Manganese ores, reduced

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-02-2010 to 26-04-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP and to a modern guideline. Read-across from FeMn slag to sinter ore is justified on the basis of similar constituents, very poor water solubility and leaching in the TDp study. Sinter ore has a lower solubility and leaching compared to FeMn slag.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Initial test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/l
Definitive test: 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
- Sample storage conditions before analysis: stored at -20°C

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus)
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Laboratory culture (originally sourced from the Culture collection of Algae and Protozoa (CCAP), Scotland)
- Method of cultivation: In culture media in conical flasks with an initial cell density of approximately 10^3 cells/mL

ACCLIMATION
- Acclimation period: Not stated
- Culturing media and conditions (same as test or not):The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24±1°C

pH:

7.6 - 7.9

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured concentrations of manganese to be less than the limit of quantitation (LOQ) of the analytical method employed. This does not infer that no test item was in solution, just that that which was present was at a concentration below the LOQ.
Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): closed
- Aeration: No
- Initial cells density:4.29x10^3 cells/mL
- Control end cells density: 3.15x10^5 cells/mL
- No. of vessels per concentration (replicates):Three
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: 7000 lux 380-730 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were determined using a Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
- Range finding study:
Initial range finding test: 10, 100 mg/L
Second range finding test: 1.0, 10 mg/L
Third range finding test: 0.010, 0.10, 1.0, 10, 100 mg/L
- Test concentrations:0, 0.32, 10, 32, 100 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 90 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate: Water Accommodated Fraction (WAF)

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 39 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate: Water Accommodated Fraction (WAF)

Basis for effect:

biomass

Remarks on result:

other: 34-46

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate: Water Accommodated Fraction (WAF)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate: Water Accommodated Fraction (WAF)

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None reported
- Unusual cell shape: None reported
- Colour differences: None reported
- Flocculation: None reported
- Adherence to test vessels:None reported
- Aggregation of algal cells:None reported

Reported statistics and error estimates:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups.

Range finding test:

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding tests showed no effect on growth at 0.010 mg/l loading rate WAF. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/l loading rate WAF

From the data , it is clear that the growth rate (r) and yield (y) and of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test item over the 72-Hour exposure period.

Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.4	3.39E+03	1.14E+04	4.31E+04	2.75E+05	7.8
	R2	7.4	4.41E+03	1.09E+04	3.99E+04	3.13E+05	7.8
	R3	7.4	3.62E+03	1.14E+04	3.93E+04	2.73E+05	7.8
	R4	7.4	5.18E+03	1.09E+04	4.07E+04	3.49E+05	7.8
	R5	7.4	5.12E+03	1.24E+04	3.95E+04	3.35E+05	7.8
	R6	7.4	4.01E+03	1.18E+04	4.76E+04	3.43E+05	7.8
	Mean		4.29E+03	1.15E+04	4.17E+04	3.15E+05
0.32	R1	7.3	3.18E+03	1.19E+04	3.90E+04	3.43E+05	7.9
	R2	7.3	4.00E+03	1.13E+04	3.81E+04	3.24E+05	7.9
	R3	7.3	4.03E+03	1.19E+04	4.53E+04	3.33E+05	7.8
	Mean		3.74E+03	1.17E+04	4.08E+04	3.33E+05
1.0	R1	7.2	4.19E+03	5.64E+03	2.01E+04	3.45E+05	7.7
	R2	7.2	3.42E+03	8.09E+03	2.92E+04	3.51E+05	7.7
	R3	7.2	3.53E+03	3.74E+03	1.27E+04	3.51E+05	7.7
	Mean		3.71E+03	5.82E+03	2.07E+04	3.49E+05
3.2	R1	7.2	4.10E+03	6.14E+03	2.76E+04	2.85E+05	7.7
	R2	7.2	3.82E+03	8.33E+03	3.01E+04	3.12E+05	7.7
	R3	7.3	3.98E+03	6.94E+03	1.36E+04	3.38E+05	7.7
	Mean		3.96E+03	7.14E+03	2.38E+04	3.12E+05
10	R1	7.3	4.18E+03	8.02E+03	2.18E+04	3.69E+05	7.6
	R2	7.4	4.42E+03	6.20E+03	3.36E+04	3.07E+05	7.6
	R3	7.4	3.82E+03	6.46E+03	2.98E+04	3.15E+05	7.7
	Mean		4.14E+03	6.89E+03	2.84E+04	3.30E+05
32	R1	7.6	4.11E+03	4.46E+03	2.34E+04	2.13E+05	7.7
	R2	7.6	4.16E+03	4.66E+03	2.07E+04	2.09E+05	7.7
	R3	7.6	4.48E+03	5.60E+03	2.26E+04	1.93E+05	7.7
	Mean		4.25E+03	4.91E+03	2.22E+04	2.05E+05
100	R1	8.8	5.44E+03	1.31E+04	2.13E+04	2.58E+04	7.9
	R2	8.8	4.59E+03	1.12E+04	2.10E+04	2.70E+04	7.9
	R3	8.8	5.18E+03	4.38E+03	3.09E+04	3.10E+04	7.8
	Mean		5.07E+03	9.55E+03	2.44E+04	2.79E+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.

R₁- R₆= Replicates 1 to 6

Inhibition of Growth Rate and Yield the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.059		2.71E+05
	R2	0.061		3.09E+05
	R3	0.059		2.69E+05
	R4	0.062	-	3.44E+05	-
	R5	0.061		3.30E+05
	R6	0.062		3.39E+05
	Mean	0.061		3.10E+05
	SD	0.001		3.32E+04
0.32	R1	0.062	[2]	3.40E+05
	R2	0.061	0	3.20E+05
	R3	0.061	0	3.29E+05
	Mean	0.061	[1]	3.30E+05	[6]
	SD	0.001		1.00E+04
1.0	R1	0.062	[2]	3.41E+05
	R2	0.062	[2]	3.47E+05
	R3	0.062	[2]	3.48E+05
	Mean	0.062	[2]	3.45E+05	[11]
	SD	0.000		3.95E+03
3.2	R1	0.059	3	2.81E+05
	R2	0.061	0	3.09E+05
	R3	0.062	[2]	3.34E+05
	Mean	0.061	0	3.08E+05	1
	SD	0.002		2.65E+04
10	R1	0.063	[3]	3.65E+05
	R2	0.060	2	3.03E+05
	R3	0.061	0	3.11E+05
	Mean	0.061	[0]	3.26E+05	[5]
	SD	0.002		3.38E+04
32	R1	0.055	10	2.09E+05
	R2	0.055	10	2.05E+05
	R3	0.054	11	1.89E+05
	Mean	0.055	10	2.01E+05	35
	SD	0.001		1.06E+04
100	R1	0.026	57	2.04E+04
	R2	0.027	56	2.24E+04
	R3	0.028	54	2.58E+04
	Mean	0.027	56	2.28E+04	93
	SD	0.001		2.75E+03

*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 effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-hour period and gave the following results:
ErL50 = 90 mg/L (Loading rate WAF)
EbL50 = 39 mg/L (Loading rate WAF)
NOAEL = 10 mg/L (Loading rate WAF) (both biomass and growth rate)

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.

Following both preliminary range-finding tests and initial experiments, Desmodesmus subspicatus was exposed toWater Accommodated Fractions(WAFs) of the test material over a range of concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) 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 the 72-hour ErL50 was determined to be 90 mg/L loading rate WAF; the 72 -hour NOEL was determined to be 10 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole the results were based on nominal loading rates only.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 90 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 39 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

biomass

Description of key information

The effect of the read-across material, ferromanganese slag, on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:

ErL50 = 90 mg/L (Loading rate WAF)

EbL50 = 39 mg/L (Loading rate WAF)

NOAEL = 10 mg/L (Loading rate WAF) (both biomass and growth rate)

Key value for chemical safety assessment

EC50 for freshwater algae:

90 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

A study was performed to assess the effect of the read-across substance, ferromanganese slag, on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the standardised guidelines OECD 201and EU Method C.3 and was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). Read-across from FeMn slag to sinter ore is justified on the basis of similar constituents, very poor water solubility and leaching in the TDp study. Sinter ore has a lower solubility and leaching compared to FeMn slag.  

Following both preliminary range-finding tests and initial experiments, Desmodesmus subspicatus was exposed toWater Accommodated Fractions(WAFs) of the test material over a range of concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) 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 the 72-hour ErL50 was determined to be 90 mg/L loading rate WAF; the 72 -hour NOEL was determined to be 10 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole the results were based on nominal loading rates only.