Ethyl ethyl(phenyl)carbamate

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:

August 22, 2017 - August 24, 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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-25ºC)
- Stability under test conditions:Yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 6.25, 12.5, 25, 50 and 100% solutions (nominal concentration).
- Sampling method: Test solutions and control sample were determined at the beginning and at the end of the test. The samples were taken from the test solutions, one from control solution, properly diluted and analysed by an HPLC-UV method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 100.0 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (ISO Medium) using ultrasonic bath (approximately 20 minutes). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the treatments).
- Controls: The dilution water (ISO medium) was used without addition of the test item

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Justification for species other than prescribed by test guideline: Daphnia magna is the standard species of the acute immobilisation test.
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
- Age of parental stock (mean and range, SD): 24 h old at the beginning of the test.
- Feeding during test: No.

ACCLIMATION
- Acclimation period: There was no acclimatization because the water used was similar to the culture water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

247 mg/L (as CACO3)

Test temperature:

20.6-21.2ºC
It was measured at the start of the test and in 24-hour intervals thereafter in each test vessel.

pH:

6.80 – 7.51
pH not varied by more than 1.5 units.

Dissolved oxygen:

8.0 – 8.6 mg/L.
It was measured in each test vessel at the start and at the end of the test.

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50 and 100% saturated solution.
Measured: 6.54, 12.69, 25.97, 54.81 and 100.17 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal in most cases, the biological results are based on the nominal test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel:Glass beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume. glass beaker, 5 mL.
- Aeration: no.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202).
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 16-hour light and 8-hour dark cycle
- Light intensity: not specified.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test.Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.
- Test concentrations: 0 (control), 0.1, 1, 10, 100 % solutions.
- Results used to determine the conditions for the definitive study: Because significant immobility was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Batch Number: A0345704), tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

84.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % conf. limits: 78.84 – 90.73 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

95.17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % conf. limits: 87.00 – 104.09 mg/L

Details on results:

- Behavioural abnormalities: No
- Mortality of control: No
- Other adverse effects control: No.
- Abnormal responses: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes
- Relevant effect levels: 24h EC50: 0.64 mg/l (95% confidence limits: 0.59 - 0.68 mg/L)
The date of the last study (Study Code: 17/058-023DA) with reference item Potassium dichromate (batch no.: A0345704) is 8- 9 March 2017

Reported statistics and error estimates:

The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by SPSS PC+ software. The 48h EC100 values of the test item were determined directly from the raw data.
For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett's Test using TOXSTAT software.

Result from the preliminary test:

Nominal concentrations [mg/L]	Untreated control	0.1	1	10	100
Number of treated / immobilised animals	10 / 0	10 / 0	10 / 0	10 / 0	10 / 8

Table No.1: Number and percentage of immobilised animals

Nominal Concentration	Number of treated animals	Immobilised animals
		24 hours		48 hours
mg/L		number	percent	number	percent
Control	20	0	0	0	0
6.25	20	0	0	0	0
12.5	20	0	0	0	0
25.0	20	0	0	0	0
50.0	20	1	5	1	5
100.0	20	11*	55	14*	70

*: statistically significantly different compared to the control values (Dunnett’s Test;alpha= 0.05)

Although 5 % immobilization could be observed at the test item group of 50.0 mg/L, this difference was notstatistically significant compared to the control and arenegligible due to that the accepted control immobilization is 10 %.

Table No.2: Immobilisation of the test animals

Nominal concentration (mg/L)	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
6.25	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
12.5	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
25.0	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
50.0	1	5	0	0
	2	5	0	0
	3	5	1	1
	4	5	0	0
100.0	1	5	4	4
	2	5	3	3
	3	5	2	3
	4	5	2	4

Table No.3: pH measured in the test vessels

Nominal concentration (mg/L)	Replicate	Measuring time
		24 h	48 h
Control	1	6.80	6.92
	2	6.80	6.93
	3	6.80	6.92
	4	6.80	6.94
6.25	1	6.84	6.92
	2	6.84	6.92
	3	6.84	6.94
	4	6.84	6.93
12.5	1	6.88	6.96
	2	6.88	6.96
	3	6.88	6.97
	4	6.88	6.98
25.0	1	7.06	7.11
	2	7.06	7.12
	3	7.06	7.11
	4	7.06	7.11
50.0	1	7.33	7.40
	2	7.33	7.39
	3	7.33	7.39
	4	7.33	7.39
100.0	1	7.48	7.50
	2	7.48	7.51
	3	7.48	7.51
	4	7.48	7.51

Table No.4:Oxygen concentrationmeasured in the test vessels (mg/L)

Nominal concentration (mg/L)	Replicate	Measuring time
		24 h	48 h
Control	1	8.6	8.5
	2	8.6	8.5
	3	8.6	8.5
	4	8.6	8.5
6.25	1	8.5	8.5
	2	8.5	8.4
	3	8.5	8.4
	4	8.5	8.4
12.5	1	8.5	8.4
	2	8.5	8.4
	3	8.5	8.4
	4	8.5	8.4
25.0	1	8.5	8.4
	2	8.5	8.4
	3	8.5	8.4
	4	8.5	8.4
50.0	1	8.5	8.4
	2	8.5	8.4
	3	8.5	8.4
	4	8.5	8.4
100.0	1	8.1	8.0
	2	8.1	8.0
	3	8.1	8.0
	4	8.1	8.0

Table No.5: Temperature measured in the test vessels (°C)

Nominal concentration (mg/L)	Replicate	Measuring time
		0 h	24 h	48 h
Control	1	20.1	20.6	20.8
	2	20.1	20.6	20.8
	3	20.1	20.6	20.8
	4	20.1	20.6	20.8
6.25	1	21.1	20.6	20.8
	2	21.1	20.6	20.8
	3	21.1	20.6	20.8
	4	21.1	20.6	20.8
12.5	1	21.1	20.6	20.8
	2	21.1	20.6	20.8
	3	21.1	20.6	20.8
	4	21.1	20.6	20.8
25.0	1	21.1	20.6	20.8
	2	21.1	20.6	20.9
	3	21.1	20.6	20.9
	4	21.2	20.6	20.8
50.0	1	21.1	20.6	20.9
	2	21.1	20.6	20.9
	3	21.2	20.6	20.9
	4	21.2	20.6	20.9
100.0	1	21.2	20.6	20.9
	2	21.2	20.6	20.9
	3	21.2	20.6	20.9
	4	21.2	20.6	20.9

Validity criteria fulfilled:

yes

Remarks:

There was no immobilization in the control group and the dissolved oxygen concentration at the end of the test is control and test vessels was more than 3 mg/L.

Conclusions:

The EC50 (48h) of the test item tested on Daphnia magna was 84.58 mg/L, NOEC was detected as 50.0 mg/L and LOEC value was 100 mg/L, based on mobility.

Executive summary:

In accordance with OECD TG 202 and GLP study, the acute immobilisation test on Daphnia magna was performed. Test was performed in static condition, during 48 hours on 20 Daphnia divided into 4 groups of five animals.Control group was used also. The choice of the test concentration was made on the basis of the results of the preliminary range-finding test, where significant immobility was observed at the examined concentration level and therefore five test concentrations in a geometric series (factor 2.0) and one control were used in the main tests under static conditions. Control and test concentrations (nominal and measured) were analytically determined (validated method) at the beginning and at the end of the experiment and did not deviate more than 20% and because of that, the biological results are based on the nominal test item concentrations.The nominal concentrations were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L and measured concentrations: 6.54, 12.69, 25.97, 54.81 and 100.17 mg/L. Positive controls (potassium dichromate) are performed at least twice a year to ensure the quality of the results. At the nominal concentration of 50 mg/L was observed 5% of immobilisation (no significant different from control group) and at the nominal concentration of 100mg/L 55% of immobilisation during 24 hours and 70% during 48 hours (significant difference from control group). All validity criteria were met. The EC50 (48 h) of the test item in Daphnia magna was 84.58 mg/L ,LOEC value was 100.0 mg/L and the NOEC was 50 mg/L. Results were based on the mobility.

Description of key information

Short-term toxicity to aquatic invertebrates (daphnia)

Key study: OECD 202, GLP study. The EC50 (48h) of the test item tested on Daphnia magna was 84.58 mg/L, NOEC was detected as 50.0 mg/L and LOEC value was 100 mg/L, based on mobility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

84.58 mg/L

Additional information

Short-term toxicity to aquatic invertebrates (daphnia)

Key study: In accordance with OECD TG 202 and GLP study, the acute immobilisation test on Daphnia magna was performed. Test was performed in static condition, during 48 hours on 20 Daphnia divided into 4 groups of five animals and one control group.The choice of the test concentration was made on the basis of the results of the preliminary range-finding test. Control and test concentrations(nominal and measured) were analytically determined at the beginning and at the end of the experiment and did not deviate more than 20% and because of that,the biological results are based on the nominal test item concentrations.The nominal concentrations were:6.25, 12.5, 25.0, 50.0 and 100.0 mg/L and measured concentrations:6.54, 12.69, 25.97, 54.81 and 100.17 mg/L. Positive controls (potassium dichromate) are performed at least twice a year to ensure the quality of the results. All validity criteria were met. At the nominal concentration of 50 mg/L was observed 5% of immobilisation (no significant different from control group) and at the nominal concentration of 100 mg/L 55% of immobilisation during 24 hours and 70% during 48 hours (significant difference from control group). The EC50 (48 h) of the test item in Daphnia magna was 84.58 mg/L ,LOEC value was 100.0 mg/L and the NOEC was 50 mg/L. Results were based on the mobility.