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:

25 April 2010 - 14 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:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition 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/L) of test item in culture medium for a period of 48 hours prior to removing any undissolved test item present by filtration (0.2 µm Gelman Acrocap, first approximate 500 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:

- Concentrations: The concentration and stability of the test material in the test solutions were verified by chemical analysis.
- Sampling method: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test

An amount of test item (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item 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 22 mg/L. A series of dilutions was made from this saturated solution to give further stock solutions of 2.2, 0.22 and 0.022 mg/L. An aliquot (200 mL) of each of the stock solutions was separately inoculated with algal suspension (3 mL) to give the required test concentrations of 0.022, 0.22, 2.2 and 22 mg/L.

Definitive test

An amount of test item (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 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test item 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 0-Hour measured concentration of 14 mg/L. A series of dilutions was made from this saturated solution to give stock solutions of 6.8, 4.0, 2.4 and 1.1 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 10 mL algal suspension to give the test concentrations of 1.1, 2.4, 4.0, 6.8 and 14 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test solutions were verified by chemical analysis at 0 and 72 hours.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): NDA
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

ACCLIMATION
- Acclimation period: NDA
- Culturing media and conditions (same as test or not): The culture medium used for the studies was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: NDA

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

NDA

Test temperature:

24 ± 1 °C

pH:

pH 7.3 – 7.9
The pH values of the control cultures were observed to increase from pH 7.3 at 0 hours to pH 7.8 — 7.9 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.
At the start of the test the test item vessels showed a concentration dependant increase in pH with increasing concentration in the range of pH 7.3 at 1.1 mg/L through to pH 9.9 at 14 mg/L. 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 cultures prior to test organism exposure.

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.1 to 14 mg/L. A concentration dependant decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.73 mg/L) to 13 mg/L.
Given that magnesium is known to be stable this decline was considered to be due to adsorption to the algal cells present. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not prelude long term adsorption over the test period.
Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 mL glass conical flasks each containing 100 mL of test preparation
- Aeration: NDA
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 4.00 x 10E3 cells per mL
- Control end cells density: 1.99 x 10E5 cells per mL
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

NaNO3 25.5 mg/L
MgCI2.6H20 12.164 mg/L
CaCI2.2H20 4.41 mg/L
MgSO4.7H20 14.7 mg/L
K2HP04 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCI2.4H20 0.415 mg/L
ZnCI2 0.00327 mg/L
FeCI3.6H20 0.159 mg/L
CoCI2.6H20 0.00143 mg/L
Na2MoO4.2H20 0.00726 mg/L
CuCI2.2H20 0.000012 mg/L
Na2EDTA.2H20 0.30 mg/L
Na2SeO3.5H20 0.000010 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was determined at initiation of the study and after 72 h exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: NDA
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI
- Photoperiod: continuous illumination
- Light intensity and quality: light intensity approximately 7000 lux provided by warm white lighting (380 - 730 nm)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Chlorophyll measurement: none
- Other: The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: NDA
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study -Test concentrations: 0.002, 0.22, 2.2 and 22 mg/L.
- Results used to determine the conditions for the definitive study: test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 11

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 12

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

The following data show that the cell concentration of the control cultures increased by a factor of 76 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 3.73 x 10^3 cells per mL
Mean cell density of control at 72 hours : 2.85 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 32% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 — 72 h) was 7% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

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, 1.1, 2.4, 4.0 and 6.8 mg/L test cultures were observed to be green dispersions whilst the 14 mg/L test cultures were observed to be very pale green dispersions.

Temperature was maintained at 24 ± 1°C throughout the test.

The pH values of the control cultures were observed to increase from pH 7.3 at 0 hours to pH 7.8 — 7.9 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.

At the start of the test the test item vessels showed a concentration dependant increase in pH with increasing concentration in the range of pH 7.3 at 1.1 mg/L through to pH 9.9 at 14 mg/L. 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 cultures prior to test organism exposure.

Information provided by the Sponsor indicated that the test item contained 31.3% magnesium. Analysis of the test preparations was conducted for magnesium, from which the equivalent test item concentrations were determined.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.1 to 14 mg/L. A concentration dependant decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.73 mg/L) to 13 mg/L.

Given that magnesium is known to be stable this decline was considered to be due to adsorption to the algal cells present. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not prelude long term adsorption over the test period. Overall the decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. This effect was considered to be due to there being greater numbers of algal cells in the lower concentrations and hence greater surface area for adsorption to occur.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.37 mg/L) was used to enable calculation of the geometric mean measured concentration.

The use of the geometric mean measured test concentrations in the calculation of the EC50 and NOEC values had no significant effect on the outcome of the study.
Care should be taken in the interpretation of the results of the study based on geometric mean measured test concentrations as, whilst there was a significant decline in measured test concentrations over the test period, the test item was known to be stable in aqueous medium and hence the decline was considered to be due to adsorption to algal cells. As such it may be considered that the algal cells were exposed to nominal concentrations of test item throughout the test period.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:
ErC50 (0 - 72 h): 0.49 mg/L*
EyC50 (0 - 72 h): 0.18 mg/L, 95% confidence limits 0.16 - 0.21 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.
* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Statistical analysis of the growth rate 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, 1.1, 2.4, 4.0 and 6.8 mg/L test concentrations (P≥0.05), however the 14 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 6.8 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 14 mg/L.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control, 1.1, 2.4, 4.0 and 6.8 mg/ILtest concentrations (P≥0.05), however the 14 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 6.8 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 14 mg/L.

Any other information on results incl. tables

REFERENCES

Dunnett, C W (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc 50, 1096-1121.

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.

SAS/STAT Proprietary Software Release 8.02 (1999 - 2001), SAS Institute Inc, Cary, NC, USA.

Sokal, R R and Rohlf, F J (1981) Biometry. New York: W H Freeman and Company.

Xlfit, ID Business Solutions Ltd.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable EC50 (mg/L) NOEC (mg/L) LOEC (mg/L)

Growth Rate >14* 6.8 14
Yield 10 6.8 14


Exposure of Desmodesmus subspicatus to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable EC50 (mg/L) NOEC (mg/L) LOEC (mg/L)

Growth Rate >13* 6.3 13
Yield 9.6 6.3 13

* It was not possible to calculate an E,C50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.

Executive summary:

A 72 h study was performed to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Exposure of Desmodesmus subspicatus to the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>14*	6.8	14
Yield	10	6.8	14

 

Exposure of Desmodesmus subspicatus to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>13*	6.3	13
Yield	9.6	6.3	13

 

* It was not possible to calculate an E,C50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.