MoVNbTe mixed oxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2021-09-24 to 2021-11-18, with the definitive exposure phase from 2021-11-09 to 2021-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2016)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

(2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item Mo10V3TeNbO42

Batch number EX.14402.600

CAS No. 146569-48-4

Active ingredient
Niobium trivanadium decamolybdenum tellurium dotetracontaoxide

Purity 87.6%

Mo content 47.87 % (stoichiometric content)

Water solubility of Mo 11.498 mg/L (at pH 8.5, after 4 d shaking at 100 rpm of 100 mg/L Mo10V3TeNbO42; Affolter, 2020)

Appearance Black solid powder

Water solubility < 0.2 g/L practically insoluble

Stability under test conditions Not specified

Expiry date 2026-03-10

Recommended storage Store tightly closed, cool and dry / 20 °C

Analytical monitoring:

yes

Remarks:

via ICP-OES at the start (0 hours) and at the end of the exposure (72 hours) without algae

Details on sampling:

Chemical Analysis of the Test Item Concentrations

The molybdenum concentration was determined via element analysis of Mo, processed at the test site. Test item samples were labelled with the test number, the concentration, the code for the sampling times, the replicate number, the date and the sampler, according to the test facility SOP and delivered to the test site for the analytical delegated phase. A full chain of custody was maintained. A phase plan with the description of the method used for the delegated phase was added to the study plan.

Sampling schedule
All loadings and the control were analytically verified via ICP-OES at the start (0 hours) and at the end of the exposure (72 hours) without algae.

Sampling and pre-treatment
At the start and at the end of the exposure phase, samples were taken from one additional replicate of each test item loading and the control without algae. These replicates were incubated under test conditions until sampling. 50 mL of each loading rate and the control were filled into a glass vial. All samples were taken in duplicate. The samples were stored cool in a fridge (2 – 8 °C) at the test facility. The first replicate was shipped pooled to the test site at the end of the exposure phase for analysis. The second replicate was stored cool in a fridge (2 – 8 °C) at the test facility. The principle investigator was informed by the study director prior to shipment. The study monitor was informed about the shipment. Analysis was performed as described in the phase plan. The analytical method, the results of the method validation as well as the results of the measured test samples were added to the final report.

Vehicle:

no

Details on test solutions:

Preparation of the Saturated Solution
A saturated solution with a nominal loading rate of 100 mg test item/L was prepared once at room temperature 96 ± 2 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred into a glass flask with an appropriate amount of demineralized water. The saturated solution was stirred with approximately 1100 rpm for 96 ± 2 hours at room temperature. After a separation phase of 30 minutes, the saturated solution was removed by siphoning from the approximate center of the glass bottle. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, were used in the test. During filtration, the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.
After measurement of the Tyndall the components of the dilution water were added to the saturated solution.


Preparation of the test item solutions
Out of the saturated solution 3 concentrations were tested in a geometric series with a dilution factor of √10: 10.0 – 31.6 – 100% of the saturated solution.
The dilution levels are based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates of dilution water (see above) without test item were exposed under the same conditions as the test groups.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test organism Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)

Synonyms
Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

Culture medium Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Remarks:

According to the guidelines without the chelating agent Na2EDTA  2 H2O, due to the chemical properties of the test item

Limit test:

no

Total exposure duration:

72 h

Hardness:

see section "Any other information on results incl. tables" below.

Test temperature:

see section "Any other information on results incl. tables" below.

pH:

see section "Any other information on results incl. tables" below.

Dissolved oxygen:

see section "Any other information on results incl. tables" below.

Conductivity:

see section "Any other information on results incl. tables" below.

Nominal and measured concentrations:

A saturated solution with a nominal loading rate of 100 mg test item/L was prepared. Out of the saturated solution 3 concentrations were tested in a geometric series with a dilution factor of √10: 10.0 – 31.6 – 100% of the saturated solution.

Details on test conditions:

Test method Static procedure

Duration of the test 72 hours

Replicates Six replicates for the control, three replicates per loading rate
 
Test container
Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.

Test volume 100 mL

Ultrapure water
Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm).

Dilution water According to the guidelines without the chelating agent
Na2EDTA  2 H2O, due to the chemical properties of the test item.

Preculture
A four days old preculture, prepared in dilution water, was used as inoculum.

Initial cell density
Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6864 cells/mL

Application
Application was carried out by adding an appropriate amount of algae inoculum to the test solutions.

Incubation The flasks were positioned randomly and repositioned daily.

Temperature Nominal range: 21 - 24 °C, controlled at ± 2 °C

Agitation
Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

 
Light intensity (target)
Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

Light regime 24 hours/day light

Light homogeneity Within ± 15% over incubation area


Type and Frequency of Measurements
Biological Parameters

Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the saturated solution (100 mg/L).

Microscopic evaluation
The algae cells were evaluated microscopically at the start and the end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Physico-chemical Properties

The pH-value at the start of the exposure was measured in one additional replicate of each test item loading rate and the control. At the end of the exposure, it was measured in a pooled sample of the test item loading rates and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

Reference substance (positive control):

yes

Remarks:

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2021-11-01 to 2021-11-04.

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

calculated nominal loading from a saturated solution of 100 mg/L

Effect conc.:

73.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL20

Remarks:

calculated nominal loading from a saturated solution of 100 mg/L

Effect conc.:

32.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Remarks:

calculated nominal laoding from a saturated solution of 100 mg/L

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Remarks:

calculated nominal loading from a saturated solution of 100 mg/L

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

calculated nominal loading from a saturated solution of 100 mg/L

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Remarks:

calculated nominal loading from a saturated solution of 100 mg/L

Effect conc.:

56.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

calculated nominal loading from a saturated solution of 100 mg/L

Basis for effect:

growth rate

Details on results:

Biological Data

Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities. All effect values given are based on the nominal test item loading rates of the test item Mo10V3TeNbO42.

Physico-chemical Data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were visually clear throughout the exposure period (possible turbidity related to algae growth not taken into account).

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2021-11-01 to 2021-11-04. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values

Reported statistics and error estimates:

EL-values and statistical analyses
EL10-,EL20- and EL50- values of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t distribution wherever possible. In the case that the confidence limits could not be calculated by the software, the respective confidence limits were empirically derived from the observation data. All calculations were carried out from the best-fit values with the software GraphPad Prism.

NOEL, LOEL and The NOEL / LOEL was determined by calculation of statistical
statistical analyses significant differences of growth rate and yield using Dunnett’s Method. A Shapiro-Wilk’s Normality test and a Brown-Forsythe’s Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.01.
 
Software The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Saturated solution with a nominal loading of	Replicate	Cell density [cells/mL]
100 mg/L
[%]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6864	22762	118288	528060
	2	6864	24440	120852	454203
	3	6864	19269	106206	490277
	Mean	6864	22157	115115	490847
31.6	1	6864	21808	144934	1010943
	2	6864	29603	144988	1030195
	3	6864	29932	187332	947521
	Mean	6864	27114	159085	996220
10.0	1	6864	26057	166669	1185960
	2	6864	26115	153058	1048232
	3	6864	27370	181476	1139451
	Mean	6864	26514	167068	1124548
Control	1	6864	28812	162773	1258693
	2	6864	25242	159688	1268229
	3	6864	30465	195348	1286221
	4	6864	28742	174594	1298815
	5	6864	26772	201672	1103332
	6	6864	27174	177275	1175840
	Mean	6864	27868	178558	1231855

 

Evaluation after 72 hours
Statistically significant differences of growth rates and yield compared to
control values are marked (s), not significant differences are marked (ns).

Saturated solution with a nominal loading of	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
100 mg/L
[%]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.45	16		521196	57
	2		1.40	19		447339	63
	3		1.42	18		483413	61
	Mean	(s)	1.42	18	(s)	483983	60
31.6	1		1.66	4		1004079	18
	2		1.67	3		1023331	16
	3		1.64	5		940657	23
	Mean	(s)*	1.70	4	(s)	989356	19
10.0	1		1.72	1		1179096	4
	2		1.68	3		1041368	15
	3		1.70	1		1132587	8
	Mean	(ns)	1.70	2	(ns)	1117684	9
Control	1		1.74			1251829
	2		1.74			1261365
	3		1.74			1279357
	4		1.75			1291951
	5		1.69			1096468
	6		1.71			1168976
	Mean		1.73			1224991

* = statistical significant but biologically not relevant (< 5%)

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 - 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean	SD	CV	Mean CV [%]
		section-by-section			(0 - 72 hours)	±	[%]
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.44	1.73	2.05	1.74	0.306	17.6	17.7
	2	1.30	1.85	2.07	1.74	0.396	22.7
	3	1.49	1.86	1.89	1.74	0.221	12.6
	4	1.43	1.80	2.01	1.75	0.292	16.7
	5	1.36	2.02	1.70	1.69	0.329	19.4
	6	1.38	1.88	1.89	1.71	0.293	17.1
				Mean	1.73
				SD ±	0.02
				CV [%]	1.23

SD = Standard deviation CV = Coefficient of variation

 

Environmental Conditions

Saturated solution with a				pH-values
nominal loading of 100 mg/L
[%]				Start; 0 hours	End; 72 hours
100				7.91	8.22
31.6				7.97	8.64
10.0				8.05	8.78
Control				8.03	8.98
Room temperature [°C]:		min.: 22.0		max.: 22.5		mean value: 22.3
	n = 24		mean value: 5436				CV [%]: 6.73
Light intensity			range of the measured values: 4671 - 6005
[lux]			equalling -14.1 to 10.5

CV = Coefficient of variation n = number of measuring points

 

Water parameters of the Dilution Water

Parameters of the dilution water (measured on 2021-11-09)
Conductivity	Total hardness	Acidity	Alkalinity	Total organic carbon
[µS/cm]	[mg CaCO3/L]	[mmol/L]	[mmol/L]	[mg C/L]
136	40	0.2	0.6	< LOQ*

*Limit of quantification = 2.00 mg C/L

 

Validity Criteria

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	179-fold
		(specific growth rate 1.70 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	17.7%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	1.23%

 

Results

The purpose of this study was the determination of the concentration of molybdenum in test medium with dissolved test item Mo10V3TeNbO42 prepared by Noack Laboratorien (study no. SO21063 / SPO19517). Therefore, the provided sample solutions were acidified and analysed by inductively coupled plasma and optical emission spectrometry (ICP-OES).

The validity of the applied analytical method was proven (specificity, linearity, limit of quantification (LOQ), accuracy and precision). The validation results meet the specifications according to SANTE/2020/12830, Rev.1.

The mean results of the provided sample solutions are summarized in the following table:

Date	Sample	Sample description Nominal Sample concentration (mg test item/L)	Mean measured	Recovery^1)
	code		Mo concentration
[yyyy-mm-dd]			[mg/L]	[%]
30.09.2021	-	Test medium	< 0.01	-
28.09.2021	A0	Control	< 0.01	-
29.09.2021	E0	Control	< 0.01	-
28.09.2021	A0	10	0.2701	5.64
29.09.2021	E0	10	0.2635	5.50
28.09.2021	A0	31.6	0.829	5.48
29.09.2021	E0	31.6	0.845	5.59
28.09.2021	A0	100	2.63	5.49
29.09.2021	E0	100	2.65	5.54

1) Mo recovery related to Mo content (47.87%, stoichiometric) of the test item

 

1st Level (0.03 mg/L, LOQ level):
Data for the evaluation of the recovery and repeatability of Mo concentration 1st level using spiked matrix samples (0.03 mg/L)

Validation	Nominal Mo	Intensity, mean	Intensity value 1	Intensity value 2	Intensity value 3
sample no.	concentration
	[mg/L]	[counts/s]	[counts/s]	[counts/s]	[counts/s]
1	0.03	23.01	23.06	22.64	23.32
2	0.03	24.67	24.14	25.34	24.52
3	0.03	24.14	25.13	22.99	24.31
4	0.03	24.54	24.82	24.79	24.01
5	0.03	23.28	23.07	23.76	23.00

 

Validation	Nominal Mo	Measured Mo	Uncorrected recovery	Matrix effect	Corrected recovery
sample no.	concentration	concentration
	[mg/L]	[mg/L]	[%]	[%]	[%]
1	0.03	0.03639	121.30	14.71	106.59
2	0.03	0.03880	129.33	22.98	106.35
3	0.03	0.03803	126.77	20.34	106.43
4	0.03	0.03861	128.70	22.33	106.37
5	0.03	0.03678	122.60	16.05	106.55
Mean					106.46
SD					0.11
%RSD					0.10

 

2nd Level (0.30 mg/L, 10 x LOQ level):
Data for the evaluation of the recovery and repeatability of Mo concentration 2nd level
using spiked matrix samples (0.30 mg/L)

Validation	Nominal Mo	Measured Mo	Recovery	Intensity mean	Intensity value 1	Intensity value 2	Intensity value 3
sample no.	concentration	concentration
	[mg/L]	[mg/L]	[%]	[counts/s]	[counts/s]	[counts/s]	[counts/s]
6	0.30	0.30565	101.88	208.8	207.2	209.2	210.1
7	0.30	0.30420	101.40	207.8	206.9	208.5	208.2
8	0.30	0.30087	100.29	205.5	207.0	205.1	204.3
9	0.30	0.30507	101.69	208.4	208.5	207.1	209.6
10	0.30	0.30710	102.37	209.8	209.4	209.1	211.0
Mean			101.53
SD			0.78
%RSD			0.77

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, Mo10V3TeNbO42 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal loading rate of the test item): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L and 73.4 mg/L (CI: 31.6 – 100 mg/L), respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L and 10.0 mg/L.

Executive summary:

The toxicity of Mo10V3TeNbO42 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2021-09-24 to 2021-11-18, with the definitive exposure phase from 2021-11-09 to 2021-11-12 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6864 cells/mL. A saturated solution was prepared as described with demineralized water 96 ± 2 hour prior to the start of the exposure. The test media were visually clear throughout the test period. Three test loadings were tested in a geometrical series with a dilution factor of √10: 10.0 - 31.6 - 100% of the saturated solution. Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

The molybdenum concentration was determined via element analysis of Mo, processed at the Test Site (WeylChem InnoTec GmbH). All loading rates of the test item and the control were analysed via ICP-OES at the start (0 hours) and at the end of the exposure (72 hours) without algae. The analytical method, the results of the method validation as well as the results of the measured test samples were added to the final report. For details, see phase report in Annex I.

NOEL, LOEL and ELx-values (and Confidence Intervals empirically derived from the observation data) of Mo10V3TeNbO42 (0 - 72 hours)
based on the nominal loading rates of the test item [mg/L]

	Inhibition of Growth Rate
	(Nominal loading rates of the test item [mg/L])
NOEL	31.6*
LOEL	100
ErL10	56.3* (CI: 31.6 – 100)
ErL20	> 100
ErL50	> 100
	Inhibition of Yield
	(Nominal loading rates of the test item [mg/L])
NOEL	10.0*
LOEL	31.6*
EyL10	15.0* (CI: ≤10 – 31.6)
EyL20	32.6* (CI: 10 – 100)
EyL50	73.4* (CI: 31.6 – 100)

*calculated nominal loading from a saturated solution of 100 mg/L

Description of key information

In this study, Mo10V3TeNbO42 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal loading rate of the test item): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L and 73.4 mg/L (CI: 31.6 – 100 mg/L), respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L and 10.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

31.6 mg/L