2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Remarks:

activated sludge respiration and nitrification inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug - 07 Nov 2016

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:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item was weighed directly into 300 mL glass Erlenmeyer flasks to 130 mL deionised water and was stirred overnight for 17 hours. 8 mL of the synthetic medium and 100 mL of activated sludge were added. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C. The same design was performed for the determination of the heterotrophic oxidation with the addition of 1.25 mL ATU solution (2.32 g/L N-allythiourea diluted with 1 L deionised water). For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water.
- Differential loading: 10, 32, 100, 320 and 1000 mg/L. Two sets of reaction mixtures were prepared, one without ATU and one with ATU.
- Controls: yes (6 replicates for control and 6 for ATU control)

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: mixed population of aquatic microorganisms of the aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim), collection date: 04 Oct 2016
- Preparation of inoculum for exposure: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20 °C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Pretreatment: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- Initial biomass concentration: 800 mg/L suspended solids (Test concentration of the activated sludge)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18.5 - 20.3 °C (controls)
18.7 - 19.8 °C (test item)
19.0 - 20.0 °C (reference item)

pH:

7.2 - 7.4 (adjusted at test start, test item, control)
7.0 - 7.5 (adjusted at test start, reference compound)
7.4 - 8.4 (at test end)

Nominal and measured concentrations:

control, 10, 32, 100, 320, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks; 250 mL of sludge with the test item (or control or reference compound)
- Aeration: test system was stirred and aerated through a glass tube at 50 - 100 L/h with clean oil-free air
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3 g (dry weight)/L
- Nutrients provided for bacteria: A synthetic waste water feed was prepared (medium according to the guideline)
- Nitrification inhibitor used: N-allylthiourea
- Biomass loading rate: 800 mg/L suspended solids (Test concentration of the activated sludge)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Particulate matter: Temperature and pH were measured at test start and test end.

OTHER TEST CONDITIONS
- Adjustment of pH: for the reference compound pH was adjusted to pH 7 ± 0.5 with HCl at test start, for test item concentrations and control pH was adjusted to pH 7.2 - 7.4 with NaOH at test start

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After the 3-hour contact time, the oxygen consumption was recorded
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study:
- Test concentrations: 100 mg/L, 3 replicates
- Results used to determine the conditions for the definitive study: see"Any other information on material and methods incl. tables"

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

test item = act. ingr.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

- Blank controls oxygen uptake rate: In the present test oxygen uptake rate was 53.2 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour for the test set without ATU and 46.4 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour for the test set with ATU
- Coefficient of variation of oxygen uptake rate in control replicates: 8.6% for the test set without ATU and; 3.9% for the test set with ATU
- The effect value relates to a nominal concentration, since no analytical monitoring was performed.
- The physico-chemical oxygen consumption has been determined at 1000 mg/L test item concentration. As nearly no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations.
- All validity criteria were met.
- Results in detail see Table 1 and 2 in "Any other information on results incl. tables"

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the EC50 of the reference compound 3,5-Dichlorophenol was 17.8 mg/L for total respiration and 15.7 mg/L for heterotrophic respiration.
- Relevant effect levels: the EC50 was between 20- 40 mg/L

Table 1: Main test, Test without ATU (Total respiration). Respiration rates after 3 hours incubation period, percentage inhibition, temperature and pH values 

Treatment [mg/L]		Respiration rate	Mean Temp.	pH-	Inhibition
		[mg/L · h]	[°C]	value	[%]
Control 1		48.341	20.3	8.3	--
Control 2		44.835	20.3	8.3	--
Control 3		42.952	20.1	8.4	--
Control 4		39.226	18.5	8.4	--
Control 5		41.466	18.7	8.3	--
Control 6		38.608	18.7	8.4	--
Control, mean		42.571	--	--	--
Test item	10	40.024	19.3	8.3	5.983
Test item	10	39.649	18.8	8.4	6.864
Test item	10	41.163	19.0	8.3	3.309
Test item	32	40.938	18.9	8.4	3.837
Test item	32	41.773	190	8.4	1.876
Test item	32	43.749	19.0	8.4	0.000
Test item	100	43.895	19.3	8.3	0.000
Test item	100	40.648	18.8	8.4	4.518
Test item	100	45.439	19.1	8.4	0.000
Test item	320	41.796	19.1	8.3	1.821
Test item	320	42.447	19.0	8.3	0.293
Test item	320	40.149	18.9	8.4	5.690
Test item	1000	36.331	19.0	8.4	14.658
Test item	1000	41.132	19.0	8.4	3.382
Test item	1000	40.169	18.7	8.4	5.642
Test item, mean	10	40.279	--	--	5.385
Test item, mean	32	42.153	--	--	1.904
Test item, mean	100	43.327	--	--	1.506
Test item, mean	320	41.464	--	--	2.601
Test item, mean	1000	39.211	--	--	7.894
Physico-chemical oxygen consumption control	1000	0.489	18.7	7.6	--
Reference compound	2.5	41.316	19.7	8.4	2.948
Reference compound	5	39.845	19.6	8.3	6.404
Reference compound	10	33.479	20.0	8.3	21.357
Reference compound	20	16.809	19.9	8.4	60.514
Reference compound	40	8.662	19.5	8.4	79.654

Table 2: Main test, Test with ATU (Heterotrophic respiration). Respiration rates after 3 hours incubation period, percentage inhibition, temperature and pH values

Treatment [mg/L]		Respiration rate	Mean Temp.	pH-	Inhibition
		[mg/L · h]	[°C]	value	[%]
Control 1		35.717	19.5	8.2	--
Control 2		37.066	19.5	8.2	--
Control 3		35.705	19.1	8.2	--
Control 4		38.580	18.5	8.3	--
Control 5		39.119	18.8	8.3	--
Control 6		36.565	19.1	8.3	--
Control, mean		37.125	--	--	--
Test item	10	38.148	19.5	8.3	0.000
Test item	10	34.990	18.8	8.4	5.753
Test item	10	36.497	19.0	8.3	1.693
Test item	32	36.728	19.0	8.3	1.070
Test item	32	37.967	19.2	8.3	0.000
Test item	32	37.358	19.2	8.4	0.000
Test item	100	37.028	19.4	8.1	0.261
Test item	100	37.237	19.0	8.3	0.000
Test item	100	37.567	19.1	8.3	0.000
Test item	320	44.513	19.8	8.3	0.000
Test item	320	40.525	19.6	8.3	0.000
Test item	320	39.761	19.5	8.4	0.000
Test item	1000	38.175	19.2	8.3	0.000
Test item	1000	39.572	19.4	8.4	0.000
Test item	1000	38.380	18.8	8.4	0.000
Test item, mean	10	36.545	--	--	2.482
Test item, mean	32	37.351	--	--	0.357
Test item, mean	100	37.278	--	--	0.087
Test item, mean	320	41.600	--	--	0.000
Test item, mean	1000	38.709	--	--	0.000
Physico-chemical oxygen consumption control 1000	0.724	18.9	7.4	--
Reference compound	2.5	35.157	19.0	8.2	5.301
Reference compound	5	36.849	19.2	8.2	0.745
Reference compound	10	26.371	19.6	8.2	28.967
Reference compound	20	11.233	19.4	8.3	69.744
Reference compound	40	8.385	19.3	8.3	77.413

 

Table 3: Validity criteria for OECD 209

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	The oxygen uptake rate was 53.2 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour for the test set without test substance and 46.4 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour for the test set with test substance.	yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	The CV was 8.6% for the test set without test substance and; 3.9% for the test set with test substance.	yes

 

 

Validity criteria fulfilled:

yes

Remarks:

See Table 3 in "Any other information on results incl. tables".

Description of key information

EC50 (total, heterotrophic and nitrification respiration) > 1000 mg/L (nominal) for activated sludge (OECD 209)

NOEC (total, heterotrophic and nitrification respiration) ≥ 1000 mg/L (nominal) for activated sludge (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

One key study is available for tefuryltrione, in which the toxicity to microorganisms was investigated. The study was conducted according to GLP and OECD guideline 209. The activated sludge was exposed to the test item at five test item concentrations of 10, 32, 100, 320 and 1000 mg/L for 3 h. Test concentrations are given as nominal concentrations. The respiration rate (oxygen consumption) is classified into two processes of oxidation, the oxidation of organic carbon (heterotrophic oxidation) and the ammonium oxidation (nitrification). By measuring the oxygen consumption rates in the presence and absence of a specific inhibitor of nitrification ATU (N-allylthiourea), total, heterotrophic and nitrification respiration rates were calculated. Nitrification inhibition could not be calculated as the differences between total and heterotrophic respiration rates were too small. As no inhibition was observed for total and heterotrophic respiration up to the highest tested concentration of 1000 mg/L, the same effect values for nitrification were chosen. No effect was detected up to a test item concentration of 1000 mg/L. Therefore, the EC50s and NOECs for total respiration, heterotrophic respiration, and nitrification respiration were determined as >1000 mg/L and ≥1000 mg/L, respectively.