Propanoic acid, 2-hydroxy-, ammonium salt (1:1), (2S)-

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:

2016-10-20 to 2017-02-16

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

Version / remarks:

July 22nd, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item: 714 mg of the test item were added directly into the test vessels to obtain a final concentration of 1000 mg/L Ammonium lactate. The pH of the test solution was measured before addition of inoculum.
Reference item: 25.0 mg of 3,5-dichlorophenol were dissolved in deionised water and filled up to 25.0 mL using a magnetic stirrer to get a stock solution with 1.0 g/L. 0.5, 5 and 15 mL of the stock solution were added to the reference vessels in order to obtain final concentrations of 1, 10 and 30 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 26 October 2016. The activated sludge was kept aerated and fed with synthetic sewage (50 mL per litre and day) until use. Dry solid of the activated sludge was determined as 5.45 g/L by weight measurements before and after 3 h drying at 105 °C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in aerated tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

21.3 to 21.7 °C

pH:

7.1 to 7.3

Dissolved oxygen:

ca. 6 mg/L

Nominal and measured concentrations:

1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers, 1000 ml, Schott, Mainz, Germany.
- Aeration: All vessels were stirred and aerated throughout the contact time with room air at a rate of 0.5-1 litre per minute using a membrane pump. Before starting the test the aeration rate was measured in all test vessels.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (blank)
- Sludge concentration (weight of dry solids per volume): 3 g/l dry solids
- Weight of dry solids per volume of reaction mixture: 1.5 g/L dry solids
- Nutrients provided for bacteria: peptone 16 g/L, meat extract 11 g/L, urea 3 g/L, NaCl 0.7 g/L, CaCl2 * 2 H2O 0.4 g/L, MgSO4* 7 H2O 0.2 g/L, K2HPO4 2.8 g/L
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water

OTHER TEST CONDITIONS
- Adjustment of pH: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): oxygen consumption: The measurement time was 10 minutes in which every 60 seconds an oxygen measurement was taken. The respiration rate is calculated from oxygen measurements using the linear portion of the oxygen-time-curves. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

TEST CONCENTRATIONS
- Range finding study: yes, no further study needed, as no inhibition was observed. See OECD 209: “Alternatively, the absence of inhibition of oxygen consumption by the test substance in a preliminary test may demonstrate that a definitive test is unnecessary, but triplicates at the highest tested concentration of the preliminary test (typically 1000 mg/L, but dependent on the data requirement) should be included.”
- Test concentrations: 1000 mg/L

Experimental setup
The different vessels must be started separately with an interval of exactly 15 min in order to be able to measure the respiration rates one by one while making sure that the aeration time is 3 h in every vessel. At time "0", 16 ml synthetic sewage and the test item or reference compound were added to the test vessel and made up to 250 ml with tap water. After measurement and adjustment of pH 250 ml of activated sludge (3 g/l dry solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/l dry solids. For the controls the same procedure was made as with the test vessels, however without test or reference substance. All vessels were stirred and aerated throughout the contact time with room air at a rate of 0.5-1 litre per minute using a membrane pump. Before starting the test the aeration rate was measured in all test vessels. After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer. The measurement time was 10 minutes in which every 60 seconds an oxygen measurement was taken. The respiration rate is calculated from oxygen measurements using the linear portion of the oxygen-time-curves. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

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:

oxygen consumption

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

Remarks:

oxygen consumption

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): not reported
- Blank controls oxygen uptake rate: The respiration rate from five of six blanks were within 20.3–24.7 mg/(l*h).
- Coefficient of variation of oxygen uptake rate in control replicates: the coefficient of variation (CV) was 9 %.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorphenol was calculated as 14.69 mg/L and was therefore within the acceptable range of 2 to 25 mg/L. The pH in the reference vessels was between 7.4 and 7.5.

Reported statistics and error estimates:

The EC50 calculation was performed with the ToxRat software by probit analysis using linear max. likelihood regression (ToxRat Standard Version 2.09, © ToxRat Solutions GmbH). The EC50 is calculated from the slope of the curve.

Validity criteria fulfilled:

yes

Conclusions:

The inhibition observed in the test vessels with 1000 mg/L test concentration was between -1 and 34 % but the mean respiration rates of the test vessels were not significantly lower than the respiration rate of the controls (Student's t-test).
EC50 > 1000 mg/L
NOEC ≥ 1000 mg/L

Executive summary:

To investigate potential harmful effects of ammonium-S-lactate on microorganisms, an activated sludge respiration inhibition test was performed with domestic sewage sludge according to OECD guideline 209 and under GLP. The inhibition observed in the test vessels with 1000 mg/L test concentration was between -1 and 34 % but the mean respiration rates of the test vessels were not significantly lower than the respiration rate of the controls. Hence, an EC₅₀ of > 1000 mg/L and a NOEC of ≥ 1000 mg/L ammonium lactate were determined.

EC₅₀ (3 h, nominal) > 1000 mg/L

NOEC (3 h, nominal) ≥ 1000 mg/L

Description of key information

To investigate potential harmful effects of ammonium-S-lactate on microorganisms, an activated sludge respiration inhibition test was performed with domestic sewage sludge according to OECD guideline 209 and under GLP. The inhibition observed in the test vessels with 1000 mg/L test concentration was between -1 and 34 % but the mean respiration rates of the test vessels were not significant lower than the respiration rate of the controls. Therefore, an EC50 of > 1000 mg/L and a NOEC of ≥ 1000 mg/L were determined.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L