3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-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:

4 January 2018 to 9 February 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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item information
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear to transparent yellow liquid
Purity/Composition: Not indicated
Test item storage: At room temperature

Additional information
Test Facility test item number: 207396/C
Purity/Composition correction factor: Contains 74% water
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name: Bis-Aminopropyl Diglycol Dimaleate
CAS number: 11629579-82-3
Molecular formula: C18H32N2O11
Molecular weight: 452.46
Volatile: Not indicated
Specific gravity / density: Not indicated
Solubility in water: Highly
Stability in water: Stable

Test Item Characterization: The Sponsor provided to the Test Facility documentation of the identity and stability for the test item.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test Item – Preparation and Test Concentrations:
The batch of Bis-Aminopropyl Diglycol Dimaleate tested was a clear to transparent yellow liquid with an unknown purity. A correction was made for the water content (74%) of the test item. A correction factor of 3.85 was used. All further concentrations reported are based on the pure test item.
A stock solution of 10 g/L was prepared by adding 19.2978 g test item to 500 mL Milli-RO and (combined limit/range-finding test) or 38.98682 g (final test) test item to 1000 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for approximately 15 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 2.7 and 2.6 for the combined limit/range-finding and final 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. Any residual volumes were discarded.

Reference Item – Preparation and Test Concentrations:
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol. A 1.0 g/L stock solution was prepared by dissolving 250.2 mg 3,5-dichlorophenol in Milli-RO water and making up to a total volume of 250 mL. The pH as used for the final test was 8.0. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15°c) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

- Preparation of the sludge: The sludge was coarsely sieved (1 mm) 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.

- 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

- Rationale: Recognized by international guidelines as the recommended test system.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Oxygen consumption was recorded for approximately 10 minutes after the 3-hour exposure period.

Hardness:

CaCl2.2H2O 0.4g/L

Test temperature:

19 - 22 °C (within the 20 °C ± 2 °C requirement of the study plan)

pH:

7.5 ± 0.5

Dissolved oxygen:

> 5mg/L at start (60-70% saturation)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Combined limit/range-finding test: 10, 100, and 1000 mg/L (nominal)
Final test: 100, 180, 320, 560, and 1000 mg/L (nominal)

Details on test conditions:

Reference Item Information
Identification number: RS92
Container: F1
Identification: 3,5-Dichlorophenol
Molecular formula: C6H4Cl2O
Molecular weight: 163.00
CAS Number: 591-35-5
Description: Grey crystals
Batch: MKBZ0947V
Purity: 100.0%
Expiry date: 25 October 2022
Storage conditions: At room temperature
Stability under storage conditions: Not indicated
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany

Combined Limit/Range-Finding Test:
In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested.

Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- A 1.0 g/L stock solution was prepared by dissolving 250.8 mg 3,5-dichlorophenol in Milli-RO water and making up to a total volume of 250 mL. The pH as used for the combined limit/range-finding test was 7.9.

Final Test – Test Concentrations:
- Bis-Aminopropyl Diglycol Dimaleate: Five concentrations: 100, 180, 320, 560 and 1000 mg/L.
- Replicates: 5 replicates per test group and 6 replicates for the control.
- Control: Test medium without test item, treated in the same way as the test item solutions.

Test Procedure and Conditions:
- Contact time: 3 hours, during which aeration and stirring took place.
- Vessels: All glass open bottles/vessels.
- Milli-RO / Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Synthetic medium (=sewage feed):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
- Air supply: Clean, oil-free air.
- Aeration: The aeration was 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.
- Test set up: See paragraph 4.7.2.
- Performance of the test: The synthetic medium (=sewage feed,16 mL) and an appropriate amount of the test item stock were mixed and made up to 250 mL with Milli-RO water. The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. 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. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
- Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 509 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 484 - 536 mg/l

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 180 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 294 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 259 - 322 mg/l

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 355 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 323 - 381 mg/l

Details on results:

Combined limit/range-finding test:
The combined limit/range-finding test showed 5%, no inhibition and 93% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was between a concentration of 100 and 1000 mg/L.
There was no significant oxygen uptake from abiotic processes.
All test conditions and acceptability criteria prescribed by the study plan were met.

Final test:
Inhibition of the respiration rate:
The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and ECx values.
No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 180 mg Bis-Aminopropyl Diglycol Dimaleate per L. At higher concentrations the inhibitory effect of Bis-Aminopropyl Diglycol Dimaleate on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 10% inhibition at 320 mg/L to 91% at 1000 mg/L. It should be noted that the pH in the highest two concentrations was below 5.0, which could lead to an effect of the pH on the respiration rate.

Results with reference substance (positive control):

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

Table 3: Final 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)¹
Control	0	42.86	28.57
T1	100	42.99	28.66	-0.31
T2	180	40.58	27.05	5.31
T3	320	38.47	25.65*	10.23
T4	560	16.74	11.16*	60.94
T5	1000	3.81	2.54*	91.11

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Determination of effect parameter

Table 4 shows the effect parameters based on Bis-Aminopropyl Diglycol Dimaleate concentrations.

Table 4: Effect Parameters

Parameter	Bis-Aminopropyl Diglycol Dimaleate Concentration (mg/L)
NOEC	180
EC10	294 (259 – 322 mg/L)
EC20	355 (323 – 381 mg/L)
EC50	509 (484 – 536 mg/L)

(  ) Between brackets the 95% confidence intervals are given.

Experimental conditions

The pH in all test vessels, before addition of sludge was between 4.0 and 7.5. After the 3 hour exposure period the pH was between 5.5 and 8.3.

The temperature continuously measured in the temperature control vessels ranged between 19 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of this present test Bis-Aminopropyl Diglycol Dimaleate was not toxic to waste water bacteria (activated sludge) at or below a concentration of 180 mg/L (NOEC).
The EC10 was 294 mg/L (95% confidence interval: 259 - 322 mg/L).
The EC20 was 355 mg/L (95% confidence interval: 323 - 381 mg/L).
The EC50 was 509 mg/L (95% confidence interval: 484 - 536 mg/L).

Executive summary:

The objective of the study was to evaluate Bis-Aminopropyl Diglycol Dimaleate for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no-observed effect concentration(NOEC).

The influence of Bis-Aminopropyl Diglycol Dimaleate on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study has been performed to the standardised guideline OECD 209, under GLP conditions.

The batch of Bis-Aminopropyl Diglycol Dimaleate tested was a clear to transparent yellow liquid of an unknown purity. A correction was made for the water content (74%) of the test item. A correction factor of 3.85 was used. All further concentrations reported are based on the pure test item.

The test started with the preparation of a 10 g/L stock solution. Magnetic stirring for approximately 15 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution corresponding to the test concentration were then added to the test media. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 100 to 1000 mg/L and increasing with a factor 1.8. Five replicates per concentration and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 180 mg Bis-Aminopropyl Diglycol Dimaleate per L. At higher concentrations the inhibitory effect of Bis-Aminopropyl Diglycol Dimaleate on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 10% inhibition at 320 mg/L to 91% at 1000 mg/L. It should be noted that the pH in the highest two concentrations was below 5.0, which could lead to an effect of the pH on the respiration rate.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Bis-Aminopropyl Diglycol Dimaleate was not toxic to waste water (activated sludge) bacteria at a concentration of 180 mg/L (NOEC).

The effect parameters for the inhibition of the respiration rate are presented below.

Parameter	Bis-Aminopropyl Diglycol Dimaleate Concentration (mg/L)
NOEC	180
EC10	294 (259 – 322 mg/L)
EC20	355 (323 – 381 mg/L)
EC50	509 (484 – 536 mg/L)

(  ) Between brackets the 95% confidence intervals are given.

Description of key information

In conclusion, under the conditions of this present test Bis-Aminopropyl Diglycol Dimaleate was not toxic to waste water bacteria (activated sludge) at or below a concentration of 180 mg/L (NOEC).

The EC10 was 294 mg/L (95% confidence interval: 259 - 322 mg/L).

The EC20 was 355 mg/L (95% confidence interval: 323 - 381 mg/L).

The EC50 was 509 mg/L (95% confidence interval: 484 - 536 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

509 mg/L