Phosphoric acid, 2-ethylhexyl 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:

supporting study

Study period:

June - September 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Ecological Method No.103 (ETAD): A screening test for the assessment of the possible inhibitory effect of a chemical substance on aerobic wastewater bacteria.

Deviations:

yes

Remarks:

less concentrations used

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: ARA Birsig, Therwil/ Swi tzerland
- Pretreatment: not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

180 min

pH:

7.8

Nominal and measured concentrations:

Nominal concentrations: 10 and 100 mg/L
Concentrations not measured

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks (> 500 mL)
- Aeration: yes, with "Pasteur" pipette (0.7 L/min)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per positive control (replicates): 1
- Sludge concentration (weight of dry solids per volume): not specified
- Biomass loading rate: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not specified

EFFECT PARAMETERS MEASURED:
After 180 minutes of aeration, the samples were taken for oxygen measurement over a period of 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The apparatus used for oxygen measurements was the WTW 0X1 91 (Wissenschaftlich-Technische Werkstaetten, Weilheim/Federal Republic of Germany). The pH was determined with the pH 90 (WTW, Weilheim/FRG).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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 inhibition of the respiration rate of activated sludge after the incubation period of three hours was 23.5% for 10 mg/L and 35.5 for 100 mg/L compared to the control.

Results with reference substance (positive control):

Respiration rate was inhibited to a minor extent (47.1 %) at concentrations of the standard up to 3.2 mg/L. The IC 50 was found to be about 5 mg/L.

The test is regarded as valid since:

 

- the coefficient of variation of oxygen uptake rate in control replicates was 4% and therefore, less than 30%.

 

- the EC₅₀(3-Hour) of the reference item 3,5-dichlorophenol was about 5 mg/L and therefore, in the range 2 to 25 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The test item has slightly to moderate inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at a test item concentration of 100 mg/L.

Executive summary:

The toxicity of the test substance to bacteria in sewage sludge was tested in a static system in accordance with Ecological Method No 103. The respiration rate of activated sludge from a domestic wastewater treatment plant was determined in the absence of test substance. Then the corresponding respiration rate of the sludge exposed to nominal test concentrations of 10 and 100 mg/L for 3 hours was determined and compared to the respiration rate of untreated sludge. The microbial activity of the sludge was inhibited at all test concentrations and reduced by 23.5% to 35.3%. The IC50 value for inhibition was higher than 100 mg/L after 3 hours. The sensitivity of the activated sludge was tested and confirmed with the reference substance 3,5 -dichlorophenole.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2019-07-08 to 2019-09-12

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 (Carbon and Ammonium Oxidation))

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Ecological Effects Test Guidelines of the United States Environmental Protection Agency (EPA 712-C-014): OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test", January 2012.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test item: Defined amounts of the test item (1 x 3; 1 x 30 mg and 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly weighed (administered) into each test flask.
- Reference item: 0.1 g 3,5-Dichlorophenol was dissolved in warm purified, deionized water. The pH of the stock solution (after cooling) was adjusted with 1N NaOH to 7.41. After pH setting the volume of this solution was completed ad 100 mL, in a volumetric flask.
- Nitrification inhibitor: The N-allylthiourea solution was prepared by dissolving 0.232 g of N-allylthiourea in 100 mL of purified, deionized water.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: sewage plant for domestic sewage in Balatonfüred, Hungary
- Preparation of inoculum for exposure: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.081 g wet weight), dried and the ratio of wet sludge to dry weight (0.4037 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (24 g dry weight that was equivalent to 302 g wet sludge) was suspended in isotonic saline solution (ad. 8 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was checked after preparation to be 7.31, therefore, pH adjustment was considered not necessary.
- Initial biomass concentration: 1.5 g per litre on dry weight basis

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.1 - 22.0 °C

pH:

6.93 - 8.14

Nominal and measured concentrations:

Nominal concentrations: 10, 100, 1000 mg/L
No measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel (material, size, headspace, fill volume): Erlenmeyer glass bottles of approximately 300 mL volume
- Aeration: aerated with compressed air (0.5 L/minute)
- No. of vessels per concentration (replicates): 1 for 10 and 100 mg/L, 3 for 1000 mg/L
- No. of vessels per control (replicates): 8
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g per litre on dry weight basis
- Nutrients provided for bacteria: no
- Nitrification inhibitor used: N-allylthiourea , 2 replicate at 11.6 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water used for the test solutions. Synthetic sludge feed prepared according to the OECD guideline.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 5.5 - 10 minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Specific Respiration Rates in the Test Item Concentrations

In the experiment abiotic oxygen consumption of the test substance was not observed (in average 0.30 mg/Lh); therefore, the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations. The measured nitrification potential of the applied activated sludge did not necessitate further testing.

The observed oxygen consumption rates and consequently the specific respiration rates in the test item treatment groups were in the range of the blank controls. No inhibitory effect of the test item was observed. The observed, in average 4.23 % (at the highest examined concentration of 1000 mg/L) inhibition was within the biological variability of the applied test system.

The specific respiration rates of the highest dose, 1000 mg/L were compared with the blank control values using 2-Sample t-Test (2-sided, α=0.05). No statistically significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/L.

Based on measured oxygen consumption values and calculated specific respiration rates it is concluded that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L.

The EC50 value was determined as: EC50 > 1000 mg/L.

Toxicity of 3,5-Dichlorophenol

The positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item at concentration levels of 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 10.23 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 21.77 % and 79.26 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 16.06 mg/L, (95 % confidence limits: 9.15 - 32.08 mg/L).

Nitrification Controls

An additional nitrification control was examined in the test with two parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 47.45 mg/Lh, the heterotrophic respiration (RH) was 46.50 mg/Lh, the nitrification respiration (RN): 0.95 mg/Lh was calculated according to the equation: RN = RT - RH.

The obtained 0.95 mg/Lh was considered as not significantly different, being within a biological variability range of the applied test system, and lower than the 5 % of RT (2.37 mg/Lh) in blank controls. According to the above calculations and based on the OECD 209 guideline it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of the test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.

Executive summary:

A study according to OECD 209, Regulation (EC) No 440/2008 Method C.11 and OCSPP 850.3300 was performed with the test item as a preliminary test. The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration.

In parallel with the test item treatments, 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification controls and abiotic controls were investigated. Abiotic controls (investigated in three parallels) were prepared containing the test item in the concentration of 1000 mg/L, synthetics sewage feed, but no inoculum. In this test no abiotic oxygen consumption was noticed.

All validity criteria of the study were met. The average specific respiration rate of the blank was 31.64 mg O2 / g activated sludge (based on dry weight) in an hour with a coefficient of variation of 4.73 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 16.06 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 30.30 mg O2/gh). No inhibitory effect of the test item was observed.

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested. The specific respiration rates at the examined highest test item concentration of 1000 mg/L were compared with the blank control values using 2-Sample T-test (α=0.05). No statistically significant differences were observed, compared to the blank control values.

In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L, therefore, in line with OECD 209 a definite test is not required.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of the test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Key:

A study according to OECD 209, Regulation (EC) No 440/2008 Method C.11 and OCSPP 850.3300 was performed with the test item as a preliminary test. The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration.

In parallel with the test item treatments, 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification controls and abiotic controls were investigated. Abiotic controls (investigated in three parallels) were prepared containing the test item in the concentration of 1000 mg/L, synthetics sewage feed, but no inoculum. In this test no abiotic oxygen consumption was noticed.

All validity criteria of the study were met. The average specific respiration rate of the blank was 31.64 mg O2 / g activated sludge (based on dry weight) in an hour with a coefficient of variation of 4.73 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 16.06 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 30.30 mg O2/gh). No inhibitory effect of the test item was observed.

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested. The specific respiration rates at the examined highest test item concentration of 1000 mg/L were compared with the blank control values using 2-Sample T-test (α=0.05). No statistically significant differences were observed, compared to the blank control values.

In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L, therefore, in line with OECD 209 a definite test is not required.

Supporting:

The toxicity of the test substance to bacteria in sewage sludge was tested in a static system in accordance with ETAD ( Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers) Ecological Method No 103. The respiration rate of activated sludge from a domestic wastewater treatment plant was determined in the absence of test substance. Then the corresponding respiration rate of the sludge exposed to nominal test concentrations of 10 and 100 mg/L for 3 hours was determined and compared to the respiration rate of untreated sludge. The microbial activity of the sludge was inhibited at all test concentrations and reduced by 23.5% to 35.3%. The IC50 value for inhibition was higher than 100 mg/L after 3 hours. The sensitivity of the activated sludge was tested and confirmed with the reference substance 3,5 -dichlorophenole.