Neodymium trinitrate

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:

From 18 February 2020 to 14 May 2021

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

GLP compliance:

yes (incl. QA statement)

Remarks:

As it is not a test guideline requirement, no analysis was conducted to determine the test item formulation homogeneity/concentration/stability. This is an exception with regard to GLP that is considered not to affect the study integrity/validity.

Analytical monitoring:

no

Remarks:

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

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was supplied as an aqueous solution containing 55.08% anhydrous neodymium trinitrate and therefore the test concentrations were corrected for this. The test item was dissolved directly in water. Nominal amounts of test item (4539, 2542, 1452, 817 and 454 mg as supplied (five replicates of each)) were each dissolved in water and the volume adjusted to 1 L of deionized reverse osmosis water to give stock solutions of 2500, 1400, 800, 450 and 250 mg/L neodymium trinitrate. An aliquot (200 mL) of each of the 2500, 1400, 800, 450 and 250 mg/L neodymium trinitrate 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 1000, 560, 320, 180 and 100 mg/L neodymium trinitrate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions. The pH of the test item stock solutions were measured and adjusted to between pH 7.0 to pH 8.0 using 1.0 M NaOH.
- Controls: The control group (six replicates) was maintained under identical conditions but not exposed to the test item.
- Test concentration separation factor: 1.8
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire,UK for the range-finding test. Aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK for the definitive test.
- Method of cultivation: 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 liters) was fed synthetic sewage (500 mL).
- Initial biomass concentration: 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 paper using a Buchner funnel which was then rinsed three times with 10 mL of deionized 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

Hardness:

No data available

Test temperature:

Between 19 and 21 °C

pH:

pH values at the start and end of the exposure period:

Control T0h: between 7.3 and 7.9
Control T3h: between 7.4 and 8.1

Test preparations T0h: between 6.9 and 7.7
Test preparations T3h*: between 7.3 and 7.8
(*one replicate were not taken into account because of too low value of pH combined with lower oxygen consumption rates)

Ref. substance T0h: 7.5
Ref. substance T3h: between 7.7 and 7.9

Dissolved oxygen:

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.

Salinity:

Not concerned

Conductivity:

No data available

Nominal and measured concentrations:

Nominal concentrations: 100, 180, 320, 560 and 1000 mg/L neodymium trinitrate

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Fill volume: 500 mL (200 mL test solution + 16 mL synthetic sewage + 250 mL activated sludge + water)
- Aeration: clean, oil-free compressed air at a rate of 0.5 to 1.0 liter per minute
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria: For each liter of water - 16.0 g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O, 0.2 g MgSO4 x 7H2O, and 2.8 g K2HPO4
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item stock solutions were adjusted to between pH 7.0 to pH 8.0 using 1.0 M NaOH
- Photoperiod and Light intensity: normal laboratory lighting
- Details on termination of incubation: 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).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
At the end of the 3-hour exposure, the rate of respiration was 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.
The respiration rate R expressed in 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 in 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).
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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations and test concentrations: 1.8
- Range finding study: 10, 100 and 1000 mg/L neodymium trinitrate (nominal)
- Results used to determine the conditions for the definitive study: Statistically significant toxic effects were shown at the test concentration of 1000 mg/L neodymium trinitrate. 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.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Neodymium Trinitrate

Basis for effect:

inhibition of total respiration

Remarks on result:

other: i.e.: = 140 mg/L as neodymium element

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

373 mg/L

95% CI:

332 - 408

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Neodymium Trinitrate

Basis for effect:

inhibition of total respiration

Remarks on result:

other: i.e.: 163 mg/L as neodymium element (CI95: 145 – 178 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

863 mg/L

95% CI:

820 - 914

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Neodymium Trinitrate

Basis for effect:

inhibition of total respiration

Remarks on result:

other: i.e.: 377 mg/L as neodymium element (CI95: 358 – 399 mg/L)

Results with reference substance (positive control):

- Results with reference substance is valid.
- Relevant effect levels: 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.

Reported statistics and error estimates:

For the test item 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.
All statistical analyses were conducted using the ToxRat Professional computer software package Version 3.2.1.

Table 1             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 (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	1	4.4	3	2.1	46.00	-
	2	4.6	3	2.3	46.00	-
	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	-
Neodymium trinitrate (mg/L)	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	48.00	[11]
	180 R4	4.6	3	2.3	46.00	[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	5	1.8	32.40	25^*
	1000 R1	7.9	10	6.7	7.20	83^†
	1000 R2	6.3	8	4.1	16.50	62^*
	1000 R3	6.1	8	3.8	17.25	60^*
	1000 R4	6.3	8	3.9	18.00	59^*
	1000 R5	4.9	6	2.8	21.00	52^*
3,5-dichlorophenol (mg/L)	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

[ ] 'Inhibition' percentages that are between brackets correspond to situations in which there is actually no inhibition, but rather an increase in O2 consumption rates as compared to the mean control value.

^*  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 BonferroniHolm.

^†  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.

 

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.

Validity criteria fulfilled:

yes

Remarks:

All validation criteria for the study were satisfied (see above).

Conclusions:

Exposure of activated sludge to the test item neodymium trinitrate during 3 hours gave the following results based on nominal concentrations of neodymium trinitrate and considering inhibition of the total respiration:
-NOEC: 320 mg/L
-EC10: 373 mg/L (CI95%: 332 – 408)
-EC50 : 863 mg/L (CI95%: 820 - 914)

Executive summary:

A study was performed to assess the effect of Neodymium Trinitrate on the respiration of activated sewage sludge. The test was conducted according to the OECD Guidelines for the Testing of Chemicals (2010) No. 209.

Following a preliminary range-finding test a definitive test to determine the NOEC and the 3h-EC50 value for the test item was performed.  Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 100, 180, 320, 560 and 1000 mg/L nominal neodymium trinitrate 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 item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge micro-organisms is shown in the table below:

	Neodymium Trinitrate		Neodymium		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence  Limits (mg/L)	ECx (3 Hours) (mg/L)
EC10	373	332 - 408	163	145 – 178	-
EC50	863	820 - 914	377	358 – 399	7.8
NOEC	320	-	140	-	-

All validation criteria for the study were satisfied.

Description of key information

Under the conditions of this study, toxic effects on the respiration of activated sewage sludge have been observed:

- 3h-NOEC = 320 mg/L

- 3h-EC10 was 373 mg/L

- 3h-EC50 was 863 mg/L

 (Concentrations expressed in mg/L neodymium trinitrate)

Key value for chemical safety assessment

EC50 for microorganisms:

863 mg/L

EC10 or NOEC for microorganisms:

373 mg/L

Additional information

An experimental study performed under GLP compliance and in accordance with OECD TG 209 was flagged as a key study and assigned a Klimisch score of 1. It revealed adverse effects on the respiration of activated sewage sludge.