Manganese dioxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2010 to 26 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(see "Principles of method if other than guideline")

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

(see "Principles of method if other than guideline")

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/L) of test material in culture medium for a period of 48 hours prior to removing any undissolved test material present by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15/09/09 Date of signature: 26/11/

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Standard solutions of manganese were prepared in 5% concentrated nitric acid in culture medium (v/v) at a nominal concentration of 1.0 mg/L

- Sampling method:
The concentration of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours.

The test samples were analysed following addition of nitric acid (1 mL per 20 mL of sample or equivalent).

- Sample storage conditions before analysis:
Samples were stored at approximately 4°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Range finding test:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
An amount of test material (200 mg) was dispersed in 2 litres of culture medium with the aid of magnetic stirring at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 ml discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (5.9 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Definitive test:
An amount of test material (250 mg) was dispersed in 2.5 litres of culture medium with the aid of magnetic stirring at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 ml discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (11.8 ml) to give the required test concentration of 100% v/v saturated solution.

The concentration of the test material in the test solutions was verified by chemical analysis at 0 and 72 hours

- Controls:
A positive control used potassium dichromate as the reference item.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: Strain CCAP 276/20
- Source: Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum : Not recorded
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.
Prior to the start of the test, sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 - 10^5 cells/mL.


ACCLIMATION
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture Medium:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test temperature:

The temperature within the incubator was recorded daily. Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.4 – 7.5 at 0 hours to pH 7.6 – 7.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250mL glass conical flasks
- Type : closed
- Fill volume: 100mL
- Aeration: yes
- Initial cells density: The algal suspension gave an initial nominal cell density of 4 x 10^3 cells per mL
- Control end cells density: 1.98 x 10^5 cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: (see appendix 3 for details)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl
- Photoperiod: constant illumination
- Light intensity and quality: (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): no
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control and test cultures were observed to be green dispersions.

- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project Number: 0039/1127) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 12 January 2010 and 15 January 2010.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100% v/v saturated solution test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P>0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

Verification of test concentrations:

The test item contains a theoretical manganese content of approximately 63% w/w. The test samples were analysed for manganese only. Analysis of the test preparations at 0 and 72 hours (see Appendix 4) showed measured test material concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.10 mg/L. This does not infer that no test material was in solution, just that that which was present was at a concentration of less than 0.10 mg/L.

Growth data:

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at a nominal test concentration of 100% v/v saturated solution over the 72 hour exposure period. Accordingly the following results were determined from the data:

 

Inhibition of growth rate:

E_rC₁₀(0 - 72 h)           : > 100% v/v saturated solution
E_rC₂₀(0 - 72 h)           : > 100% v/v saturated solution
E_rC₅₀(0 - 72 h)           : > 100% v/v saturated solution

where E_rC_xis the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (i.e. P > 0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

 

Inhibition of yield:

E_yC₁₀(0 - 72 h)          : > 100% v/v saturated solution
E_yC₂₀(0 - 72 h)          : > 100% v/v saturated solution
E_yC₅₀(0 - 72 h)          : > 100% v/v saturated solution

where E_yC_xis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as above. There were no statistically significant differences (i.e. P > 0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.

 

Table 1: Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	3.68E+03	9.00E+04
	R2	4.47E+03	8.08E+04	-	-
	Mean	4.08E+03	8.54E+04
0.10	R1	4.58E+03	1.48E+05
	R2	4.48E+03	1.60E+05	[17]	[84]
	Mean	4.53E+03	1.54E+05
1.0	R1	5.33E+03	7.46E+04
	R2	4.03E+03	7.45E+04	10	14
	Mean	4.68E+03	7.45E+04
10	R1	4.91E+03	9.52E+04
	R2	4.14E+03	8.17E+04	2	[3]
	Mean	4.53E+03	8.85E+04
100	R1	5.09E+03	9.52E+04
	R2	4.70E+03	1.08E+05	0	[19]
	Mean	4.89E+03	1.02E+05

 

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2: Cell Densities and pH Values in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities*(cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	4.62E+03	1.15E+04	4.11E+04	2.75E+05	7.7
	R2	7.5	4.35E+03	1.14E+04	3.08E+04	1.88E+05	7.7
	R3	7.5	3.86E+03	1.06E+04	3.16E+04	1.20E+05	7.7
	R4	7.4	3.86E+03	9.35E+03	3.72E+04	2.55E+05	7.7
	R5	7.4	4.98E+03	1.20E+04	3.34E+04	1.63E+05	7.7
	R6	7.4	4.62E+03	1.05E+04	3.27E+04	1.87E+05	7.6
	Mean		4.38E+03	1.09E+04	3.45E+04	1.98E+05
100	R1	7.3	4.54E+03	1.10E+04	4.84E+04	2.24E+05	7.6
	R2	7.3	4.22E+03	1.12E+04	2.12E+04	2.30E+05	7.6
	R3	7.3	4.74E+03	9.10E+03	2.30E+04	1.96E+05	7.6
	R4	7.2	4.48E+03	1.09E+04	4.80E+04	2.09E+05	7.6
	R5	7.2	3.97E+03	9.69E+03	2.79E+04	2.18E+05	7.6
	R6	7.2	4.72E+03	1.16E+04	2.97E+04	1.22E+05	7.6
	Mean		4.45E+03	1.06E+04	3.30E+04	2.00E+05

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

 

Table 3: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.044	0.053	0.079
	R2	0.044	0.041	0.075
	R3	0.041	0.045	0.056
	R4	0.035	0.058	0.080
	R5	0.046	0.043	0.066
	R6	0.040	0.047	0.073
	Mean	0.042	0.048	0.072

R₁- R₆= Replicates 1 to 6

 

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.059		2.70E+05
	R2	0.053		1.83E+05
	R3	0.047		1.16E+05
	R4	0.058	-	2.51E+05	-
	R5	0.051		1.58E+05
	R6	0.053		1.82E+05
	Mean	0.054		1.94E+05
	SD	0.004		5.78E+04
100	R1	0.056	[4]	2.19E+05
	R2	0.056	[4]	2.26E+05
	R3	0.054	0	1.91E+05
	R4	0.055	[2]	2.05E+05
	R5	0.056	[4]	2.14E+05
	R6	0.048	11	1.18E+05
	Mean	0.054	[1]	1.95E+05	[1]
	SD	0.003		4.00E+04

 

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration (NOEC) was 100% v/v saturated solution. This study showed that there were no toxic effects at saturation.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. It was therefore considered that the most appropriate method of preparation for the test material was as a saturated solution.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a nominal test concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.  Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Under the conditions of the study, exposure of Desmodesmus subspicatus to the test material gave EC₅₀ values of greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. Therefore, under the conditions of the study there were no toxic effects up to the limit of solubility of the test material in the test medium.

Description of key information

No toxic effects up to the limit of solubility of the test substance in the test medium. (72 hour EC50 > 100%v/v saturated soultion; 72 hour NOEC = 100%v/v saturated soultion), OECD 201, EU Method C.3, Vryenhoef & Mullee (2010)

Additional information

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. It was therefore considered that the most appropriate method of preparation for the test material was as a saturated solution.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a nominal test concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.  Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Under the conditions of the study, exposure of Desmodesmus subspicatus to the test material gave EC₅₀ values of greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.Therefore, under the conditions of the study there were no toxic effects up to the limit of solubility of the test material in the test medium.