Ethyl (S)-2-hydroxypropionate

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:

2014-01-22 to 2014-05-20

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Batch:1007002470
- Purity: 98%

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

The batch of Ethyl-(S)-2-hydroxypropionate tested was a clear colourless liquid with a purity of 98%. No correction was made for the purity/composition of the test substance. A stock solution of 10 g/l was prepared by adding 2.5 g test substance to 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 5- 9 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution corresponding to the test concentration were added to the test media within 8 minutes after preparation. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) was included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.
In the limit test a concentration of 1000 mg/l was tested in triplicate. In addition, a blank control (6 replicates) was included. Contact time 3 hours, during which aeration and stirring took place.

PREPARATION OF THE SLUDGE
The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test). The pH was 7.6 (combined limit/range-finding test) and 7.7 (limit test) on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium (= sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

Test temperature:

Combined limit/range-finding test: 19.2–20.6 °C (min-max, continuously measured).
Limit test: 19.4–22.7 °C (min-max, continuously measured).

pH:

pH before addition of sludge: 7.5–7.6
pH after 3 h contact time: 7.8–8.2

Dissolved oxygen:

32 - 45 mg O2/L.h

Nominal and measured concentrations:

Nominal concentrations: loading rates of 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Type: open
- Material, size, fill volume: glass, 1 L, 250 mL fill volume
- Aeration: stirring and oxygen consumption was continuously recorded
- No. of vessels per concentration (replicates): 3 replicate at 1000 mg/L, 1 replicate at 100 and 10 mg/L
- No. of vessels per control (replicates): abiotic control 1 replicate; blank control 6 replicates
- Biomass loading rate: 3 g/L of sludge; the sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium.

OTHER TEST CONDITIONS
- Adjustment of pH: 7.8 to 7.6

EFFECT PARAMETERS MEASURED:
- Respiration rate: the oxygen consumption of aerobic activated sludge or waste water microorganisms expressed generally as mg O2 per litre per hour.

Medium: Adjusted ISO medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

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

Remarks:

respiration rate

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:

respiration rate

Details on results:

The mean blank control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The coefficient of variation of oxygen uptake in blank control replicates did not exceed 30% at the end of the definitive test. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration.
The study met the acceptability criteria prescribed by the test guidelines and was considered valid. Under the conditions of this present test ethyl-(S)-2-hydroxypropionate was not toxic to waste water (activated sludge) bacteria at 1000 mg/l (NOEC). The EC50 was above 1000 mg/L.
There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic respiration rate was increased compared with the corresponding control.

Results with reference substance (positive control):

3,5-dichlorophenol
EC50 = 4.33 mg/L
EC50 = 6.38 mg/L

Table 1: Details on study results

	Concentration (mg/L)	Respiration rate (mg O2/L.h)	% Inhibition respiration relative to blank control
Reference substance	2	27	31
	5	17	57
	12	12	69
Test item (combined test)	10	41	-4
	100	50	-27
	1000	57	-45
		56	-42
		68	-73
Test item (limit test)	1000	66	-58
		68	-36
		63	-51

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test according to OECD 209 ethyl-(S)-2-hydroxypropionate was not toxic to waste water (activated sludge) bacteria at 1000 mg/L (NOEC). The EC50 was above 1000 mg/L.

Executive summary:

The influence of ethyl-(S)-2-hydroxypropionate on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures were based on OECD guideline 209 (2010). In addition, the procedures were designed to meet the test methods of Council Regulation (EC) No. 440/2008 of 30 May 2008, OJ L142, part C.11, and ISO standard 8192 (2007). The test organisms were exposed to the test substance under continuous aeration and stirring for 3 hours. Thereafter, oxygen consumption was recorded for approximately 10 minutes. In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate; lower concentrations consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) and three replicates with a nitrification inhibitor were tested. To confirm the results from the the combined test a limit test was performed. In the limit test a concentration of 1000 mg/L was tested in triplicate. In both tests a blank control (6 replicates) was included. The responses were compared to the corresponding controls. The combined limit/range-finding test showed an increased respiration rate at 10, 100 and 1000 mg/L. The respiration rate increased with increasing concentration. There was no significant oxygen uptake from abiotic processes. The limit test showed an increase of the total respiration rate at 1000 mg/L, confirming the results obtained in the combined limit/range-finding test. For both tests the batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the test guidelines and is considered to be valid. Under the conditions of this test ethyl-(S)-2-hydroxypropionate was not toxic to waste water (activated sludge) bacteria at 1000 mg/L (NOEC). The EC₅₀ was above 1000 mg/L.

Description of key information

Ethyl (S)-lactate does not impair microbial respiration rates at concentrations up to the limit concentration of 1000 mg/L. The substance is not hazardous for activated sludge microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In an activated sludge respiration inhibition according to OECD test guideline 209 Ethyl (S)-lactate did not impair the respiration rate of the test inoculum at concentrations up to 1000 mg/L. Therefore, Ethyl (S)-lactate is not harmful to activated sludge microorganisms within the relevant range of test concentrations. No hazard to microorganisms associated with Ethyl (S)-lactate has been identified.