N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical determination
Analytical evaluation of the test concentration of the test item and the control were carried out via LC-MS/MS from freshly prepared media after 0 and 72 h and from corresponding aged test media after 24 and 96 h.

Vehicle:

no

Details on test solutions:

Test item

Preparation of the saturated solution
The saturated solution (100 mg/L test item was weighed out) was prepared with dilution water three days prior to the start of the exposure (-72 h).

Treatment
The stock solution was stirred for 72 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). After check for Tyndall effect the filtrate was used for testing. This procedure was repeated for daily renewal of the test media.

Control
7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 2.31 cm
- Weight at study end (mean and range, SD): Average body weight: 0.171 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 67 mg/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: Glass aquaria of 3 L were used (dimensions 11.5/13/20 cm) and covered with glass plates.
- Aeration: The test vessels were gently aerated during exposure.
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control and pH-control (replicates): 1
- Biomass loading rate: 0.599 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5

- Culture medium different from test medium: no
- Intervals of water quality measurement:pH-value, temperature and oxygen saturation were measured in all test vessels every 24 hours.
Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded continuously once per hour with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 15 h light / 9 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 7 - 750 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 2, 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 18.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: not applicable

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 18.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: not applicable

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 18.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: not applicable

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

18.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 18.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation): The test solution was clear throughout the period of exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation
The LC0 after 96 h was determined directly from the raw data of the limit concentration. LC50-values do not have to be calculated in a limit test.

Software
All data were computer-generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Sublethal observations / clinical signs:

Biological Data

 

All effect levels are given based on the geometric mean measured concentration of the test item in the saturated solution. The LC₀ corresponds to the geometric mean measured concentration of  18.1 µg/, the LC₅₀ and the LC₁₀₀ to

> 18.1 µg/L (geometric mean measured concentration).

 

Observations of sublethal effects in the Test Vessels

Nominal test item concentration

Effect *)

Number of fish effected at observation time [hours]

2

24

48

72

96

Saturated solution

(1)

7/7

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

7/7

              *) The numbers in brackets correspond to the following observations: 

            (1)      = Normal behaviour                          

 

 

 

Cumulative Mortality [%] in the Test Vessels

Nominal test item concentration	Cumulative mortality at observation time [hours]
	2	24	48	72	96
Saturated solution	0	0	0	0	0
Control	0	0	0	0	0

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, the saturated solution of the test item was found to have no effect on the vitality of zebrafish after 96 hours. No effects were found when tested with the saturated solution, which is below the nominal loading level of the saturated solution of 100 mg/L and corresponds to a geometric mean measured concentration of 18.1 µg/L.

Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to the OECD Guideline for Testing of Chemicals No. 203 (1992) and the EU Council Regulation (EC) No. 440/2008/Method C.1 (2008) from 2016-01-22 to 2016-01-29 with the definitive exposure phase from 2016-01-25 to 2016-01-29 at the test facility.

 

The test item is a yellowish, liquid, but highly viscous. A saturated solution was prepared with a nominal loading rate of 100 mg test item/L. The stock solution was stirred for 72 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45µm, RC,Macherey-Nagel). This procedure was repeated for daily renewal of the test media.The filtrate, i. e. the saturated solution, was used in the test.

 

An acute toxicity test under semi-static conditions with daily renewal of the test media was performed as a limit test using the saturated solution. The test had a duration of 96 hours. Seven zebrafish (test system) were exposed to the saturated solution and the control, respectively. The water quality parameters (pH-value, temperature and oxygen saturation) measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

 

Analytical evaluation of the saturated solution of the test item and the control were carried out from freshly prepared media after 0 and 72 hours and from corresponding aged test media after 24 and 96 hours by LC-MS/MS. In the beginning of the first interval, a test item concentration of 170 µg/L was observed. In the end of the first interval, a concentration of 97.8 µg/L was measured in the saturated solution. In the beginning of the second interval, a test item concentration of 2.18 µg/L was observed. In the end of the second interval, a concentration of 2.95 µg/L was measured in the saturated solution. Although no Tyndall effect was observed in any of the freshly prepared solutions it is assumed that the measured concentrations are above the limit of the water solubility. The experimentally derived water solubility is < 0.36 mg/L, the calculated water solubility is 7.538x10^-5mg/L.

 

The measured concentrations indicated that a reproducible preparation of saturated solutions was hardly possible which led to fluctuating concentrations. In the range finding test, more uniform values were obtained from fresh and old media. As no effects on the fish had been observed neither in the range finding test nor in the definitive test, the study is considered valid.

All effect values were based on the geometric mean measured concentration of the test item.

 

In this study, no effects of the test item were observed on the test system after 96 hours. No effects were found when tested with the saturated solution, which is below the nominal loading level of 100 mg/L and corresponds to a geometric mean measured concentration of 18.1 µg/L.

 

NOEC, LOEC, EC-values and 95 % Confidence Intervals of Hostavin 3206 Liq, Impoverishedof xylene (0 - 96 hours)

based on geometric mean measured test item concentrations [µg/L]

 

	Mortality and Sublethal Effects
	Geometric mean measured concentrations [µg/L]
NOEC	18.1
LOEC	> 18.1
LC0	≥18.1
LC50	>18.1
LC100	>18.1

Description of key information

In this study, the saturated solution of the test item was found to have no effect on the vitality of zebrafish after 96 hours. No effects were found when tested with the saturated solution, which is below the nominal loading level of the saturated solution of 100 mg/L and corresponds to a geometric mean measured concentration of 18.1 µg/L.