2,2',2''-nitrilotriethanol citrate

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:

18 Jan 2018 - 09 Feb 2018

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:

2010

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity/Composition correction factor: Yes, 1.32 (for the water content)
Solubility in water: >770 g/L (not corrected for water content)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition to the test medium. For the range-finding test a stock solution of 5.8 g/L was prepared by adding 3.79947 g test item to 500 mL of Milli-RO water (tap water purified by reverse osmosis). For the limit test a stock solution of 10 g/L was prepared by adding 3.31664 g test item in to 250 mL Milli-RO. Magnetic stirring for approximately 20 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear slightly yellow stock solution (pH 6.7 and 6.8 for the range-finding and limit test, respectively) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring
- Controls: Test medium without test item, treated in the same way as the test item solutions.
- A correction factor of 1.32 for water content was used in the limit test. All further concentrations reported are based on the pure test item.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm^2) and allowed to settle. The supernatant was removed and ISO-medium was added. 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 7.7 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

Test temperature:

19 - 22°C

pH:

Before addition of sludge: 7.0 - 7.5.
After the 3 hour exposure period: 7.6 and 8.3

Dissolved oxygen:

Above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) throughout the test.

Nominal and measured concentrations:

Control, test substance at 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels, containing 500 mL medium/sludge
- Aeration: Clean, oil-free air, adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6
- No. of vessels for reference substance: 1 for each concentration (4 concentrations used)
- No. of vessels per abiotic control (replicates): 1 (only in Range-Finding test)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)
- Intervals of water quality measurement: pH at the begining of the test and at the end of the measuring period. Temperature was recorded continuously in temperature control vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED
- Dissolved oxygen: After the 3-hour contact time, the oxygen consumption was recorded with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany) for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

TEST CONCENTRATIONS
- Range finding study test concentrations: 5.8, 58, and 577 mg/L
- Results used to determine the conditions for the definitive study: Yes, no respiration inhibition was observed up to 577 mg/L. There was no significant oxygen uptake from abiotic processes.

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

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

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol in the Limit test was 5.9 mg/L, which is in the accepted range of 2 to 25 mg/L for total respiration.

Reported statistics and error estimates:

ECx: For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis.
Statistical significance: Two-sample t-test Procedure, α=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Limit test - overview of the results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)
Control		42.86	28.57
T1	1000	47.63	31.76	-11.15

Validity criteria fulfilled:

yes

Remarks:

See 'Overall results' for details on validity criteria.

Conclusions:

Under the conditions of this present test 1184 ZITRONENSÄURE, TEA-SALZ was not toxic to waste water bacteria (activated sludge) at 1000 mg/L (NOEC). The EC50 was above 1000 mg/L.

Description of key information

The 3-h EC50 and 3-h NOEC values are > 1000 and 1000 mg/L as observed in a limit test of a respiration inhibition study with activated sludge of a domestic sewage.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The toxicity of the substance to microorganisms was investigated in a limit test of an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (Charles River, 2018). In this study, non-adapted activated sludge of a predominantly domestic sewage was exposed to 0 (control) and a limit concentration of 1000 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. No inhibition of respiration rates was observed at the limit concentration. Therefore, the 3-h EC50 and 3-h NOEC values were determined to be > 1000 mg/L and 1000 mg/L, respectively.