4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 17 Jan 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Analytical monitoring of water samples was performed only at the beginning of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen (29.03.1994)

Analytical monitoring:

yes

Remarks:

HPLC - UV

Details on sampling:

- Concentrations: Control, solvent control, 0.556, 0.993, 1.787, 3.178, 5.561, 9.93, 17.87, 31.78 and 55.61 mg/L (nominal).
- Sampling method: Quantitative analysis of the active ingredients in cell-free samples of the nutrient medium were made at the beginning of the experiment. Because growing algal cells can adsorb, incorporate and/or metabolize the active ingredients under study, concentrations were not determined at the end of the experiment.
- Sample storage conditions before analysis: The samples were stored deep frozen until they were analyzed.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method for preparing stock solution: A pre-solution was prepared by adding 0.0559 g of the substance to a 10 mL volumetric flask and bringing this up to volume with acetone. Stock solution was prepared by adding 0.500 mL of the pre-solution to a 5000 volumetric flask and bringing this up to volume with sterile, deionized water. Before use, the flask containing the stock solution was agitated with a magnetic stirrer for 10 min.
- Method for preparing test medium for quantitative analysis: Sterile, deionized water, 10-fold concentrated OECD medium and stock solution of the product were mixed. The medium was then divided into 2 parts. One part was used for the growth inhibition tests. A second part, used for quantitative analyses, was mixed with an appropriate amount of sterile, deionized water (instead of algal pre-culture). All operations were carried out under sterile conditions.
- Controls: control and solvent control
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: < 0.1 mL/L
- Evidence of undissolved material: no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81
- Source: Collection of Algal Cultures, Inst. Plant Physiologie, Universitat Gottingen, Germany.
- Culturing conditions: Stock cultures of the alga were grown at 23 ± 2°C under 16 h light/day in cotton plugged, 300-rnl Erlenmeyer flasks containing 50 mL of culture medium (Bringmann and Kuhn, 1980). Fresh stock cultures were prepared once a week. All operations were conducted under sterile conditions.
- Age of inoculum (at test initiation): 3 days old cultures

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

However effect results were calculated for 72 h, as provided by the guideline

Test temperature:

23 ± 2 °C

pH:

Treatments (0-72 h): 7.83 - 9.10
Control (0-72 h): 7.87 - 9.38
Solvent Control (0-72 h): 7.88 - 9.35

Nominal and measured concentrations:

Nominal concentrations: Control. Solvent Control, 0.556, 0.993, 1.787, 3.178, 5.561, 9.93, 17.87, 31.78 and 55.61 mg/L
Measured concentrations (initial): < LOQ,

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: closed (cotton wool plugs)
- Fill volume: 150 mL
- Initial cells density: 1 x 10E+04 cells/mL
- Control end cells density (72 h): 137.70 x 10E+04 cells (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201 with slight modification (80 µg/L instead of 64 µg/L FeCl3·6(H2O)).
- Culture medium different from test medium: yes, culture medium according to Bringmann and Kuhn (1980), test medium see above.
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 h/day
- Light intensity and quality: 8000 lux, 2 banks of lights containing 3 fluorescent lamps each

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were estimated photometrically at a wave length of 578 nm using a single-beam-photometer. Cell counts were made using a Thoma-Zeiss counting chamber and a microscope at a magnification of 400 times. To detect possible alterations in algal cells that might influence extinction measurements, such as unusual cell size, samples from each flask were examined under a microscope at a magnification of 400 times. Cell numbers were estimated photometrically only if alterations that might influence extinction were not detected.

TEST CONCENTRATIONS
- Range finding study: yes, but no further details in the report

Reference substance (positive control):

yes

Remarks:

0.10, 0.18, 0.32, 0.56, 1.00 and 1.80 mg/L K2Cr2O7 (16 June 1997)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.04 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.49 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.87 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.63 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.49 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.87 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: yes: 1.57 µg/L after 96 h some cells were swollen; 2.44 µg/L after 24 h cells swollen, after 48, 72 and 96 h cells slightly swollen; 4.29 µg/L after 24, 48, 72 and 96 h cells swollen; 8.02 µg/L after 24, 48, 72 and 96 h cells swollen; 15.1µg/L after 24, 48, 72 and 96 h cells swollen; 24.8 µg/L after 24, 48, 72 and 96 h cells swollen; 42.7 µg/L after 24, 48, 72 and 96 h cells swollen.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- EC50 (72 h) = 0.78 mg/L
- ErC50 (72 h) = 1.57 mg/L

Table 1: % Inhibition of growth rate at different concentrations.

Measured Concentrations [µg/L]	% Inhibition of growth rate
	24 h	48 h	72 h
Pooled control	0	0	0
0.491	0.9	0.5	2.3
0.867	10.2	8.2	6.4
1.57	17.6	17.8	15
2.44	24.2	30.6	28.9
4.29	28.5	44.6	50.6
8.02	37.7	57	76
15.1	40.4	59.7	80.4
24.8	40.2	57.5	76.8
42.7	39.6	57.2	74.8

Table 2: Analytical results.

Nominal Concentrations [µg/L]	Determination 1	Determination 2	Average	%
Stock solution 556.08	473.3	474.8	474	85
0.556	0.497	0.486	0.491	88
0.993	0.857	0.877	0.867	87
1.787	1.558	1.59	1.57	88
3.178	2.447	2.433	2.44	77
5.561	4.17	4.404	4.29	77
9.93	7.77	8.273	8.02	81
17.87	15.31	14.9	15.1	84
31.78	24.71	24.96	24.8	78
55.61	42.83	42.52	42.7	77

Table 3: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	137.908	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	8.66%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.7	yes

Validity criteria fulfilled:

yes

Remarks:

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

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 - 14 Aug 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study consists of two parts. The first part is performed according to the principles of OECD guideline 201. The second part (algae recovery test) does not follow a guideline but is well documented and meets general scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The exposure experiment (3 d) was performed based on the principles of OECD guideline 201 (First part of the test). The exposure period was finished by separating and transferring the exposed algal cells into new growth media without the testing substance for maximum 7 d in order to study their recovery (Second part of the test). The second part of the study was designed to measure growth rates at different times after exposure and to compare the maximum achievable growth rates and minimum doubling times with the growth rates of the control groups after recovering of algal cells.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen (29.03.1994)

Analytical monitoring:

yes

Remarks:

HPLC - UV

Details on sampling:

- Concentrations: Control, solvent control, 3, 6, 15, 30, 60 µg/L as well as additional 60 µg/L treatment which was incubated for one day only.
- Sampling method: Quantitative analysis of the active ingredients in cell-free samples of the nutrient medium were made at the beginning of the experiment. Because growing algal cells can adsorb, incorporate and/or metabolize the active ingredients under study, concentrations were not determined at the end of the experiment.
- Sample storage conditions before analysis: The samples were stored deep-frozen until they were analyzed.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method for preparing stock solution: A pre-solution was prepared by adding 0.606 g of the substance to a 10 mLvolumetric flask and bringing this up to volume with acetone. Stock solution was prepared by adding 0.1 mL of the pre-solution to a 1000 mL volumetric flask and bringing this up to volume with sterile, deionized water. Before use, the flask containing the stock solution was agitated with a magnetic stirrer for 30 min and disperged in an ultrasonic bath for 10 minutes.
- Method for preparing test medium for quantitative analysis: Sterile, deionized water, 10-fold concentrated OECD medium and stock solution of the product were mixed. The medium was then divided into 2 parts. One part was used for the growth inhibition tests. A second part, used for quantitative analyses, was mixed with an appropriate amount of sterile, deionized water (instead of algal pre-culture). All operations were carried out under sterile conditions.
- Controls: control and solvent control
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: < 0.1 mL/L
- Evidence of undissolved material: no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 1.81
- Source: Collection of Algal Cultures, Inst. Plant Physiologie, Universität Göttingen, Germany.
- Culturing conditions: Stock cultures of the alga were grown at 23 ± 2 °C under 16 h light/day in cotton plugged, 300 mL Erlenmeyer flasks containing 50 mL of culture medium (Bringmann and Kuhn, 1980). Fresh stock cultures were prepared once a week. All operations were conducted under sterile conditions.
- Age of inoculum (at test initiation): 3 days old cultures

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Exposure duration was 3 d according to OECD guideline 201 (first part of study). Cells in the highest test level of 60 µg/L were additionally exposed for one day only.

Post exposure observation period:

After 3 d of exposure (additional 60 µg/L treatment for 1 day) cells were transfered into new fresh media without testing substance and were incubated for further maximum 7 days in order to determine their recovery.

Test temperature:

23 ± 2 °C

pH:

Treatments (0-72 h): 7.80 - 8.16
Control (0-72 h): 7.76 - 8.54
Solvent Control (0-72 h): 7.79 - 8.24

Nominal and measured concentrations:

Nominal concentrations (72 h): 0, 3, 6, 15, 30, and 60 µg/L
Nominal concentrations (24 h): 60 µg/L
Measured concentrations (initial, 72 h): 0.779, 1.75, 5.09, 11.2, and 21.3 µg/L
Measured concentrations (initial, 24 h): 21.8 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: closed (cotton wool plugs)
- Fill volume: 150 mL
- Initial cells density: 1 x 10E+04 cells/mL
- Control end cells density (72 h): 72.12 x 10E+04 cells (solvent control), 75.56 x 10E+04 cells (control)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201 with slight modification (80 µg/L instead of 64 µg/L FeCl3·6(H2O)).
- Culture medium different from test medium: yes, culture medium according to Bringmann and Kuhn (1980), test medium see above.
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 h/day
- Light intensity and quality: 8000 lux, 2 banks of lights containing 3 fluorescent lamps each

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Cell numbers were estimated photometrically at a wave length of 578 nm using a single-beam-photometer. Cell counts were made using a Thoma-Zeiss counting chamber and a microscope at a magnification of 400 times. To detect possible alterations in algal cells that might influence extinction measurements, such as unusual cell size, samples from each flask were examined under a microscope. Cell numbers were estimated photometrically only if alterations that might influence extinction were not detected.
- Recovery phase: Growth rate was measured at different times after exposure and compared to the maximum achievable growth rates and minimum doubling times with the growth rates of the control groups after recovering of algal cells.

TEST CONCENTRATIONS
- Range finding study: yes but no further details in the report.

Reference substance (positive control):

yes

Remarks:

0.10, 0.18, 0.32, 0.56, 1.00 and 1.80 mg/L K2Cr2O7 (16 June 1997)

Remarks on result:

other: For details on results see "Details on results" and "Any other information on results incl. tables" below

Details on results:

Temporary exposure (three days) of nominal test item concentrations (measured) of 3 to 60 µg/L (0.779 to 21.3 µg/L) had no impact on the ability of Selenastrum capricornutum to recover and to reproduce sustainably in product-free growth medium. For further details see field any other information on results incl. tables.

Results with reference substance (positive control):

- EbC50 (72 h) = 0.91 mg/L
- ErC50 (72 h) = 1.81 mg/L

Table 1: Growth rate inhibition of algae during exposure time.

Nominal concentration [mg/L]	0-24 h	0-48 h	0-72 h
control	0	0	0
3	-4.6	6	3.6
6	10.6	20.3	16.4
15	-29.5	46.6	49.8
30	24.3	65.4	73.5
60	8.6	67.7	75.7

Table 2: Growth rates (µ) and doubling times (G) in S. capricornutum cultures after exposure with the test item.

Nominal exposure level in µg/L (µg initial measured/L)	Exposure time in days	Growth rates µ max after recovering of algal cells in L/day (mean (var. of replicates))	Doubling times Gmin after recovering of algal cells in hours (mean (var. of replicates))
0 (control)	3	0.57 (0.53-0.62)	29.2 (26.8-31.4)
0 (solvent control)	3	0.73 (0.50-1.09)	22.7 (15.3-33.3)
pooled controls	3	0.67 (0.50-1.09)	26.6 (15.3-33.3)
3 (0.779)	3	0.69 (0.66-0.72)	24.1 (23.0-25.1)
6 (1.75)	3	0.76 (0.72-0.84)	22.0 (19.9-23.2)
15 (5.09)	3	0.74 (0.61-0.90)	23.0 (18.5-27.4)
30 (11.2)	3	0.93 (0.54-1.31)	20.3 (12.7-30.7)
60 (21.3)	3	0.90 (0.79-1.01)	18.6 (16.4-21.1)
60 (21.8)	1	0.69 (0.52-0.94)	24.3 (17.7-32.0)

In the controls, 3, 6 and 60 (1 day exposure) µg/L test levels the maximum achievable growth rates and the minimum doubling times respectively, were reached immediately (0 -3 d). At the test level 15, 30 and 60 µg/L the rates were delayed by one day per each increasing concentration until up to 5 days. It is shown that a temporary exposure (3 days) of the algae to nominal test item concentrations (measured) of 3 to 60 µg/L (0.779 to 21.3 µg/L) had no impact on their ability to recover and to reproduce sustainably in product-free growth medium. There were no significant differences in growth rates at all test levels compared with the pooled controls.

Table 3: Analytical results.

Concentration	Measured Concentration (µg/L)
	Determination 1	Determination 2	Average	% of nominal
Nominal (measured initial)
3.00 (2.98)	0.923	0.635	0.779	26
6.00 (5.96)	1.761	1.749	1.75	29
15.0 (14.9)	5.033	5.144	5.09	34
30.0 (29.8)	10.76	11.57	11.2	37
60.0 (59.6)	21.26	21.37	21.3	36
60.0 (59.6) (1 day)	20.37	23.28	21.8	37

Validity criteria fulfilled:

not applicable

Description of key information

ErC50 (72 h) = 6.04 µg/L, NOEC (72 h) = 0.49 µg/L (initial measured, OECD 201, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

6.04 µg/L

EC10 or NOEC for freshwater algae:

0.49 µg/L

Additional information

Two experimental studies are available giving evidence on the toxicity of the substance towards algae.

The first study with Pseudokirchneriella subcapitata as the test organism was performed according to the OECD guideline 201 and GLP standards (1997). Algae were exposed to nominal test item concentrations of 0.56, 1.00, 1.80, 3.20, 5.60, 10.0, 18.0, 32.0, and 56.0 µg/L. Acetone was used as a vehicle and therefore both, an untreated and a vehicle control, were run in parallel. The concentration of the solvent in bioassay water was below the recommendation of 0.1 mL/L as given by the OECD (2018) and had no influence on the outcome of the study. Samples were analyzed via HPLC-UV method for the test substance at the beginning of the test. Quantitative analyses of the treated, cell-free culture media showed recoveries of the substance between 77 and 88%. Therefore, all results were expressed based on initial measured concentrations. Based on inhibition of growth rate of the algae, an ErC50 (72 h) of 6.04 µg/L and a NOErC (72 h) of 0.49 µg/L (initial measured) were determined in this study.

A follow up study (1998) under GLP conditions investigated the recovery (in terms of growth rate and doubling times) of the algae Pseudokirchneriella subcapitata after exposure to nominal test item concentrations of 3, 6, 15, 30, and 60 µg/L (initial measured concentrations 0.779, 1.75, 5.09, 11.2, and 21.3 (21.8) µg/L). The study was performed in two steps. In the first part algae were exposed to the nominal concentrations for 72 h (highest test concentration was incubated additionally for one day only) based on the principles of the OECD guideline 201. The exposure period was finished by separating and transferring the algal cells into new growth media without the testing substance for maximum 7 d in order to study their recovery (second part of the test). Growth rates at different times after exposure were measured and the maximum achievable growth rates as well as the minimum doubling times were compared with the growth rates of the control groups after recovering of algal cells. In the controls, 3, 6, and 60 (1 day exposure) µg/L test levels the maximum achievable growth rates and the minimum doubling times respectively, were reached in 0-3 d. At the test level 15, 30, and 60 µg/L the rates were delayed by one day per each increasing concentration until up to 5 days. The study revealed that a temporary exposure (3 days) of the algae to nominal test item concentrations (measured) of 3 to 60 µg/L (0.779 to 21.3 µg/L) has no impact on their ability to recover and to reproduce sustainably in product-free growth medium. There were no significant differences in growth rates at all test levels compared with the pooled controls.

According to the above mentioned results, 6.04 µg/L (ErC50) and 0.49 µg/L (NOErC) represent the key values for acute and chronic toxicity, respectively, based on growth rate. These values are the most sensitive effect values found for aquatic organisms and are thus also used for classification and assessment purposes.