Diniobium pentaoxide

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:

19 Mar - 13 Apr 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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordheim-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Remarks:

ICP-MS

Details on sampling:

- Concentrations: fresh and aged samples of control and all treatment groups
- Sampling method: 15 mL fresh samples (test and retain samples) were taken at the beginning of the test and at each media renewal prior to addition of daphnids and feeding algae. 15 mL aged samples were taken before media renewal and at test termination. Aged samples were filtered (0.45 µm PES filter) and stabilised with HNO3.
- 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). 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.
- 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 settlement 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): not applicable

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

300 - 310 mg CaCO3

Test temperature:

18. 8 - 19.6 °C

pH:

8.10 – 9.17 (pH did not vary by more than 1.5 units within one treatment and 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:

6.74 mg/L and 11.80 mg/L (corresponds to > 80.4% dissolved oxygen concentration)

Nominal and measured concentrations:

Nominal: 0.0 (control), 0.10, 0.316, 1.00, 3.16 and 10.0 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL, glass beakers (acid washed (10% HNO3)), 50 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 exchange: three times per week; test specimens were transferred by means of a bore pasteur pipette.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified tap water from the Schmallenberg district water production plants; 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.
- Non-purgeable organic carbon: 1.1 mg/L
- Metals: measured
- Pesticides: not measured
- Chlorine: 0.02 mg/L
- Alkalinity: 1.6 - 1.8 mmol/L
- Ca/Mg ratio: 2.7 - 9
- Conductivity: 251-257 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: regularly

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: 590 - 702 lux
- Oxygen concentration, pH value, and temperature were checked directly before adding the animals and at each water renewal in new and aged test solutions. Hardness was measured in fresh and aged samples of the control and the highest test concentrations once per week. In order to additionally investigate the particle sizes introduced to the test, a determination of the hydrodynamic diameter with a Zetasizer Nano ZS was included (for more details see field "Details on analytical methods").

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: 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

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

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 loading rates 0.1 and 1.0 mg test item/L (nominal), no significant effects 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 35.1% compared to the control. Mobility was not significantly affected up to the highest loading rate. The test fulfilled the validity criteria according to OECD 211.

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

			Nb concentrations
Sample	Nominal concentration [mg Nb/L]	Filtration	[µg Nb/L]
0h fresh	10.0	Not filtered	12.3
0h fresh	10.0	0.45 µm	5.41
0h fresh*	10.0	Not filtered	2.13
0h fresh*	10.0	0.45 µm	1.60
72h aged	10.0	Not filtered	3.53
72h aged	10.0	0.45 µm	0.62

*measured after 72 h

 

DEFINITIVE TEST

No significant effects on the assessed parameters compared to control were observed for daphnids exposed to diniobium pentaoxide for 21 d. Considering the high PDI and the low count rate below 30 kcps, the dynamic light scattering (DLS) results are not reliable, and it can be concluded that the test item concentrations were not necessarily appropriate for DLS measurements. Furthermore, the analytically measured niobium concentration in the test medium showed a low reproducibility between the pre-tests, the range finder, and main test. For example, measured niobium concentrations in the 10 mg/L treatment group (nominal, stirred for 96 h, 0.45 µm filtered) ranged from 1.6-18.4 µg/L in the preliminary, range finder and main tests. The test item is a powder consisting of particles in the µm-range, including particles with a particle size smaller than the pore size of the used filter (0.45 µm, PES filter).

Niobium metal powder is a powder with an even wider particle size distribution in the lower mm-size range and with larger particles than those of diniobium pentaoxide. Niobium metal powder was tested with the same application method as diniobium pentaoxide in a daphnia long-term test according to OECD 211. As for diniobium pentaoxide, the analytically measured niobium concentration in the test medium were not reproducible, showing a huge variation between the pre-tests, the range finder, and main tests. In this niobium metal test, dynamic light scattering revealed the presence of niobium particles in the test medium. Thereby, it was demonstrated that non-dissolved niobium passed the 0.45 µm filter, meaning that the test organisms were not only exposed to dissolved niobium but also to a non-reproducible amount of non-dissolved niobium. Thus, the measured niobium concentrations did not reflect the dissolved fraction of niobium in the test medium only and the daphnids were exposed to non-dissolved niobium.

Since, i) the particle sizes of diniobium pentaoxide were even smaller than niobium metal powder, including particles smaller than the used filter pore size and ii) the measured niobium concentrations in the test medium of the recent daphnia long-term test with diniobium pentaoxide were also not reproducible, it is assumed that non-dissolved diniobium pentaoxide also passed the filter.

Thus, as for niobium, it is concluded that the measured niobium concentrations did not reflect the dissolved fraction of niobium in the test medium, do not represent the actual exposure concentration and that daphnids were exposed to non-dissolved niobium. Therefore, it is concluded that the measured niobium test medium concentrations were not reliable and all endpoints were evaluated based on the nominal concentrations.

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

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

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

*one daphnid died accidently during handling, therefore one replicate was excluded

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	4.48 ± 0.31	-
0.10	4.25 ± 0.30 (-)	5.1 (-)
0.316	4.26 ± 0.29 (-)	4.7 (-)
1.00	4.19 ± 0.31 (+)	6.3 (+)
3.16	4.57 ± 0.34 (-)	-2.1 (-)
10.0	4.50 ± 0.30 (-)	-0.6 (-)

(+) 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	Inhibition of development rate	Inhibition of intrinsic rate r
[mg/L]	[%]	[%]	[%]
Control	-	-	-
0.10	-3.3 (-)	-2.0 (-)	-0.2 (-)
0.316	-7.8 (-)	-9.3 (-)	-7.5 (-)
1.00	6.8 (-)	-0.8 (-)	2.8 (-)
3.16	10.9 (-)	-1.5 (-)	3.0 (-)
10.0	-7.2 (-)	4.7 (-)	3.4 (-)

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

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.	79.2	yes

- Coefficient of variation around the mean number of living offspring produced per parent animal in the control(s): 15.5%

-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 diniobium pentaoxide (CAS 1313-96-8) to freshwater invertebrates is available.

 

Effects on the chronic toxicity to the freshwater invertebrate Daphnia magna were investigated after exposure to saturated solutions of diniobium pentaoxide (CAS 1313-96-8) 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 with the aim of testing the dissolved fraction of diniobium pentaoxide (0 (control), 0.10, 0.316, 1.00, 3.16 and 10.0 mg/L (nominal)). For the preparation of the test solutions, an appropriate amount of diniobium pentaoxide was weighed individually into test vessels and filled up with the respective volume of test medium, which was previously adjusted to pH 8.5 (as requested in the ECHA decision CCH-D-2114517435-52-01/F). After stirring for 96 h, the saturated solutions were filtered (0.45 µm) and used in the daphnia test. The test item concentration in the test medium was analytically monitored at test initiation and at the end of each renewal period by means of inductively coupled plasma mass spectrometry (ICP-MS) in control and all treatment groups.

 

An excessive effort was done to optimize the preparation of test solutions and analytics (for details see the robust study summary), by performing several 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 homogenous and reproducible test item concentrations and to reliably measure the real exposure concentrations (dissolved Nb) in the test medium. The analytically measured niobium concentration in the test medium were not reproducible, showing a low reproducibility between the pre-tests, the range finder, and main test. For example, measured niobium concentrations in the 10 mg/L treatment group (nominal, stirred for 96 h, 0.45 µm filtered) ranged from 1.6-18.4 µg/L in the preliminary, range finder and main tests.

The test item is a powder consisting of particles in the µm-range, including particles with a particle size smaller than the pore size of the used filter (0.45 µm, PES filter).

The similar substance niobium metal powder is a powder with an even wider particle size distribution in the lower mm-size range and with larger particles than those of diniobium pentaoxide. Niobium metal powder was tested with the same application method as diniobium pentaoxide in a daphnia long-term test according to OECD 211. As for diniobium pentaoxide, the analytically measured niobium concentration in the test medium were not reproducible, showing a huge variation between the pre-tests, the range finder, and main test. For example, measured niobium concentrations in the 10 mg/L (nominal) treatment group varied between 3.88 µg/L and 110 µg/L in the preliminary, range finder and main tests. Therefore, effect concentrations were based on nominal instead of measured concentrations. In this daphnia test with niobium, dynamic light scattering measurements of the fresh and aged test medium (1.0 – 10.0 mg/L (nominal)) were carried out throughout the test (0-16 d). These measurements demonstrated that niobium particles were present in the test medium. Thereby, it was demonstrated that non-dissolved niobium passed the 0.45 µm filter, meaning that the test organisms were not only exposed to dissolved niobium but also to a non-reproducible amount of non-dissolved niobium. Thus, the measured niobium concentrations did not reflect the dissolved fraction of niobium in the test medium only and the daphnids were exposed to non-dissolved niobium.

Since, i) the particle sizes of diniobium pentaoxide were even smaller than niobium metal powder and ii) the measured niobium concentrations in the test medium of the recent daphnia long-term test with diniobium pentaoxide were also not reproducible, it is assumed that non-dissolved diniobium pentaoxide also passed the filter.

This assumption is confirmed by similar observations, which were made in a water solubility test with diniobium pentaoxide (OECD 105) and by the results of the transformation dissolution test with diniobium pentaoxide (OECD GD 29).

In the water solubility test with diniobium pentaoxide, the water phase was also filtered with a 0.45 µm filter. As for the daphnia test, a low reproducibility of measured niobium concentration in the water phase after this filtration step was observed (205 – 412 µg Nb/L). A subsequent membrane filtration (0.1 µm) combined with centrifugation (9000 RPM, 20 min) still revealed a low reproducibility but a much lower niobium concentration (11.3 – 43.6 µg Nb/L). These results demonstrate, that non-dissolved diniobium pentaoxide was present in the water solubility test after the 0.45 µm filtration step.

Although the measured initial niobium test medium concentrations in the daphnia test are not considered to represent reliably the real exposure concentrations in the fresh test medium (0.59 - 9.40 µg Nb/L), these high measured Nb concentrations do not match the overall picture from the the available TD tests with diniobium pentaoxide. These T/D results show a very low bioavailability of resleased niobium species in environmental media (28 d, pH 8, 1 mg Nb2O5/L: ≤ 0.011 µg Nb/L; 7 d, pH 8, 100 mg Nb2O5/L: 0.25 µg Nb/L).

Thus, as for niobium metal, the measured niobium concentrations from diniobium pentaoxide did not reflect the dissolved fraction of the substance in the test medium, do not represent the actual exposure concentration and that daphnids were exposed to non-dissolved diniobium pentaoxide. Therefore, it is concluded that the substance is a difficult to test substance and in spite of extensive efforts, the measured niobium test medium concentrations were not reliable and therefore all endpoints were evaluated based on the nominal concentrations.

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