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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 March 2020 to 07 January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Testing was conducted on a soluble form of the substance as instructed by ECHA.
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Water samples were taken from the control and all surviving test groups from the freshly prepared bulk test preparation on Days 0, 7, 14, 21, 28 and 32 and from the corresponding aged media on Days 1, 8, 15, 22, 29 and 33 (from one replicate per concentration changing systematically amongst replicates throughout the test duration) for quantitative analysis of dissolved erbium. With the exception of the Day 1 old samples which were analyzed on the day of sampling, all samples were stored frozen prior to analysis.
- Duplicate samples were taken at each time point and stored frozen for further analysis if necessary.
- All samples were bottled for analysis following filtration through 0.45 µm cellulose acetate filters (first approximate 10 mL discarded in order to pre-condition the filter).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test material (12.6 mg erbium trinitrate pentahydrate) was dissolved in test water and the volume adjusted to 1 litre to give a 10 mg/L as erbium trinitrate stock solution. A series of dilutions was made from this stock solution to give the required test concentrations of 0.32, 0.10, 0.032, 0.010 and 0.0032 mg/L as erbium trinitrate. The 10 mg/L stock solution and its dilutions were prepared at the beginning of the definitive test and before each renewal period. 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.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnows
- Source: The test was initiated with newly fertilised eggs of fathead minnows. The adult fathead minnows that produced the eggs for the test were bred at Covance Laboratories Limited on 9 June 2020 and maintained in dechlorinated tap water with a Tropic Marine Centre 2500 recirculation system.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilised. Fertilised eggs were collected from the breeding tanks on 11 November 2020 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
other: reconstituted water (ISO medium)
Limit test:
no
Total exposure duration:
33 d
Hardness:
252 to 260 mg/L as CaCO3 at the start of the test and from 264 to 286 mg/L as CaCO3 at termination of the test
Test temperature:
23.6 to 25.9 °C
pH:
5.6 to 8.0
Dissolved oxygen:
The dissolved oxygen concentration was maintained at least at 6.8 mg/L (equivalent to 75 % ASV)
Nominal and measured concentrations:
Nominal: 0.0032, 0.010, 0.032, 0.10, and 0.32 mg/L as erbium trinitrate
Geometric mean measured test concentrations: 0.0030, 0.0076, 0.024, 0.076 and 0.25 mg/L as erbium trinitrate, 0.0015, 0.0036, 0.011, 0.037 and 0.12 mg/L as erbium, and 0.0039, 0.0094, 0.030, 0.096 and 0.31 mg/L as erbium trinitrate pentahydrate.
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 litre glass vessels were used from Day 0 to Day 15 and from Day 16 to the end of the test 5 litre glass vessels were used.
- Material, size, headspace, fill volume: 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 15 and 4000 mL from Day 16 to Day 33 to account of organism growth.
- The vessels were covered to reduce evaporation.
- Renewal rate of test solution: A semi-static test regime was employed in the test involving a daily renewal of the test preparations in an attempt to maintain near nominal concentrations and to prevent the build-up of nitrogenous waste products. There was no media renewal on Day 3 in order to prevent premature hatching of the eggs. During media renewal eggs were transferred to new test vessels containing fresh media via a wide bore glass pipette. Hatched fish were retained in the test vessel with the majority of media (90 %) being carefully poured off prior to transferring to freshly prepared media in clean vessels (days 4 to 15), or were captured in a fine sieve standing in a 10 L glass aquarium prior to transfer to fresh media in clean vessels (day 16 to 32).
- The test vessels were aerated via narrow bore glass tubes from Day 8 onwards.
- No. of fertilized eggs/embryos per vessel: 20. Based on the numbers of fry present at test termination totalled with the number of egg and fry mortalities recorded throughout, it can be considered that 21 eggs were inadvertently added to the nominal 0.032 mg/L as erbium trinitrate replicates R1 and R3 and the nominal 0.10 mg/L as erbium trinitrate replicate R2, and that 22 eggs were added to the nominal 0.10 mg/L as erbium trinitrate replicate R3
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water had a pH of 6.0 ± 0.1 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.
- Reconstituted Water – ISO Medium: CaCl2.2H2O: 294 mg/L, MgSO4.7H2O: 123 mg/L, NaHCO3: 65 mg/L and KCl: 5.8 mg/L.
- Culture medium different from test medium: no
- 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 used aged or old test preparations. The pH and dissolved oxygen concentration was measured using a calibrated digital 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 all test concentrations at the start of the test (from bulk preparations of test media) and in each surviving test vessel at the end of the test. The light intensity was recorded daily throughout the test using an ATP Instrumentation Lux meter.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, 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
- Feeding: The larvae were fed freshly hatched brine shrimp nauplii from Day 7 (two days post-hatch) to Day 32 at a rate of between 2.0 and 40 µL/fry. Feed was given as a single portion on days 7 and 32 and as two portions on all other days.

EFFECT PARAMETERS MEASURED
- 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 heartbeat, white opaque colouration 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 was determined.

RANGE-FINDING TESTS
- Preliminary Assessment of Solubility: The test material was found, by visual inspection, to be readily soluble in test media at a concentration of 100 mg/L as erbium trinitrate pentahydrate.
- The test concentrations to be used in the definitive test were determined by preliminary range finding tests. The initial range-finding test was conducted by exposing newly fertilised eggs to a series of nominal test concentrations of 0.010, 0.10 and 1.0 mg/L as erbium trinitrate for a period of 7 days.
- A nominal amount of test material (12.6 mg erbium trinitrate pentahydrate) was dissolved in test water and the volume adjusted to 1 litre to give a 10 mg/L as erbium trinitrate stock solution from which a series of dilutions was made to give required test concentrations of 1.0, 0.10 and 0.010 mg/L erbium trinitrate. The 10 mg/L test 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 test concentrations were inverted several times to ensure adequate mixing and homogeneity.
- The test was conducted in 1 litre glass vessels each containing approximately 400 mL of test preparation. Two replicate vessels were used for the control and each 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 7 days under semi static test conditions. The test preparations were renewed daily with the exception of Day 3 (to prevent premature hatching of eggs). During media renewal eggs were transferred to new test vessels containing fresh media via a wide bore glass pipette, whilst hatched fish were retained in the test vessel with the majority of media (90 %) being carefully poured off prior to transferring to freshly prepared media in clean vessels. 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.
- Samples of the fresh test preparations were taken on Day 0 and of the corresponding expired test preparations on Day 1. The Day 0 samples were stored frozen prior to analysis alongside the Day 1 samples which were analysed on the day of sampling. Duplicate samples were taken on each occasion and stored frozen for further analysis if required. All samples were bottled for analysis following filtration through 0.45 µm cellulose acetate filters (first approximate 10 mL discarded in order to pre-condition the filter).
- On Day 7 the test was terminated due to significant mortalities in all test groups, which did not allow defining the concentration range to be tested in the definitive test. A second range-finding test was therefore conducted at with a concentration range including lower concentrations: of 0.00010, 0.0010, 0.010 and 0.10 mg/L as erbium trinitrate for a period of 15 days.
- A nominal amount of test material (12.6 mg erbium trinitrate pentahydrate) was dissolved in test water and the volume adjusted to 1 liter to give a 10 mg/L as erbium trinitrate stock solution from which a dilution was made to give a further stock solution of 1.0 mg/L as erbium trinitrate. A series of dilutions was made from this stock solution to give the required test concentrations of 0.10, 0.010, 0.0010 and 0.00010 mg/L erbium trinitrate. The 10 and 1.0 mg/L stock solutions and subsequent dilutions were prepared at the beginning of the range-finding test and before each renewal period. The stock solutions and each of the prepared test concentrations were inverted several times to ensure adequate mixing and homogeneity. The test was conducted in 1 litre glass vessels each containing approximately 400 mL of test preparation from Day 0 to Day 6, and 800 mL from Day 7 to the end of the test to account of organism growth. Two replicate vessels were used for the control and each 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 daily with the exception of Day 3 (to prevent premature hatching of eggs). During media renewal eggs were transferred to new test vessels containing fresh media via a wide bore glass pipette, whilst hatched fish were retained in the test vessel with the majority of media (90%) being carefully poured off prior to transferring to freshly prepared media in clean vessels. 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 larvae were fed freshly hatched brine shrimp nauplii from Day 7 onwards at a rate of 2.0 to 8.0 µL per fry/larvae.
- Samples of the fresh test preparations were taken on Day 0 and of the corresponding expired test preparations on Day 1. All samples were stored frozen prior to analysis. Duplicate samples were taken on each occasion and stored frozen for further analysis if required. All samples were bottled for analysis following filtration through 0.45 µm cellulose acetate filters (first approximate 10 mL discarded in order to pre-condition the filter).
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate pentahydrate
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.011 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium
Basis for effect:
other: Post-hatch Survival
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.024 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate
Basis for effect:
other: Post-hatch Survival
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate pentahydrate
Basis for effect:
other: Post-hatch Survival
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.037 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.076 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.096 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate pentahydrate
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.037 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.076 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.096 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Erbium trinitrate pentahydrate
Basis for effect:
weight
Details on results:
RANGE-FINDING TESTS
-The results of the initial range-finding test showed a significant difference between the control and all test groups (0.01, 0.1 and 1 mg/L as erbium trinitrate) occurred in terms of hatching and fish survival. As such the test was terminated on Day 7.
-Chemical analysis of the test preparations taken from the initial range-finding test on Day 0 showed measured concentrations to range from 0.026 to 0.88 mg/L as erbium trinitrate (82 to 255 % of nominal), equivalent to 0.012 to 0.41 mg/L as erbium, and 0.032 to 1.1 mg/L as erbium trinitrate pentahydrate respectively. Analysis of the corresponding aged preparations on Day 1 showed measured concentrations to range from 0.0083 to 0.86 mg/L as erbium trinitrate (79 to 86 % of nominal), equivalent to 0.0040 to 0.41 mg/L as erbium, and 0.011 to 1.1 mg/L as erbium trinitrate pentahydrate respectively.
-The results of the second range-finding test showed that there was no difference between the control and test concentrations of 0.00010, 0.0010 and 0.010 mg/L as erbium trinitrate in terms of hatching, survival and growth (wet weight), however, differences were observed at the test concentration of 0.10 mg/L as erbium trinitrate.
-A number of underdeveloped fish were observed throughout the test in the control and test vessels. This was considered to be due to a natural effect rather than due to exposure to the test material as the effects were not dose related.
-Based on the results of the second range-finding test, test concentrations of 0.0032, 0.010, 0.032, 0.10, and 0.32 mg/L as erbium trinitrate were selected for the definitive test.
-Chemical analysis of the test preparations taken from the second range-finding test on Day 0 showed measured test concentrations to range from less than the LOQ to 0.082 mg/L as erbium trinitrate (
DEFINITIVE TEST
Verification of Test Concentrations
-Analysis of the freshly prepared test solutions on Days 0, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0020 to 0.31 mg/L as erbium trinitrate (64 to 621 % of nominal), equivalent to 0.00096 to 0.15 mg/L as erbium, and 0.0026 to 0.39 mg/L as erbium trinitrate pentahydrate respectively.
-Analysis of the freshly prepared 0.0032 and 0.010 mg/L test samples on Day 28 showed measured test concentrations of 0.022 and 0.016 mg/L as erbium trinitrate, equivalent to 697 and 158 % of nominal respectively. Analysis of duplicate samples showed measured test concentrations of 0.018 and 0.015 mg/L as erbium trinitrate, equivalent to 546 to 145 % of nominal respectively. Such values indicate a potential dosing error on this day. As the duplicate measurements were similar to those obtained from the initial analysis it was considered appropriate to use the mean of the two measurements for dose level calculations. The extremely high values obtained from the potential dosing error on Day 28 are not thought to have affected the outcome of the study. The high values are observed at a single time point towards the end of the study in two test concentrations, and both these concentrations are observed to be below the lowest NOEC observed in the study, (see below). These short-term spikes in concentration (as indicated in the Day 29 old analyses) were shown to cause no adverse effects in the test fish at these concentrations.
- Similarly, analysis of the freshly prepared 0.0032 and 0.010 mg/L test samples on Day 32 showed measured test concentrations of 0.0039 and 0.015 mg/L as erbium trinitrate, equivalent to 121 and 150 % of nominal respectively. Analysis of duplicate samples showed measured test concentrations of 0.0026 and 0.0086 mg/L as erbium trinitrate, equivalent to 80 and 86 % of nominal respectively. As the duplicate measurements were more in line with the expected measured concentrations it was these values that were used for dose level calculations.
- Analysis of the corresponding aged preparations on Days 1, 8, 15, 22, 29 and 33 showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0020 mg/L to 0.23 mg/L as erbium trinitrate (31 to 103 % of nominal), equivalent to less than the LOQ to 0.11 mg/L as erbium, and less than the LOQ to 0.29 mg/L as erbium trinitrate pentahydrate respectively.
-Analysis of the aged 0.0032 mg/L test samples on Day 8 showed measured test concentrations of less than the LOQ, determined to be 0.0020 mg/L as erbium trinitrate was obtained. Analysis of the duplicate sample showed a measured test concentration of 0.0033 mg/L as erbium trinitrate, equivalent to 102 % of nominal indicating that the initial value was erroneous and it was thus the value from the duplicate sample that was used for dose level calculations.
- Given the decline in measured test concentrations in the aged preparations, and also the slight variability in the measured test concentrations obtained from the freshly prepared test samples within each period of media renewal it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.0030, 0.0076, 0.024, 0.076 and 0.25 mg/L as erbium trinitrate, 0.0015, 0.0036, 0.011, 0.037 and 0.12 mg/L as erbium, and 0.0039, 0.0094, 0.030, 0.096 and 0.31 mg/L as erbium trinitrate pentahydrate respectively.

Number of Eggs Hatching
- The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed. The start of egg hatching for the control and all test concentrations was observed on Day 3 of the test and completion of hatching, with the exception of one egg in the control which observed to be dead on Day 8, was observed on Day 6 of the test. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.
- 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 test groups with the mean hatching rates ranging from 79 to 89 %. Therefore the NOEC based on the number of eggs hatching was greater than or equal to ≥0.25 mg/L as erbium trinitrate. Correspondingly the LOEC based on the number of eggs hatching could not be determined.

Post-hatch Survival
- A number of discrepancies in the numbers of live larvae present were noted throughout the duration of the test. It was considered that the number of fish present in each vessel at the end of the test which were weighed and measured was accurate. This value totaled with the number of egg and larvae mortalities was considered to be the represent the number of organisms present from Day 0. Where the total was less than 20 it was considered that mortalities occurred which were not observed, likely due to rapid decomposition. Where the total exceeded 20 it can be considered that more than 20 eggs were inadvertently added at test initiation. The number of dead larvae were observed to be low throughout the duration of the test with the exception of the 0.25 mg/L as erbium trinitrate test group where all hatched larvae were observed to die by Day 8.
- There were no statistically significant differences between the control and the 0.0030, 0.0076, 0.0024 and 0.076 mg/L as erbium trinitrate test concentrations (P≥0.05) with mean post-hatch survival rates of 99 , 96, 95, 97 and 85 % respectively. The 0.25 mg/L as erbium trinitrate test concentration was significantly different (P<0.05) with 0 % of the hatched fish surviving and therefore the NOEC based on post-hatch survival was 0.076 mg/L as erbium trinitrate. Correspondingly the LOEC based on post-hatch survival was 0.25 mg/L as erbium trinitrate.

Total Length and Weight Data
- There were no significant effects on total length or wet weight in any of the test concentrations surviving to the end of the test.
- There were no statistically significant differences (P≥0.05), between the control and all surviving test groups and therefore the NOEC based on total length was greater than or equal to ≥0.076 mg/L as erbium trinitrate. Correspondingly the LOEC based on total length could not be determined.
- There were no statistically significant differences (P≥0.05), between the control and all surviving test groups and therefore the NOEC based on wet weight was greater than or equal to ≥0.076 mg/L as erbium trinitrate. Correspondingly the LOEC based on wet weight could not be determined.

Observations
- The number of fish recorded with abnormal observations was low and did not follow a dose response. Therefore, these observations are considered most likely to be due to defects occurring in a natural population, and not necessarily attributable to exposure to the test material.

Validity Criteria
The following validity criteria were achieved during the test:
- Dissolved oxygen: ≥75 % ASV (Required ≥60 % ASV)
- Water temperature (Between test chambers): ± 1.8 °C (Required ± 1.5 °C)
- Water temperature (Between successive days): ± 1.8 °C (Required ± 1.5 °C)
- Control hatching success rate: 86 % (Required >70 %)
- Control post-hatch survival: 99 % (Required >75 %)
A minor deviation was observed as the variation in water temperature exceeded ±1.5 oC on occasion between successive days and between test chambers. All other validity criteria were met and this minor deviation was considered not have affected the outcome or integrity of the study.

Water Quality Criteria
- The temperature measurements recorded by a Testo temperature logger in a surrogate vessel placed within the same water bath as the test were shown to have been maintained at between 23.3 and 25.7 °C.
- The temperature recorded in each test vessel at the start and end of the test and before and after each media renewal ranged from 23.6 to 25.9 °C. Whilst the temperature variation both between test chambers and between successive days exceeded the required limit of ±1.5 °C, as no adverse effects on hatching, or the growth of those fish which survived to the end of the test was observed, it was considered that the variability of ±1.8 °C had no adverse effect on the outcome or integrity of the test. It should be noted that in the 0.25 mg/L as erbium trinitrate test group, prior to 100 % mortality on Day 8, the test vessel temperatures deviated by no more than ±1.3 °C and as such failure to meet the required validity criteria was not the cause for the effects seen in this group.
- The dissolved oxygen concentration was maintained at least at 6.8 mg/L (equivalent to 75% ASV) and the pH ranged from 5.6 to 8.0. Throughout the test the light intensity was observed to be in the range 612 to 722 Lux.
- The water hardness values were observed to range from 252 to 260 mg/L as CaCO3 at the start of the test and from 264 to 286 mg/L as CaCO3 at termination of the test.

Observations on Test Material Solubility
- After dosing all test concentrations were observed to be clear, colourless solutions by visual inspection throughout the duration of the test.

Summary of Results

Endpoint

Geometric Mean Measured Concentration

Erbium

(mg/L)

Erbium trinitrate

(mg/L)

Erbium trinitrate pentahydrate

(mg/L)

Number Hatched1

LC10

>0.12

>0.25

>0.31

LC20

>0.12

>0.25

>0.31

LC50

>0.12

>0.25

>0.31

NOEC

≥0.12

≥0.25

≥0.31

LOEC

Not determined

Not determined

Not determined

Post-hatch Survival2

LC10

0.010

0.022

0.028

LC20

0.018

0.037

0.046

LC50

0.048

0.10

0.13

NOEC

0.011

0.024

0.0030

LOEC

0.037

0.076

0.096

Body Length3

EC10

>0.037

>0.076

>0.096

EC20

>0.037

>0.076

>0.096

EC50

>0.037

>0.076

>0.096

NOEC

≥0.037

≥0.076

≥0.096

LOEC

Not determined

Not determined

Not determined

Wet Weight3

EC10

>0.037

>0.076

>0.096

EC20

>0.037

>0.076

>0.096

EC50

>0.037

>0.076

>0.096

NOEC

≥0.037

≥0.076

≥0.096

LOEC

Not determined

Not determined

Not determined

1 Calculated by Probit Analysis using Linear Maximum Likelihood Regression or theChi22x2 Table Test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts.

2Calculated by Probit Analysis using Linear Maximum Likelihood Regression or theStep-down Cochran Armitage Test with Tarone’s Test incorporating Qualitative Trend Analysis by Contrasts.

3Calculated by the 3-parameter Normal Cumulative Distribution Function Test incorporating Shapiro Wilk’s Test on Normal Distribution orDunnett’s Multiple Sequential t-test incorporating Trend Analysis by Contrasts and Levene’s test for Homogeneity of Variance.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study exposure of fathead minnow larvae to the test material gave the following results based on the geometric mean measured test concentrations:
Number hatched: LC50: Erbium >0.12 mg/L, Erbium trinitrate >0.25 mg/L and Erbium trinitrate pentahydrate >0.31 mg/L. NOEC: Erbium ≥0.12 mg/L, Erbium trinitrate ≥0.25 mg/L and Erbium trinitrate pentahydrate ≥0.31 mg/L.
Post-hatch survival: LC50: Erbium 0.048 mg/L, Erbium trinitrate 0.10 mg/L and Erbium trinitrate pentahydrate 0.13 mg/L. NOEC: Erbium 0.011 mg/L, Erbium trinitrate 0.024 mg/L and Erbium trinitrate pentahydrate 0.0030 mg/L.
Body length: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.
Wet weight: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.
Executive summary:

The long term toxicity of the test material to fish was investigated in accordance with the standardised guideline OECD 210, under GLP conditions in a Fish, Early-Life Stage Toxicity Test.

Based on the results of preliminary range-finding tests, newly fertilised fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to solutions of the test material at nominal concentrations of 0.0032, 0.010, 0.032, 0.10 and 0.32 mg/L as anhydrous erbium trinitrate for a period of 33 days (28 days post hatch) 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. In order to maximise the dissolved concentrations of the test material, larvae 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 freshly prepared test solutions on Days 0, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0020 to 0.31 mg/L as erbium trinitrate (64 to 621 % of nominal), equivalent to 0.00096 to 0.15 mg/L as erbium, and 0.0026 to 0.39 mg/L as erbium trinitrate pentahydrate respectively (the extremely high value of 621 % of nominal concentration is attributed to a dosing error at single timepoint for a single test concentration and is not thought to have affected the outcome of the study). Analysis of the corresponding aged preparations on Days 1, 8, 15, 22, 29 and 33 showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0020 mg/L to 0.23 mg/L as erbium trinitrate (31 to 103 % of nominal), equivalent to less than the LOQ to 0.11 mg/L as erbium, and less than the LOQ to 0.29 mg/L as erbium trinitrate pentahydrate respectively.

Given the decline in measured test concentrations in the aged preparations, and also the slight variability in the measured test concentrations obtained from the freshly prepared test samples within each period of media renewal it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.0030, 0.0076, 0.024, 0.076 and 0.25 mg/L as erbium trinitrate, 0.0015, 0.0036, 0.011, 0.037 and 0.12 mg/L as erbium, and 0.0039, 0.0094, 0.030, 0.096 and 0.31 mg/L as erbium trinitrate pentahydrate respectively.

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

Number hatched: LC50: Erbium >0.12 mg/L, Erbium trinitrate >0.25 mg/L and Erbium trinitrate pentahydrate >0.31 mg/L. NOEC: Erbium ≥0.12 mg/L, Erbium trinitrate ≥0.25 mg/L and Erbium trinitrate pentahydrate ≥0.31 mg/L.

Post-hatch survival: LC50: Erbium 0.048 mg/L, Erbium trinitrate 0.10 mg/L and Erbium trinitrate pentahydrate 0.13 mg/L. NOEC: Erbium 0.011 mg/L, Erbium trinitrate 0.024 mg/L and Erbium trinitrate pentahydrate 0.0030 mg/L.

Body length: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.

Wet weight: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.

Key results for safety assessment and classification and labelling are as follows:

Post-hatch Survival: LC 10 = 0.010 mg/L as Erbium. This equates to LC10 = 0.011 mg/L as Dierbium Trioxide.

Post-hatch Survival: No Observed Effect Concentration (NOEC) = 0.011 mg/L as Erbium. This equates to NOEC = 0.013 mg/l as Dierbium Trioxide

Description of key information

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

Number hatched: LC50: Erbium >0.12 mg/L, Erbium trinitrate >0.25 mg/L and Erbium trinitrate pentahydrate >0.31 mg/L. NOEC: Erbium ≥0.12 mg/L, Erbium trinitrate ≥0.25 mg/L and Erbium trinitrate pentahydrate ≥0.31 mg/L.

Post-hatch survival: LC50: Erbium 0.048 mg/L, Erbium trinitrate 0.10 mg/L and Erbium trinitrate pentahydrate 0.13 mg/L. NOEC: Erbium 0.011 mg/L, Erbium trinitrate 0.024 mg/L and Erbium trinitrate pentahydrate 0.0030 mg/L.

Body length: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.

Wet weight: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.

Key results for safety assessment and classification and labelling are as follows:

Post-hatch Survival: LC10 = 0.010 mg/L as Erbium. This equates to LC10 = 0.011 mg/L as Dierbium Trioxide.

Post-hatch Survival: No Observed Effect Concentration (NOEC) = 0.011 mg/L as Erbium. This equates to NOEC = 0.013 mg/l as Dierbium Trioxide

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC10
Remarks:
Converted for Dierbium Trioxide.
Effect concentration:
0.011 mg/L
Fresh water fish
Dose descriptor:
NOEC
Remarks:
Converted for Dierbium Trioxide.
Effect concentration:
0.013 mg/L

Additional information

The long term toxicity of the test material to fish was investigated in accordance with the standardised guideline OECD 210, under GLP conditions in a Fish, Early-Life Stage Toxicity Test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on the results of preliminary range-finding tests, newly fertilised fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to solutions of the test material at nominal concentrations of 0.0032, 0.010, 0.032, 0.10 and 0.32 mg/L as anhydrous erbium trinitrate for a period of 33 days (28 days post hatch) 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. In order to maximise the dissolved concentrations of the test material, larvae 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 freshly prepared test solutions on Days 0, 7, 14, 21, 28 and 32 showed measured test concentrations to range from 0.0020 to 0.31 mg/L as erbium trinitrate (64 to 621 % of nominal), equivalent to 0.00096 to 0.15 mg/L as erbium, and 0.0026 to 0.39 mg/L as erbium trinitrate pentahydrate respectively (the extremely high value of 621 % of nominal concentration is attributed to a dosing error at single timepoint for a single test concentration and is not thought to have affected the outcome of the study). Analysis of the corresponding aged preparations on Days 1, 8, 15, 22, 29 and 33 showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0020 mg/L to 0.23 mg/L as erbium trinitrate (31 to 103 % of nominal), equivalent to less than the LOQ to 0.11 mg/L as erbium, and less than the LOQ to 0.29 mg/L as erbium trinitrate pentahydrate respectively.

Given the decline in measured test concentrations in the aged preparations, and also the slight variability in the measured test concentrations obtained from the freshly prepared test samples within each period of media renewal it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.0030, 0.0076, 0.024, 0.076 and 0.25 mg/L as erbium trinitrate, 0.0015, 0.0036, 0.011, 0.037 and 0.12 mg/L as erbium, and 0.0039, 0.0094, 0.030, 0.096 and 0.31 mg/L as erbium trinitrate pentahydrate respectively.

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

Number hatched: LC50: Erbium >0.12 mg/L, Erbium trinitrate >0.25 mg/L and Erbium trinitrate pentahydrate >0.31 mg/L. NOEC: Erbium ≥0.12 mg/L, Erbium trinitrate ≥0.25 mg/L and Erbium trinitrate pentahydrate ≥0.31 mg/L.

Post-hatch survival: LC50: Erbium 0.048 mg/L, Erbium trinitrate 0.10 mg/L and Erbium trinitrate pentahydrate 0.13 mg/L. NOEC: Erbium 0.011 mg/L, Erbium trinitrate 0.024 mg/L and Erbium trinitrate pentahydrate 0.0030 mg/L.

Body length: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.

Wet weight: EC50: Erbium >0.037 mg/L, Erbium trinitrate >0.076 mg/L and Erbium trinitrate pentahydrate >0.096 mg/L. NOEC: Erbium≥0.037 mg/L, Erbium trinitrate≥0.076 mg/L and Erbium trinitrate pentahydrate≥0.096 mg/L.