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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2009 - 7 June 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:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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/l) 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 Gelman Acrocap, first approximate 100 mL 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 (replicates R1 - R4 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.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

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. This was equivalent to a test item concentration of 22.7 mg/L based on a theoretical magnesium content of 31.3% as supplied by the Sponsor.
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)

Range-finding test

The results obtained from a pre-study media preparation trial conducted using reconstituted water (the results of which have not been reported due to problems later established with analysing for the test item when prepared in reconstituted water) indicated that a dissolved test item concentration of approximately 6.8 mg/L could be obtained using a saturated solution method of preparation stirred for a period of 24 hours. Given that this result was unreliable the concentrations used in the range-finding test are based on % v/v saturated solution.
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
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. The test item was prepared as a saturated solution.
An amount of test item (1100 mg) was added to 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm at 19°C to 22°C for 24 hours. After the stirring period, the undissolved test item was removed by filtration 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. Serial dilutions were prepared in reconstituted water from the 100% v/v saturated solution to give the remainder of the test series of 10, 1.0 and 0.10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test

Based on the results of the pre-study media preparation trial and the range-finding test a "Limit test" was conducted at a nominal test 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.

Experimental preparation

An initial experiment (the results of which have not been reported) was conducted using reconstituted water as the diluent. Analysis of the test samples from this test showed highly variable results. As the test concentrations were analysed for magnesium, it was considered that the analytical problems were due to the high levels of magnesium in the reconstituted water diluent interfering with the analysis of the test item. It was therefore considered justifiable to change the diluent to dechlorinated tap water which contains little magnesium. The range-finding test was not repeated as experience has shown that the diluent used should not affect the results.
For the purpose of the definitive test the test item was prepared as a saturated solution.
An amount of test item (1100 mg) was added to 11 litres of dechlorinated tap water and
stirred using a propeller stirrer at approximately 1500 rpm at 19°C to 22°C for 24 hours.
After the stirring period, the undissolved test item was removed by filtration through a
0.2 µm Gelman Acrocap filter (first approximate 100 mL discarded to pre-condition the filter) to give a saturated solution.
Due to a technical oversight, the amount of volume discarded for the saturated solution prior to use during the definitive test was 100 mL as opposed to 500 mL as in the range-finding test and the pre-study media preparation trial. However, no significant difference was observed in the saturated solution concentrations after either 500 mL or 1 litre discard when using dechlorinated tap water during the pre-study media preparation trial and hence it was considered that this discrepancy had no impact on the outcome or validity of the test.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: 1st instar Daphnia magna
- Source: derived from in-house laboratory cultures. Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Age at study initiation (mean and range, SD): Adult
- Weight at study initiation (mean and range, SD): N/A
- Length at study initiation (length definition, mean, range and SD): N/A
- Valve height at study initiation, for shell deposition study (mean and range, SD): N/A
- Peripheral shell growth removed prior to test initiation: N/A
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before the initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were then removed from the cultures and used for testing.
- Feeding during test
- Food type: The daphnids received no food during the exposure period.
- Amount: NDA
- Frequency: NDA

ACCLIMATION
- Acclimation period: NDA.
- Acclimation conditions (same as test or not): Same as test conditions
- Type and amount of food: Mixed algae (predominantly Chlorella spp.)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): NDA

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

N/A

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

21 - 22 °C
A single temperature was measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

pH:

pH of control 7.6 - 8.2.
pH of test solution 9.3 - 9.0.
A difference in pH between the control and 56 mg/L test concentration was observed throughout the test.
At the start of the test the test item vessels showed a value of pH 9.0. Given that this was considered to be due to an intrinsic property of the test item no attempt was made to alter the pH of the test media prior to test organism exposure.

Dissolved oxygen:

8.6 - 9.2 mg O2/L
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.

Salinity:

N/A

Nominal and measured concentrations:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a mean measured concentration of 56 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): covered to reduce evaporation.
- Material, size, headspace, fill volume: 250 mL glass jars contaning approximately 200 mL of test preparation
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- 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): N/A
- Biomass loading rate: NDA

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Stock Solutions
a) CaCI2.2H20 11.76 g/L
b) MgSO4.7H20 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCI 0.23 g/L

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 HCI 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.

- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded daily and dissolved oxygen concentrations and pH were recorded at the start and termination of the study.

OTHER TEST CONDITIONS
- Adjustment of pH: if necessary with NaOH or HCI
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: NDA

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: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.1, 1.0, 10 and 100% v/v saturated solution.
No significant immobilisation was observed at the nominal test concentrations of 0.1, 1.0, 10 and 100% v/v saturated solution.
Definitive Test
Based on the results of the pre-study media preparation trial and the range-finding test a "Limit test" was conducted at a nominal test 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.
Analysis of the test preparations at 0 hours showed measured concentrations of 51.5 and 55.7 mg/L. Analysis of the old media at 48 hours showed measured concentrations of 60.6 and 56.9 mg/L. During the pre-study media preparation trial a measured concentration of approximately 18 mg/L was attained from a saturated solution prepared in dechlorinated tap water. The difference in measured concentration was considered to be due to the differences in temperature at which the saturated solutions were stirred (approximately 14°C in the pre-study media preparation trial compared to approximately 21°C in the definitive test). The higher temperature was considered to have possibly aided the dissolution of the test item in the medium.
Given that no decline in measured concentration was observed over the test period, it was considered justifiable to base the results on mean measured test concentration. This was calculated to be 56 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 56 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Zero Immobilisation

Details on results:

- Behavioural abnormalities: None recorded
- Observations on body length and weight: NDA
- Other biological observations: None recorded
- Mortality of control: 0%
- Other adverse effects control: None recorded
- Abnormal responses: None recorded

Observations on test material solubility: The test media preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements: Temperature was maintained at 21 - 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

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 supersaturating the diluent and was considered not to have had an impact on the outcome or validity of the test as no adverse effects were observed in the control daphnids.

Analysis of the test preparations at 0 hours showed measured concentrations of 51.5 and 55.7 mg/L. Analysis of the old media at 48 hours showed measured concentrations of 60.6 and 56.9 mg/L. During the pre-study media preparation trial a measured concentration of approximately 18 mg/L was attained from a saturated solution prepared in dechlorinated tap water. The difference in measured concentration was considered to be due to the differences in temperature at which the saturated solutions were stirred (approximately 14°C in the pre-study media preparation trial compared to approximately 21°C in the definitive test). The higher temperature was considered to have possibly aided the dissolution of the test item in the medium.
Given that no decline in measured concentration was observed over the test period, it was considered justifiable to base the results on mean measured test concentration. This was calculated to be 56 mg/L.

Results with reference substance (positive control):

Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1149) during the positive control are given below.
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:

Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 1.5 1.3 – 1.6
48 1.2 1.1 – 1.3

The No Observed Effect Concentrations after 24 and 48 hours were 1.0 and 0.32 mg/L respectively. 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 1.2 mg/L (sd = 0).

Reported statistics and error estimates:

Evaluation of data for the positive control
The EC50 value and associated confidence limits at 24 and 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Any other information on results incl. tables

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)	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
	24 Hours			48 Hours
	No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control     R1                  R2                  R3                  R4	0 0 0 0	0	0	0 0 0 0	0	0
56             R1                  R2                  R3                  R4	0 0 0 0	0	0	0 0 0 0	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	0	0	0	0	0	2	2	10
1.0	0	0	0	0	1	1	2	10
1.8	9	8	17	85	10	10	20	100
3.2	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 to 2

REFERENCES

Environment Directorate, Organisation for Economic Co-operation and Development (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) Monograph No. 26 (1996) Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances.

Finney, D J (1971) Statistical Method in Biological Assay. London : Griffin and Company Ltd.

Hamilton, M A, Russo, R C and Thurston, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol 11, 714-719.

ToxCalc Version 5.0.23C (1999), Tidepool Scientific Software, McKinleyville, CA 95519, USA.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was greater than 56 mg/L and correspondingly the No Observed Effect Concentration was 56 mg/L.
This study showed that there were no toxic effects at saturation.

Executive summary:

 The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was greater than 56 mg/L and correspondingly the No Observed Effect Concentration was 56 mg/L.

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

The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.