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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 - 11. Nov 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
no
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:
- Test start: all treatment and control solutions, prior to addition of algae (n = 2; 15 mL)
- Test termination: all treatment and control replicates (n = 2; 15 mL), algae were removed by filtration (0.45 µm PES filter)
- Sampling method: Before sampling the test solution was shaken and samples were taken from the middle of the respective flask using a pipette.
- Sample storage conditions before analysis: one sample was stored (4 ± 3 °C) until further analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Test item solutions were prepared as highly saturated test solutions according to OECD GD No. 23:
- Test medium: OECD growth medium adjusted to pH 8.5
- > 1 mg/L: Individually prepared; Amounts of 1.00, 3.17, 10.01, 31.62 and 100.08 mg of the test item were transferred into sterile glass flasks and 1 L sterile modified growth medium was added. The test solutions were stirred vigorously for about 96 hours at room temperature. After stirring, the prepared test solutions were filtered through a 0.45 µm filter (PES) to remove undissolved test item.
- Controls: growth medium without test item; the growth medium was treated in the same way as the test solutions
- Test concentration separation factor: approx. 3.16
- Evidence of undissolved material: In the pre test on solubility of the test item following observations were made: Directly after stirring (0h), undissolved test item was floating on the water surface and greyish particles appeared on the bottom of the glass flask at both loading rates.

PRELIMINARY EXPERIMENTS - SEPARATION METHOD AND STIRRING TIME
a) Additional settling time of 24 h after 96 h of stirring: 10 and 100 mg/L (nominal, loading) prepared as described in 'Preparation and application of test solution' without filtration step; visual observation of undissolved test item after 0, 2, 4, 6 and 24 h
b) 0.45 µm filtration after 96 h of stirring: 10 and 100 mg/L (nominal, loading) prepared as described in 'Preparation and application of test solution'; After stirring, fresh samples (0h) were taken before filtration and again after filtration through 0.45 µm and 0.2 µm PES filter. The test solutions were then distributed to replicates and incubated under test conditions of an algal growth inhibition test (OECD 201). Samples for analytical measurements of Nb were taken at 24 h, 48 h and 72 h from individually prepared replicates per test concentration. 0.20 µm filtered samples were only measured 0 h and 72 h after the stirring period.

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, 37073 Göttingen, Catalog No 61.81 SAG
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: according to OECD 201 in culture medium recommended by Bringmann and Kühn (1980); Precultures: OECD growth medium

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.0 °C throughout the whole test
pH:
Control/ treatment group test initiation: 8.21 (mean 72 h, n= 8) / 8.26 - 8.29 (n =5)
Control/ treatment group test termination: 8.03 (mean 72 h, n= 8) / 8.01 - 8.03 (n= 5)
Nominal and measured concentrations:
Nominal: 0.0 (control), 1.00, 3.17, 10.0, 31.6 and 100 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks, acid-washed (10% HNO3) and rinsed six times with purified water
- Type: covered with air permeable silicone-sponge caps, sterile
- Material, size, headspace, fill volume:100 mL
- Aeration: no
- Initial cells density: 10 000 cells/mL
- Control end cells density: 43.13 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 4 (randomly placed in the incubator)
- No. of vessels per control (replicates): 8 (randomly placed in the incubator)


GROWTH MEDIUM
- Standard medium used: yes, OECD medium according to OECD 201, prepared with purified water, but the pH was modified to a pH of 8.5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water (ELGA „PURELAB Ultra“)
- Culture medium different from test medium: no, pH culture medium: about 8.1

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, 8.5
- Photoperiod: continuously illuminated (OSRAM 'day light')
- Light intensity and quality: 108.91 to 110.84 µE m-2 s-1 (direct light above the tray of the incubator was measured)
- Temperature: measured continuously during the test in an additional test vessel
- pH measurement: 0 h: one replicate; 72 h: all replicates

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 hours in the test cultures during the growth test; electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany)
- Microscopic observation: 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

RANGE FINDING TEST
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations:1, 10, 100 mg/L (nominal loading)
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
- Details on culturing apparatus used: Test vessels were placed on an orbital laboratory shaker (150 rpm) (INFORS, Switzerland) in an incubator.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.03 mg/L
95% CI:
> 2.48 - < 5.78
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no, normal appearance of algal cells at all concentrations, less visible cells at concentrations of 10, 31.6 and 100 mg/L

Results with reference substance (positive control):
- Results with reference substance valid: yes (February 2022)
- EC50: ErC50 value of 3.40 mg/L (nominal; 95% confidence limits: 2.80 – 4.13 mg/L)
Reported statistics and error estimates:
Biological data were statistically analysed to determine EC50 and EC10 values together with 95 % confidence intervals, where possible. For yield the data was analysed using non-linear regression procedures (3 – parametric normal). For growth rate, the data showed a significant lack of fit for the tested 3-parametric non-linear regression procedures (3-parametric normal, logistic and Weibull) and therefore, the ECx values were calculated based on linear regression procedures (Probit).

The NOEC for growth rate and yield was determined using the Williams Multiple Sequential t-test Procedure (significance level of 0.05, one-sided smaller).

The computer program ToxRat® was used for statistical evaluations.

RANGE FINDING TEST

No significant and no concentration dependent effects were observed in the treatment groups 1 to 100 mg/L (nominal) after 72 h of exposure. Growth rate was inhibited by 0.8, 10.3 and 4.9% compared to the growth rate of the controls, respectively.

Table: Measured Nb concentrations in the non-GLP range finding test.

Sample

Nominal concentration [µg Nb/L]

Filtration

Measured concentration [µg Nb/L]

Mean Nb conc. [µg/L]

Deviation from 0h samples [%]

Recovery of filtered 3d samples compared to 0h sample
[%]

Test initiation
„direct measurement“
(August 23, 2021)

100,000

filtered 0.45 µm

18.3

-

-

-

Test
termination

(August 26, 2021)

100,000

filtered 0.45 µm

19.8

-

-

108

not filtered

24.6

-

August 23, 2021: LOQ = 0.0013; August 26, 2021: LOQ = 0.0047 µg/L

Based on the results of the (analytical) pre-tests and the non-GLP range finder it was decided that the test design of the non-GLP range finder in terms of test media preparation appeared to be appropriate for the GLP main test.

DEFINITIVE TEST

After exposure of algae to the test solutions for 72 h the growth rate of algae was significantly inhibited compared to the control group in all treatment groups in a concentration dependent manner, ranging from 3.6 to 36.4% inhibition. The 72 hour EC10 and EC50 values for growth rate were determined as 4.03 and > 100 mg Nb2O5/L (nominal), respectively. A NOEC for growth rate of below the lowest nominal concentration of 1.00 mg test item/L was determined. However, due to the low inhibition of 3.6% at a nominal concentration of 1.00 mg test item/L and 7.7% at 3.17 mg test item/L, the NOEC was set to be at 3.17 mg test item/L, since effects below 10% compared to the control are generally not considered to be ecotoxicologically relevant.

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 100 mg/L treatment group (nominal, stirred for 96 h, 0.45 µm filtered) ranged from 18.3 – 327 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 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 an algae long-term test according to OECD 201. 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 observed effects are caused by direct physical effects of the non-dissolved niobium.

Since, i) the particle sizes of diniobium pentaoxide were even smaller than niobium metal powder, containing also particles smaller than the pore size of the filter and ii) the measured niobium concentrations in the test medium of the recent algae 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 metal, it is concluded that the measured niobium concentrations in the test medium were not reliable and therefore, all endpoints were evaluated based on the nominal concentrations. Furthermore, it is concluded that direct physical effects of the non-dissolved diniobium pentaoxide caused the observed effects on the growth rate of algae.

 

TEST ITEM CONCENTRATION IN THE TEST MEDIUM - DEFINITIVE TEST

Table: Results of the chemical analysis of Nb concentration in the test media at test initiation and test termination after 72 hours exposure (* values are considered as not reliable: potential error due to carry over during the analytical measurement of > 25%)

Sampling

Nominal
concentration
[µg test item/L]

Nominal
Nb concentration
[µg Nb/L]

Measured Nb concentration
[µg Nb/L]

Measured Nb – background concentration
[µg Nb/L]

% of nominal
[%]

% of initial
[%]

0d
(Analytical
sample)

Control

Control

0.386*

-

-

-

1000

699

1.47

1.09

0.16

-

3170

2216

11.6

11.2

0.51

-

10000

6990

29.6

29.2

0.42

-

31600

22088

57.6

57.2

0.26

-

100000

69900

328

327

0.47

-

3d

Control

Control

0.468*

 

 

1000

699

0.961

0.493

0.07

65.1

3170

2216

9.01

8.544

0.39

77.6

10000

6990

19.6

19.2

0.27

66.5

31600

22088

38.3

37.8

0.17

66.4

100000

69900

45.1*

44.7

0.06

13.8

*: Value not reliable (potential error of > 25 %).

GROWTH RATES - DEFINITIVE TEST

Table: Inhibition of growth rate and yield compared to controls after 72 hours.

Nominal
concentration
[mg test item/L]

Growth rate

Yield

Growth rate
[1/d]

%
Inhibition

Yield
[cells x 104/mL]

%
Inhibition

Control

1.252

-

42.1

-

1.00

1.207

3.6 (+)

36.4

13.5 (+)

3.17

1.155

7.7 (+)

31.2

26.0 (+)

10.0

1.014

19.0 (+)

20.1

52.3 (+)

31.6

0.977

21.9 (+)

17.8

57.8 (+)

100

0.796

36.4 (+)

9.9

76.4 (+)

(+) significantly/ (-) not significantly different from controls, Williams Multiple Sequential t-test Procedure, significance level 0.05, one-sided smaller.

CELL NUMBERS - DEFINITIVE TEST

Table: Cell numbers (x 104) per mL dependent on nominal concentrations and time.

Nominal
concentration
[mg test item/L]

Control

1.00

3.17

10.0

31.6

100

0 h

1

1

1

1

1

1

24 h

3.265

3.463

3.235

3.403

2.875

2.584

 

3.184

2.824

3.596

2.841

2.970

2.829

 

3.260

3.172

3.207

2.817

3.274

2.453

 

3.398

2.861

3.373

2.757

2.988

2.796

 

3.165

 

 

 

 

 

 

3.262

 

 

 

 

 

 

3.309

 

 

 

 

 

 

2.903

 

 

 

 

 

Mean:

3.218

3.080

3.353

2.955

3.027

2.666

Std.Dev.:

0.146

0.299

0.178

0.301

0.172

0.179

n:

8

4

4

4

4

4

CV:

4.5

9.7

5.3

10.2

5.7

6.7

 

 

 

 

 

 

 

48 h

11.57

13.35

11.46

8.43

6.97

5.59

 

12.57

10.38

11.76

7.59

6.62

5.45

 

10.72

11.08

7.38

6.23

6.79

5.48

 

12.85

10.98

11.55

7.65

6.99

5.17

 

11.65

 

 

 

 

 

 

10.86

 

 

 

 

 

 

10.47

 

 

 

 

 

 

12.54

 

 

 

 

 

Mean:

11.65

11.45

10.54

7.47

6.84

5.42

Std.Dev.:

0.92

1.31

2.11

0.91

0.17

0.18

n:

8

4

4

4

4

4

CV:

7.9

11.4

20.0

12.2

2.5

3.3

 

 

 

 

 

 

 

72 h

37.65

41.82

36.91

24.79

19.82

11.73

 

54.42

35.96

34.00

21.43

18.62

11.89

 

41.86

35.08

27.57

18.06

18.50

10.30

 

41.08

36.91

30.25

20.04

18.14

9.77

 

44.58

 

 

 

 

 

 

37.09

 

 

 

 

 

 

38.43

 

 

 

 

 

 

49.93

 

 

 

 

 

Mean:

43.13

37.44

32.18

21.08

18.77

10.92

Std.Dev.:

6.21

3.01

4.11

2.83

0.73

1.05

n:

8

4

4

4

4

4

CV:

14.4

8.0

12.8

13.4

3.9

9.6

Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

Cell number at test start: 10 000 cells/mL

Table: Validity criteria for OECD 201 - Definitive test

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 43.1

 yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

7.9%

 yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitataandDesmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

3.7%

 yes

Validity criteria fulfilled:
yes
Remarks:
For details please refer to field "any other information on results incl. tables"

Description of key information

ErC50 (72h) > 100 mg Nb2O5/L (nominal) for Raphidocelis subcapitata (OECD 201, static)

No long-term intrinsic toxicity by the test item towards Raphidocelis subcapitata (OECD 201, static; Observed effects resulting into an ErC10 of 4.03 mg/L are attributed to physical effects)

Key value for chemical safety assessment

Additional information

One key study investigating the toxicity of diniobium pentaoxide (CAS 1313-96-8) to freshwater algae is available.

 

Effects on the growth rate of the freshwater algae Raphidocelis subcapitata were investigated after exposure to saturated solutions of diniobium pentaoxide for 72 h in a test according to OECD 201 (GLP). Five saturated solutions of diniobium pentaoxide (adjusted to pH 8.5) were prepared with the aim of testing the dissolved fraction of diniobium pentaoxide (0 (control), 1.00, 3.17, 10.0, 31.6 and 100.0 mg/L (nominal)). For the preparation of the test solutions, appropriate amounts of diniobium pentaoxide were weighed individually into the 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 these solutions were filtered (0.45 µm) and used in the algae test.

The test item concentration in the test medium was analytically monitored at test initiation and termination 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 (dissolved Nb) exposure concentration in the test medium. For example, measured niobium concentrations in the 100 mg/L (nominal) treatment group ranged from 18.3 – 327 µg Nb/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 an algae test according to OECD 201. 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 (measured concentrations at 100 mg/L (nominal): 86.7 – 870 µg Nb/L). In this algae test with niobium, dynamic light scattering measurements of the test medium (10-100 mg/L (nominal)) were carried out throughout the test (0-72 h). These measurements demonstrated that niobium particles with a particle size below around 200 nm 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. This observation shows that measured niobium concentrations did not reflect the dissolved fraction of niobium in the test medium only and that direct physical effects of the non-dissolved niobium caused the observed effects.

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 algae 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 algae 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 algae test are not considered to represent reliably the real exposure concentrations in the test medium (1.09 – 327 µg/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 released 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, it is concluded that 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 concentrations and that algae 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.

 

After exposure of algae to the test solutions for 72 h the growth rate of algae was significantly inhibited compared to the control group in all treatment groups in a concentration dependent manner, ranging from 3.6 to 36.4% inhibition. The 72 hour EC10 and EC50 values for growth rate were determined as 4.03 and > 100 mg Nb2O5/L (nominal), respectively. As explained above, algae were exposed to non-dissolved diniobium pentaoxide. Therefore, it is concluded that the observed effects are linked to mechanical/physical effects and not to the intrinsic toxic properties of the test item, showing that diniobium pentaoxide is neither acutely nor chronically toxic to aquatic algae.