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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 27, 2021 to September 29, 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

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/or test solutions will be taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.

Samples were taken and analysed, as follows :
Control : 0 hr, 72 hrs
Solvent Control : 0 hr, 72 hrs
C1 (1.25 mg/L nominal) : 0 hr, 48 hrs, 72 hrs
C2 (2.50 mg/L nominal) : 0 hr, 48 hrs, 72 hrs
C3 (5.00 mg/L nominal) : 0 hr, 48 hrs, 72 hrs
C4 (10.0 mg/L nominal) : 0 hr, 48 hrs, 72 hrs
C5 (20.0 mg/L nominal) : 0 hr, 48 hrs, 72 hrs

C3 (5.00 mg/L) : at 72 hours, including i) Stability control without algae stored in light; ii) Stability control without algae stored in the dark, iii) Volatility control sample with algae but without removal of test solutions at 24 and 48 hours.

Solvent control and selected test solutions will be sampled in triplicate. The triplicate samples will be kept separately as a reserve. The volume of each sample will be recorded.

Samples of a defined volume (50 mL of water samples if not advised otherwise) will be 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 will not be combined prior to sampling, the test solution will be directly transferred into the extraction and sample vessels (e.g. pipetting using a glass pipette, 50 mL). The extraction vessels will immediately be processed as described below, and the sampling bottles will be immediately tightly closed by a screw-cap with PTFE lining, and stored in the freezer (≤ -18 °C).

Extraction procedure:
Each sample will be stabilised by adding 1-N hydrochloric acid (1% of sample volume). To each sample, 5 mL of the extraction solvent (toluene) will be added. The two phases will be mixed vigorously, e.g. for 30 seconds on a vortex, and manually shaken afterwards. After phase separation (e.g. allow to stand for several minutes, if the separation will occur slowly the extracts may be transferred to a centrifuge beaker and centrifuged at about 3000 rpm, at
10°C for at least 5 minutes), 1.0 to 2.0 mL of the upper (organic) phase will be transferred (e.g. using a glass pipette) into a glass vial of appropriate volume. Afterwards the vials containing the solvent extracts (toluene) will be tightly closed, and stored in the freezer (≤ -18 °C). The water phase after extraction will be disposed.

Samples from the stock solution and dilution solutions in acetone (e.g. 0.5 mL if not advised otherwise) will be stored frozen as reserve samples.

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

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

Test solutions

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

Based on the results of a preliminary non-GLP range finding test conducted at 0.16, 0.80, 4.00 and 20.0 mg test item/L, the following concentration levels in a geometrical series (spacing factor: 2.0) were tested in the definitive test:

1.25, 2.50, 5.00, 10.0 and 20.0 mg test item/L.

Additionally, the test organisms were exposed under control conditions (untreated test medium, mod. OECD medium) and to a solvent control (test medium water plus solvent; 0.1 mL solvent per litre test medium) using the same amount of solvent as used for the treated test solution, for the same period of time.

The test item was applied once at the beginning of the exposure period. Three replicate vessels were used per test item concentration, and for the control, and six replicate vessels for the solvent control.

Preparation of Stock, Application and Test Solutions :

A stock solution at a nominal concentration of 200000 mg test item/L was prepared by dissolving 2.00088 g test item in 10 mL acetone. This stock solution (S5) was stirred shortly (approximately 5 minutes at 500 rpm) using a magnetic stirrer at ambient temperature.

This stock solution was used to to prepare a dilution series in acetone (application solutions S1–S4). This application solutions (S1–S4) were stirred shortly (approximately 5 minutes at 500 rpm) using a magnetic stirrer at ambient temperature.

The test solutions were prepared by adding the solvent stock solution (S5) or the corresponding dilution solutions (application solutions, S1–S4) to the test medium. Before application of the solvent solutions the glass beakers were nearly completely filled with test medium by weighing, to minimise the head-space while application/preparation of the test solutions, and the beaker were tightly closed by a PTFE-lined screwcap with septum.

The solvent solutions (S1–S5) were slowly spiked into the test medium using a syringe (Hamilton syringe, volume: 100 µL) in a sealed glass bottle, closed with a screw-cap with septum and PTFE lining. Therefore, the tip of the syringe was punctured through the septum and the tip was placed below the water surface. While adding the solvent to the test medium the solution was intensively stirred (750 rpm) using a magnetic stirrer. The maximum amount of 0.1 mL solvent/L test medium was used to prepare the test solutions.
After preparation of the test solutions, the preparation flasks were shortly opened, and the approximate volume needed to inoculate the algae cells was removed from the test solutions. Directly thereafter, the inoculum was added to the glass beaker resulting in a cell concentration of 0.25×104 cells per mL.

An additional preparation flask for the intermediate concentration level was prepared, but no algae cells were added to this additional flask. This solution was later be used in order to assess test item stability without the presence of algae. The preparation flasks were be closed by a PTFE-lined screwcap with septum.

The volume of the stock solutions was large enough to prepare the test solutions and all analytical samples and solutions used for conditioning.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pre-Culture Conditions: to adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
- Species: Pseudokirchneriella subcapitata (SAG 61.81), currently valid species name: Raphidocelis subcapitata
- Supplier: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany
- Date of receipt: February 24, 2016
- Charge No. (ECT): A/240216/A
- pH-value of the algal medium: 8.0
- Culture medium: OECD medium (OECD 201, EN ISO 8692)
- Volume of liquid stock culture per pre-culture vessel: 100±5 mL
- Pre-culture vessels: 300 mL Erlenmeyer flasks
- Number of replicates: 2
- Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip “daylight”
- Light intensity: 60–120 µE m-2s-1; (measured: 73.5–78.1 µE m-2s-1)
- Shaker: 100±5 oscillations/min
- Temperature in the test room: 21–24 C; (measured: 21.9–22.2 °C)

Study design

Test type:

static

Water media type:

freshwater

Remarks:

OECD medium

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.7–22.0, mean 21.8 °C

pH:

pH of test medium: 7.9
pH in test solutions: 8.3–9.4

Nominal and measured concentrations:

Based on the results of a preliminary non-GLP range finding test conducted at 0.16, 0.80, 4.00 and 20.0 mg test item/L, the following concentration levels in a geometrical series (spacing factor: 2.0) were tested in the definitive test: 1.25, 2.50, 5.00, 10.0 and 20.0 mg test item/L.
The concentrations of the test item measured during the test can be summarised Table 1: Summary of measured concentrations at the beginning and at the end of the test.

Details on test conditions:

TEST SYSTEM / TEST CONDITIONS
Test vessels: 60 mL glass vials (nominal), air-tightly closed by a screw-cap with PTFE lining
Volume of test solution per test vessel: 60 mL (nominal), filled to the rim
Age of the pre-culture: 4 days
Number of cells per mL in the pre-culture before inoculating the test solutions: 257×104
Number of cells per mL test solution at the beginning of the test: 0.25×104
Number of replicates per test item concentration: 3
Number of replicates in the control: 3
Number of replicates in the solvent control: 6
Number of replicates for potential chemical analyses (after 24 and 48 hours of exposure): In total 5 per treatment level
Number of replicates for stability check (without algae; light and dark): 3 (treatment level: C3)
Number of replicates for volatility check (with algae): 3 (treatment level: C3 at light and in dark)
Test medium: OECD medium + additional NaHCO3 (modified)
pH of test medium: 7.9
pH in test solutions: 8.3–9.4; for details see section 18.1
Light regime: 24 h light/0 h dark
Type of light: Econlux LED Sunstrip “daylight”
Light intensity: 77.6–95.1 µE m–2s–1; mean 85.3 µE m–2s–1
Temperature: 21.7–22.0, mean 21.8 °C
Shaker: 100±5 oscillations/min; the test vessels were placed randomly on a horizontal shaker, so that each test vessel was rotated around its own axis
Test duration (exposure): 72 hours
Counting of algae: Daily

MATERIAL AND METHODS
Test organism: Pseudokirchneriella subcapitata
Test medium: OECD-Medium + additional NaHCO3 (modified)
Endpoints: ECx (e.g. EC50, EC20, EC10), NOEC/LOEC
Biological parameters: Inhibition of growth in relation to control (growth rate & yield)
Test duration: 72 hours
Temperature (target): 21 - 24 °C, controlled at ±2 °C
Light regime: Permanent illumination
Light intensity (target): 60 - 120 µE m-2s-1, within ±15% over the incubation area
Test units: 60 mL glass vials, air-tightly closed by a screwcap with PTFE lining
Test concentrations (nominal): 1.25, 2.50, 5.00, 10.0 and 20.0 mg test item/L plus a control and a solvent control
No. of replicates in the control: 3
No. of replicates in the solvent control: 6
No. of replicates per test concentration: 3
Renewal of test solution during exposure: None (static system)
Chemical analysis of test concentrations: At start (0 hours), 48 hours and at the end of exposure period (72 hours)
Data evaluation: Shapiro-Wilk’s on Normal Distribution; Levene’s and Cochran’s Test on Variance Homogeneity; Student-t test; Williams t-test; 3-parametric normal; CDF analysis

GROWTH MEDIUM
The growth medium was OECD medium as described in the test guideline, modified to optimise growth of Pseudokirchneriella subcapitata. The OECD growth medium was supplemented with 250 mg/L sodium bicarbonate (in addition to the normal content of 50.0 mg/L NaHCO3), marked below in bold (Mayer et al. (2000), ISO 14442 (2006)).
NaHCO3 300.0 mg/L
NH4Cl 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
KH2PO4 1.6 mg/L
FeCl3 × 6 H2O 0.064 mg/L
Na2EDTA × 2 H2O 0.100 mg/L
H3BO3 0.1850 mg/L
MnCl2 × 4 H2O 0.4150 mg/L
ZnCl2 0.0030 mg/L
CoCl2 × 6 H2O 0.0015 mg/L
Na2MoO4 × 2 H2O 0.0070 mg/L
CuCl2 × 2 H2O 0.00001 mg/L

Justification for modification of growth medium: The use of a sealed system result in culture growth being limited by CO2 depletion, thus additional NaHCO3 is used to serve as a source of CO2.
Before use, the pH of the medium was adjusted to 8.1±0.2 and the medium was aerated (sterile) for 30 minutes. The algal medium was sterilised (filtered, pore size 0.2 µm) before use. The vessels used were sterilised by autoclaving for 20 minutes at 121°C. There was no indication of interaction between this medium and the test item.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 6.40 - 8.80 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 12.2 - 17.7 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI : 2.22 - 9.72 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI : 3.14 - 20.4 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.
The EyC50 of Pseudokirchneriella subcapitata was determined to be 10.5 mg test item/L based on nominal concentrations, equivalent to 8.24 mg 2-methyl-3-(p-tolyl)propionaldehyde/L based on measured concentrations.
The EyC10 value determined was 6.59 mg/L test item/L based on nominal concentrations, equivalent to 4.65 mg 2-methyl-3-(p-tolyl)propionaldehyde/L based on measured concentrations.
The NOEC based on nominal concentration was 5.00 mg test item/L, equivalent to 2.64 mg 2-methyl-3-(p-tolyl)propionaldehyde/L based on geometric mean measured concentrations.

The ErC50 of Pseudokirchneriella subcapitata was determined to be 16.6 mg test item/L based on nominal concentrations, equivalent to 14.7 mg 2-methyl-3-(p-tolyl)propionaldehyde/L based on measured concentrations. The ErC10 value determined was 9.78 mg/L test item/L based on nominal concentrations, equivalent to 7.50 mg 2-methyl-3-(p-tolyl)propionaldehyde/L based on measured concentrations.

Results with reference substance (positive control):

Two reference tests using potassium dichromate (K2Cr2O7) as reference item were performed in separate studies. One was performed using a standard test (study number: IAO2102, February 2021) design and the other using a closed-bottle system (study number: IAO2011-CB, November 2020). The latter was performed to confirm that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system.
Result:
Standard: Growth rate ErC50 (0–72 h): 0.825 mg/L (0.785 – 0.864 mg/L; 95%-CL)
Closed-Bottle: Growth rate ErC50 (0–72 h): 0.880 mg/L (0.817 – 0.937 mg/L; 95%-CL)

Based on an international ring test (ISO (2004). Water Quality – Freshwater algal growth inhibition test with unicellular green algae. European Standard EN ISO 8692, October 2004.) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories was 1.19 mg/L with a standard deviation of 0.27 mg/L.

The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.825 mg/L (standard system) and 0.880 mg/L (closed system). These values are within the historical range of the general reference test results (standard open test system) of our laboratory (mean ErC50 value = 1.046 ± 0.335 mg/L) and within the narrow upper (1.373 mg/L) and lower (0.721 mg/L) warning limits. The narrow warning limits were calculated as one standard deviation from the historical mean of the EμC50 values. The calculation of the warning limits was not conducted in compliance with Good Laboratory Practice Principles (all calculated EμC50 using the log concentration; according to Environment Canada (2005), Guidance Document on Statistical Methods for Environmental Toxicity Tests, Method Development and Applications Section, Environment Canada, Ottawa, ON, Report EPS 1/RM/46). Furthermore, the EμC50 value (standard system) is within the refined range of 1.10 ± 0.48 mg/L, recommended based on an international ring test (Pattard, M.; Römbke, J.; Moser, T. (2009). Range of Reference Tests in Aquatic Tests. [in] Moser, H.; Römbke, J. “Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test”, chapter 5, pp. 61-70.). Therefore, the results of this reference test are acceptable and the test conditions are reliable.

Any other information on results incl. tables

Analytical findings

Samples from the test solutions were analysed using GC-MS to determine actual levels of the test item at start of exposure period (time 0 hours) and following 48 and 72 hours of exposure. At time 0 hours the measured concentrations ranged between 94.4–101% of the nominal concentration and decreased to values between 25.8% and 108% after 48 hours and <LOD and 93.5% after 72 hours exposure. Therefore, the test item concentration based on nominal and initial measured concentrations were not stable within ±20% at the end of the exposure period. As a consequence, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level.

Biological findings

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. The resulting endpoints are presented in Table 2 and Table 3, respectively, based on geometric mean measured concentrations in mg of the substance/L and based on nominal concentrations expressed in mg test item/L.

The following ECx, NOEC and LOEC values for the parameters cell number and biomass expressed in yield and growth rate, based on statistical evaluation of biological results and measured geometric mean concentrations of the test item.

Table 2: Summary of biological results based on geometric mean measured test item concentrations in mg 2-methyl-3-(p-tolyl)propionaldehyd/L.

Endpoints (mg 2-methyl-3-(p-tolyl)propionaldehyde/L)
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cl)	4.65 (2.22 - 9.72)	5.66 (2.76 - 11.7)	8.24 (3.14 - 20.4)	2.64	7.71
Growth Rate (95% cl)	7.50 (6.40 - 8.80)	9.46 (8.14 - 11.0)	14.7 (12.2 - 17.7)	2.64	7.71

cl: confidence limit

NOEC, LOEC and ECx values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and nominal concentrations in mg test item/L are given in the following table.

Table 3: Summary of biological results based on nominal concentrations in mg test item/L.

Endpoints (mg test item/L)
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cl)	6.59 (3.92 - 11.1)	7.73 (4.67 - 12.8)	10.5 (5.42 - 19.8)	5.00	10.0
Growth Rate (95% cl)	9.78 (8.63 - 11.1)	11.7 (10.4 - 13.2)	16.6 (14.3 - 19.1)	5.00	10.0

Validity of the test

All validity criteria were fulfilled as required by the study plan:

Required	Found
Mean biomass increase in the control cultures: factor of at least 16 within the 72-hour test period	186.2
Mean coefficient of variation for section-by-section specific growth rates in the control cultures: up to 35%	19.0%
Coefficient of variation of average specific growth rates during test period in replicate control cultures: up to 7%	0.6%

Required	Found
Mean biomass increase in the solvent control cultures: factor of at least 16 within the 72 -hour test period	207.2
Mean coefficient of variation for section-by-section specific growth rates in the solvent control cultures: up to 35%	15.6%
Coefficient of variation of average specific growth rates during test period in replicate solvent control cultures: up to 7%	0.6%

The study is therefore considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the ErC50 and ErC10 values for 72 hours of 2-methyl-3-(p-tolyl)propionaldehyde were found to be 14,7 mg/L and 7,50 mg/L respectively.
The results observed in the present study meets all the validity criteria as per OECD 201 Test guideline.

Executive summary:

The objective of this study was to assess the Growth Inhibition Study of 2-methyl-3-(p-tolyl)propionaldehyde in Freshwater Alga (Pseudokirchneriella subcapitata) followed by the period of 72 hours observation. The study was performed in compliance with following regulatory guideline: OECD Guidelines for the Testing of Chemicals, Section.2. Number 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” Adopted on 23 March 2006 Annexure 5 corrected: 28 July 2011.

The aim of the study was to determine the toxicity of the test item towards the green algae, Pseudokirchneriella subcapitata. To achieve this, a series of concentrations of the test item in aqueous solution were prepared using a dilution series of solvent solutions at different concentration levels for addition of the test item. The test organisms were exposed to these concentrations, as well as to controls without the test item, for a test period of 72 hours in a

completely filled, closed-bottle system. The closed-bottle system is intended to reduce losses of volatile components of the test item during exposure. The study was conducted under static conditions; i.e., the test solutions were not renewed during the test and without air exchange (no headspace).

The data obtained were analysed in order to estimate the concentration that caused a x% growth inhibition, i.e. ECX (e.g. EC50, EC20 or EC10). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated.

Growth was determined daily based on determination of the cell number per volume test solution (cell concentration).

To verify the nominally applied concentration, samples were taken from the test solution and analytically measured. The samples taken from the test vessels were transferred to the analytical test site and analysed under the responsibility of the principal investigator for analysis.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.

Based on the results of this study, the EC50 and ErC10 values for 72 hours of 2-methyl-3-(p-tolyl)propionaldehyde were found to be 14,7 mg/L and 7,50 mg/L respectively.

The results observed in the present study meets all the validity criteria as per OECD 201 Test guideline.