Reaction mass of 1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

List of study plan deviations
1. Range-finding test: Samples for possible analysis were taken during the test.
Evaluation: To verify the initial concentration and maintenance of the exposure concentration during the test.
2. Final test: Samples, with and without algae, were taken at 48 hours of exposure.
Evaluation: Extra samples were taken in order to calculate a more accurate TWA.
The study integrity was not adversely affected by these deviations.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

257-942-2

EC Name:

1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

52475-86-2

Molecular formula:

C14H22O

IUPAC Name:

1-methyl-4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

Constituent 2

Reference substance name:

1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

257-941-7

EC Name:

1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

52474-60-9

Molecular formula:

C14H22O

IUPAC Name:

1-methyl-3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

Specific details on test material used for the study:

Test item: 205226/D
Identification: Precyclemone B
Appearance: Colourless to pale yellow liquid
Batch: SC00019213
Purity/Composition: 99.7% (sum of two isomers)
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 26 September 2017 (expiry date)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t=24 h, t=48 h and t=72 h
Volume: 15.0 mL
Storage: Samples were stored in a freezer (≤ -15°C) until analysis.

At the end of the exposure period, samples were taken from 1 replicate of each test concentration and the control. Except at the end of the test for the 1.0% SS of which all three replicates were sampled. This, because of the variation in cell densities.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24, 48 and 72 hours of exposure.
Additionally, reserve samples of 15.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Details on test solutions:

Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one day where after the clear and colourless Saturated Solution (SS) was siphoned out and used as the highest test concentration. Lower concentrations were prepared by subsequent dilutions of the SS in test medium.
After preparation, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an
internationally accepted species.

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2 /s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104
cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium Adjusted M2; Standard M2 according to the OECD 201 Guideline but with a larger amount of NaHCO3, the addition of
HEPES buffer and a lower pH.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

Monitored continuously in a temperature control vessel. During the exposure period the temperature measured in the incubator was maintained
between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

Monitored at the beginning and at the end of the test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).

Nominal and measured concentrations:

Range finding (nominal): Exponentially growing algal cultures were exposed to 0.10, 1.0, 10 and 100% of a SS at 100 mg/L
Final Test (nominal): 1.0, 3.2, 10, 32 and 100% of a SS at 100 mg/L
Final Test (measured): 0.050, 0.15, 0.53, 1.7 and 5.5 mg/L in 1.0, 3.2, 10, 32 and 100% of a SS at 100 mg/L, respectively

Details on test conditions:

Control: Test medium without test item or other additives
Replicates:
3 replicates of each test concentration;
6 replicates of the control;
2 extra replicates of the control and each test concentration for sampling purposes;
1 or 3 replicates of each test concentration without algae.

Test vessels: 120 mL, airtight closed with limited headspace to prevent loss of the test item due to volatilization.
Medium: Adjusted M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 84 µE.m-2.s-1
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 1.6 to 2.1 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 0.14 to 0.24 mg/L

Details on results:

Effects based on yield were also reported (see attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table. Furthermore, the preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus, only the EC10 is presented in the above "effects concentration" table. The NOEC values are available in the full study report.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Precyclemone B inhibited both growth rate and yield of this fresh water algae species significantly at TWA concentrations of 0.29 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.8 mg/L with a 95% confidence interval ranging from 1.6 to 2.1 mg/L. The corresponding 72h-ERC10 was 0.19 mg/L with a 95% confidence interval ranging from 0.14 to 0.24 mg/L.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 1.8 mg/L and 0.19 mg/L respectively.