Manganese dioxide

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:

15 February 2010 to 23 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 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 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 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 material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15/09/2009. Date of signature: 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 to give the 100% v/v saturated solution (equivalent to 4.0 mg/L as test material based on the mean measured test concentration).

- Sampling method:
The concentration of the test material 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 (especially for difficult test substances)
- Method: For the purpose of the definitive test the test material was prepared as a saturated solution.
An amount of test material (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 material was removed by filtration twice 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 material based on the mean measured test concentration).

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

- Controls: The control group was maintained under identical conditions but not exposed to the test material.

- Positive control: 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.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Source: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): 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. For the definitive test, the daphnids were placed in the test vessels at 14.30. Isolation of the young occurred at 14.10 the previous day. This meant that a few of the daphnia young may have been slightly in excess of 24 hours old. As the daphnia used were less than 30 minutes older than the required 24 hours old this deviation was considered not to have affected the integrity or outcome of the study.
- 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.).

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 controlled at 20°C to 22°C

pH:

pH was recorded at the start and termination of the test. The pH using a WTW pH/Oxi 340I meter

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentrations were measured using a WTW pH/Oxi 340I meter

Nominal and measured concentrations:

Range-finding: 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.
Definitive test: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars.
- Type : covered
- 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): 4
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (see appendix 2 - attached background material).
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 covered to reduce evaporation and maintained in a temperature controlled room at 20°C to 22°C 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:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. 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.
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

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

Details on results:

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

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

Analysis of the 100% v/v saturated solution (see Appendix 3 - attached background material) showed a measured concentration of manganese of 0.0186 mg/L (equivalent to 0.0294 mg/L as test material).

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 material contained a theoretical manganese content of 63% w/w. The test samples were analysed for manganese only. Analysis of the fresh test preparations (see Appendix 3) at 0 hours showed measured concentrations of 0.0647 and 0.0653 mg/L as test material. Analysis of the old media at 48 hours showed measured concentrations of 0.0767 and 0.0873 mg/L as test material.
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 material only. This was calculated to be 0.0735 mg/L.

Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.
There was no immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution (equivalent to 0.0735 mg/L as test material 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 0.0735 mg/L as test material based on the mean measured test concentration). The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

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 20°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Some of the temperatures were observed to be slightly in excess of the range given in the study plan of 20 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperature range was within guideline specification.

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 material).

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:
24 hour EC50 (mg/L): 0.84 (95% confidence limits 0.72 - 0.97 mg/L).
48 hour EC50 (mg/L): 0.65 (95% confidence limits 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).

Table 1: Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Cumulative Immobilised Daphnia (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	0

Table 2: Cumulative Immobilisation Data in the Definitive Test

Mean Measured Concentration (mg/L as Test Material)		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
0.0735	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

R₁– R₄= Replicates 1 to 4

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 0.0735 mg/l. Correspondingly the No Observed Effect Concentration was 0.0735 mg/l.

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

Executive summary:

The short term toxicity of the test material to aquatic invertebrates was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202 and EU Method C.2.

As the test material is considered to be poorly soluble in water the study was conducted with a saturated solution of test material.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material 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 material solution was prepared by stirring an excess (100 mg/L) of test material in reconstituted water using a magnetic stirrer at approximately 100 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm filter to give a saturated solution of the test material. 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 magnawere 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.
No daphnia immobilisation was observed over the 48 hour test period. The 48-hour EC50 was therefore determined to be greater than 100% v/v saturated solution. This study showed that there were no toxic effects at the limit of solubility of the test material in the test medium.

Description of key information

No toxic effects at the limit of solubility of the test material in the test meidum, OECD 202, EU Method C.2, Goodband & Mullee (2010)

Additional information

The short term toxicity of the test material to aquatic invertebrates was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202 and EU Method C.2.

As the test material is considered to be poorly soluble in water the study was conducted with a saturated solution of test material.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material 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 material solution was prepared by stirring an excess (100 mg/L) of test material in reconstituted water using a magnetic stirrer at approximately 100 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm filter to give a saturated solution of the test material. 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 were 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.
No daphnia immobilisation was observed over the 48 hour test period. The 48-hour EC50 was therefore determined to be greater than 100% v/v saturated solution. This study showed that there were no toxic effects at the limit of solubility of the test material in the test medium.