Manganese oxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2010 and 13 March 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and current guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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 items, 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 item in cases where the test item 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 item in reconstituted water for a period of 48 hours prior to removing any undissolved test material present by filtration to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
For the purpose of the definitive test the test item was prepared as a saturated solution.
An amount of test item (250 mg) was added to 2.5 litres of reconstituted water and stirred using a magnetic stirrer at approximately 100 rpm at approximately 21°C for 48 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 μm Gelman AcroCap filter (first approximate 500 ml discarded to pre-condition the filter) to give the 100% v/v saturated solution (equivalent to 4.0 mg/l as test item based on the mean measured test concentration).

- Sampling method:
The concentration of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours.

Water samples were taken from the control (replicates R1 – R2 pooled) and the 100% v/v saturated solution test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
For the purpose of the definitive test the test item was prepared as a saturated solution.
An amount of test item (250 mg) was added to 2.5 litres of reconstituted water and stirred using a magnetic stirrer at approximately 100 rpm at approximately 21°C for 48 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 μm Gelman AcroCap filter (first approximate 500 ml discarded to pre-condition the filter) to give the 100% v/v saturated solution (equivalent to 4.0 mg/l as test item based on the mean measured test concentration).

- Eluate:
Test water - The reconstituted water for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Controls:
A positive control conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at approximately 20°C.

Test organisms (species):

Daphnia magna

Details on test organisms:

EXAMPLE:
TEST ORGANISM
- Common name:
Water Flea

- Source:
1st instar Daphnia magna derived from in-house laboratory cultures.

- Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

- Feeding during test:
Each culture was fed daily with a suspension of algae (Chlorella sp.).

ACCLIMATION

- Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20°C.


- Type and amount of food:
Each culture was fed daily with a suspension of algae (Chlorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation:
No mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at approximately 20.0°C throughout the test.

pH:

pH was recorded at the start and termination of the test. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH meter.
pH range for test samples: 7.8-8.6

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH meter.
The oxygen concentration in some of the test vessels was observed to have an air
saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Salinity:

Not applicable.

Nominal and measured concentrations:

Range-finding test:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Definitve test:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass jars containing approximately 200 ml of test preparation were used.
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: 250 ml
- Aeration: Test vessels not aerated.
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS (see attached background material Appendix 2).
Reconstituted water.

i) Stock Solutions
a) CaCl2.2H2O: 11.76 g/l
b) MgSO4.7H2O: 4.93 g/l
c) NaHCO3: 2.59 g/l
d) KCl: 0.23 g/l

ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 μS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.


OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
- Photoperiod: The test vessels were maintained in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal 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 results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution.

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 48-Hour EC50 of greater than 100% v/v saturated solution.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on the mean measured test concentration as test item the 48-Hour EC50 was estimated to be greater than 4.0 mg/l.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The No Observed Effect Concentration was 100% v/v saturated solution

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on the mean measured test concentration the No Observed Effect Concentration was 4.0 mg/l.

Details on results:

RANGE-FINDING TEST:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No significant immobilisation was observed at any of the test concentrations employed in the test.

A single immobilised daphnid was observed at the 100% v/v saturated solution test concentration after 48 hours exposure. This was considered to be possibly due to natural causes rather than a toxic effect given that no other immobilisation was observed.

Analysis of the 100% v/v saturated solution (see Appendix 3) showed a measured concentration of manganese of 3.17 mg/l (equivalent to 4.09 mg/l as test item).
Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.

DEFINITIVE TEST:

Verification of test concentrations:
The test item contained a theoretical manganese content of 77% w/w as manganese. The test samples were analysed for manganese only. Analysis of the fresh test preparations (see Appendix 3) at 0 hours showed measured concentrations of 3.96 and 4.10 mg/l as test item. Analysis of the old media at 48 hours showed measured concentrations of 3.92 and 4.12 mg/l as test item.
Given that no decline in measured concentration was observed over the test period, the results are based on the mean measured test concentration as test item only. This was calculated to be 4.0 mg/l.


Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

There was no significant immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution (equivalent to 4.0 mg/l as test item based on the mean measured test concentration) for a period of 48 hours.

The No Observed Effect Concentration after 24 and 48 hours exposure was 100% v/v saturated solution (equivalent to 4.0 mg/l as test item based on the mean measured test concentration). The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

A single immobilised daphnid was observed at the 100% v/v saturated solution test concentration after 48 hours exposure. This was considered to be possibly due to natural causes rather than a toxic effect given that no other immobilisation was observed.

Observations on test item solubility:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements:
The results of the physico-chemical measurements are given in Appendix 4 (see attached background material).. Temperature was maintained at approximately 20°C throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the control and 100% v/v saturated solution test concentration at 0 Hours.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Results with reference substance (positive control):

Cumulative immobilisation data from the exposure of Daphnia magna to the reference item during the positive control are
given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 (see attached background information).

Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:

24h EC50 (mg/l): 0.84 (95% CL 0.72 - 0.97 mg/l)
48h EC50 (mg/l): 0.65 (95% CL 0.58 - 0.72 mg/l)

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).

Reported statistics and error estimates:

None reported.

Table 1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	0	1[1]

 

[1]Immobilisation considered to be possibly due to natural causes rather than a toxic effect given that no other immobilisation was observed.

Table 2              Cumulative Immobilisation Data in the Definitive Test

Mean Measured Concentration (mg/l as Test Item)		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
4.0	R1	0	0	0	0	1[1]	5
	R2	0			1
	R3	0			0
	R4	0			0

R₁– R₄= Replicates 1 to 4

[1]Immobilisation considered to be possibly due to natural causes rather than a toxic effect given that no other immobilisation was observed.

Table 3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	1	1	2	10	3	2	5	25
1.0	7	7	14	70	10	10	20	100
1.8	10	10	20	100	10	10	20	100
3.2	10	10	20	100	10	10	20	100

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.
Based on the mean measured test concentration as test item the 48-Hour EC50 was estimated to be greater than 4.0 mg/l. Correspondingly the No Observed Effect Concentration was 4.0 mg/l.
This study showed that there were no toxic effects at saturation.

Executive summary:

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality to fulfil the data requirement

 

The study report includes the entire information requirement as appropriate for OECD 202 (Daphnia sp. Acute Immobilisation Test). The test fulfilled the following validity criteria:

• In the control the percentage of daphnids immobilised was below 10%
• The dissolved oxygen concentration at the end of the end was ≥ 3 mg/L in control and test vessels. 
• The results of the positive control with potassium dichromate were within the normal range for this reference item.

Therefore the quality criteria of the study have been fulfilled as the study was conducted to guideline and to GLP.

 

This study showed that there were no toxic effects at saturation. Lack of any observed effect in the study supports the lack of classification and labelling of this substance.

Introduction.

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Information provided by the Sponsor indicated that the test item was insoluble in water.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on information supplied by the Sponsor it was considered that the most appropriate method of preparation for the test item was as a saturated solution.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 20°C under static test conditions. The test item solution was prepared by stirring an excess (100 mg/l) of test item in reconstituted water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test item was removed 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 item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna was exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a

temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

The test item contained a theoretical manganese content of 77% w/w as manganese. The test samples were analysed for manganese only. Analysis of the fresh test preparations at 0 hours showed measured concentrations of 3.96 and 4.10 mg/l as test item. Analysis of the old media at 48 hours showed measured concentrations of 3.92 and 4.12 mg/l as test item.

Given that no decline in measured concentration was observed over the test period, the results are based on the mean measured test concentration as test item only. This was calculated to be 4.0 mg/l.

The 48-Hour EC50 for the test item to Daphnia magna, based on the mean measured test concentration as test item, was greater than 4.0 mg/l and correspondingly the No Observed Effect Concentration was 4.0 mg/l.

This study showed that there were no toxic effects at saturation.

The 48-Hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.65 mg/l with 95% confidence limits of 0.58 – 0.72 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

Based on the mean measured test concentration as test item the 48-Hour EC50 was estimated to be greater than 4.0 mg/l. Correspondingly the No Observed Effect Concentration was 4.0 mg/l.

This study showed that there were no toxic effects at saturation.

Additional information

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality to fulfil the data requirement

 

The study report includes the entire information requirement as appropriate for OECD 202 (Daphnia sp. Acute Immobilisation Test). The test fulfilled the following validity criteria:

• In the control the percentage of daphnids immobilised was below 10%
• The dissolved oxygen concentration at the end of the end was ≥ 3 mg/L in control and test vessels. 
• The results of the positive control with potassium dichromate were within the normal range for this reference item.

Therefore the quality criteria of the study have been fulfilled as the study was conducted to guideline and to GLP.

 

This study showed that there were no toxic effects at saturation. Lack of any observed effect in the study supports the lack of classification and labelling of this substance.

Introduction.

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Information provided by the Sponsor indicated that the test item was insoluble in water.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on information supplied by the Sponsor it was considered that the most appropriate method of preparation for the test item was as a saturated solution.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 20°C under static test conditions. The test item solution was prepared by stirring an excess (100 mg/l) of test item in reconstituted water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test item was removed 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 item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna was exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a

temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

The test item contained a theoretical manganese content of 77% w/w as manganese. The test samples were analysed for manganese only. Analysis of the fresh test preparations at 0 hours showed measured concentrations of 3.96 and 4.10 mg/l as test item. Analysis of the old media at 48 hours showed measured concentrations of 3.92 and 4.12 mg/l as test item.

Given that no decline in measured concentration was observed over the test period, the results are based on the mean measured test concentration as test item only. This was calculated to be 4.0 mg/l.

The 48-Hour EC50 for the test item to Daphnia magna, based on the mean measured test concentration as test item, was greater than 4.0 mg/l and correspondingly the No Observed Effect Concentration was 4.0 mg/l.

This study showed that there were no toxic effects at saturation.

The 48-Hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.65 mg/l with 95% confidence limits of 0.58 – 0.72 mg/l. The No Observed Effect Concentration was 0.32 mg/l.