4-isopropyl-1-methylcyclohexene

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:

The study was conducted between 19 August 2017 and 28 September 2017 (biological phase).

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The pH in the test medium exceeded the required upper limit also the temperature during the first 49 hours while preparing the stock solution was lower than 30°C. These deviations had no influence on the integrity of the study results.

Principles of method if other than guideline:

Deviations:
1. Deviating from the study plan the pH in the test medium exceeded the required upper limit (pH = 9) by 0.6 pH units. This deviation has no influence on the integrity of the study results, This deviation has no influence on the integrity of the study results, since all cell growth related validity criteria were fulfilled and the pH drift throughout all well growing treatment levels were always below 1.4 pH units.
2. Deviating from the study plan the temperature during the first 49 hours while preparing the stock solution was lower than 30°C due to a malfunction of the heating chamber. The mean temperature was 22.6°C. This deviation has no influence on the integrity of the study results, since the analytical measurements of the stock solution at start of the test show comparable high concentration (0.603 mg Menthene,-1,para/L) as observed in previous non-GLP test (up to 0.799 mg Menthene,-1,para/L at 100% of a saturated solution, freshly prepared).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-isopropyl-1-methylcyclohexene

EC Number:

226-841-5

EC Name:

4-isopropyl-1-methylcyclohexene

Cas Number:

5502-88-5

Molecular formula:

C10H18

IUPAC Name:

1-methyl-4-(propan-2-yl)cyclohex-1-ene

impurity 1

Reference substance name:

rel-(1R,4R)-1-isopropyl-4-methylcyclohexane

Cas Number:

1678-82-6

Molecular formula:

C10H20

IUPAC Name:

rel-(1R,4R)-1-isopropyl-4-methylcyclohexane

impurity 2

Reference substance name:

rel-(1S,4S)-1-isopropyl-4-methylcyclohexane

Cas Number:

6069-98-3

Molecular formula:

C10H20

IUPAC Name:

rel-(1S,4S)-1-isopropyl-4-methylcyclohexane

Test material form:

liquid

Specific details on test material used for the study:

Lot No.: VE00502099
Aspect: colourless to pale yellow liquid
Purity: 87.5% (major peak)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical findings
Samples from the test solutions were analysed after 0, 24, 48 and 72 hours to determine actual levels of the test item in solution during the exposure period. The measured concentrations decreased over the exposure period. After 72 hours exposure, the test concentrations remained unstable at between 14–51 % of the time 0 hours measured initial concentration. Therefore, the test item concentration based on initial measured concentration remained unstable within ±20% throughout the exposure period.

Since all concentration levels were measured after 0, 24, 48 and 72 hours, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level. Details are reported in the analytical phase report and in section 18.3 of the study report.

Biological findings
A weak concentration-response relationship was observed for the biological parameter yield during the exposure period, whereas no meaningful concentration-response relationship was observed for the biological parameter growth rate. The resulting endpoints are presented in Table 1 and Table 2 of the study report, respectively, based on geometric mean measured concentrations and based on nominal concentrations expressed as % of a saturated solution at a loading of 200 mg/L.

The ECx, NOEC and LOEC values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and measured geometric mean concentrations of the test item (Menthene,-1, para).

Test organisms

Test organisms (species):

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

Details on test organisms:

The test system used in this study was the freshwater algae Pseudokirchneriella subcapitata (SAG 61.81).
The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.

Study design

Test type:

static

Water media type:

other: OECD medium (modified) + additional NaHCO3

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.1 to 22.5 °C

pH:

8.2

Nominal and measured concentrations:

Test Concentrations (nominal): 19.8, 29.6, 44.4, 66.7 and 100 % of a saturated solution at a loading rate of 200 mg/L, and a control

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit 0.1103 - 0.4200 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

* Note: The %-inhibition seen for the parameter yield was <20%. Therefore, the EC20 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).
** Note: The %-inhibition seen for the parameter growth rate was <10%. Therefore, the EC10, EC20 and the E50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).

Any other information on results incl. tables

Analytical findings

Samples from the test solutions were analysed after 0, 24, 48 and 72 hours to determine actual levels of the test item in solution during the exposure period. The measured concentrations decreased over the exposure period. After 72 hours exposure, the test concentrations remained unstable at between 14–51 % of the time 0 hours measured initial concentration. Therefore, the test item concentration based on initial measured concentration remained unstable within ±20% throughout the exposure period.

 

Since all concentration levels were measured after 0, 24, 48 and 72 hours, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level. Details are reported in the analytical phase report and in section 18.3.

 

Biological findings

A weak concentration-response relationship was observed for the biological parameter yield during the exposure period, whereas no meaningful concentration-response relationship was observed for the biological parameter growth rate. The resulting endpoints are presented in Table 1 and Table 2, respectively, based on geometric mean measured concentrations and based on nominal concentrations expressed as % of a saturated solution at a loading of 200 mg/L.

 

The following EC_x, NOEC and LOEC values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and measured geometric mean concentrations of the test item (Menthene,-1, para).

Table1: Summary of biological results based on measured test item concentrations in mg Menthene,-1, para/L.

	Endpoints [mg Menthene,-1, para/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cI)	0.2152 (0.1103-0.4200)	> 0.179	> 0.179	≥0.179	> 0.179
Growth Rate (95% cI)	> 0.179	> 0.179	> 0.179	≥0.179	> 0.179

cl: confidence limit

 

NOEC, LOEC and EC_xvalues 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 % of a saturated solution at a loading of 200 mg/L were given in the following table.

 

Table2: Summary of biological results based on nominal concentrations in % of a saturated solution.

	Endpoints [% of a saturated solution at a loading of 200 mg/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cI)	52.6 (33.0-83.8)	> 100	> 100	≥100	> 100
Growth Rate (95% cI)	> 100	> 100	> 100	≥100	> 100

cl: confidence limit

 

* Note: The %-inhibition seen for the parameter yield was <20%. Therefore, the EC₂₀was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).

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	132.0
Mean coefficient of variation for section-by-section specific growth rates in the control cultures:up to 35%	12.0%
Coefficient of variation of average specific growth rates during test period in replicate control cultures:up to 7%	0.7%

The study is therefore considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Analytical findings
Samples from the test solutions were analysed after 0, 24, 48 and 72 hours to determine actual levels of the test item in solution during the exposure period. The measured concentrations decreased over the exposure period. After 72 hours exposure, the test concentrations was unstable at between 14-51% of the time 0 hours measured initial concentration. Therefore, the test item concentration based on initial measured concentration
did not remain stable within ±20% throughout the exposure period.
Since all concentration levels were measured after 0, 24, 48 and 72 hours, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level. Details are reported in the analytical phase report and in section 18.3.

Biological findings
A slight concentration-response relationship was observed for the biological parameter yield during the exposure period, whereas no meaningful concentration-response relationship was observed for the biological parameter growth rate. The resulting endpoints are presented in Table 11 and Table 12 of the study report, respectively, based on geometric mean measured concentrations and based on nominal concentrations expressed as % of a saturated solution at a loading of 200 mg/L.
The following ECx, NOEC and LOEC values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and measured geometric mean concentrations of the test item (Menthene,-1, para).
Summary of biological results based on measured test item concentrations in mg Menthene,-1, para/L.
Endpoints [mg Menthene,-1, para/L]
Parameter EC10 E20 EC50 NOEC LOEC
Yield 0.2152 > 0.179* > 0.179* ≥ 0.179 ≥ 0.179
(95% cl) (0.1103-0.4200)

Growth Rate > 0.179** > 0.179** > 0.179** ≥ 0.179 ≥ 0.179
(95% cl)
cl: confidence limit
n.d.: not determined due to mathematical reasons (% inhibition were lower than the effect concentration, e.i. EC10, EC20, EC50)
* Note: The %-inhibition seen for the parameter yield was <20%. Therefore, the EC20 and the EC50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).
** Note: The %-inhibition seen for the parameter growth rate was <10%. Therefore, the EC10, EC20 and the EC50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).

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 % of a saturated solution at a loading of 200 mg/L were given in the following table (Table 12 of the study report): Summary of biological results based on nominal concentrations in % of a saturated solution.
Endpoints [% of a saturated solution at a loading of 200 mg/L]
Parameter EC10 EC20 EC50 NOEC LOEC
Yield 52.6 > 100 * > 100* ≥ 100 ≥ 100
(95% cl) (33.0 - 83.8)
Growth Rate > 100 ** > 100 ** > 100 ** ≥ 100 ≥ 100
(95% cl)

cl: confidence limit
* Note: The %-inhibition seen for the parameter yield was <20%. Therefore, the EC20 and the EC50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).
** Note: The %-inhibition seen for the parameter growth rate was <10%. Therefore, the EC10, EC20 and the EC50 was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 200 mg/L).

Executive summary:

The impact of the test item Menthene,-1, para on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline for the Testing of Chemicals, No. 201 (2011).

Based on measured concentrations, the 72h EC50 of Pseudokirchneriella subcapitata was found to be > 0.179 mg/L. The corresponding NOEC is ≥ 0.179 mg/L.