Neodymium oxide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 2020 to 02 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Remarks:

As it was not a requirement of the Test Guideline, no analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

RANGE-FINDING TEST
- Test Material Preparation
The test material was supplied as an aqueous solution containing 55.08 % anhydrous test material and therefore the test concentrations were corrected for this.
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1 000 mg/L test material (single replicates of the 10 and 100 mg/L test material and three replicates of the 1 000 mg/L test material concentrations). The test material was dissolved directly in water.
A nominal amount of test material (4 539 mg as supplied) was dissolved in water with the aid of ultrasonication for 10 minutes, and the volume was adjusted to 1 L to give a 2 500 mg/L test material stock solution from which dilutions were made to give 250 and 25 mg/L test material stock solutions. An aliquot (200 mL) of each of the 2 500, 250 and 25 mg/L test material stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 1 000, 100 and 10 mg/L test material. The 1 000 mg/L concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.

Reference Item Preparation
- A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L (one replicate per concentration) in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for 15 minutes. The pH of this stock solution was measured to be pH 5.9 and was adjusted to pH 7.4 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of
the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.

DEFINITIVE TEST
- Test Material Preparation:
The test material was dissolved directly in water.
Nominal amounts of test material (4 539, 2 542, 1 452, 817 and 454 mg as supplied (five replicates of each)) were each dissolved in water and the volume adjusted to 1 L of deionised reverse osmosis water to give stock solutions of 2 500, 1 400, 800, 450 and 250 mg/L test material. An aliquot (200 mL) of each of the 2 500, 1 400, 800, 450 and 250 mg/L stock solutions was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentrations of 1 000, 560, 320, 180 and 100 mg/L test material. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
As it was not a requirement of the Test Guideline, no analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The control group (six replicates) was maintained under identical conditions but not exposed to the test material.

- Reference Item Preparation
For the purpose of the definitive test a reference item, 3,5-dichlorophenol was tested in parallel under identical test conditions. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for 20 minutes.
The pH of this stock solution was measured to be pH 5.5 and adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL give the final concentrations of 3.2, 10 and 32 mg/L (one replicate per concentration). The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: A mixed population of activated sewage sludge micro-organisms was obtained on 03 March 2020 for the range-finding test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK and on 30 September 2020 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection, the activated sewage sludge (10 L) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.3 measured using a calibrated Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter pape (Rinsed 3 times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed three times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19 °C - 21 °C

pH:

6.1 - 8.1

Dissolved oxygen:

5.84 - 7.97 mg O2/L

Nominal and measured concentrations:

100, 180, 320, 560 and 1 000 mg/L test material.

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Material, size, headspace, fill volume: At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum.
- Aeration: The mixture was aerated with clean, oil-free compressed air at a rate of 0.5 to 1.0 L per minute.
- No. of vessels per concentration: Five
- No. of vessels per control: One for the positive control and six for the untreated control.
- Nutrients provided for bacteria: A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate: 16 g Peptone, 11 g Meat extract, 3 g Urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4.
Each constituent was dissolved in a final volume of 1 L of water with the aid of ultrasonication.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

OTHER TEST CONDITIONS
- pH: The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range-finding and definitive tests was between pH 7.1 and 7.4. The pH values were measured using a calibrated Hach HQ40d Flexi handheld meter or a calibrated Hach HQ30d Flexi handheld.
The pH of the test material stock solutions were measured using a calibrated Hach HQ30d Flexi hand held meter and adjusted to between pH 7.0 to pH 8.0 using 1.0 M NaOH.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED:
- Observations were made on the test preparations throughout the test period. Observations of the test vessels at 0 hour were made prior to addition of activated sewage sludge.
- pH Measurements: The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a calibrated Hach HQ30d Flexi handheld.
- Oxygen Concentration: The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- Measurement of the Respiration Rates: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a calibrated Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10-minute period.
- Data evalution:
Calculation of the Oxygen Uptake Rates: The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:

R = [(Q1 - Q2) / Δt] x 60

Where:
Q1 = The oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2 = The oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt = The time interval between the beginning and end of the selected section of the linear phase (minute).
The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:

RS = R / SS

Where:
SS = The concentration of suspended solids in the test mixture (g dry weight/L).

Calculation of Percentage of Inhibition: The percentage inhibition was calculated according to the following equation:

Percentage inhibition = [1 – (R/Rbc)] x 100

Where:
Rbc = The mean respiration rate of the blank controls.


- Range finding study
- Test concentrations: 1 000, 100 and 10 mg/L test material
- Results used to determine the conditions for the definitive study: Yes. The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L.
Statistically significant toxic effects were shown at the test concentration of 1 000 mg/L test material, equivalent to 440 mg/L neodymium. In this test group, the respiration rate was inhibited by 71 % (mean of the three replicates, standard deviation 1.53) as compared to the control group.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain both a NOEC and ECX/EC50 values for the test material. Test concentrations of 100, 180, 320, 560 and 1 000 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

863 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: 95 % confidence limits: 820 - 914 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

377 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Nd

Basis for effect:

growth inhibition

Remarks on result:

other: 95 % confidence limits: 358 - 399 mg/L Nd

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

140 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Nd

Basis for effect:

growth inhibition

Details on results:

Inhibition of Respiration Rate
No statistically significant toxic effects were observed at the test concentrations of 100, 180 and 320 mg/L test material, equivalent to 44, 79 and 140 mg/L neodymium respectively; however statistically significant toxic effects (P < 0.05) were observed at the test concentration of 560 and 1 000 mg/L test material, equivalent to 245 and 437mg/L neodymium. In these test groups, the respiration rates were inhibited by 27 % (mean of the five replicates, standard deviation 3.54) and 58 % (mean of four replicates, standard deviation 4.35, due to the pH of the test preparation of 1 000 mg/L test material replicate R1 at 0 hours and 3 hours were outside the range of 7.0 to 8.0 at the beginning and end of the exposure period. Therefore this replicate was therefore not included in the statistical calculations of the ECx and NOEC, respectively, as compared to the control group. Based on these results the NOEC after 3 hours exposure was 320 mg/L test material, equivalent to 140 mg/L neodymium.
In some instances, the initial and final dissolved oxygen concentrations of the linear section selected for calculation were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). These were mostly only slightly outside of the recommended range and are considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L.

Reported statistics and error estimates:

All statistical analyses were conducted using the ToxRat Professional computer software package (TOXRAT).
For the test material ECx values were calculated by Probit analysis using linear maximum likelihood regression and the NOEC was assessed using Shapiro-Wilk’s test on normal distribution, Levene’s test on variance homogeneity and Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm.

For the reference item ECx values were calculated by 3-parameter normal cumulative distribution function.

Validation Criteria

The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 7.8 mg/L and therefore within the acceptance range of 2 to 25 mg/L for total respiration.

The specific respiration rate of the controls was 28.93 mg oxygen per gram dry weight of sludge per hour and therefore greater than the threshold of 20 mg oxygen per gram dry weight of sludge per hour.

The coefficient of variation of oxygen uptake in the control vessels was 11.34 % and therefore less than the threshold of 30 %.

All validation criteria for the study were therefore satisfied.

 

 

Dissolved Oxygen Concentrations and Saturation of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Control	1	6.01	68
	2	5.92	67
	3	5.93	67
	4	6.14	699
Test material (mg/L)	10	5.60	63
	100	5.72	64
	1 000 R1	6.09	68
	1 000 R2	6.76	76
	1 000 R3	5.88	66
3,5-dichlorophenol (mg/L)	3.2	6.31	71
	10	6.52	73
	32	7.98	90

R = Replicate

 

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration		Initial O2 Reading (mg O2/L)	Measurement Period (Min)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/h)	Inhibition (%)
Control	1	4.7	4	2.1	39.00	-
	2	4.5	4	2.0	37.50	-
	3	4.6	4	2.1	37.50	-
	4	4.3	4	2.0	34.50	-
Test material (mg/L)	10	4.5	4	2.0	37.50	[1]
	100	4.6	4	2.2	36.00	3
	1 000 R1	7.2	9	5.6	10.67	71*
	1 000 R2	7.2	9	5.5	11.33	69*
	1 000 R3	6.8	7	5.6	10.29	72*
3,5-dichlorophenol (mg/L)	3.2	6.6	10	1.9	28.20	24
	10	7.1	10	4.2	17.40	53
	32	8.1	10	7.3	4.80	87**

- = Not applicable

R = Replicate

*= Statistically different compared to the control, assessed using Shapiro-Wilk’s test on normal distribution, Levene’s test on variance homogeneity, Trends analysis by contrasts and Williams Multiple sequential t-test Procedure.

**= Williams Multiple sequential t-test Procedure could not be performed on the result.

[ ] = Increase in respiration rate as compared to controls

 

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration		pH
		0 h	3 h
Control	1	7.4	7.4
	2	7.0	7.5
	3	7.4	7.6
	4	7.4	7.5
Test material (mg/L)	10	7.3	7.6
	100	7.0	7.5
	1 000 R1	7.2	7.1
	1 000 R2	7.3	7.2
	1 000 R3	7.3	7.1
3,5-dichlorophenol (mg/L)	3.2	7.1	7.7
	10	7.8	7.8
	32	7.4	7.8

 

Observations on the Test Preparations throughout the Test Period in the Range-Finding Test

Nominal Concentration		Observations on Test Preparations
		0 h*	30 Min Contact Time	3 H Contact Time
Control	1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test material (mg/L)	10	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	100	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R1	Pale yellow/brown cloudy dispersion	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R2	Pale yellow/brown cloudy dispersion	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R3	Pale yellow/brown cloudy dispersion	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
3,5-dichlorophenol (mg/L)	3.2	Pale yellow/brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Pale yellow/brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Pale yellow/brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible

*= Observations made prior to the addition of activated sewage sludge

R = Replicate

 

pH Values of the Test Item Stock Solutions prior to the Addition of Inoculum in the Definitive Test

Nominal Concentration of Test Material (mg/L)	pH
	Prior to Adjustment	After Adjustment
250	4.8	7.2
450	4.6	7.2
800	4..3	7.2
1 400	4.1	7.3
2 500	3.8	(6.1) 7.6*

Figure in brackets was the pH of the stock solution used to prepare 1 000 mg/L replicate R1, the pH of the stock solution was re-adjusted before preparing replicates R2 to R5.

 

 

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test

Nominal Concentration		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Control	1	6.73	76
	2	5.96	67
	3	6.75	76
	4	5.88	66
	5	6.23	70
	6	6.18	69
Test material	100 R1	5.93	67
	100 R2	6.42	72
	100 R3	6.25	70
	100 R4	6.14	69
	100 R5	6.08	68
	180 R1	5.76	65
	180 R2	5.89	66
	180 R3	6.04	68
	180 R4	6.67	75
	180 R5	6.01	68
	320 R1	6.12	69
	320 R2	6.11	69
	320 R3	5.92	67
	320 R4	6.04	68
	320 R5	5.87	66
	560 R1	6.07	68
	560 R2	6.44	72
	560 R3	6.23	70
	560 R4	5.84	66
	560 R5	5.77	65
	1 000 R1	7.92	89
	1 000 R2	6.95	78
	1 000 R3	7.13	80
	1 000 R4	6.88	77
	1 000 R5	6.73	76
3, 5-dichlotophenol	3.2	6.59	74
	10	7.54	85
	32	7.97	90

R = Replicate

 

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal Concentration		Initial O2 Reading (mg O2/L)	Measurement Period (Min)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/h)	Inhibition (%)
Control	1	4.4	3	2.1	46.00	-
	2	4.6	3	2.3	46.0	-
	3	3.7	2	2.1	48.00	-
	4	4.0	3	1.7	46.00	-
	5	5.0	5	1.8	38.40	-
	6	5.0	5	2.0	36.00	-
Test material	100 R1	4.1	3	1.8	46.00	[6]
	100 R2	4.8	4	1.7	46.50	[7]
	100 R3	4.4	3	2.1	46.00	[6]
	100 R4	4.5	3	2.1	48.00	[11]
	100 R5	4.2	3	1.9	46.00	[6]
	180 R1	4.3	3	2.0	46.00	[6]
	180 R2	4.2	3	1.9	46.00	[6]
	180 R3	4.2	3	1.8	46.00	[11]
	180 R4	4.6	3	2.3	48.00	[6]
	180 R5	4.7	3	2.4	46.00	[6]
	320 R1	5.2	5	2.1	37.20	14
	320 R2	4.3	3	2.0	46.00	[6]
	320 R3	4.3	3	2.2	42.00	3
	320 R4	4.6	4	1.8	42.00	3
	320 R5	4.5	4	1.7	42.00	3
	560 R1	5.9	8	1.9	30.00	31*
	560 R2	4.6	5	2.0	31.20	28*
	560 R3	5.3	6	1.9	34.00	22*
	560 R4	5.5	7	1.9	30.86	29*
	560 R5	4.5		1.8	32.40	25*
	1 000 R1	7.9	10	6.7	7.20	38**
	1 000 R2	6.3	8	4.1	16.50	62*
	1 000 R3	6.1	8	3.8	17.25	60*
	1 000 R4	6.3	8	3.9	18.00	59*
	1 000 R5	4.9	6	2.8	21.00	52*
3, 5-dichlotophenol	3.2	6.1	8	2.0	30.75	29*
	10	6.7	10	3.3	20.40	53*
	32	8.0	10	7.1	5.40	88*

R = Replicate

*= Statistically different compared to the control, assessed using Shapiro-Wilk’s test on normal distribution, Levene’s test on variance homogeneity and Multiple sequentially-rejective Welsh t test after Bonferroni-Holm.

**= The values obtained for the pH of the test preparation of 1000 mg/L neodymium trinitrate replicate R1 at 0 hours and 3 hours were outside the range of 7.0 to 8.0 at the beginning and end of the exposure period. Therefore this replicate was therefore not included in the statistical calculations of the ECx and NOEC.

 

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Definitive Test

Nominal Concentration		pH
		0 H	3 H
Control	1	7.5	7.4
	2	7.5	7.5
	3	7.5	7.4
	4	7.3	7.5
	5	7.9	8.1
	6	7.8	8.1
Test material	100 R1	7.2	7.4
	100 R2	7.2	7.4
	100 R3	7.2	7.4
	100 R4	7.2	7.4
	100 R5	7.2	7.3
	180 R1	7.0	7.3
	180 R2	7.1	7.4
	180 R3	7.2	7.4
	180 R4	7.2	7.5
	180 R5	7.6	7.6
	320 R1	7.0	7.6
	320 R2	7.1	7.6
	320 R3	7.3	7.6
	320 R4	7.6	7.7
	320 R5	7.7	7.8
	560 R1	7.0	7.6
	560 R2	7.0	7.6
	560 R3	6.9	7.6
	560 R4	7.0	7.7
	560 R5	7.2	7.7
	1 000 R1	6.1*	6.8*
	1 000 R2	7.5	7.5
	1 000 R3	7.1	7.5
	1 000 R4	7.2	7.6
	1 000 R5	7.0	7.6
3, 5-dichlotophenol	3.2	7.5	7.9
	10	7.5	7.7
	32	7.5	7.7

* = The values obtained for the pH of the test preparation of 1 000 mg/L neodymium trinitrate replicate R1 at 0 hours and 3 hours were outside the range of 7.0 to 8.0 at the beginning and end of the exposure period. This may be considered to be a contributing factor to the lower oxygen consumption rates observed in this replicate (7.2 mg O2/L/h) as compared to the other four replicates (16.5 to 21 mg O2/L/h) and this replicate was therefore not included in the statistical calculations of the ECx and NOEC.

R = Replicate

 

Observations on the Test Preparations throughout the Test Period in the Definitive Test

Nominal Concentration		Observations on the Test Preparations
		0 H*	30 Min Contact Time	3 H Contact Time
Control	1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	5	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	6	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test material	100 R1	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	100 R2	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	100 R3	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	100 R4	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	100 R5	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	180 R1	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	180 R2	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	180 R3	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	180 R4	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	180 R5	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	320 R1	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	320 R2	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	320 R3	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	320 R4	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	320 R5	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	560 R1	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	560 R2	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	560 R3	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	560 R4	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	560 R5	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R1	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R2	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R3	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R4	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1 000 R5	Pale yellow/brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
3, 5-dichlotophenol	3.2	Pale yellow/brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Pale yellow/brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Pale yellow/brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible

* Observations made prior to the addition of activated sewage sludge.

R =Replicate

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 863 mg/L test material, 95 % confidence limits 820 to 914 mg/L test material (equivalent to 377 mg/L neodymium, 95 % confidence limits equivalent to 358 to 399 mg/L neodymium).
The NOEC after 3 hours exposure was 320 mg/L test material (equivalent to 140 mg/L neodymium).

Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge according to OECD Test Guideline 209 and in compliance with GLP.

The test material was supplied as an aqueous solution containing 55.08 % anhydrous test material and therefore the test concentrations were corrected for this. The concentrations are shown in the report expressed as the test material and neodymium. 

In a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 100 and 1 000 mg/L test material (single replicates of the 10 and 100 mg/L test material and three replicates of the 1 000 mg/L test material concentrations) for a period of 3 hours at a temperature of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

Following the preliminary range-finding test, at the request of the Sponsor, a test to determine the No Observed Effect Concentration (NOEC) and the 3-Hour EC50 value for the test material was performed. Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 100, 180, 320, 560 and 1 000 mg/L test material (five replicates) for a period of 3 hours at a measured temperature of between 19 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. The reference material, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 7.8 mg/L which confirmed the suitability of the activated sludge and the method used.

Under the conditions of the study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 863 mg/L test material, 95 % confidence limits 820 to 914 mg/L test material (equivalent to 377 mg/L neodymium, 95 % confidence limits equivalent to 358 to 399 mg/L neodymium).

The NOEC after 3 hours exposure was 320 mg/L test material (equivalent to 140 mg/L neodymium).

Description of key information

Under the conditions of the study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 863 mg/L test material, 95 % confidence limits 820 to 914 mg/L test material (equivalent to 377 mg/L neodymium, 95 % confidence limits equivalent to 358 to 399 mg/L neodymium).

The NOEC after 3 hours exposure was 320 mg/L test material (equivalent to 140 mg/L neodymium).

Key value for chemical safety assessment

EC50 for microorganisms:

863 mg/L

EC10 or NOEC for microorganisms:

320 mg/L

Additional information

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge according to OECD Test Guideline 209 and in compliance with GLP.

The test material was supplied as an aqueous solution containing 55.08 % anhydrous test material and therefore the test concentrations were corrected for this. The concentrations are shown in the report expressed as the test material and neodymium. 

In a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 100 and 1 000 mg/L test material (single replicates of the 10 and 100 mg/L test material and three replicates of the 1 000 mg/L test material concentrations) for a period of 3 hours at a temperature of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

Following the preliminary range-finding test, at the request of the Sponsor, a test to determine the No Observed Effect Concentration (NOEC) and the 3-Hour EC50 value for the test material was performed. Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 100, 180, 320, 560 and 1 000 mg/L test material (five replicates) for a period of 3 hours at a measured temperature of between 19 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. The reference material, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 7.8 mg/L which confirmed the suitability of the activated sludge and the method used.

Under the conditions of the study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 863 mg/L test material, 95 % confidence limits 820 to 914 mg/L test material (equivalent to 377 mg/L neodymium, 95 % confidence limits equivalent to 358 to 399 mg/L neodymium).

The NOEC after 3 hours exposure was 320 mg/L test material (equivalent to 140 mg/L neodymium).