2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

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 2019-05-08

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))

Version / remarks:

July 22, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ecological Effects Test Guidelines of the United States Environmental Protection Agency (EPA 712-C-014): OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test", January 2012.

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

In the main test the test item concentration (as a limit concentration) of 1000 mg/L was examined with five replicates. The defined amount (300 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 1000 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:

- Species: Activated sludge, microorganisms from a domestic waste water treatment plant
- Name and location of sewage treatment plant where inoculum was collected: domestic sewage in Balatonfüred, Hungary, on 25.09.2017 (one day before the test)
- Preparation of inoculum for exposure: Coarse particles were removed after 10 min of settling and the upper layer of finer solids were decanted. The sludge was washed by centrifugation (at 3488 g) after adding isotonic saline solution and the supernatant was decanted. The solid material was re-suspended in isotonic saline solution with shaking and centrifuged again. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight was determined. Based on this ratio, wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to about 3 g/L (on dry weight basis).
- Pretreatment: continuous aeration (2 L / minute) at test temperature for 24h and fed with 50 mL synthetic sewage / L activated sludge.
- Initial biomass concentration: 1000 mg/L
- Adjustment of pH: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

19.0 - 21.7 °C

pH:

7 - 8

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)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass bottles, 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: 3 g/L (on dry weight basis)
- Nutrients provided for bacteria: synthetic sewage
- Nitrification inhibitor used: N-allylthiourea (in preliminary experiment only)

EFFECT PARAMETERS MEASURED: respiration rate

TEST CONCENTRATIONS
- Justification for using fewer concentrations than requested by guideline: no effects in preliminary test
- Range finding study: yes
- Test concentrations: 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes

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

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

Details on results:

The observed oxygen consumption rates and, consequently, the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed (the observed slight in average 2.76 % inhibition was within the biological variability of the applied test system).
The specific respiration rates of the 1000 mg/L concentration were compared with the blank control values. No statistical significant differences were observed in the comparison of test item treatment group and blank control. Based on the results of this study the NOEC was determined to be 1000 mg/L.
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 higher than 1000 mg/L.
The EC50 value was determined as: EC50 > 1000 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 16.82 mg/L

Table 1: The Specific Respiration Rate (R_S) 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	1000 mg Test Item/L	35.64	36.54 ^NS CV(%)= 2.33
T1B		37.16
T1C		37.60
T1D		35.78
T1E		36.51

C_B: blank control

C_N: nitrification control n-allylthiourea

R: reference control 3,5 –dichlorophenol

T: test substance

ns: not-significant (t-test vs blank control)

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 according to OECD guideline one 209 , the EC10 and EC50 values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 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 1000 mg/L.

Executive summary:

A 3-hour test to evaluate the influence of the test item on the activity of activated sludge by measuring the respiration rate under defined conditions was performed. 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. The test item was investigated at the nominal concentrations of 10, 100 and 1000 mg/L. These test item concentrations were chosen according to the referred OECD 209 guideline requirements. 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. 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 1000 mg/L: 36.54 mg O₂/gh), no remarkable inhibitory effect of the test item was observed (the obtained 2.76 % inhibition at 1000 mg/L was considered as being within the range of biological variability of the applied test system). Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 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 1000 mg/L.

Description of key information

The toxicity of the substance on microorganisms was investigated in an activated sludge respiration inhibition test according to OECD Test Guideline 209 under GLP-conditions.

In a limit test with the nominal concentration of 1000 mg/L no (statistically significant) inhibitory effect of the substance was observed as compared to the blank control after 3 hours of incubation (reference 6.1.7-1).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A 3-hour test to evaluate the influence of the test item on the activity of activated sludge by measuring the respiration rate under defined conditions was performed. 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. The test item was investigated at the nominal concentrations of 10, 100 and 1000 mg/L. These test item concentrations were chosen according to the referred OECD 209 guideline requirements. 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. 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 1000 mg/L: 36.54 mg O₂/gh), no remarkable inhibitory effect of the test item was observed (the obtained 2.76 % inhibition at 1000 mg/L was considered as being within the range of biological variability of the applied test system). Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 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 1000 mg/L.