Trimanganese bis(orthophosphate)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-28 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with an acceptable guideline (OECD 209) with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: >450°C
- Water solubility (under test conditions): The water solubility of the manganese hydrogen phosphate has been determined to be at least 211 g/l of solution at 20.0 ± 0.5°C in un-buffered glass double-distilled water, resulting in sample solution pH’s of approximately 2.6. However, it was demonstrated that the water solubility of the test material varied with nominal loading rate and as such may increase should the loading rate be increased further.

- Stability in water: dissociates to manganese(II) cations and phosphate anions

Analytical monitoring:

no

Details on sampling:

- Concentrations:
10, 100 and 1000 mg/l

- Sampling method:
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference item and test item preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

- Sample storage conditions before analysis:
not specified in report

Vehicle:

no

Details on test solutions:

The test item was dispersed directly in water.
Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


- Eluate:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as described above. Finally two further control vessels were prepared.

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L and 2.0 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
The test was conducted under normal laboratory lighting in a temperature controlled room at 20 ± 2 ºC.
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time

- Differential loading:
Not applicable.

- Controls:
The control group was maintained under identical conditions but not exposed to the test item.

- 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):
See table 5.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (7 liters) was fed synthetic sewage sludge (350 mL). The pH of the sample on the day of the test was 7.9 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 deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 (see in attached section).

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 Deg C.

pH:

The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 340I pH and dissolved oxygen meter and adjusted to between pH 7.0 and 8.0.

Dissolved oxygen:

The dissolved oxygen concentrations after 30 minutes contact time in some of the vessels were below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were measured over the linear portion of the traces.

Salinity:

Not applicable.

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/l

Details on test conditions:

RANGE-FINDING TEST
In the range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 340I pH and dissolved oxygen meter and adjusted to between pH 7.0 and 8.0.
The control group was maintained under identical conditions but not exposed to the test item.
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 5.4 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.



TEST MEDIUM / WATER PARAMETERS
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).


OTHER TEST CONDITIONS
- Adjustment of pH:
The pH values of the test preparations at the start and end of the exposure period are given in the tables.

- Photoperiod:
3 hours.

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:
R = [(Q1 - Q2) / Δt] x 60
Where:
Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt = the time interval between the beginning and end of the selected section of the linear phase (min).
The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:
RS = R / SS
Where:
SS = the concentration of suspended solids in the test mixture (g/L).
The percentage inhibition was calculated according to the following equation:

% inhibition = [1 – (R/Rbc)] x 100
Where:
Where:
Rbc = the mean respiration rate of the blank controls.

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
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 oxygen consumption data after 3 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).
The results of the study are considered valid if (i) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L (ii) the specific respiration rate of the blank controls should not be less than 20 mg oxygen per gram dry weight of sludge per hour (iii) the coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of the test.

TEST CONCENTRATIONS
10, 32, 100, 320 and 1000 mg/l

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

Remarks on result:

other: not specified

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

Remarks on result:

other: not specified

Details on results:

No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The coefficient of variation of oxygen uptake in the control vessels was 5.6% and the specific respiration rate of the controls was 28.17 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied.
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 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.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes.

- Relevant effect levels:
The reference material gave a 3-Hour EC50 value of 11 mg/l, 95% confidence limits 9.2 - 13 mg/l.

- Other:
None.

Reported statistics and error estimates:

None.

Table 1.pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test             

Nominal Concentration (mg/L)	pH*
	Prior to Adjustment	After Adjustment
10	7.0	7.2
100	6.4	7.2
1000 R1	5.4	7.2
1000 R2	5.4	7.3
1000 R3	5.5	7.3

*Measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

R1 – R3 = Replicates 1 to 3

Table 2.Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)	Dissolved Oxygen Concentration (mg O2/L) *
Control
R1	4.8
R2	5.8
R3	5.4
R4	4.0
Test Item
10	5.7
100	4.1
1000 R1	2.9
1000 R2	5.0
1000 R3	5.5
3,5-dichlorophenol
3.2	6.0
10	6.6
32	7.2

* Measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

R1 – R4 = Replicates 1 to 4

 

Table 3. Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Timein the Range-Finding Test

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	4.2	3	2.3	44.00	-
	R2	4.9	4	2.3	39.00	-
	R3	4.0	3	1.8	44.00	-
	R4	3.1	2	1.7	42.00
Test Item	10	4.0	3	1.9	42.00	1
	100	3.8	2	2.4	42.00	1
	1000 R1	3.7	2	2.3	42.00	1
	1000 R2	4.0	3	2.0	40.00	5
	1000 R3	4.4	4	1.8	39.00	8
3,5-dichlorophenol	3.2	4.7	4	2.2	37.50	11
	10	6.7	10	3.2	21.00	50
	32	7.5	10	6.0	9.00	79

 

R1 – R4 = Replicates 1 to 4

Table 4.  pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.8	8.1
	R2	7.7	8.1
	R3	7.7	8.1
	R4	7.7	8.0
Test Item	10	7.7	8.1
	100	7.7	8.1
	1000 R1	7.5	8.0
	1000 R2	7.5	8.0
	1000 R3	7.6	8.1
3,5-dichlorophenol	3.2	7.8	8.3
	10	7.8	8.4
	32	7.8	8.4

 

R1– R4= Replicates 1 to 4

Table 5. Observations on the Test Preparations throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Pale yellow/brown cloudy dispersion	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Pale yellow/brown cloudy dispersion. Undissolved test item on bottom of flask	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R1	Pale yellow/cream cloudy dispersion. Undissolved test item dispersed throughout and on bottom of flask	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	1000 R2	Pale yellow/cream cloudy dispersion. Undissolved test item dispersed throughout and on bottom of flask	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	1000 R3	Pale yellow/cream cloudy dispersion. Undissolved test item dispersed throughout and on bottom of flask	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
3,5-dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

 

R1– R4 = Replicates 1 to 2

*Observations made prior to the addition of synthetic sewage and activated sewage sludge

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the analogue justification attached to chapter 13.

Reason / purpose for cross-reference:

read-across source

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

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

Description of key information

Not toxic to microorganisms.

Additional information

Experimental data on the toxicity to microorganisms are not available for trimanganese bis(orthophosphate). In accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across to the structurally related substances substance manganese hydrogen phosphate (CAS 51349-94-1) was conducted to fulfil the standard information requirements. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for aquatic toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across). According to Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 substances can be considered to be similar if their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

The effect of manganese hydrogen phosphate (CAS 51349-94-1) on the respiration of activated sewage sludge was investigated in a guideline study following OECD Guidelines 209. Non-adapted activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L. The respiration of the sludge was determined over a period of 3 hours at a temperature of 21 ± 1°C. Significant effects on the respiration rate of the activated sludge were not observed. The determined EC50 (3h) was >1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000mg/L.

Based on the data on the read across substance manganese hydrogen phosphate (CAS 51349-94-1) a toxicity of the structural similar substancetrimanganese bis(orthophosphate)to microorganisms is not anticipated.