Manganese sulphide

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:

from 2010-02-28 to 2010-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted to GLP and to current guidelines with no deficiencies.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative 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:

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 item (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 item 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 (7.3 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

In the definitive test an amount of test item (300 mg) was dispersed in 3 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 item 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 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 5.9 mL algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (equivalent to 0.32, 0.63, 1.3, 2.6 and 5.2 mg/L as test item based on the mean measured test concentrations obtained). The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration of the test item in the test solutions were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source: Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- 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 1.0E+3 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 1.0E+4 - 1.0E+5 cells/mL.

ACCLIMATION
- Acclimation period: yes
- Culturing media and conditions the same as the test conditions.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

not reported

Test temperature:

24 ± 1ºC

pH:

7.2-7.6 (Table 2)

Dissolved oxygen:

not reported

Salinity:

not reported

Nominal and measured concentrations:

In the range-finding test a series of nominal concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution was used. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (equivalent to 0.32, 0.63, 1.3, 2.6 and 5.2 mg/L as test item based on the mean measured test concentrations obtained).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: 250 mL flasks containing of 100 mL of test preparation
- Mean cell density of control at 0 hours: 4.27E+03 cells per mL
- Mean cell density of control at 72 hours: 2.15E+05 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP-medium (US. EPA)

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

TEST CONDITIONS
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 1.0E+03 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 1.0E+04 - 1.0E+05 cells/mL.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell concentration, samples were taken at 0, 24, 45 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: 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.
- Test concentrations: In the definitive study nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were used.
- Results used to determine the conditions for the definitive study: The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 1. The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Details on results:

The growth rate (r) and yield (y) of Desmodesmus subspicatus were affected by the presence of the test item over the 72 hour exposure period. The following results were determined from the data based on the mean measured test concentrations:
Growth rate
ErC10 (0 - 72 h) : 3.4 mg/L
ErC20 (0 - 72 h) : 3.9 mg/L
ErC50 (0 - 72 h) : 4.9 mg/L
No Observed Effect Concentration (NOEC) = 2.6 mg/L
Lowest Observed Effect Concentration (LOEC) = 5.2 mg/L
Yield
EyC10 (0 - 72 h) : 2.5 mg/L
EyC20 (0 - 72 h) : 2.9 mg/L
EyC50 (0 - 72 h) : 3.6 mg/L
No Observed Effect Concentration (NOEC) = 2.6 mg/L
Lowest Observed Effect Concentration (LOEC) = 5.2 mg/L

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility
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, 6.25, 12.5, 25 and 50% v/v saturated solution test cultures were observed to be pale green dispersions whilst the 100% v/v saturated solution test cultures were observed to be clear colourless solutions.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h): 0.49 mg/L*
EyC50 (0 – 72 h): 0.18 mg/L, 95% confidence limits 0.16 – 0.21 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/L
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

Statistical analyses of the growth rate and yield data were carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5, 25 and 50% v/v saturated solution test concentrations (P≥0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate as well as based on yield was 50% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate as well as on yield was 100% v/v saturated solution.

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 1. The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

Table1            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	3.86E+03	1.98E+05
	R2	4.30E+03	1.77E+05	-	-
	Mean	4.08E+03	1.88E+05
0.10	R1	4.10E+03	2.78E+05
	R2	3.81E+03	3.02E+05	[13]	[56]
	Mean	3.96E+03	2.90E+05
1.0	R1	3.84E+03	2.75E+05
	R2	3.38E+03	2.65E+05	[13]	[45]
	Mean	3.61E+03	2.70E+05
10	R1	3.93E+03	2.07E+05
	R2	4.13E+03	2.47E+05	[6]	[21]
	Mean	4.03E+03	2.27E+05
100	R1	4.22E+03	3.01E+04
	R2	3.86E+03	1.56E+04	55	90
	Mean	4.04E+03	2.29E+04

 

 Table 2 Cell Densities and pH Values in the Definitive Test

Mean Measured Test Item Concentration (mg/L)		pH	Cell Densities*(cells per mL)				pH
		0 h	0 h	24 h	45 h	72 h	72 h
Control	R1	7.3	4.46E+03	1.29E+04	5.43E+04	2.09E+05	7.6
	R2	7.3	4.25E+03	1.29E+04	2.81E+04	2.06E+05	7.6
	R3	7.3	4.18E+03	1.31E+04	2.52E+04	2.05E+05	7.6
	R4	7.3	4.08E+03	1.28E+04	2.86E+04	2.11E+05	7.5
	R5	7.3	4.22E+03	1.15E+04	3.45E+04	2.27E+05	7.6
	R6	7.3	4.44E+03	1.04E+04	2.89E+04	2.32E+05	7.5
	Mean		4.27E+03	1.22E+04	3.33E+04	2.15E+05
0.32	R1	7.3	4.00E+03	1.07E+04	3.21E+04	2.66E+05	7.5
	R2	7.3	4.29E+03	9.28E+03	3.08E+04	2.20E+05	7.6
	R3	7.3	4.04E+03	1.09E+04	3.06E+04	1.78E+05	7.6
	Mean		4.11E+03	1.03E+04	3.12E+04	2.21E+05
0.63	R1	7.2	4.28E+03	5.01E+03	3.84E+04	2.16E+05	7.5
	R2	7.2	4.41E+03	4.81E+03	3.91E+04	2.11E+05	7.5
	R3	7.2	3.97E+03	8.40E+03	4.88E+04	2.26E+05	7.5
	Mean		4.22E+03	6.07E+03	4.21E+04	2.18E+05
1.3	R1	7.2	3.99E+03	5.90E+03	1.36E+04	1.75E+05	7.5
	R2	7.2	4.22E+03	7.63E+03	2.61E+04	1.81E+05	7.5
	R3	7.2	4.34E+03	8.43E+03	2.08E+04	1.98E+05	7.5
	Mean		4.18E+03	7.32E+03	2.02E+04	1.85E+05
2.6	R1	7.2	4.06E+03	7.10E+03	2.28E+04	2.06E+05	7.4
	R2	7.2	4.19E+03	7.13E+03	1.88E+04	1.80E+05	7.4
	R3	7.2	4.46E+03	7.21E+03	3.03E+04	1.89E+05	7.4
	Mean		4.24E+03	7.14E+03	2.40E+04	1.92E+05
5.2	R1	7.2	4.08E+03	9.48E+03	1.62E+04	1.62E+04	7.4
	R2	7.2	4.14E+03	9.42E+03	1.36E+04	1.30E+04	7.4
	R3	7.2	4.41E+03	8.22E+03	1.96E+04	4.51E+04	7.4
	Mean		4.21E+03	9.04E+03	1.65E+04	2.47E+04

 

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		0.049		0.069		0.050
		R2		0.049		0.037		0.074
		R3		0.049		0.031		0.078
		R4		0.048		0.038		0.074
		R5		0.044		0.052		0.070
		R6		0.040		0.049		0.077
		Mean		0.047		0.046		0.071

 

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

Mean Measured Test Item Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.055		2.05E+05
	R2	0.055		2.02E+05
	R3	0.055		2.01E+05
	R4	0.055	-	2.07E+05	-
	R5	0.056		2.23E+05
	R6	0.056		2.27E+05
	Mean	0.055		2.11E+05
	SD	0.001		1.12E+04
0.32	R1	0.058	[5]	2.62E+05
	R2	0.056	[2]	2.16E+05
	R3	0.053	4	1.74E+05
	Mean	0.056	[1]	2.17E+05	[3]
	SD	0.003		4.40E+04
0.63	R1	0.055	0	2.11E+05
	R2	0.055	0	2.07E+05
	R3	0.056	[2]	2.22E+05
	Mean	0.055	[1]	2.14E+05	[1]
	SD	0.001		7.92E+03
1.3	R1	0.053	4	1.71E+05
	R2	0.053	4	1.77E+05
	R3	0.054	2	1.93E+05
	Mean	0.053	3	1.80E+05	14
	SD	0.001		1.14E+04
2.6	R1	0.055	0	2.02E+05
	R2	0.053	4	1.76E+05
	R3	0.054	2	1.84E+05
	Mean	0.054	2	1.88E+05	11
	SD	0.001		1.35E+04
5.2	R1	0.019	65	1.21E+04
	R2	0.016	71	8.81E+03
	R3	0.034	38	4.07E+04
	Mean	0.023	58	2.05E+04	90
	SD	0.010		1.75E+04

[ ]

*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]

*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

R₁- R₆= Replicates 1 to 6

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

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 (0 - 72 h) of MnS was 4.9 mg/L. The No Observed Effect Concentration (NOEC) of MnS was 2.6 mg/L.

Executive summary:

The toxicity of the test material to algae was assessed according to OECD Test Guideline 201 and EU Method C.3 and in compliance with GLP.

The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 % v/v saturated solution. However, growth was observed to be reduced at 100 % v/v saturated solution. The ErC50 (0 - 72 h) of the test material was 4.9 mg/L. The No Observed Effect Concentration (NOEC) was 2.6 mg/L.

Description of key information

The  ErC50 (0 - 72 h) of the test material was  4.9 mg/L. The No Observed Effect Concentration (NOEC) was  2.6 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.9 mg/L

EC10 or NOEC for freshwater algae:

2.6 mg/L

Additional information

The toxicity of the test material to algae was assessed according to OECD Test Guideline 201 and EU Method C.3 and in compliance with GLP.

The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 % v/v saturated solution. However, growth was observed to be reduced at 100 % v/v saturated solution. The ErC50 (0 - 72 h) of the test material was 4.9 mg/L. The No Observed Effect Concentration (NOEC) was 2.6 mg/L.