Dimethyl (p-methoxybenzylidene)malonate

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:

2016-2017

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

Qualifier:

according to guideline

Guideline:

other: OECD 126 “Short Guidance on the threshold approach for acute fish toxicity"

Version / remarks:

2010

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical determination
Analytical evaluation of the test concentration of the test substance was carried out after 0 h and 72 h and corresponding 24 h aged test media after 24 and 96 h via UPLC on a reversed phase column using external standard calibration.

Vehicle:

no

Details on test solutions:

Test concentration
2.00 mg/L was tested as a threshold concentration in a limit test.
The selection of the test concentration was based on the derivation of a threshold concentration (TC) from the results of an alga toxicity test (EYC50 (0-72 h) = 1.11 (0.816 – 1.47) mg/L), based on nominal concentrations of the test item (SCHEERBAUM, 2016) and of an acute daphnia toxicity test (EC50 (0-48 h) = 1.68 mg/L) provided by the sponsor.


Preparation of the test medium
An appropriate amount of the test item was weighed and transferred to an aquaria filled with an appropriate volume of the dilution water. The test media was mixed with an ultraturrax (1 min with 17000 rpm, afterwards 45 min with 8500 rpm). This procedure was repeated for each renewal.


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: 2.74 cm
- Weight at study end (mean and range, SD): Average body weight: 0.284 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: 71 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 plates
- Material, size, headspace, fill volume: Glass aquaria of 13 L were used, filled with 10 L of dilution water (dimensions: 25.5/18/28 cm, depth of water: 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: 0.199 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 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 10 - 100 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

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 1.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 1.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 1.23 mg/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

The environmental conditions (pH-value, temperature, O2saturation) were determined to be within the acceptable limits. The continuous measuring of water temperature was in the valid range of 23 ± 2 °C. The measured light intensity was in the given range of 10 – 100 Lux(mean value 82 Lux).

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 test item concentration.

The LC₀after 96 hours was ≥1.23 mg/L.

 

Observations in the Test Vessels

Geometric mean measured test item concentration [mg/L]	Effect *	Number of fish effected at observation time [hours]
		2	24	48	72	96
1.23	(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

Geometric mean measured concentration [mg/L]	Cumulative mortality at observation time [hours]
	2	24	48	72	96
1.23	0	0	0	0	0
Control	0	0	0	0	0

 

 

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the geometric mean measured test item concentration. The geometric mean measured test item concentration of 1.23 mg/L was found to have no effect on the vitality of zebrafish.
In conclusion, the LC0 corresponds to the geometric mean measured test item concentration ≥ 1.23 mg/L. The LC50 and the LC100 correspond to the geometric mean measured test item concentration > 1.23 mg/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) from 2016-04-04 to 2017-01-18 at the test facility with the definitive exposure phase from 2016-12-19 to 2016-12-23.

 

A nominal test item concentration of 2.00 mg/L (corresponding to a geometric mean measured test item concentration of 1.23 mg/L) was tested as a threshold concentration in a limit test under semi-static conditions.The test had a duration of 96 hours. 7 test organisms were exposed to the limit concentration 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.

 

The concentration of the test item in the limit concentration and in the control was analytically verified by UPLC analysis in the freshly prepared media after 0 and 72 hours and in the corresponding 24 hours aged media after 24 and 96 hours. The measured concentrations of the test item in freshly prepared media after 0 and 72 hours of exposure were 58 and 74 % of the nominal values. Corresponding 24 h aged test media showed measured concentrations of 50 to 66 % of the nominal values. Therefore all effect levels given were based on the geometric mean measured test item concentration.

 

No effect on the test organism was observed at the geometric mean measured test item concentration of 1.23 mg/L after 96 hours. Therefore, the LC0 corresponds to ≥ 1.23 mg/L. The NOEC corresponds to ≥1.23 mg/L and the LOEC corresponds to > 1.23 mg/L.

 

At the geometric mean measured test item concentration of 1.23 mg/L of the test item, no effects on vitality were observed in Danio rerio. The LC0 corresponds to a geometric mean measured test item concentration of ≥ 1.23 mg/L.

Description of key information

96-LC50 (Danio rerio) > 1.23 mg/L (geom. mean measured, OECD 203, 2017)

Note: A key value was inserted in order to enable the application of the PNEC calculator. The inserted concentration is higher than the effect concentration used as basis for the PNEC derivation (i.e. 48h-EC50 (Daphnia magna, immobilisation) = 1.68 mg/L) and therefore does not bias the PNEC calculation performed by the PNEC calculator. The nominal test concentration used in the fish acute toxicity test was taken as surrogate (2 mg/L), because the measured concentration -- with no mortality observed -- was 1.23 mg/L and thus lower than the actual effect concentration used as basis for the PNEC calculation (i.e. 1.68 mg/L).  

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2 mg/L

Additional information

In a reliable key study, acute toxicity of the submission substance to fish (zebrafish) was determined according to the OECD 203 (1992) and in compliance with GLP (RL1). A nominal test concentration of 2.00 mg/L was tested as a threshold concentration in a limit test under semi-static conditions during 96 hours. A control was run in parallel. The test concentration and control were analytically verified by UPLC analysis. Effect levels given were based on the geometric mean measured test item concentration. No mortality or sublethal effects were observed in neither treatment group nor control during exposure. The derived effect concentrations were therefore: 96h-LC50 > 1.23 mg/L, 96h-NOEC > 1.23 mg/L. The study was considered reliable and adequate for the environmental hazard assessment for aquatic organisms.