2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 December 2017 to 14 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on June 07th to June 10th, 2016 and July 13th 2016 / signed on 03.01.2017

Specific details on test material used for the study:

- Stability under test conditions: Stable in water at all pHs after 28 days at 20 and 40°C (sponsor information)
- Water solubility: 13.2 mg/L (20 °C, pH 6.5)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via HPLC-DAD in fresh media at the start of exposure (0 h) and in old media at the end of the exposure (48 h)
- Sampling method: At the start of the exposure (0 hours), samples were taken after preparation of the test item concentrations and analyzed. At the end of the exposure (48 hours), samples of the old media were taken directly from the test vessels.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Preparation of the saturated solution: A saturated solution (100 mg/L) was prepared with dilution water one day before the start of the exposure (at approximately -24 hours). An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. A slow stirring procedure was applied. Gentle stirring was carried out for 24 ± 1 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was stand for 1 hour for separation of undissolved test item. Thereafter, the saturated solution was removed by siphoning of the homogenous liquid phase from the approximate center of the water body. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water.
- Controls: dilution water without test item was incubated under the same conditions as the test groups.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut fur Wasser-, Boden- und Lufthygiene (WaBoLu)
- Culture: In glass vessels (2-3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination; light intensity of max. 1500 lx
- Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL
- Age at study initiation and method of breeding: Less than 24 h old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels at the latest 24 hours before the start of the exposure. The juveniles born within the following period of max 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: Daphnids were not fed during the study

ACCLIMATION
- Acclimation period: Acclimatization is not necessary, because the composition of the dilution water is equivalent to the culture medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

169 mg CaCO3/L

Test temperature:

During the test period, the temperature in the incubator was 18.5 – 19.5 °C.

pH:

Comprised between 7.36 to 8.19

Dissolved oxygen:

Comprised between 7.77 to 8.73 mg/L

Salinity:

Not applicable

Conductivity:

428 µS/cm

Nominal and measured concentrations:

Nominal: 1.94 - 4.27 - 9.39 - 20.7 - 45.5 - 100% of the saturated solution.
The geometric mean measured concentrations are 0.0750 – 0.212 – 0.454 – 1.02 – 2.19 – 4.34 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw were used and filled up to the top with the test solutions.
- Type (delete if not applicable): Closed
- Test volume: approximately 130 mL
- Renewal of the test solutions: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Application: The test vessels were filled up to the top with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps.

TEST MEDIUM / WATER PARAMETERS
- Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L
- Preparation of dilution water: Same composition as the culture medium

OTHER TEST CONDITIONS
- Photoperiod: 16 h/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED:
Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other relevant observations were not made.

RANGE-FINDING STUDY
A non-GLP preliminary range finding test was conducted at the test facility under static conditions over a period of 48 hours with a saturated solution of the test item with a nominal loading of 100 mg/L prepared with dilution water and two further dilution levels prepared by diluting the saturated solution by factor 10 and 100 with dilution water. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). The saturated solution was prepared one day prior to the start of the exposure (at -24 hours) and used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water by factor 10 and 100. The dilution levels 1 and 10% of the saturated solution were visually clear throughout the exposure period, whereas turbidity (at the start of the exposure) and undissolved material (white sediment at the bottom of the test vessels) was observed in the saturated solution. In the range finding test, two replicates per concentration level and control, each with ten daphnids, were tested.
- Test concentrations: control, 1, 10 and 100% saturated solutions
- Results used to determine the conditions for the definitive study: 0% immobilisation after 48h in the control and at 1% saturated solution; 50% immobilisation after 48h at 10% saturated solution and 60% immobilisation after 48h at 100% saturated solution. See tables in "Any other information on result incl. tables".

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.02 - >4.34 mg/L

Details on results:

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

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits during the study.
All tested concentration levels were visually clear throughout the exposure period. No immobility or any adverse effects were observed at the control.
The concentrations of the test item were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the test (48 hours) in all concentration levels and in the control. The measured concentrations in the old media at the end of the test (48 hours) were in the range of 92 to 102% of the initially measured concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 12-06 to 2017-12-07.
24h-EC50 = 1.64 mg/L (95% CL: 1-4 mg/L).
Valid range: 0.6-2.4 mg/L according to AQS P 9/2 (02/2000); clone 5

Table 6.1.3/1: Immobilization rates in the non-GLP preliminary range-finding test

Dilution level of the saturated solution [%]	IMMOBILIZATION [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	60	50	55	70	50	60
10	40	40	40	50	50	50
1	0	0	0	0	0	0
Control	0	0	0	0	0	0

Table 6.1.3/2: Measured concentrations of the test substance during the non-GLP preliminary range-finding test (HPLC-DAD)

Sampling date	2017-08-29 Start of the exposure, 0 hours	2017-08-31 End of the Exposure, 48 hours	Geometric mean of measured test item concentrations [mg/L]	% to initial concentrations
Dilution level of the saturated solution [%]	Test substance
	Meas. conc. [mg/L]	Meas. conc. [mg/L]
100	23.4	22.9	23.1	99
10	2.04	2.07	2.05	101
1	0.186	0.194	0.190	102
Control	< 0.0500^1)	< 0.0500^1)

* = saturated solution

Meas. conc.  = measured concentration of the test item, singledeterminations, dilution factors taken into account

% = percent of the initially measured concentration of the test item

¹⁾  = lowest calibration Level (0.0500 mg/L of the test item)

Table 6.1.3/3: Immobilization rates after 24 and 48 hours of exposure in the definitive test

Geometric mean measured test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
4.34	0	40	40	20	25	60	60	60	60	60
2.19	0	40	20	20	20	40	60	40	40	45
1.02	0	0	20	0	5	40	20	40	60	40
0.454	0	0	20	0	5	60	40	20	20	35
0.212	0	20	0	0	5	0	20	0	20	10
0.0750	0	0	0	0	0	0	20	0	0	5
Control	0	0	0	0	0	0	0	0	0	0

Table 6.1.3/4: Measured concentrations of the test substance during the definitive test (HPLC-DAD)

Sampling date	2017-12-12 Fresh media, 0 hours	2017-12-14 Old media, 48 hours
Start of analysis	2017-12-12	2017-12-14
Dilution level of the saturated solution [%]	Test substance			Geometric mean measured concentration [mg/L]^1
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100*	4.32	4.37	101	4.34
45.5	2.28	2.11	92	2.19
20.7	1.02	1.02	100	1.02
9.39	0.463	0.446	97	0.454
4.27	0.210	0.214	102	0.212
1.94	< LOQ	< LOQ		0.0750
Control	< LOQ	< LOQ

Meas. conc.  = measured concentration of the test item, enrichment and dilution factors taken into account

%    = percentage of the initially measured concentration of the test item

LOQ   = limit of quantification of the analytical method (0.150 mg test item/L)

*  = saturated solution

¹⁾    = If the measured concentration was < LOQ, 1/2 LOQ (0.0750 mg test item/L) was used

   for the calculation of the geometric mean measured concentration.

Validity criteria fulfilled:

yes

Conclusions:

According to the results of this study, the 48h-EC50 for Daphnia magna was 2.18 mg/L (95% CL: 1.02 - >4.34 mg/L), based on geometric mean measured concentrations.

Executive summary:

The short-term toxicity of the test substance to aquatic invertebrates Daphnia magna was determined according to the principles of OECD Guideline 202 and EU Method C.2 with GLP statement. The study was conducted in a closed system (sealed glass flasks) without headspace under static conditions over a period of 48 hours with the undiluted saturated solution of the test substance and further five dilution levels prepared from the saturated solution in a geometric series with a separation factor of 2.2: 1.94, 4.27, 9.39, 20.7, 45.5 and 100% of the saturated solution. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the test substance were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the test (48 hours) in all concentration levels and in the control.

The measured concentrations in the old media at the end of the test (48 hours) were in the range of 92 to 102% of the initially measured concentrations. The geometric mean measured concentrations are: 0.0750 – 0.212 – 0.454 – 1.02 – 2.19 – 4.34 mg/L.

All tested concentration levels were visually clear throughout the exposure period. No immobility or any adverse effects were observed at the control.

According to the results of this study, the 48h-EC50 for Daphnia magna was 2.18 mg/L (95% CL: 1.02 - >4.34 mg/L), based on geometric mean measured concentrations.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 2.18 mg/L (95% CL: 1.02 - >4.34 mg/L) based on geometric mean measured concentrations

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.18 mg/L

Additional information

One experimental key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates.

In this study (NOACK, 2018), the acute toxicity of the registered substance to the aquatic invertebrates Daphnia magna was determined according to OECD Guideline 202, EU Method C.2 with GLP statement. The study was conducted in a closed system (sealed glass flasks) without headspace under static conditions over a period of 48 hours with the undiluted saturated solution of the test substance and further five dilution levels prepared from the saturated solution in a geometric series with a separation factor of 2.2: 1.94, 4.27, 9.39, 20.7, 45.5 and 100% of the saturated solution. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the test substance were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the test (48 hours) in all concentration levels and in the control. The measured concentrations in the old media at the end of the test (48 hours) were in the range of 92 to 102% of the initially measured concentrations. The geometric mean measured concentrations are: 0.0750 – 0.212 – 0.454 – 1.02 – 2.19 – 4.34 mg/L. All tested concentration levels were visually clear throughout the exposure period. No immobility or any adverse effects were observed at the control.

According to the results of this study, the 48h-EC50 for Daphnia magna was 2.18 mg/L (95% CL: 1.02 - >4.34 mg/L), based on geometric mean measured concentrations.