2-methyl-3-(p-tolyl)propionaldehyde

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 27, 2021 to September 20, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-3-(p-tolyl)propionaldehyde

EC Number:

255-410-4

EC Name:

2-methyl-3-(p-tolyl)propionaldehyde

Cas Number:

41496-43-9

Molecular formula:

C11H14O

IUPAC Name:

2-methyl-3-(4-methylphenyl)propanal

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

by GC-MS

Details on sampling:

Samples of selected stock, application and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.

All fresh and aged samples (24 hours old) were sampled and analysed (Fresh : 0 hrs, 24 hrs; Aged : 24 hrs and 48 hrs).

Solvent control and selected test solutions were sampled in triplicate. One extract sample and one water sample of each test solution were kept separately as a reserve. The volume of each sample was recorded.

Samples of a defined volume (50 mL of water samples if not advised otherwise) were taken from the designated test solution, and transferred to a glass tube.
Note: Due to the potential volatility of the test item the test solutions were not combined prior to sampling, the test solution was directly transferred into the extraction and sample vessels (pipetting using a glass pipette, 50 mL). The extraction vessels were immediately processed
as described below, and the sampling bottles were immediately tightly closed by a screw-cap with PTFE lining, and stored in the freezer (≤ -18 °C).

Extraction procedure:
Each sample was stabilised by adding 1-N hydrochloric acid (1% of sample volume). To each sample, 5 mL of the extraction solvent (toluene) was added. The two phases were mixed vigorously, for 30 seconds on a vortex, and manually shaken afterwards. After phase separation (allow to stand for several minutes), 1.0 to 2.0 mL of the upper (organic) phase was transferred (using a glass pipette) into a glass vial of appropriate volume. Afterwards the vials containing the solvent extracts (toluene) were tightly closed, and stored in the freezer (≤ - 18 °C). The water phase after extraction was disposed.

Samples from the stock solution and dilution solutions (S1-S5) in acetone (0.5 mL if not advised otherwise) were stored frozen as reserve samples.

Further samples from the stabilised test solutions without extraction solvent were stored frozen as reserve samples.

After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of ≤ -18 °C if not advised otherwise. A record was kept for each sample.

Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis under the required storage conditions.

Analysis was performed according to an analytical method validated by the test site for chemical analysis.

Test solutions

Vehicle:

yes

Remarks:

solvent (acetone)

Details on test solutions:

As the test item is poorly water-soluble, the following approach was used to prepare stock and test solutions, taking into account aspects of OECD 2019, Guidance Document on AqueousPhase Aquatic Toxicity Testing of Difficult Test Chemicals. OECD Series on Testing and Assessment Number 23 (Second Edition). ENV/JM/MONO(2000)6/REV1.

An application solution at a nominal concentration of 120 g test item/L was prepared once on day 0 of the test by directly adding 1.20145 g test item to 10 mL acetone. This stock solution (S5) was slowly stirred (5 minutes) using a magnetic stirrer (250 rpm) at ambient temperature in the dark.
No particles were visible, and the solvent stock solution was used directly to prepare further dilution solutions (application solutions).

See Table 1 - Day 0: Preparation of application solutions.
These application solutions (S1 – S4) were stirred shortly (5 minutes) using a magnetic stirrer (250 rpm) at ambient temperature in the dark.
For the test solutions C0L (solvent control), C1, C2, C3, C4 and C5 a 1 L glass bottle (nominal volume: 1000 mL) containing a magnetic stir bar was filled nearly to the brim with M4 medium. These volumes were weighed, and air-tightly closed by a screwcap with PTFE-lined septum. Afterwards the corresponding volume of the solvent and application solutions S1 to S5 were added to the test solutions (see Table 4 and Table 5) by piercing through the septum with a Hamilton syringe (100 μL). During stirring (750 rpm) the solvent control and application solutions were slowly applied to the corresponding glass bottles filled with test medium.
The application solutions (S1 – S5) were sealed with parafilm and stored in a refrigerator overnight.
These test solutions were further stirred on a magnetic stirrer with 750 rpm for 10 minutes at ambient temperature.

DAY 0: See Table 2 - Day 0: Preparation of test solutions

DAY 1: See Table 3 - Day 1: Preparation of test solutions
The application solutions S1 to S5 of day 0 were adapted to room temperature before usage. A new set of test solutions was prepared as described Table 3. The freshly prepared final test solutions were distributed to the respective test vessels. Samples of the final test solutions were taken for analysis.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

• Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
• Origin: The organisms were originally supplied by KU Leuven, Belgium.

Study design

Test type:

semi-static

Water media type:

freshwater

Remarks:

Elendt medium M4

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water hardness: 14.0°dH / 250 mg/L CaCO3.
Determined before use.

Test temperature:

Within required ranges (20 +/- 2 °C)

pH:

Range : 7.4 - 7.9

Dissolved oxygen:

Range :
9.4 - 9.7 mg O2/L (107 - 110%) in fresh samples
6.4 - 9.4 mg O2/L (74 - 108%) in aged samples

Conductivity:

673 µS/cm.
Determined before use.

Nominal and measured concentrations:

Based on the results of a preceding non-GLP range finding acute toxicity test to Daphnia magna, the following nominal concentrations in a geometrical series (spacing factor: 1.8) were tested in the definitive test:
12.0, 6.67, 3.70, 2.06 and 1.14 mg test item/L.
The concentrations of the test item measured during the test can be summarised Table 4: Summary of time-weighted mean measured concentrations during the test.

Details on test conditions:

TEST SYSTEM / CONDITIONS
Material of stock vessel: Glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: Approximately 14.5 cm
Number of daphnids kept as stock per culture vessel (batch size): Approximately 20
Separation of adult and young daphnids: 2–5 times per week
Medium: Elendt medium M4
Renewal of medium: Twice per week
Temperature: Within required ranges (20  2 °C)
Photoperiod (light/dark) 16/8 h
Light intensity: Within required ranges (50 – 1000 lx)
Aeration: None
Food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 3–4 times per week

MATERIAL AND METHODS
Test system: Daphnia magna (Straus)
Age of exposed daphnids at start < 24 hours
Medium: Elendt Medium M4
Endpoint: ECx (e.g. EC50, EC20, EC10), NOEC/LOEC
Biological parameters: Immobilisation
Test duration: 48 h
Test units: glass vials (nominal volume 60-mL) air-tightly closed by a PTFE lined screwcap
Volume of test solution per test vessel: 60-65 mL (vessels filled to the brim)
Number of daphnids per test vessel: 5
Target temperature: 18 - 22°C
Photoperiod: 16 h light / 8 h dark
Aeration: none
Feeding during exposure: none
Test concentrations (nominal): Control; Solvent control
12.0, 6.67, 3.70, 2.06 and 1.14 mg test item/L.
Number of replicates per concentration: 4
Number of replicates in the control: 4
Number of replicates in the solvent control: 4
Renewal of test solution during exposure: Semi-static, renewal after 24 hours of exposure
Chemical analysis of test concentrations: At start, after 24 h (in fresh and aged media), and at end of exposure
Data evaluation: Evaluation of immobility. Probit analysis

TEST MEDIUM / WATER PARAMETERS
Elendt medium M4 was used to dilute the test item and to keep the daphnids during the period of the test. The medium consists of trace elements, vitamins and macro-nutrients in the following final concentrations (in mg/L):

Trace elements:
H3BO3 2.86
MnCl2 × 4 H2O 0.361
LiCl 0.306
RbCl 0.071
SrCl2 × 6 H2O 0.152
NaBr 0.016
Na2MoO4 × 2 H2O 0.063
CuCl2 × 2 H2O 0.017
ZnCl2 0.013
CoCl2 × 6 H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA × 2 H2O 2.5
FeSO4 × 7 H2O 1.0

Macronutrients:
CaCl2 × 2 H2O 293.8
MgSO4 × 7 H2O 123.3
KCl 5.8
NaHCO3 64.8
Na2SiO3 × 9 H2O 10.0
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184

Vitamins:
Thiamine hydrochloride 0.075
Cyanocobalamin 0.001
Biotin 0.00075

The required volume of medium was prepared within one month prior to use. During storage, the medium was aerated.

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI : 1.24 - 11.8 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Sublethal observations on the mobile daphnids were concentration-related. After 24 h daphnids showing reduced swimming activity were observed at treatment of (nominally) 2.06 mg test item/L in one test vessel. At 3.70, 6.67 and 12.0 mg test item/L (nominal) daphnids showed reduced swimming activity in all test vessels after 24 h. After 48 h mobile daphnids showed reduced swimming activity in all test vessels at 3.70, 6.67 and 12.0 mg test item/L.

Results with reference substance (positive control):

The most recent reference test with potassium dichromate performed in a separate study (Study No. IDA2105; May 2021) resulted in an EC50(24 h) of 1.352 mg/L.
According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L.
The toxicity of the reference item is within this range. Therefore, the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Any other information on results incl. tables

Analytical findings:
The concentrations of the test item measured during the test can be summarised as follows:

Summary of time-weighted mean measured concentrations during the test.

Nominal concentration [mg test item/L]	Concentration Determined in Fresh Samples (0 hour) [mg test item/L]	Concentration Determined in Fresh Samples (24 hour) [mg test item/L]	Concentration Determined in Aged Samples (24 hour) [mg test item/L]	Concentration Determined in Aged Samples (48 hour) [mg test item/L]	Time-weighted mean measured concentration over 48 h [mg test item/L]
0 (solvent control)	0 (<LOD)	n.d.	0 (<LOD)	n.d.	n.a.
1.14	1.20	1.06	0.921	<LOQ (0.297)	0.827
2.06	1.92	1.84	1.63	0.779	1.50
3.70	3.56	3.41	2.85	1.74	2.84
6.67	6.22	6.11	4.58	4.17	5.22
12.0	11.2	10.7	9.54	9.80	10.3

n.d.: not determined; n.a.: not applicable; Limit of quantification (LOQ): 0.5 mg/L; Limit of detection (LOD): 0.15
mg/L.

Biological findings:

A concentration-response relationship was observed. The following EC50 and NOEC values were determined based on nominal test item concentrations in mg/L  and on time-weighted mean (TWM) measured in mg/L:

Immobility (48 h)	Endpoints / Nominal [mg test item/L]	Endpoints / TWM measured [mg test item/L]
EC50 (95%-confidence limits)	4.89 (1.72‒13.9)	3.83 (1.24‒11.8)
NOEC	2.06	1.50

Validity of the test

All validity criteria were fulfilled as required by the test guideline:

Required:	Found:
Dissolved oxygen concentration measured in test solutions and control at the end of the test: (required: at least 3 mg/L)	≥ 6.4 mg/L
Immobility in the controls and solvent controls (required: not more than 10%): Control Solvent Control	0% 10%
Daphnids in the control and solvent control showing signs of disease or stress (required: not more than 10%):	0%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the EC50 value for 48 hours of 2-methyl-3-(p-tolyl)propionaldehyde was found to be 3.83 mg/L with 95% confidence interval ranging from 1.24 mg/L to 11.8 mg/L.
The results observed in the present study meets all the validity criteria as per OECD 202 Test guideline.

Executive summary:

The objective of this study was to assess the Acute Immobilization Study of 2-methyl-3-(p-tolyl)propionaldehyde in Daphnia sp (Daphnia magna). The study was performed in compliance with following regulatory guideline: OECD Guideline for the Testing of Chemicals. Test Guideline 202: Daphnia sp. Acute Immobilization Test, Adopted: 13 April 2004.

Based on the results of a preceding non-GLP range finding acute toxicity test to Daphnia magna, the following nominal concentrations in a geometrical series (spacing factor: 1.8) were tested in the definitive test:

12.0, 6.67, 3.70, 2.06 and 1.14 mg test item/L.

The test organisms were exposed to these concentrations, as well as to controls (blank control, solvent control) without the test item, for a period of 48 hours under semi-static test conditions in a completely filled, closed-bottle system to minimise losses of the test item due to its volatility. Those animals not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile.

The data obtained was analysed in order to estimate the concentration that would cause an x% immobilisation compared to the control, i.e. ECx (e.g. EC50), and, where possible, a No-observed Effect concentration (NOEC).

A concentration-response relationship was observed.

Based on the results of this study, the EC50 value for 48 hours of 2-methyl-3-(p-tolyl)propionaldehyde was found to be 3.83 mg/L with 95% confidence interval ranging from 1.24 mg/L to 11.8 mg/L.

The results observed in the present study meets all the validity criteria as per OECD 202 Test guideline.