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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 October 2017 to 22 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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 from freshwater prepared media at the start (0 hours - with algae) and the end of the exposure (72 hours - with algae).
- Sampling method: The control and the three lowest concentrations (6.25% to 25.0%) were analyzed undiluted and the concentrations 50.0% till 100% of the test item solution were diluted with dilution medium prior to analysis.
- 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
- Preparation of the saturated solution: A saturated solution with a nominal loading of 200 mg/L test item were prepared once with dilution water 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item were weighed out. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24 ± 1 hour. After a separation phase of at least 1 hour at room temperature the saturated solution was taken from the homogeneous phase and was used for testing.
- Controls: Six replicates (without test item) were exposed under the same test conditions as the test vessels.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three to four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE/m2/s for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Dilution water medium has a nominal hardness of 0.24 mmol Ca+Mg/L

Test temperature:

From 22 to 23°C

pH:

From 8.09 to 9.51

Dissolved oxygen:

None

Salinity:

None

Nominal and measured concentrations:

6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution
Geometric mean measured test substance concentrations: 0.391 - 0.779 - 1.55 - 3.19 - 6.49 mg/L.
The measured concentrations of the test substance in the old media (3 d) were in the range of 86 to 96% of the initially measured values.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 59 mL, with aluminium tops with PFTE seals
- Test volume: 59 mL
- Initial cells density: Nominal: approximately 5000-50000 cells/mL; Current: 6839 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Application: Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Incubation: The flasks were positioned randomly and repositioned daily.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the OECD guideline 201

OTHER TEST CONDITIONS
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux; Current (mean value): 5308 lux
- Light homogeneity: Within ± 15 % over incubation area

EFFECT PARAMETERS MEASURED:
- Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the range finding test at the saturated solution with a nominal loading of 200 mg/L.
- Microscopic evaluation: Microscopic evaluation of the cells was carried out at the start and end of the incubation. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.
Physical Chemical Parameters:
- The pH-value at the beginning and end of the test were measured from pooled replicates per concentration and control after measurement of the cell density, respectively. The room temperature was measured continuously. Light intensity was measured prior to test start.

TEST CONCENTRATIONS
- Range finding study:
A non-GLP preliminary range finding test without headspace in a closed bottle system (in order to avoid losses of test item to the headspace) under static conditions over a period of 72 hours were conducted at the test facility. A saturated solution of the test item with a nominal loading level of 100 mg/L was prepared and two further dilution levels prepared by diluting the saturated solution by factor 10 and 100 with dilution water. A second saturated solution of the test item with a nominal loading level of 10.0 mg/L was prepared and was tested undiluted. The saturated solutions and the dilution levels of the saturated solutions were visually clear throughout the exposure period. In the range finding test, two replicates per concentration and four for the control, were tested. The results are presented in the Tables in "Any other information on results incl. tables".
As the initial measured concentration at 100% saturated solution with a nominal loading rate of 100 mg/L (=8.60 mg/L) is lower than the measured water solubility value (13.2 mg/L), the final test was conducted with a nominal loading of 200 mg/L instead of 100 mg/L, to be sure than we test the maximum attainable test substance concentration.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.65 - >6.49

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 6.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Microscopic evaluation of the cells at the start and at the end of the exposure revealed no morphological abnormalities in the test item treatments and the control.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-10-10 to 2017-10-13 (with headspace) and 2017-10-09 to 2017-10-12 (without headspace), respectively. The reference item toxicity is in the valid range following test facility SOPS.
See table in "Any other information on results incl. tables"

Table 6.1.5/1: Results of the Range finding test (0 -72 hours)

Saturated Solution (nominal loading 100 mg/L) [%]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	44	87
10.0	2	6
1.00	-3	-16
100 (nominal loading 10.0 mg/L)	7	22

Negative values = growth stimulation

Table 6.1.5/2: Measured Exposure Concentrations during non-GLP Preliminary Range-finding test

	Measured concentration (mg/L)
Saturated Solution (nominal loading 100 mg/L) [%]	0 hours Start of the exposure (with algae)	72 hours End of the exposure (with algae)
100	8.60	9.13
10.0	0.920	0.946
1.00	0.0986	0.104
100 (nominal loading 10.0 mg/L)	1.22	1.23
Control	< LCL	< LCL

LCL = lowest calibration standard = 0.0500 mg/L

Table 6.1.5/3: Cell densities in the definitive test

Geometric mean measured test item concentration (mg/L)	Replicate	Cell density (cells/mL)
		0 hours	24 hours	48 hours	72 hours
6.49	1	6839	31356	152922	459199
	2	6839	31619	113486	399099
	3	6839	25507	95719	371448
	Mean	6839	29494	120709	409915
3.19	1	6839	29428	164644	636426
	2	6839	32020	170145	617261
	3	6839	31611	145274	521874
	Mean	6839	31020	160021	591854
1.55	1	6839	38083	228197	698867
	2	6839	40927	221824	728215
	3	6839	28158	191159	695590
	Mean	6839	35723	213727	707557
0.779	1	6839	43417	255912	777401
	2	6839	45436	265983	773919
	3	6839	41149	227307	699306
	Mean	6839	43334	249734	750209
0.391	1	6839	51569	312869	831678
	2	6839	55036	302862	800926
	3	6839	56455	315239	842007
	Mean	6839	54353	310323	824870
Control	1	6839	40977	206375	705568
	2	6839	42779	222579	712473
	3	6839	49155	222608	740914
	4	6839	44043	233142	722626
	5	6839	44418	230801	715545
	6	6839	45611	194062	608395
	Mean	6839	44497	218261	700920

Table 6.1.5/4: Evaluation of growth rates after 72 hours

Geometric mean measured test item concentration (mg/L)	Replicate	Growth rate inhibition (%)
6.49	1	9
	2	12
	3	14
	Mean	12
3.19	1	2
	2	3
	3	6
	Mean	4
1.55	1	0
	2	-1
	3	0
	Mean	0
0.779	1	-2
	2	-2
	3	0
	Mean	-1
0.391	1	-4
	2	-3
	3	-4
	Mean	-4

Table 6.1.5/5: Measured exposure Concentrations during the definitive test

Sampling date	2017-12-19 0 d fresh medium	2017-12-22 3 d old medium
Start of analysis	2017-12-19	2017-12-22
Dilution level of the saturated solution [%]	Test substance			Geometric mean measured concentration [mg/L]
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100	6.62	6.36	96	6.49
50.0	3.37	3.02	90	3.19
25.0	1.61	1.50	93	1.55
12.5	0.803	0.755	94	0.779
6.25	0.422	0.363	86	0.391
Control	< LOQ	< LOQ

Meas. conc.  = measured concentration of the test item, mean value of two injections, dilution factor taken into account

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

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

Table 6.1.5/6: EC50 values of the reference item

Set-up and Experimental Date	with Headspace	without Headspace	Valid Range (average ± 3 x SD)
	Growth Rate Inhibition
ErC50	0.535	0.498	0.754±0.558
95 % confidence interval	0.523 - 0.547	0.459 - 0.541

Validity criteria fulfilled:

yes

Conclusions:

The 72h-ErC50 value was determined to be greater than the maximum attainable test substance concentration in the test conditions (geometric mean concentration 6.49 mg/L), and the 72h-ErC10 value can be determined at 5.80 mg/L (95% CL: 4.65 ->6.49 mg/L) based on geometric mean measured concentrations.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD Guideline 201 and EU Method C.3 with GLP statement. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

 

A saturated solution with a nominal loading of 200 mg/L test item was prepared once with dilution water 24±1 hour prior to the start of the exposure. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24±1 hour. After a separation phase of at least 1 hour at room temperature the saturated solution was taken from the homogeneous phase and was used for testing.

With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to geometric mean measured test item concentrations after 72 hours of 0.391 – 0.779 – 1.55 – 3.19 – 6.49 mg/L, respectively. Three replicates were tested for each test item concentration and six replicates for the control. The study was conducted under static conditions with an initial cell density of 6839 cells/mL.The environmental conditions were within the acceptable limits. The test media were clear and the test item was dissolved throughout the test period.

 

The concentrations of the test substance were determined at the start of the exposure in the fresh media (0 hours) and at the end of the exposure in the old media (72 hours) of all tested concentration levels and the control via HPLC-DAD. The measured concentrations of the test substance in the old media (72 hours) were in the range of 86 to 96% of the initially measured values. All effect values were based on the geometric mean measured test item concentrations.

According to this test, the 72h-ErC50 value was determined to be greater than the maximum attainable test substance concentration in the test conditions (geometric mean concentration 6.49 mg/L), and the 72h-ErC10 value can be determined at 5.80 mg/L (95% CL: 4.65 ->6.49 mg/L) based on geometric mean measured concentrations.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokircheriella subcapitata) > 6.49 mg/L, corresponding to the maximum attainable test substance concentration in the test conditions;

72h-ErC10 (Pseudokircheriella subcapitata) = 5.80 mg/L (95% CL: 4.65 - >6.49 mg/L), based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

5.8 mg/L

Additional information

One experimental key study is available to assess the toxicity of the registered substance to algae.

In this study (NOACK, 2018), the toxicity of the registered substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD Guideline 201, EU Method C.3 with GLP statement. The study was performed under static conditions during 72 hours. Glass flasks without headspace were used to reduce losses of the test substance. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to geometric mean measured test item concentrations after 72 hours of 0.391 – 0.779 – 1.55 – 3.19 – 6.49 mg/L, respectively. The environmental conditionswere within the acceptable limits. The test media were clear and the test item was dissolved throughout the test period. The measured concentrations of the test substance in the old media (72 hours) were in the range of 86 to 96% of the initially measured values. Therefore, all effect values were based on the geometric mean measured test item concentrations. According to this test, the 72h-ErC50 value was determined to be greater than the maximum attainable test substance concentration in the test conditions (corresponding to 6.49 mg/L), and the 72h-ErC10 value can be determined at 5.80 mg/L (95% CL: 4.65 ->6.49 mg/L) based on geometric mean measured concentrations.