3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

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:

08 February 2017 to 10 March 2017

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
No correction was made for the purity/composition of the test material. For the combined limit/range-finding test, a stock solution of 10 g/L was prepared by adding 4.9886 g test material to 500 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). For the final test, a stock solution of 10 g/L was prepared by adding 5.0621 g test material to 500mL of Milli- RO water. After sonicating for 15 minutes and magnetic stirring for approximately 1 hour, volumes of the turbid, dark red stock solution (pH 3.5, in a glass pipette the stock solution was a clear dark red solution) corresponding to the test concentration were added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO water), under continuous stirring of the stock solution. Thereafter, 250 mL of activated sludge was added, resulting in the required concentrations. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of municipal sewage treatment plant where inoculum was collected: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: 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. The pH was 7.8 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.
- Initial biomass concentration: Suspended solids 3.0 g/L of sludge

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18 to 21 °C

pH:

7.6 to 8.3

Nominal and measured concentrations:

- Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Bottles/vessels
- Type: Open
- Material, size, headspace, fill volume: All glass, filled with 500 mL. The synthetic medium (16 mL) and appropriate amount of test material solution were mixed and made up to 250 mL with Milli- RO water. Thereafter, 250 mL activated sludge was added.
- Aeration: Yes, with clean, oil-free air. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60 to 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): 5 vessels
- No. of vessels per control (replicates): 6 vessels
- No. of vessels per abiotic control (replicates): 1 vessel (combined limit/rangefinding test)
- Sludge concentration: Suspended solids 3.0 g/L
- Nutrients provided for bacteria: Synthetic medium (sewage feed) consisted of 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water; tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Composition of medium: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: 211.5 mg/L CaCl2.2H2O, 88.8 mg/L MgSO4.7H2O, 46.7 mg/L NaHCO3 and 4.2 mg/L KCl.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Not specified
- Details on termination of incubation: After the 3-hour contact time, the oxygen consumption was recorded. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
The pH was determined in the remaining part of the reaction mixture after oxygen consumption measurements. The medium temperature was recorded continuously in temperature control vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2
- Range finding study: Yes. a combined limit/range-finding test was carried out prior to the final test. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes; the combined limit/range-finding test showed 29 % inhibition of the respiration rate at 10 mg/L, 81 % at 100 mg/L and 98 % at 1000 mg/L. Therefore, the expected EC50 was between 10 and 100 mg/L. There was no oxygen uptake from abiotic processes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95 % confidence interval 26 to 41 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

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 3.2 mg/L. At higher concentrations the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 9.2 % inhibition at 10 mg/L to 83 % at 100 mg/L.

Results with reference substance (positive control):

The EC50 for 3,5-dichlorophenol was determined to be 9.02 mg/L (95 % CL 5.71 to 12.42 mg/L).

Reported statistics and error estimates:

Evaluation was based on the inhibition of the total respiration.
Calculation of ECx values of the test material was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test material.
The calculation of the EC50 value of the reference material was based on a 3- parameter logistic cumulative distribution function (CDF) using non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference material.
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test Procedure, α = 0.05, one-sided, smaller).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Final test results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean)
		(mg O2/L.h)	(mg O2/g.h)¹
Control	-	37.04	24.70	-
T1	1.0	39.62	26.41	-7.0
T2	3.2	38.57	25.71	-4.1
T3	10	33.63	22.42	9.2*
T4	32	17.61	11.74	52*
T5	100	6.30	4.20	83*

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

*Statistically significantly different compared to control

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material was not toxic to waste water bacteria (activated sludge) at or below a concentration of 3.2 mg/L (NOEC). The EC50 was 33 mg/L (95 % confidence interval 26 to 41 mg/L).

Executive summary:

The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 3 hours in accordance with the standardised guidelines OECD 209, EU Method C.11 and ISO Standard 8192 under GLP conditions.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 1.0 to 100 mg/L and increasing with a factor of 3.2. Five replicates per concentration and six replicates for an untreated control group were tested.

A stock solution was prepared at 10 g/L. After sonicating for 15 minutes and magnetic stirring for 56 minutes, volumes of the turbid, dark red stock solution corresponding to the test concentration were then added to the test media under continuous stirring of the stock solution. In a glass pipette the stock solution appeared to be a clear dark red solution. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 3.2 mg/L. At higher concentrations the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 9.2 % inhibition at 10 mg/L to 83 % at 100 mg/L.

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

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

Under the conditions of this study, the test material was not toxic to waste water bacteria (activated sludge) at or below a concentration of 3.2 mg/L (NOEC). The EC50 was 33 mg/L (95 % confidence interval 26 to 41 mg/L).

Description of key information

Under the conditions of this study, the test material was not toxic to waste water bacteria (activated sludge) at or below a concentration of 3.2 mg/L (NOEC). The EC50 was 33 mg/L (95 % confidence interval 26 to 41 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

33 mg/L

EC10 or NOEC for microorganisms:

3.2 mg/L

Additional information

The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 3 hours in accordance with the standardised guidelines OECD 209, EU Method C.11 and ISO Standard 8192 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 1.0 to 100 mg/L and increasing with a factor of 3.2. Five replicates per concentration and six replicates for an untreated control group were tested.

A stock solution was prepared at 10 g/L. After sonicating for 15 minutes and magnetic stirring for 56 minutes, volumes of the turbid, dark red stock solution corresponding to the test concentration were then added to the test media under continuous stirring of the stock solution. In a glass pipette the stock solution appeared to be a clear dark red solution. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 3.2 mg/L. At higher concentrations the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 9.2 % inhibition at 10 mg/L to 83 % at 100 mg/L.

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

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

Under the conditions of this study, the test material was not toxic to waste water bacteria (activated sludge) at or below a concentration of 3.2 mg/L (NOEC). The EC50 was 33 mg/L (95 % confidence interval 26 to 41 mg/L).