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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The range-finding test was conducted between 13 September 2006 and 18 September 2006 and the definitive test between 15 October 2006 and 20 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 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 29/03/07

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:
Nominal Concentration of 12.5mg/l

- Sampling method:
Samples were taken from the control and each test group (replicates R1-R3 pooled at 0+72 hours. The test samples were analysed following filtration through Sartorius Minisart 0.45 µm filters to remove algal cells.

- Sample storage conditions before analysis:
Samples were used immediately. Duplicate samples were taken at each occasion and stored at aproximately -20°C for further analysis.

Test solutions

Vehicle:

no

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 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 culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 19 to 22°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 Gelman AcroCap filter (first approximate 500 ml discarded in order to pre-condition the filter) to give a saturated solution with a measured concentration of 9.89 mg/l. A series of dilutions was made from this saturated solution to give further stock solutions of 6.38, 3.90, 2.35 and 1.58 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with 2.5 ml algal suspension to give the required test concentrations of 1.58, 2.35, 3.90, 6.38 and 9.89 mg/l.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours (see Appendix 1).
* Concentration determined by chemical analysis of the saturated solution prepared for use in the definitive test.

- Differential loading:
Not applicable.

- Controls:
Test media control.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

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

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Not stated.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Green algae.

- Strain:
CCAP 276/20

- Source (laboratory, culture collection):
The Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum (at test initiation):
Not applicable.

- Method of cultivation:
Cultures were maintained in the laboratory by the periodic replenishment of culture medium (as used in the test). The culture was maintained in the laboratory at a temperature of 21 ± 1ºC under continuous illumination (intensity approximately 7000 lux) and constant aeration.

ACCLIMATION
- Acclimation period:
Not applicable.

- Culturing media and conditions (same as test or not):
Same as test.

- Any deformed or abnormal cells observed:
None observed.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

Not stated.

Test conditions

Hardness:

Not stated.

Test temperature:

24 ± 1 deg C. The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures (see Table 2 which is attached) were observed to increase from pH 7.0 – 7.1 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
The test material vessels showed an increase in pH at 0 hours with increasing test concentration. After the 72-hour test period the pH of the 2.35, 3.90, 6.38 and 9.89 mg/l test cultures was observed to have decreased.

Dissolved oxygen:

Not stated.

Salinity:

Not applicable.

Nominal and measured concentrations:

Range finding test:
Nominal concentrations: 0.07, 0.7, 7.0mg/l
Measured concentrations: Not stated.

Definitive test:
Nominal concentrations: 6.25,12.5,25,50,100mg/l
Measured concentrations: 1.58, 2.35, 3.90, 6.38, 9.89mg/l

Details on test conditions:

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

- Material, size, headspace, fill volume:
Glass conical flask, 150ml headspace, 100ml fill volume.

- Aeration:
Not stated.

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

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

- Initial cells density:
1.61 x 10^6 cells per ml prior to use.

- Control end cells density:
4.5 x 10^5 cells per ml.

- No. of organisms per vessel:
Not applicable

- No. of vessels per concentration (replicates):
Range finding test: 2
Definitive test: 3

- No. of vessels per control (replicates):
Range finding test: 2
Definitive test: 3

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

GROWTH MEDIUM
- Standard medium used:
yes
- Detailed composition:
Culture Medium

NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reverse osmosis purified deionised water (Elga Optima 15+)

- Total organic carbon:
Not stated.

- Particulate matter:
At 48h counts indicated a large amount of small particulate matter was present in test cultures.

- Metals:
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
CuCl2.2H2O 0.000012 mg/l

- Pesticides:
Not stated.

- Chlorine:
Not stated.

- Alkalinity:
Not stated.

- Ca/mg ratio:
MgCl2.6H2O 12.164 mg/l
MgSO4.7H2O 14.7 mg/l
CaCl2.2H2O 4.41 mg/l


- Conductivity:
Not stated.

- Culture medium different from test medium:
No.

- Intervals of water quality measurement:
72 hours.

OTHER TEST CONDITIONS
- Sterile test conditions:
yes

- Adjustment of pH:
None within the test period.
pH of culture medium adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Photoperiod:
Constant illumination.

- Light intensity and quality:
Approximately 7000 Lux.

- Salinity (for marine algae):
Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Cell density determined using a Coulter® Multisizer Particle Counter. Counts conducted at 48h indicated that a large amount of small particulate matter was present in the test cultures that interfered with the coulter counter. It was therefore considered appropriate to determine cell densities at 48 and 72 hours using a haemocytometer and light microscope in order to give an accurate count of the number of algal cells present.

- Chlorophyll measurement:
Not stated.

- Other:
See remarks section.

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
-The results of the media preparation trial indicated that a dissolved test material concentration of approximately 7.0 mg/l was obtained following a 48 hour preparation period with any undissolved test material removed by filtration through a 0.2 µm AcroCap filter (approximately 500 ml discarded in order to pre-condition the filter).
An amount of test material (2200 mg) was dispersed in 22 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 19 to 24°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 Gelman AcroCap filter (first approximate 500 ml discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 7.0 mg/l*. A series of dilutions was made from this saturated solution to give further stock solutions of 0.70 and 0.070 mg/l. An aliquot (250 ml) of each of the stock solutions was separately inoculated with algal suspension (1.25 ml) to give the required test concentrations of 0.070, 0.70 and 7.0 mg/l.

- Results used to determine the conditions for the definitive study:
Yes.Based on the results of the range-finding test, test material solutions for the definitive test were prepared by stirring an excess (100 mg/l) of test material in culture medium for a period of time and then removing any undissolved test material by filtration. The saturated solution was then further diluted, as necessary, to produce the remaining test groups.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Statistical

Basis for effect:

growth rate

Remarks on result:

other: Value determined from the equation for the fitted line as no concentration tested resulted in greater than 50% inhibition of growth rate.

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% CL

Details on results:

- Exponential growth in the control (for algal test):
yes the mean cell density @ 72h was 4.5 x 10^5 cells per ml.

- Observation of abnormalities (for algal test):
- Unusual cell shape:
None stated.

- Colour differences:
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.58, 2.35 and 3.90 mg/l test cultures were observed to be green dispersions. The 6.38 mg/l test concentrations were observed to be pale green dispersions whilst the 9.89 mg/l test cultures were observed to be very pale green dispersions.

- Flocculation:
Not stated.

- Adherence to test vessels:
Not stated.

- Aggregation of algal cells:
Cell clumping was observed in the test cultures of 6.38 and 9.89 mg/l.

- Other:

- Any stimulation of growth found in any treatment:
None stated.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
The analytical procedure used for the quantification of dissolved test material concentrations in the test preparations was based on the determination of magnesium by ion chromatography as the test material predominately existed as a magnesium hydroxide complex.
Analysis of the test preparations at 0 hours (see Appendix 1) showed measured test concentrations to range from 1.58 to 9.89 mg/l. Analysis of the test preparations 72 hours (see Appendix 1) showed measured concentrations to range from 1.85 to 18.3 mg/l.
During validation of the method of analysis it was evident that a significant amount of magnesium was present in the test medium and as such all test sample concentrations were corrected for the concentration found in the control samples. Furthermore it was also observed that an increase in magnesium concentration occurred over the test period which was considered to be due to the chelating properties of the EDTA present in the test medium (Vogel 5th Edition). Whilst a significant proportion of the magnesium present at 0 hours was bound to the EDTA and hence undetected the decrease in pH of the test cultures over the test period was considered to reduce the chelating properties of the EDTA resulting in a much greater proportion of the magnesium present being available for detection at 72 hours.
Additional studies conducted on this test material (Acute Toxicity to Rainbow Trout and Acute Toxicity to Daphnia magna Safepharm Laboratories Project numbers: 1456/0073 and 1456/0074) using both dechlorinated tap water and reconstituted water in which no EDTA was present did not show such an effect.
Whilst the test material concentrations were prepared as serial dilutions the 0-Hour measured concentrations were non-linear. This was considered to be due to subsequent dilutions of the saturated solution resulting in a decrease in pH which reduced the chelating properties of the EDTA thus increasing the proportion of magnesium present. This decrease in pH was considered to be due to the changes in the buffering capacity of the test medium over time.
As such an apparent increase in test material concentrations was observed over the 72-Hour test period.
Given this increase in measured test concentrations it was considered justifiable to base the results on the 0-Hour measured test concentrations only in order to give a "worst case" analysis of the data.


- 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 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 culture medium 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. Filtration was considered an appropriate method for the removal of any undissolved test material as values close to the water solubility of the test material were obtained for samples prepared in both dechlorinated tap water and reconstituted water indicating that minimal adsorption to filters occurred.
The results obtained from chemical analysis (see Appendix 1) showed measured concentrations of 6.56 and 7.40 mg/l were obtained following filtration with either approximately 100 or 500 ml discarded in order to pre-condition the filter respectively. These measured concentrations were significantly different to the water solubility value of 46.4 mg/l. This was considered to be due to the culture medium which contained EDTA having a significant effect on the measured concentrations of test material present (see Section 5.3.5).
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.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Not stated.

Any other information on results incl. tables

DefinitiveTest

Growth data

From the data given in Tables 2 and 3 which are attached, it is clear that both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.

The mean cell densities versus time for the definitive test are presented in Figure 1 as attached. Percentage inhibition values are plotted against test concentration in Figures 2 and 3 as attached.

Accordingly the following results were determined from the data:

E_bC₅₀ (72 h)                  : 4.1 mg/l
E_rC₅₀ (0 - 72 h)             : 10 mg/l*

where E_bC₅₀ is the test concentration that reduced biomass by 50% and E_rC₅₀ is the test concentration that reduced specific growth rate by 50%.

Statistical analysis of the area under the growth curve data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 1.58 and 2.35 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) was 2.35 mg/l.

It was not possible to calculate 95% confidence limits for the EC₅₀values as the data generated did not fit the models available for the calculation of confidence limits

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria as set out above were met.

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72-Hour period and gave an EbC50 (72 h) value of 4.1 mg/l and an ErC50 (0 - 72 h) value of 10 mg/l*. The No Observed Effect Concentration at 72 hours was 2.35 mg/l.
* Value determined from the equation for the fitted line as no concentration tested resulted in greater than 50% inhibition of growth rate.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 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 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 was therefore prepared by stirring an excess (100 mg/l) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of 19 to 22°C for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Gelman 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 measured concentration of 9.89mg/l*. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using either a Coulter^®Multisizer Particle Counter or a haemocytometer and light microscope.

Results.  

Exposure of Scenedesmus subspicatus to the test material gave an E_bC₅₀ (72 h) value of 4.1 mg/l and an E_rC₅₀ (0 - 72 h) value of 10 mg/l**. The No Observed Effect Concentration was 2.35 mg/l.

The analytical procedure used for the quantification of dissolved test material concentrations in the test preparations was based on the determination of magnesium by ion chromatography as the test material predominately existed as a magnesium hydroxide complex. 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.58 to 9.89 mg/l. Analysis of the test preparations 72 hours showed measured concentrations to range from 1.85 to 18.3 mg/l.

During validation of the method of analysis it was evident that a significant amount of magnesium was present in the test medium and as such all test sample concentrations were corrected for the concentration found in the control samples. Furthermore it was also observed that an increase in magnesium concentration occurred over the test period which was considered to be due to the chelating properties of the EDTA present in the test medium (Vogel 5^thEdition). Whilst a significant proportion of the magnesium present at 0 hours was bound to the EDTA and hence undetected the decrease in pH of the test cultures over the test period was considered to reduce the chelating properties of the EDTA resulting in a much greater proportion of the magnesium present being available for detection at 72 hours.

Additional studies conducted on this test material (Acute Toxicity to Rainbow Trout and Acute Toxicity to Daphnia magna Safepharm Laboratories Project numbers: 1456/0073 and 1456/0074) using both dechlorinated tap water and reconstituted water in which no EDTA was present did not show such an effect.

As such an apparent increase in test material concentrations was observed over the 72-Hour test period. 

Given this increase in measured test concentrations it was considered justifiable to base the results on the 0-Hour measured test concentrations only in order to give a "worst case" analysis of the data.

*Concentration determined by chemical analysis of the saturated solution prepared for use in the definitive test.

**Value determined from the equation for the fitted line as no concentration tested resulted in greater than 50% inhibition of growth rate.