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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The range-finding test was conducted on 30 June 2006 and the definitive test on 1 November 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 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 12/01/07

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:
Definitive test only:
Nominal concentrations: 34, 1000 mg/l.

- Sampling method:
Not stated.

- Sample storage conditions before analysis:
Not stated.

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 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 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.
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)
This solubility work is described in Appendix 1. Whilst chemical analysis of the test preparations in the range-finding and definitive tests was not appropriate to the Test Guideline, chemical analysis was conducted in the solubility work in order to determine the dissolved test material concentration.

For the purpose of the definitive test, the test material was dispersed directly in water for the test concentration of 1000 mg/l and prepared using a saturated solution of the test material for the test concentration of 34 mg/l.
To obtain the test concentration of 1000 mg/l, an amount of test material (500 mg) was dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentration of 1000 mg/l. This method of preparation was performed for three separate replicate test vessels.
Additionally the definitive test also consisted of a single test concentration prepared using a saturated solution of the test material.
An amount of test material (1100 mg) was dispersed in water (11 litres) with the aid of propeller stirring (approximately 1500 rpm, 48 hours) at approximately 21ºC to give an initial test material dispersion of 100 mg/l. After the stirring period, any undissolved test material was removed by filtration through a 0.2 µm Sartorious Sartopore filter (discarding the initial 1 litre to pre-condition the filter) to give a saturated solution of 38 mg/l*. Due to the limitations of having to add inoculum and synthetic sewage to the test vessels the maximum test concentration that could be attained when using this method of preparation was 90% of that of the saturated solution i.e. 34 mg/l.
An aliquot (450 ml) of the 38 mg/l saturated solution was dispersed with activated sewage sludge (equivalent to 800 mg suspended solids), synthetic sewage (16 ml) and water, to a final volume of 500 ml, to give the required concentrations of 34 mg/l.
The control group was maintained under identical conditions but not exposed to the test material.
Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.


- Differential loading:
Not applicable.

- Controls:
Test medium control.
Positive control (3,5-dichlorophenol).

- 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):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
A mixed population of activated sewage sludge micro-organisms was obtained on 30 June 2006 for the range-finding test and on 1 November 2006 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

- Method of cultivation:
Not applicable, inoculum was used on the day of collection.

- Preparation of inoculum for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 8.0 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids was equal to 4.0 g/l prior to use.
In order to minimise dilution of the initial test material stock saturated solution by the addition of the relatively large volumes of activated sewage sludge required to give an initial loading rate of 1.6 g/l, the activated sewage sludge was centrifuged (3500 rpm, 5 minutes) and the supernatant discarded prior to addition of the activated sewage sludge 'pellets' to the test vessels. Using this approach the maximum attainable test concentration was 90% of the saturated solution concentration i.e 34 mg/l attained from a 38 mg/l stock solution

- Pretreatment:
As above.

- Initial biomass concentration:
Not stated.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not stated.

Test conditions

Hardness:

Approximately 100 mg/l as CaCO3 for the range-finding test and approximately 140 mg/l as CaCO3 for the definitive test.

Test temperature:

21 ± 1 deg C.

pH:

pH of the sample was 8.0 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. See Table 4.

Dissolved oxygen:

See Tables 1-3.

Salinity:

Not applicable.

Nominal and measured concentrations:

Range finding test:
Nominal: 100, 1000 mg/l.
Measured: Not appropriate to test guideline.

Definitive study:
Nominal: 34, 1000 mg/l
Measured: Not stated.

Details on test conditions:

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

- Material, size, headspace, fill volume:
500ml glass conical flasks filled to 500ml.

- Aeration:
Throughout at a rate of approximately 0.5 – 1 litre per minute.

- 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:
Not applicable.

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

Definitive test:
34 mg/l concentration: no replicate.
100 mg/l concentration: 3

- No. of vessels per control (replicates):
Range finding:
Test material control: 2
3,5-dichlorophenol: 1

Definitive test:
Test material control: 2
3,5-dichlorophenol: 1

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

- Biomass loading rate:
Not stated.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
See Appendix 2.

- Total organic carbon:
See Appendix 2.

- Particulate matter:
See Appendix 2.

- Metals:
See Appendix 2.

- Pesticides:
See Appendix 2.

- Chlorine:
See Appendix 2.

- Alkalinity:
See Appendix 2.

- Ca/mg ratio:
See Appendix 2.

- Conductivity:
Not stated.

- Culture medium different from test medium:
No.

- Intervals of water quality measurement:
30 minutes.


OTHER TEST CONDITIONS
- Adjustment of pH:
See Appendix 2.

- Photoperiod & Light intensity:
The test was conducted under normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
30 minutes at 0 hours and 3 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Not applicable.

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

- Range finding study
- Test concentrations:
100 & 1000 mg/l

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

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- 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. 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 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.
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).

No significant effect on respiration was observed in the range finding test. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.
In order to satisfy the requirements of all Competent Authorities worldwide a single test concentration of 34 mg/l, prepared using a saturated solution of the test material was also included in the study. This concentration was included in the definitive test as pre-study solubility work had indicated that under the conditions of the test this was the highest test concentration that could be attained in solution.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes.

- Relevant effect levels:
EC50 at 3 hours: 7.6 mg/l, 95% confidence limits 6.0-9.7 mg/l.

- Other:

Reported statistics and error estimates:

None given.

Any other information on results incl. tables

Definitive test results.

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the definitive test are given in Tables 2 and 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4, and observations made on the test preparations throughout the study are given in Table 5.

Percentage inhibition is plotted against concentration for the reference material, 3,5‑dichlorophenol only (Figures 1 and 2).

The following results were derived for the test material dispersed directly in water with the aid of ultrasonication:

MOS-HIGE:

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	>1000	-	>1000	-
EC50	>1000	-	>1000	-
EC80	>1000	-	>1000	-
NOEC	1000	-	1000	-

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The following results were derived for the test material from the single vessel prepared at the limit of water solubility of the test material using a saturated solution:

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	>34	-	>34	-
EC50	>34	-	>34	-
EC80	>34	-	>34	-
NOEC	34	-	34	-

3,5-dichlorophenol:

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	3.6	-	2.5	-
EC50	9.1	7.4 - 11	7.6	6.0 - 9.7
EC80	23	-	23	-

Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 2%, and ± 1% after 3 hours contact time.

The validation criteria for the control respiration rates and reference material EC₅₀values were therefore satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O₂/l and 2.5 mg O₂/l respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

The test material vessels at 1000 mg/l showed significantly higher pH values (see Table 4) than those in the control vessels. This was considered to be attributed to the high concentration of test material in these vessels as this effect was not seen in the 34 mg/l test vessel.

Observations made throughout the test period (see Table 5) showed that at the test concentration of 34 mg/l no undissolved test material was visible.

The 1000 mg/l test concentration vessels contained cloudy light brown dispersions with particles of test material dispersed homogenously throughout.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the test vessels prepared at a concentration in excess of the test material solubility, the effect of the test material on the respiration of activated sewage sludge gave a 3 Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.
In the test vessel prepared at a concentration of 34 mg/l, the effect of the test material on the respiration of activated sewage sludge gave a 3 Hour EC50 of greater than 34 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 34 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", EEC Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

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. A solution of 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[1].

In order to satisfy the requirements of all Competent Authorities worldwide, the definitive test was therefore conducted using two methods of addition of the test material to the test system.

The first method of preparation involved exposing activated sewage sludge to a dispersed test material concentration of 1000 mg/l (three replicate flasks) prepared by direct dispersion of the test material in water.

Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge micro-organisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge. Additionally the effect of the test material as a whole on a waste water treatment facility and any possible contact toxicity can be determined.

The second method employed in the definitive test involved exposing activated sewage sludge to a nominal test concentration of 34 mg/l. This test concentration was prepared from a saturated solution of the test material and was the maximum concentration that could be attained from a stock concentration of 38 mg/l* after the addition of synthetic sewage and inoculum. Using this approach the effect of the test material at the highest attainable test concentration that was in solution can be determined.

In each method the activated sewage sludge was exposed to the test material for a period of 3 hours at 21ºC with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

In the test vessels prepared at a concentration in excess of the test material solubility, the effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀ of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

In the test vessel prepared at a concentration of 34 mg/l, the effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀ of greater than 34 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 34 mg/l.

The reference material gave a 3-Hour EC₅₀ value of 7.6 mg/l, 95% confidence limits
6.0 – 9.7 mg/l.

[1]Concentration determined by chemical analysis of the saturated solution during pre-study solubility work.

*Concentration determined by chemical analysis of the saturated solution during pre-study solubility work.