Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

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:

2017

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))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant of 75175 Pforzheim, Germany, collected from the aeration tank was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage. The sludge was used one day after collection. It was settled and the upper layer with finer solids was decanted and discarded. Before starting the test, the remaining sludge was washed two times with chlorine free tap water by centrifugation (10 minutes at 3000 rpm equals 1966 g). After centrifuging, the supernatant was decanted and discarded and the sludge was resuspended in chlorine free tap water. This procedure was repeated once. The mixed liquor suspended solids (MLSS) in the sludge inoculum were adjusted to a concentration of 3.0 g/L (± 10 %). The activated sludge was continuously aerated at the test temperature, the solids did not settle down.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

The test was performed in a synthetic sewage feed that was prepared with the following amounts of substances in 1 litre of deionised water:
16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2 H2O, MgSO4 × 7 H2O, 2.8 g K2HPO4.

Test temperature:

Temperature in the main test: 18.4 – 20.8 °C (measured during O2-determination after 3 h of exposure)

pH:

pH of all test item solutions, the controls and reference item in the main test: 7.31 – 7.94

Dissolved oxygen:

The test solutions without microbial inoculum were aerated for some minutes to saturate the solutions with O2 above 60 - 70 %. Then the test was started (t0 = 0 hours) by adding 250 mL of microbial inoculum. During the incubation period of 3 hours all test solutions were continuously aerated by compressed air.

Salinity:

The test was performed in a synthetic sewage feed that was prepared with the following amounts of substances in 1 litre of deionised water:
16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2 H2O, MgSO4 × 7 H2O, 2.8 g K2HPO4.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (DCP)

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

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. (total fraction)

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

The EC50 of the total oxygen uptake for DCP was estimated (following the normal distribution) to be 2.11 mg/L. This is within the accepted range of 2 to 25 mg/L for total respiration.

Reported statistics and error estimates:

The statistical evaluation was performed for the respiration rate of the control and the test highest item concentration using SAS® (2002–2010). The data were tested on normal distribution by the Shapiro-Wilk’s statistics and for homogeneity of variance by using F-test. The NOEC and LOEC were determined by using the Satterthwaite t-test. The statistical EC50 estimation of the toxic reference item was performed using probit analysis following the normal distribution.

Validity Criteria of the Study

Control oxygen uptake:

The oxygen uptake rate RS in the blank controls (without test item or reference substance) should be not less than 20 mg O2/(g×h). In this study the mean control oxygen uptake was determined to be 24 mg O2/(g×h) after 3 hours.

Coefficient of variation:      

The coefficient of variation of the total oxygen uptake rate in control replicates should be ≤ 30% at the end of the test. In this test, the coefficient of variation was 9 % after 3 hours.

Reference item EC50:       

The EC50 of the total oxygen uptake for DCP was estimated (following the normal distribution) to be 2.11 mg/L. This is within the accepted range of 2 to 25 mg/L for total respiration.

Biological Results

The specific respiration rate of the range-finding test in a limit-test design and the percentage inhibition are summarised in below. No statistical significant differences to the control were determined up to 1000 mg/L after 3 hours for the total oxygen uptake so no main test was required. The NOEC was therefore determined to be at 1000 mg/L. The EC50 for total oxygen uptake was above 1000 mg/L. No chemical oxygen depletion was observed in the abiotic control. The EC50 of the total oxygen uptake for DCP was estimated (following the normal distribution) to be 2.11 mg/L. This is within the accepted range of 2 to 25 mg/L for total respiration.

Specific respiration rate and inhibition of total oxygen uptake

Test assay nominal [mg/L]	Mean specific respiration rates after 3 h [mg O2/(g × h)]	Inhibition after 3 h [%]
Control (mean)	23.97	-
10.0	22.17	7.5
100	23.61	1.5
1000 (mean of three replicates)	20.98	12.5
DCP 0.2	18.31	23.6
DCP 1	13.93	41.9
DCP 5	10.76	55.1
DCP 25	4.42	81.6

Validity criteria fulfilled:

yes

Conclusions:

In a Klimisch 1 study according to OECD 209 guideline the test item had no statistically significant inhibitory effect on the total oxygen uptake of activated sludge microorganisms after 3 hours. The NOEC is therefore determined to be 1000 mg/L. All validity criteria according to OECD Guideline for Testing Chemicals 209 were fulfilled.

Executive summary:

In a Klimisch 1 study according to OECD 209 guideline the test item had no statistically significant inhibitory effect on the total oxygen uptake of activated sludge microorganisms after 3 hours. The NOEC is therefore determined to be 1000 mg/L. The EC50 for total oxygen uptake was above the highest test item concentration of 1000 mg/L. All validity criteria according to OECD Guideline for Testing Chemicals 209 were fulfilled.

Description of key information

In a Klimisch 1 study according to OECD 209 guideline the test item had no statistically significant inhibitory effect on the total oxygen uptake of activated sludge microorganisms after 3 hours. The NOEC is therefore determined to be 1000 mg/L. All validity criteria according to OECD Guideline for Testing Chemicals 209 were fulfilled.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L