Vetiveria zizanioides, ext., acetylated

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:

2018

Report date:

2018

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

rel-((3aR,6R)-7,7-dimethyl-8-methyleneoctahydro-1H-3a,6-methanoazulen-3-yl)methyl acetate

Molecular formula:

C17H26O2

IUPAC Name:

rel-((3aR,6R)-7,7-dimethyl-8-methyleneoctahydro-1H-3a,6-methanoazulen-3-yl)methyl acetate

Constituent 2

Reference substance name:

2-(8,8a-dimethyl-3,4,6,7,8,8a-hexahydronaphthalen-2(1H)-ylidene)propyl acetate

Molecular formula:

C17H26O2

IUPAC Name:

2-(8,8a-dimethyl-3,4,6,7,8,8a-hexahydronaphthalen-2(1H)-ylidene)propyl acetate

Constituent 3

Reference substance name:

rel-(8R,8aS)-8,8a-dimethyl-2-(propan-2-ylidene)-1,2,3,7,8,8a-hexahydronaphthalene

Molecular formula:

C15H22

IUPAC Name:

rel-(8R,8aS)-8,8a-dimethyl-2-(propan-2-ylidene)-1,2,3,7,8,8a-hexahydronaphthalene

Constituent 4

Reference substance name:

rel-(5R,10R)-6,10-dimethyl-2-(propan-2-ylidene)spiro[4.5]dec-6-en-8-one

Cas Number:

22196-46-9

Molecular formula:

C15H22O

IUPAC Name:

rel-(5R,10R)-6,10-dimethyl-2-(propan-2-ylidene)spiro[4.5]dec-6-en-8-one

Constituent 5

Reference substance name:

rel-(4R,4aS)-4,4a-dimethyl-6-(propan-2-ylidene)-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one

Cas Number:

16981-90-1

Molecular formula:

C15H22O

IUPAC Name:

rel-(4R,4aS)-4,4a-dimethyl-6-(propan-2-ylidene)-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one

Constituent 6

Reference substance name:

rel-(1R,8aS)-1,8a-dimethyl-7-(propan-2-ylidene)-1,2,3,7,8,8a-hexahydronaphthalene

Molecular formula:

C15H22

IUPAC Name:

rel-(1R,8aS)-1,8a-dimethyl-7-(propan-2-ylidene)-1,2,3,7,8,8a-hexahydronaphthalene

Constituent 7

Reference substance name:

2-methoxy-4-prop-1-enylphenyl acetate

EC Number:

202-236-1

EC Name:

2-methoxy-4-prop-1-enylphenyl acetate

Cas Number:

93-29-8

Molecular formula:

C12H14O3

IUPAC Name:

2-methoxy-4-(prop-1-en-1-yl)phenyl acetate

Constituent 8

Reference substance name:

rel-(1R,4aS,8aR)-1,4a-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,8a-octahydronaphthalene

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,4aS,8aR)-1,4a-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,8a-octahydronaphthalene

Constituent 9

Reference substance name:

rel-(3aR,6R,8aR)-7,7-dimethyl-8-methylenehexahydro-1H-3a,6-methanoazulen-3(2H)-one

Cas Number:

64550-95-4

Molecular formula:

C14H20O

IUPAC Name:

rel-(3aR,6R,8aR)-7,7-dimethyl-8-methylenehexahydro-1H-3a,6-methanoazulen-3(2H)-one

Constituent 10

Reference substance name:

rel-(1R,4aS,8aR)-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,8a-hexahydronaphthalene

Cas Number:

31983-21-8

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,4aS,8aR)-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,8a-hexahydronaphthalene

Constituent 11

Reference substance name:

6-isopropyl-4,8a-dimethyl-1,2,3,7,8,8a-hexahydronaphthalene

Cas Number:

473-14-3

Molecular formula:

C15H24

IUPAC Name:

6-isopropyl-4,8a-dimethyl-1,2,3,7,8,8a-hexahydronaphthalene

Constituent 12

Reference substance name:

rel-(4R,4aR)-4,4a-dimethyl-6-(prop-1-en-2-yl)-1,2,3,4,4a,7-hexahydronaphthalene

Molecular formula:

C15H22

IUPAC Name:

rel-(4R,4aR)-4,4a-dimethyl-6-(prop-1-en-2-yl)-1,2,3,4,4a,7-hexahydronaphthalene

Constituent 13

Reference substance name:

rel-(1R,8aR)-1-isopropyl-4,7-dimethyl-1,2,3,5,6,8a-hexahydronaphthalene

Cas Number:

317819-82-2

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,8aR)-1-isopropyl-4,7-dimethyl-1,2,3,5,6,8a-hexahydronaphthalene

Constituent 14

Reference substance name:

Likely sesquiterpine, MW 202

Molecular formula:

C15H22

IUPAC Name:

Likely sesquiterpine, MW 202

Constituent 15

Reference substance name:

Likely acetylated sesquiterpenes and acetylated sesquiterpene alcohols

IUPAC Name:

Likely acetylated sesquiterpenes and acetylated sesquiterpene alcohols

Constituent 16

Reference substance name:

Likely oxygenated sesquiterpenes

IUPAC Name:

Likely oxygenated sesquiterpenes

Constituent 17

Reference substance name:

Unknown constituent, MW 260

IUPAC Name:

Unknown constituent, MW 260

Test material form:

liquid

Specific details on test material used for the study:

Test Item: Vetiveryl Acetate
Givaudan Code No.: 0519103
Batch number: Ve00464206
CAS No.: 84082-84-8
Content: complex mixture
Appearance: yellow to brownish yellow liquid
Expiry date: January 07, 2019
Recommended sotrage: Dry, well ventilated, preferably full, hermetically sealed, amient temperature (10-30 °C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Specific chemical analysis was performed at the start of exposure (0 hours) to check for consistent preparation of the WAFs and at the end of the exposure phase (72 hours) to assess the stability of the WAFs during the test.

Fortified samples were prepared in algae dilution water.
For extraction the samples and the control were prepared in a headspace vial and homogenized. Afterwards cyclohexane and 10% (w/w) sodium chloride were added (approx. 2 g for 20 mL). The samples were shaken for 2 min. The cyclohexane phase was removed with a pipette and transferred into a vial containing sodium sulfate for drying the extract.

Sample storage: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction (WAF):
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble and multi-component test items, a modification of the standard method for the preparation of aqueous media is performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media is prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms are exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure is expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
Five water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L (spacing factor 2). The loading levels are based on the results of a preliminary range finding test (non GLP, closed bottles without headspace).
For each loading level, an appropriate amount of the test item was pipette into a brown glass flask with an appropriate amount of dilution water (Table 2). These dispersions were shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour at room temperature, the aqueous phase or WAFs were removed by siphoning (from the approximate bottom of the glass flask). The resulting water accommodated fractions (WAF) were used in the test.

Test loading
Per definition of the WAF, all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Control
Six replicates (without test item) were exposed under the same conditions as the test loadings.

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

Total exposure duration:

72 h

Test conditions

Test temperature:

Nominal range: 21 - 24°C, controlled at ± 2°C. The room temperature was measured continuously.

pH:

The pH-values at the start of exposure were measured in one additional replicate of each test item loading and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item loading and the control.

Nominal and measured concentrations:

Range finding test: nominal loadings of 100, 10.0, 1.00 mg/L
Definitive test: nominal loadings of 100, 50, 25, 12.5, 6.25 mg/L
measured concetrations of 74.5, 44, 20.6, 12.1, 5.86 mg/L at 0 hours (fresh medium)
measured concentrations of 27.3, 26.3, 11.2, 7.76, 4.23 mg/L at 72 hours (old medium)

The measured concentrations of the test item at the start of the exposure increased with the applied dose and were in the range of 5.86 to 74.5 mg/L. At test end, the test item concentrations were in the range of 37 to 72% of the initially measured concentrations indicating some instability of WAF components.

Details on test conditions:

Test method A static test was carried out. With regard to the volatility of the test item glass flasks without headspace were used to reduce losses of test item.
Duration of the test: 72 hours
Replicates Three replicates per loading level and six for the control. Separate replicates for each measuring time were prepared.
Test container: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals.
Test volume: 59 mL
Preculture: A four days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL Actual: 6839 cells/mL
Application: Application was carried out by adding appropriate volumes of the water accommodated fractions to the replicate test vessels.
Incubation: The flasks were positioned randomly and repositioned daily.
Temperature (target): Nominal range: 21 - 24°C, controlled at ± 2°C
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity (target): Approximately 4440 to 8880 lux, corresponding to 60 to 120 pE*nrr2*S'1
Light regime: 24 hours/day light
Light homogeneity (target): Within ± 15% over incubation area

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

76.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 66.9 - 86.4

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

22.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL <6.25 - 31.2

Details on results:

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 VETIVERYL ACETATE 112 EXTRA.

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, the EC10 has been selected as the key value for long-term (chronic) aquatic hazard classification.

Results with reference substance (positive control):

The toxicity of potassium dichromate (Sigma-Aldrich, 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.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, VETIVERYL ACETATE 112 EXTRA was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal item loadings): The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 76.6 (66.9 - 86.4) mg/L and 32.6 (26.9 - 38.1) mg/L, respectively. The EL10-values with 95% confidence intervals for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 22.1 (< 6.25 - 31.2) and < 6.25 mg/L. The NOEL- values for inhibition of growth rate and yield after 72 hours were 6.25 and < 6.25 mg/L, respectively.

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 v1.1). 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 EL50 and EL10 based on growth rate are used for classification purposes, which were determined in this study to be 76.6 mg/L and 22.1 mg/L respectively.