Magnesium hydroxide sulfate (Mg6(OH)10(SO4)), hydrate (1:3)

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2010 - 25 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

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/I) of test item in dechlorinated tap water for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-390-9

EC Name:

-

Cas Number:

12508-61-1

Molecular formula:

H16Mg6O17S MgSO4 • 5Mg(OH)2 • 3H2O

IUPAC Name:

Magnesium hydroxide sulphate trihydrate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Pre-study media preparation trial
A determination of the General Physico-Chemical Properties study conducted on the test item (Harlan Laboratories Ltd. Project Number: 0656/0397) showed the water solubility value of the test item was 9.46 mg/L (with respect to the magnesium content) using the shake-flask method of preparation.
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (1100 mg) was dispersed, in duplicate, in 11 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
Filtration through a 0.2 pm Sartorius Sartopore filter (approximate 500 mL discarded in order to pre-condition the filter)
Filtration through a 0.2 pm Sartorius Sartopore filter (approximate 1 litre discarded in order to pre-condition the filter)

Experimental Preparation
An amount of test item (2250 mg) was added to 22.5 litres of dechlorinated tap water and stirred using a propeller stirrer at approximately 1500 rpm at approximately 14°C for 24 hours. After the stirring period, the undissolved test item was removed by filtration through a 0.2 pm Sartorius Sartopore filter (first approximate 1 litre discarded to pre-condition the filter) to give a saturated solution with a mean measured test concentration of 33 mg/L.
This method of preparation was conducted in duplicate to give replicates R1 and R2.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media).
- Eluate:
Not applicable

- Differential loading:
Not applicable

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

- Chemical name of vehicle:
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material:
None recorded

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name:
Rainbow trout

- Strain:
Not applicable

- Source:
Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK

- Age at study initiation (mean and range, SD):
juvenile (age not specified)

- Length at study initiation (length definition, mean, range and SD):
mean standard length of 5.8 cm (sd = 0.2) at the end of the definitive test.

- Weight at study initiation (mean and range, SD):
mean weight of 2.61 g (sd = 0.19) at the end of the definitive test.

- Method of breeding:
Not applicable

- Feeding during test
Discontinued approximately 24 hours prior to the start of the definitive test.


ACCLIMATION
- Acclimation period:
Fish were acclimatised to test conditions from 28 April 2010 to 10 May 2010

- Acclimation conditions (same as test or not):
Same as test

- Type and amount of food:
The stock fish were fed commercial trout pellets. Amount not specified

- Feeding frequency:
Not recorded

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

QUARANTINE (wild caught)
Not applicable

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not stated

Test conditions

Hardness:

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

Test temperature:

The water temperature was recorded daily throughout the test.

The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations.

The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Temperature was maintained at 14.0 °C to 15.0 °C throughout the test

pH:

The pH was recorded daily throughout the test.

The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations.

The pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

While there were no treatment related differences in pH between the control and the 38 mg/l test concentration were observed throughout the test.
A difference in pH between the control and 33 mg/L test concentration was observed throughout the test: pH range of 7.8 – 8.3 for the control; pH range of 8.8 – 9.5 for the two replicates.

Dissolved oxygen:

The dissolved oxygen concentration was recorded daily throughout the test.

The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations.

The dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen 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.

The dissolved oxygen level ranged from 8.7 - 11.0 mg/L in the control and 8.6 - 11.3 mg/L in the test vessels.

Salinity:

Not recorded

Nominal and measured concentrations:

The results obtained from the pre-study media preparation trial conducted indicated that a preparation period of 24 hours resulted in the maximum dissolved test item concentration.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to removal of any undissolved test item by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded) to give a nominal test concentration of approximately 18 mg/L.

Analysis of the freshly prepared test preparations at 0 24, 48 and 72 hours showed measured concentrations of test item to range from 28.0 to 35.2 mg/L with the exception of a single analysis which showed a measured concentration of 20.3 mg/L. Analysis of the corresponding duplicate sample, stored frozen prior to analysis, showed a measured concentration of 29.3 mg/L indicating that the initial result was erroneous.
Analysis of the old media at 24, 48, 72 and 96 hours showed measured concentrations of test item to range from 28.9 to 36.3 mg/L.
The measured concentrations in the saturated solutions prepared during the definitive test where higher than that obtained during the pre-study media preparation trial of 18 mg/L. This was considered to be possibly due slight variations in stirring speeds and/or water quality despite every effort being made to keep preparation conditions standard. The slight variation in measured concentration of the saturated solutions observed within the definitive test was considered to be within the normal variation associated with the preparation of saturated solutions.
Given that no decline in measured concentration was observed over each dosing period it was considered justifiable to base the results on mean measured test concentrations only. The mean measured test concentration was calculated to be 33 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Closed (covered to reduce evaporation)
- Material, size, headspace, fill volume: 20 litre glass exposure vessels
- Aeration: The test vessels were aerated via narrow bore glass tubes

- Type of flow-through (e.g. peristaltic or proportional diluter): Not recorded.
- 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 test concentration of test material remained near nominal and to prevent build up of nitrogenous waste products.

- No. of organisms per vessel: 7

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 1

- No. of vessels per vehicle control (replicates): Not applicable

- Biomass loading rate: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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.

- Total organic carbon: 1.018 mg/L
- Particulate matter:
- Metals: As attached in Appendix 1 as part of typical water quality characteristics
- Pesticides: 0.001 mg/L
- Chlorine: 0.248mg/L
- Alkalinity: pH=7.624
- Ca/mg ratio:Not recorded
- Conductivity: 386.192 µS/cm at 20 °C
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not recorded

OTHER TEST CONDITIONS
- Adjustment of pH: No

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

- Light intensity: Not recorded.
EFFECT PARAMETERS MEASURED:
An estimate of the LC50 values was given by inspection of the mortality data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None

- Justification for using less concentrations than requested by 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 dechlorinated tap water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 14 °C for a period of 24 hours prior to removing any undissolved test material present 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 material of approximately 33 mg/L.

- Range finding study
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 series of nominal test concentrations of 1.8 and 18 mg/L. The test item was prepared as a saturated solution.
An amount of test item (2250 mg) was added to 22.5 litres of dechlorinated tap water and stirred using a propeller stirrer at approximately 1500 rpm at approximately 14°C for 24 hours. After the stirring period, the undissolved test item was removed by filtration through a 0.2 pm Sartorius Sartopore filter (first approximate 1 litre discarded to pre-condition the filter) to give a saturated solution with a nominal test concentration of 18 mg/L. An aliquot (2 litres) of the 18 mg/L test concentration was diluted in a final volume of 20 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 1.8 mg/L test concentration. The volume of this prepared concentration was then adjusted to 18 litres.
In the range-finding test three fish were added to each 20 litre test and control vessel, containing 18 litres of 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.
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.
Based on the results of the media preparation trial and the range-finding test a "Limit test" was conducted at a nominal test concentration of 18 mg/L to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
No sublethal effects of exposure observed in 14 fish exposed to a mean measured test concentration of 33 mg/L for a period of 96 hours.

- Observations on body length and weight:
Not stated.

- Other biological observations:
There were no mortalities

- Mortality of control:
None.

- Other adverse effects control:
None stated.

- Abnormal responses:
None stated.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
None stated.

- Effect concentrations exceeding solubility of substance in test medium:
A determination of the General Physico-Chemical Properties study conducted on the test item (Harlan Laboratories Ltd. Project Number: 0656/0397) showed the water solubility value of the test item was 9.46 mg/L (with respect to the magnesium content) using the shake-flask method of preparation.
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (1100 mg) was dispersed, in duplicate, in 11 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
Filtration through a 0.2 µm Sartorius Sartopore filter (approximate 500 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Sartorius Sartopore filter (approximate 1 litre discarded in order to pre-condition the filter)

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not stated

Any other information on results incl. tables

Sublethal observations / clinical signs:

Range-finding Test

Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in the table below. There were no sub-lethal effects of exposure during the range-finding test.

The results showed no mortalities at the nominal test concentrations of 1.8 and 18 mg/L.

A single fish in the control was observed dead next to the exposure vessel having jumped out, despite the vessel being covered, after approximately 52.5 hours exposure. This was considered not to have affected the outcome of the test.

Based on this information, a single nominal test concentration, in duplicate, of 18 mg/L 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.

Table 1: Cumulative Mortality

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0*	1	1
1.8	0	0	0	0	0	0
18	0	0	0	0	0	0

 

*A single fish was observed dead next to the exposure vessel having jumped out, despite the vessel being covered, after approximately 52.5 hours exposure. This was considered not to have affected the outcome of the test.

Definitive Test

Mortality data

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in the following table.

Table 2: % Mortality

Mean Measured Concentration (mg/L)	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
33 R1	0	0	0	0	0	0	0
33 R2	0	0	0	0	0	0	0

 

There were no mortalities in 14 fish exposed to a mean measured test concentration of 33 mg/L for a period of 96 hours. Inspection of the mortality data gave the following results:

Table 3: LC50 data

Time (h)

LC50 (mg/L)

3 6 24 48 72 96

>33 >33 >33 >33 >33 >33

 

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 33 mg/L, the lowest test concentration resulting in 100% mortality to be greater than 33 mg/L and the No Observed Effect Concentration (NOEC) to be 33 mg/L. 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.

Applicant's summary and conclusion

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 33 mg/L. Correspondingly the No Observed Effect Concentration was 33 mg/L.
This study showed that there were no toxic effects at saturation.

Executive summary:

The 96-Hour LC50 based on mean measured test concentrations was greater than 33 mg/L and correspondingly the No Observed Effect Concentration was 33 mg/L. This study showed that there were no toxic effects at saturation. The study performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss) followed the method 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.