methyl 2-hydroxybut-3-enoate

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:

2021-09-07 to 2021-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008 (updated on 1st March 2016)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test

Version / remarks:

January 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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. The subsequent calculations refer to the initial weighed nominal concentration. Concentrations in excess of nominal 1000 mg test item/L (the limit concentration according to OECD 209) were not tested.
- Eluate: no
- Controls: blank control, abiotic control, reference control, nitrification control
- Evidence of undissolved material: no

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 06 September 2021 (one day before the test).
- 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 used for this study 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 (5.1186 g wet weight), dried and the ratio of wet sludge to dry weight (0.3917 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (18 g dry weight that was equivalent to 235.2 g wet sludge) was suspended in isotonic saline solution (ad. 6 L) to yield a concentration equivalent to about 3 g/L (on dry weight basis). In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g/L on dry weight basis. The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature (20 ± 2 °C, actually: 20.5-21.3 °C) for about 24 hours (one day) and fed with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.36). pH adjustment of the inoculum was considered not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

post exposure observation period

Hardness:

not specified

Test temperature:

20.7 - 21.3 °C

pH:

7 - 8

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 10, 100 and 1000 mg/L; measured values not available

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: glass, approximately 300 mL volume
- Aeration: mixtures were aerated at 0.5 L/min
- No. of vessels per concentration: 3
- No. of vessels per blank control: 8
- No. of vessels per abiotic control: 3
- No. of vessels per nitrification control: 1
- No. of vessels per reference control: 3
- Sludge concentration: 150 mL per vessel, 1.5 g/L on dry weight basis
- Weight of dry solids per volume of reaction mixture per unit of time: 1.5 g/L
- Nutrients provided for bacteria: 9.6 mL synthetic sewage per treatment
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified, deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: darkness

EFFECT PARAMETERS MEASURED : respiration inhibition

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Triplicates were examined at the highest tested concentration of 1000 mg/L (as a limit concentration) and additionally two lower concentration levels of 10 and 100 mg/L were examined with one vessel each. Blank, abiotic (three abiotic controls with the highest concentration of the test item) and reference controls were included. The nitrification potential of sludge was examined with additional control mixture (nitrification control in three parallels) that contained N-allylthiourea at 11.6 mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

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

Details on results:

- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption: no

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 43.92 mg/Lh, the heterotrophic respiration (RH) was 44.10 mg/Lh, the nitrification respiration (RN): -0.18 mg/Lh was calculated according to the equation: RN = RT - RH. The obtained -0.18 mg/Lh was considered as being within a biological variability range of the applied test system, and lower than the 5 % of RT (2.2 mg/Lh) in blank controls. According to the above calculations and based on the OECD 209 guideline the heterotrophic oxygen uptake equals the total uptake.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 11.06 mg/L being will within the range of 2 mg/L to 25 mg/L, that is required for total respiration.

Reported statistics and error estimates:

The EC50 value of the reference item was calculated using Probit analysis.

Table 1: Oxygen concentration

Identification	Concentration (mg/L)	Oxygen concentration (mg O2/L)
		Start of the 3-hour aeration	End of the 3-hour aeration
CBA	0	8.11	7.03
CBB		7.95	7.19
CBC		7.94	7.02
CBD		7.97	7.34
CBE		7.89	7.23
CBF		7.91	7.01
CBG		7.94	7.04
CBH		7.94	7.2
CNA	11.6 mg ATU/L	8.3	7.29
CNB		7.99	7.29
CR1	2 mg 3,5-DCP/L	8.27	7.11
CR2	7 mg 3,5-DCP/L	8.29	7.31
CR3	24.5 mg 3,5-DCP/L	8.26	7.25
T1	10 mg Test Item/L	8.04	7.08
T2	100 mg Test Item/L	7.99	7.25
T3/A	1000 mg Test Item/L	8.03	7.17
T3/B		8.06	7.49
T3/C		8.14	7.69
CAA	1000 mg Test Item/L	8.44	8.02
CAB		8.42	8.19
CAC		8.36	8.37

3,5-DCP: 3,5-dichlorophenol
ATU: N-allylthiourea

 

Table 2.:

Identification	Concentration (mg/L)	Specific Respiration Rate (mg O2/gh)	Avergare Rs (mg O2/gh)
CBA	0	26.61	29.28 (CV(%) = 7.52
CBB		27.04
CBC		31.20
CBD		31.63
CBE		28.23
CBF		27.20
CBG		31.20
CBH		31.14
CNA	11.6 mg ATU/L	30.46	29.40 CV(%) = 5.10
CNB		28.34
CR1	2 mg 3,5-DCP/L	26.35	26.35
CR2	7 mg 3,5-DCP/L	20.20	20.20
CR3	24.5 mg 3,5-DCP/L	6.95	6.95
T1	10 mg Test Item/L	28.97	28.97
T2	100 mg Test Item/L	29.66	29.66
T3/A	1000 mg Test Item/L	26.93	28.79 NS CV(%) = 6.45
T3/B		28.80
T3/C		30.65
CAA	1000 mg Test Item/L	-0.04	-0.24 CV(%) = -76.38^#
CAB		-0.28
CAC		-0.40

3,5-DCP: 3,5-dichlorophenol
ATU: N-allylthiourea

CV: Coefficient of variation

NS: There is no statistically significant difference when compared to the control (2-sample t-Test; 2-sided, alpha = 0.05)

#: The high CV value was caused by the low calculated mean value and a standard deviation of 0.18

Validity criteria fulfilled:

yes

Conclusions:

In a test according to OECD 209 the 3-hour EC10 and EC50 values of the test item were greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

Executive summary:

The purpose of the test was to evaluate the influence of the test item on the activity of an 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 pre-test is used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption or to serve as stand-alone (limit test) according to OECD 209. 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 at 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 at 1000 mg/L, synthetics sewage feed, but without inoculum. Under these test conditions no abiotic oxygen consumption was noticed. The experiment was performed without pH adjustment. The test item did not have any influencing effect on the pH within the test system and additional neutralization step of test item containing mixtures before inoculum addition was therefore not necessary. All validity criteria of the study were met. The average specific respiration rate of the blank was 29.28 mg O2 /gh activated sludge (based on dry weight) in an hour (unequivocally higher than 20 mg/gh) with a coefficient of variation of 7.52 % (clearly below 30 % as required in the validity criteria). The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 11.06 mg/L being will within the range of 2 mg/L to 25 mg/L, that is required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary as the parallel investigated nitrification control, containing N-allylthiourea confirmed the equality of heterotrophic and total oxygen uptake). 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 was 28.79 mg O2/gh. This corresponds to 1.67 % inhibition which is well within the biological variability range of the applied test system. Consequently, no inhibitory effect of the test item up to 1000 mg/L (the limit concentration according to OECD 209) 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.

Description of key information

In a test according to OECD 209 the 3-hour EC10 and EC50 values of the test item were greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

Additional information

The purpose of the test was to evaluate the influence of the test item on the activity of an 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 pre-test is used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption or to serve as stand-alone (limit test) according to OECD 209. 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 at 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 at 1000 mg/L, synthetics sewage feed, but without inoculum. Under these test conditions no abiotic oxygen consumption was noticed. The experiment was performed without pH adjustment. The test item did not have any influencing effect on the pH within the test system and additional neutralization step of test item containing mixtures before inoculum addition was therefore not necessary. All validity criteria of the study were met. The average specific respiration rate of the blank was 29.28 mg O2 /gh activated sludge (based on dry weight) in an hour (unequivocally higher than 20 mg/gh) with a coefficient of variation of 7.52 % (clearly below 30 % as required in the validity criteria). The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 11.06 mg/L being will within the range of 2 mg/L to 25 mg/L, that is required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary as the parallel investigated nitrification control, containing N-allylthiourea confirmed the equality of heterotrophic and total oxygen uptake). 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 was 28.79 mg O2/gh. This corresponds to 1.67 % inhibition which is well within the biological variability range of the applied test system. Consequently, no inhibitory effect of the test item up to 1000 mg/L (the limit concentration according to OECD 209) 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.