2-morpholinoethanesulphonic acid

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:

2017-09-26 to 2018-05-08

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:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the main test the test item concentration (as a limit concentration) of 1080 mg/L was examined with five replicates. The defined amount (323 mg) of the test item was (administered) measured directly into empty containers that were filled up with water and synthetic sewage, just before the inoculation. Concentrations in excess of nominal 1080 mg test item/L were not tested. Based on the preliminary experience, the test item does not affect adversely pH within the test system; therefore any pH adjustment was not performed prior to inoculum addition.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Source: 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 used for this study was washed by centrifugation (at 4000 rpm that corresponds to 3488 g at the given centrifuge) after adding isotonic saline solution 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, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to about 3 g per litre (on dry weight basis). The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 24 hours and fed with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.32), pH adjustment of the inoculum was considered not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

not specified

Test temperature:

20.1 - 21.0 °C

pH:

before addition of inoculum: 7.65 - 7.68
at the start of the 3h aeration: 7.41 - 7.94
at the end of the 3h aeration: 7.85 - 8.29
(across all controls groups)

Test substance:
before addition of inoculum: 5.39 - 5.48
at the start of the 3h aeration: 6.09 - 6.24
at the end of the 3h aeration: 7.36 - 7.48

Dissolved oxygen:

at the beginning of the selected section of the linear phase: 7.01 - 7.58
at the end of the selected section of the linear phase: 2.00 - 5.41
(across all controls and test substance groups)

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal conc.: 1080 mg/L
Measured conc.: Not specified

Details on test conditions:

TEST SYSTEM
- Test vessel (type and size): Erlenmeyer glass bottles of approximately 300 mL volume
- Aeration: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L (based on dry weight)
- Nutrients provided for bacteria: synthetic sewage
- Nitrification inhibitor used: N-allylthiourea

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: activated sludge respiration rate

TEST CONCENTRATIONS:
- Range finding study: yes
- Test concentrations: 1000 mg/L (nominal)
- 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:

EC50

Effect conc.:

> 1 080 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 080 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.:

> 1 080 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: none
- Effect concentrations exceeding solubility of substance in test medium: no
- Blank controls oxygen uptake rate: approx. 56 mg O2/Lh
- Coefficient of variation of oxygen uptake rate in control replicates: 2.7%

Results with reference substance (positive control):

- Results with reference substance 3,5-Dichlorophenol valid? Yes
- Relevant effect levels: EC50: 16.82 mg/L (95 % confidence limits: 10.21 - 31.42 mg/L)

Reported statistics and error estimates:

- 2-sample test to compare the test substance with control
- variability measure: coefficient of variation (CV)

Validity of the Study

The specific respiration rate of the blank controls (without the test substance or reference substance) was 37.57 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 2.71 %. The 3-hour EC₅₀ of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 16.82 mg/L and, thus,  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).

 

Toxicity of 3,5-Dichlorophenol

The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 9.39 % 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 19.50 % and 76.86 %, respectively. The 3-hour EC₅₀ of 3,5-Dichlorophenol was calculated to be 16.82 mg/L, (95 % confidence limits: 10.21-31.42 mg/L).

 

Nitrification Controls

An additional nitrification control was examined in the test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 56.36 mg/Lh, the heterotrophic respiration (RH) was 56.36 mg/Lh, the nitrification respiration (RN): 0.00 mg/Lh was calculated according to the equation: RN= RT-RH. There was no difference between the total and heterotrophic respiration, the obtained zero value was consequently lower than the 5 % of RT (2.82 mg/Lh) in blank controls. In this test the heterotrophic oxygen uptake equals the total uptake.

 

Table 1: The Specific Respiration Rate (RS) in the Test Item and Control Groups

Identification	Concentration (mg/L)	Specific Respiration Rate  (mg O2/gh)	Average RS (mg O2/gh)
cba	0.00	39.20	37.57 CV(%)= 2.71
cbb		37.45
cbc		37.31
cbd		38.32
cbe		37.60
cbf		35.60
cbg		37.75
cbh		37.36
cna	11.6 mg ATU/L	37.53	37.58   CV(%)= 0.78
cnb		37.31
cnc		37.89
R1a	2 mg 3,5-DCP/L	35.27	34.04   CV(%)= 4.35
R1b		34.47
R1c		32.40
R2a	7 mg 3,5-DCP/L	30.71	30.25   CV(%)= 4.96
R2b		28.57
R2c		31.47
R3a	24.5 mg 3,5-DCP/L	8.68	8.69   CV(%)= 2.53
R3b		8.48
R3c		8.92
T1a	1080 mg Test Item/L	37.38	37.13 ^NS   CV(%)= 1.12
T1b		36.58
T1c		37.38
T1d		36.80
T1e		37.53

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; α = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was determined to be 1080 mg/L.

Executive summary:

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 in a study according to OECD TG 209 under GLP. 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. A preliminary test was performed to estimate the range of concentrations of the test item needed in the definite (main) test for determining the inhibition of oxygen consumption.  In the preliminary test of this study the test item was investigated at the nominal concentrations of 10.8; 108 and 1080 mg/L. These test item concentrations were chosen taking into account a correction for the water content of the test item. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. Based on the absence of any inhibitory effect in the preliminary experiment, the test item was investigated at the limit concentration of nominal 1080 mg/L in the main test. Similarly to the preliminary test, defined amounts of the test item were added directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control at concentration levels of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1080 mg/L and no remarkable abiotic oxygen consumption was noticed. In accordance to the preliminary experiment, the test item does not affect adversely the pH within the test system; therefore, the main test was performed without additional pH adjustment. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in the examined test item (limit) concentration remained in the range of the blank controls (the average specific respiration rate at 1080 mg/L: 37.13 mg O₂/gh), no remarkable inhibitory effect of the test item was observed (the obtained 1.17 % inhibition was considered as being within the range of biological variability of the applied test system). The specific respiration rates at the examined test item concentration of 1080 mg/L were compared with the blank control values using 2-Sample T-test (α = 0.05). No statistical significant differences were noticed in the comparison of blank control and test item treated groups. Under the test conditions, the EC₁₀ and EC₅₀ values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was 1080 mg/L. In conclusion this study demonstrated the absence of inhibition of oxygen consumption by the test item up to and including the limit concentration of 1080 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC₁₀ and EC₅₀ values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was 1080 mg/L (reference 6.1.7-1).

Key value for chemical safety assessment

EC50 for microorganisms:

1 080 mg/L

EC10 or NOEC for microorganisms:

1 080 mg/L

Additional information

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 in a study according to OECD TG 209 under GLP. 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. A preliminary test was performed to estimate the range of concentrations of the test item needed in the definite (main) test for determining the inhibition of oxygen consumption.  In the preliminary test of this study the test item was investigated at the nominal concentrations of 10.8; 108 and 1080 mg/L. These test item concentrations were chosen taking into account a correction for the water content of the test item. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. Based on the absence of any inhibitory effect in the preliminary experiment, the test item was investigated at the limit concentration of nominal 1080 mg/L in the main test. Similarly to the preliminary test, defined amounts of the test item were added directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control at concentration levels of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1080 mg/L and no remarkable abiotic oxygen consumption was noticed. In accordance to the preliminary experiment, the test item does not affect adversely the pH within the test system; therefore, the main test was performed without additional pH adjustment. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in the examined test item (limit) concentration remained in the range of the blank controls (the average specific respiration rate at 1080 mg/L: 37.13 mg O₂/gh), no remarkable inhibitory effect of the test item was observed (the obtained 1.17 % inhibition was considered as being within the range of biological variability of the applied test system). The specific respiration rates at the examined test item concentration of 1080 mg/L were compared with the blank control values using 2-Sample T-test (α = 0.05). No statistical significant differences were noticed in the comparison of blank control and test item treated groups. Under the test conditions, the EC₁₀ and EC₅₀ values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was 1080 mg/L. In conclusion this study demonstrated the absence of inhibition of oxygen consumption by the test item up to and including the limit concentration of 1080 mg/L.