Manganese oxide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2010 to 21 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to GLP and guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 dechlorinated tap water for a period of 48 hours prior to removing any undissolved test item 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: 26/11/09

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Nominal concentration of 100% v/v saturated solution.

- Sampling method:
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis.

- Sample storage conditions before analysis:
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) 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 item was prepared as a saturated solution.

An amount of test item (2100 mg) was added to 21 litres of dechlorinated tap water and stirred using a magnetic stirrer at approximately 100 rpm at approximately 14°C for 48 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded to pre-condition the filter) to give the 100% v/v saturated solution.

This method of preparation was conducted in duplicate to give replicates R1 and R2.

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

- Controls:
Control groups were maintained under identical conditions but not exposed to the test material.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Strain: Not reported
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): Juvenile.
- Length at study initiation (length definition, mean, range and SD): Fish had a mean standard length of 4.3 cm (sd = 0.2)
- Weight at study initiation (mean and range, SD): Mean weight of 0.85g (sd = 0.16) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.30g bodyweight/litre.
- Method of breeding: Not reported.
- Feeding during test: discontinued 24 hours prior to the start of the definitive test
- Food type:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

ACCLIMATION
- Acclimation period:
Fish were acclimatised to test conditions from 24 February 2010 to 8 March 2010.

- Acclimation conditions (same as test or not):
Fish were maintained in a glass fibre tank with a "single pass" water renewal system. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14°C with a dissolved oxygen content of greater than or equal to 10.1 mg O2/l. These parameters were recorded daily.

- Health during acclimation (any mortality observed):
There was zero mortality in the 7 days prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure.

Hardness:

The water had a total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

Temperature maintained at 12 to 15°C throughout the test.
The water temperature was recorded daily throughout the test.

pH:

pH was recorded daily throughout the test.
pH range 7.7-8.3.
Differences in pH were observed between the control and 1.2 mg/l test concentration in the freshly prepared media.

Dissolved oxygen:

The dissolved oxygen concentrations were recorded daily throughout the test
The dissolved oxygen concentration was measured using a dissolved oxygen meter.
No treatment related differences for oxygen concentration were observed.

Salinity:

Not reported.

Nominal and measured concentrations:

Nominal test concentration of 100% v/v saturated solution in range-finding test and definitive test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 litre glass exposure vessels
- Type (delete if not applicable): covered
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate): A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 2
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (see attached background material).


OTHER TEST CONDITIONS
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

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.
In the range-finding test fish were exposed to a nominal test concentration of 100% v/v saturated solution. A single concentration was used as results from the range-finding test for the Acute Toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 2702/0143) indicated that toxicity was not expected at this concentration. The test item was prepared as a saturated solution.
In the range-finding test 3 fish were added to each test and control vessel, containing 18 litres of test preparation, and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.
The control group was maintained under identical conditions but not exposed to the test item.
Data from the control group was shared with similar concurrent studies.
Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

- 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 mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

other: LC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 as test item >1.2 mg/l

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: NOEC as test item 1.2 mg/l

Details on results:

Range-finding Test:
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the test concentration of 100% v/v saturated solution.
Analysis of the 100% v/v saturated solution (see Appendix 3 - attached background material) showed a measured concentration of manganese of 1.16 mg/l (equivalent to 1.49 mg/l as test item) was attained.
Based on this information, a single test concentration, in duplicate, 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 mortalities or sub lethal effects of exposure were observed.

Definitive Test:
Mortality data:-
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2.
There were no mortalities in 14 fish exposed to a test concentration of 100% v/v saturated solution (equivalent to 1.2 mg/l as test item based on the mean measured test concentration) for a period of 96 hours.

The results of the definitive test showed the No Observed Effect Concentration (NOEC) to be 100% v/v saturated solution (equivalent to 1.2 mg/l as test item based on the mean measured test concentration). The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

Sub-lethal effects:
There were no sub-lethal effects of exposure observed in 14 fish exposed to a test concentration of 100% v/v saturated solution (equivalent to 1.2 mg/l as test item based on the mean measured test concentration) for a period of 96 hours.

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 12°C to 15°C throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the control and 1.2 mg/l test concentration in the freshly prepared media.
A single temperature during the definitive test was observed to be slightly below the range given in the protocol of 14 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as no sub-lethal effects or mortality were observed over the test period.
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):

Not applicable.

Sublethal observations / clinical signs:

Verification of test concentrations

The test item contained a theoretical manganese content of 77% w/w. The test samples were analysed for manganese only.   

Initial analysis of the test preparations (see Appendix 3 - attached background material) at 0 (fresh media) and 24 hours (old media) showed measured concentrations of 0.363 and 0.360 mg/l as test item and 8.39 and 9.04 mg/l as test item for the 100% v/v saturated solution Replicates R₁and R₂respectively. Analysis of the corresponding duplicate samples, stored frozen prior to analysis, confirmed these results. Analysis of the remaining samples at 24 (fresh media), 48 (old and fresh media), 72 (fresh media) and 96 hours (old media) showed measured concentrations to range from 0.479 to 2.14 mg/l as test item. Given this range it was considered that the high measured concentrations in the 100% v/v saturated solution Replicate R₂was possibly due to contamination from an unknown source. This was considered not to have affected the outcome of the study given that no mortalities or sub-lethal effects were observed throughout the duration of the test.

Result Tables:

Table 1              Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0
100	0	0	0	0	0	0

Table 2              Cumulative Mortality Data in the Definitive Test

Mean Measured Concentration (mg/l as Test Item)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
1.2 R1	0	0	0	0	0	0	0
1.2 R2	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 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 concentrations as test item the acute toxicity of the test item to rainbow trout gave a 96-Hour LC50 value of greater than 1.2 mg/l. The No Observed Effect Concentration was 1.2 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 203 (Fish, Acute Toxicity Test). The test fulfilled the following validity criteria:

• The mortality in the control(s) did not exceed 10 per cent at the end of the test.
• The dissolved oxygen concentration was greater than 60 % of the air saturation value throughout the test.

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 rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 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 fish were exposed, in two groups of seven, to an aqueous solution of the test item, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of 12ºC to 15°C under semi-static test conditions. The test item solution was prepared by stirring an excess (100 mg/l) of test item in dechlorinated tap water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 14°C for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution of the test item. 

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC₅₀based 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. The test samples were analysed for manganese only.   

Initial analysis of the test preparations at 0 (fresh media) and 24 hours (old media) showed measured concentrations of 0.363 and 0.360 mg/l as test item and 8.39 and 9.04 mg/l as test item for the 100% v/v saturated solution Replicates R₁and R₂respectively. Analysis of the corresponding duplicate samples, stored frozen prior to analysis, confirmed these results. Analysis of the remaining samples at 24 (fresh media), 48 (old and fresh media), 72 (fresh media) and 96 hours (old media) showed measured concentrations to range from 0.479 to 2.14 mg/l as test item. Given this range it was considered that the high measured concentrations in the 100% v/v saturated solution Replicate R₂was possibly due to contamination from an unknown source. This was considered not to have affected the outcome of the study given that no mortalities or sub-lethal effects were observed throughout the duration of the test.

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. The high results from the 100% v/v saturated solution Replicate R₁at 0 and 24 hours were not included in the calculation. This was calculated to be 1.2 mg/l.

The 96-Hour LC₅₀based on the mean measured test concentrations as test item was greater than 1.2 mg/l and correspondingly the No Observed Effect Concentration was 1.2 mg/l.

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

Description of key information

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 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 concentrations as test item the acute toxicity of the test item to rainbow trout gave a 96-Hour LC50 value of greater than 1.2 mg/l. The No Observed Effect Concentration was 1.2 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 203 (Fish, Acute Toxicity Test). The test fulfilled the following validity criteria:

• The mortality in the control(s) did not exceed 10 per cent at the end of the test.
• The dissolved oxygen concentration was greater than 60 % of the air saturation value throughout the test.

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 rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 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 fish were exposed, in two groups of seven, to an aqueous solution of the test item, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of 12ºC to 15°C under semi-static test conditions. The test item solution was prepared by stirring an excess (100 mg/l) of test item in dechlorinated tap water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 14°C for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution of the test item. 

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC₅₀based 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. The test samples were analysed for manganese only.   

Initial analysis of the test preparations at 0 (fresh media) and 24 hours (old media) showed measured concentrations of 0.363 and 0.360 mg/l as test item and 8.39 and 9.04 mg/l as test item for the 100% v/v saturated solution Replicates R₁and R₂respectively. Analysis of the corresponding duplicate samples, stored frozen prior to analysis, confirmed these results. Analysis of the remaining samples at 24 (fresh media), 48 (old and fresh media), 72 (fresh media) and 96 hours (old media) showed measured concentrations to range from 0.479 to 2.14 mg/l as test item. Given this range it was considered that the high measured concentrations in the 100% v/v saturated solution Replicate R₂was possibly due to contamination from an unknown source. This was considered not to have affected the outcome of the study given that no mortalities or sub-lethal effects were observed throughout the duration of the test.

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. The high results from the 100% v/v saturated solution Replicate R₁at 0 and 24 hours were not included in the calculation. This was calculated to be 1.2 mg/l.

The 96-Hour LC₅₀based on the mean measured test concentrations as test item was greater than 1.2 mg/l and correspondingly the No Observed Effect Concentration was 1.2 mg/l.

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