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:

The range-finding test was conducted between 11 September 2006 and 15 September 2006 and the definitive test between 2 October 2006 and 6 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 04/04/07

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:
rangefinding: 0.038, 0.38 3.8 and 38 mg/l
definitive: 38 mg/l

- Sampling method:
Not stated.

- Sample storage conditions before analysis:
Samples analysed immediately after collection.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
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. Given this, in order to obtain a solution of the material the test concentration used in the definitive test was prepared as a saturated solution prepared from an initial test material dispersion at a concentration of 100 mg/l.
An amount of test material (1100 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°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 AcroCap filter (first approximate 500 ml discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 38 mg/l*. The use of filtration to remove undissolved test material was considered appropriate as 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.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 1).

- Eluate:
Reconstituted Water
i)Stock Solutions
a)CaCl2.2H2O 11.76 g/l
b)MgSO4.7H2O 4.93 g/l
c)NaHCO3 2.59 g/l
d)KCl 0.23 g/l

ii)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 HCl 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.

- Differential loading:
Not applicable.

- Controls:
Rangefinding test:
Test medium control (1 replicate).

Definitive test:
Test medium control (4 replicates).

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable, no vehicle used.

- Concentration of vehicle in test medium (stock solution and final test solution):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
None stated.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
Water flea

- Strain:
not stated.

- Source:
In-house laboratory cultures.

- Age at study initiation (mean and range, SD):
<24 hours.

- Weight at study initiation (mean and range, SD):
Not stated.

- Length at study initiation (length definition, mean, range and SD):
Not stated.

- Valve height at study initiation, for shell deposition study (mean and range, SD):
Not stated.

- Peripheral shell growth removed prior to test initiation:
Not stated.

- Method of breeding:
Parthenogenesis.

- Feeding during test
None.

ACCLIMATION
Not applicable.

QUARANTINE (wild caught)
Not applicable.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None.

Test conditions

Hardness:

Approximate theoretical total hardness of 250mg/l CaCO3.

Test temperature:

18.9-21.3 deg C.

pH:

Control:
7.9 at 0 hours
7.8 at 48 hours

Definitive test (38mg/l):
10.5 at 0 hours
8.8 at 48 hours

Dissolved oxygen:

9.5 at 0 hours
9.0 at 48 hours

Salinity:

Not applicable.

Nominal and measured concentrations:

Range finding test:
Nominal:0.038, 0.38, 3.8 and 38 mg/l.
Measured:

Definitive test:
Nominal: 38 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable):
closed

- Material, size, headspace, fill volume:
250ml glass test container, filled with 200ml of test concentration.

- Aeration:
None.

- Type of flow-through (e.g. peristaltic or proportional diluter):
Not applicable.

- Renewal rate of test solution (frequency/flow rate):
Not applicable.

- No. of organisms per vessel:
10

- No. of vessels per concentration (replicates):
Range finding test: 1 replicate.
Definitive test: 4 replicates.

- No. of vessels per control (replicates):
Range finding test: 1 replicate.
Definitive test: 4 replicates.

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

- Biomass loading rate:
Not stated.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted Water
i)Stock Solutions
a)CaCl2.2H2O 11.76 g/l
b)MgSO4.7H2O 4.93 g/l
c)NaHCO3 2.59 g/l
d)KCl 0.23 g/l

ii)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 HCl 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.

- Total organic carbon:
Not stated.

- Particulate matter:
None.

- Metals:
As above.

- Pesticides:
None.

- Chlorine:
None.

- Alkalinity:
As above.

- Ca/mg ratio:
As above.

- Conductivity:
Not stated.

- Culture medium different from test medium:
No.

- Intervals of water quality measurement:
48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH:
As above.

- Photoperiod:
16 hours light/8 hours darkness

- Light intensity:
Not stated.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation:
measured at 24 hour interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Range finding test: x10
Definitive test: not applicable.

- Justification for using less concentrations than requested by guideline:
Not applicable.

- Range finding study
- Test concentrations:
0.038, 0.38, 3.8 & 38 mg/l.

- Results used to determine the conditions for the definitive study:
Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

immobilisation

Effect conc.:

> 38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

other: 95% confidence limits not given.

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

other: 95% confidence limits not given.

Details on results:

- Behavioural abnormalities:
- Immobilisation:
Range finding test:
No immobilisation was observed at the test concentrations of 0.038, 0.38, 3.8 and 38 mg/l.

Definitive test:
There was no immobilisation in 20 daphnids exposed to a test concentration of 38 mg/l for a period of 48 hours.

- Observations on body length and weight:
Not stated.

- Other biological observations:
None stated.

- Mortality of control:
There was no mortality in the control.

- 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:
Not applicable.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes.

- Mortality:
Not stated.

- EC50/LC50:
Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
3 > 3.2 -
24 0.88 0.75 - 1.0
48 0.60 0.53 - 0.68

- Other:
The mean 48-Hour EC50 value calculated from all positive controls was 0.82 mg/l (sd = 0.24).

Reported statistics and error estimates:

The slope and standard error of the response curve at 24 hours was 6.3 (SE = 1.2). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response curve at 48 hours.

Any other information on results incl. tables

Pre-Study 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 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.

Given this, a saturated solution was prepared in reconstituted water at an initial loading rate of 100 mg/l in order to determine whether a dissolved test material concentration close to that provided by the Sponsor could be obtained.

The results obtained from chemical analysis (see Appendix 1) showed measured concentrations of 36 and 38 mg/l were obtained following filtration with either 100 or 500 ml 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 500 ml discarded in order to pre-condition the filter). The use of filtration to remove undissolved test material was considered appropriate as 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.

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.

No immobilisation was observed at the test concentrations of 0.038, 0.38, 3.8 and 38 mg/l.

Based on this information, a single test concentration of four replicates, 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 immobilisation or adverse reactions to exposure were observed.

DefinitiveTest

 Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.

There was no immobilisation in 20 daphnids exposed to a test concentration of 38 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:

Time (h)	EC50(mg/l)	95% Confidence limits (mg/l)
24	>38	-
48	>38	-

The No Observed Effect Concentration after 24 and 48 hours exposure was 38 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

Observations on test material 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 3. Temperature was maintained at approximately 20°C throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the control and the 38 mg/l test concentration throughout the test.

The oxygen concentration in the test vessels at 0 hours (fresh media) 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.

Verification of test concentrations

Analysis of the test preparations at 0 and 48 hours (see Appendix 1) showed measured test concentrations to range from 95% to 115% of nominal and so it was considered justifiable to estimate the EC₅₀values in terms of the nominal test concentrations only.

Positive Control

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Safepharm Laboratories Project No: 0039/0799) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2.

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit methods (Finney 1971) at 24 hours and 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)
3	> 3.2		-
24	0.88	0.75	-	1.0
48	0.60	0.53	-	0.68

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and standard error of the response curve at 24 hours was 6.3 (SE = 1.2). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response curve at 48 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC₅₀ value calculated from all positive controls was 0.82 mg/l (sd = 0.24).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 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 Daphnia magna. 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 Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

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 therefore prepared by stirring an excess (100 mg/l) of test material in reconstituted water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm AcroCap filter, first approximate 500 ml 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[1]. 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 38 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21.2°C to 21.9°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀for the test material to Daphnia magna based on nominal test concentrations was greater than 38 mg/l and correspondingly the No Observed Effect Concentration was 38 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 95% to 115% of nominal and so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.60 mg/l with 95% confidence limits of 0.53 – 0.68 mg/l. The No Observed Effect Concentration was 0.32 mg/l.