Pyridinium, 1-(phenylmethyl)-, ethyl methyl derivs., chlorides

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:

3 hour

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The solubility was tested in water and it was found that a stock solution could not be prepared at a sufficiently high concentration to treat the test vessels at the proposed test concentrations. The test substance was therefore added directly to the test vessels.

Duplicate vessels were treated at nominal MK92K nominal test concentrations of 1, 10, 100 and 1000 mg/L (equivalent to 0.75, 7.5, 75 and 750 mg/L active ingredient) in the range-finder test and 1.9, 6.1, 19.5, 62.5 and 200 mg/L (equivalent to 1.4, 4.6, 14.6, 46.9 and 150 mg/L active ingredient) in the definitive test.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was collected from one of the sludge return lines at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic waste water catchment. The activated sludge used in this study was not deliberately acclimatised or adapted before exposure to the test substance.

Preparation and Maintenance
On arrival at Covance, activated sludge was aerated using a compressed air supply. The suspended solids concentration of the activated sludge was determined by filtering a sub-sample (25 mL) through a pre-dried and pre weighed glass microfibre filter (Whatman GF/C). The filtered and retained solids were then dried at 105°C (nominal) in a conventional oven, re-weighed and the contribution made by the sludge solids determined by difference. The suspended solids concentration for the sludge collected for the range-finder, determined on the day of collection was 3.9 g/L and was within the permitted limits of 4 g/L ± 10% specified by the test guideline. The suspended solids concentration of the sludge collected for the definitive test, determined on the day of collection was 4.5 mg/L and required adjustment to the permitted limits of 4 g/L ±10%.

During the interval between preparation and final use, the inoculum was aerated at all times. Sludge was fed before each overnight period with synthetic sewage concentrate at a rate of 50 mL/L. The activated sludge used to inoculate cultures was blended to remove large particulate matter before use.

Range-finder Test Inoculum
The suspended solids concentration of the blended activated sludge was determined before the range-finder test. The concentration was 4.3 g/L and was within the permitted limits of 4 g/L ± 10%. The pH of the inoculum was pH 6.02 and was within the acceptable range of pH 6 to 8.

Definitive Test Inoculum
The suspended solids concentration of the blended activated sludge was determined before the definitive test. The concentration was 4.6 g/L and was outside the permitted limits of 4 g/L ± 10% and required adjustment to the permitted limits. The pH of the inoculum was pH 6.22 and was within the acceptable range of pH 6 to 8.

Test type:

static

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

At the end of the first 3-hour incubation period, a portion of the test mixture was transferred to fill a sample bottle (ca 250 mL in volume) containing a PTFE coated stirrer bar. The DO probe was inserted in the sample bottle, taking care to avoid displacing too much liquid, which would create a headspace. The probe was sealed against the bottle neck to ensure that the sample could not become re oxygenated by contact with the atmosphere. The bottle was centred on a stirrer drive unit, the magnetic stirrer started and the chart set to run. Measurements were taken over a period of approximately 10 minutes, or until the DO concentration was less than 1 mg O2/L.

The procedure was repeated for each subsequent sample, rinsing the sample bottle and probe thoroughly with RO water between samples. The stirrer speed was kept the same for all measurements to ensure that the DO meter response was not influenced artificially. Respiration rates were subsequently derived from a linear portion of each valid trace by measuring the decline in oxygen concentration over time. The traces were assessed to ensure the measured linear portion was typically between 2.5 and 6.5 mg O2/L. This criterion is set to avoid the respiration rate being influenced by high or low dissolved oxygen concentrations.

Test temperature:

20 ± 2°C

pH:

pH remained within the acceptable range of pH 6 to 8.

Nominal and measured concentrations:

Range-finding test: nominal test concentrations of 1, 10, 100 and 1000 mg/L (equivalent to 0.75, 7.5, 75 and 750 mg/L active ingredient).
Definitive test: nominal test concentrations of 1.9, 6.1, 19.5, 62.5 and 200 mg/L (equivalent to 1.4, 4.6, 14.6, 46.9 and 150 mg/L active ingredient).

Details on test conditions:

Preparation of Test Vessels
All mixtures contained synthetic sewage (16 mL), which provided a uniform respiration substrate, the test or reference substance as required, and sufficient volumes of dechlorinated tap water to achieve a volume of 300 mL. Inoculation entailed addition of activated sludge (200 mL) prepared as described previously, giving a final volume of 500 mL. The test mixtures were inoculated sequentially at timed intervals and aerated by means of a compressed air supply delivered to each test system via a Pasteur pipette. The rate of aeration was sufficient to keep the test preparations adequately mixed. At the end of the incubation, dissolved oxygen (DO) measurements were made from a sub-sample of each preparation using a DO meter and probe.

Treatment of Test Substance Vessels
Duplicate vessels were treated at each nominal MK92K test concentration.

Treatment of Reference Substance Vessels
Single preparations were tested containing the reference substance, (3,5-DCP), at nominal concentrations of 5, 15 and 45 mg/L.

Blank Control Vessels
Four blank controls were prepared to enable measurement in two vessels at the start and two vessels at the end of each test series.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (equivalent to 13.6 mg/L active ingredient)

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (equivalent to 25.8 mg/L active ingredient)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

117 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (equivalent to 88.0 mg/L acitve ingredient)

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (equivalent to >150 mg/L active ingredient)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (equivalent to 4.6 mg/L active ingredient)

Results with reference substance (positive control):

The EC50 estimate for inhibition of respiration resulting from the presence of 3,5 DCP was estimated as 15 mg/L and the control respiration rates used to obtain the mean were within 15% of each other. The definitive test can therefore be considered valid.

Range-finder Test Respiration Rate and Inhibition Data

Treatment	Respiration Rate (mg O2/L/h)	% Inhibition
Blank Control
	72.41^1	NA
	76.36^1	NA
	73.47^2	NA
	70.59^2	NA
Mean	73.21	NA
1 mg MK92K/L
	72.00	1.65
	72.73	0.66
Mean	72.36*	1.15
10 mg MK92K/L
	68.85	5.95
	70.00	4.38
Mean	69.43	5.17
100 mg MK92K/L
	33.87	53.73
	30.25	58.68
Mean	32.06	56.21
1000 mg MK92K/L
	14.22	80.57
	14.63	80.01
Mean	14.43	80.29
5 mg 3,5-DCP/L	56.76	22.47
15 mg 3,5-DCP/L	32.56	55.53
45 mg 3,5-DCP/L	5.93	91.89
^1start-of-series control ^2end-of-series control *Mean respiration rate within range of blank controls NA: not applicable Note: Test concentrations are nominal test concentrations and are equivalent to 0.75, 7.5, 75 and 750 mg/L active ingredient.

Definitive Test Respiration Rate and Inhibition Data

Treatment	Respiration Rate (mg O2/L/h)	% Inhibition
Blank Control
	85.71^1	NA
	81.82^1	NA
	87.80^2	NA
	87.80^2	NA
Mean	85.79	NA
1.9 mg MK92K/L
	85.71	0.08
	92.31	-7.60
Mean	89.01*	-3.76
6.1 mg MK92K/L
	82.76	3.53
	84.00	2.08
Mean	83.38*	2.80
19.53 mg MK92K/L
	78.26	8.77
	80.00	6.74
Mean	79.13	7.76
62.5 mg MK92K/L
	51.43	40.05
	52.17	39.18
Mean	51.80	39.62
200 mg MK92K/L
	33.71	60.71
	32.97	61.57
	33.34	61.14
5 mg 3,5-DCP/L	67.92	20.82
15 mg 3,5-DCP/L	51.43	40.05
45 mg 3,5-DCP/L	9.09	89.40
^1start-of-series control ^2end-of-series control *Mean respiration rate within or above range of blank controls NA: not applicable Note: Test concentrations are nominal test concentrations and are equivalent to 1.4, 4.6, 14.6, 46.9 and 150 mg/L active ingredient.

 

Validity criteria fulfilled:

yes

Conclusions:

No notable inhibition was observed at the 1.9 or 6.1 mg/L nominal test concentrations (equivalent to 1.4, 4.8 mg/L active ingredient), however 8%, 40% and 61% inhibition was observed at nominal test concentrations of 19.5, 62.5 and 200.0 mg/L (equivalent to 14.7, 46.9 and 150.0 mg/L active ingredient), respectively.
Based on Probit analysis, the EC10, EC20 and EC50 were 18, 34 and 117 mg/L (equivalent to 13.6, 25.8 and 88.0 mg/L active ingredient), respectively. The EC80 could not be reliably determined but can be said to be greater than the highest test concentration, 200 mg/L (equivalent to >150 mg/L active ingredient). The NOEC is considered to be 6.1 mg/L (equivalent to 4.6 mg/L active ingredient).
The guideline validity criteria relating to the reference substance response and the variation between the blank control cultures were met. The results of this study are therefore considered valid.

Executive summary:

The impact on the respiration rate of activated sludge was assessed according to the OECD Guideline 209 (Adopted 1984).

Samples of activated sludge were exposed, for three hours, to a range of concentrations. Each preparation contained dechlorinated tap water and a synthetic sewage preparation that provided a uniform respiration substrate. At the end of the exposure period, the respiration rate of the activated sludge microbes was determined by measuring the decline in oxygen concentration of the test culture. The inhibitory effect of the test substance was calculated by comparing the respiration rate of cultures containing test substance to that of the blank control cultures containing only activated sludge, dechlorinated tap water and synthetic sewage. A series of cultures containing a reference substance, 3,5‑dichlorophenol (3,5-DCP) confirmed that the sensitivity of the sludge was within acceptable limits.

A range-finder test was conducted at nominal concentrations of 1, 10, 100 and 1000 mg/L (equivalent to 0.75, 7.5, 75 and 750 mg/L active ingredient). The range of mean respiration rates observed for all of the test substance concentrations was 14 to 72 mg O₂/L/h. The mean respiration rate at the 1 mg/L test concentration was within the range observed for the blank control samples at 71 to 76 mg O₂/L/h. Inhibition of respiration relative to the blank controls was therefore not observed at this concentration. However, the respiration rates at the 10, 100 and 1000 mg/L test concentrations were 69, 32 and 14 mg O₂/L/h, equivalent to 5%, 56% and 80% inhibition, respectively. A definitive test was therefore performed.

In the definitive test, activated sludge was exposed to nominal concentrations of 1.9, 6.1, 19.5, 62.5 and 200.0 mg/L (equivalent to 1.4, 4.6, 14.6, 46.9 and 150.0 mg/L active ingredient). The mean respiration rates observed at each of the test concentrations were 89, 83, 79, 52 and 33 mg O₂/L/h, respectively. The mean respiration rates for the 1.9 and 6.1 mg/L test concentrations were above or within the range observed for the blank control samples suggesting no inhibitory effect. However, at the 19.5, 62.5 and 200 mg/L test concentrations an inhibitory effect of 8%, 40% and 61% respectively, was observed.

Based on Probit analysis, the effective concentrations of MK92K that caused a 10%, 20% and 50% reduction in respiration rate relative to the untreated controls (EC₁₀, EC₂₀and EC₅₀) were 18, 34 and 117 mg/L equivalent to 13.6, 25.8 and 88.0 mg/L active ingredient, respectively. The EC₈₀could not be reliably determined but can be said to be >200 mg/L (equivalent to >150 mg/L active ingredient). The no observed effect concentration (NOEC) is considered to be 6.1 mg/L (equivalent to 4.6 mg/L active ingredient).

The EC₅₀estimate for inhibition of respiration resulting from the presence of 3,5‑DCP was estimated as 15 mg/L and the control respiration rates used to obtain the mean were within 15% of each other. The definitive test can therefore be considered valid.

Description of key information

No notable inhibition was observed at the 1.9 or 6.1 mg/L nominal test concentrations (equivalent to 1.4, 4.8 mg/L active ingredient), however 8%, 40% and 61% inhibition was observed at nominal test concentrations of 19.5, 62.5 and 200.0 mg/L (equivalent to 14.7, 46.9 and 150.0 mg/L active ingredient), respectively.
Based on Probit analysis, the EC10, EC20 and EC50 were 18, 34 and 117 mg/L (equivalent to 13.6, 25.8 and 88.0 mg/L active ingredient), respectively. The EC80 could not be reliably determined but can be said to be greater than the highest test concentration, 200 mg/L MK92K (equivalent to >150 mg/L active ingredient). The NOEC is considered to be 6.1 mg/L MK92K (equivalent to 4.6 mg/L active ingredient).
The guideline validity criteria relating to the reference substance response and the variation between the blank control cultures were met. The results of this study are therefore considered valid.

Key value for chemical safety assessment

EC50 for microorganisms:

117 mg/L

EC10 or NOEC for microorganisms:

6.1 mg/L

Additional information

The activated sludge respiration inihibition test was performed according to OECD guideline 209 (Activated Sludge, Respiration Inhibition Test).