Essential oil of Cedarwood obtained from the wood of Cupressus funebris (Cupressaceae) by steam distillation and washing - Terpenes

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:

08 August 2017 to 29 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

rel-(3R,3aS,7S,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulene

Cas Number:

22567-43-7

Molecular formula:

C15H24

IUPAC Name:

rel-(3R,3aS,7S,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulene

Constituent 2

Reference substance name:

rel-(3R,3aR,7R,8aS)-3,8,8-trimethyl-6-methyleneoctahydro-1H-3a,7-methanoazulene

Cas Number:

1404220-60-5

Molecular formula:

C15H24

IUPAC Name:

rel-(3R,3aR,7R,8aS)-3,8,8-trimethyl-6-methyleneoctahydro-1H-3a,7-methanoazulene

Constituent 3

Reference substance name:

rel-(3R,3aS,7S,8aS)-3,8,8-trimethyl-6-methyleneoctahydro-1H-3a,7-methanoazulene

Cas Number:

65450-98-8

Molecular formula:

C15H24

IUPAC Name:

rel-(3R,3aS,7S,8aS)-3,8,8-trimethyl-6-methyleneoctahydro-1H-3a,7-methanoazulene

Constituent 4

Reference substance name:

rel-(1R,9S,E)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene

Cas Number:

17627-40-6

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,9S,E)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene

Constituent 5

Reference substance name:

rel-(1aR,4aR,8aR)-2,4a,8,8-tetramethyl-1,1a,4,4a,5,6,7,8-octahydrocyclopropa[d]naphthalene

Cas Number:

32435-95-3

Molecular formula:

C15H24

IUPAC Name:

rel-(1aR,4aR,8aR)-2,4a,8,8-tetramethyl-1,1a,4,4a,5,6,7,8-octahydrocyclopropa[d]naphthalene

Constituent 6

Reference substance name:

1,8-dimethyl-4-(propan-2-ylidene)spiro[4.5]dec-7-ene

Molecular formula:

C15H24

IUPAC Name:

1,8-dimethyl-4-(propan-2-ylidene)spiro[4.5]dec-7-ene

Constituent 7

Reference substance name:

1-methyl-4-(1,2,2-trimethylcyclopentyl)benzene

Cas Number:

51773-81-0

Molecular formula:

C15H22

IUPAC Name:

1-methyl-4-(1,2,2-trimethylcyclopentyl)benzene

Test material form:

liquid

Details on test material:

UVCB

Specific details on test material used for the study:

CEDRENE WASHED
Batch No. : SH00085512
Purity : Conform to Chromatogram (UVCB)
Expiry Date : 09 July 2018

The test item is a multi-component complex mixture isolated from a natural source and is, consequently, defined as an UVCB.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

All loading levels and the control were analytically verified via GC-MS after 0 (start of exposure), 24 and 72 hours (end of the
exposure). Separate replicates for the test item analysis after 0, 24 and 72 hours were prepared with algae at the start of the exposure and incubated under test conditions until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Five water accommodated fractions (WAF) were prepared with a
accommodated fraction nominal loading of the test item of 6.25 - 12.5 - 25.0 - 50.0 – 100 mg/L (spacing factor 2). A slow stirring procedure with a minimized headspace was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24 ± 1 hour. After a separation phase of four hours at room temperature the WAFs were taken from the bottom of the flask and were used for testing. Each WAF was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The resulting water accommodated fractions (WAF) were used in the test. Additionally, the loading level 3.125 mg/L was tested. Therefore, the WAF with a loading of 6.25 mg/L was diluted with dilution water. The loading levels are based on the results of the first test.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test organism
Pseudokirchneriella subcapitata HINDAK, SAG 61.81

Synonyms
Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin
Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux corresponding to 35-70 pE -nr2 S’1 for 24 hours per day.

Culture medium
Nutrient medium Z according to Lüttge et al. (1994)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Nominal range: 21 - 23°C, Mean value = 22.5 °C

pH:

Nominal Test Item Loading pH Value Start (0 hours) pH value End (72 hrs)
100 mg/L 7.96 9.00
50.0 mg/L 8.00 9.02
25.0 mg/L 8.04 9.06
12.5 mg/L 8.08 9.11
6.25 mg/L 8.02 9.18
3.13 mg/L 8.16 9.33
Control 8.08 9.17

Nominal and measured concentrations:

The measured concentrations of the test item at the start of the exposure were in the range of 0.0207 to 0.120 mg/L. At test end, the test item concentrations were in the range of 0.0146 to 0.116 mg/L.

Nominal Loading Rate (mg/L) Geometric Mean Measured Concn. (mg/L)
100 0.0893
50.0 0.124
25.0 0.0297
12.5 0.0587
6.25 0.0390
3.13 0.0192
Control < LOQ (LOQ = 0.001 mg/L test item)

Per definition of the WAF, all terms related to concentration level were given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations, therefore, all effect values given are based on the nominal test item loadings of CEDRENE WASHED.

Details on test conditions:

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Since the test item is an UVCB five water accommodated fractions (WAF) with the nominal test item loadings 3.13 - 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L (spacing factor of 2) were prepared with dilution water and slowly stirred for 24 hours (20 rpm, room temperature). Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (SIGMA, Batch NO. MKBV0900V, Purity 99.0%)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Test item is an UVCB

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Test item is an UVCB

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Test item is an UVCB

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Test item is an UVCB

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

3.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Test item is an UVCB

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Test item is an UVCB

Details on results:

The NOEL and LOEL were determined by calculation of statistical significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations.

Results with reference substance (positive control):

The toxicity of potassium dichromate (Sigma, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-10-10 to 2017-10-13 (with headspace) and 2017- 10-09 to 2017-10-12 (without headspace), respectively. The reference item toxicity is in the valid range following test facility SOPs.

ErC50 (72 hours) :
Test system with Hedaspace = 0.535 mg/L;
Test system without headspace = 0.498 mg/L
Valid Range = 0.754 mg/L +/- 0.558 mg/L

EyC50 (72 hours) :
Test system with Hedaspace = 0.320 mg/L;
Test system without headspace = 0.392 mg/L
Valid Range = 0.406 mg/L +/- 0.281 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Cedrene washed was found to inhibit the growth of the algae after 72 hours with the following effect values (based on nominal item loadings):
The EL50-values for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L.
The EL10-values for both inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were, respectively, > 100 mg/L and 3.31 mg/L.

The NOEL values for inhibition of growth rate and yield after 72 hours were 25.0 mg/L, respectively.

Executive summary:

In this study, Cedrene washed was found to inhibit the growth of the algae after 72 hours with the following effect values (based on nominal item loadings):

The EL₅₀-values for both inhibition of growth rate (E_rL₅₀) and yield (E_yL₅₀) after 72 hours were > 100 mg/L.

The EL₁₀-values for both inhibition of growth rate (E_rL₁₀) and yield (E_yL₁₀) after 72 hours were, respectively, > 100 mg/L and 3.31 mg/L.

The NOEL values for inhibition of growth rate and yield after 72 hours were 25.0 mg/L, respectively.