Niobium

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Feb - 09 Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Principles of method if other than guideline:

Evidence of particles in the test medium, indicating that effects are linked to physical effects; Analytically measured niobium concentrations in the test medium were not reliable and are expected to i) not reflect the exposure concentrations during the test and ii) to not reflect the dissolved niobium test medium concentration. Thus effect values were based on nominal concentrations.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

ICP-MS

Details on sampling:

- Concentrations: Fresh and aged samples of control and all treatment groups
- Sampling method: One test sample and one retain sample (20 mL) sample
- Sample storage conditions before analysis: 4 ± 3 °C until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item solutions were prepared freshly 96 hour before each renewal as highly saturated test solutions according to OECD 23 (2018).
- Dilution medium: Purified tap water; regularly analysed; water hardness adjusted to 250 - 350 mg CaCO3/l by addition of CaCl2; The pH of the test media was adjusted to 8.5 prior to test item addition.
- Treatment groups > 1 mg/L: The test item was directly weighed (weights are given in the field 'Any other information on materials and methods incl. tables') and transferred to the dilution medium and stirred for 96 h at room temperature. Thereafter, the test media were filtered through a 0.45 µm PES filter to remove undissolved test item.
- Treatment groups < 1 mg/L: Test concentrations were prepared by dilution of the lowest test solution with a loading of > 1.0 mg test item/L.
- Test medium exchange: three times per week; test specimens were transferred by means of a bore pasteur pipette.
- Controls: Dilution water only
- Test concentration separation factor: 3.16
- Evidence of undissolved material: Sedimenting particles were observed by means of dynamic light scattering (DLS) in the test medium of the 1.0, 3.16 and 10 mg/L (nominal loading) treatment groups during the course of the experiment.

PRELIMINARY EXPERIMENTS - SEPARATION METHOD AND STIRRING TIME (non-GLP)
- i) 10.0 and 100.0 mg/L directly weighed into the test vessels and filled up with 1 L dilution water (adjusted to pH 8.5); stirred for 2, 24 and 96 h; sampling time: prior and after filtration through 0.45 and 0.2 µm PES; pH: 7.84 - 8.15
- ii) 10.0 mg/L in dilution medium (adjusted to pH 8.5); stirred for 96 h; 24 h settling time; visual observation of settelment of undissolved test item at 0, 2, 4, 6 and 24 h
- iii) 1 and 10.0 mg/L in dilution medium (adjusted to pH 8.5); stirred for 96 h; After stirring, fresh samples (0h) were taken before filtration and again after filtration through 0.45 µm PES filter. In order to investigate the influence of dilution of test solutions, the solution with a nominal loading rate of 10 mg/L was further diluted (1:10). The test solutions were then distributed to replicates and incubated under test conditions of a chronic toxicity test with Daphnia magna (OECD 211). Samples for analytical measurements of Nb were taken at 24 h, 48 h and 72 h from individually prepared replicates per test concentration.

REFERENCES:
- OECD Series on testing and assessment, Number 23. Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. ENV/JM/MONO(2000)6/REV1, 6-Jul-2018.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age at study initiation (mean and range, SD): neonates, 4 - 24 h old
- Method of breeding: Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water (purified well water) for one week; fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL GmbH & Co. KG); Water change: three times per week
- Source: Origin of the cladocerans was the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test were bred in the laboratory of the Fraunhofer IME for over 15 years.
- Age of parental stock: at least 3 weeks
- Feeding during test : yes
- Food type: Desmodesmus subspicatus suspension
- Amount: 0.2 mg C/Daphnia * day
- Frequency: daily

ACCLIMATION
- Acclimation period: no
- Acclimation conditions (same as test or not): yes

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
- Seperation of neonates: sieving (first generation was discarded)
- Transferation of neonates: bore pasteur pipette

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

250 - 350 mg CaCO3/L

Test temperature:

18.5 - 21.7 °C

pH:

8.16 – 9.15 (did not vary by more than 1.5 units; Since the pH was > 9.0 in aged test media only a few times in all treatments and did not vary more than 1.5 units from test start on, this is not considered to be critical.)

Dissolved oxygen:

6.93 - 12.44 mg/L (> 83.4%)

Nominal and measured concentrations:

0, 0.10, 0.316, 1.00, 3.16 and 10.0 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 -100 mL, glass beakers (acid washed (10% HNO3)), 50 - 100 mL fill volume
- Type: covered with glass panes
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 15
- No. of vessels per control (replicates): 15

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water, hardness adjusted by adition of CaCO3
- Non-purgeable organic carbon: 1.1 - 1.4 mg/L
- Metals: measured
- Pesticides: not measured
- Chlorine: 0.02 mg/L
- Alkalinity: 1.4 - 1.6 mmol/L
- Ca/mg ratio: 4.5 - 9
- Conductivity: 257 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: regularly

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: 719 – 778 lx (corresponding to 9.6 – 10.4 µE/(m² *s))
- Oxygen concentration, pH value, and temperature of the test solutions: checked directly before adding the animals and at each water renewal in new and aged test solutions
- Hardness: fresh and aged samples of the control and the highest test concentrations once per week.

EFFECT PARAMETERS MEASURED
- Mobility: daily
- Appearance and behavior: daily
- Number of newborn daphnids per beaker: New born daphnids were removed daily until all control daphnids start to reproduce. After that, newborn daphnids were counted and removed at each water renewal.
- Abnormalities in conditions (e.g. male sex of adults, presence of winter eggs): assessed
- Body length of adults (digital photography and image analysis): study termination

RANGE FINDING TEST
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations, range finder: 0, 0.1, 1.0 and 10.0 mg test item/L
- Results used to determine the conditions for the definitive study: yes

FIRST REPETITION OF MAIN TEST
- Prior to the definitive test (results reported in this robust study summary) a first repetition of the main test was performed in the same way as the definitive test (except that no particles size measurements were conducted)
- Test concentrations: 0, 0.1, 0.316, 1.00, 3.16, 10.0 mg/L (nominal)

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Sedimenting particles were observed by means of dynamic light scattering (DLS) in the test medium of the 1.0, 3.16 and 10 mg/L (nominal loading) treatment groups during the course of the experiment.
- Effect concentrations exceeding solubility of substance in test medium: Measured Nb concentrations in the test medium were found to exceed the Nb concentrations released in transformation dissolution tests according to OECD GD No. 29 with different Nb materials (≥100 mg/L loading rate, 7 d, pH 8, dissolved Nb: 2.77 - 3.5 µg/L; 1 - 10 mg/L loading rate, 7 or 28 d, pH 8: dissolved Nb concentration < LOQ of 0.01 or 0.4 µg/L; for details please refer to IUCLID section 4.8).

Reported statistics and error estimates:

For each endpoint, the NOEC, LOEC, and, if possible, the EC50 and EC10 was determined. A LOEC was calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test. The computer software ToxRat® was used for statistical evaluations. The evaluation of the concentration-effect-relationships and the calculations of effect concentrations was based on nominal concentrations.

RANGE FINDING

In the two lower loadings (0.1 and 1.0 mg test item/L (nominal)), no significant effect on the reproduction or immobility of parent daphnids was observed. In the highest loading rate (10 mg test item/L), the number of juveniles per introduced parent was significantly decreased by 78.7 % compared to the control. In addition, 50 % immobility occurred in the highest loading rate.The test fulfilled the validity criteria according to OECD 211.

Table: Measured concentrations (initial and aged) of niobium of the Range Finding Test.

			Nb concentrations
Sample	Nominal concentration [mg Nb/L]	Filtration	[µg Nb/L]	[%] of nominal	[%] of initial
0h fresh	10.0	Not filtered	113	1.13	-
0h fresh	10.0	0.45	110	1.10	-
72h aged	10.0	Not filtered	25.5	0.26	22.5
72h aged	10.0	0.45	23.0	0.23	20.9

 

Table: Survival data and percent reduction of survival compared to the control after 21 days of the Range Finding Test.

Nominal concentration [mg test item/L]	Total Introduced	Parental survival	Reduction of survival [%] (immobility)
Control	10	10	0
0.10	6	6 (-)	0 (-)
1.00	6	5 (-)	16.7 (-)
10.0	6	3 (-)	50.0 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments

 

 

Table: Reproduction data of the Range Finding Test.

Nominal Concentration	Cumulative offspring per introduced parent	Cumulative offspring per survived parent	Reduction of reproduction per introduced parent	Reduction of reproduction per survived parent
[mg test item/L]	Mean ± SD [Ind.]	Mean ± SD [Ind.]	[%]	[%]
Control	65.0 ± 16.53	65.0 ± 16.53	-	-
0.10	71.5 ± 18.54 (-)	71.5 ± 18.54 (-)	-10.0 (-)	-10.0 (-)
1.00	54.5 ± 7.82 (-)	56.6 ± 6.58 (-)	16.2 (-)	12.9 (-)
10.0	13.8 ± 17.94 (+)	25.0 ± 20.52 (+)	78.7 (+)	61.5 (+)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

DEFINITIVE TEST

No significant effects on the assessed parameters compared to control were observed for daphnids exposed to niobium for 21 d. Although the DLS measurements are not considered reliable, they indicate that particles were present in the aqueous media which had a particle size below around 200 nm.

The test item is a powder consisting of a broad range of particle sizes. Due to this inhomogeneity, it was not possible to apply homogenous and reproducible aliquots of this powder, in terms of particle size distribution, to each treatment group. It is assumed that due to this property, different amounts of particles e.g. of < 0.45 µm are available at each test item application, resulting in a huge variation of the measured test medium concentrations. Furthermore, due to the dynamic behaviour of the particles in the test medium (sedimentation/agglomeration) it is assumed that the measured concentrations do not reflect the real exposure situation in the test vessels. This was confirmed by the measured niobium concentration in the test solutions at test initiation and test termination which varied significantly between various analytical pre-tests, the range finder and a 1st main test. For example, measured niobium concentrations in the 10 mg/L (nominal, stirred for 96 h, 0.45 µm filtered) ranged from 3.88 to 110 µg/L in the preliminary, range finder and main tests.

 

Thus, based on following facts, it is concluded that the measured concentrations were not reliable and all endpoints were evaluated based on the nominal concentrations:

- Evidence of non-dissolved niobium material in the test medium demonstrates that the measured niobium concentrations do not only reflect the dissolved niobium fraction in the test medium.

- Sedimentation of particles demonstrates a dynamic exposure system, therefore it is expected that e.g. sedimented material was not recorded during sampling.

- Wide particle size distribution of the test item and the presence of particles < 0.45 µm (filter pore size) did not allow a reproducible and homogeneous application of the test item, resulting in a high variation of measured niobium concentrations in the preliminary, range finder and the main tests.

Table: Survival data and percent reduction of survival compared to the control after 21 days.

Nom. Conc. [mg/L]	Total Introduced	Parental survival	Reduction of survival [%] (immobility)
Control	15	15	0
0.10	15	15 (-)	0 (-)
0.316	15	14 (-)	6.70 (-)
1.00	15	15 (-)	0 (-)
3.16	15	15 (-)	0 (-)
10.0	15	15 (-)	0 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments

Table: Growth data and percent reduction of length compared to controls after 21 days.

Nom. conc. [mg/L]		Length on day 21 Mean ± SD [mm]	Decrease in length [%]
Control		4.32 ± 0.236	-
0.10		4.31 ± 0.181 (-)	0.3 (-)
0.316		4.16 ± 0.275 (-)	3.8 (-)
1.00		4.25 ± 0.275 (-)	1.8 (-)
3.16		4.30 ± 0.275 (-)	0.4 (-)
10.0		4.29 ± 0.217 (-)	0.9 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

Table: Reproduction data.

Nom. Conc.	Cumulative offspring per introduced parent	Cumulative offspring per survived parent	Age of first brood	Development rate	Intrinsic rate r
[mg/L]	Mean±SD [Ind.]	Mean±SD [Ind.]	Mean± SD [days]	Mean± SD [Ind.]	Mean± SD [Ind.]
Control	74.3 ± 12.3	74.3 ± 12.3	10.6 ± 1.19	0.095 ± 0.010	0.305 ± 0.031
0.10	79.1 ± 16.92 (-)	79.1 ± 16.92 (-)	10.2 ± 1.05 (-)	0.099 ± 0.009 (-)	0.309 ± 0.024 (-)
0.316	72.3 ± 12.89 (-)	73.6 ± 12.41 (-)	10.3 ± 1.01 (-)	0.098 ± 0.009 (-)	0.303 ± 0.026 (-)
1.00	71.6 ± 12.12 (-)	71.6 ± 12.12 (-)	10.7 ± 1.32 (-)	0.095 ± 0.011 (-)	0.299 ± 0.030 (-)
3.16	73.3 ± 13.24 (-)	73.3 ± 13.24 (-)	10.6 ± 1.22 (-)	0.096 ± 0.011 (-)	0.302 ± 0.028 (-)
10.0	76.3 ± 21.31 (-)	76.3 ± 21.31 (-)	9.8 ± 0.59 (-)	0.103 ± 0.006 (-)	0.313 ±0.038(-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

Table:       Percent reduction of reproduction compared to controls after 21 days.

Nom. Conc.	Reduction of reproduction per introduced parent	Reduction of reproduction per survived parent	Inhibition of development rate	Inhibition of intrinsic rate r
[mg test item/L]	[%]	[%]	[%]	[%]
Control	-	-	-	-
0.10	-6.6 (-)	-6.6 (-)	-4.3(-)	-1.3(-)
0.316	2.6 (-)	0.9 (-)	-3.0(-)	0.6(-)
1.00	3.6 (-)	3.6 (-)	0.4(-)	1.9(-)
3.16	1.3 (-)	1.3 (-)	-0.7(-)	0.8(-)
10.0	-2.7 (-)	-2.7 (-)	-8.0(-)	-2.7(-)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments

Table: Mean measured concentrations (initial and aged) of Nb (All values, except control values and the lowest treatment group are considered as reliable; reliable values: potential error due to carry over during the analytical measurement of < 25%).

Nominal concentrations	Nb
[mg/L]	[µg/L]	[%] of nominal	[%] of initial
	Mean measured initial concentrations
Control	-	-	-
0.10	1.87 ± 0.96	1.97	-
0.316	6.06 ± 3.07	2.00	-
1.00	20.6 ± 10.07	2.11	-
3.16	28.6 ± 11.87	0.922	-
10.0	43.5 ± 18.60	0.457	-
	Mean measured aged concentrations
Control	-	-	-
0.10	0.754 ± 0.44	0.754	40.3
0.316	2.96 ± 1.60	0.938	48.9
1.00	12.3 ± 6.23	1.229	59.6
3.16	19.8 ± 9.99	0.627	69.3
10.0	30.6 ± 13.77	0.306	70.4

Standard deviations calculated based on the measured concentrations of the fresh and aged test solutions of the sampling days.

Table:        Particle size distribution of the test item in test medium in the three highest test concentration throughout the test.

Date	Nominal conc. [mg test item/L]	Z-Average	PdI	Peak 1 Mean Int	Peak 1 Area Int	Peak 2 Mean Int	Peak 2 Area Int	Mean Count Rate
		d.nm		d.nm	%	d.nm	%	kcps
Day 0 – fresh	10.0	1117	0.873	176.4	100	0	0	117.2
		904.5	0.701	334.7	70.1	112	29.9	122.1
		337.9	0.607	180.3	70	403.5	30	99.4

Day 0 – fresh	3.16	2954	1.000	489.1	100	0	0	21.4
		19.07	0.930	692.6	63.2	3.346	36.8	16.5
		0	0.000	0	0	0	0	15.7
	1.00	21590	0.720	0	0	0	0	79.2
		3988	1.000	64.81	100	0	0	68.9
		1996	1.000	205.7	100	0	0	63.9
Day 2 – aged	10.0	1122	0.863	1.04	52.8	267.7	37	15.9
		272.2	0.400	516	45.4	13.98	30.1	15.0
		305.7	0.851	863	52	1.002	23.5	16.2
	3.16	1684	0.974	242.7	78.4	0.7002	21.6	21.9
		836.3	0.771	236	77.2	5379	22.8	20.9
		500.3	0.461	156.3	47.9	5031	27.1	16.4
	1.00	2897	1.000	77.63	57.8	1.295	42.2	17.1
		529.5	0.550	221.9	54.1	0.6707	22.9	17.9
		378.4	0.963	399.1	45.2	1.339	29.3	16.4
Day 7 – fresh	10.0	281.7	0.331	100.5	89.5	1.155	6.9	21.3
		120.1	0.492	124.4	76.6	4058	14.6	22.1
		129.9	0.363	116.4	83.8	4280	16.2	20.9
	3.16	305.6	0.329	295.3	67.1	4790	16.8	20.0
		155.4	0.504	126.5	58.6	874.2	41.4	23.4
		139.5	0.300	113.5	81	4056	19	21.8
	1.00	417.2	0.415	261.2	54.3	0.7097	24.4	19.0
		342.5	0.413	265.1	60.1	4615	24.1	21.8
		271.7	0.409	328.1	70.6	1.532	15.2	21.5
Day 9 – aged	10.0	5412	0.816	433.7	83.5	3.54	11	20.3
		1367	0.859	197.5	91.4	36.6	8.6	17.9
		632.1	0.560	0.9986	41.2	160.4	34	16.5
	3.16	3015	1.000	130.6	100	0	0	21.7
		870.3	0.586	518.5	88.1	5381	11.9	21.7
		566.5	0.487	439.8	63.4	4265	29.9	20.1
	1.00	6511	1	1.102	80.1	7.104	19.9	17.2
		41.56	0.973	899.2	66.5	13.21	18	16.2
		0	0	0	0	0	0	16.4

Day 14 – fresh	10.0	2481	1.000	100.6	100	0	0
		603.3	0.625	194.7	70.9	66.92	29.1	85.6
		307.8	0.415	96.04	51.5	281.3	48.5	75.3
	3.16	470	0.397	320.2	88.8	5225	11.2	23.7
		416.8	0.354	665.6	100	0	0	24.6
		391.8	0.331	591.1	100	0	0	18.3
	1.00	1001	0.854	273.9	92.5	5.584	7.5	20.7
		215.5	0.447	213.4	58.7	3522	26.3	16.9
		162.8	0.78	2217	43.8	150.1	34.5	16.3
Day 16 – aged	10.0	882.9	0.678	363.2	100	0	0	24.0
		773.9	0.548	502.8	80.1	5118	19.9	21.8
		423	0.546	499	72.6	3770	27.4	19.0
	3.16	1216	0.855	267.6	91.4	24.54	8.6	18.5
		124.2	0.567	6.326	39.7	449.1	35.1	16.2
		85	0.914	433.7	35.6	7.902	35.3	16.2
	1.00	2135	1	231.7	94.4	2.024	5.6	19.5
		780.3	0.58	548.3	52.9	4695	26.8	19.1
		538.9	0.671	399.7	56	4539	30.6	20.1

PDI: Polydispersity index; Int: Intensity; kcps: kilo counts per second Italic values are out of the range of the detection limit ranging from 0.6 to 6 000 nm.

Table: Validity criteria for OECD 211 (main test)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.	0%	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	74.3	yes

- Coefficient of variation around the mean number of living offspring produced per parent animal in the control(s): 16.6%

- No clinical signs as physical/pathological symptoms, changes in behaviour or winter eggs were observed.

Validity criteria fulfilled:

yes

Remarks:

For details please refer to field "any other information on results incl. tables"

Description of key information

 NOEC (21 d)≥10 mg/L (nominal) for Daphnia magna (OECD 211, static renewal test)

Additional information

One key study investigating the chronic toxicity of niobium (CAS 7440-03-1) to freshwater invertebrates is available.

 

Effects on the chronic toxicity to the freshwater invertebrate Daphnia magna were investigated after exposure to saturated solutions of niobium (CAS 7440-03-1) for 21 d under a semi static test regime (water renewal three times per week) in a test according to OECD 211 (GLP). Five saturated solutions of niobium were prepared in the assumption to test the dissolved fraction of niobium (0, 0.10, 0.316, 1.00, 3.16 and 10.0 mg/L (nominal)). For this, an appropriate amount of niobium was weighed into test vessels and filled up with the specific volume of test medium, which was previously adjusted to pH 8.5 (as requested in the ECHA decision CCH-D-2114517391-56-01/F). After stirring for 96 h, the saturated solutions were filtered (0.45 µm) and used in the daphnia test.

The test item medium concentration was analytically monitored in fresh samples and immediately before the medium renewal by means of inductively coupled plasma mass spectrometry (ICP-MS) in control and treatment groups. Hydrodynamic diameters of niobium particles were analysed in the test medium in the three highest test concentrations throughout the test by means of dynamic light scattering (DLS).

An excessive effort was done to optimize the application method (for details see the robust study summary), by performing preliminary tests. Nevertheless, due to the particulate properties (sedimentation of particles, wide particle size distribution including particles sized < 0.45 µm) of the test item it was not possible to apply a homogenous and reproducible test item concentrations and to reliably measure the real exposure concentration in the test medium. For example, measured niobium concentrations in the 10 mg/L (nominal) treatment group from 3.88 µg/L – 110 µg/L in the preliminary, range finder and main tests. Therefore, effect concentrations were based on nominal instead of measured concentrations.

Dynamic light scattering analysis, demonstrated that non-dissolved niobium particles were available in the test medium, demonstrating exposure of daphnids to non-dissolved niobium.

The 21 d NOEC values for the reproductive performance, immobilisation, growth (adult length at test termination), development rate and intrinsic rate r of population increase were determined as ≥ 10 mg/L (nominal). Therefore, it is concluded that niobium is not chronically toxic to aquatic invertebrates.