Sodium metaphosphate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate:26 November 2009

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 32, 100, 320 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:

For the purpose of the test, the test item was dispersed directly in water.
An amount of test item (2000 mg) was dispersed in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock dispersion from which serial dilutions were made to give 200 and 20 mg/l stock solutions. An aliquot (250 ml) of the 20 mg/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg/l. Similarly, aliquots (80 and 250 ml) of the 200 mg/l stock solution and aliquots (80 and 250 ml) of the 2000 mg/l stock dispersion were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/l respectively. The volumetric flasks containing the stock solutions/dispersion were inverted several times to ensure homogeneity of the stocks.
The control group was maintained under identical conditions but not exposed to the test item.
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 300 ml with water and 200 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 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference item being added. The test item vessels were prepared as described above. Finally a second control was prepared.

As each vessel reached 30 minutes 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 6.5 mg O2/l and 2.5 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. After measurement the contents of the BOD bottle were returned to the test vessel.
This procedure was repeated after 3 hours contact time. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 deg C.
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.

- 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):
Observations made throughout the test period (see Table 3) showed that at 0 hours, 30 minutes and 3 hours that the control vessels contained a dark brown dispersion and the reference item vessels contained a dark brown dispersion with no undissolved reference item visible.
Observations made of the test item vessels at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that all test concentrations contained a clear colourless solution.
Observations made of the test item vessels after 30 minutes and 3 hours contact time showed that all test concentrations contained a dark brown dispersion with no undissolved test item visible.

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 21ºC and was used on the day of collection. The pH of the sample was 7.2 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 concentration was equal to 3.9 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.

Test temperature:

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

pH:

The pH values of the test preparations at the start and end of the exposure period are given in Table 2.

Dissolved oxygen:

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.

Salinity:

Not applicable.

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 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 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference item being added. The test item vessels were prepared as described in "details of test solutions". Finally a second control was 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 6.5 mg O2/l and 2.5 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 21±1degC.
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.


TEST MEDIUM / WATER PARAMETERS
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.


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 Table 2

- Photoperiod:
3 hours.

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference items the respiration rate was expressed as a percentage of the two control respiration rates.

% inhibition = [ 1 – 2 RS ] x 100
RC1 + RC2
where
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2
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 EC50 value for the reference item only using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
The No Observed Effect Concentration (NOEC) was determined by inspection of the inhibition of respiration rate data.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.


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:

Definitive Test
Oxygen consumption rates and percentage inhibition values for the control, test and items in the definitive test are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3.
Percentage inhibition is plotted against concentration for the reference item, 3,5 dichlorophenol only (Figure 1).

The following results were derived:
Sodium metaphosphate
ECx (30 Hours) (mg/l) 95% Confidence Limits (mg/l)
EC20 >1000 -
EC50 >1000 -
EC80 >1000 -
NOEC 1000 -

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

3,5-dichlorophenol:
ECx (3 Hours) (mg/l) 95% Confidence Limits (mg/l)
EC20 2.8 -
EC50 8.3 6.5 - 11
EC80 25 -
NOEC 2.3 -
Variation in respiration rates of controls 1 and 2 was ± 0% after 3 hours contact time.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

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 8.3 mg/l, 95% confidence limits 6.5 - 11 mg/l.

- Other:
None.

Reported statistics and error estimates:

None.

Table 1. Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurent Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	5.6	6	2.2	0.57	-
	R2	5.9	6	2.5	0.57	-
Test Item	10	6.1	6	2.7	0.57	0
	32	5.6	5	2.8	0.56	2
	100	6.0	6	2.6	0.57	0
	320	5.4	5	2.4	0.60	[5]
	1000	6.0	7	2.4	0.51	11
3,5-dichlorophenol	3.2	6.1	8	2.5	0.45	21
	10	7.6	10	5.4	0.22	61
	32	8.2	10	7.3	0.09	84

[Increase in respiration rate as compared to controls]

R₁– R₂= Replicates 1 to 2

Table 2.  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	7.3	7.7
	R2	7.4	7.7
Test Item	10	7.4	7.8
	32	7.4	7.7
	100	7.4	7.7
	320	7.4	7.7
	1000	7.5	7.7
3,5-dichlorophenol	3.2	7.3	7.9
	10	7.4	8.0
	32	7.4	8.0

R₁– R₂= Replicates 1 to 2

Table 3. Observations on the Test Preparations Throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours	30 Minutes Contact Ti	3 Hours Contact Ti
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Clear colourless solution*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	32	Clear colourless solution*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Clear colourless solution*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	320	Clear colourless solution*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000	Clear colourless solution*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
3,5-dichlorophenol	3.2	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

R₁– R₂= Replicates 1 to 2

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

Concentration-Response Curve: 3,5-dichlorophenol – 3 Hours Contact Time see attached figure 1

See atached Appendix1 For Typical Water Quality Characteristics

 REFERENCES

Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experints. J Pharmacol Exp Ther96, 99-113.

Xlfit, ID Business Solutions Ltd.

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 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition the study is considered to be acceptable for classification and labelling in accordance with Regulation (EC) No 1272/2008 (EU CLP).

Executive summary:

Introduction.A study was perford to assess the effect of the test item on the respiration of activated sewage sludge. Thethod followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods. Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of 21 ± 1°C with the addition of a synthetic sewage as a respiratory substrate.

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

Results.The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

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

The reference item gave a 3-Hour EC₅₀value of 8.3 mg/l, 95% confidence limits 6.5 - 11 mg/l.

Description of key information

Two studies are available to assess the toxicity of sodium metaphosphate to STP microorganisms. The key study (Clark, 2010) has been conducted to OECD guideline 209 and under the conditions of GLP. 
The additional supporting data (Post, 1963) discusses the effects of sodium metaphosphate on selected bacteria and is therefore not considered to fulfil the endpoint for toxicity to microorganisms.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The effect of the test item on the respiration of activated sewage sludge micro-organisms 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.