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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

7 Oct - 5 Dec 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Preparation of stock solution: 10 mg of test substance was weighed and was dissolved in 1 mL of the solvent (acetone). This stock solution was then diluted with acetone for each stock solution. 0.1 mL of the stock solution was added into the medium.
- Chemical name of vehicle: acetone
- Controls: untreated control and vehicle control
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 1 mL acetone/L for test concentration 10 mg/L; 0.1 mL acetone/L for test concentration 1 mg/L; 0.01 mL acetone/L for test concentration 0.1 mg/L; 0.001 mL acetone/L for test concentration 0.01 mg/L; 0.0001 mL acetone/L for test concentration 0.001 mg/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Laboratory, Hino Institute, NBA.
- Age: within 24 h old.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

23 - 23.7 °C

pH:

7.3 - 7.4

Dissolved oxygen:

89-100%

Nominal and measured concentrations:

Nominal: 0 (vehicle control and untreated control), 0.001, 0.01, 0.1, 1, 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 300 mL.
- Fill volume: 100 mL
- No. of organisms per vessel: > 30


EFFECT PARAMETERS MEASURED:
Fleas were examined for general behavior and mortality after 15, 30, 60, 120 and 180 min as well as 24 and 48 h..

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Table 1: Monitoring of test medium conditions.

Concentration	Temper. [°C]	pH	D.O. %
10	23.0 - 23.7	7.3- 7.4	100 - 89
vehicle control	23.0 - 23.5	7.3- 7.4	100 - 90

Table 2: Toxicological findings.

Concentration [mg/L]	No of flea	Duration							Other findings
		15 min	30 min	60 min	120 min	180 min	24 h	48 h	Symptoms	Medium
10	40	0/-*	0/a	0/a	0/a	0/a	17/a	40/-	A, B, C	1
1	33	0/-	0/-	0/-	0/a	0/a	10/a	26/a	A, B, C	1
0.1	39	0/-	0/-	0/-	0/-	0/-	3/a	17/a	A, B, C	1
0.01	33	0/-	0/-	0/-	0/-	0/-	0/-	6/a	A, B	0
0.001	40	0/-	0/-	0/-	0/-	0/-	0/-	0/-	-	0
veh. control	36	0/-	0/-	0/-	0/-	0/-	0/-	0/-	-	0
control	32	0/-	0/-	0/-	0/-	0/-	0/-	0/-	-	-

* Cumulative No. of death/ a: appearance of symptoms, b: disappearance of symptoms

** A: decrease of swimming movements, B: lie at the bottom, C: decrease of antennae movements

*** 0: clear, 1: homogeneous/ unhomogeneous

A decrease of swimming movements and sometimes lying daphnids at bottom was observed at all concentration groups. This appeared from 30 min. after the exposure at 10 mg/L group and from 2 h at 1 mg/L group.

Validity criteria fulfilled:

not applicable

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 Jan - 4 Feb 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: test the acute toxicity of the substance towards freshwater shrimp
- Short description of test conditions: temperature: 21-24 °C, lighting: 18 h, test concentrations: 10, 2, 0.4, 0.08 mg/L and control and vehicle control, vehicle: acetone, exposure duration: 96 h.
- Parameters analysed / observed: Shrimps were examined daily for general behavior and mortality. Water temp., pH, and D.O., in the test medium were monitored.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution containing 10 mg of the test substance in 10 mL of acetone was prepared. Test solutions were made by adding each adequate volumes of stock solution into 1 L of the test water.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Controls: control and vehicl control
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
10 mL acetone/L for test concentration 10 mg/L; 2 mL acetone/L for test concentration 2 mg/L; 0.4 mL acetone/L for test concentration 0.4 mg/L; 0.08 mL acetone/L for test concentration 0.08 mg/L

Test organisms (species):

other aquatic crustacea: Paratya compressa improvisa

Details on test organisms:

TEST ORGANISM
- Common name: Nuka shrimp
- Size: Larva: body length 5-8 mm, Adult: body length 25-30 mm
- Source: National Institute for Environmental Studies
- Age of parental stock (mean and range, SD): Larva: 10-14 days; Adult: 40 - 50 days
- Feeding during test: no

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): only healthy shrimp were used for the final test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21-24°C

Nominal and measured concentrations:

Nominal concentrations: 0 (control, vehicle control), 0.08, 0.4, 2 and 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L glass aquaria
- Fill volume: 1 L
- No. of organisms per vessel: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtrated underground water.
- Intervals of water quality measurement: The water temp., pH, and D.O., in the test medium were monitored. Intervals are not mentioned.

OTHER TEST CONDITIONS
- Photoperiod: 18 h light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Shrimps were examined daily for general behavior and mortality.

VEHICLE CONTROL PERFORMED: yes

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Larva

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Larva

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: adults

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: adults

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: larva

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: larva

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: adults

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: adults

Table 1: Toxicological findings: Larva 10 - 14 d

Concentration [mg/L]	No of Shrimp	Duration in h					Mortality %
		3	24	48	72	96	48 h	96 h
10	10	0/0*	2/5	10/10	0/0	0/0	100	100
2	10	0/0	0/1	0/4	1/4	0/0	0	10
0.4	10	0/0	0/0	0/0	0/0	0/0	0	0
0.08	10	0/0	0/0	0/0	0/0	0/0	0	0
vehicle control	10	0/0	0/0	0/0	0/0	0/0	0	0

* No of dead/No of shrimps showing symptoms

Table 2: Toxicological findings: adult 40 - 50 d

Concentration [mg/L]	No of Shrimp	Duration in h					Mortality %
		3	24	48	72	96	48 h	96 h
10	10	0/1*	0/6	2/6	4/8	7/10	20	70
2	10	0/0	0/0	0/0	0/0	0/0	0	0
0.4	10	0/0	0/0	0/0	0/0	0/0	0	0
0.08	10	0/0	0/0	0/0	0/0	0/0	0	0
Vehic. Control	10	0/0	0/0	0/0	0/0	0/0	0	0

* No of dead/No of shrimps showing symptoms

Concerning general behavior of larvae and adult shrimp:

- Lying on side at the bottom in the test aquaria and a decreased reflex activity by the physical stimulation were observed.

- Throughout the observation period, no abnormal malting activity was seen in both aged stages of shrimps.

From the frequency and degree of these findings, it was considered that larva seem to be more sensitive to the test substance than adult shrimps.

A small age difference to freshwater shrimp was suspected.

Validity criteria fulfilled:

not specified

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines of November 25, 1965

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

dimethyl sulfoxide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Activated carbon-filtered tap water was used as the fresh water source of the test. Tap water was aerated more than 24 h prior to use. Test water was prepared by adding seawater to freshwater at specific gravity of 1.005 (23 °C). The test substance was then weighed 2018 mg (active ingredient 2000 mg). The test substance was added to the 20 mL of special grade dimethyl sulfoxide to 10% (w/v) solution. This solution was added to the test water at the concentrations of 1, 3, 10, 30 and 100 ppm.
- Controls: 10 mL of the above special grade dimethyl sulfoxide was added to the control water.
- Chemical name of vehicle: dimethyl sulfoxide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 1 mL DMSO/L for test concentration 100 mg/L; 0.3 mL DMSO/L for test concentration 30 mg/L; 0.1 mL DMSO/L for test concentration 10 mg/L; 0.03 mL DMSO/L for test concentration 3.0 mg/L; 0.01 mL DMSO/L for test concentration 1.0 mg/L
- Evidence of undissolved material: At the concentrations of 30 and 100 ppm, the test water became muddy with the test substance.

Test organisms (species):

other aquatic mollusc: Corbicula japonica

Details on test organisms:

TEST ORGANISM
- Common name: basket clam
- Source: Miyakoda river, Japan

ACCLIMATION
- Acclimation period: 6 d
- Acclimation conditions (same as test or not): Specific gravity of water in Miyakoda river was 1.005 (23 °C). So, the test water was prepared by adding seawater to well activated carbon-filtered tap water at specific gravity of 1.005 (23 °C). Corbicula were maintained in an acclimatization tank in the testing facility and acclimatized to the test water at the temperature of 23 °C.

Test type:

static

Water media type:

other: Seawater was added to freshwater at specific gravity of 1.005 (23 °C).

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23 ± 2 °C

pH:

6.7 - 6.9

Dissolved oxygen:

7.3 - 8.2 ppm

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0 (vehicle control), 1, 3, 10, 30, 100 ppm

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass test chambers (300 mm width x 200 mm depth x 250 mm height).
- Fill volume: 10 L
- Aeration: yes
- No. of organisms per vessel: 10
- Biomass loading rate: 3 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Seawater was added to freshwater at specific gravity of 1.005 (23 °C).
- Intervals of water quality measurement: Dissolved oxygen and pH levels were measured before the beginning of the test and at 24, 48, 72 and 96 h.

OTHER TEST CONDITIONS
- Photoperiod: 13 h/d

EFFECT PARAMETERS MEASURED:
The number of dead or affected animals in each test chamber were counted at 1, 3, 6, 24, 48, 72 and 96 h after the beginning of the test. Death was determined by the lack of reaction to gentle prodding with a glass rod and/or not opening the shell after 30 minutes in the water with no test substance.

Reference substance (positive control):

yes

Remarks:

PCP-Na (2, 3, 4, 5, 7 and 10 ppm)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

shut the shell

Details on results:

- At concentrations between 1 - 10 ppm neither dead nor affected animal was observed throughout the test period. At concentrations of 30 and 100 ppm after 24 to 96 h, all animals shut the shell and evaded the test substance. No dead animals were detected in these groups.

Results with reference substance (positive control):

- Relevant effect levels: LC50 (24 h) > 10 ppm, LC50 (48 h) = 9.4 ppm, LC50 (72 h) = 8 ppm, LC50 (96 h) = 4 ppm

Corbicula used were 2.1 +/- 0. 1 cm in length, 2.0 +/- 0.1 cm high and 3.2 +/- 0.8 g in weight of the shell at the end of test.

Table 1: Acute toxicity of the substance to Corbicula.

Concentration  [ppm]	Mortality %
	24 h	48 h	72 h	96 h
0	0	0	0	0
1	0	0	0	0
3	0	0	0	0
10	0	0	0	0
30	0	0	0	0
100	0	0	0	0

Validity criteria fulfilled:

not specified

Description of key information

EC50 (48 h) = 0.1 mg/L (nominal, similar to OECD 202, Daphnia magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.1 mg/L

Additional information

Three experimental studies are available testing the acute toxicity of the test substance towards aquatic invertebrates. None of the studies was conducted according to GLP.

One available study was conducted with Daphnia magna as the test organism (1994b). The study procedure is similar to OECD guideline 202. Daphnids were exposed to nominal test item concentrations of 0.001, 0.01, 0.1, 1, and 10 mg/L for 48 h. Acetone was used as a vehicle and therefore a vehicle control as well as an untreated control were run in parallel. The concentration of the solvent in bioassay water was in part above the recommendation of 0.1 mL/L as given by the OECD (2018): 1 mL/L in the highest test concentrations. In the lower test concentrations the concentration of solvent was 0.1 mL/L or below. No mortalities occurred, but effects were observed at the concentration level of 0.01 mg/L and higher. However, no symptoms or mortalities were observed in both controls. Moreover, no oxygen depletion was observed. The study resulted in an EC50 (48 h) of 0.1 mg/L (nominal). Effects were observed within and above the water solubility (2.5 mg/L). Thus, effects above the water solubility could be of physical nature and the result can be considered as a worst case.

Another available study was performed with the clam Corbicula japonica as the test organism (1996d). The clams were exposed for 96 h to the nominal test item concentrations of 1, 3, 10, 30, and 100 mg/L as well as to an untreated and a vehicle control. Dimethyl sulfoxide (DMSO) was used as a vehicle. The concentration of the solvent in bioassay water was in part above the recommendation of 0.1 mL/L as given by the OECD (2018): 1 mL/L and 0.3 mL/L in the two highest test concentrations, respectively. In the lower test concentrations the concentration of solvent was 0.1 mL/L or below. No oxygen depletion was observed. Neither mortalities nor effects were observed at the concentration levels of 1 to 10 mg/L. At the concentration of 30 mg/L and above, all animals shut the shell and evaded the test substance after 96 h. However, no mortalities were determined. No mortalities were observed in the untreated and vehicle control as well. Therefore a LC50 (96 h) > 100 mg/L was derived.

One further study is available investigating the short term toxicity of the substance towards freshwater shrimp (1994c). Larvae (10-14 d) as well as adult shrimps (40-50 d) of the species Paratya compressa improvisa were exposed to nominal test item concentrations of 0 (control, vehicle control), 0.08, 0.4, 2, and 10 mg/L for 96 h. Acetone was used as a vehicle. The concentration of the solvent in bioassay water was above the recommendation of 0.1 mL/L as given by the OECD (2018) in the three highest test concentrations. In the lowest test concentrations the concentration of solvent was below 0.1 mL/L. No oxygen depletion is reported. Mortalities occurred at the two highest test item concentrations for larvae and only at the highest tested concentration for adults. The study showed that larvae are more sensitive to the substance compared to adult shrimps. A 48 and 96 h LC50 of 4.5 and 4 mg/L were derived for larvae while the 48 and 96 h LC50 for adult shrimps was >10 and 6.5 mg/L (nominal), respectively. No mortalities were observed in the untreated and vehicle control as well. Effects were observed within (only larvae) and above (larvae, adults) the water solubility (2.5 mg/L). Thus, effects above the water solubility could be of physical nature and the result can be considered as a worst case.

All results are used in a weight-of-evidence approach. The lowest effect concentration (EC50 (48 h) = 0.1 mg/L) derived by the experimental study with Daphnia magna was used as key value for this trophic level.