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:

The range-finding test was conducted between 30 September 2006 and 6 October 2006 and the definitive test between 21 October 2006 and 27 October 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 18/01/07

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

IUPAC Name:

hexamagnesium(2+) trihydrate decahydroxide sulfate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The test samples were diluted with water* to give a final theoretical concentration of 7.6 mg/l.
Saturated solutions of the test material prepared by stirring an excess of test material (100 mg/l) in dechlorinated tap water for 48 hours prior to removal of the undissolved test material by filtration (0.2 µm Sartorius filter) using either 500 ml or 1 litre to precondition the filter.
Sample Treatment Measured Concentration (mg/l)
Control Filtered - 500 ml discarded 37.7
Filtered - 1 litre discarded 38.2

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

- Sample storage conditions before analysis:
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.
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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Media preparation trial
Information provided by the Sponsor indicated the water solubility value of the test material to be 30 mg/l. A Study to Determine the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 1456/0064) gave a water solubility value for the test material of 46.4 mg/l at 20.0 ± 0.5°C. Pre-study solubility work conducted indicated that despite the use of ultrasonication and high sheer mixing it was not possible to obtain a stock solution of the test material. In addition, the test material did not dissolve in auxiliary solvents.

Given this, a saturated solution was prepared in dechlorinated tap water at an initial loading rate of 100 mg/l in order to determine whether a dissolved test material concentration close to that predicted could be obtained. Filtration was used to remove the undissolved test material as, due to the volume of media required, centrifugation would not be practical.

An amount of test material (2250 mg) was dispersed in 22.5 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C for 48 hours. After the stirring period samples were removed for chemical analysis after the following pre-treatments:

* Filtered through a 0.2 µm Sartorius Sartopore filter (approximately 500 ml discarded in order to pre-condition the filter)
* Filtered through a 0.2 µm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)

Experimental Preparation
An amount of test material (2250 mg) was dispersed in 22.5 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 38 mg/l*. Filtration was used to remove the undissolved test material as, due to the volume of media required, centrifugation would not be practical. In addition, the results of the pre-study solubility work indicated that when using this method of preparation the dissolved test material concentration in the filtrate was in line with the reported water solubility of the test material.

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

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media), 24 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 4.3 cm (sd = 0.4) at the end of the definitive test.

- Weight at study initiation (mean and range, SD):
mean weight of 0.98 g (sd = 0.26) 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 11 October 2006 to 23 October 2006

- 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 13.4 deg C to 15.1 deg C throughout the test

A single temperature at 0 hours (fresh media) during the definitive test was observed to be slightly in excess of the range given in the protocol of 14 ± 1 deg C. This small 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.

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 either a WTW pH 320 pH meter or 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.

Dissolved oxygen:

The dissolved oxygen concentrations 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 either a YSI 550 dissolved oxygen meter or a WTW pH/Oxi 340I pH and dissolved oxygen meter.

While there were no treatment related differences for oxygen concentration between the control and the 38 mg/l test concentration were observed throughout the test.

The oxygen concentration in the 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 recorded

Nominal and measured concentrations:

Range-finding test:
- Nominal: 38 mg/l.
- Measured: Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 86% to 110% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only

Definitive test:
- Nominal: 38 mg/l.
- Measured: Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 86% to 110% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only

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:
1.573mg/l

- Particulate matter:
- Metals: As attached in Appendix 2
- Pesticides:0.066mg/l
- Chlorine:0.287mg/l
- Alkalinity:pH=7.564
- Ca/mg ratio:Not recorded
- Conductivity:Not stated
- 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 for a period of 48 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 38 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 nominal test concentration of 38 mg/l. A single test concentration was used as results from the range-finding test for the Acute Toxicity to Daphnia magna study (Safepharm Laboratories Project Number: 1456/0074) indicated that toxicity was not expected at this concentration. The test material was prepared as a saturated solution.

An amount of test material (2250 mg) was dispersed in 22.5 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 38 mg/l.

In the range-finding test 3 fish were added to each 20 litre test and control vessel 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 material.

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 range-finding test a "Limit test" was conducted at a concentration of 38 mg/l. The test material solution for the definitive test was prepared by stirring an excess (100 mg/l) of test material in dechlorinated tap water for a period of time and then removing any undissolved test material by filtration to give the 38 mg/l test concentration.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: It was not possible to calculate the 95% CL

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: It was not possible to calculate the 95% CL

Details on results:

- Behavioural abnormalities:
No sublethal effects of exposure observed in 14 fish exposed to a test concentration of 38 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:

Information provided by the Sponsor indicated the water solubility value of the test material to be 30 mg/l. A Study to Determine the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 1456/0064) gave a water solubility value for the test material of 46.4 mg/l at 20.0 ± 0.5°C. Pre-study solubility work conducted indicated that despite the use of ultrasonication and high sheer mixing it was not possible to obtain a stock solution of the test material. In addition, the test material did not dissolve in auxiliary solvents. Given this, a saturated solution was prepared in dechlorinated tap water at an initial loading rate of 100 mg/l in order to determine whether a dissolved test material concentration close to that predicted could be obtained. Filtration was used to remove the undissolved test material as, due to the volume of media required, centrifugation would not be practical. The results obtained from chemical analysis (see Appendix 1) showed measured concentrations of 38 mg/l were obtained following filtration with either 500 ml or 1 litre discarded in order to pre-condition the filter respectively. Therefore for the purposes of testing it was considered appropriate to prepare the saturated solution using a 48-Hour stirring period followed by removal of any undissolved test material by filtration (first approximate 1 litre discarded in order to pre-condition the filter). Filtration was used to remove the undissolved test material as, due to the volume of media required, centrifugation would not be practical. In addition, the results of the pre-study solubility work indicated that when using this method of preparation the dissolved test material concentration in the filtrate was in line with the reported water solubility of the test material.

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 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 38 mg/l.

Based on this information, a single test concentration, in duplicate, of 38 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 of 38 mg/l, no mortalities or sub‑lethal effects of exposure were observed.

DefinitiveTest

Mortality data

Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given in Table 2.

There were no mortalities in 14 fish exposed to a test concentration of 38 mg/l for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)	LC50(mg/l)	95% Confidence limits (mg/l)
3	>38	-
6	>38	-
24	>38	-
48	>38	-
72	>38	-
96	>38	-

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 38 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 38 mg/l and the No Observed Effect Concentration (NOEC) to be 38 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 (Section5.3.2).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Validity criteria as stated above were fulfilled.

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 38 mg/l. Correspondingly the No Observed Effect Concentration was 38 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Information provided by the Sponsor indicated the water solubility value of the test material to be 30 mg/l. A Study to Determine the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 1456/0064) gave a water solubility value for the test material of 46.4 mg/l at 20.0 ± 0.5°C. Pre-study solubility work conducted indicated that despite the use of ultrasonication and high shear mixing it was not possible to obtain a stock solution of the test material. In addition, the test material did not dissolve in auxiliary solvents. The test material 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 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a nominal test concentration of 38 mg/l.

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 38 mg/l for a period of 96 hours at a temperature of 13.4ºC to 15.1°C under semi-static test conditions. 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 LC₅₀ based on nominal test concentrations was greater than 38 mg/l and correspondingly the No Observed Effect Concentration was 38 mg/l.

nalysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 86% to 110% of nominal and so the results are based on nominal test concentrations only.