4-(octadecylamino)-4-oxoisocrotonic acid

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:

19 Nov - 27 Nov 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

For further information see section "Any other information on material and methods incl tables".

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Mainz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and test item concentration
- Sampling method: Concentration was measured at the beginning and at the end of the test and on at medium renewal after 24 h in the new and the old solutions.
- Sample storage conditions before analysis: Samples were frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: A saturated solution was prepared for the test. This was done by weighing the nominal load of 100 mg/L, adding the corresponding amount of tap water and shaking vigorously for 24 h. The resulting solution was filtrated through 0.45 μm cellulose ester filters. On each day of the experiment, the necessary solutions were freshly prepared.
- Controls: Yes, medium only

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Source: Dehner Garten Center, Neustadt, Germany (date of arrival 05 Jul 2018)
- Length at study initiation: 2 ± 1 cm
- Method of breeding: Vessels: polyethylene aquaria; Medium: tap water; Feeding: two times a day with warmwater fishfood, totalling about 1-2% of body weight per day; Photoperiod 12/12 h; Temperature 23 ± 2 °C

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: same as test conditions
- Type and amount of food during acclimation: warmwater fishfood , totalling about 1 - 2% of body weight per day
- Feeding frequency during acclimation: three times a day
- Health during acclimation: mortality was observed after day 7 (3 of 18 animals were found dead -corresponding to 16.7%)

FEEDING DURING TEST: no feeding during the test; 24 h before the start of the test, the test fish were no longer fed.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

0.88 (medium) mmol/L

Test temperature:

23.1 – 24.0 °C (deviation within on test 1 °C)

pH:

7.8 - 7.9 (Control)
7.7 - 7.9 (100 mg/L)

Dissolved oxygen:

8.3 - 9.1 mg/L (Control)
8.2 - 9.1 mg/L (100 mg/L)

Conductivity:

210 µS/cm at 25 °C (tap water)

Nominal and measured concentrations:

nominal: Control and 100 mg/L
The measured concentrations are listed in section "Any other information on results incl tables" in table 4-5

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: Open
- Material, size, headspace, fill volume: glass, 10 L, headspace: 3 L, fill volume: 7 L
- Aeration: Accomplished with glass tubes
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates):1
- Biomass loading rate: 1 fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap/Drinking water see analysis in table 1 in section "Any other information on material and methods incl tabels"
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 12/12 hours using neon tubes

EFFECT PARAMETERS MEASURED: Mortality was recorded every 24 h

TEST CONCENTRATIONS
- Range finding study : No pre-test was performed

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 32.23 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 32.23 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: No mortality was observed
- Other biological observations: The toxicity against Danio rerio was tested using a semi-static test design. The study was performed as a limit test using the concentration 100 mg/L (nominal). In the treatment and the control, no mortality was observed.

Sublethal observations / clinical signs:

Biological Results:

Table 1: pH-Values

Nominal Concentration in mg/L	pH at t in h (old/new test solution)
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
Blank control	7.8	7.8	7.8	7.8	7.9	7.8	7.8	7.8
100	7.7	7.8	7.8	7.9	7.9	7.8	7.8	7.8

 Table 2: O2 Values

Nominal Concentration in mg/L	O2 in mg/L at t in h (old/new test solution)
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
Blank control	9.1	8.7	8.6	8.5	9.0	8.7	9.0	8.3
100	8.9	8.6	8.6	8.4	9.1	8.6	9.0	8.2

 Tabel 3: Temperature

Nominal Concentration in mg/L	Temperature in °C (old/new test solution)
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
Blank control	24.0	23.3	23.4	23.1	23.3	23.2	23.1	23.2
100	23.3	23.1	23.3	23.2	23.3	23.4	23.2	23.3

 Table 4: Measured Concentrations

Nominal Conc.  Test Item		Measured Conc.  t = 0 h		Measured Conc.  t = 24 h old solution		Measured Conc.  t = 24 h new solution		Measured Conc.  t = 48 h old solution		Measured Conc.  t = 48 h new solution
mg/L		µg/L		µg/L		µg/L		µg/L		µg/L
Blank control		n. m.		--		--		--		--
100		n. m.		22.19		103.15		80.94		30.04

n. m. = not measured because of a technical defect of the originally planned LC-MS-MS

  Table 5: Measured Concentrations, continuance

Nominal Conc.  Test Item	Measured Conc.  t = 72 h old solution	Measured Conc.  t = 72 h new solution	Measured Conc.  t = 96 h old solution	Geometric Mean	Measured Conc. additional new solution
mg/L	µg/L	µg/L	µg/L	µg/L	µg/L
Blank control	--	--	n. d.	--	--
100	12.26	16.44	0.64*	32.23	94.83

*Implausible low measured concentration, an error in sampling was suspected, value excluded from evaluation.

n. d. = not detectable

Analytical Results:

At each measurement the recovery of the QC samples was taken into account because the recovery was not in the range of 97% - 103% of the nominal value. At the beginning the measurement was not yet stable. This can be seen from the area values of the QC sample. Deviating from the study plan, the recovery of QC samples was taken into account although the recovery was not in the range of 97% - 103% of the nominal value. This can be stated as uncritical because it was clear to see that the measurement stabilized later and an additional later measurement was therefore not necessary.

The measured concentration in the freshly prepared solutions was between 16.44 μg/L and 103.15 μg/L and in the old solutions between 0.64 μg/L and 80.94 μg/L. A sampling error is assumed as reason of the implausible low measured concentration of 0.64 μg/L at the end of the test. This value was excluded from evaluation. Reason for the scattering measured concentrations in the test solutions might be the presence of the fish and their excrements.

Table 6: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	No mortality was observed	Yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	92%	Yes

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

LC50 (96h) >32.23 µg/L (Danio rerio, OECD 203, measured geometric mean)

Additional information

One experimental study is available investigating the toxicity of 4-(octadecylamino)-4-oxoisocrotonic acid (CAS 3077-27-8) to fish. The study was conducted according to OECD 203 (GLP) using the zebrafish Danio rerio as test species (LAUS GmbH, 2018c) under semi-static conditions with daily medium renewal. A nominal test item concentration of 100 mg/L was tested in a limit test. The test solution was prepared by adding appropriate amounts of test item to test medium, followed by intensive stirring and filtration. During the study the actual concentrations in the test medium was measured by HPLC/MS. The measured concentration in the freshly prepared solutions was between 16.44 μg/L and 103.15 μg/L and in the aged test solutions between 0.64 μg/L and 80.94 μg/L, respectively. Therefore the results are given based on the geometric mean of the measured concentrations. Hence the LC50 (96 h) was determined to be > 100 mg/L (nominal) and > 32.23 μg/L (geom. mean measured concentration). The measured water solubility for the test item was determined to be 5.9 mg/L at 25 °C, pH 5. The discrepancy between the low recovery in the test solutions and the water solubility may have been caused by the different test media, pH-values and temperature.