concentration	0.13	0.31

 

 

 

Validity criteria fulfilled:

yes

Remarks:

Dissolved oxygen ≥ 8.7 mg /L, No ephippia , Control group: mortality: 10%, Mean number of live young surviving adult: 178, Coefficient of variation: 8.9%, pH: 5.9 -7.7 (Variation 1.8) without effect on validity (see above).

Conclusions:

Exposure of Daphnia magna to the test item Neodymium Trinitrate during 21 days gave the following results based on the geometric mean measured test concentrations of neodymium trinitrate.
No significant effect were founded on immobilisation of parental Daphnia, NOEC ≥ 0.31 mg/L.
Neodymium Trinitrate had chronic toxic effects on Daphnia magna reproduction with an EC10 of 0.18 mg/L (CI: 0.081 – 0.41) and a NOEC of 0.13 mg/L. Chronic effects on body length were also observed at 21 days with a EC10 of 0.18 mg/L (CI: 0.091 – 0.36) and a NOEC of 0.070 mg/L.

Executive summary:

The effect of the test item Neodymium trinitrate on the survival and reproduction of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2012), referenced as Method C.20 of Commission Regulation (EC) No. 440/2008. Daphnids were exposed to control, and test chemical at nominal concentration of 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L. To these nominal concentrations corresponded the following geometric mean measured test concentrations: 0.028, 0.045, 0.070, 0.13 and 0.31 mg/L neodymium trinitrate. Test solutions were renewed daily. In order to maximize the dissolved concentrations of the test item, daphnids were exposed in test media adjusted to pH 6.0.

 

The mortality, body length and reproduction of the daphnids were compared with the corresponding parameters in the control and symptoms of toxicity were recorded.

 

The test item Neodymium trinitrate had no effect on survival of Daphnia magna after the exposure period of 21 days at the concentration of 0.31 mg/L.

However, effects on reproduction and body length have been observed. Thus, it can be concluded that neodymium trinitrate exerts chronic toxic effects on Daphnia magna reproduction with an EC10 of 0.18 mg/L (CI: 0.081 – 0.41) and a NOEC of 0.13 mg/L as neodymium trinitrate. Chronic effects on body length were also observed at 21 days with an EC10 of 0.18 mg/L (CI: 0.091 – 0.36) and a NOEC of 0.070 mg/L as neodymium trinitrate.

 

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Description of key information

Under the conditions of this study, chronic toxic effects on Daphnia magna have been observed:
- Based on reproduction: 21d NOEC = 0.13 mg/L, 21d EC10 = 0.18 mg/L.
- Based on body length: 21d NOEC = 0.07 mg/L, 21d EC10 = 0.18 mg/L.
(Concentrations expressed in mg/L neodymium trinitrate)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

Effects on reproduction and body length - 21 days

Effect concentration:

0.18 mg/L

Additional information

An experimental study performed under GLP compliance and in accordance with OECD TG 211 was flagged as a key study and assigned a Klimisch score of 1. It revealed adverse effects on daphnids reproduction and body length.