Phosphoric acid, butyl ester, sodium salt

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:

weight of evidence

Study period:

from 2007-02-22 to 2007-05-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

no

Remarks:

The study was conducted in accordance with ISO/IEC 17025.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Final test concentrations were prepared with test medium by dilution of stock solutions of the test substance.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: Activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not pre-conditioned
- Name and location of sewage treatment plant where inoculum was collected: ARA Werdhölzli, CH-8048 Zürich, Switzerland (5 March 2007, 8.45 a.m.)
- Concentration: 1.6 g/L dry matter in the final mixture
- The activated sludge was used immediately after sampling from the treatment plant without adaptation. lf the sludge was used on the following day, 50 mL of the synthetic sewage concentrate were added per liter of activated sludge and the sludge aerated overnight. Prior to the test, the sludge was washed twice with tap water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

1.64 - 30.2 mmol/L

Test temperature:

22 ± 0.5 °C

pH:

7.5 ± 0.5

Conductivity:

288 - 614 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 125, 250, 500, 1000 and 2000 mg/L
No measured concentrations.

Details on test conditions:

- Test unit: 1000 mL flasks containing a total volume of test suspension of 500 mL;
- Aerated by shaking (120 r.p.m.)
- Measuring apparatus: 300 mL flat bottom BOD-flask; polarographic oxygen electrode, connected to a potentiometric recorder
- Duration/contact time: 3 hours in a temperature-controlled room
- Test medium: Synthetic sewage feed prepared with tap water - 16 mL of 100-fold sewage concentrate per 500 mL of final test mixture
- Controls: 2 blank controls with inoculated sample without test substance

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

At the applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls. The variation between the two controls was ±1.7 %. Therefore, the test was considered valid.

Results with reference substance (positive control):

The EC50 value of the reference substance 3,5-dichlorophenol was estimated to be 3.7 mg/L which was low but in the range of 5 mg/L, thus, confirming suitability of activated sludge and test conditions.

Table 1: Definitive test: activated sludge respiration rates after 3 h of exposure to the test material and calculation of the inhibition.

Nominal concentration (mg/L)	Code	O2 consumption rates (mg O2 / L.h)	Respiration inhibition (%)
Control                      0 0	B1 B2	36.1 37.4	- -
125	T1	38.7	-5.4
250	T2	38.9	-6.0
500	T3	38.0	-3.4
1000	T4	38.8	-5.6
2000	T5	39.2	-6.6
Reference                 3 10 30	R1 R2 R3	21.0 8.9 4.4	42.9 75.7 88.0

 

Table 2: Chemical composition of the tap water supplying the laboratory

Parameter ^a)	Unit	Minimum	Maximum
pH		7.3	7.6
Conductivity (20 °C)	µS/cm	288	614
Total Hardness	°F	16.4	30.2
CO3 - Hardness	°F	14.9	26.3
Rest – Hardness	°F	1.5	3.9
Total Hardness	mmol/L	1.64	3.02
Acid consumption	mmol/L	2.88	5.26
E. Coli	Per 100 mL	0	0
Colony counts: (20 °C) (30 °C)	Per mL	0 0	22 57
Ammonium	mg/L	<0.01	<0.01
Nitrite	mg/L	<0.002	0.002
Nitrate	mg/L	3.5	12.0
Chloride	mg/L	5.0	9.0
Phosphate as PO4-P	mg/L	0.004	0.035
Sulfate	mg/L	13.5	18.5

^a) Analysis of 18.09.06; conducted by KANTONALES LABOR ZÜRICH, CH-8032 Zürich, Switzerland.

 

Table 3: Composition of the synthetic sewage feed; 100-fold concentrate OECD synthetic sewage

Dissolved amount per liter of tap water:

Substance	Concentration
Peptone	16 g
Meat extract	11 g
Urea	3 g
Sodium chloride	0.7 g
Calcium chloride dihydrate	0.4 g
Magnesium sulfate heptahydrate	0.2 g
Dipotassium hydrogen phosphate	2.8 g

 

Validity criteria fulfilled:

yes

Conclusions:

At the applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. Based on this data the EC20, EC50 and EC80 values were determined to be >2000 mg/L.

Executive summary:

The toxicity to microorganisms of the test item was determined according to OECD 209 (1984). The test item was tested in concentration levels of 125, 250, 500, 1000 and 2000 mg/L. A concurent control ran parallel. The respiration inhibition of the test item was determined with measuring the O2 concentration after 3 hours contact time with activated sludge from a domestic waste water treatment plant. The activated sludge was treated according to the guideline and then test solution in different concentrations were added to it in BOD bottles. After 3 h exposure time no signifficant effects were observed in any of the treated replicates. Based on this data the EC20, EC50 and EC80 values were determined to be >2000 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 1989-05-10 to 1989-07-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Proposed Method for the Determination of the Biodegradability of Surlactants used in Synthetic Detergents"

Version / remarks:

(OECD Technical Report of June 11, 1976)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

After 180 minutes of aeration at 22 °C, the first sample was taken for O2 measurement over a period of up to 10 minutes. Thereafter, in 15 minutes intervals, the rest of the samples were submitted to measurement of O2 concentration.

Vehicle:

no

Details on test solutions:

A stock solution of the test item prepared by dissolving the test item in distilled water at a concentration of 500 mg/L.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Inoculum: aerobic activated sludge fed with an excess but standard amount of biodegradable food
- Name and location of sewage treatment plant where inoculum was collected: ARA, Sissach, Switzerland
- Preparation of inoculum for exposure:
An aliquot of the final sludge suspension was weighed, dried and the ratio of the wet sludge (mL) to its dry weight (g) determined. Based on this ratio, a volume of washed sludge suspension corresponding to 4 g dry material were made up with Soerensen buffer (pH = 7) to 1 L. To that mixture 50 mL OECD synthetic sewage feed were addedd. Thereafter, the sludge was incubated overnight at 20 - 25 °C under aeration.
- Pretreatment:
The sludge was coarsely sieved, centrifuged, the supernatant liquid phase decanted, the solid material resuspended in tap water and again centrifuged. This procedure was repeated two times more.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

22 °C

pH:

7.7

Nominal and measured concentrations:

Nominal concentrations: 10 and 100 mg/L
No measured concentrations.

Details on test conditions:

The tests were carried out in glass-flasks (>500 mL) aerated with 0.2 L/minute. Flasks were filled up to 500 mL final volume.

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol sodium salt, 3.2, 10 and 50 mg/L

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The respiration rate was not inhibited up to the highest tested concentration of 100 mg/L. The IC50 of the test item was found to be >100 mg/L.

Results with reference substance (positive control):

At a concentration of the standard of 3.2 mg/L, the respiration rate was moderately inhibited to 43.6 %. The IC50 was found to be about 5.2 mg/L. Almost complete inhibition of 96.2 % was observed at 50 mg/L.

Table 1. Influence of the test item on oxygen consumption of activated sludge

Flask No.	Chemical	Concentration of test item (mg/L)	Oxygen consumption (mg O2/L min.)	Inhibition (%)	pH
1 11	Control Control	- -	0.79 0.77	- -	7.7 7.7
Mean ± s			0.78 ± 0.01 (1 %)
5 6	Test item Test item	100 10	0.83 0.86	-6.4 -10.3	7.7 7.7

Table 2. Influence of 3,5-dichlorophenol sodium salt on oxygen consumption of activated sludge

Flask No.	Chemical	Concentration of ref. item (mg/L)	Oxygen consumption (mg O2/L min.)	Inhibition (%)	pH
1 11	Control Control	- -	0.79 0.77	- -	7.7 7.7
Mean ± s			0.78 ± 0.01 (1 %)
2 3 4	3,5-dichlorophenol 3,5-dichlorophenol 3,5-dichlorophenol	50 10 3.2	0.03 0.38 0.44	96.2 61.3 43.6	7.7 7.7 7.7

Validity criteria fulfilled:

yes

Conclusions:

The test item showed no inhibition of the respiration rate up to a concentration of 100 mg/L. The IC50 was determined to be >100 mg/L.

Executive summary:

The toxicity to microorganisms of the test item was determined according to OECD 209 (1984) and OECD Technical report "Proposed Method for the Determination of the Biodegradability of Surlactants used in Synthetic Detergents" (1976). The test item was tested in concentration levels of 10 and 100 mg/L. A concurent two controls ran parallel and as reference item 3,5 -dichlorophenol was tested in three different concentrations - 3.2, 10 and 50 mg/L. The respiration inhibition of the test item was determined with measuring the O2 concentration after 3 hours contact time with activated sludge from a domestic waste water treatment plant. The activated sludge was treated according to the guideline and then test solution in different concentrations were added to it in glass flasks. After 3 h exposure time no signifficant effects were observed in any of the treated replicates. Based on this data the IC50 for the test item was determined to be >100 mg/L.

Description of key information

In a weight of evidence approach was shown that the test item is not toxic to microorganisms and the EC50 was determined to be >2000 mg/L. The NOEC was determined to be 2000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

2 000 mg/L

Additional information

Two studies were performed with the test item. In the first it was tested up to 2000 mg/L and in the second up to 100 mg/L. In both studies no significant toxic effects to the activated sludge were observed. Based on this data the EC50 value was determined to be >2000 mg/L and >100 mg/L, respectively. The NOEC was determined to be 2000 mg/L.

In the first study (2007), the toxicity to microorganisms of the test item was determined according to OECD 209 (1984). The test item was tested in concentration levels of 125, 250, 500, 1000 and 2000 mg/L. A concurent control ran parallel. The respiration inhibition of the test item was determined with measuring the O2 concentration after 3 hours contact time with activated sludge from a domestic waste water treatment plant. The activated sludge was treated according to the guideline and then test solution in different concentrations were added to it in BOD bottles. After 3 h exposure time no signifficant effects were observed in any of the treated replicates. Based on this data the EC20, EC50 and EC80 values were determined to be >2000 mg/L. The NOEC was determined to be 2000 mg/L.

In an additional study (1989), the toxicity to microorganisms of the test item was determined according to OECD 209 (1984) and OECD Technical report "Proposed Method for the Determination of the Biodegradability of Surlactants used in Synthetic Detergents" (1976). The test item was tested in concentration levels of 10 and 100 mg/L. A concurent two controls ran parallel and as reference item 3,5 -dichlorophenol was tested in three different concentrations - 3.2, 10 and 50 mg/L. The respiration inhibition of the test item was determined with measuring the O2 concentration after 3 hours contact time with activated sludge from a domestic waste water treatment plant. The activated sludge was treated according to the guideline and then test solution in different concentrations were added to it in glass flasks. After 3 h exposure time no signifficant effects were observed in any of the treated replicates. Based on this data the IC50 for the test item was determined to be >100 mg/L. This result supported the results obtained from the other study.

In conclusion, the test item is not toxic towards microorganisms and the EC50 can be stated as >2000 mg/L.