Phosphoric acid, C4 and C12-14(even-numbered) Alkyl Esters, Ammonium Salts

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:

The study was conducted between 24 May 2011 and 25 May 2011.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations:10, 32, 100, 320 and 1000 mg/l.- Sampling method: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.

Vehicle:

no

Details on test solutions:

Test item preparation:For the purpose of the test, the test item was dispersed directly in water.Amounts of test item (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximise the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentrations of 10, 32, 100, 320 and 1000 mg/l.The control group was maintained under identical conditions but not exposed to the test item. Reference item (control) preparation:For the purpose of the test a reference item, 3,5-dichlorophenol was used. Two stock solutions of 50 and 160 mg/I were prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. Aliquots (10 and 100 ml) of the 160 mg/I stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/1. Similarly, a 100 ml aliquot of the 50 mg/I stock solution was used to prepare the 10 mg/I concentration. The volumetric flasks containing the reference item were inverted several times to ensure homogeneity of the solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test Species:A mixed population of activated sewage sludge micro-organisms was obtained on 25 May 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.Preparation of inoculumThe 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 7.4 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 3.6 g/l prior to use.* rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

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 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.

Test temperature:

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

pH:

The pH values of the test preparations at the start and end of the exposure period are given in Table 2 (see results section).Test item pH ranged from 6.1 - 7.7.Values obtained for the pH of the test preparations at the end of the exposure period (see Table 2), showed a significantly lower pH value at the test concentration of 1000 mg/l compared to the other test vessels. This may be considered to be a contributing factor to the toxic nature of the test item but is considered not to affect the integrity of the study, as the purpose was to determine the effect that the test item would have on a waste water treatment facility where pH adjustment of the incoming effluent would not occur.

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:

Nominal Concentrations (mg/l) 10, 32, 100, 320 and 1000

Details on test conditions:

Preparation of 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 Section "Test item preparation" (see details on 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±1ºC.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 Water:The test water used for the 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. Synthetic Sewage:A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:16 gPeptone11 gMeat extract3 gUrea0.7 gNaCl0.4 gCaCl2.2H2O0.2 gMgSO4.7H2O2.8 gK2HPO4dissolved in 1 litre of water with the aid of ultrasonication.EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

420 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence limits 330 - 530 mg/l

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not specified

Details on results:

Please see Any other information on results incl tables section

Results with reference substance (positive control):

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.The reference item gave a 3-Hour EC50 value of 6.6mg/l, 95% confidence limits 5.1-8.5 mg/l.

RESULTS

Oxygen consumption rates and percentage inhibition values for the control, test and reference items 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 test and reference items (see attached Figures 1 to 2 - attached background material).

The following results were derived:

	Reactionmass of bis(2 -ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate		3,5-dichlorophenol
	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	190	-	2.1	-
EC50	420	330 - 530	6.6	5.1 - 8.5
EC80	790	-	21	-
NOEC	150	-	1.7	-

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 0%.

The validation criteria for the control respiration rates and reference item EC₅₀ values 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.

Values obtained for the pH of the test preparations at the end of the exposure period (see Table 2), showed a significantly lower pH value at the test concentration of 1000 mg/l compared to the other test vessels. This may be considered to be a contributing factor to the toxic nature of the test item but is considered not to affect the integrity of the study, as the purpose was to determine the effect that the test item would have on a waste water treatment facility where pH adjustment of the incoming effluent would not occur.

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	4.5	6	1.6	0.48	-
	R2	5.5	8	1.7	0.48	-
Test Item	10	4.7	6	1.8	0.48	0
	32	4.5	6	1.6	0.48	0
	100	4.0	5	1.8	0.44	8
	320	5.0	10	2.0	0.30	38
	1000	8.1	10	7.6	0.05	90
3,5-dichlorophenol	3.2	5.7	7	3.3	0.34	29
	10	7.5	10	5.8	0.17	65
	32	8.2	10	7.6	0.06	88

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	7.4	7.8
	R2	7.4	7.7
Test Item	10	7.5	7.7
	32	7.5	7.6
	100	7.5	7.5
	320	7.1	7.3
	1000	6.1	6.3
3,5-dichlorophenol	3.2	7.4	8.0
	10	7.4	8.1
	32	7.5	8.1

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 Time	3 Hours Contact Time
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 water column with few very small globules of test item visible floating on the surface*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	32	Clear colourless water column with few small globules of test item visible floating on the surface *	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Very slightly cloudy dispersion, small globules of test item visible dispersed throughout and floating on surface*	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	320	Slightly cloudy dispersion with globules of test item visible dispersed throughout and floating on surface*	Dark brown dispersion few globules of test item visible dispersed throughout and floating on surface	Dark brown dispersion, few globules of test item visible dispersed throughout and floating on surface. Few globules of test item adhered to side of flask
	1000	Cloudy dispersion with large globules of test item visible floating on surface*	Dark brown dispersion, globules of test item visible dispersed throughout and floating on surface	Dark brown dispersion, globules of test item visible dispersed throughout and floating on surface. Globules/scum of test item adhered to side of flask
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

Validity criteria fulfilled:

yes

Remarks:

The results of the study were considered valid as the two control respiration rates were within 15% of each other and the EC50 (3 hour contact time) for 3,5-dichlorophenol was within the range 5 - 30 mg/l.

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of 420 mg/l, 95% confidence limits 330 - 530 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 150 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with 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 dispersion 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 time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Conclusion.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC₅₀ of 420 mg/l, 95% confidence limits 330 - 530 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was150mg/l.

The reference item gave a 3-Hour EC₅₀value of 6.6 mg/l, 95% confidence limits 5.1 – 8.5 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:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar ecotoxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Source: Phosphoric acid, 2-methylpropyl ester, potassium salt [CAS 90605-13-3]
Target: Phosphoric acid, C4 and C12-14(even numbered) alkyl esters, Ammonium salts [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Available data for 2-ethylhexyl- and 2-methylpropyl- ester, potassium salt analogues of the target demonstrate low toxicity to aquatic microorganisms. This low toxicity is expected for the target substance also.
4. DATA MATRIX
Source: Phosphoric acid, 2-ethylhexyl ester; ASRI - 3-h EC50 = 420 mg/L; NOEC = 150 mg/L
Source: Phosphoric acid, 2-methylpropyl ester, potassium salt; ASRI – 3-h EC50 > 1000 mg/L; NOEC = 1000 mg/L
Target: ASRI - no data

Reason / purpose for cross-reference:

read-across source

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

420 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence limits 330 - 530 mg/l

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not specified

Reference 3

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:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar ecotoxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Source: Phosphoric acid, 2-methylpropyl ester, potassium salt [CAS 90605-13-3]
Target: Phosphoric acid, C4 and C12-14(even numbered) alkyl esters, Ammonium salts [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Available data for 2-ethylhexyl- and 2-methylpropyl- ester, potassium salt analogues of the target demonstrate low toxicity to aquatic microorganisms. This low toxicity is expected for the target substance also.
4. DATA MATRIX
Source: Phosphoric acid, 2-ethylhexyl ester; ASRI - 3-h EC50 = 420 mg/L; NOEC = 150 mg/L
Source: Phosphoric acid, 2-methylpropyl ester, potassium salt; ASRI – 3-h EC50 > 1000 mg/L; NOEC = 1000 mg/L
Target: ASRI - no data

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

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-26

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Sludge was taken from the activation basin of the ESN (Stadtentsorung Neustadt) sewage treatment plant, Im Altenschemel, 67435 NW-Laken-Speyerdorf
- Pretreatment: The sludge was washed with tap water three times, then centrifuged and re-suspended in tap water. 200 mL of the sludge were added to each test vessel.
- Initial biomass concentration: Dry matter in the test = 1518 mg suspended solids/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

not applicable

Hardness:

no data

Test temperature:

20 +/- 2 deg C

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

nominal: 1, 10, 100, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Aeration: yes, approx 0.75 L/min
- No. of vessels per concentration (replicates): 2 for 1000 mg/L, 1 for 1-100 mg/L
- No. of vessels per control (replicates): 2
- No. of vessels per abiotic control (replicates):
- Sludge concentration (weight of dry solids per volume): 1518 mg/L
- Nutrients provided for bacteria: Synthetic sewage
- Biomass loading rate:
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED: Respiration rate
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

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

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

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Coefficient of variation of oxygen uptake rate in control replicates: Maximum of 8%, which is below the recommended limit of 15%

Results with reference substance (positive control):

- Results with reference substance valid? yes, EC50 = 5.1 mg/L which is within the recommended range of 5-30 mg/L

Reported statistics and error estimates:

not applicable

The test item showed very little inhibition in the experiment. In the highest treatment (1000 mg/L) no inhibition could be detected. In the the lower treatments, inhibition values stayed below 5%. Therefore, these values were not attributed to an inhibitory effect of the test item.

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50 > 1000 mg/L
The 3-h NOEC = 1000 mg/L

Executive summary:

The inhibitory effect of the test item on the respiration rate of activated sludge microorganisms was determined according to OECD TG 209. The test item was tested using 5 concentrations ranging from 1 to 1000 mg/L. Duration of the test was 3 hours. Activated sludge taken from a domestic sewage treatment plant was used as inoculum. After washing, the dry matter was determined as 3.79 g suspended solids/L, giving a concentration of 1.52 g suspended solids/L in the test.

3,5 -dichlorophenol was used as the reference item. Five concentrations were tested; an EC50 of 5.1 was determined, which lies within the range of 5 -30 mg/L.

At 1000 mg/L no inhibition occurred. Therefore the pre-test can be taken as a limit test.

3h NOEC = 1000 mg/L

3h EC50 > 1000 mg/L

Description of key information

Phosphoric acid, 2-ethylhexyl ester; ASRI - 3-h EC50 = 420 mg/L; NOEC = 150 mg/L

Phosphoric acid, 2-methylpropyl ester, potassium salt; ASRI – 3-h EC50 > 1000 mg/L; NOEC = 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

420 mg/L

EC10 or NOEC for microorganisms:

150 mg/L