5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 Mar - 31 Mar 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data based on the inhibition control of a ready biodegradability study. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2012).

Qualifier:

according to guideline

Guideline:

other: OOECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted in 1992

Deviations:

yes

Remarks:

At test start, the inorganic carbon content of the test substance suspension in medium was 15% of total carbon and not < 5% as recommended.

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, date of inspection 23 Mar 1998

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained on 01 Mar 1999 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
- Storage conditions: continuous aeration upon receipt
- Preparation of inoculum for exposure: A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Nominal and measured concentrations:

Initial test substance concentrations: 10 mg TOC/L (and 10 mg TOC/L reference substance sodium benzoate)
Toxicity control: test material: 10 mg TOC/L, reference substance sodium benzoate: 10 mg TOC/L

Details on test conditions:

TEST CONDITIONS
- Composition of medium: standard medium as recommended in the guideline
- Test temperature: 21 °C
- Suspended solids concentration: 30 mg suspended solids/L
- Continuous darkness: yes
- Oxygen conditions: aerobic

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels containing 3 L of solution
- Measuring equipment: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water. The samples were analysed for CO2 using an Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser. 40 - 50 µl of the samples were injected into the inorganic carbon channel of the TOC analyser.

SAMPLING
- Sampling frequency: the first CO2 absorber vessel was sampled on days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29; the second absorber vessel was sampled on days 0 and 29
- Sample storage before analysis: instant analysis

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 vessels
- Toxicity control: 1 vessel (20 mg carbon/L)
- Reference control: 2 vessels

Reference substance (positive control):

yes

Remarks:

benzoic acid, sodium salt

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 25.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of CO2 evolution

Remarks on result:

other: Biodegradation toxicity control

Details on results:

- The toxicity control attained 75% degradation after 28 days confirming the test material to be non-toxic to the microorganisms within the water solubility.
- The total CO2 evolution in the inoculum blank was 28.8 mg/L after 28 days (required: ≤ 40 mg/L).

Results with reference substance (positive control):

The reference substance attained 74% degradation after 28 days (77% after 10 days) confirming the suitability of the test system.

The toxicity control attained > 35% degradation after 14 days (46% after 14 d) of incubation.

"If in a toxicity test, containing both the test and the reference substance, less than 35% based on total DOC or less than 25% based on total ThOD or ThCO2 occurred within 14 days, the test substance can be assumed to be inhibitory." (OECD Guideline 301)

Since more than 25% degradation occurred in the toxicity control, the substance is with high probability not toxic to aquatic microorganisms.The test item concentration in the toxicity control of 10 mg/L TOC can be used as NOEC value for the toxicity to aquatic microorganisms.

Table 1. Percentage biodegradation values

Day	% Degradation the test item plus sodium benzoate toxicity control
0	0
1	5
2	19
3	27
6	38
8	35
10	36
14	46
16	47
20	68
22	69
24	76
27	74
28	75
29	78

Table 2: Validity criteria for OECD 301 B.

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	CV at day 29 = 3.39%	Yes
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	77%	Yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	46%	Yes
The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.	15% in replicate 1 and 14% in replicate 2	No
The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.	28.8 mg/L	Yes

Validity criteria fulfilled:

no

Remarks:

See Table 2 in 'any other information on results incl.tables'

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Jan 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Insoluble test substance was visible during the test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted 1984

Deviations:

yes

Remarks:

In some instances, the initial and final oxygen concentrations were below those recommended in the test guideline.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, date of inspection 28 Feb 2000

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 500 mg of test substance was separately dispersed in approximately 250 mL of water and subjected to ultrasonication (approximately 30 minutes). Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentration of 1000 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of inoculum: Activated sludge was obtained on 07 Jan 2002 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Method of cultivation: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at 21°C and was used on the day of collection.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

21 °C

pH:

7.6 - 8.0 at the end of exposure time

Nominal and measured concentrations:

Nominal: 0 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flasks
- Aeration: with compressed air at approx. 0.5 - 1 L/min
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water, dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approx. 100 mg/L as CaCO3.
- Total organic carbon: 1.1 mg/L
- Particulate matter: 3.8 g/L
- Pesticides: < 0.01 µg/L
- Chlorine: 0.16 mg/L
- Conductivity: 366 µS/cm
- Intervals of water quality measurement: pH values were determined at test termination

OTHER TEST CONDITIONS
- Light: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Respiration rates of activated sludge microorganisms were determined after 30 min and after 3 h of incubation.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.70 mg/L (limit of water solubility), 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No significant effect on respiration was observed at any of the test concentrations employed.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: Insoluble test substance was visible.

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: Effect concentrations are above the water solubility of the test item.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations made throughout the study period showed that at the test concentration of 1000 mg/L, white particles of undissolved test material were visible on the surface and dispersed throughout the test media at 0 h, 30 min and 3 h contact time.

Results with reference substance (positive control):

The reference substance attained 81% inhibition of respiration after 3 h.
3,5-dichlorophenol EC50 (30 minutes): 20 mg/L
3,5-dichlorophenol EC50 (3 hours): 13 mg/L

Test substance EC50 (30 minutes): >1000 mg/L

Variation in respiration rates of controls 1 and 2 after 30 minutes: ± 1%.

Variation in respiration rates of controls 1 and 2 after 3 hours: ± 8%.

Table 1: Oxygen consumption rates and percentage inhibition values after 30 minutes contact time

Substance	Nominal conc. (mg/L)	Initial O2 reading (mg/L)	Time of measurement (min)	Final O2 reading (mg/L)	O2 consumption rates (mg/L*min)	% Inhibition
Control	0 A	6.1	7	2.7	0.49	-
	0 B	6.4	8	2.4	0.50	-
Test substance	1000 A	5.6	6	2.2	0.57	-15
	1000 B	5.6	6	2.4	0.53	-7
	1000 C	5.8	7	2.3	0.50	-1
Reference substance	3.2	6.2	7	2.8	0.49	1
	10	6.0	9	1.8	0.47	5
	32	7.6	10	6.4	0.12	76

Table 2: Oxygen consumption rates and percentage inhibition values after 3 hours contact time

Substance	Nominal conc. (mg/L)	Initial O2 reading (mg/L)	Time of measurement (min)	Final O2 reading (mg/L)	O2 consumption rates (mg/L*min)	% Inhibition
Control	0 A	6.5	8	2.5	0.50	-
	0 B	6.1	9	2.2	0.43	-
Test substance	1000 A	6.0	8	2.0	0.50	-8
	1000 B	5.9	7	2.4	0.50	-8
	1000 C	6.4	9	2.3	0.46	1
Reference substance	3.2	6.4	9	2.5	0.43	8
	10	6.7	10	3.8	0.29	38
	32	7.9	10	7.0	0.09	81

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.	Not reported	not applicable
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	17.2%	yes

Validity criteria fulfilled:

not specified

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC50 (3 h) > 1000 mg/L (nominal, OECD 209, respiration rate)

NOEC (3 h) ≥ 1000 mg/L (nominal, OECD 209, respiration rate)

No inhibitory effect on activated sludge respiration was observed in the range of water solubility of the test substance.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The respiration inhibition of the test substance was tested in a 3-hour respiration inhibition test using activated sludge from a plant treating predominantly domestic sewage and following OECD guideline 209 and GLP. No inhibitory effect of the substance was observed in the limit test at 1000 mg/L. During the test, white particles of undissolved material were visible on the surface and dispersed throughout the test media.

Furthermore, in the toxicity control of a biodegradation study according to OECD 301 B, no inhibitory effects were caused by the test item. Test item concentration in the toxicity control was 25.4 mg test item/L (equivalent to 10 mg/L TOC) and the TOC concentration of the reference substance was 10 mg/L. The reached biodegradation was > 35% within 14 days (46% after 14 d). Thus, the test substance is not inhibitory to aquatic microorganisms. According to ECHA Guidance R.7b (ECHA, 2017) the test item concentration can be used as NOEC value for the toxicity to aquatic microorganisms. Thus, a NOEC (14 d) of ≥ 25.4 mg/L resulted.