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REACH

Neodymium oxide

EC number: 215-214-1 | CAS number: 1313-97-9

Life Cycle description
Uses advised against

Ecotoxicological information

Long-term toxicity to fish

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Administrative data

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2020 to 08 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

GLP compliance:

yes (incl. QA statement)

Details on sampling:

Water samples were taken and filtered through a 0.45 µm cellulose acetate filter from the control and all surviving test groups from the freshly prepared bulk test preparation on Days 0, 6, 13, 20, 27 and 32 and from the corresponding old or aged media on Days 1, 7, 14, 21, 28 and 33 (cycling between replicates) for quantitative analysis. The fresh samples were stored frozen prior to analysis with the corresponding old samples which were analysed on the day of sampling. Samples were analysed for dissolved neodymium concentrations from which were calculated corresponding test material concentrations.
Duplicate samples were taken and stored frozen for further analysis if necessary

Vehicle:

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For preparation of fresh test solutions on Days 0 to 14 a nominal amount of test material (9.0 mg) was dissolved in modified ISO test media and the volume adjusted to 5 L to give a 1.0 mg/L test material stock solution from which a series of dilutions were made to give the required test concentrations of 0.0128, 0.032, 0.080, 0.20 and 0.50 mg/L test material. The 0.50 mg/L test concentration was not prepared after Day 7 as there were no surviving fish.
For the preparation of fresh test solutions on Day 15 until end of the test a nominal amount of test material (18 mg) was dissolved in modified ISO test media and the volume adjusted to 10 L to give a 1.0 mg/L neodymium stock solution from which a series of dilutions were made to give the required test concentrations of 0.0128, 0.032, 0.080 and 0.20 mg/L test material. The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The prepared concentrations were, depending upon the volume required, either inverted several times, or stirred via magnetic stirrer for a period of 5 minutes to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Lighting: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Temperature: In the 7 days preceding the start of the test, the water temperature was controlled at 25 to 26 °C.
- Dissolved oxygen content: Greater than or equal to 7.0 mg O2/L.
- Breeding stock feeding: The breeding stock fish were fed daily with ZM small granular fish food and frozen brine shrimp. The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
- Breeding tank: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 06 August 2020 and used for the definitive test.
- Health: The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.

POST-HATCH FEEDING
- Start date: From Day 7 (2 dph) until the end of the test.
- Type/source of feed: The larvae were fed freshly hatched brine shrimp nauplii.
- Amount given: 2.0 to 40 µL per fry/larvae split into two portions except for Day 7 and 32 where the larvae were only fed one portion of feed.

Test type:

semi-static

Water media type:

freshwater

Limit test:

Total exposure duration:

33 d

Hardness:

246 to 252 mg/L as CaCO3 at the start of the test and from 252 to 264 mg/L as CaCO3 at termination of the test.

Test temperature:

24 to 26 °C

pH:

5.9 to 8.3

Dissolved oxygen:

At least at 6.0 mg/L (equivalent to 73 % ASV).

Conductivity:

513 μS/cm at 20 °C

Nominal and measured concentrations:

Nominal concentrations: 0.0128, 0.032, 0.080, 0.20 and 0.50 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 the tests.
A nominal amount of test material (90.8 mg) was dispersed in 5 L of test water to give a nominal concentration of 10 mg/L anhydrous 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 5.5 to 6.2 and a sample taken for chemical analysis at 0, 24 and 72 hours. Duplicate samples were taken and stored frozen for further analysis if required. This investigation confirms that the test material was completely soluble and stable over 72 hours in pH adjusted test media to a nominal concentration of 10 mg/L as test material.
From these results it was evident that there was no significant decline in the measured concentration of test material over the 72-Hour period. At the Sponsors request a further trial was conducted to assess whether the solution was a true solution. An amount of test material (36 mg) was dissolved in 2 L of ISO media at pH 6.0 to give a 10 mg/L test material stock solution. Samples were taken for chemical analysis of neodymium after the following pre-treatments:
• Untreated; these samples were expected to reflect total neodymium concentration
• Filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded); these samples were expected to reflect dissolved neodymium concentration
An additional trial was conducted to determine whether the test material formed a true solution.
A nominal amount of test material (36.3 mg) was dispersed in 2 L of test water to give a nominal concentration of 10 mg/L anhydrous test material. The stock solution was inverted several times to ensure homogeneity and a sample taken for chemical analysis after the following pre-treatments: 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. This investigation showed no difference of measured concentrations between untreated and filtered samples and confirmed that true solutions were prepared. However, as a precautionary step samples taken for chemical analysis in the range-finding and definitive tests were filtered prior to analysis.
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 longer 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.

TEST SYSTEM
- Test vessel: 1 L glass vessels were used from Day 0 to Day 14 and from Day 15 to the end of the test 5 L glass vessels were used.
- Type: Closed. The vessels covered to reduce evaporation.
- Material, size, headspace, fill volume: Glass, 1 L and 5 L. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 6, 800 mL from Day 7 to Day 14 and 4 000 mL from Day 15 to Day 33. Glass vessel size and test preparation volume were increased during the test to account for organism growth.
- Aeration: The test vessels were aerated via narrow bore glass tubes from Day 8 onwards.
- Renewal rate of test solution: Daily test medium renewal, with the exception of Day 3 to avoid premature hatching. For the medium renewals during the egg stage, eggs were gently transferred from the test vessel to a new test vessel containing freshly prepared test medium. For the medium renewals during the larval stage, approximately 90 % of the medium were gently removed and replaced with fresh test medium.
- No. of fertilized eggs/embryos per vessel: At the start of the test, twenty eggs were placed into each replicate test vessel.
- No. of vessels per concentration: Four replicates
- No. of vessels per control: Four replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the stability and filtration trial, range-finding and definitive tests was ISO medium. This medium is suitable when metal toxicity is investigated as it does not contain chelators such as EDTA. The pH of the media was adjusted to 6.0 on order to aid solubilisation and stability of the test material.
ISO medium was composed of: 294 mg/L CaCl2·2H2O, 123 mg/L MgSO4·7H2O, 65 mg/L NaHCO3, 5.8 mg/L 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 and was stored at approximately 25 °C. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
- Intervals of water quality measurement: Dissolved oxygen concentrations, pH and water temperature were recorded before and after each test media renewal. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations. The measurements taken prior to each test media renewal, and on termination of the test, represent those of the aged or old test preparations. The pH and dissolved oxygen concentration was measured using a calibrated Hach Flexi handheld meter. The temperature was measured using a calibrated digital thermometer. The temperature was also monitored approximately every hour in a surrogate test vessel using a Testo temperature logger.
The water hardness, using methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993), was measured in the bulk test preparation at the start and in each surviving replicate at the end of the test.
The light intensity was recorded daily throughout the test using an ATP Instrumentation Lux meter.
- Metals: Typical Dechlorinated Water Characteristics indicated < 6 μg/L aluminium, < 35 μg/L iron, 1.5 μg/L manganese.
- Pesticides: Typical Dechlorinated Water Characteristics indicated < 0.004 μg/L.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days (28 dph).
- Light intensity: 546 to 625 Lux.

EFFECT PARAMETERS MEASURED
- Test Organism Observations:The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. Eggs which show a marked loss of translucency and change in colour (caused by coagulation and/or precipitating of protein) and embryos which show no body movement are considered to be dead. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus.
Visible abnormalities of the larvae and juvenile fish such as abnormal appearance (body form) and behaviour (e.g., uncoordinated swimming, atypical quiescence and atypical behavior) were recorded daily.
At the end of the test the total length and wet weight of each surviving fish were determined.

RANGE-FINDING STUDY
- Test concentrations: Nominal test concentrations of 0.010, 0.10, 1.0 and 10 mg/L
- The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing newly fertilised eggs to a series of nominal test concentrations of 0.010, 0.10, 1.0 and 10 mg/L test material for a period of 15 days, with daily test media renewal (apart from Day 3 in order to avoid premature hatching of eggs).
A nominal amount of test material (36 mg) was dispersed in modified ISO media and the volume adjusted to 2 L to give a 10 mg/L stock solution from which a series of dilutions were made to give required test concentrations 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.
Each of the stock solutions were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 1 L glass vessels each containing approximately 400 mL of test preparation from Day 0 to 7, increasing to approximately 800 mL from Day 8 to 15 to account for organism growth. Two replicate flasks were used for each control and test concentration.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ±1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 15 days under semi-static test conditions. The test preparations were renewed on Days 0, 1 and from Day 4 onwards the test preparations were renewed daily. For the medium renewals during the egg stage, eggs were gently transferred from the test vessel to a new test vessel containing freshly prepared media. For the medium renewals during the larval stage, approximately 90 % of the medium were gently removed and replaced with fresh test media.The control group was maintained under identical conditions but not exposed to the test material. The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily.
The fish were fed freshly hatched brine shrimp nauplii from Day 8 until the end of the test at a rate of 2.0 to 8.0 µL per fry/larvae split into 2 portions excepts on Days 8 and 14 where the larvae were only fed one portion of feed.
Samples of the bulk fresh test preparations were taken after filtration through an 0.45 µm cellulose acetate filter (initial 10 mL discarded) on Days 0 and 1 and of the equivalent expired test preparations (cycling between each replicate) taken after filtration through an 0.45 µm cellulose acetate filter (initial 10 mL discarded) on Days 1 and 4. The samples on Day 0 were analysed on the day of sampling whilst all other samples were stored frozen prior to analysis. Duplicate samples were taken on each occasion and stored frozen for further analysis if required
- Results used to determine the conditions for the definitive study: Yes.
The results showed there was no difference between the control and test concentrations of 0.010 and 0.10 mg/L test material in terms of hatching, survival and growth (wet weight), however, 100 % mortality was observed at test concentrations of 1.0 and 10 mg/L test material.
No sub-lethal effects were observed at all the test concentrations.
Based on this information test concentrations of 0.0128, 0.032, 0.080, 0.20 and 0.50 mg/L test material were selected for the definitive test.
Chemical analysis of the fresh test preparations on Days 0 and 1 showed measured test concentrations to range from 0.0087 to 9.2 mg/L as test material (78 % to 112 % of nominal), equivalent to 0.0038 to 4.0 mg/L as neodymium. Measured test concentration of the corresponding aged test preparations on Days 1 and 4 showed measured test concentrations to range from 0.0082 to 9.1 mg/L as test material (75 % to 154 %), equivalent to 0.0036 to 4.0 mg/L as neodymium indicating that the test material was stable under test conditions.

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

> 0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

> 0.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

LC50

Effect conc.:

> 0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

LC50

Effect conc.:

> 0.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

0.035 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

Post-hatch survival

Remarks on result:

other: 95 % confidence limits: 0.029 - 0.04 mg/L.

Key result

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

0.81 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post-hatch survival

Remarks on result:

other: 95 % confidence limits: 0.04 - 0.11 mg/L.

Duration:

32 d

Dose descriptor:

LC50

Effect conc.:

0.063 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

Post-hatch survival

Remarks on result:

other: 95 % confidence limits: 0.058 - 0.069 mg/L.

Duration:

32 d

Dose descriptor:

LC50

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post-hatch survival

Remarks on result:

other: 95 % confidence limits: 0.11 - 0.19.

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

Post-hatch survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.061 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post-hatch survival

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

length

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

length

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

length

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.061 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: 95 % confidence limits: 0.002 - 97 mg/L

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

weight

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Mortality/survival at embryo and larval stages:
Post-hatch Survival: Analysis of the post-hatch survival data at Day 33 (28 days post-hatch) by Weibull Analysis using Linear Maximum-Likelihood regression analysis based on the geometric mean measured test concentrations gave the following results expressed as test material:
LC10 (Post-hatch survival): 0.081 mg/L; 95% confidence limits 0.040 – 0.11 mg/L
LC20 (Post-hatch survival): 0.10 mg/L; 95% confidence limits 0.063 – 0.13 mg/L
LC50 (Post-hatch survival): 0.15 mg/L; 95% confidence limits 0.11 – 0.19 mg/L
Statistical analysis of the post-hatch survival data was carried out for the control and all test concentrations. There were no statistically significant difference between the control and 0.010, 0.024 and 0.061 mg/L test material concentrations (P≥0.05), (corresponding to 0.0046, 0.010 and 0.027 mg/L neodymium) with mean post-hatch survival rates ranging from 87 % to 93 %. However, the two other test concentrations were significantly different (P < 0.05) with mean post-hatch survival rates of 28 % and 0 % in the 0.17 and 0.49 mg/L test material concentration groups, respectively (corresponding to 0.073 and 0.22 mg/L neodymium). Therefore, the NOEC based on post-hatch survival was 0.061 mg/L test material (corresponding to 0.027 mg/L neodymium). Correspondingly the LOEC based on post-hatch survival was 0.17 mg/L test material (corresponding 0.073 mg/L neodymium).

- Days to hatch and numbers hatched: The start of egg hatching for the control and all test concentrations was observed on Day 4 of the test and completion of hatching was observed on Day 6 of the test. Analysis of the hatching data at Day 6 by Probit Analysis using Linear Maximum-Likelihood regression based on the geometric mean measured test concentrations gave the following results expressed as test material:
LC10 (Hatching): >0.49 mg/L
LC20 (Hatching): >0.49 mg/L
LC50 (Hatching): >0.49 mg/L. It was not possible to calculate 95 % confidence limits for the LC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Statistical analysis of the hatching data was carried out for the control and all test concentrations. There were no statistically significant differences (P≥0.05), between the control and all of the test groups with mean hatching rates ranging from 8 5% to 93 %. Therefore the NOEC based on the number of eggs hatching was ≥ 0.49 mg/L test material (corresponding to ≥ 0.22 mg/L neodymium). Correspondingly the LOEC based on the number of eggs hatching could not be determined

- Data for length and weight of surviving fish:
No body length or wet weight was generated at 0.49 mg/L as test material (corresponding to 0.22 mg/L neodymium) due to 100 % mortality.
Analysis of body length data at Day 33 (28 days post-hatch) by 2-Parameter Normal Cumulative Distribution Function analysis based on the geometric mean measured test concentrations gave the following results expressed as test material:
EC10 (Total Length): >0.17 mg/L
EC20 (Total Length): >0.17 mg/L
EC50 (Total Length): >0.17 mg/L. It was not possible to calculate 95 % confidence limits for the LC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Statistical analysis of the fish total length data was carried out for the control, 0.010, 0.024,0.061 and 0.17 mg/L test material groups. There were no statistically significant differences between the control, 0.010, 0.024 and 0.061 mg/L test material concentrations (P≥0.05) (corresponding to 0.0046, 0.010 and 0.027 mg/L neodymium). However, the test concentration of 0.17 mg/L test material (corresponding to 0.073 mg/L neodymium) was significantly different (P<0.05) with total length being reduced by 4.2 % as compared to control. Therefore the NOEC based on total length was 0.061 mg/L test material (corresponding to 0.027 mg/Lneodymium). Correspondingly the LOEC based on total length was 0.17 mg/L test material (corresponding to 0.073 mg/L neodymium). Analysis of the fish wet weight data at Day 33 (28 days post-hatch) by 2-Parameter Logistic Cumulative Distribution Function analysis based on the geometric mean measured test concentrations gave the following results expressed as test material:
EC10 (Wet Weight): 0.16 mg/L
EC20 (Wet Weight): >0.17 mg/L
EC50 (Wet Weight): >0.17 mg/L. It was not possible to calculate 95 % confidence limits for the EC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Statistical analysis of the fish wet weight data was carried out for the control, 0.010, 0.024, 0.061 and 0.17 mg/L test material groups. There were no statistically significant differences between the control, 0.010 and 0.024 mg/L test material concentration (P≥0.05) (corresponding to 0.0046 and 0.010 mg/L neodymium. However, the two other test concentrations of 0.061 and 0.17 mg/L test material (corresponding to 0.027 and 0.073 mg/L neodymium) were significantly different (P<0.05)with wet weight being reduced by 7.6 % and 9.9 % as compared to control, respectively. Therefore, the NOEC based on wet weight was 0.024 mg/L test material (corresponding to 0.010 mg/L neodymium). Correspondingly the LOEC based on wet weightwas 0.061 mg/L test material (corresponding to 0.027 mg/L neodymium).

- Type of and number with morphological abnormalities: The majority of the abnormal observations were observed at 0.17 and 0.49 mg/L test material (equivalent to 0.073 and 0.22 mg/L neodymium) and included the following observations; abnormal development, pale fish, under developed, lethargic and kinked and curved spine. Abnormal observations were also observed in the control, 0.010, 0.024 and 0.061 mg/L test material (equivalent 0.0046, 0.010 and 0.027 mg/L neodymium). The observations were curved spine, abnormal development, kinked spine, small fish and pale fish. Therefore, these observations are considered to be due to defects occurring in a natural population, and not attributable to exposure of the test material.

- Observations on test material solubility: After dosing all test concentrations were observed to be clear, colorless solutions by visual inspection.

Reported statistics and error estimates:

ECx Calculations
The LC/ECx values in terms of hatching were calculated by Probit Analysis using Linear Maximum-Likelihood regression. The LCx values and associated confidence limits and the slope of response curve and its standard error in terms of post-hatch survival were calculatedby Weibull Analysis using Linear Maximum-Likelihood regression. The ECx values in terms of the weights of the fish were calculated by 2-Parameter Logistic Cumulative Distribution Function and the length of the fish were calculated by 2-Parameter Normal Cumulative Distribution Function. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Lowest Observed Effect Concentration and No Observed Effect ConcentrationDeterminations
For the estimation of the LOEC and the NOEC values the hatching data for the control and each test group were compared using the Chi2 2x2 Table Test with Bonferroni Correctionincorporating Qualitative Trend Analysis by Contrasts and the post-hatch survival data for the control and each test group were compared using Multiple Sequentially-rejective Fisher Test after Bonferroni-Holm incorporating Tarone’s test and Qualitative Trend Analysis by Contrasts. The total length and wet weight data for the control and each surviving test group obtained on termination of the test were compared using Williams Multiple Sequential t-testincorporating Trend Analysis by Contrasts, Levene’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal Distribution. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Verification of Test Concentrations

Analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.0084 to 0.61 mg/L as test material (69 % to 121 % of nominal), equivalent 0.0039 to 0.27 mg/L as neodymium. Analysis of the old test preparations on Days 1, 7, 14, 21, 28 and 33 showed measured concentrations to range from less than the Limit of Quantification (LOQ), which was determined to be 0.0050 mg/L to 0.49 mg/L as test material (19.5 % to 99 % of nominal, equivalent to less than the LOQ, which was determined to be 0.0022 mg/L to 0.21 mg/L as neodymium It was noted that on Day 6 the 0.0128 mg/L as test material group measured approximately 330 % of nominal, however given that the Day 7 analysis of this test concentration measured concentrations within the range of the old analysis sample it can be considered that this measured concentration on Day 6 is an analytical anomaly/error. As this result did not affect the geometric mean measured test concentrations it was included within the calculation. Given the decline in measured concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.

Validity Criteria

The following validity criteria were achieved during the test and the test was considered as valid:

	Required	Achieved
Dissolved oxygen	Equal to or greater than 60 %
Water temperature:
Between test chambers	± 1.5 °C	± 1.3 °C
Between successive days	± 1.5 °C	± 1.8 °C
Control hatching success rate	> 70 %	87.5 %
Control post-hatch survival	> 75 %	87.1 %

A minor deviation was observed as the variation in water exceeded ±1.5 °C on occasion between successive days. All other validity criteria were met and this minor derivation is not though to have affect the outcome of the study

Summary of Results from the 15-Day Range-Finding Test

Nominal Concentration (mg/Test Material)		Number Eggs Hatched	Number of Fish Surviving to End of Test	Cumulative Mortalities	Average weight of fish surviving at the end of the test (mg)
Control	R₁	18	18	2	5.5
Control	R₂	20	19	1	6.3
0.010	R₁	20	19	1	5.6
0.010	R₂	20	20	0	6.2
0.10	R₁	18	18	2	5.9
0.10	R₂	19	18	2	5.2
1.0	R₁	3	0	20	-
1.0	R₂	3	0	20	-
10	R₁	0	0	20	-
10	R₂	0	0	20	-

R= Replicate

- = No average weight determined as no fish survived until end of test.

Number of Dead Eggs in the Definitive Test

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Dead Eggs (Initial Population = 20)
		Days						Total
		1	2	3	4	5	6*
Control	R₁	2	0	0	0	0	0	2
	R₂	0	0	0	0	1	0	1
	R₃	3	0	0	0	0	0	3
	R₄	2	0	0	0	2	0	4
0.010	R₁	1	0	0	1	0	0	2
	R₂	2	0	0	0	0	0	2
	R₃	0	0	0	0	1	0	1
	R₄	1	0	0	0	0	0	1
0.024	R₁	2	0	0	0	0	0	2
	R₂	0	1	0	1	1	0	3
	R₃	0	0	0	0	0	0	0
	R₄	3	0	0	0	0	0	3
0.061	R₁	1	0	1	0	0	0	2
	R₂	2	0	0	0	0	0	2
	R₃	1	0	0	1	0	0	2
	R₄	1	0	0	0	0	0	1
0.17	R₁	3	0	0	0	0	0	3
	R₂	2	0	0	0	0	0	2
	R₃	1	0	0	0	0	0	1
	R₄	1	1	0	0	0	0	2
0.49	R₁	3	0	0	0	1	0	4
	R₂	1	0	0	1	3	0	5
	R₃	0	0	0	0	0	0	0
	R₄	3	0	0	0	0	0	3

*All surviving eggs hatched by day 6

R= Replicate

Number of Dead Fish in the Definitive Test

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Dead Fish (Non-cumulative)
		Day
		1	2	3	4	5	6	7	8	9	10	11
Control	R₁	0	0	0	0	0	2	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	3	2	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.010	R₁	0	0	0	1	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	1	0	1	0	1	0
	R₃	0	0	0	0	0	1	0	0	0	0	0
	R₄	0	0	0	0	0	2	0	0	1	1	0
0.024	R₁	0	0	0	0	0	0	0	1	0	0	0
	R₂	0	0	0	0	0	0	0	2	0	1	0
	R₃	0	0	0	0	0	0	0	1	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.061	R₁	0	0	0	0	1	0	0	0	1	0	1
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	1	0	0	0	0	0
0.17	R₁	0	0	0	0	0	7	0	0	0	4	0
	R₂	0	0	0	0	0	6	0	0	0	3	0
	R₃	0	0	0	0	0	6	0	2	0	2	0
	R₄	0	0	0	0	0	7	0	3	0	0	0
0.49	R₁	0	0	0	0	0	16	AD	AD
	R₂	0	0	0	0	1	12	2
	R₃	0	0	0	1	6	13	AD
	R₄	0	0	0	0	4	13	AD

*All surviving eggs hatched by day 6

R= Replicate

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Dead Fish (Non-cumulative)
		Day
		12	13	14	15	16	17	18	19	20	21	22
Control	R₁	0	0	0	0	0	0	1	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	1	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.010	R₁	0	1	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.024	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	1	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	1	0	0	0	0	0	0	0	0	0
0.061	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.17	R₁	0	1	0	1	0	0	0	0	0	0	0
	R₂	1	3	0	1	0	0	0	0	0	0	0
	R₃	0	0	0	2	0	0	1	0	0	0	0
	R₄	0	1	1	0	0	0	0	0	0	0	0

R = Replicate

AD = All fish dead

Geometric Mean Measured Concentration (m/L Test Material)		Number of Dead Fish (Non-cumulative)
		Day
		23	24	25	26	27	28	29	30	31	32	33
Control	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.010	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.024	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	1	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.061	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	1	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.17	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0

R = Replicate

Number of Hatched (Live) Fish in the Definitive Test

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Live Fish
		Day
		1	2	3	4	5	6	7	8	9	10	11
Control	R₁	0	0	0	1	18	16	16	16	16	16	16
	R₂	0	0	0	5	17	19	19	16	14	14	14
	R₃	0	0	0	10	17	17	17	17	17	17	17
	R₄	0	0	0	6	15	16	16	16	16	16	16
0.010	R₁	0	0	0	3	17	17	17	17	17	17	17
	R₂	0	0	0	5	18	17	17	16	16	15	15
	R₃	0	0	0	5	19	18	18	18	18	18	18
	R₄	0	0	0	2	15	17	17	17	16	15	15
0.024	R₁	0	0	0	4	17	18	18	17	17	17	17
	R₂	0	0	0	5	17	17	17	15	15	14	14
	R₃	0	0	0	6	20	20	20	19	19	19	19
	R₄	0	0	0	2	12	17	17	17	17	17	17
0.061	R₁	0	0	0	0	16	17	17	17	16	16	15
	R₂	0	0	0	5	17	18	18	18	18	18	18
	R₃	0	0	0	6	18	18	18	18	18	18	18
	R₄	0	0	0	5	19	18	18	18	18	18	18
0.17	R₁	0	0	0	5	17	10	10	10	10	6	6
	R₂	0	0	0	6	17	12	12	12	12	9	9
	R₃	0	0	0	6	19	13	13	11	11	9	9
	R₄	0	0	0	10	15	11	11	8	8	8	8
0.49	R₁	0	0	0	12	16	AD	AD
	R₂	0	0	0	8	14	2
	R₃	0	0	0	12	12	AD
	R₄	0	0	0	11	12	AD

R= Replicate

AD = All fish dead

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Live Fish
		Day
		12	13	14	15	16	17	18	19	20	21	22
Control	R₁	16	16	16	16	16	16	15	15	15	15	15
	R₂	14	14	14	14	14	14	14	14	14	14	14
	R₃	17	17	16	16	16	16	16	16	16	16	16
	R₄	16	16	16	16	16	16	16	16	16	16	16
0.010	R₁	17	16	16	16	16	16	16	16	16	16	16
	R₂	15	15	15	15	15	15	15	15	15	15	15
	R₃	18	18	18	18	18	18	18	18	18	18	18
	R₄	15	15	15	15	15	15	15	15	15	15	15
0.024	R₁	17	17	17	17	17	17	17	17	17	17	17
	R₂	14	13	13	13	13	13	13	13	13	13	13
	R₃	19	19	19	19	19	19	19	19	19	19	19
	R₄	17	16	16	16	16	16	16	16	16	16	16
0.061	R₁	15	15	15	15	15	15	15	15	15	15	15
	R₂	18	18	18	18	18	18	18	18	18	18	18
	R₃	18	18	18	18	18	18	18	18	18	18	18
	R₄	18	18	18	18	18	18	18	18	18	18	18
0.17	R₁	6	5	5	4	4	4	4	4	4	4	4
	R₂	8	5	5	4	4	4	4	4	4	4	4
	R₃	9	9	9	7	7	7	6	6	6	6	6
	R₄	8	7	6	6	6	6	6	6	6	6	6

R= Replicate

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Live Fish
		Day
		23	24	25	26	27	28	29	30	31	32	33
Control	R₁	15	15	15	15	15	15	15	15	15	15	15
	R₂	14	14	14	14	14	14	14	14	14	14	14
	R₃	16	16	16	16	16	16	16	16	16	16	16
	R₄	16	16	16	16	16	16	16	16	16	16	16
0.010	R₁	16	16	16	16	16	16	16	16	16	16	16
	R₂	15	15	15	15	15	15	15	15	15	15	15
	R₃	18	18	18	18	18	18	18	18	18	18	18
	R₄	15	15	15	15	15	15	15	15	15	15	15
0.024	R₁	17	17	17	17	17	17	17	17	17	17	17
	R₂	13	13	13	13	13	13	13	13	13	13	13
	R₃	18	18	18	18	18	18	18	18	18	18	18
	R₄	16	16	16	16	16	16	16	16	16	16	16
0.061	R₁	15	15	15	15	15	15	15	15	15	15	15
	R₂	18	18	18	18	18	18	18	18	18	18	18
	R₃	18	18	18	18	18	18	18	18	17	17	17
	R₄	18	18	18	18	18	18	18	18	18	18	18
0.17	R₁	4	4	4	4	4	4	4	4	4	4	4
	R₂	4	4	4	4	4	4	4	4	4	4	4
	R₃	6	6	6	6	6	6	6	6	6	6	6
	R₄	6	6	6	6	6	6	6	6	6	6	6

R= Replicate

Hatching and Post Hatch Survival in the Definitive Test

Geometric Mean Measured Concentration (mg/Test Material)		Hatching Rate (%)	Mean Hatching Rate (%)	Survival Rate (%)	Mean Survival Rate (%)
Control	R1	90	88	83	88
	R2	95		74
	R3	85		94
	R4	80		100
0.010	R1	90	93	89	87
	R2	90		83
	R3	95		95
	R4	95		79
0.024	R1	90	90	94	89
	R2	85		76
	R3	100		90
	R4	85		94
0.061	R1	90	91	83	93
	R2	90		100
	R3	90		94
	R4	95		95
0.17	R1	85	90	24	28*
	R2	90		22
	R3	95		32
	R4	90		33
0.49	R1	80	85	0	0*
	R2	75		0
	R3	100		0
	R4	85		0

*Statistically significant from the control.

R = Replicate

Fish	Control
	R₁		R₂		R₃		R₄
	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)
1	18.91	58.1	20.28	73.0	20.37	71.4	21.27	81.7
2	20.03	69.2	22.23	91.7	20.73	78.5	20.50	65.9
3	22.25	97.8	20.82	80.0	20.33	67.7	19.28	54.1
4	19.76	60.4	20.92	91.7	21.03	80.3	21.83	90.0
5	20.58	74.7	20.48	70.6	21.05	80.7	21.64	77.8
6	22.03	99.8	20.67	79.0	21.27	83.3	20.73	74.5
7	20.43	73.9	21.31	79.2	19.93	64.1	20.88	79.8
8	21.08	83.7	19.55	61.6	19.59	57.0	20.78	80.1
9	20.01	72.8	20.79	70.8	19.94	70.6	20.19	63.3
10	20.99	81.4	20.98	81.4	21.55	87.1	20.40	69.8
11	20.58	64.1	20.44	79.9	20.75	69.9	20.22	68.7
12	20.61	79.5	22.12	94.7	21.11	74.5	19.10	52.4
13	19.38	49.9	19.68	60.0	19.55	64.3	21.14	73.0
14	22.87	101.8	20.62	65.0	20.85	75.0	21.45	76.9
15	21.10	77.6			19.57	54.4	21.80	84.6
16					19.53	64.5	20.62	77.3
17
18
19
20
Average	20.71	76.3	20.78	77.0	20.45	71.5	20.74	73.1
SD	1.07	15.26	0.76	10.94	0.69	9.28	0.80	10.36

Group Mean Total Length:	20.67 mm
Group Mean Wet Weight:	74.5 mg

Fish	0.010 mg/L
	R₁		R₂		R₃		R₄
	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)
1	21.09	88.1	22.23	72.6	21.41	82.6	21.35	81.8
2	20.52	68.4	21.19	83.8	20.99	67.7	20.56	68.1
3	18.62	53.5	21.72	89.3	21.54	79.8	21.63	90.4
4	20.71	72.5	19.66	58.1	19.91	61.4	21.48	86.6
5	21.79	81.2	21.55	88.3	20.96	76.1	21.67	86.2
6	21.82	83.2	21.22	84.1	20.84	84.9	21.59	79.7
7	18.41	48.9	20.86	70.4	21.17	76.0	21.24	80.6
8	20.08	64.1	19.93	73.6	21.15	77.1	19.82	66.1
9	19.83	63.7	21.92	80.4	20.69	72.2	21.63	73.5
10	18.89	53.4	22.16	98.3	18.67	51.4	23.09	110.7
11	21.72	84.5	21.69	87.2	18.72	58.1	20.26	72.9
12	22.48	93.9	20.14	67.4	20.43	69.1	18.94	61.5
13	19.96	62.0	20.18	78.9	20.45	80.6	21.53	90.1
14	21.23	72.8	19.72	68.6	20.83	76.3	21.49	86.9
15	21.20	79.9	21.40	79.1	21.70	96.7	20.12	72.5
16	21.09	71.9			19.37	59.5
17					20.68	79.7
18					21.31	78.8
19
20
Average	20.59	71.4	21.04	78.7	20.60	73.8	21.09	80.5
SD	1.21	13.24	0.90	10.37	0.89	11.02	1.00	12.27

Group Mean Total Length:	20.83 mm
Group Mean Wet Weight:	76.2 mg

Fish	0.024 mg/L
	R₁		R₂		R₃		R₄
	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)
1	19.54	60.4	21.61	78.0	20.80	79.0	20.29	75.7
2	22.72	101.2	21.49	72.1	21.64	85.6	17.89	50.3
3	20.59	72.4	20.28	80.7	21.68	94.0	21.71	89.6
4	20.90	79.0	21.04	79.8	19.57	64.6	22.28	95.6
5	19.24	54.2	21.75	99.5	21.33	80.3	19.03	50.8
6	19.42	72.2	22.50	71.4	21.31	78.8	20.61	76.3
7	18.76	58.4	20.30	83.3	20.67	78.1	20.78	78.3
8	19.72	74.8	21.00	95.2	22.92	113.2	20.12	66.3
9	19.26	71.3	22.95	95.5	20.46	76.9	21.09	85.7
10	22.08	93.8	22.00	61.4	19.77	62.4	22.31	94.4
11	21.02	79.2	20.30	69.7	19.96	66.5	21.31	84.2
12	21.74	84.6	20.38	69.8	20.13	69.6	19.85	64.7
13	20.47	70.3	20.47	78.9	20.83	69.4	23.79	121.6
14	19.34	55.6			21.13	77.6	20.62	72.3
15	21.80	91.0			20.75	74.8	19.36	62.2
16	19.50	59.7			19.32	60.3	19.59	59.8
17	20.08	69.6			21.29	83.1
18					19.94	76.0
19
20
Average	20.36	73.4	21.24	79.6	20.75	77.2	20.66	76.7
SD	1.18	13.67	0.90	11.40	0.89	12.38	1.44	18.49

Group Mean Total Length:	20.75 mm
Group Mean Wet Weight:	76.7 mg

Fish	0.061 mg/L
	R₁		R₂		R₃		R₄
	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)
1	20.86	80.6	20.80	73.6	19.33	55.5	16.87	40.1
2	20.64	76.1	19.85	65.7	20.38	64.7	20.20	68.0
3	20.59	66.9	21.13	74.9	21.19	70.8	21.06	75.1
4	22.47	93.1	21.77	90.6	19.03	49.8	21.02	81.2
5	20.42	75.8	20.91	82.5	19.79	60.0	21.10	76.4
6	20.21	69.9	20.06	69.3	20.50	59.9	20.14	77.1
7	18.33	53.5	19.20	54.9	20.93	67.0	17.07	39.2
8	19.97	75.0	19.13	58.1	19.02	55.0	20.16	67.0
9	21.26	74.4	21.19	78.8	21.10	83.5	19.15	62.4
10	21.72	82.2	19.78	61.3	19.67	54.1	20.92	81.7
11	21.94	85.7	21.89	88.5	19.03	54.1	22.26	98.1
12	21.86	89.1	22.50	93.8	18.54	43.7	20.22	66.4
13	18.80	54.6	19.67	59.0	20.43	67.1	20.23	69.4
14	22.32	96.4	17.19	41.1	22.49	86.2	21.38	77.2
15	18.45	46.6	20.21	71.8	21.86	76.5	18.91	55.9
16			20.99	81.8	21.09	66.3	17.88	55.7
17			19.80	62.5	20.38	65.9	20.82	66.0
18			19.39	54.3			21.10	47.7
19
20
Average	20.66	74.7	20.30	70.1	20.28	63.5	20.03	66.9
SD	1.34	14.54	1.25	14.29	1.10	11.44	1.50	15.13

Group Mean Total Length:	20.32 mm
Group Mean Wet Weight:	68.8 mg

Fish	0.17 mg/L
	R₁		R₂		R₃		R₄
	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)	Total Length (mm)	Wet Weight (mg)
1	19.09	60.1	20.55	75.7	19.92	70.0	20.26	70.6
2	20.38	79.2	20.81	82.3	18.36	52.0	20.45	66.8
3	16.47	40.4	20.33	72.9	16.39	37.1	18.93	55.1
4	21.78	85.9	19.65	57.0	19.70	62.9	20.61	74.8
5					20.59	75.9	21.26	79.0
6					20.84	81.0	19.48	56.1
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Average	19.43	66.4	20.34	72.0	19.30	63.2	20.17	67.1
SD	2.26	20.49	0.50	10.73	1.67	16.32	0.83	9.78

Group Mean Total Length:	19.81 mm
Group Mean Wet Weight:	67.2 mg

Number of Larvae with Abnormal Observations in the Definitive Test

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Larvae with Abnormal Observations
		Day
		1	2	3	4	5	6	7	8	9	10	11
Control	R₁	0	0	0	0	1 CS	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	1 KS
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.010	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	1 AD	0	0	0	0	0	0
	R₃	0	0	0	0	1 AD	0	0	0	0	0	0
	R₄	0	0	0	0	0	1 AD	1 AD	1 CS	0	0	0
0.024	R₁	0	0	0	0	1 AD	1 AD	1 AD	0	0	0	0
	R₂	0	0	0	0	1 AD	1 AD	1 AD	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.061	R₁	0[2]	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	1 AD	0	0	0	0	0	0
0.17	R₁	0	0	0	0	5 P	2 AD	2 AD	2 CS	2 CS	0	1 L
	R₂	0	0	0	0	3 P 1 AD	3 AD	3 AD	3 CS	3 CS	0	2 L 1 KS
	R₃	0	0	0	0	4 P	3 AD	3 AD	0	0	0	0
	R₄	0	0	0	0	2 P 1 AD	0	0	0	0	0	2 KS
0.49	R₁	0^*	0	0	0	14 P	0	All Dead
	R₂	0	0	0	0	11 P	2 AD
	R₃	0	0	0	0	5 P + U	0
	R₄	0	0	0	0	10 P + U	0

R= Replicate

CS = Curved Spine

1= Fine layer of clean particles stuck to the eggs. Eggs otherwise look healthy.

AD = Abnormal Development

P = Pale

U = Underdeveloped

L = Lethargic

KS = Kinked Spine

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Larvae with Abnormal Observations
		Day
		12	13	14	15	16	17	18	19	20	21	22
Control	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	1 KS + P	1 KS + P	1 KS + P	1 KS + P	1 KS + P	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.010	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.024	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	1 CS	1 CS	1 S	1 S	1 S
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.061	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.17	R₁	6 L	0	0	0	0	0	4 L	4 L	0	0	0
	R₂	1 KS 3 L	0	0	0	0	0	4 L	4 L	0	0	0
	R₃	4 L	4 L	0	0	0	0	6 L	6 L	0	0	0
	R₄	2 KS	1 KS	0	0	0	0	6 L	6 L	0	0	0

R= Replicate

CS = Curved Spine

1= Fine layer of clean particles stuck to the eggs. Eggs otherwise look healthy.

AD = Abnormal Development

P = Pale

U = Underdeveloped

L = Lethargic

KS = Kinked Spine

S = Small

Geometric Mean Measured Concentration (mg/L Test Material)		Number of Larvae with Abnormal Observations
		Day
		23	24	25	26	27	28	29	30	31	32	33
Control	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.010	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.024	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.061	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0
0.17	R₁	0	0	0	0	0	0	0	0	0	0	0
	R₂	0	0	0	0	0	0	0	0	0	0	0
	R₃	0	0	0	0	0	0	0	0	0	0	0
	R₄	0	0	0	0	0	0	0	0	0	0	0

Statistical Analysis: Analysis of Number of Eggs Hatching

Analysis of the number of eggs successfully hatching from the control and each test group were compared using the Probit analysis using Linear Maximum Likelihood Regression (LCx determinations) and Chi squared 2x2 Table test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts (LOEC/NOEC determinations). The total number of eggs hatched from the control and each test group were used for this analysis and are given in the table below:

Geometric Mean Measured Test Concentration (mg/L Test Material)		Eggs Hatched
Control	R1 – R4	70
0.010	R1 – R4	74
0.024	R1 – R4	72
0.061	R1 – R4	73
0.17	R1 – R4	72
0.49	R1 – R4	68

R1 – R4 = Replicates 1 to 4

No significant differences (P≥0.05) were found between the control and each test group using the above methods of statistical analysis and therefore the NOEC based on the number of eggs hatching was greater than or equal to 0.49 mg/L as test material. Percentage of eggs of which failed to hatch (arithmetic axis) were plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation. LCx values were then determined from the equation for the fitted line as follows:

Endpoint	Geometric Mean Measured Test Concentration (mg/L Test Material)
LC10	> 0.49
LC20	> 0.49
LC50	> 0.49

Analysis of Post-Hatch Survival

Analysis of the post-hatch survival from the control and each test group were compared using the Weibull analysis using Linear Maximum Likelihood Regression (LCx determinations) and Multiple Sequentially-rejective Fisher Test After Bonferroni-Holm incorporating Tarone’s test and Qualitative Trend Analysis by Contrasts (LOEC/NOEC determinations). The total post-hatch survival of the control and each test group were used for this analysis and are given in the table below:

Geometric Mean Measured Test Concentration (mg/L Test Material)		Post-Hatch Survival
Control	R1 – R4
0.010	R1 – R4
0.024	R1 – R4
0.061	R1 – R4
0.17	R1 – R4
0.49	R1 – R4

1 – R4 = Replicates 1 to 4.

There were no statistically significant differences between the control and the 0.010, 0.024 and 0.061 mg/L as test material concentrations (P≥0.05), however the 0.17 and 0.49 mg/L as test material concentrations were significantly different (P0.05), however the 0.17 and 0.49 mg/L as test material concentrations were significantly different (P

Endpoint	Geometric Mean Measured Test Concentration (mg/L Test Material)
LC10	0.081 (0.04 – 0.11)
LC20	0.10 (0.063 – 0.13)
LC50	0.15 (0.11 – 0.19)

Analysis of Total Length Data

Analysis of the total length data obtained at termination of the test from the control, 0.010, 0.024, 0.061 and 0.17 mg/L test groups were compared using 2-Parameter Normal Cumulative Distribution Function (ECx determinations) and Williams Multiple Sequential t-test incorporating Trend Analysis by Contrasts, Levene’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal Distribution (LOEC/NOEC determinations). The 0.49 mg/L test group was excluded from the length data as there were no fish that survived until the end of the test. Mean, standard deviation and percentage reduction values from the total length data of each test group were used for this analysis and are given in the table below:

Geometric Mean Measured Concentration (mg/L Test Material)		Total Length		Percentage Reduction in Total Length (%)
		Mean	Standard Deviation	Percentage Reduction in Total Length (%)
Control	R1 – R4	20.67	0.15	-
0.010	R1 – R4	20.83	0.27	-0.8
0.024	R1 – R4	20.75	0.37	-0.4
0.061	R1 – R4	20.32	0.26	1.7
0.17	R1 – R4	19.81	0.52	4.2

Negative percentage reflects an increase in length as compared to control.

There were no statistically significant differences between the control and the 0.010, 0.024 and 0.061 mg/L as test material concentrations (P≥0.05) however the 0.17 mg/L as test material concentration was significantly different (P< 0.05) and, therefore the NOEC based on total length was 0.061 mg/L as test material. Correspondingly the LOEC based on total length was 0.17 mg/L as test material. Percentage reduction in total length (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation. ECx values were then determined from the equation for the fitted line as follows:

Endpoint	Geometric Mean Measured Test Concentration (mg/L Test Material)
EC10	> 0.17
EC20	> 0.17
EC50	> 0.17

R1 – R4 = Replicates 1 to 4.

Analysis of Fish Wet Weight Data Analysis of the fish weight data obtained at termination of the test from the control, 0.010, 0.024, 0.064 and 0.17 mg/L test groups were compared using 2-Parameter Logistic Cumulative Distribution Function (ECx determinations) and Williams Multiple Sequential t-test incorporating Trend Analysis by Contrasts, Levene’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal Distribution (LOEC/NOEC determinations). The 0.49 mg/L test group was excluded from the wet weight data as there were no fish that survived until the end of the test. Mean, standard deviation and percentage reduction values from the fish wet weight data of the control and each test group were used for this analysis and are given in the table below:

Geometric Mean Measured Concentration (mg/L Test Material)		Fish Weight (mg)		Percentage Reduction in Total Length (%)
		Mean	Standard Deviation	Percentage Reduction in Total Length (%)
Control	R1 – R4	74.5	2.61	-
0.010	R1 – R4	76.2	4.31	-2.3
0.024	R1 – R4	76.7	2.55	-3.0
0.061	R1 – R4	68.8	4.77	7.6
0.17	R1 – R4	67.2	3.64	9.8

Negative percentage reflects an increase in wet weight as compared to control.

There were no statistically significant differences between the control and the 0.010 and 0.024 mg/L as test material concentrations (P≥0.05), however the 0.061, and 0.17 mg/L as test material concentrations were significantly different (P < 0.05) and, therefore the NOEC based on wet weight was 0.024 mg/L as test material. Correspondingly the LOEC based on wet weight was 0.061 mg/L as test material. Percentage reduction in wet weight (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation. ECx values were then determined from the equation for the fitted line as follows:

Endpoint	Geometric Mean Measured Test Concentration (mg/L Test Material)
EC10	0.16 (0.0002 – 97)
EC20	> 0.17
EC50	> 0.17

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, exposure of fathead minnow larvae to the test material gave the following results based on the geometric mean measured test concentrations:
The LC10 based on the number hatched was > 0.22 mg/L as Nd and > 0.49 mg/L as test material.
The LC50 based on the number hatched was > 0.22 mg/L as Nd and > 0.49 mg/L as test material.
The NOEC based on the number hatched was ≥0.22 mg/L as Nd and ≥ 0.49 mg/L as test material.
The LOEC based on the number hatched was not determined.
The LC10 based on post-hatch survival was 0.035 mg/L as Nd (95 % confidence limits of 0.029 – 0.04 mg/L) and 0.081 mg/L as test material (95 % confidence limits of 0.04 – 0.11 mg/L).
The LC50 based on post-hatch survival was 0.063 mg/L as Nd (95 % confidence limits of 0.058 – 0.069 mg/L) and 0.15 mg/L as test material (95 % confidence limits of 0.11 – 0.19 mg/L).
The NOEC based on post-hatch survival was 0.027mg/L as Nd and 0.061 mg/L as test material.
The LOEC based on post-hatch survival was 0.073mg/L as Nd and 0.17 mg/L as test material.
The EC10 based on body length was > 0.073 mg/L as Nd and > 0.17 mg/L as test material.
The EC50 based on body length was > 0.073 mg/L as Nd and > 0.17 mg/L as test material.
The NOEC based on body length was 0.027 mg/L as Nd and 0.061 mg/L as test material.
The LOEC based on body length was 0.073 mg/L as Nd and 0.17 mg/L as test material.
The EC10 based on wet weight was 0.068 mg/L as Nd and 0.16 mg/L (95 % confidence limits of 0.002 – 97) as test material.
The EC50 based on wet weight was > 0.073 mg/L as Nd and > 0.17 mg/L as test material.
The NOEC based on wet weight was 0.01 mg/L as Nd and 0.024 mg/L as test material.
The LOEC based on wet weight was 0.027 mg/L as Nd and 0.061 mg/L as test material.

Executive summary:

A study was performed to assess the effects of the test material on the early life stages of the fathead minnow (Pimephales promelas) according to OECD Test Guideline 210 and in compliance with GLP.

Based on the results of a preliminary range-finding test, newly fertilised fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to solutions of the test material at nominal concentrations of 0.0128, 0.032, 0.080, 0.20, 0.50 mg/L test material for a period of 33 days (28 days post-hatch) at a temperature of 24 to 26 ºC under semi-static test conditions. The test solutions were renewed daily throughout the test with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs. On Days 0 to 14 an initial stock solution was prepared by dispersing 9.0 mg of test material in a final volume of 5.0 L of test water to give a 1.0 mg/L test material stock solution from which a series of dilutions were made to give the required test solutions of 0.0128, 0.032, 0.080, 0.20 and 0.50 mg/L test material. The 0.50 mg/L test concentration was not prepared after Day 7 as there were no surviving fish. On Day 15 until end of test an initial stock solution was prepared by dispersing 18 mg of test material in a final volume of 10 L of test water to give a 1.0 mg/L test material stock solution from which a series of dilutions were made to give the required test solutions of 0.128, 0.032, 0.080 and 0.20 mg/test material. In order to maximize the dissolved concentrations of the test material, fish were exposed in test media adjusted to pH 6.0.

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post‑hatch). At test termination the total length and wet weight of the surviving fish were measured.

Analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.0084 to 0.61 mg/L as test material (69 % to 121 % of nominal), equivalent 0.0039 to 0.27 mg/L as neodymium. Analysis of the old test preparations on Days 1, 7, 14, 21, 28 and 33 showed measured concentrations to range from less than the Limit of Quantification (LOQ), which was determined to be 0.0050 mg/L to 0.49 mg/L as test material (19.5 % to 99 % of nominal, equivalent to less than the LOQ, which was determined to be 0.0022 mg/L to 0.21 mg/L as neodymium. Given the decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.010, 0.024, 0.061, 0.17 and 0.49 mg/L as test material (equivalent to 0.0046, 0.010, 0.027, 0.073 and 0.22 mg/L as neodymium).

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

The LC10 based on the number hatched was > 0.22 mg/L as Nd and > 0.49 mg/L as test material.

The LC50 based on the number hatched was > 0.22 mg/L as Nd and > 0.49 mg/L as test material.

The NOEC based on the number hatched was ≥ 0.22 mg/L as Nd and ≥ 0.49 mg/L as test material.

The LOEC based on the number hatched was not determined.

The LC10 based on post-hatch survival was 0.035 mg/L as Nd (95 % confidence limits of 0.029 – 0.04 mg/L) and 0.081 mg/L as test material (95 % confidence limits of 0.04 – 0.11 mg/L).

The LC50 based on post-hatch survival was 0.063 mg/L as Nd (95 % confidence limits of 0.058 – 0.069 mg/L) and 0.15 mg/L as test material (95 % confidence limits of 0.11 – 0.19 mg/L).

The NOEC based on post-hatch survival was 0.027mg/L as Nd and 0.061mg/L as test material.

The LOEC based on post-hatch survival was 0.073mg/L as Nd and 0.17mg/L as test material.

The EC10 based on body length was > 0.073 mg/L as Nd and > 0.17 mg/L as test material.

The EC50 based on body length was > 0.073 mg/L as Nd and > 0.17 mg/L as test material.

The NOEC based on body length was 0.027 mg/L as Nd and 0.061 mg/L as test material.

The LOEC based on body length was 0.073 mg/L as Nd and 0.17 mg/L as test material.

The EC10 based on wet weight was 0.068 mg/L as Nd and 0.16 mg/L (95 % confidence limits of 0.002 – 97) as test material.

The EC50 based on wet weight was > 0.073 mg/L as Nd and > 0.17 mg/L as test material.

The NOEC based on wet weight was 0.01mg/L as Nd and 0.024 mg/L as test material.

The LOEC based on wet weight was 0.027 mg/L as Nd and 0.061 mg/L as test material.

Description of key information