Trimanganese tetraoxide

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:

2010-02-27 to 2010-03-29

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 guidelines and the study was conducted under GLP conditions.

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 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 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 of test material in dechlorinated tap 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:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 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.
- Sampling method: The test samples were analysed following addition of nitric acid (1.0 mL per 20 mL of sample).
- 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 (2200 mg) was added to 22 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 material 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.

- Eluate: 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 4.9 cm (sd = 0.7)
- Weight at study initiation (mean and range, SD): Mean weight of 1.38g (sd = 0.63) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.48g 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 10 March 2010 to 22 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:

Approximately 14°C

pH:

pH range 7.7-8.5.

Dissolved oxygen:

10.2-10.6 mg/L - Dissolved oxygen

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.


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. 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 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: 2702/0173) indicated that toxicity was not expected at this concentration.
- 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)

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)

Details on results:

Range-finding Test:
Cumulative mortality data from the exposure of rainbow trout to the test material 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. Increased pigmentation was observed in two of the fish at 96 hours. However, it was considered that this was possibly a stress response due to bullying by the remaining fish.
Analysis of the 100% v/v saturated solution (see Appendix 3) showed a measured concentration of manganese of less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.050 mg manganese/L). 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.

Verification of test concentrations:
The test material contained a theoretical manganese content of 72% w/w. The test samples were analysed for manganese only. Analysis of the test preparations (see Appendix 3) at 0, 24 and 96 hours showed measured concentrations of manganese to be less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.050 mg manganese/L). These results do not infer that no test item was present in the saturated solution merely that any test material present was below the LOQ of the analytical method.

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 significant mortalities in 14 fish exposed to a test concentration of 100% v/v saturated solution for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h) -3 =>100 LC50 (% v/v Saturated Solution)
Time (h) -3 =>100 LC50 (% v/v Saturated Solution)
Time (h) -6 =>100 LC50 (% v/v Saturated Solution)
Time (h) -24 =>100 LC50 (% v/v Saturated Solution)
Time (h) -48 =>100 LC50 (% v/v Saturated Solution)
Time (h) -72 =>100 LC50 (% v/v Saturated Solution)
Time (h) -96 =>100 LC50 (% v/v Saturated Solution)
The results of the definitive test showed the No Observed Effect Concentration (NOEC) to be 100% v/v saturated solution. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration .This study showed that there were no toxic effects at saturation.

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 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. Temperature was maintained at approximately 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH. The oxygen concentration in the majority 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.

Sublethal observations / clinical signs:

Table1: 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 DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)	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
100 R1	0	0	0	0	0	0	0
100 R2	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material 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. This study showed that there were no toxic effects at the limit of solubility in the test medium.

Executive summary:

The acute toxicity of the test material to fish was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 203 and EU Method C.1.

As the test material is considered to be poorly soluble in water, a saturated solution of test material was prepared for use in the study.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test material, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material 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 material was removed by filtration through a 0.2 µm filter to give a saturated solution of the test material. 

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.

There were no mortalities or sub-lethal effects over the 96 hour test period. The 96-hour LC50 value was therefore considered to be greater than 100% v/v saturated solution.

Therefore, under the conditions of the study there were no toxic effects up to the limit of solubility of the test material in the test medium.

Description of key information

No toxic effects up to the limit of solubility of the test material in the test medium, OECD 203, EU Method C.1, Priestly & Mullee (2010)

Additional information

The acute toxicity of the test material to fish was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 203 and EU Method C.1.

As the test material is considered to be poorly soluble in water, a saturated solution of test material was prepared for use in the study.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test material, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material 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 material was removed by filtration through a 0.2 µm filter to give a saturated solution of the test material. 

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.

There were no mortalities or sub-lethal effects over the 96 hour test period. The 96-hour LC50 value was therefore considered to be greater than 100% v/v saturated solution.

Therefore, under the conditions of the study there were no toxic effects up to the limit of solubility of the test material in the test medium.