(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

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:

Study initiation date: October 08, 2019; Experimental start date: October 09, 2019; Experimental completion date: October 23, 2019

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

Version / remarks:

adopted 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 2016/266, C.11. (2015)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of decision 2018-01-10; Swiss Ordinance relating to GLP, adopted May 18, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of GLP, as revised in 1997 and adopted November 26, 1997 by decision of the OECD Council [C(97)186/Final].

Specific details on test material used for the study:

Appearance: Beige solid
Expiration Date: End of February 2021
Storage Conditions: < 30°C

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and finally again two blank control vessels. Based on the results of a solubility/dispersibility pre-experiment, no appropriate stock solution (e.g. of 5 g/L) could be prepared due to the low water solubility of the test item. Therefore, the test item amounts were weighed by means of an analytical balance and transferred to the designated test vessels with 234 mL deionized water (Table 1). The test item was mixed into the deionized water by intense stirring for approximately 10 minutes at room temperature to finely disperse the test item. No emulsifiers or solvents were used. After stirring, the appearance of the test media was evaluated and the pH measured. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.
- Controls: See above (Method)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of the test item under the test conditions) was reached at a concentration of <10 mg/L.
- Other relevant information: Except for the lowest test concentration (10 mg/L), all loading rates were clearly above the water solubility limit of the test item under the test conditions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: ARA Birs (Birsfelden / Switzerland)
- Method of cultivation: Not applicable
- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in chlorine free tap water. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
- Pretreatment: A calculated amount of wet sludge was suspended in chlorine free tap water to obtain a concentration equivalent to 3 g dry material per litre. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed per litre and was kept at room temperature under continuous aeration until use. Before use, on the next day, the pH of the activated sludge inoculum was adjusted from pH 6.9 to pH 7.4 with a 1M NaOH solution.
- Initial biomass concentration: At the start of the test, 16 mL synthetic sewage feed were made up to 250 mL with deionized water and then 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to this first control vessel. Due to this 1:2 dilution an initial biomass concentration of 1.5 g/L (dry weight) was achieved.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

The exposure duration was chosen as recommended by the test guideline.

Post exposure observation period:

Not performed.

Hardness:

Not determined.

Test temperature:

The test was conducted at a temperature of 20 to 22 ºC. The temperature was recorded in one of the control vessels at the start and at the end of the incubation period.

pH:

The pH of the complete incubation mixture was measured in each vessel at the start and end of the incubation period. At start the pH was in the range of 7.2 to 7.5 (no pH adjustment) and at the end in the range of 7.7 to 8.1
For the test item incubation mixtures the pH was measured also before the addition of synthetic sewage feed and activated sludge. The pH was in the range of 7.2 to 7.5.

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 60-70% saturation and the sludge flocs were maintained in suspension during the incubation period. The dissolved oxygen concentration in the incubation mixture measured in each vessel at the start of the incubation period (i.e. after the addition of activated sludge and a few minutes of stirring) was in the range of 8.2 to 8.4 mg/L. Just before measurement of the respiration rates, at the end of the incubation period, the dissolved oxygen concentrations were at least 7.1 mg/L.
For the measurement of the dissolved oxygen (DO) an oxygen meter (Oxi 7310P from WTW GmbH, Weilheim, Germany) with a selfstirring DO sensor (StirrOx® G from WTW GmbH) and a 250 mL BOD bottle were used.

Salinity:

Not determined.

Conductivity:

Not determined.

Nominal and measured concentrations:

Nominal test item concentrations were 10, 32, 100, 320 and 1000 mg/L.
All test results were based on nominal concentrations of the test and reference item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L beakers
- Type: Open
- Fill volume: 500 mL of incubation mixture
- Aeration: Magnetic stirrers and followers, covered with inert material, were used to assure the aeration and mixing during incubation.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6; six blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the definitive test. As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).
- No. of vessels per abiotic control (replicates): No abiotic control was performed.
- Sludge concentration (weight of dry solids per volume): Nominally 1.5 g/L (dry weight)
- Nutrients provided for bacteria: Synthetic Sewage Feed was prepared to contain the following constituents per litre: 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 filled up to the final volume with deionized water. The pH of this solution was pH 7.2.
- Biomass loading rate: Not determined
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water, containing less than 1 mg/L DOC (dissolved organic carbon)
- Particulate matter: Not determined
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Throughout the whole exposure period of 3 h
- Light intensity: The test vessels was incubated under normal laboratory lighting.
- Details on termination of incubation: After the incubation period the respiration rate was measured.
EFFECT PARAMETERS MEASURED:
- Measurement of the respiration rates: For measurement of the respiration rate, a well-mixed sample of each incubation mixture was poured into a 250 mL glass BOD bottle after three hours incubation time, and was not further aerated. Subsequently, the dissolved oxygen (DO) concentration was measured with a self-stirring DO sensor attached to an oxygen meter and was continuously recorded for a period of up to 10 minutes or until the oxygen concentration fell below 2 mg/L. The oxygen uptake rate of each sample was determined from the linear part of the respiration curve normally in the range of 2-7 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: Not applicable as the number of treatments is in line with the test guideline recommendations.
RANGE FINDING STUDY
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: 2.5, 4.7 and 72.8 (mean of 73.1, 73.5 and 71.7) % inhibition, respectively

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Sigma-Aldrich) CAS 591-35-5; Batch MKBS0425V; Purity 99.7 % (GC); Expiration Date: 2020-12-29 (i.e. 5 years after opening); Storage Conditions: Cool, dry, tightly closed container in a well ventilated place.

Key result

Duration:

3 h

Dose descriptor:

EC10

Remarks:

Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of the test item under the test conditions) was reached at a concentration of <10 mg/L.

Effect conc.:

119 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95 % confidence limits 8.2 – 218 mg/L; NOEC 100 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Remarks:

Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of the test item under the test conditions) was reached at a concentration of <10 mg/L.

Effect conc.:

181 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95 % confidence limits 29 – 295 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Remarks:

Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of the test item under the test conditions) was reached at a concentration of <10 mg/L.

Effect conc.:

404 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95 % confidence limits 224 – 745 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Remarks:

Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of the test item under the test conditions) was reached at a concentration of <10 mg/L.

Effect conc.:

901 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95 % confidence limits 550 – > 1000 mg/L

Details on results:

The inhibition of the respiration rates for individual replicates of all tested concentrations was in the range of -3.9 to 80% compared to the mean control. For three lower tested concentration of 10, 32 and 100 mg/L the inhibition of the respiration rate of activated sludge after the incubation period of 3 hours was lower than 10 % (i.e. mean values of 7.3, 4.4 and 02 %, respectively) compared to the mean control. Therefore, up to and including the concentration of 100 mg/L the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours. At the higher test item concentrations of 320 and 1000 mg/L the test item had an increasing statistically significant inhibitory effect, with mean values of 45 and 80 % inhibition, respectively. A clear concentration-effect relationship on the inhibition of the respiration rate was observed.
- Effect concentrations exceeding solubility of substance in test medium: Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of the test item under the test conditions) was reached at a concentration of <10 mg/L.
- Adsorption (e.g. of test material to the walls of the test container): Not observed
- Blank controls oxygen uptake rate: The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 42 and 36 mg O2 per dry weight of activated sludge per hour, for the two blank controls prepared after the reference item group were 37 and 43 mg O2/gh and for the two replicates prepared at the end of the test were 41 and 45 mg O2/gh. All six values are higher than the 20 mg O2/gh stipulated in the guidelines.
- Coefficient of variation of oxygen uptake rate in control replicates: The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 9 % and is therefore, as stipulated in the guidelines, not more than 30 % (Table 3).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 5.2 mg/L with 95 % confidence limits from 4.7 to 5.7 mg/L. The 3 hour EC50 is within the guidelines-recommended range of 2 to 25 mg/L, confirming the suitability of the activated sludge used.
- Other: As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).

Reported statistics and error estimates:

The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration and statistically analysed using a suitable software (ToxRat® Professional, (Version 3.3.0), ToxRat® Solutions GmbH).
The NOEC was derived by per-vessel statistics using the Welch t-test (one sided smaller, α = 0.05).
The 3-hour EC10, EC20, EC50 and EC80 values and their 95 % confidence limits were, as far as possible, derived by Probit Analysis.
The 3 hour EC50 of the reference item 3,5 dichlorophenol was derived by Probit Analysis.

Table 3: Definitive Test: Influence of the Test Item and 3,5 Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

Vessel no.	Nominal concentration of test chemical	Oxygen consumption rate		Inhibition		pH values		Oxygen concentration
		Respiration rate                ( R)	Specific respiration rate (RS)
								[mg O2/L]
	[mg/L]	[mg O2/Lh]	[mg O2/gh]	%		start*	end*	start*	end*
	Control
1	0	62.73	41.82			7.4	7.9	8.4	7.6
2	0	53.90	35.93			7.2	7.9	8.3	7.7
6	0	55.20	36.80			7.4	7.8	8.3	7.1
7	0	64.93	43.29			7.3	7.8	8.3	7.2
23	0	61.09	40.73			7.2	7.8	8.4	7.5
24	0	67.92	45.28			7.2	7.8	8.3	7.3
Mean		60.96	40.64
Standard Deviation		5.48	3.66
Coefficient of Variation		9.00%	9.00%
	Test item				Mean
8	10	55.30	36.87	9.3		7.3	7.8	8.4	7.6
9	10	54.90	36.60	9.9	7.3	7.4	7.9	8.4	7.8
10	10	59.35	39.56	2.7		7.3	7.8	8.3	7.5
11	32	55.40	36.93	9.1		7.3	7.9	8.4	7.6
12	32	56.10	37.40	8.0	4.4	7.3	7.9	8.4	7.6
13	32	63.36	42.24	-3.9		7.3	7.7	8.2	7.1
14	100	57.60	38.40	5.5		7.4	7.7	8.3	7.3
15	100	63.12	42.08	-3.5	0.2	7.2	7.7	8.4	7.3
16	100	61.73	41.16	-1.3		7.3	7.7	8.3	7.1
17	320	32.94	21.96	46.0		7.3	7.7	8.2	8.0
18	320	33.96	22.64	44.3	44.8^S	7.3	7.7	8.2	7.5
19	320	34.02	22.68	44.2		7.4	7.7	8.3	7.4
20	1000	12.55	8.36	79.4		7.3	7.8	8.3	8.0
21	1000	12.38	8.25	79.7	79.58^S	7.2	7.8	8.4	8.0
22	1000	12.49	8.33	79.5		7.2	7.8	8.3	8.0
	Reference item
3	3.2	39.00	26.00	36.0		7.5	8.1	8.3	8.0
4	10	18.93	12.62	69.0		7.4	8.0	8.3	8.5
5	32	5.51	3.67	91.0		7.3	7.9	8.4	8.6

*: Start and end of the 3-hour incubation period.

^S: Significantly different compared to the control (Welch t-test, one-sided smaller,α= 0.05).

- % inhibition: Negative value due to increased oxygen consumption rate relative to the control.

Validity criteria fulfilled:

yes

Conclusions:

A clear concentration-effect relationship on the inhibition of the respiration rate was observed. An EC10 of 119 mg/L (95 %-confidence limits 8.2 – 218 mg/L) and an EC50 of 404 mg/L (95 %-confidence limits 224 – 745 mg/L) have been established.

Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010 and to the Commission Regulation (EU) No. 2016/266, C.11. (2015). The validity criteria were met and the study can be considered relevant and adequate for the endpoint.

Based on the results of a range-finding test a definitive test with the following nominal test item concentrations was performed: 10, 32, 100, 320 and 1000 mg/L, with three replicates per concentration. As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates). Six blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the definitive test. The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and finally again two blank control vessels.

The inhibition of the respiration rates for individual replicates of all tested concentrations was in the range of -3.9 to 80% compared to the mean control. For three lower tested concentration of 10, 32 and 100 mg/L the inhibition of the respiration rate of activated sludge after the incubation period of 3 hours was lower than 10% (i.e. mean values of 7.3, 4.4 and 0.2%, respectively) compared to the mean control. Therefore, up to and including the concentration of 100 mg/L the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours (result of a Welch t-test, one-sided smaller, α = 0.05). At the higher test item concentrations of 320 and 1000 mg/L the test item had an increasing statistically significant inhibitory effect, with mean values of 45 and 80% inhibition, respectively. A clear concentration-effect relationship on the inhibition of the respiration rate was observed.

In conclusion, the substance had a clear concentration-effect relationship on the inhibition of the respiration rate of activated sludge. An EC10 of 119 mg/L and an EC50 of 404 mg/L have been established.

Description of key information

3-hour EC10 = 119 mg/L (activated sewage sludge, OECD TG 209, GLP)

3-hour EC50 = 404 mg/L (activated sewage sludge, OECD TG 209, GLP)

Key value for chemical safety assessment

EC50 for microorganisms:

404 mg/L

EC10 or NOEC for microorganisms:

119 mg/L