Ethyl diphenylcarbamate

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 29, 2017 - August 31, 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)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 6.25, 12.5, 25, 50 and 100% saturated solutions. The corresponding measured geometric mean test item concentrations were: 1.75, 3.42, 7.02, 14.94 and 29.70 mg/L.
- Sampling method: Test solutions and control sample were determined at the beginning and at the end of the test. The samples were properly diluted and analysed by an HPLC-UV method.
- Sample storage conditions before analysis: Saturated solution and about 30 fold diluted saturated solution were stored at test conditions. At the end of the storage period formulations were analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: method according to the Guidance Document on Aquatic Toxicity Testing of Difficult substances and Mixtures, OECD No. 23.
A saturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30ºC and then equilibrated for about 24 hours at approximately 20ºC.
- Controls: The dilution water (ISO medium) was used without addition of the test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Yes, the non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solution.

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:

20.4 - 21.1ºC

pH:

7.73 - 7.86

Dissolved oxygen:

7.9 - 8.8 mg/L

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50 and 100% saturated solution.
Measured: 1.75, 3.42, 7.02, 14.94 and 29.70 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume. glass beaker, 5 mL fill volume
- 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 of 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.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0.1, 1, 10, 100 % saturated solutions.
- Results used to determine the conditions for the definitive study: Because significant immobility was observed at the highest 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 is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

16.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

29.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

7.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

14.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

26.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No abnormal behaviour, or appearance of test animals was detected.
- Mortality of control: 0/20
- 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
The date of the last study (Study Code: 17/058-023DA) with reference item Potassium dichromate (batch no.: A0345704) is 8- 9 March 2017
- Relevant effect levels: 24h EC50: 0.64 mg/l (95% confidence limits: 0.59 - 0.68 mg/L)

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.

Table 4:Number and percentage of immobilised animals

Concentration		Number of treated animals	Immobilised animals
Nominal	Measured		24 hours		48 hours
(% sat. sol.)	(mg/L)		number	percent	number	percent
Control	0.0	20	0	0	0	0
6.25	1.75	20	0	0	0	0
12.5	3.42	20	0	0	0	0
25.0	7.02	20	0	0	0	0
50.0	14.94	20	0	0	6	30*
100.0	29.70	20	14	70*	20	100*

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

Table 5:Temperature measured in the test vessels (°C)

Concentration		Replicate	Measuring time
Nominal (% sat. sol.)	Measured (mg/L)		0 h	24 h	48 h
Control	0.0	1	21.0	20.4	20.7
		2	21.0	20.4	20.7
		3	21.0	20.4	20.7
		4	21.0	20.4	20.7
6.25	1.75	1	21.0	20.4	20.7
		2	21.0	20.4	20.7
		3	21.0	20.4	20.7
		4	21.0	20.4	20.7
12.5	3.42	1	21.0	20.4	20.7
		2	21.0	20.4	20.7
		3	21.0	20.4	20.7
		4	21.0	20.4	20.7
25.0	7.02	1	21.0	20.4	20.7
		2	21.0	20.4	20.7
		3	21.0	20.4	20.7
		4	21.0	20.4	20.8
50.0	14.94	1	21.0	20.4	20.7
		2	21.0	20.4	20.8
		3	21.0	20.4	20.8
		4	21.0	20.4	20.8
100.0	29.70	1	21.0	20.4	20.8
		2	21.0	20.4	20.8
		3	21.0	20.4	20.8
		4	21.1	20.4	20.8

 

Table 6:Oxygen concentrationmeasured in the test vessels (mg/L)

Concentration		Replicate	Measuring time
Nominal (% sat. sol.)	Measured (mg/L)		24 h	48 h
Control	0.0	1	8.3	8.5
		2	8.3	8.5
		3	8.3	8.5
		4	8.3	8.5
6.25	1.75	1	8.3	8.5
		2	8.3	8.5
		3	8.3	8.4
		4	8.3	8.5
12.5	3.42	1	8.3	8.5
		2	8.3	8.5
		3	8.3	8.8
		4	8.3	8.6
25.0	7.02	1	8.3	8.6
		2	8.3	8.6
		3	8.3	8.5
		4	8.3	8.5
50.0	14.94	1	8.3	8.5
		2	8.3	8.5
		3	8.3	8.6
		4	8.3	8.6
100.0	29.70	1	7.9	8.3
		2	7.9	8.3
		3	7.9	8.4
		4	7.9	8.3

 

Table 7:pHmeasured in the test vessels

Concentration		Replicate	Measuring time
Nominal (% sat. sol.)	Measured (mg/L)		24 h	48 h
Control	0.0	1	7.73	7.81
		2	7.73	7.82
		3	7.73	7.82
		4	7.73	7.83
6.25	1.75	1	7.78	7.85
		2	7.78	7.86
		3	7.78	7.86
		4	7.78	7.86
12.5	3.42	1	7.79	7.83
		2	7.79	7.82
		3	7.79	7.83
		4	7.79	7.84
25.0	7.02	1	7.78	7.83
		2	7.78	7.84
		3	7.78	7.82
		4	7.78	7.82
50.0	14.94	1	7.81	7.83
		2	7.81	7.84
		3	7.81	7.81
		4	7.81	7.82
100.0	29.70	1	7.83	7.84
		2	7.83	7.84
		3	7.83	7.85
		4	7.83	7.85

 

Table 8:Immobilisation of the test animals

Concentration		Replicate	Number of treated animals	Number of immobilised animals
Nominal (% sat. sol.)	Measured (mg/L)			24 h	48 h
Control	0.0	1	5	0	0
		2	5	0	0
		3	5	0	0
		4	5	0	0
6.25	1.75	1	5	0	0
		2	5	0	0
		3	5	0	0
		4	5	0	0
12.5	3.42	1	5	0	0
		2	5	0	0
		3	5	0	0
		4	5	0	0
25.0	7.02	1	5	0	0
		2	5	0	0
		3	5	0	0
		4	5	0	0
50.0	14.94	1	5	0	1
		2	5	0	2
		3	5	0	2
		4	5	0	1
100.0	29.70	1	5	4	5
		2	5	3	5
		3	5	4	5
		4	5	3	5

 

 

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 48h-EC50 of the test item in Daphnia magna was 16.75 mg/L (measured) and the NOEC was 7.02 mg/L (measured), both based on mobility.

Executive summary:

An acute immobilisation test on Daphnia magna was performed in accordance with OECD Guideline 202 and EU method C.2, following GLP. Due to the low solubility of the test item in water, the test solution was prepared using a saturated method according to OECD No. 23 (Guidance document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the results obtained from a preliminary range-finding test, where significant immobility was observed at the highest examined concentration level, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions. Control and test concentrations were analytically determined (validated method) at the beginning and at the end of the experiment and did not deviate more than 20%. The following concentrations were tested: 6.25, 12.5, 25, 50 and 100% saturated solution. The corresponding measured geometric mean test item concentration were: 1.75, 3.42, 7.02, 14.94 and 29.70 mg/L. Twenty animals, divided into four groups (glass beaker) of five animals each were exposed to the different test item concentrations or the control for 48 h. Positive controls (potassium dichromate) are performed at least twice a year to ensure the quality of the results. All validity criteria were met. Significant immobilisation was observed at 48 h at the dose level of 14.94 mg/L (measured) and at 24 h and 48 h at the dose level of 29.70 mg/L (measured). The 48h-EC50 of the test item in Daphnia magna was 16.75 mg/L (measured) and the NOEC was 7.02 mg/L (measured), both based on mobility.

Description of key information

Key study: OECD 202 and EU method C.2. GLP study. The 48h-EC50 of the test item in Daphnia magna was 16.75 mg/L (measured) and the NOEC was 7.02 mg/L (measured), both based on mobility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

16.75 mg/L

Additional information

Key study: An acute immobilisation test on Daphnia magna was performed in accordance with OECD Guideline 202 and EU method C.2, following GLP. Based on the results obtained from a preliminary range-finding test, where significant immobility was observed at the highest examined concentration level, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions. Control and test concentrations were analytically determined (validated method) at the beginning and at the end of the experiment and did not deviate more than 20%. The following concentrations were tested: 6.25, 12.5, 25, 50 and 100% saturated solution. The corresponding measured geometric mean test item concentration were: 1.75, 3.42, 7.02, 14.94 and 29.70 mg/L. Twenty animals, divided into four groups (glass beaker) of five animals each were exposed to the different test item concentrations or the control for 48 h. Positive controls (potassium dichromate) are performed at least twice a year to ensure the quality of the results. All validity criteria were met. The 48h-EC50 of the test item in Daphnia magna was 16.75 mg/L (measured) and the NOEC was 7.02 mg/L (measured), both based on mobility.